! I* "[■ '■' QK 551 9< THE METRIC SYSTEM 10 6entImeiter rule The upper edge is in millimeters, the lower in centimeters, and half centimeters. UNITS. The most commonly used divisions and multiples. (Centimeter (era.), o.oi Meter; Millimeter (mm.), o.ooi Meter: Micron i/j.), TI ? p "tL - o.ooi Millimeter ; the Micron is the unit in Micrometry (J 166). ' ' ' ^Kilometer, iooo Meters ; used in measuring roads and other long distances. THE GRAM FOR ( Milligram { mg.) , o.ooi Gram. "WEIGHT. . . / Kilogram, iooo Grams, used for ordinary masses, like groceries, etc. the liter for J Cubic Centime ler ( cc. ), o.ooi Liter. This is more common than the correct capacity. . \ form, Milliliter. Divisions of the Units are indicated by the Latin prefixes : deci, o.i ; centi, o.oi ; Milli, o.ooi ; Micro, one millionth (o.oooooi) of any unit. Multiples are designated by the Greek prefixes : deka, io times ; hecto, ioo times ; kilo, iooo timse ; myria, 10,000 times ; Mega, one million (i,ooo,ooo) times any unit. TABLE OF METRIC AND ENGLISH MEASURES Meter (Unit of length )=ioo centimeters ; 1,000 millimeters ; 1,000,000 microns (// ) 39.3704 inches ; 3.2S08 feet ; 1.0936 yards. Centimeter (cm. )=io millimeters ; 10,000 microns (u) 0.01 meter ; 0.3937 (I) inch. Millimeter (mm.)=i,ooo microns (/') ; 0.1 cm. ; 0.001 meter ; 0.03937 (o- 1 -) inch. Micron (u) (Unit of measure in micrometry (§ i66)=o.ooi millimeter ; one mil- lionth of a meter ; 0.00003937 ( rrimr) lncn - Yard=3 feet ; 36 inches ; 0.91439 meter ; 91.4399 centimeters. Foot=i 2 inches ; 30.4799 centimeters ; 304.799 millimeters. Inch^yV foot ; 3 l 6 yard ; 25.3999 millimeters (2.54 centimeters). Liter (Unit of capacity) = 1,000 cubic centimeters (milliliters) ; (1 quart — . ) Cubic centimeter=o.ooi liter (milliliter) ; (^ cub. inch.) Fluid ounce (8 fluidrachms) =29.574 cubic centimeters (30 cc. ). Gram (Unit of weight)= 1 cc. of water ; 15.432 grains. Kilogram=i, 000 grams ; 2.2046 (2^) lbs. avoirdupois. Ounce avoirdupois=437J- grains ; 28.349 grams. 1 ms Ounce Troy or apothecaries=4So grains ; 31.103 grams) J 5 • trf TEMPERATURE To change Centigrade to Farenheit : (CX I) +32 =F. For example, to find the equivalent of io° Centigrade, C.= io°X f+ 3 2 = 5°° E. To change Farenheit to Centigrade : ( F. — 32 ) x | = C. For example to re- duce 50 Farenheit to Centigrade, F.= 50°, and (50 — 32°)X § = IOC. ; or — 40 Farenheit to Centigrade, F.= — 40 ( — 40 — 32°)= — 72 , whence — 72°Xf = — 40 C. Address of American Opticians : For the price of microscopes and microscopical supplies the student is advised to obtain a catalog of one or more of the opticians. Nearly all of them import foreign apparatus. For all institutions entitled to duty free importation, American microscopes are sold at duty free rates. For the foreign opticians see the table of tube-length p. 14. Q The Bausch and Lomb Optical Co., New York, Rochester, and Chicago. Eimer and Amend, 205-211 3d Ave., New York. The Franklin Laboratory Supply Co , Harcourt St., Boston, Mass. The Gundlach Optical Company, Rochester, N. Y. Wffl, Krafft (Representative of Leitz in America), 411 West 59th St., New York. Edward Pennock, , 3609 Woodland Ave., Philadelphia, Pa. Queeu & Company, 1010 Chestnut St., Philadelphia, Pa. Richards & Co., 12 East iSth St., New York, and 108 Lake St., Chicago, Ills. Spencer Lens Company, 367-373 Seventh St., Buffalo, N. Y- Williams, Brown & Earle, 91S Chestnut St., Philadelphia, Pa. G. S. Woolman (Queen & Co. in New York), 116 Fulton St., New York. Joseph Zentmayer, 226-22S South 15th St., Philadelphia, Pa. Besides the names here given, nearly every large city has one or more dealers in microscopes and microscope supplies. Cornell University Library QM 551.G13 1901 The microscope; an Introduction to micros 3 1924 001 037 062 The original of this book is in the Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://archive.org/details/cu31924001037062 THE MICROSCOPE AN INTRODUCTION TO MICROSCOPIC METHODS AND TO HISTOLOGY BY SI M O N HENRY GAG E PROFESSOR OF MICROSCOPY, HISTOL- OGY AND EMBRYOLOGY IN CORNELL UNIVERSITY, AND THE NEW YORK STATE VETERINARY COLLEGE. EIGHTH EDITION REVISED, ENLARGED AND ILLUSTRATED BY OVER TWO HUNDRED FIGURES COMSTOCK PUBLISHING COMPANY ITHACA, NEW YORK 1901 PREFACE TO THE EIGHTH EDITION AS the preface to the sixth edition of this work expresses accurately what should be said to-day it is appended : "The rapid advance in microscopical knowledge, and the great strides in the sci- ences employing the microscope as an indispensable tool, have reacted upon the microscope itself, and never before were microscopes so excellent, convenient and cheap. Indeed, the financial reason for not possessing a microscope can no longer be urged by any high school or academy, or by any person whose profession de- mands it. Naturally, to get the greatest good from instruments, tools, or machines of any kind, the one ■who uses them must understand the principles upon which their action depends, their possibilities and limitations. That the student may acquire a just comprehension of some of the fundamental principles of the microscope, and gain a working acquaintance with it and its ap- plications, this book has been prepared. It is a growth of the laboratory, and has been modified from time to time to keep pace with optical improvements and ad- vancing knowledge." In rewriting this edition the different chapters have been recast, new figures added and in most cases the matter considerably increased. A new chapter has been added upon class demonstrations. The general availability of the constant electric current, and the improvement in apparatus have made micro-projection practicable and satisfactory. It has served the writer so well in his teaching of histology and embryology that it seemed worth while to give the benefit of his ex- perience to his fellow workers. It is hoped that the book as it now stands will serve more completely than ever before the needs of the class-room and of the laboratory. "Simply reading a work on the microscope, and looking a few times into an instrument completely adjusted by another, is of very little value in giving real knowledge. In order that the knowledge shall be made alive, it must become a part of the student's experience by actual experiments carried out by the student himself. Consequently, exercises illustrating the principles of the microscope and the methods of its employment have been made an integral part of the work. "In considering the real greatness of the microscope, and the truly splendid service it has rendered, the fact has not been lost sight of that the microscope is, after all, only an aid to the eye of the observer, only a means of getting a larger image on the retina than would be possible without it ; but the appreciation of iv PREFACE the retinal image, whether it is made with or without the aid of a microscope, must always depend upon the character ami training of the seeing and appreciat- ing brain behind the eye. The microscope simply aids the eye in furnishing raw material, so to speak, for the brain to work upon. ( From 3d ed. ) Grateful acknowledgment is made to the opticians and instrument makers for the loan of cuts and for courteous and complete answers to numerous question ; to the directors of laboratories in different parts of the country, to his colleagues in the departments of Physics, Chemistry and Electrical Engineering in Cornell University ; and finally to his pupils past and present wdio have given their sup- port and encouragement. In closing I would like to urge those who are interested in Microscopy to take same microscopical journal, ami if possible to become a member of some microscopical club or society. One can do very little alone, but by helping others ami being helped in return, the workers in any field of human endeavor can accomplish great things. SIMON HENRY GAGE, Cornell University, October /, /go/. Ithaca, N. Y., U. S. A. CONTENTS CHAPTER I PAGE \ l- 59 — The Microscope and its parts i- 33 CHAPTER II | 60-128 — Lighting and Focusing; Manipulation of Dry, Adjustable ami Immersion Objectives ; Care of the Microscope and of the Eyes. Laboratory Microscopes 34- 89 CHAPTER HI % 129-153 — Interpretation of Appearances. 90-102 CHAPTER IV # 154-176 — Magnification and Micrometry 103-121 CHAPTER V I 177-187 — Drawing with the Microscope 122-133 CHAPTER VI 'i 188-233 — The Microspectroscope and Polariscope ; Micro-Chemistry ; Textile Fibers and Food Products ; Micro-Metallography 134-160 CHAPTER VII (J 234-335 — Slides and Cover-Glasses ; Mounting ; Isolation ; Sectioning by the Collodion and the Paraffin Methods ; Serial Sections ; Labeling and Storing Microscopical Preparations ; Reagents and their Preparation 161-204 CHAPTER VIII i 336-392 — Photographing objects with a Vertical Camera ; Photograph- ing Large Transparent Objects ; Photographing with a Mi- croscope ; (A) Transparent Objects ; (B) Opaque Objects, and the .Surfaces of Metals and Alloys ; Enlarging Nega- tives ; Photographing Petri Dishes and Culture Tubes 205-242 CHAPTER IX i 393-4 2 6 — Class-Room Demonstrations with the Microscope ; With the Projection Microscope; with the Episcope 243.267 CHAPTER X \ 427-438 — The Abbe Test- Plate and Apertometer ; Equivalent Focus of Objectives and Oculars ; Drawings for Photo-Engravings ; Wax Models ; Some Apparatus for Imbedding and Section- ing 268-281 BOOKS AND PERIODICALS 282-28S INDEX 289 THE MICROSCOPE IN SECTION r. Huygenian ocular (see p. 102 for positive ocular). 2. Draw-tube by which the tube is length- ened or shortened. 3. Main tube or body containing the draw- tube, and attached to the pillar by the arm 4. Society screw in the lower end of the draw-tube. 5. Society screw in the lower end of the tube, (1 ( >bjective in position. 7. Stage, under which is the substage with the substage condenser. 8. Spring clip for holding the specimen. 9. Screw for centering, and handle for the iris diaphragm in the achromatic con- denser (see Fig. 41 ). 10 Iris diaphragm outside the principal focus of the condenser for use in cen- tering (§81) Mirror with plane aud concave faces. Horse-shoe base. Rack and pinion for the substage cou- denser i,|. Jointed pillar. is. Part of pillar with spiral spring of fine adjustment. Screw of line adjustment. Milled head of coarse adjustment. THE MICROSCOPE AND MICROSCOPICAL METHODS CHAPTER I THE MICROSCOPE AND ITS PARTS APPARATUS AND MATERIAL FOR THIS CHAPTER A simple microscope ($ 2, II) ; A compound microscope with nose-piece (Figs. 70-80); eye-shade (Fig. 60), achromatic (J 20), apochromatic (\ 22), dry (S17), immersion {\ iS), unadjustable and adjustable objectives (J 23, 24) ; Huygenian or negative (| 38), positive (£37) and compensation oculars (§ 39) ; stage microme- ter (Ch. IV) ; homogeneous immersion liquid ( i? 18) ; mounted letters or figures ( '/■ 53 ) ; ground-glass and lens paper ( i, 53) . A MICROSCOPE (jr. A Microscope is an optical apparatus with which one may obtain a clear image of a near object, the image being always larger than the object ; that is, it enables the eye to see an object under a greatty increased visual angle, as if the object were brought very close to the eye without affecting the distinctness of vision. Whenever the microscope is used for observation, the eye of the observer forms an integral part of the optical combination (Figs. 16, 21). £ 2. A Simple Microscope. — With this an enlarged, erect image of an object may be seen. It always consists of one or more converging lenses or lens-systems (Figs. 16-20), and the object must be placed within the principal focus (I 11). The simple microscope may be held in the hand or it may be mounted in some way to facilitate its use (Figs 17-20). MICROSCOPE AND ACCESSORIES [CIS. I Figs. 1-9, showing the Principal Optic Axis and the Optical Center of various forms of Lenses. Axis. The Principal Optic Axis, c-c' . Centers of curvature of the two sur- faces of the lens, c.l. Optical center of the lens, r-r' . Radii of curvature of the two lens surfaces, t-t' ' . Tangents in Eig. 4. ? /: 3. Principal Optic Axis. — In spherical lenses, i. e. % lenses whose surfaces are spherical, the Axis is a line joining the centers of curvature and indefinitely extended. In the lens it is the unbroken part of the line c-c' in the figures. In lenses with one plane surface (Figs. 3, 6, 7) the radius of the plane surface is any line at right angles to it, but in determining the axis it must be the one which is continuous with the radius of the curved surface, consequently the axis in such lenses is on the radius of the curved surface which meets the plane surface at right angles. # 4. Optical Center. — The optical center of a lens is the point through which rays pass without angular deviation, that is, the emergent ray is parallel to the [CH. I MICROSCOPE AND ACCESSORIES incident ray. It is determined geometrically by drawing parallel radii of the curved surfaces, r-r' in Figs. 4-9, and joining the peripheral ends of the radii. The optical center is the point on the axis cut by the line joining the peripheral ends of the parallel radii of the two lens surfaces. In Figs. 4-5 it is within the lens ; in 6-7 it is at the curved surface, and in the meniscus (8, 9) it is wholly out- side the lens, being situated on the side of the greater curvature. In determining the center in a lens with a plane surface, the conditions can be satisfied only by using the radius of the curved surface which is continuous with the axis of the lens, then any line at right angles to the plane surface will be parallel with it, and may be considered part of the radius of the plane surface. (That is, a plane surface may be considered part of a sphere with infinite radius, hence any line meeting the plane surface at right angles may be considered as the peripheral part of the radius. ) In Figs. 6, 7, (r') is the radius of the curved sur- face and (r) of the plane surface ; and the point where a line joining the ends of these radii crosses the axis is at the curved surface in each case. By a study of Fig. 4 it will be seen that if tangents be drawn at the peripheral ends of the parallel radii, the tangents will also be parallel and a ray incident at one tangential point and traversing the lens and emerging at the other tangential point acts as if traversing, and is practically traversing a piece of glass which has parallel sides at the point of incidence and emergence, therefore the emergent ray will be parallel with the incident ray. This is true of all rays traversing the center of the lens. Thick Lenses. — In all of the diagrams of lenses and the course of rays through them in this book the lenses are treated as if they were infinitely thin. In thick lenses like those figured, while there would be no angular deviation for rays trav- ersing the center of the lens, there would be lateral displacement. This is shown in Fig. 57 illustrating the effect of the cover-glass. \ 5. Secondary Axis. — Every raj- traversing the center of the lens, except the principal axis, is a secondary axis ; and every secondary axis is more or less oblique to the principal axis. In Fig. 14, line (2), is a secondary axis, and in Fig. 15, line (1 ). See also Fig. 5S. Figs. 10, 11. — Sectional views of a concave or diverging and a convex or converging lens to show that in the con- cave lens the principal focus is virtual as indicated by the dotted lines, while with the convex lens the focus is real and on the side of the lefts opposite to that from which the light comes. \ 6. Principal Focus. — This is the point where rays parallel with the axis and traversing the lens cross the axis ; and the distance from the focus to the center of the lens measured along the axis is the Principal Focal Distance. In the diagrams, Fig. 10 is seen to be a diverging lens and the rays cross the axis only by being pro- jected backward. Such a focus is said to be virtual, as it has no real existence. In MICROSCOPE AND ACCESSORIES [CH. I Fig. II the rays do cross the axis and the focus is said to be real. If the light came from the opposite direction it would be seen that there is a principal focus on the other side, that is there are two principal foci, one on each side of the lens. These two foci are both principal foci, but they will be equally distant from the center of the lens only when the curvature of the two lens surfaces are equal. There may be foci on secondary axes also, each focus on a secondary axis has its conjugate. In the formation of images the image is the conjugate of the object and conversely the object is the conjugate of the image. Fig. 12. — Double Convex Lens, Showing Chromatic Aberration. The ray of white light (w) is represented as dividing into the short waved, bine [b) and the long waved, red (r) light. The blue (b) ray comes to a focus nearer the lens and the red ray (r) farther from the lens than the principal focus ( _/). Principal focus ( /') for rays very near the axis ; f and f" , foci of blue and red light coming from near the edge of the lens. The intermediate wave lengths would have foci all the way between f and f". '£ 7. Chromatic Aberration — This is due to the fact that ordinary light con- sists of waves of varying length, and as the effect of a lens is to change the direc- tion of the waves, it changes the direction of the short waves more markedly than the long waves. Therefore, the short waved, blue light will cross the axis sooner than the long waved, red light, and there will result a superposition of colored images, none of which are perfectly distinct (Fig. 12). Fig. 13. The ray (0) near the edge of the lens is brought to a focus nearer the lens than the ray (/). Both are brought to a focus sooner than rays very near the axis, (f) Principal focus for rays very near the axis; (f) Focus for the ray (*'), and [f") Focus for the ray (o). Intermediate rays would cross the axis all the "way from if" tof). 'i S. Spherical Aberration— This is due to the unequal turning of the light in different zones of a lens. The edge of the lens refracts proportionally too much and hence the light will cross the axis or come to a focus nearer the lens than a ray which is nearer the middle of the lens. Thus, in Fig. 13, if the focus Fig. 13. Double Convex Lens, showing Spherical Aberration. ch. r MICROSCOPE AND ACCESSORIES of parallel rays very near the axis is at/", rays {o i), nearer the edge, would come to a focus nearer the lens, the focus of the ray nearest the edge being nearest the lens. | 9. Correction of Chromatic and of Spherical Aberration. — Every simple lens has the defect of both chromatic and spherical aberration, and to overcome this, kinds of glass of different refractive power and different dispersive power are combined, concave lenses neutralizing the defects of convex lenses. If the concave lens is not sufficiently strong to neutralize the aberrations of the convex lens, the combination is said to be under-corrected, while if it is too strong and brings the marginal raj's or the blue rays to a focus beyond the true principal focus, the combination is over-corrected. In Newton's time there was supposed to be a direct proportion between the refractive power of any transparent medium and its dispersive power ( i. e. its power to separate the light into colors). If this were true then the contention of Newton that it would be impossible to do away with the color without at the same time doing away with the refraction would be true and useful achromatic combinations would be impossible. It was found by experiment, however, that there is not a direct ratio between the refractive and dispersive powers for the different colors in different forms of glass, so that it is possible to do away largely with chromatic aberration and retain sufficient refraction to make the combination serve for the production of images. ( See also the discussion under apochromatic objectives \ 22 ) Probably no higher technical skill is used in any art than is requisite in the preparation of microscopical objectives, oculars and illuminators. Figs. 14 and 15. 14. Convex lens showing the position of the object (A-B ) outside the principal focus {F i, and the course of the rays in the formation of real images. To avoid confusion the rays are drawn from only one point. A B. Object outside the principal focus. B' A'. Real, enlarged image on the opposite side of the lens. Axis. Principal optic axis. 1,2,3. Rays after traversing the lens. They are converging, and consequently form a real image. The dotted line and the line 1 2 ) give the direction of the rays as if unaffected by the lens. (F). The principal focus. Fig. 15. — Convex lens, showing the position of the object (A B) within the principal focus and the course of rays in the formation of a virtual image. A B. The object placed between the lens and its focus ; A formed by tracing the rays backward. the object, and is erect (\ 11). Axis. The principal optic axis of the lens. F. The principal focus. /,2,j. Rays from the point B of the object. They are diverging after trav- ersing the lens, but not so divergent as if no lens were present, as is shown by the 14 15 B' virtual image It appears on the same side of the lens as MICROSCOPE AND ACCESSORIES \CH. I dotted lines. Ray (/) traverses the center of the lens, and is therefore not deflected. It is a secondary axis (§ 5) . J 10. Geometrical Construction of Images. — As shown in Figs. 14-15, for the determination of any point of an image, or the image being known, to determine the corresponding part of the object, it is necessary to know the position of the principal focus (and there is one on each side of the lens, \ 6), and the optical center (Figs. 1-9 of the lens). Then a secondary axis (2) in Fig. 14, ( 1) in Fig. 15, is drawn from the extremity of the object and prolonged indefinitely above the lens, or below it for virtual images. A second line is drawn from the extremity of the object, (3) in Fig. 14, (2) in Fig. 15, to the lens parallel with the principal axis. After traversing the lens it must be drawn through the principal focal point. If now it is prolonged it will cross the secondary axis above the lens for a real image and below for a virtual image. The crossing point of these lines determines the position of the corresponding part of the image. Commencing with any point of the object the corresponding point of the image may be determined as just described, and conversely commencing with the image, corresponding points of the object may be determined. SIMPLE MICROSCOPE : EXPERIMENTS § 11. Employ a tripod or other simple microscope, and for object a printed page. Hold the eye about two centimeters from the upper surface of the magnifier, then alternately raise and lower the magnifier until a clear image may be seen. (This mutual arrangement of micro- scope and object so that a clear image is seen, is called focusing). When a clear image is seen, note that the letters appear as with the unaided eye except that they are larger, and the letters appear erect or right side up, instead of being inverted, as with the compound microscope (§ 12). Fig. 16. Diagram of the simple microscope show- ing the course of the rays and all the images, and that the eye forms an integral part of it. A'B 1 . The object within the principal focus. A> B 3 . The virtual image on the same side of the lens as the object. It is indicated with dotted lines, as it has no actual existence. B- A-. Retinal image of "the object (A 1 S 1 ). The virtual image is simply a projection of the retinal image in the field of vision. Axis. The principal optic axis of the micro- scope and of the eye. Cr. Cornea of the eye. L. Crystalline lens of the eye. R. Ideal refracting surface at which all the refractions of the eve may be assumed to lake place. A<- U-^s 3 CH. I] MICROSCOPE AND ACCESSORIES Hold the simple microscope directly toward the sun and move it away from and toward a piece of printed paper until the smallest bright point on the paper is obtained. This is the burning point or focus, and as the rays of the sun are nearly parallel, the burning point represents approximately the principal focus (Fig. n). With- out changing the position of the paper or the magnifier, look into the magnifier and note that the letters are very in- distinct or invisible. Move the magnifier a centimeter or two farther from the paper and no image can be seen. Now move the magnifier closer to the paper, that is, so that it is less than the focal distance from the paper, and the letters will appear distinct. This shows that in order to see a distinct image with a simple microscope, the object must always be nearer to it than its principal focal point. Or, in other words, the object must be within the principal focus. Com- pare (§53). After getting as clear an image as possible with a simple micro- scope, do not change the position of the microscope but move the eye nearer and farther from it, and note that when the eye is in one posi- tion, the largest field may be seen. This position corresponds to the eye-point (Fig. 30) of an ocular, and is the point at which the largest number of rays from the microscope enter the eye. Note that the image appears on the same side of the magnifier as the object. Fig. 17. Tripod Magnifier. Fig. 19, Lens-holder ( The Bausch & Lomb Optical Co. ) Fig. 18. The Hastings Aplan- atic Triplet. ( The Bausch & Lomb Optical Co. ) MICROSCOPE AND ACCESSORIES [CM. / Vic,. 20. Dissecting Microscope. This is simply a device for holding the lens and the object to be observed. ( The Bausch &= Lomb Optical Co. ) Simple microscopes are very convenient when only a small mag- nification (Ch. IV) is desired, as for dissecting. Achromatic triplets are excellent and convenient for the pocket. For use in conjunction with a compound microscope, the tripod magnifier (Fig. 17) is one ot the best forms. For many purposes a special mechanical mounting is to be preferred. COMPOUND MICROSCOPE § 12. A Compound Microscope. — This enables one to see an enlarged, in- verted image. It always consists of two optical parts — an objective, to produce an enlarged, inverted, real image of the object, and an ocular acting in general like a simple microscope to magnify this real image (Fig. 21 ). There is also usually present a mirror, or both a mirror and some form of condenser or illuminator for lighting the object. The stand of the microscope consists of certain mechanical arrangements for holding the optical parts and for the more satisfactory use of them. (See frontispiece. ) jj 13. The Mechanical Parts of a laboratory, compound microscope are shown injthe frontispiece, and are described in the explanation of that figure. The stu- CH. /] MICROSCOPE AND ACCESSORIES dent should study the figure with a microscope before him and become thoroughly familiar with the names of all the parts. See also the cuts of microscopes at the end of Ch. II. OPTICAL PARTS '',. 14. Microscopic Objective. — This consists of a converging lens or of one or more converging lens-systems, which give an enlarged, inverted, real image of the object (Figs. 14,21). And as for the formation of real images in all cases, the object must be placed outside the principal focus, instead of within it, as for the simple microscope. (See {i; 11, 53, Figs. 16, 2 [ ) Modern microscopic objectives usu- ally consist of two or more systems or combinations of lenses, the one next the object being called the front com- bination or lens, the one farthest from the object and nearest the ocular, the back combination or system. There may be also one or more intermediate sys- tems. Each combination is, in general, composed of a convex and a concave lens. The combined action of the sys- tem serves to produce an image free from color and from spherical distor- tion. In the ordinary achromatic ob- jectives the convex lenses are of crown and the concave lenses of flint glass (Figs. 22, 23). Fig. 21. Diagram showing the principle of a compound microscope with the course of the rays from the object {A B) through the objective to the real image { B' A' '), thence through the ocu- lar and into the eye to the retinal image (A 2 B 2 ), and the projection of the retinal image into the field of vision as the virtual image ( B^A 1 ). A B. The object. A 2 B=. The retinal image of the inverted real image, (B'A<), formed by the objective. B'A^. The inverted virtual image, a projection of the retinal image. MICROSCOPE AND ACCESSORIES [CM. I Axis. The principal optic axis of the microscope and of the eye. Cr. Cornea of the eye. L. Crystalline lens of the eye. R. Single, ideal, re- fracting surface at which all the refractions of the eye may be assumed to take place. F. F. The principal focus of the positive ocular and of the objective. Mirror. The mirror reflecting parallel rays to the object. The light is central. See Ch. II. Pos. Ocular. An ocular in -which the real image is formed outside the ocular. Compare the positive ocular with the simple microscope (Fig. 16). 15- NOMENCLATURE OR TERMINOLOGY OF OBJECTIVES Equivalent Focus. — In America, England, and sometimes also on the Continent, objectives are designated by their equivalent focal length. This length is given either in inches ( usually contracted to in. ) or in millimeters (mm. ) Thus : An objective designated T \, in. or 2 mm., indicates that the objective produces a real image of the same size as is produced by a simple converging lens whose principal focal distance is fa inch or 2 millimeters (Fig. II). An objective marked 3 in. or 75 mm., produces approximately the same sized real image as a simple converging lens of 3 inches or 75 millimeters focal length. And in accord- ance with the law that the relative size of object and image vary directly as their distance from the center of the lens (Figs. 14, 15, see Ch. IV,) it follows that the less the focal distance of the simple lens or of the equivalent focal distance of the objective, the greater is the sizeof the real image, as the tube-length remains con- stant and the image in all cases is found at about 160 or 250 mm. from the objective. ''1, 16. Numbering or Lettering Objectives. — Instead of designating objectives by their equivalent focus, many Continental opticians use letters or figures for this purpose. With this method the smaller the number, or the earlier in the alpha- bet the letter, the lower is the power of the objective. ( See further in Ch. IV, for the power or magnification of objectives). This method is entirely arbitrarv and does not, like the one above, give direct information concerning the objective. \ 17. Air or Dry Objectives. — These are objectives in which the space be- tween the front of the objective and the object or cover-glass is filled with air (Fig. 22). Most objectives of low and medium power (z. e,, \ in. or 3 mm. and lower powers) are dry. Fig. 22. Sec/ion of a dry objective showing working distance and lighting by refected light. Axis. The principal optic axis of the ob- jeclive. B C. Back Combination, composed of a plano-concave lens of flint glass (F '), and a double convex lens of crown glass (c). F C. Front Combination. C, O, si. The cover-glass, object and slide. Mirror. The minor is represented as above the stage, and as reflecting parallel rays from its plane face upon the object. Stage. Section of the stage of the microscope. CH. /] MICROSCOPE AND ACCESSORIES IV. The Working Distance, that is the distance from the front of the objective to the object when the objective is in focus. \ iS. Immersion Objectives. — An immersion objective is one with which there is some liquid placed between the front of the objective and the object or cover-glass. The most common immersion objectives are those ( A) in which water is used as the immersion fluid, and (B) where some liquid is used having the same refractive and dispersive power as the front lens of the objective. Such a liquid is called homogeneous, as it is optically homogeneous with the front glass of the objective. It may consist of thickened cedar wood oil or of glycerin contain- ing some salt, as stannous chlorid in solution. When oil is used as the immersion fluid the objectives are frequently called oil immersion objectives. The disturb- ing effect of the cover-glass (Fig. 57) is almost wholly eliminated by the use of homogeneous immersion objectives, as the rays undergo very little or no refraction on passing from the cover-glass through the immersion medium and into the ob- jective ; and when the object is mounted in balsam there is practically no refrac- tion in the ray from the time it leaves the balsam till it enters the objective. Fig. 23. Sectional view of an Immersion, Ad- justable Objective, and the object lighted with axial or central and with oblique light. Axis. The principal optic axis of the objective. B C, M C, EC. The hack, middle and front combination of the objective. In this case the front is not a combination, but a single plano- convex lens. A, B. Parallel rays reflected by the mirrot axially or centrally upon the object. C. Ray reflected to the object obliquely. I. Immersion fluid between the front of the objective and the cover glass or object ( 0). Mirror. The mirror of the microscope. O. Object. It is represented without a cover- . glass. Ordinarily objects are covered whether ex- amined with immersion or with dry objectives. Stage. Section of the stage of the microscope. I 19. Non-Achromatic Objectives. — These are objectives in which the chro- matic aberration is not corrected, and the image produced is bordered by colored fringes. They show also spherical aberration and are used only on very cheap microscopes. (|| 7, 8, Figs. 12, 13). '',. 20. Achromatic Objectives. — In these the chromatic and the spherical aber- ration are both largely eliminated by combining concave and convex lenses of dif- ferent kinds of glass "so disposed that their opposite aberrations shall correct each other." All the better forms of objectives are achromatic and alsoaplanatic. That is the various spectral colors come to the same focus. I 21. Aplanatic Objectives, etc. — These are objectives or other pieces of optical apparatus (oculars, illuminators, etc.), in which the spherical distortion is 12 MICROSCOPE AND ACCESSORIES [CH. I wholly or nearly eliminated, and the curvatures are so made that the central and marginal parts of the objective focus rays at the same point or level. Such pieces of apparatus are usually achromatic also. I 22. Apochromatic Objectives. — A term used by Abbe to designate a form of objective made by combining new kinds of glass with a natural mineral ( Calcium fluorid, Fluorite, or Fluor spar). The name, Apochromatic, is used to indicate the higher kind of achromatism in which rays of three spectral colors are com- bined at one focus, instead of rays of two colors as in the ordinary achromatic ob- jectives. At the present time ( 1901 ) several opticians make apochromatic ob- jectives without using the fluorite. Some of the early apochromatics deteriorated rather quickly in hot moist climates. Those now made are quite permanent. The special characteristics of these objectives, when used with the "compen- sating oculars" are as follows : ( 1 ) Three rays of different color are brought to one focus, leaving a small ter- tiarv spectrum onlv, while with objectives as formerly made from crown and flint glass, only two different colors could be brought to the same focus. (2) In these objectives the correction of the spherical aberration is obtained for kvo different colors 111 the brightest part of the spectrum, and the objective shows the same degree of chromatic correction for the marginal as for the central part of the aperture. In the old objectives, correction of the spherical aberration was confined to raws of one color, the correction being made for the central part of the spectrum, the objective remaining under-corrected spherically for the red rays and tfzvr-corrected for the blue rays ( ''/, 9). (3) The optical and chemical foci are identical, and the image formed by the chemical rays is much more perfect than with the old objectives, hence the new objectives are well adapted to photography. (4 ) These objectives admit of the use of very high oculars, and seem to be a considerable improvement over those made in the old way with crown and flint glass. According to Dippel (Z. w. M. 18S6, p. 300) dry apochromatic objectives give as clear images as the same power water immersion objectives of the old form. '/, 23. Non-Adjustable or Unadjustable Objectives. — Objectives in wdiich the lenses or lens systems are permanently fixed in their mounting so that their rela- tive position always remains the same. Low power objectives and those with homogenous immersion are mostly non-adjustable. For beginners and those un- skilled in manipulating adjustable objectives ( \ 24), non- adjustable ones are more satisfactory, as the optician has put the lenses in such a position that the most satisfactory results may be obtained when the proper thickness of cover-glass and tube-length are employed. (See table of tube-length and thickness of cover-glass below, p. 14. ) \ 24. Adjustable Objectives. — An adjustable objective is one in which the dis- tance between the systems of lenses (usually the front and the back systems) may- be changed by the observer at pleasure. The object of this adjustment is to cor- rect or compensate for the displacement of the rays of light produced bv the mounting medium and the cover-glass after the rays have left the object. It is also to compensate for variations in "tube-length." See §29, As the displace- ment of the rays by the cover-glass is the most constant and important, these ob- jectives are usually designated as having cover-glass adjustment or correction. ( Fig. 23, See also practical work with adjustable objectives, Ch. II ). CH. I] MICROSCOPE AND ACCESSORIES 13 5 25. Parachromatic, Pantachromatic and Semi-apochromatic Objectives. — These are trade names for objectives, most of them containing one or more lenses of the new glass [\ 22). They are said to approximate much more closely to the apochromatics than to the ordinary objectives. \ 26. Variable Objective. — This is a low power objective of 36 to 26 mm. equivalent focus, depending upon the position of the combinations. By means of a screw collar the combinations may be separated, diminishing the power, or ap- proximated and thereby increasing it. \ 17. Projection Objectives. — These are designed especially for projecting an image on a screen and for photo-micrography. They are characterized by having a flat, sharp field brilliantly lighted. (See Ch. IV, IX.) \ 2S. Illuminating or Vertical Illuminating Objectives. — These are designed for the study of opaque objects with good reflecting surfaces, like the rulings on metal bars and broken or polished and etched surfaces of metals emplo}'ed in micro-metallography. The light enters the side of the tube or objective and is reflected vertically downward through the objective and thereby is concentrated upon the object. The object reflects part of the light back into the microscope thus enabling one to see a clear image. \ 29. Tube-Length and Thickness of Cover-Glasses. — "In the construction of microscopic objectives, the corrections must be made for the formation of the image at a definite distance, or in other words the tube of the microscope on which the objective is to be used must have a definite length. Consequently the microscopist must know and use this distance or 'microscopical tube-length' to obtain the best results in using any objective in practical work." Unfortunately different opticians have selected different tube-lengths and also different points between which the distance is measured, so that one must know what is meant by the tube-length of each optician whose objectives are used. See table. The thickness of cover-glass used on an object (See Ch. VII, on mounting), except with homogeneous immersion objectives, has a marked effect on the light passing from the object (Fig. -57 ). To compensate for this the position of the sys- tems composing the objective are closer together than they would be if the object were uncovered. Consequently, in non-adjustable objectives some standard thick- ness of cover-glass is chosen by each optician and the position of the systems arranged accordingly. With such an objective the image of an uncovered object would be less distinct than a covered one, and the same result would follow the use of a cover-glass much too thick. 14 MICROSCOPE AND ACCESSORIES \CH. I Length in Millimeters and Paris included in Various Opticians* Pts 'Tube-Length" by * C3 Tube-Length" Millimeters. Fir,. 24. Thickness of i',r mm. vP mm. '-Mi 8 mm. - : hf- mm. ] -r o's- mm. rtfV mm. - r iu-}j- mm. ucluded 'Tube- length." See Diagram. I' E. Leitz, Wetzlar 17° mm. I Natchetet Fils, Paris 160 mm. Powell and Lealand, London 254 mm. C. Reichert, Vienna 160 to 1S0 mm. Spencer Lens Co., Buffalo 160 mm. W. Wales, New York 254 mm. f Bausch & LombOpt. Co., Rochester. __ 160 or 216mm. I Bezu, Hausser et Cie, Paris 1S0 mm. I Klonne und Miiller, Berlin 160-1S0 or 254mm. -! \V. & H. Seibert, Wetzlar 170 mm. Swift & Son, London 165 to 22S 1 , mm. I C. Zeiss, Jena 160 or 250 mm. I R. Winkel, Gottingen 192 mm. . Gundlach Optical Co., Rochester 254 mm. . Ross & Co., London 254 or 160 mm. . Queen & Co., Philadelphia 160 mm. . R. & J. Beck, London 254 or 160 mm. . Hartnack, Potsdam, Germany i6oand iSomm. Verick (Stiassnie) Paris 160-200 mm. Watson & Sons, London 160-250 mm. J. Zentmayer, Philadelphia 160-235 mm. Cover-Glass for Which Non-Adjustable Objectives are Corrected by Various Opticians, f Powell and Lealand, London, i W. Wales, New York. Watson & Sons, London. I E. Leitz, Wetzlar. I. R. Winkel, Gottingen, Germany. Ross & Co., London. C Klonne und Miiller, Berlin. I Spencer Lens Co., Buffalo. I Bausch & Lomb Optical Co., Rochester. I Queen & Co., Philadelphia. C. Zeiss, Jena. C. Reichert, Vienna. ( Gundlach Optical Co., Rochester. W. and H. Seibert, Wetzlar. |_R. and J. Beck, London. J. Zentmayer, Philadelphia. I Nachet et Fils, Paris. [ Bezu, Hausser et Cie, Paris. Swift and Son, London. E. Hartnack, Potsdam, Germany, *The information contained in these tables was very kindly furnished by the opticians named, or obtained by consulting catalogs. In most of the later catalogs the information is definite, and many makers now not only put their names and the equivalent focal length on their objectives, but they add the numerical aperture ( 'i 31 ) and the tube-length for which the objective is corrected. This is in accord- ance with the recommendations of the author in the original paper on "tube- length," (Proc. Amer. Soc. Micr., Vol. IX., p. 16S, also by Bausch, Vol. XII, p. 43). If the table in this edition is compared with the original table or with that in the previous edition of this book some differences will be noted, the changes beintr Owing to information received after the table on p. 14 was printed it is neces- sary to replace that table by one containing the latest information. In this revised table tube-length b-d of the diagram greatly preponderates, and the great majority of unadjustable objectives are corrected for a thickness of cover-glass falling be- tween fifteen and twenty one hundredths of a millimeter (0.15-0.20 mm.). Length in Millimeters and Parts included in the "Tube- Length" by Various Opticians. Pts. included in "Tube- length." See Diagram. "Tube-Length" Millimeters. I 150 or 250 mm. 1 60 or 2 1 6 mm . .160 or 220 mm. . 1S0 mm. . 160 or 250 mm. 170 mm. 160 or 254 mm. .170 mm. 160 or 22S mm. 160 or 250 mm. _ 192 mm. . 160 or 250 mm. 170 mm. Fig. 24. o 20 mm 0.25 mm | Chas. Baker, London, England _ The Bausch & Lomb Optical Co., Rochester, N. Y. R. & J. Beck, London, England Bezu, Hausser & Cie, Paris, France, __ Klonne und Miiller, Berlin, Germany. _ Queen & Co., Incorporated, Phila., Pa. Ross, Ltd , London, England W. und H. Seibert, Wetzlar, Germany, .Swift & Son, London, England . .. Watson & Sons, London, England R. Winkel, Goettingen, Germany I Carl Zeiss, Jena, Germany I Ernst Leitz, Wetzlar, Germany Nachet et Fils, Paris, France 160 mm. j Powell & Lealand, London, England 254 mm. I C. Reichert, Vienna, Austria 160-180 mm. I Spencer Lens Company, Buffalo, N. Y 160 mm. I W. Wales, New York 254 mm. The Gundlach Opt. Co., Rochester, N. Y. 254 mm. E. Hartnack, Potsdam, Germany 160 mm. Dollond & Co., London, England __. 165, 240 mm. Verick (Stiassnie) Paris, France 160- 200 mm. P. Waechter, Berlin-Friedenau, Germany, 160 mm. J. Zentmayer, Philadelphia, Pa. 160 or 235 mm. kness of Cover-Glass for Which Non- Adjustable Objectives are Corrected by Various Opticians I The Bausch & Lomb Optical Co., Rochester, N. Y. ] Klonne und Miiller, Berlin, Germany. | Queen & Co., Incorporated, Philadelphia, Pa. I The Spencer Lens Co., Buffalo, N. Y. j Ernst Leitz, Wetzlar, Germany. ■ P. Wachter, Berlin-Friedenau, German}'. I R. Winkel, Goettingen, Germany, f Chas. Baker, London, England. I R. &. J. Beck, Ltd., London, England. 1 Gundlach Optical Co., Rochester, N. Y. ( W. und H. Seibert, Wetzlar, German}'. 1 E. Hartnack, Potsdam, Germany. I C. Reichert, Vienna, Austria. ( Ross, Ltd., London, England. - Vdrick (Stiassnie), Paris, France. I Carl Zeiss, Jena, Germany. J. Zentmayer, Philadelphia, Pa. 1 Dollond & Co., London, England. I Nachet et Fils, Paris, France. Bezu Hausser & Cie, Paris, France. ( Powell & Lealand, London, England. I Swift & Son, London, England. Watson & Sons, London, England. W. Wales, New York. Tin o. 18 mm. o. 17 mm o. 15 mm. o. 15-0. 18 mm. 0.15-0.20 mm. o. 12-0. 17 mm. o. 10-0. 15 mm. o. 10-0. 12 mm. o. 10 mm. CH. /] MICROSCOPE AND ACCESSORIES '5 § 30. Aperture of Objectives. — The angular aperture or angle of aperture of an objective is the "angle contained, in each case, be- tween the most diverging of the rays issuing from the axial point of an object [i.e., a point in the object situated on the optic axis of the microscope], that can enter the objective and take part in the formation of an image." (Carpenter). in the direction of uniformity and in general in the direction recommended by the writer and Mr. Bausch and the committee of the American Microscopical Society. The recommendations of the committee, published in the Proceedings, Vol. XII., p. 250, are as follows : "Believing in the desirabilit}' of a uniform tube-length for microscopes, we unanimously recommend : 1. That the parts of the microscope included in the tube-length should be the same by all opticians, and that the parts included should be those between the upper end of the tube where the ocular is inserted and the lower end of the tube where the objective is inserted. 2. That the actual extent of tube length as defined in section 1 — Be, for the short or continental tube, 160 mm., or 6.3 inches, and 216 mm., or 8)4 inches, for the long tube, and that the draw tube of the microscope possess two special marks indicating these standard lengths. 3. That oculars be made par-focal, and that the par-focal plane be coincident w-ith that of the upper end of the tube. 4. That the mounting of all object- ives of 6 mm. ( % inch) and shorter focus should be such as to bring the optical center of the objective i}£ inches below the shoulder, and that all objectives be marked with the tube-length for which they are corrected. 5. That non-adjustable objectives be corrected for cover-glass from T \j% to ffo mm. ( t i 5 to T jij inch) in thickness. These recommendations give a dis- tance of 10 inches (254 mm.) between the par-focal plane of the ocular and the op- tical center of the objective for the long tube, and are essentially in accord with the actual practice of opticians. At the request of the committee, a joint conference was held with the opti Fig. 25. The tube of a microscope with ocular micrometer and nose piece in position to show that in measuring tube-length one must measure from the eye lens to the place where the ob- jective is attached. {Zeiss' Catalog.) cians belonging to the Society and present at the meeting. They expressed their belief in the entire practicability of the above recommendations and a willingness to adopt them." (Signed) Simon H. Gage, A. Clifford Mercer, Charles E. Barr. 16 MICROSCOPE AND ACCESSORIES \_CH. I In general the angle increases with the size of the lenses forming the objective and the shortness of the equivalent focal distance (ji 15). If all objectives were dry or all water or all homogeneous immersion a comparison of the angular aper- ture would give one a good idea of the relative number of image forming rays Fig. 26. Diagram illustrating the angular aperture of a microscopic objective. Only the front lens of the objective is shown. Axis. The principal optic axis of the objective. B A, B C, the most divergent rays that can enter the objective, they mark the angular aperture. A B D or C B D half the angular aperture. This is designated by u in making Numerical Aperture computations. See the table, \ 33. transmitted by different objectives ; but as some are dry, others water and still others homogeneous immersion, one can see at a glance that, other things being equal, the dr3' objective (Fig. 27) receives less light than the water immersion, and the water im- mersion (Fig. 28) less than the homogeneous immersion (Fig. 29). In order to render comparison accurate between different kinds of objectives, Professor Abbe takes into consideration the rays actually passing from the back combi- nation of the objectives to form the real image ; he thus takes into account the medium in front of the objective as well as the angular aperture. The term "Numerical Aperture," {N. A. ) was introduced by Abbe to indicate the capacity of an optical instrument "for receiving rays from the object and transmitting them to the image. I 31. Numerical Aperture (abbreviated N. A. ), as now employed for micro- scope objectives, is the ratio of the semi-diameter of the emergent pencil to the focal length of the lens. Or as the factors are more readily obtainable it is sim- pler to utilize the relationship shown in the La Grange- Helmholtz-Abbe formula, and indicate the aperture by the expression : N. A. =n sin u. In this formula n is the index of refraction of the medium in front of the objective (air, water or homogeneous liquid), and sin u is the sine of half the angle of aperture ( Fig. 26, D B A). For the mathematical discussion showing that the expressions semi-diameter of emergent pencil — 1 — rr- ., -r\\- , = n sin u, the student is referred to the Journal local length of the lens J of the Royal Microscopical Society, iS8i,pp. 392-395, 1S9S, p. 363. For example, take three objectives each of 3 mm. equivalent focus, one being a dry, one a water immersion, and one a homogeneous immersion. Suppose that the dry objective has an angular aperture of 106°, the water immersion of 94 and the homogeneous immersion of 90 . Simply compared as to their angular aper- ture, without regard to the medium in front of the objective, it would look as if the dry objective would actually take in and transmit a wider pencil of light than either of the others. However, if the medium in front of the objective is con- sidered, that is to say, if the numerical instead of the angular apertures are compared, the results would be as follows : Numerical Aperture of a dry objective of ro6°, N. h.—n sin u. In the case of dry objectives the medium in front of the objective being air, the index of refraction is unity, whence ;/=l. Half the angular aperture isA$£°=53°. By consulting a table of natural sines it will be found that the sine of 53 is 0.799, whence N. A. =« or 1 \ sin u or 0.799=0.799.* ' I: ''i 32. Interpolation. — In practice, as in solving problems similar to those on the following pages and rhose in refraction if one cannot find a sine exactly CH. /.] MICROSCOPE AND ACCESSORIES 17 Figs. 27-29 are somewhat modified from Ellenberger, and are introduced to illustrate the relative amount of utilized light, with dry, water immersion and homogeneous immer- 27 sion objectives of the same equivalent focus. The point from which the rays emanate is in air in each case. If Canada balsam were be- neath the cover-glass in place of the air there would be practically no refraction of the rays on entering the cover glass (§ /i "=< \*W mm/ 1 wwl \l//A»"\ e., sin ^=0.731, whence N. A. =11 or 1.33 x3m u found to be 0.731, or 0.731=0.972. corresponding to a given angle ; or if one has an angle which does not correspond to any sine or angle given in the table, the sine or angle may be closely approxi- mated by the method of interpolation, as follows : Find the sine in the table nearest the sine whose angle is to be determined. Get the difference of the sines of the angles greater and less than the sine whose angle is to be determined. That will give the increase of sine for that region of the arc for 15 minutes. Divide this increase by 15 and it will give with approximate accuracy the increase for 1 minute. Now get the difference between the sine whose angle is to be determined and the sine just below it in value. Divide this difference by the amount found necessary for an increase in angle of 1 minute and the quotient will give the number of minutes the sine is greater than the next lower sine whose angle is known. Add this num- ber of minutes to the angle of the next lower sine and the sum will represent the desired angle of the sine. Or if the sine whose angle is to be found is nearer in size to the sine just greater, proceed exactly as before, getting the difference in the sines, but subtract the number of minutes of difference and the result will give the angle sought. For example take the case in Section 97 where the sine of the angle of 28° 54' is given as 0.48327. If one consults the table the nearest sines found are 0.4S099, the sine of 2S 45', and 0.4S481, the sine of 29 . Evidently 1 8 MICROSCOPE AND ACCESSORIES [CH. I With the oil immersion in the same way N. A.= n sin u ; n or the index of refraction of the homogeneous fluid in front of the objective is 1.52, and the semi-angle of aperture is j y ) -°=45°. The sine of 45 is 0.707, whence N. A.=n or 152 X sin 21 or 0.707=1.074. By comparing these numerical apertures : Dry 0.799, water 0.972, homogeneous immersion 1.074, the same idea of the real light efficiency and image power of the different objectives is obtained, as in the graphic representations shown in Figs. 27-29. If one knows the numerical aperture (N. A.) of an objective the angular aperture is readily determined from the formula ; and one ■can determine the equivalent angles of objectives used in different media (z. e., dry or immersion). For example, suppose each of three objectives has a numerical aperture (N. A.) of 0.80, what is the an- gular aperture of each ? Using the formula of N. A.=« sin u, one has N. A.= 0.80 for all the objectives. For the dry objective n = 1 (Refractive index of air). " water immersion objective 72 = 1.33 (Refractive index of water), homogeneous immersion objective 72=1. 52 (Refractive index of homogeneous liquid ). And 2 21 is to be found in each case. For the dry objective, substituting the known values the formula becomes 0.80 = 1 sin 22, or sin it = 0.80. By inspecting the table of natural sines (3d page of cover; it will be found that 0.80 is the sine of 53 degrees and 8 minutes. As this is half the angle the entire angular aperture of the dry objective must be 53 8'X2 = 106 16'. For the water immersion objective, substituting the known values in the formula as before : 0.80= 1.33 sin 72, or sin 22 = -- - =0.6015. Consulting the table of sines as before, it will be found that 0.6015 is the sine of 36 59' whence the angular aperture (water angle) is 36 5 9 'X2 = 73° 58'. For the homogeneous immersion objective, substituting the known values, the formula becomes: 0.80 = 1.52 sin 72 whence sin it = — — = 0.5263. And by consulting the table of sines it will be found then the angle sought must lie between 2S 45', and 29 . If the difference between 0.484S1 and 0.48099 be obtained, 0.48481 — 0.48099 = 0.00382, and if this increase for 15' be divided by 15 it will give the increase for 1 minute ; 0.003S2 -.- 15 .= 0.000254. Now the difference between the sine whose angle is to be found and the next lower sine is 0.48327—0.48099 = 0.00228. If this difference be divided by. the amount found necessary for 1 minute it will give the total minutes above 2S 45'; 0.00228 -f- 0.000254 = 9. That is, the angle sought is 9 minutes greater than 28°45' 28=54'. [CH. I MICROSCOPE AND ACCESSORIES 19 that this is the sine of 31 45V whence 2 u or the entire angle (balsam or oil angle) is 63 31'. That is, three objectives of equal resolving powers, each with a numerical aperture of 0.80 would have an angular aperture of 106 16' in air, 73° 58' in water and 63 31' in homogeneous immersion liquid. For the apparatus and method of determining aperture, see appendix. § 33. Table of a Group of Objectives with the Numerical Aperture (N. A) and the method of obtaining it. Half the aiigular aperture is designated by u and the index of refraction of the medium in front of the objective by n. For dry objectives this is air and n = 1, jot water immersions n = 1-33, and for homogeneous immersions n = 1 .52. {For a table of natural sines, see third page of cover. ) Objective. Angular Aperture (2«) Natural Sine of half the angular aperture (sin 11. ) Index of Refraction NUMERICAL APERTURE of the medi- um in front oftheobjec- ( N. A. ) = n sin u tive («). ! 25 mm. (Dry.) 20° Sin - - =0.1736 2 ;; = 1 N.A.= I X 0.1736 =0.173 2s mm. (Dry. ) 40° „. 40 Sin — = 0.3420 11 = 1 N.A.= I X0.3420 =0.342 12 K mm. (Dry. ) 42° Sin — = 0.3584 n = 1 N.A.= 1 X0.35S3 -0.35S 12 'i mm. (Dry.) IOO° ■ 100 Sin = 0.7660 2 n = 1 N.A.= I X 0.7660 = 0.766 6 mm. (Dry.) 75° Sin— - = 0.6087 2 n = 1 N.A.= 1 X 0.6087 =0.609 6 mm. (Dry.) 136° • 136 Sin -— = 0.9272 11 = 1 N. A.= IX0.9272 =0.927 3 mm. (Dry.) 115° Sin — - =0.8434 n = 1 N. A.= 1X0.8434=0.843 3 mm. ( Dry. ) 163° Sin- -=0.9890 n = 1 N. A.= 1X0.9S90 = 0.989 2 mm. Water. Immersion. 96° I 2' 96° I 2' Sin — - — =0.7443 "= i-33 N. A.= 1.33X 0.7443 =0.99 2 mm. Homogeneous Immersion. no°38 / iio^S' Sin —=0.8223 11 = 1.52 N. A.= 1.52x0.8223 = 1.25 2 mm. Homogeneous Immersion. i 3 4°io / „. i34°io' Sin — =0.9211 2 n ~ 1.52 N. A.= 1.52x0.9210 = 1.40 20 MICROSCOPE AND ACCESSORIES [CRT. I § 34. Significance of Aperture. — As to the real significance of aperture in microscopic objectives, it is now an accepted doctrine that — the corrections in spherical and chromatic aberration being the same — (1 ) Objectives vary directly as their numerical aperture in their ability to define or make clearly visible minute details (resolving power J. For example an objective of 4 mm. equivalent focus and a numerical aper- ture of 0.50 would define or resolve only half as many lines to the millimeter or inch as a similar objective of 1.00 N.A. So also an objective of 2 mm. focus and 1.40 N.A. would resolve only twice as many lines to the millimeter as a 4 mm. objective of 0.70 N.A. Thus it is seen that defining power is not a result of magnification but of aperture, otherwise the 2 mm. objective would resolve far more than twice as many lines as the 4 mm. objective. Taking the results of the researches of Abbe as a guide to visibility with the microscope, one has the general formula 2/lxN.A. That is twice the number of wave lengths of the light used multiplied by the numerical aperture of the objective. From this general statement it will be seen that the shorter the wave lengths of the light, the more there will be in an inch or centimeter and therefore the greater the number of lines visible in a given space. That is the kind of light used is one ele- ment and the objective the other in determining the number of lines visible under the microscope. Following Mr. E. M. Nelson (Jour. Roy. Micr. Soc, 1S93, P- : 5) it is believed that not more than ^ths of the numerical aperture of an objective is really available for microscopic stud}-, with a central, solid cone of light. To determine the number of lines visible in a given space with a given light the formula would become 2/Yx J^ths N. A. =3/2 AN. A. To determine the working-resolving power of any objective it is only necessary to know the number of light waves in a given space, say an inch or a centimeter and to multiply this number by 3/2 N. A. For example suppose one uses ordinary daylight and assumes the average wavelength is 1/46666 in., then there must be 46,666 per inch and 46,666x3/2 = 70,000 approximately. If the N. A. is 1, then the objective will resolve or make visible 70,000 lines to the inch, or ap- proximately 28,000 to the centimeter. If blue light were used the number would be 32,000 per centimeter, or 80,000 per inch. It will be seen that the number of lines here given is smaller than that in the table of Carpenter-Dallinger, because in the latter the full aperture is supposed to be employed and the light is of the greatest available obliquity. CH.I] MICROSCOPE AND ACCESSORIES 21 (2) The illuminating power of an objective of a given focus is found to vary directly as the square of the numerical aperture (N. A. )". Thus if two 4 mm. objectives of N.A. 0.20 and N. A. 0.40 were compared as to their illuminating power it would be found from the above that they would vary as o.20 2 :o.40 ? = 0.0400:0.1600 or 1:4. That is the objective of 0.20 N.A. would have but J^th the illuminating power of the one of 0.40 N.A. (3) The penetrating power, that is the power to see more than one plane, is found to vary as the reciprocal of the numerical aperture— — -r- so that in an objective of a given focus the greater the aperture the less the penetrating power. Of course when equivalent focus and numerical aperture both differ the problem becomes more complex. While all microscopists are agreed that the fineness of detail which can be seen depends directly on the numerical aperture of the objective used, the general theory of microscopic vision has two interpretations : (A.) That it is as with the unaided eye, the telescope and the photographic camera. This is the original view and the one which many are favoring at the present day (see Mercer, Proceedings of the Amer. Micr. Soc. 1896, pp. 321-396). (B) The other view originated with Professor Abbe, and in the words of Carpenter-Dallinger, pp. 62, 43 : "What this is becomes ex- plicable by the researches of Abbe. It is demonstrated that micro- scopic vision is sui generis. There is and can be, no comparison between microscopic and macroscopic vision. The images of minute objects are not delineated microscopically by means of the ordinary laws of refraction ; they are not dioptrical results, but depend entirely on the laws of diffraction. These come within the scope of and demonstrate the modulatory theory of light, and involve a characteristic change which material particles or fine structural details, in proportion to their minuteness, effect in transmitted rays of light. The change consists generally in the breaking up of an incident ray into a group of rays with large angular dispersion within the range of which periodic alter- nations of dark and light occur." For a consideration of the aperture question, its history and sig- nificance, see J. D. Cox, Proc. Amer. Micr. Soc, 1S84, pp. 5-39; Jour. Roy. Micr. Soc, 1881, pp. 303, 348, 365, 388 ; 1SS2, pp. 300, 460 ; 1883, p. 790 ; 1884, p. 20 ; 1896, p. 681 ; 1897, p. 71 ; 1898, pp. 354, 362, 592 ; Mercer, Proceedings Amer. Micr. Soc, 1896, pp. 321- MICROSCOPE AND ACCESSORIES [CH. I o - 1/ nW-^ \4 ;■ / ■- i\ i / * V / ^ ,J\ T^ -4-t 1 ill ; lilt 396 ; Lewis Wright, Philos. Mag., June, 1898, pp. 480-503 ; Carpen- ter-Dallinger, Chapter II ; Nelson, Jour. Quekett Micr. Club, VI, pp. 14-38. THE OCULAR I 35. A Microscopic Ocular or Eye-Piece consists of one or more converging lenses or lens systems, the combined action of which is, like that of a simple microscope, to magnify the real image formed by the objective. Fig. 30. Sectional view of a Huygenian ocular to show the formation of the Eye-Point. Axis .Optic axis of the ocular. D. Diaphragm of the ocular. E. L. Eye-Lens. F. L. Field-Lens. E. P. Eye-point. As seen in- section, it appears some- thing like an hour-glass. When seen as looking into the ocular, i. e., in transection, it appears as a circle of light. It is at the point cohere the most rays cross. Depending upon the relation and action of the different lenses forming oculars, they are divided into two great groups, negative and positive. \ 36. Negative Oculars are those in which the real, inverted image is formed within the ocular, the lower or field-lens serving to collect the image-forming rays somewhat, so that the real image is smaller than as if the field-lens were absent (Fig. 21 ). As the field-lens of the ocular aids in the formation of the real image it is considered by some to form a part of the objective rather than of the ocular. The upper or eye-lens of the ocular magnifies the real image. \ 37. Positive Oculars are those in which the real, inverted image of the object is formed outside the ocular, and the entire system of ocular lenses magnifies the real image like a simple microscope (Fig. 16). Positive and negative oculars may be readily distinguished, as a positive ocular may be used as a simple microscope, while a negative ocular cannot be so used when its field-lens is in the natural position toward the object. By turning the eye-lens toward the object and looking into the field-lens an image may be seen, however. In works and catalogs concerning the microscope and microscopic apparatus, and in articles upon the microscope in periodicals, various forms of oculars or eye- pieces are so frequently mentioned, without explanation or definition, that it seems worth while to give a list, with the French and German equivalents, and a brief statement of their character. Achromatic Ocular ; Fr. Oculaire achromatique ; Ger. achromatisches Okular. Oculars in which chromatic aberration is wholly or nearly eliminated. — Aplanatic Ocular ; Fr. Oculaire aplanatique ; Ger. aplanatisches Okular (see \ 21). — Binocu- lar, stereoscopic Ocular ; Fr. Oculaire binoculaire stereoscopique ; Ger. stereosko- pisches Doppel-Okular. An ocular consisting of two oculai's about as far apart as the two eyes. These are connected with a single tube which fits a monocular mi- croscope. By an arrangement of prisms the image forming rays are divided, half CH. /] MICROSCOPE AND ACCESSORIES 23 being sent to each eye. The most satisfactory form was worked out by Tolles and is constructed on true stereotomic principles, both fields being equally illuminated. His ocular is also erecting. — Campani's Ocular (see Huygenian Ocular). — Com- pound Ocular ; Fr. Oculaire compose ; Ger. zusammengesetztes Okular. An ocu- lar of two or more lenses, e. g., the Huygenian (see Fig. 30). — Continental Ocular. An ocular mounted in a tube of uniform diameter as in Fig. 31. — Deep Ocular, see high ocular. — Erecting Ocular ; Fr. Oculaire redresseur ; Ger. bildumkeh- rendes Okular. An ocular with which an erecting prism is connected so that the image is erect as with the simple microscope. Such oculars are most common on dissecting microscopes. — Filar micrometer Ocular; Screw m. o., Cobweb m. o., Ger. Okular-Schraubenmikrometer. A modification of Ramsden's Telescopic Cob- web micrometer ocular. — Goniometer Ocular ; Fr. Oculaire a goniometre ; Ger. Goniometer-Okular. An ocular with goniometer for measuring the angles of minute crystals. — High Ocular, sometimes called a deep ocular. One that magnifies the real image considerably, i. e., 10 to 20 fold. — Huygenian Ocular, Huygens' O., Campani's 0.,Airy's 0.; Fr. Oculaire d'Huygens, o. de Campani ; Ger. Huy- gens'sches Okular, Campaniches Okular, see ? 38. — Index Ocular; Ger. Spitzen- O. An ocular with a minute pointer or two pointers at the level of the real image. The points are movable and serve for indicators and also, although not satisfac- torily, for micrometry. — Kellner's Ocular, see orthoscopic ocular — Low ocular, also called shallow ocular. An ocular which magnifies the real image only moder- ately, i. e., 2 to 8 fold. — Micrometer or micrometric Ocular ; Fr. Oculaire microme- trique ou a micrometre ; Ger. Mikrometer-Okular, Mess Okular, Beneches 0., Jackson m. o., see ? 41. — Microscopic Ocular ; Fr. Oculaire microscopique ; Ger. mi- kroskopisches Okular. An ocular for the microscope instead of one for a telescope. — Negative Ocular, see \ 36. — Nelson' s screw-micrometer ocular. A modification of the Ramsden's screw or cob-web micrometer in which positive compensating ocu- lars may be used. — Orthoscopic Oculars; also called Kellner's Ocular ; Fr. Ocu- laire orthoscopique ; Ger. Kellner'sches oder orthoskopisches Okular. An ocular with an eye-lens like one of the combinations of an objective (Figs. 22, 23) and a double convex field lens. The field-lens is in the focus of the eye-lens and there is no diaphragm present. The field is large and flat. — Par-focal Oculars, a series of oculars so arranged that the microscope remains in focus when the oculars are interchanged (Pennock, Micr. Bulletin, vol. iii, p. 9, 31 ). — Periscopic Ocular ; Fr. Oculaire periscopique ; Ger. periskopisches Okular. A positive ocular devised by Gundlach. It consists of a double convex field-lens and a triplet eye-lens. It gives a large, flat field. — Positive Ocular, see 2 37. — Projection Ocular; Fr. Ocu- laire de projection ; Ger. Projections-Okular, see \ 40. — Ramsden's Ocular; Fr. Oculaire de Ramsden ; Ger. Ramsden'sches Okular. A positive ocular devised by Ramsden. It consists of two plano-convex lenses placed close together with the convex surfaces facing each other. Only the central part of the field is clear. Searching Ocular ; Fr. Oculaire d'orientation ; Ger. Sucher-Okular, see § 39, Shallow Ocular, see low ocular. — So/ia 'Ocular, holosteric 0.; Fr. Oculaire holo- stere ; Ger. holosterisches Okular, Vollglass-Okular. A negative eye-piece de- vised by Tolles. It consists of a solid piece of glass with a moderate curvature at one end for a field-lens, and the other end with a much greater curvature for an eye-lens. For a diaphragm, a groove is cut at the proper level and filled with black pigment. It is especially excellent where a high ocular is desired. — Spectral 24 MICROSCOPE AND ACCESSORIES [CM. I or spectroscopic Ocular ; Fr. Oculaire spectroscopique ; Ger, Spectral-Okular, see Microspectroscope, Ch. Vl.—Stauroscopic Ocular ; Fr. Oculaire Stauroscopique. Ger. Stauroskop-Okular. An ocular with a Bertraud's quartz plate for mineralog- ical purposes. — Working Ocular; Fr. Oculaire de travail; Ger. Arbeits-Okular, see 'i 39. I 38. Huygenian Ocular — A negative ocular designed by Huygens for the telescope, but adapted also to the microscope. It is the one now most commonly employed. It consists of a field-lens or collective (Fig. 30), aiding the objective in forming the real image, and an eye-lens which magnifies the real image. While Ocular Ho 2 Fig. 31. Compensating Oculars of Zeiss, with section removed to show the con- struction. The line A- A is at the level of the upper end of the tube of the micro- scope while B-B represents the lower focal points. It will be seen that the mount- ing is so arranged that the lower focal points in all are in the same plane and therefore the microscope remains in focus upon changing oculars. ( The oculars are par-focal ). The lower oculars, 2, 4 and 6 are negative, and the higher ones, S, 12, iS, arc positive. The numbers 2, /, 6, S, 12, 18, indicate the magnification of the ocular. From Zeiss' Catalog. ) the field-lens aids the objective in the formation of the real, inverted image, and increases the field of view, it also combines with the eye-lens in rendering the image achromatic. (See £46). 2 39. Compensating Oculars. — These are oculars specially constructed for use with the apochromatic objectives. They compensate for aberrations outside the axis which could not be so readil\- eliminated in the objective itself. An ocu- lar of this kind, magnifying but twice, is made for use with high powers, for the sake of the large field in finding objects; it is called a searching ocular; those ordinarily used for observation are in contradistinction called working oculars. Part of the compensating oculars are positive and part negative. ( Fig. 31. ) # 40. Projection Oculars. — These are oculars especially designed for project- ing a microscopic image on the screen for class demonstrations, or for photo- graphing with the microscope. While they are specially adapted for use with apochromatic objectives, they mav also be used, with ordinary achromatic objectives of large numerical aperture. ch. n MICROSCOPE AND ACCESSORIES 25 Fig. 32. Projection Oculars with section re- moved to show the construction. Below are shown the upper ends with graduated circle to indicate the amount of rotation found necessary to focus the diaphragm on the screen. No. 2, No. 4. The numbers indicate the amount the ocular magnifies the image formed by the objective as with the compensation oculars. (Zeiss' Catalog.) '',. 41. Micrometer Ocular. — This is an ocular connected with an ocular micrometer. The micrometer may be removable, or it may be permanently in connection with the ocular, and arranged with a spring and screw, by which it maj' be moved back and forth across the field. (See Ch. IV.) Fig- 33 Fig. 34 Figs. 33-34. Ocular Micrometer and movable scale. Fig. 33 is a side view of the ocular while Fig . 34 gives a sectional end view, and shows the ocular micrometer in position. In both the screw -which moves the micrometer is sho~wn at the left. (From Bausch elf Lomb Opt. Co. ) i 42. Spectral or Spectroscopic Ocular. — (See Micro-Spectroscope, Ch. VI). DESIGNATION OF OCULARS '/. 43. Equivalent Focus. — As with objectives, some opticians designate the oculars by their equivalent focus ( j 15 ). With this method the power of the ocular, as with objectives, varies inversely as the equivalent focal length, and therefore the greater the equivalent focal length the less the magnification. This seems as desirable a mode for oculars as for objectives and is coming more and more into use by the most progressive opticians. It is the method of designation advo- cated by Dr. R. H. Ward for many years, and was recommended by the committee of the American Microscopical Society, (Proc. Amer. Micr. Soc, 18S3, p. 175, 1884, p. 22S). MICROSCOPE AND ACCESSORIES \_CH.I Fig. 35. Ocular Screw- Micrometer with compensation ocular 6. The upper figure shows a sectional view of the ocular and the screw for moving the micrometer at the right. At the left is shown a clamping screw to fasten the ocular to the upper part of the mi- croscope tube. Below is a face view, showing the graduation on the wheel. An ocular micrometer like this is in general like the cob-web micrometer and may be used for measuring objects of varying sizes very accu- rately. With the ordinary ocular -micrometer very small objects frequently fill but a part of an interval of the micrometer, but with this the movable cross lines traverse the object [or rather its real image) regardless of the minute- ness of the object. (Zeiss' Catalog). \ 44. Numbering and Lettering. — Oculars like objectives may be numbered or lettered arbitrarily. When so designated, the smaller the number, or the earlier the letter in the alphabet, the lower the power of the ocular. I 45. Magnification. — The compensating oculars are marked with the amount they magnify the real image. Thus an ocular marked X 4. indicates that the real image of the objective is magnified four fold by the ocular. The projection oculars are designated simply by the amount they multiply the real image of the objective. Thus for the short or 160 mm. tube-length they are, > 2, > 4 ; and for the long or 250 mm. tube, they are • 3 and • 6. That is, the final image on the screen or the ground glass of the photographic camera will be 2, 3, 4, or 6 times greater than it would be if no ocular were used. See Ch. VIII. iJ 46. Standard Size Oculars. — The Royal Microscopical Society of London took a very important step (Dec. 20, 1S99) m establishing standard sizes for ocu- lars and sub-stage condensers. To quote from the Journal of the Royal Micro- scopical Society for 1900, p. 147 : Resolved, "That the standard size for the inside diameter of the substage fit- ting be 1.527 in. =38.786 mm. That the gauges for standardizing eye-pieces be the internal diameters of the draw-tubes, the tightness of the fit being left to the discretion of the manufacturers." The sizes for oculars are four in number, 1 and 2 being most common. ( 1 ) 0.9173 inch = 23.300 mm. This is the Continental size. This is the size used by the English Opticians for student and small microscopes. Medium size binoculars (English. ) Long tube binoculars. For the history of the Huygenian Ocular, and a discussion of formulae for its construction, see Nelson, J. R. M. S., 1900, p. 162-169. EXPERIMENTS § 47. Putting an Objective in Position and Removing it. — Elevate the tube of the microscope by means of the coarse adjustment (2) 1.04 inch = 26.416 mm. (3) 1-27 (4) I.4I inch = 32.25S mm. inch = 35.S14 mm. CH. /] MICROSCOPE AND ACCESSORIES 27 (frontispiece) so that there may be plenty of room between its lower end and the stage. Grasp the objective lightly near its lower end with two fingers of the left hand, and hold it against the nut at the lower end of the tube. With two fingers of the right hand take hold of the milled ring near the back or upper end of the objective and screw it into the tube of the microscope. Reverse this operation for removing the objective. By following this method the danger of dropping the objective will be avoided. § 48. Putting an Ocular in Position and Removing it. — Ele- vate the body of the microscope with the coarse adjustment so that the objective will be 2 cm. or more from the object — grasp the ocular by the milled ring next the eye-lens (Fig. 21), and the coarse adjustment or the tube of the microscope and gently force the ocular into position. In removing the ocular, reverse the operation. If the above precau- tions are not taken, and the oculars fit snugly, there is danger in in- serting them of forcing the tube of the microscope downward and the objective upon the object. § 49. Putting an Object under the Microscope. — This is so placing an object under the simple microscope, or on the stage of the compound microscope, that it will be in the field of view when the microscope is in focus (§ 50). With low powers, it is not difficult to get an object under the microscope. The difficulty increases, however, with the power of the microscope and the smallnessof the object. It is usually necessary to move the object in various directions while looking into the micro- scope, in order to get it into the field. Time is usually saved by get- ting the object in the center of the field with a low objective before putting the high objective in position. This is greatly facilitated by using a nose-piece, or revolver. (See Figs. 36-363, and the pictures of microscopes, Ch. II.) Fig. 36. Triple nose-piece or revol- ver for quickly changing objectives. ( The Spencer Lens Co. ) Fig. 36a. Triple nose-piece or re- volver for quickly changing objectives. ( The Bausch & Lomb Optical Co. ) 2S MICROSCOPE AND ACCESSORIES [CH. I § 50. Field or Field of View of a Microscope. — This is the area visible through a microscope when it is in focus. When properly lighted and there is no object under the microscope, the field appears as a circle of light. When examining an object it appears within the light circle, and by moving the object, if it is of sufficient size, differ- ent parts are brought successively into the field of view. In general, the greater the magnification of the entire microscope, whether the magnification is produced mainly by the objective, the ocular, or by increasing the tube length, or by a combination of all three (see Ch. IV, under magnification), the smaller is the field. The size of the field is also dependent, in part, without regard to magnification, upon the size of the opening in the ocular diaphragm. Some oculars, as the orthoscopic and periscopic, are so constructed as to eliminate the ocular diaphragm, and in consequence, although this is not the sole cause, the field is considerably increased. The exact size of the field may be read off directly by putting a stage micrometer under the microscope and noting the number of spaces required to measure the diameter of the light circle. Fig. 37. Figures showing approximately the actual size of the field with ob- jectives op S5 mm., 15 mm., ij mm., 5 mm., and 2 vim., equivalent focus, and ocular of '37% vim., equivalent focus in each ease. This figure shows graphically what is a/so 'eery clearly indicated in the /aide ( \ 52). §51. The size of the field of the microscope as projected into the field of vision of the normal human eye (z. e., the virtual image) may be determined by the use of the camera lucida with the drawing surface placed at the standard distance of 250 millimeters (Ch. IV.) § 52. Table showing the actual size in millimeters of the field of a groicp of commonly used objectives and oculars. Compare with the graphic representation in Fig. jj . See also $ jo. CH. 7] MICROSCOPE AND ACCESSORIES 29 Equivalent Focus and N. A. of Objective Diameter of Field in mm. Equivalent Focus of Ocular Kind of Ocular S5 mm 15-4 io.6 8.3 37^4 mm. 25' I2>2 " Huygenian 45 mm 7.0 4.0 37^2 mm. 25 123-2 " Hu3'genian 17 mm. N. A. = 0.25 3-° 2.0 1.6 5-7 2.S 1.4 0.97 37 yi mm. 25 / " 1 2 } 2 " Huygenian 1S0 mm. 45 15 10 ", Compensation 5 mm N. A. = 0.92 0.541 0.371 0.290 37^4 mm. 25 1 2 % " Huygenian 0.S50 0.501 0.250 Q-I73 1 So mm. 45 15 10 Compensation 2 mm. . N. A. = 1.25 1 0.270 0.186 0.147 37 % mm. 25 12% " Huygenian 0.450 0.251 0.125 0.0SS I So mm. 45 15 10 Compensation FUNCTION OF AN OBJECTIVE S S3- Put a 50 mm. objective on the microscope or screw off the front combination of a 16 mm.,( 2 3-in.), and put the back combination on the microscope for a low objective. Place some printed letters or figures under the microscope, and light well. In place of an ocular put a screen of ground glass, or a piece of lens paper, over the upper end of the tube of the microscope. * *Ground glass may be very easily prepared by placing some fine emery or carborundum between two pieces of glass, wetting it with water and then rubbing the glasses together for a few minutes. If the glass becomes too opaque, it may be rendered more translucent by rubbing some oil upon it. 30 MICROSCOPE AND ACCESSORIES \_CH. I Lower the tube of the microscope by means of the coarse adjust- ment until the objective is within 2-3 cm. of the object on the stage. Look at the screen on the top of the tube, holding the head about as far from it as for ordinary reading, and slowly elevate the tube by means of the coarse adjustment until the image of the letter appears on the screen. The image can be more clearly seen if the object is in a strong light and the screen in a moderate light, i. e., if the top of the micro- scope is shaded. The letters will appear as if printed on the ground glass or paper, but will be inverted (Fig. 21). If the objective is not raised sufficiently, and the head is held too near the microscope, the objective will act as a simple microscope. If the letters are erect, and appear to be down in the microscope and not on the screen, hold the head farther from it, shade the screen, and raise the tube of the microscope until the letters do appear on the ground glass. To demonstrate that the object must be outside the principal focus with the compound microscope, remove the screen and turn the tube of the microscope directly toward the sun. Move the tube of the micro- scope with the coarse adjustment until the burning or focal point is found (§ 6). Measure the distance from the paper object on the stage to the objective, and it will represent approximately the principal focal distance (Figs. 10, 11). Replace the screen over the top of the tube, no image can be seen. Slowly raise the tube of the microscope and the image will finally appear. If the distance between the object and the objective is now taken, it will be found considerably greater than the principal focal distance (compare § 11). § 54. Aerial Image. — After seeing the real image on the ground- glass, or paper, use the lens paper over about half of the opening of the tube of the microscope. Hold the eye about 250 mm. from the microscope as before and shade the top of the tube by holding the hand between it and the light, or in some other way. The real image can be seen in part as if on the paper and in part in the air. Move the paper so that the image of half a letter will be on the paper and half in the air. Another striking experiment is to have a small hole in the paper placed over the center of the tube opening, then if a printed word extends entirely across the diameter of the tube its central part may be seen in the air, the lateral parts on the paper. The advantage of the paper over part of the opening is to enable one to accommodate the CH. I] MICROSCOPE AND ACCESSORIES 31 eyes for the right distance. If the paper is absent the eyes adjust themselves for the light circle at the back of the objective, and the aerial image appears low in the tube. Furthermore it is more difficult to see the aerial image in space than to see the image on the ground- glass or paper, for the eye must be held in the right position to receive the rays projected from the real image, while the granular surface of the glass and the delicate fibres of the paper reflect the rays irregularly, so that the image ma}- be seen at almost any angle, as if the letters were actually printed on the paper or glass. § 55. The Function of an Objective, as seen from these experi- ments, is to form an enlarged, inverted, real image of an object, this image being formed on the opposite side of the objective from the object (Fig. 21 ). FUNCTION OF AN OCULAR § 56. Using the same objective as for § 53, get as clear an image of the letters as possible on the lens paper screen. Look at the image with a simple microscope (Fig. 17 or 18) as if the image were an object. Observe that the image seen through the simple microscope is merely an enlargement of the one on the screen, and that the letters remain inverted, that is they appear as with the naked eye (§ n). Remove the screen and observe the aerial image with the tripod. Put a 50 mm. (A, No. 1 or 2 in.), ocular i. § 5S. The field-lens of a Huygenian ocular makes the real image smaller and consequently increases the size of the field ; it also makes the image brighter by contracting the area of the real image. (Fig. 30.) Demonstrate this by screwing off the field-lens and using the eye-lens alone as an ocular, refocusing if necessary. Note also that the image is bordered by a colored haze (§7). When looking into the ocular with the field-lens removed, the eye should not be held so close to the ocular, as the eye-point is consider- ably farther away than when the field-lens is in place. § 59. The eye-point. — This is the point above the ocular or simple microscope where the greatest number of emerging rays cross. Seen in profile, it may be likened to the narrowest part of an hour glass. Seen in section (Fig. 30), it is the smallest and brightest light circle above the ocular. This is called the eye-point, for if the pupil of the eye is placed at this level, it will receive the greatest number of rays from the microscope, and consequently see the largest field. Demonstrate the eye-point by having in position an objective and ocular as above (§ 53). Light the object brightly, focus the micro- scope, shade the ocular, then hold some ground-glass or a piece of the lens paper above the ocular and slowly raise and lower it until the smallest circle of light is found. By using different oculars it will be CH. /.] MICROSCOPE AND ACCESSORIES 33 seen that the eye-point is nearer the eye-lens in high than in low ocu- lars, that is the eye-point is nearer the eye-lens for an ocular of small equivalent focus than for one of greater focal length. REFERENCES FOR CHAPTER I In chapter X will be given a bibliography, with full titles, of the works and periodicals referred to. For the subjects considered in this chapter, general works on the microscope may be consulted with great advantage for different or more exhaustive treatment. The most satisfactory work in English is Carpenter-Dallinger, Sth Ed. For the history of the microscope, Mayall's Cantor Lectures on the microscope are very satisfactory. For a continuation of the history begun by Mayall in the Cantor Lectures see Nelson, Journal of the Queckett Micr. Club, and the Jour. Roy. Micr. Soc, 1S97-1901-1-. Carpenter-Dallinger, Sth Ed. Petri, Das Mikroskop. The following special articles in periodicals may be examined with advantage : Apochrornatic Objectives, etc. Dippel in Zeit. wiss. Mikr., 1SS6, p. 303 ; also in the Jour. Roy. Micr. Soc, 1SS6, pp. 316, 849, 1110, ; same, 1890, p. 4S0 ; Zeit. f. Instrumentenk., 1890, pp. 1-6 ; Micr. Built., 1S91, pp. 6-7. Tube-length, etc. Gage, Proc. Amer. Soc. Micrs., 1SS7, pp. 168-172 ; also in the Microscope, the Jour. Roy. Micr. Soc, and in Zeit, wiss. Mikr., 1887-8. Bausch, Proc Amer. Soc. Micrs., 1890, pp. 43-49 ; also in the Microscope, 1S90, pp. 2S9-296. Aperture. J. D. Cox, Presidential Address, Proc. Amer. Soc. Micrs., 1S84, pp, 5-39, Jour. Roy. Micr. Soc, 1SS1, pp. 303, 34S, 365, 38S ; 1SS2, pp. 300, 460; 1883, p. 790 ; 1SS4, p. 20. Czapski, Theorie der optischen Instrumente nach Abbe. See also references in (j 34. The Barnes Disserting Microscope ( The Bausch & Lomb Optical Company). CHAPTER II LIGHTING AND FOCUSING; MANIPULATION OF DRY, ADJUSTABLE AND IMMERSION OBJECTIVES ; CARE OF THE MICROSCOPE AND OF THE EYES ; LABORATORY MICROSCOPES APPARATUS AND MATERIAL FOR THIS CHAPTER Microscope supplied with plane and concave mirror, Achromatic and Abbe condensers, dry, adjustable and immersion objectives, oculars, triple nose-piece. Microscope lamp and movable condenser (bull's eve or other form, Fig. 53), Homogeneous immersion liquid ; Benzin, alcohol, distilled water ; Mounted prep- aration of fly's wing(§ 70); Mounted preparation of Pleurosigma (\ 77, 78). Stage or ocular micrometer [\ 92); Glass slides and cover-glasses (Ch. VII); 10 per ct. solution of salicylic acid in 95 per ct. alcohol {\ 92); Preparation of stained bac- teria ('i 108); Vial of equal parts olive or cotton seed oil or liquid vaselin and benzin (? 112): Double eye shade (Fig. 60); Screen for whole microscope (Fig. 59). FOCUSING \ 60. Focusing is mutually arranging an object and the microscope so that a clear image may be seen. With a simple microscope (| 11) either the object or the microscope or both may be moved in order to see the image clearly, but with the compound microscope the object more conveniently remains stationary on the stage, and the tube or body of the microscope is raised or lowered (frontispiece). In general, the higher the power of the whole microscope whether simple or compound, the nearer together must the object and objective be brought. With the compound microscope, the higher the objective, and the longer the tube of the microscope, the nearer together must the object and the objective be brought. If the oculars are not par-focal, the higher the magnification of the ocular, the nearer must the object and objective be brought. \ 61. Working Distance. — By this is meant the space between the simple mi- croscope and the object, or between the front lens of the compound microscope and the object, when the microscope is in focus. This working distance is always con- siderably less than the equivalent focal length of the objective. For example, the front-lens of a 6 mm. or '4 th in. objective would not be 6 millimeters or if th inch from the object when the microscope is in focus, but considerably less than that distance. If there were no other reason than the limited working distance of high objectives, it would be necessary to use a very thin cover-glass over the CH. II ' LIGHTING AND FOCUSING 35 object. (See i 24, 29. ) If too thick covers are used it may be impossible to get an objective near enough an object to get it in focus. For objects that admit of examination with high powers it is always better to use thin covers. LIGHTING WITH DAYLIGHT \. 62. Unmodified sunlight should not be employed except in special cases. North light is best and most uniform. When the sky is covered with white clouds the light is most favorable. To avoid the shadows produced by the hands in manipulating the mirror, etc., it is better to face the light ; but to protect the eyes and to shade the stage of the microscope some kind of screen should be used. The one figured in (Fig. 62) is cheap and efficient. If one dislikes to face the window or lamp it is better to sit so that the light will come from the left as in reading. It is of the greatest importance and advantage for one who is to use the microscope for serious work that he should comprehend and appreciate thoroughly the various methods of illumination, and the special appearances due to different kinds of illumination. Depending on whether the light illuminating an object traverses the object or is reflected upon it, and also whether the object is symmetrically lighted, or lighted more on one side than the other, light used in microscopy is designated as reflected and transmitted, axial and oblique. Figs. 39-40. F01 39 "or j nil explanation see Figs. 40 22 and 23. \ 63. Reflected, Incident or Direct Light. — By this is meant light reflected upon the object in some way and then irregularly reflected from the object to the microscope. By this kind of light objects are ordinarily seen by the unaided eye, and the objects are mostly opaque. In Vertebrate Histology, reflected light is but 36 LIGHTING AND FOCUSING [CM. II little used ; but in the study of opaque objects, like whole insects, etc., it is used a great deal. For low powers, ordinary daylight that naturally falls upon the object, or is reflected or condensed upon it with a mirror or condensing lens, answers very well. For high powers and for special purposes, special illuminating apparatus has been devised (\ 28). (See also Carpenter-Dallinger, Ch. IV). \ 64. Transmitted Light. — By this is meant light which passes through an object from the opposite side. The details of a photographic negative are in many cases only seen or best seen by transmitted light, while the print made from it is best seen by reflected light. Almost all objects studied in Vertebrate Histology are lighted by transmitted light, and they are in some way rendered transparent or semi-transparent. The light traversing and serving to illuminate the object in working with a compound microscope is usually reflected from a plane or concave mirror, or from a mirror to a condenser [\ SS), and thence transmitted to the object from below (Figs. 48-51). \ 65. Axial or Central Light. — By this is understood light reaching the object, the rays of light being parallel to each other and to the optic axis of the micro- scope, or a diverging or converging cone of light whose axial ray is coincident with the optic axis of the microscope. In either case the object is symmetrically illuminated. \ 66. Oblique Light. — This is light in which parallel rays from a plane mirror form an angle with the optic axis of the microscope (Fig. 40). Or if a concave mirror or a condenser is used, the light is oblique when the axial ray of the cone of light forms an angle with the optic axis (Fig. 40). DIAPHRAGMS \ 67. Diaphragms and their Proper Employment. — Diaphragms are opaque disks with openings of various sizes, which are placed between the source of light or mirror and the object. In some cases an iris diaphragm is used, and then the same one is capable of giving a large range of openings. The object of a dia- phragm in general, is to cut off all adventitious light and thus enable one to light the object in such a way that the light finally reaching the microscope shall all come from the object or its immediate vicinity. The diaphragms of a condenser serve to vary its aperture to the needs of each object and each objective. \ 68. Size and Position of Diaphragm Opening. — When no condenser is used the size of the opening in the diaphragm should be about that of the front lens of the objective. For some objects and some objectives this rule may be quite widely departed from ; one must learn by trial. When lighting with a mirror the diaphragm should be as close as possible to the object in order, (a) that it may exclude all adventitious light from the object ; (b) that it may not interfere with the most efficient illumination from the mirror by cutting off a part of the illuminating pencil. If the diaphragm is a considera- ble distance below the object, ( 1 ) it allows considerable adventitious light to reach the object and thus injures the distinctness of the microscope image ; (2) it pre- vents the use of very oblique light unless it swings with the mirror ; (3) it cuts off a part of the illuminating cone from a concave mirror. On the other hand, even with a small diaphragm, the whole field will be lighted. CH. II] LIGHTING AND FOCUSING 37 With an illuminator or condenser (Figs. 41, 48), the diaphragm serves to narrow the pencil to be transmitted through the condenser, and thus to limit the aperture (see \ 84). Furthermore, by making the diaphragm opening eccentric, oblique light may be used, or by using a diaphragm with a slit around the edge (central stop diaphragm), the center remaining opaque, the object may be lighted with a hollow cone of light, all of the rays having great obliquity. In this way the so- called dark-ground illumination may be produced (§ 92 ; Fig. 51). ARTIFICIAL ILLUMINATION \ 69. For evening work and for certain special purposes, artificial illumina- tion is employed. A good petroleum (kerosene) lamp with flat wick has been found very satisfactory, also an incandescent electric or Welsbach light, but for brilliancy and for the actinic power necessar3' for very rapid photo-micrography (see Ch. VIII) the electric arc lamp or an acetylene lamp serves well. Whatever source of artificial light is employed, the light should be brilliant and steady. LIGHTING AND FOCUSING : EXPERIMENTS § 70. Lighting with a Mirror. — As the following experiments are for mirror lighting only, remove the substage condenser if present (see § 79, for condenser). Place a mounted fly's wing under the microscope, put the 16 mm. (^3 in.) or other low objective in position, also a low ocular. With the coarse adjustment lower the tube of the microscope to within about 1 cm. of the object. Use an opening in the diaphragm about as large as the front lens of the objective ; then with the plane mirror try to reflect light up through the diaphragm upon the object. One can tell when the field (§ 50) is illuminated, by looking at the object on the stage, but more satisfactorily by looking into the microscope. It sometimes requires considerable manipulation to light the field well. After using the plane side of the mirror turn the concave side into position and light the field with it. As the con- cave mirror condenses the light, the field will look brighter with it than with the plane mirror. It is especially desirable to remember that the excellence of lighting depends in part on the position of the diaphragm (§ 68). If the greatest illumination is to be obtained from the concave mirror, its position must be such that its focus w 7 ill be at the level of the object. This distance can be very easily determined by finding the focal point of the mirror in full sunlight. § 71. Use of the Plane and of the Concave Mirror. — The mir- ror should be freely movable, and have a plane and a concave face. The concave face is used when a large amount of light is needed, the plane face when a moderate amount is needed or when it is necessary to have parallel rays or to know the direction of the rays. 3 8 LIGHTING AND FOCUSING [CH. II § 72. Focusing with Low Objectives. — Place a mounted fly's wing under the microscope; put the 16 mm. (fi in.) objective in position, and also the lowest ocular. Select the proper opening in the diaphragm and light the object well with transmitted light (§ 64, 68). Hold the head at about the level of the stage, look toward the window, and between the object and the front of the objective ; with the coarse adjustment lower the tube until the objective is within about half a centimeter of the object. Then look into the microscope and slowly elevate the tube with the coarse adjustment. The image will appear dimly at first, but will become very distinct by raising the tube still higher. If the tube is raised too high the image will become indistinct, and finally disappear. It will again appear if the tube is lowered the proper distance. When the microscope is well focused try both the concave and the plane mirrors in various positions and note the effect. Put a high ocular in place of the low one (§43). If the oculars are not par- focal it will be necessary to lower the tube somewhat to get the micro- scope in focus.* Pull out the draw-tube 4-6 cm., thus lengthening the body of the microscope ; it will be found necessary to lower the tube of the micro- scope somewhat. (For reason, see Fig. 58.) § 73. Pushing in the Draw-Tube. — To push in the draw-tube, grasp the large milled ring of the ocular with one hand, and the milled head of the coarse adjustment with the other, and gradually push the draw-tube into the tube. If this were done without these precautions the objective might be forced against the object and the ocular thrown out by the compressed air. § 74. Focusing with High Objectives. — Employ the same object as before, elevate the tube of the microscope and, if no revolving nose-piece is present, remove the 16 mm. ( 2 3 in.) objective as indi- cated. Put the 3 mm.(}s in.) or a higher objective in place, and use a low ocular. *Par-focal oculars are so constructed, or so mounted, that those of different powers may be interchanged without the microscopic image becoming wholly out of focus (Fig. 31). When high objectives are used, while the image may be seen after changing oculars, the instrument nearly always needs slight focusing. With low powers this may not be necessary. Objectives are also now commonly mounted in the triple or double revolving nose-pieces (Figs. 36, 36 a) so that if one of the objectives is in focus either of the others will be approximately in focus when turned into position. This is a ver}' great convenience. CH. II] LIGHTING AND FOCUSING 39 Light well, and employ the proper opening in the diaphragm, etc. (§ 68). Look between the front of the objective and the object as before (§ 72), and lower the tube with the coarse adjustment till the objective almost touches the cover-glass over the object. Look into the microscope, and with the coarse adjustment, raise the tube very slowly until the image begins to appear, then turn the milled head of the fine adjustment (frontispiece), first one way and then the other, if necessary, until the image is sharply defined. In practice it is found of great advantage to move the preparation slightly while focusing. This enables one to determine the approach to the focal point either from the shadow or the color, if the object is col- ored. With high powers and scattered objects there might be no object in the small field (see § 50, Fig. 37 for size of field). By moving the preparation an object will be moved across the field and its shadow gives one the hint that the objective is approaching the focal point. It is sometimes desirable to focus on the edge of the cement ring or on the little ring made by the marker (see Figs. 61-66). Note that this high objective must be brought nearer the object than the low one, and that by changing to a higher ocular (if the ocu- lars are not par-focal) or lengthening the tube of the microscope it will be found necessary to bring the objective still nearer the object, as with the low objective. (For reason see Fig. 58.) § 75. Always Focus Up, as directed above. If one lowers the tube only when looking at the end of the objective as directed above, there will be no danger of bringing the objective in contact with the object, as may be done if one looks into the microscope and focuses down. When the instrument is well focused, move the object around in order to bring different parts into the field. It may be necessary to re-focus with the fine adjustment every time a different part is brought into the field. In practical work one hand is kept on the fine adjust- ment constantly, and the focus is continually varied. § 76. Determination of Working Distance. — As stated in § 61 , this is the distance between the front lens of the objective and the object when the objective is in focus. It is always less than the equiv- lent focal length of the objective. Make a wooden wedge 10 cm. long which shall be exceedingly thin at one end and about 20 mm. thick at the other. Place a slide on the stage and some dust on the slide. Do not use a cover-glass. Focus the dust carefully first with the low then with the high objective. When 40 LIGHTING AND FOCUSING \_CH. II the objective is in focus push the wedge under the objective on the slide until it touches the objective. Mark the place of contact with a pencil and then measure the thickness of the wedge with a rule opposite the point of contact. This thickness will represent very closely the working distance. For measuring the thickness of the wedge at the point of contact for the high objective use a steel scale ruled in iths mm. and the tripod to see the divisions. Or one may use a cover-glass measure (Ch. VIII) for determining the thickness of the wedge. For the higher powers if one has a microscope in which the fine ad- justment is graduated, the working distance may be readily determined when the thickness of the cover-glass over the specimen is known, as follows : Get the object in focus, lower the tube of the microscope, un- til the front of the objective just touches the cover-glass. Note the position of the micrometer screw and slowly focus up with the fine adjustment until the object is in focus. The distance the objective was raised plus the thickness of the cover-glass represents the working dis- tance. For example, a 3 mm. objective after being brought in contact with the cover-glass was raised by the fine adjustment a distance repre- sented by 16 of the divisions on the head of the micrometer screw. Each division represented .01 mm., consequently the objective was raised . 16 mm. As the cover-glass on the specimen used was . 15 mm. the total working distance is .16 +.15 =.31 mm. \ 76a. Free Working Distance. — In the microscope catalog of Zeiss there is given a table of the size of the field and also of the "free working-distance." This free working-distance is the space between the lower end of the objective and the cover glass of Jfo mm. thickness, when the objective is in focus on an object imme- diately under the cover. This is exceedingly practical information for a possessor of a microscope, and it is hoped that the other opticians will adopt the suggestion. Naturally, however, the free working-distance for each optician should be reckoned from the top of the cover for which his unadjustable objectives are corrected. If, for example, the thickness of cover for which an objective is corrected is fflg mm, then the free working-distance should be that between the top of this and the objective when the objective is in focus on an object under the cover. (See the table of cover-glass thickness, p. 14). CENTRAL AND OBLIQUE LIGHT WITH A MIRROR § 77. Axial or Central Light (§ 65). — Remove the condenser or any diaphragm from the substage, then place a preparation contain- ing minute air bubbles under the microscope. The preparation may be easily made by beating a drop of mucilage on a slide and covering CH. II] LIGHTING AND FOCUSING 41 it (see Ch. III). Use a 3 mm.,(^ in.) or No. 7 objective and a medium ocular. Focus the microscope and select a very small bubble, one whose image appears about 1 mm. in diameter, then arrange the plane mirror so that the light spot in the bubble appears exactly in the center. Without changing the position of the mirror in the least, replace the air bubble preparation by one of Pleurosigma angulatum or some other finely marked diatom. Study the appearance very carefully. § 78. Oblique Light (§ 66). — Swing the mirror far to one side so that the rays reaching the object may be very oblique to the optic axis of the microscope. Study carefully the appearance of the diatom with the oblique light. Compare the appearance with that where central light is used. The effect of oblique light is not so striking with histo- logical preparations as with diatoms. It should be especially noted in §§ 77, 78, that one cannot deter- mine the exact direction of the rays by the position of the mirror. This is especially true for axial light ( §77). To be certain the light is axial some such test as that given in § 77 should be applied. (See also Ch. Ill, under Air-bubbles.) CONDENSERS OR ILLUMINATORS* § 79. These are lenses or lens-systems for the purpose of illuminat- ing with transmitted light the object to be studied with the microscope. For the highest kind of investigation their value cannot be over- estimated. They may be used either with natural or artificial light, and should be of sufficient numerical aperture to satisfy objectives of the widest angle. *No one has stated more cleaily, or appreciated more truly the value of cor- rect illumination and the methods of obtaining it than Sir David Brewster, 1820, 1831. He says of illumination in general : "The art of illuminating microscopic objects is not of less importance than that of preparing them for observation. " "The eye should be protected from all extraneous light, and should not receive any of the light which proceeds from the illuminating center, excepting that portion of it which is transmitted through or reflected from the object." So likewise the value and character of the substage condenser was thoroughly understood and pointed out by him as follows : "I have no hesitation in saying that the apparatus for illumination requires to be as perfect as the apparatus for vision, and on this account I would recommend that the illuminating lens should be perfectly free of chromatic and spherical aberration, and the greatest care be taken to exclude all extraneous light both from the object and from the eye of the observer. " See Sir David Brewster's treatise on the Microscope, 1837, pp. 136, 138, 146, and the Edinburgh Journal of Science, new series, No. 11 (1831) p. 83. 42 LIGHTING AND FOCUSING [CH. II It is of the greatest advantage to have the sub-stage condenser mounted so that it may be easily moved up or down under the stage. The iris diaphragm is so convenient that it should be furnished in all cases, and there should be marks indicating the N. A. of the condenser utilized with different openings. Finally the condenser should be supplied with central stops for dark-ground illumination (§92) and with blue and neutral tint glasses to soften the glare when artificial light is used (§ 89, 93). Condensers or Illuminators fall into two great groups, the Achromatic, giving a large aplanatic cone, and Non-achromatic, giving much light, but a relatively small aplanatic cone of light. § 80. Achromatic Condenser. — It is still believed by all expert microscopists that the contention of Brewster was right, and the con- denser to give the greatest aid in elucidating microscopic structure must approach in excellence the best objectives. That is, it should be as free as possible from spherical and chromatic aberration, and there- fore would transmit to the object a very large aplanatic cone of light. Such condensers are especially recommended for photo-micrography by all, and those who believe in getting the best possible image in every case are equally strenuous that achromatic condensers should be used for all work. Unfortunately good condensers like good objectives are expensive, and student microscopes as well 'as many others are usually supplied with the non-achromatic condensers or with none. Many excellent achromatic condensers have been made, but the most perfect of all seems to be the apochromatic of Powell and Lealand (Carpenter-Dallinger, p. 302). To attain the best that was possible many workers have adopted the plan of using objectives as condensers. A special substage fitting is provided with the proper screw and the objective is put into position, the front lens being next the object. As will be seen below (§ 83-84), the full aperture of an objective can rarely be used, and for histological preparations perhaps never, so that an objective of greater equivalent focus, i. 55- N> 56. Figs. 54-56. Diagrams illustrating refraction in different media and at plane and curved surfaces. In each case the denser medium is represented by line shad- ing and the perpendicular or normal to the refracting surface is represented by the dotted Hue N-N', the refracted ray by the bent line A C. \ 97. Refraction. — Lying at the basis of microscopical optics is refraction, which is illustrated by the above figures. It means that light passing from one medium to another is bent in its course. Thus in Fig. 54 light passing from air into water does not continue in a straight line but is bent toward the normal N-N', the bending taking place at the point of contact of the air and water ; that is, the ray of light A B entering the water at B is bent out of its course, extending to C instead of C. Conversely, if the ray of light is passing from water into air, on reaching the air it is bentfrow the normal, the ray C B passing to A and not in a straight line to C". By comparing Figs. 55, 56 in which the denser medium is crown glass in- stead of water, the bending of the rays is seen to be greater as crown glass is denser than water. It has been found by physicists that there is a constant relation between the angle taken by the ray in the rarer medium and that taken by the ray in the denser medium. The relationship is expressed thus : Sine of the angle of inci- CH. II] LIGHTING AND FOCUSING 53 dence divided by the sine of the angle of refraction equals the index of refraction. CI y. a o AT" In the figures, _. %- _ , r , = index of refraction. Worked out completely in Sin C a N' Fig. 54, ABN=4o°,CBN<= 28 ° 5 V^ 1 ^^=^ = , i3 , i.e., the index of refraction from air to water is 1.33. (See \ 33.) In Figs. 55-56, illustrating refraction in crown glass, the angles being given, the problem is easily solved as just illustrated. (For table of natural sines see third page of cover ; for interpolation, \ 32). \ 98. Absolute Index of Refraction. — This is the index of refraction obtained when the incident ray passes from a vacuum into a given medium. As the index of the vacuum is taken as unity, the absolute index of any substance is always greater than unity. For many purposes, as for the object of this book, air is treated as if it were a vacuum, and its index is called unity, but in reality the index of refraction of air is about 3 ten-thousandths greater than unity. Whenever the refractive index of a substance is given, the absolute index is meant unless otherwise stated. For example, when the index of refraction of water is said to be 1.33, and of crown glass 1.52, etc., these figures represent the absolute index, and the incident ray is supposed to be in a vacuum. § 99. Relative Index of Refraction. — This is the index of refraction between two contiguous media, as for example between glass and diamond, water and glass, etc. It is obtained by dividing the absolute index of refraction of the sub- stance containing the refracted ray, by the absolute index of the substance trans- mitting the incident ray. For example, the relative index from water to glass is 1.52 divided by 1 .33. If the light passed from glass to water it would be, 1.33 divided by 1.52. By a study of the figures showing refraction, it will be seen that the greater the refraction the less the angle and consequently the less the sine of the angle, and as the refraction between two media is the ratio of the sines of the angles of incidence and refraction ( -. | , it will be seen that whenever the sine of the anide of refrac- \ sin r J tion is increased by being in a less refractive medium, the index of refraction will show a corresponding decrease and vice versa. That is the ratio of the sines of the angles of incidence and refraction of any two contiguous substances is inversely as the refractive indices of those substances. The formula is : / Sine of angle of incident ray \ / Index of refraction of refracting medium \ \ Sine of angle of refracted ray / \ Index of refraction of incident medium / , / sin i \ / index r\ _ . ., . Abbreviated I -. — I = I ; — 5 -. I ■ By means of this general formula one can V sin r J \ index l / solve any problem in refraction whenever three factors of the problem are known. The universality of the law may be illustrated by the following cases : (A) Light incident in a vacuum or in air, and entering some denser medium, as water, glass, diamond, etc. / Sine of angle made by the ray in air _ \ _ / Index of ref.of d enser med. \ \ Sine of angle made by ray in denser medium / V Index of ref . of air ( 1 J / If the dense substance were glass I . -— ) = ( ) • If the two media were 54 LIGHTING AND FOCUSING [CH. II water and glass, the incident light being in water the formula would be : ( 1 = 1 ) • If the incident ray were in glass and the refracted ray in V sin rj \ 1.33 / water ■ I 1 = 1 — - ) • And similarly for any two media ; and as stated V sin r) V. 1.52 / above if any three of the factors are given the fourth may be readily found. 5 100. Critical Angle and Total Reflection. — In order to understand the Wol- laston camera lucida (Ch. IV) and other totally reflecting apparatus, it is necessary briefly to consider the critical angle. The critical angle is the greatest angle that a ray of light in the denser of two contiguous media can make with the normal and still emerge into the less refrac- tive medium. On emerging it will form an angle of 90 with the normal, and if the substances are liquids, the refracted ray will be parallel with the surface of the denser medium. Total Reflection. — In case the incident ray in the denser medium is at an angle with the normal greater than the critical angle, it will be totally reflected at the surface of the denser medium, that surface acting as a perfect mirror. By consult- ing the figures it will be seen that there is no such thing as a critical angle and total reflection in the rarer of two contiguous media. To find the critical angle in the denser of two contiguous media : — Make the angle of refraction (/. e. , the angle in the rarer of the two media) / si" i \ / index r \ _ , ,, qo° and solve the general equation : ( . ) = I ■ — ? r I . Let the two sub- V sin r I \ index t / stances be water and air, then the sine of r (90 ) is 1, the index of air is I, that of water 1.33, whence ( ) = ( ^ ) or sin 2 = 751 + . This is the sine of 48° — , and whenever the ray in the water is at an angle of more than 4S it will not emerge into the air, but be totally reflected back into the water. The case of a ray passing from crown glass into the water : / sin i \ / index water (1.33) \ / sin i \ / 1,33 \ \ sin r ( sin 90°= 1 )/ ~~ V index glass (1.52) / \ 1 / \ 1.52/ whence sin i =.875 sine of critical angle in glass covered with water. The corresponding angle is approximately 6i°. \ 101. Color Images. — These are images of objects which are strongly col- ored and lighted with so wide an aperture that the refraction images are drowned in the light. Such images are obtained by removing the diaphragm or by using a larger opening. This method of illumination is especially applicable to the study of deeply stained bacteria. (See below. 5 108). ADJUSTABLE WATER AND HOMOGENEOUS OBJECTIVES EXPERIMENTS \ 102. Adjustment for Objectives. — As stated above ($ 24), the aberration produced by the cover-glass (Fig. 57), is compensated for by giving the combina- tions in the objective a different relative position than they would have if the objective were to be used on uncovered objects. Although this relative position cannot be changed in unadjustable objectives, one can secure the best results of CH. IT' LIGHTING AND FOCUSING 55 which the objective is capable by selecting covers of the thickness for which the objective was corrected. (See table p. 14.) Adjustment maybe made also by increasing the tube-length for covers thinner than the standard and by shortening the tube-length for covets thicker than the standard (Fig. 58). Fig. 57. Effect of the cover-glass on the rays from the object to the objective (Ross). Axis. The projection of the optic axis of the microscope. F. Focal or axial point of the objective. F' and F" . Points on the axis where rays 2 and 3 appear to originate if traced backward after emerging from the upper side of the cover-glass. In learning to adjust objectives, it is best for the student to choose some object whose structure is well agreed upon , and then to practice lighting it, shad- ing the stage and adjusting the objective, until the proper appearance is obtained. The adjustment is made by turning a ring or collar which acts on a screw and increases or diminishes the distance between the systems of lenses, usually the front and the back systems (Fig. 40). § 103. General Directions. — (A) The thinner the cover-glass, the further must the system of lenses be separated, i. , 146); Carmine, India ink, or lamp black (ji 148-150); Frog, castor oil and micro-polariscope (§ 152). INTERPRETATION OF APPEARANCES UNDER THE MICROSCOPE § 129. General Remarks. — The experiments in this chapter are given secondarily for drill in manipulation, but primarily so that the student may not be led into error or be puzzled by appearances which are constantly met with in microscopical investigation. Anyone can look into a microscope, but it is quite another matter to interpret cor- rectly the meaning of the appearances seen. It is especially important to remember that the more of the relations of an}' object are known, the truer is the comprehension of the object. In microscopical investigation every object should be scrutinized from all sides and under all conditions in which it is likely to occur in nature and in microscopical investigation. It is best also to begin with objects of considerable size whose character is well known, to look at them carefully with the unaided eye so as to see them as wholes and in their natural setting ; then a low power is used, and so on, step by step until the highest power available has been employed. One will in this way see less and less of the object as a whole, but every increase in magnifi- cation will give increased prominence to detail, detail which might be meaningless when taken alone and independent of the object as a whole. The pertinence of this advice will be appreciated when the student undertakes to solve the problems of histology ; for even after all the years of incessant labor spent in trying to make out the structure of man and the lower animals, many details are still in doubt, the same visual appearances being quite differently interpreted by eminent observers. CH. HI] INTERPRETATION OF APPEARANCES 91 Appearances which seem perfectly unmistakable with a low power may be found erroneous or very inadequate, for details of structure that were indistinguishable with the low power may become perfectly evi- dent with a higher power or a more perfect objective. Indeed the prob- lems of microscopic structure appear to become ever more complex, for difficulties overcome by improvements in the microscope simply give place to new difficulties, which in some cases render the subject more obscure than it appeared to be with the less perfect appliances. The need of the most careful observation and constant watchful- ness lest the appearances may be deceptive are thus admirably stated by Dallinger (see Carpenter-Dallinger, p. 427): "The correctness of the conclusions which the microscopist will draw regarding the nature of any object from the visual appearances which it presents to him when examined in the various modes now specified will necessarily depend in a great degree upon his previous experience in microscopic observation and upon his knowledge of the class of bodies to which the particular specimen may belong. Not only are observations of any kind liable to certain fallacies arising out of the previous notions which the observer may entertain in regard to the constitution of the objects or the nature of the actions to which his attention is directed, but even the mosf practiced observer is apt to take no note of such phenomena as his mind is not prepared to appreciate. Errors and im- perfections of this kind can only be corrected, it is obvious, by general advance in scientific knowledge ; but the history of them affords a use- ful warning against hasty conclusions drawn from a too cursor}' exam- ination. If the history of almost any scientific investigation were fully made known it would generally appear that the stability and completeness of the conclusions finally arrived at had been only attained after many modifications, or even entire alterations, of doctrine. And it is therefore of such great importance as to be almost essential to the correctness of our conclusions that they should not be finally formed and announced until they have been tested in every conceivable mode. It is due to science that it should be burdened with as few false facts [artifacts] and false doctrines as possible. It is due to other truth-seekers that they should not be misled, to the great w T aste of their time and pains, by our errors. And it is due to ourselves that we should not commit our reputation to the chance of impairment by the premature formation and publication of conclusions which may be at once reversed by other observers better informed than ourselves, or mav be proved fallacious at some future time, perhaps even by our 92 INTERPRETATION OF APPEARANCES \CH, III own more extended and careful researches. The suspension of the judg- ment whenever there seems room for doubt is a lesson inculcated by all those philosophers who have gained the highest repute for practical wisdom ; and it is one which the microscopist cannot too soon learn or too constantly practice." For these experiments no condenser is to be used except where specifically indicated. § 130. Dust or Cloudiness on the Ocular. — Employ the 16 mm. ( 2 3 in.) objective, low ocular, and fly's wing as object. Unscrew the field-lens and put some particles of lint from dark cloth on its upper surface. Replace the field-lens and put the ocular in position (§ 48). Light the field well and focus sharply. The im- age will be clear, but part of the field will be obscured by the irregular outline of the particles of lint. Move the object to make sure this appearance is not due to it. Grasp the ocular by the milled ring, just above the tube of the microscope, and rotate it. The irregular objects will rotate with the ocular. Cloudiness or particles of dust on any part of the ocular may be detected in this way. § 131. Dust or Cloudiness on the Objective. — Employ the same ocular and objective as before and the fly's wing as object. Focus and light well, and observe carefully the appearance. Rub glycerin on one side of a slide near the end. Hold the clean side of this end close against the objective. The image will be obscured, and cannot be made clear by focusing. Then use a clean slide and the image ma}' be made clear by elevating the tube slightly. The obscurity produced in this way is like that caused by clouding the front-lens of the objec- tive. Dust would make a dark patch on the image that would remain stationary while the object or ocular is moved. If a small diaphragm is employed and it is close to the object, only the central part of the field will be illuminated, and around the small light circle will be seen a dark ring (Fig. 42). If the diaphragm is lowered or a sufficiently large one employed the entire field will be lighted. S 132. Relative Position of Objects or parts of the same object. The general rule is that objects highest up come into focus last in focusing up, first in focusing down. § 133. Objects having Plane or Irregular Outlines. — As object use three printed letters in stairs mounted in Canada balsam (Fig. 89). The first letter is placed directly upon the slide, and covered with a CH. Ill] INTERPRETATION OF APPEARANCES 93 small piece of glass about as thick as a slide. The second letter is placed upon this and covered in like manner. The third letter is placed upon the second thick cover and covered with an ordinary cover-glass. The letters should be as near together as possible, but not over-lapping. Employ the same ocular and objective as above (§ 130). Fig. S9. Letters mounted in stairs to show the order of coming into focus. b c d S / X A F. . a, b, c, d. The various letters indi- cated by the oblique row of black marks in sectional view. Slide. The glass slide on which the letters are mounted. Lower the tube till the objective almost touches the top letter, then look into the microscope, and slowly focus up. The lowest letter will first appear, and then, as it disappears, the middle one will appear, and so on. Focus down, and the top letter will first appear, then the mid- dle one, etc. The relative position of objects is determined exactly in this way in practical work. For example, if one has a micrometer ruled on a cover-glass 15-25 hundredths mm. thick, it is not easy to determine with the naked eye which is the ruled surface. But if one puts the micrometer under a microscope and uses a 3 mm. ('a in.) objective, it is easily determined. The cover should be laid on a slide and focused till the lines are sharp. Xow, without changing the focus in the least turn the cover over. If it is necessary to focus up to get the lines of the micrometer sharp, the lines are on the upper side. If one must focus down, the lines are on the under surface. With a thin cover and delicate lines this method of determining the position of the rulings is of considerable practical importance. § 134. Determination of the Form of Objects. — The procedure is exactly as for the determination of the form of large objects. That is, one must examine the various aspects. For example, if one were placed in front of a wall of some kind he could not tell whether it was a simple wall or whether it was one side of a building unless in some way he could see more than the face of the wall. In other words, in order to get a correct notion of any body, one must examine more than one dimension, — two for plane surfaces, three for solids. So for micro- scopic objects, one must in some way examine more than one face. To do this with small bodies in a liquid the bodies may be made to roll over by pressing on one edge of the cover-glass. And in rolling over the various aspects are presented to the observer. With solid bodies, 94 INTERPRETATION OF APPEARANCES [CH. Ill like the various organs, correct notions of the form of the elements can be determined by studying sections cut at right angles to each other. The methods of getting the elements to roll over, and of sectioning in different planes are in constant use in Histology, and the microscopist who neglects to see all sides of the tissue elements has a very inade- quate and often a very erroneous conception of their true form. § 135. Transparent Objects having Curved Outlines. — The success of these experiments will depend entirely upon the care and skill used in preparing the objects, in lighting, and in focusing. Employ a 3 mm. ( Vs in.) or higher objective and a high ocular for all the experiments. It may be necessary to shade the object (§ 109) to get satisfactory results. When a diaphragm is used the opening should be small and it should be close to the object. § 136. Air Bubbles. — Prepare these by placing a drop of thin mucilage on the center of a slide and beating it with a scalpel blade until the mucilage looks milky from the inclusion of air bubbles. Put on a cover-glass but do not press it down. Fig. 90. Diagram show- !j^£_j^liiK? ing how to place a cover- glass upon an object with the forceps. § 137. Air Bubbles with Central Illumination.— Shade the object ; and with the plane mirror, light the field with central light (Fig. 23). Search the preparation until an air bubble is found appearing about 1 mm. in diameter, get it into the center of the field, and if the light is central the air bubble will appear with a wide, dark, circular margin and a small bright center. If the bright spot is not in the center, adjust the mirror until it is. This is one of the simplest and surest methods of telling when the light is central or axial when no condenser is used (§ 65). Focus both up and down, noting that, in focusing up, the central spot becomes very clear and the black ring very sharp. On elevating the tube of the microscope still more the center becomes dim, and the whole bubble loses its sharpness of outline. § 13.S. Air Bubbles with Oblique Illumination.— Remove the sub-stage of the microscope and all the diaphragms. Swing the mirror so that the rays may be sent very obliquely upon the object (Fig. 23, CH.III~\ INTERPRETATION OF APPEARANCES 95 C). The bright spot will appear no longer in the center but on the side away from the mirror (Fig. yi). § 139. Oil Globules. — Prepare these by beating a small drop of clove oil with mucilage on a slide and covering as directed for air bub- bles (§ 137), or use a drop of milk. § 140. Oil Globules with Central Illumination. — Use the same diaphragm and light as above (§ 137). Find an oil globule appearing about 1 mm. in diameter. If the light is central a bright spot will ap- pear in the center as with air. Focus up and down as with air, and note that the bright center of the oil globule is clearest last in focus- ing up. A » > 3 o Fig. 91. Very small Globule of Oil (O) and an Air Bubble (A) seen by Oblique Light. The arrow indicates the direction of the light rays. § 141. Oil Globules with Oblique Illumination. — Remove the sub-stage, etc., as above, and swing the mir- ror to one side and light with oblique light. The bright spot will be eccentric, and will appear to be on the same side as the mirror (Fig. 91). § 142. Oil and Air Together. — Make a prepara- tion exactly as described for air bubbles (§ 136), and add at one edge a little of the mixture of oil and mucilage (§ T 39) ! cover and examine. The sub-stage need not be used in this experiment. Search the preparation until an air bubble and an oil globule, each appearing about 1 mm. in diameter, are found in the same field of view. Light first with central light, and note that, in focusing up, the air bubble comes into focus first and that the central spot is smaller than that of the oil globule. Then, of course, the black ring will be wider in the air bubble than in the oil globule. Make the light oblique. The bright spot in the air bubble will move away from the mirror while that in the oil globule will move toward it. See Fig. 91.* § 143. Air and Oil by Reflected Light. — Cover the diaphragm or mirror so that no transmitted light (§ 64) can reach the preparation, using the same preparation as in § 142. The oil and air will appear like globules of silver on a dark ground. The part that was darkest in *It should be remembered that the image in the compound microscope is inverted (Fig. 21), hence the bright spot really moves toward the mirror for air, and away from it for oil. 96 INTERPRETATION OF APPEARANCES [CH. Ill each will be lightest, and the bright central spot will be somewhat dark.* § 144. Distinctness of Outline. — In refraction images this depends on the difference between the refractive power of a body and that of the medium which surrounds it. The oil and air were very distinct in outline as both differ greatly in refractive power from the medium which surrounds them, the oil being more refractive than the mucilage and the air less. (Figs. 54-56.) Place a fragment of a cover-glass on a clean slide, and cover it (see under mounting). The outline will be distinct with the unaided eye. Use it as object and employ the 16 mm. (-1 in. ) objective and high ocular. Light with central light. The fragment will be outlined by a dark band. Put a drop of water at the edge of the cover-glass. It will run in and immerse the fragment. The outline will remain dis- tinct, but the dark band will be somewhat narrower. Remove the cover-glass, wipe it dry, and wipe the fragment and slide dry also. Put a drop of 50% glycerin on the middle of the slide and mount the .fragment of cover-glass in that. The dark contour will be much nar- rower than before. Draw a solid glass rod out to a fine thread. Mount one piece in air, and the other in 5c 1 ,, glycerin. Put a cover-glass on each. Em- ploy the same optical arrangement as before. Examine the one in air first. There will be seen a narrow, bright band, with a wide, dark baud on each side. The one in glycerin will show a much wider bright central band, with the dark borders correspondingly narrow (Fig. 92, b). The dark contour depends also on the numerical aperture of the objective — being wider with low apertures. This can be readily understood when it is remembered that the greater the aperture the more oblique the rays of light that can be received, and the dark band simply represents an area in which the rays are so greatly bent or refracted (Figs. 54-56) that the)- cannot enter the objective and contribute to the formation of the image ; the edges are dark simply because no light from them reaches the observer. *It is possible to distinguish oil and air optically, as described above, only when quite high powers are used and very small bubbles are selected for observa- tion. If a 16 mm. (-, in. ) is used instead of a 3 mm. ( 's in.) objective, the ap- pearances will vary considerably from that given above for the higher power. It is well to use a low as well as a high power. Marked differences will also be seen in the appearances with objectives of small and of large aperture. CH.III] INTERPRETATION OF APPEARANCES 97 Fig. 92. Solid glass rod showing the appearance when viewed with transmit- ted, central light, and with an objective of medium aperture, a. Mounted in air. b. Mounted in 50 per cent, glycerin. If the glass rod or any other object were mounted in a medium of the same color and refractive power, it could not be distinguished from the medium.* A very striking and satisfactory demonstration may be made by painting a zone or band of eosin or other transparent color on a solid glass rod, and immersing the rod in a test tube or vial of cedar oil, clove oil or turpentine. Above the liquid the glass rod is very evident, as it is also at the colored zone, but at other levels it can hardly be seen in the liquid. £ 145. Highly Refractive. — This expression is often used in de- scribing microscopic objects, (medullated nerve fibers, for example), and means that the object will appear to be bordered by a wide, dark margin when it is viewed by transmitted light. And from the above (§ 144), it would be known that the refractive power of the object, and the medium in which it was mounted must differ considerably. S 146. Doubly Contoured. — This means that the object is bounded by two, usually parallel dark lines with a lighter band between them. In other words, the object is bordered by ( 1 ) a dark line, (2 J a light band, and (3 J a second dark line (Fig. 93). This may be demonstrated by coating a fine glass rod (§ 144 ) with one or more coats of collodion or celloidin and allowing it to dry, and then mounting in 50% glycerin as above. Employ a 3 mm. (^3 in.) or higher objective, light with transmitted light, and it will be seen that where the glycerin touches the collodion coating there is a dark line — next this is a light band, and finally there is a second dark line where the collodion is in contact with the glass rod.* (Fig. 93). , Fig. 93. Solid glass rod coated with col- lodion to show a double contour. Toward I one end the collodion had gathered in a fusi- form drop. *Some of the rods have air bubbles in them, and then there results a capillary tube when they are drawn out. It is well to draw out a glass tube into a fine thread and examine it as described. The central cavity makes the experiment much more complex. 9 S INTERPRETATION OF APPEARANCES [CH. Ill § 147. Optical Section. — This is the appearance obtained in examining transparent or nearly transparent objects with a microscope when some plane below the upper surface of the object is in focus. The upper part of the object which is out of focus obscures the image but slightly. By changing the position of the objective or object, a different plane will be in focus and a different optical section obtained. The most satisfactory optical sections are obtained with high objectives having large aperture. Nearly all the transparent objects studied ma}' be viewed in optical section. A striking example will be found in studying mammalian red blood-corpuscles on edge. The experiments with the solid glass rods (Fig. 92) furnish excellent and striking examples of optical sections. § 148. Currents in Liquids. — Employ the 16 mm. (^3 in.) ob- jective, and as object put a few particles of carmine on the middle of a slide, and add a drop of water. Grind the carmine well with a scalpel blade, and then cover it. If the microscope is inclined, a current will be produced in the water, and the particles of carmine will be carried along by it. Note that the particles seem to flow up instead of down — why is this ? Lamp-black rubbed in water containing a little mucilage answers well for this experiment. § 149. Velocity Under the Microscope. — In studying currents or the movement of living things under the microscope, one should not forget that the apparent velocity is as unlike the real velocity as the apparent size is unlike the real size. If one consults Fig. 37 it will be seen that the actual size of the field of the microscope with the different objectives and oculars is inversely as the magnification. That is, with great magnification only a small area can be seen. The field appears to be large, however, and if any object moves across the field it may appear to move with great rapidity, whereas if one measures the actual distance passed and notes the time, it will be seen that the actual motion is quite slow. One should keep this in mind in study- ing the circulation of the blood. The truth of what has just been said can be easily demonstrated in studying the circulation in the gills *The collodion used is a 6",, solution of gun cotton in equal parts of sulphuric ether and 95% alcohol. It is well to dip the rod two or three times in the collo- dion and to hold it vertically while drying. The collodion will gather in drops, and one will see the difference between a thick and a thin membranous covering. (Fig- 93)- CH. iff] INTERPRETATION OF APPEARANCES 99 of Necturus, or in the frog's foot, b}' using first a low power in which the field is actually of considerable diameter (Fig. 37, Table, § 51) and then using a high power. With the high power the apparent motion will appear much more rapid. For spiral, serpentine and other forms of motion, see Carpenter-Dallinger, p. 433. § 150. Pedesis or Brownian Movement. — Employ the same object as above, but a 3 mm. ('s in.) or higher objective in place of the 16 mm. Make the bod}' of the microscope vertical, so that there may be no currents produced. Use a small diaphragm and light the field well. Focus and there will be seen in the field large motionless masses, and between them small masses in constant motion. This is an indefinite, dancing or oscillating motion. This indefinite but continuous motion of small particles in a liquid is called Pe-de'sis or Brownian movement . Also, but improperly, molec- ular movement, from the smallness of the particles. The motion is increased by adding a little gum arabic solution or a slight amount of silicate of soda or soap ; sulphuric acid and various saline compounds retard or check the motion. One of the best objects is lamp-black ground up with a little gum arabic. Carmine prepared in the same way, or simply in water, is excellent ; and very finely powdered pumice-stone in water has for many years been a favorite object. Pedesis is exhibited by all solid matter if it is finely enough di- vided and in a suitable liquid. In the minds of most, no adequate explanation has yet been offered. See Carpenter-Dallinger, p. 431 ; Beale, p. 195 ; Jevons in Quart, four. Science, n. s. , Vol. VIII (1878), p. 167. In 1894, Meade Bache published a paper in the Proc. Amer. Philos. Soc, Vol. XXXIII, pp. 163-167, entitled "The Secret of the Brownian Movement." This paper is suggestive if not very satisfactory. For the orginal account of this see Robert Brown, "Botanical appendix to Captain King's voyage to Australia," Vol. II, p. 534. (1826 J. See also Dr. C. Aug. Sigm. Schultze, "Mikroskopische Unter- suchungen fiber des Herren Robert Brown Entdeckunglebender, selbst im Feuer unzerstorbarer Theilchen in alien Kbrpern." From "Die Gesellschaft fur Betbrderung der Naturwissenschaften zu Freiburg. ' ' 1828. Compare the pedetic motion with that of a current by slightly in- clining the tube of the microscope. The small particles will continue ioo INTERPRETATION OF APPEARANCES [CH.I1I their independent leaping movements while they are carried along by the current. The pedetic motion makes it difficult to obtain good photographs of milk globules and other small particles. The difficulty may be overcome by mixing the milk with a very weak solution of gelatin and allowing it to cool (see Ch. IX ). § 151. Demonstration of Pedesis with the Polarizing Micro- scope (Ch. VI). — The following demonstration shows conclusively that the pedetic motion is real and not illusive. (Ranvier, p. 173. ) Open the abdomen of a dead frog (an alcoholic or formalin specimen is satisfactory). Turn the viscera to one side and observe the small, whitish masses at the emergence of the spinal nerves. With fine forceps remove one of these and place it on the middle of a clean slide. Add a drop of water, or of water containing a little gum arabic. Rub the white mass around in the drop of liquid and soon the liquid will have a milky appearance. Remove the white mass, place a cover- glass on the milky liquid and seal the cover by painting a ring of castor oil all around it, half the ring being on the slide and half on the cover-glass. This is to avoid the production of currents by evaporation. Put the preparation under the microscope and examine with, first a low then a high power (3 mm. or '3 in. ). In the field will be seen multitudes of crystals of carbonate of lime ; the larger crystals are motionless but the smallest ones exhibit marked pedetic movement. Use the micro-polariscope, light with great care and exclude all adventitious light from the microscope by shading the object (§ 109) and also by shading the eye. Focus sharply and observe the pedetic motion of the small particles, then cross the polarizer and analyzer, that is, turn one or the other until the field is dark. Part of the large motion- less crystals will shine continuously and a part will remain dark, but small crystals between the large ones will shine for an instant, then disappear, only to appear again the next instant. This demonstration is believed to furnish absolute proof that the pedetic movement is real and not illusory. §152. Muscae Volitantes. — These specks or filaments in the eyes due to minute shreds or opacities of the vitreous sometimes appear as part of the object as they are projected into the field of vision. They may be seen by looking into the well lighted microscope when there is no object under the microscope. They may also be seen \>y looking at brightly illuminated snow or other white surface. By studying them carefully it will be seen that they are somewhat movable and float CH. ///] INTERPRETATION OF APPEARANCES 101 across the field of vision, and thus do not remain in one position as do the objects under observation. Furthermore, one may, by taking a little pains, familiarize himself with the special forms in his own eyes so that the more conspicuous at least may be instantly recognized. S 153. In addition to the above experiments it is very strong^' recommended that the student follow the advice of Beale, p. 248, and examine first with a low then with a higher power, mounted dry, then in water, lighted with reflected light, then with transmitted light, the following : Potato, wheat, rice, and corn starch, easily obtained by scraping the potato and the grains mentioned ; bread crumbs ; portions of feather. Portions of feather accidentally present in histological preparations have been mistaken for lymphatic vessels ( Beale, 288). Fibers of cotton, linen and silk. Textile fibers accidentally present have been considered nerve fibers, etc. Human and animal hairs. Study with especial care hairs from various parts of the body of the animals used for dissection in the laboratory where you work. These are liable to be present in histological preparations, and unless their character is understood there is chance for much confusion and erro- neous interpretation. The scales of butterflies and moths, especially the common clothes moth. The dust swept from carpeted and wood floors. Tea leaves and coffee grounds. Dust found in living rooms and places not frequently dusted. In the last will be found a regular museum of objects. For figures (photo-micrographs, etc. ) of the various forms of starch, see Bulletin No. 13 of the Chemical Division of the U. S. Department af Agriculture. For Hair and Wool, see Bulletin of the National Asso- ciation of Wool Growers, 1875, P- 47°' Proc. Amer. Micr. Soc, 1884, pp. 65-6S. Herzfeld, translated by Salter. — The technical testing of yarns and textile fabrics, London, 1898. For different appearances due to the illuminator, see Nelson, in Jour. Roy, Micr. Soc, 1891, pp. 90-105 ; and for the illusory appear- ances due to diffraction phenomena, see Carpenter-Dallinger, p. 434. Mercer. Trans. Amer. Micr. Soc., pp. 321-396. If it is necessary to see all sides of an ordinary gross object, and to observe it with varying illumination and under various conditions of temperature, moisture, etc., in order to obtain a fairly accurate and satisfactory knowledge of it, so much the more is it necessary not to be satisfied in microscopical observation until every means of investigation and verification has been called into service, and then of the image that falls upon the retina, only such details will be noted as the brain behind the eye is ready to appreciate. INTERPRETATION OF APPEARANCES [ CH. Ill To summarize this chapter and leave with the beginning student the result of the experience of many eminent workers : i. Get all the information possible with the unaided eye. See the whole object and all sides of it, so far as possible. 2. Examine the preparation with a simple microscope in the same thorough way for additional detail. 3. Use a low power of the compound microscope. 4. Use a higher power. 5. Use the highest power available and applicable. In this way one sees the object as a whole and progressively more and more details. Then as the object is viewed from two or more aspects, something like a correct notion may be gained of its form and structure. THE MICROSCOPE IN SECTION 1. Positive ocular. 2. Draw-tube. 3. Maiu tube or bodv. -5. Society screws in the ;draw-tube and body. 6. Objective in position. 7. Stage. 8. Spring for holding- slides. 9. Sub-stage condenser. 10. Iris diaphragm. 11. Plane and concave mir- ror. Horse-shoe base. Rack and pinion for condenser. Flexible pillar. Spiral spring of fine ad- justment. Fine adjustment Coarse adjustment. 1 3 CHAPTER IV MAGNIFICATION AND MICROMETRY APPARATUS AND MATERIAL FOR THIS CHAPTER Simple and compound microscope (f 156, 158); Steel scale or rule divided to millimeters and Iths ; Block for magnifier and compound microscope (2 156, 160); Dividers (? 156, 160); Stage micrometer (? 159); Wollaston camera lucida ( \ 160); Ocular screw-micrometers (Figs. 106-107); Micrometer ocular (Figs. 104-105). Abbe camera lucida (Fig. 101 ). Necturus red blood corpuscles (§ 168). § 154. The Magnification, Amplification or Magnifying Power of a simple or compound microscope is the ratio between the real and the apparent size of the object examined. The apparent size is ob- tained by measuring the virtual image (Figs. 21, 38), The object for determining magnification must be of known length and is designated a micrometer (§159). In practice a virtual image is measured by the aid of some form of camera lucida (Figs. 97, 101), or by double vision (§ 156). As the length of the object is known, the magnification is easily determined by dividing the apparent size of the image by the actual size of the object. For example, if the virtual image measures 40 mm. and the object magnified, 2 mm., the amplification must be 40 -r- 2 = 20, that is, the apparent size is 20 fold greater than the real size. Magnification is expressed in diameters or times linear, that is, but one dimension is considered. In giving the scale at which a micro- scopical or histological drawing is made, the word magnification is fre- quently indicated by the sign of multiplication thus : X 450, upon a drawing would mean that the figure or drawing is 450 times as large as the object. § 155. Magnification of Real Images. — In this case the mag- nification is the ratio between the size of the real image and the size of the object, and the size of the real image can be measured directly. By recalling the work on the function of an objective (§ 53), it will be remembered that it forms a real image on the ground glass placed on the top of the tube, and that this real image could be looked at with the 104 MAGNIFICATION AND MICROMETRY [CH. IV eye or measured as if it were an actual object. For example, suppose the object were three millimeters long and its image on the ground glass measured 15 mm., then the magnification must be, 15-5-3 = 5, that is, the real image is 5 times as long as the object. The real images seen in photograph}- are mostly smaller than the objects, but the magnifica- tion is designated in the same way by dividing the size of the real im- age measured on the ground glass by the size of the object. For example, if the object is 400 millimeters long and its image on the ground glass is 25 mm. long, the ratio is 25-f-400=- 1 1 ^-. That is, the image is j 1 ,. th as long as the object and is not magnified but reduced. In marking negatives, as with drawings, the sign of multiplication is put before the ratio, and in the example the designation would be rth. MAGNIFICATION OF A SIMPLE MICROSCOPE § 156. The Magnification of a Simple Microscope is the ratio between the object magnified (Fig. 16, A'B'), and the virtual image (A 3 B 3 ). To obtain the size of this virtual image place the tripod mag- nifier near the edge of a support of such a height that the distance from the upper surface of the mag- nifier to the table is 250 millimeters. Fig. 94. Tripod Magnifier. As object, place a scale of some kind ruled in millimeters on the support under the magnifier. Put some white paper on the table at the base of the support and on the side facing the light. Fig. 95. Ten Centimetei Rule. The upper edge is divided into millimeters, the lower into centimeters at the left and half centimeters at the right. Close one eye, and hold the head so that the other will be near the upper surface of the lens. Focus if necessary to make the image clear CH. IV] MA GNIFICA TION AND MICROME TR Y 1 05 (§ 11). Open the closed eye and the image of the rule will appear as if on the paper at the base of the support. Hold the head very still, and with dividers get the distance between any two lines of the image. This is the so-called method of double vision in which the microscope image is seen with one eye and the dividers with the other, the two images appearing to be fused in a single visual field. § 157. Measuring the Spread of Dividers. — This should be done on a steel scale divided to millimeters and Aths. As \ mm. cannot be seen plainly by the unaided eye, place one arm of the dividers at a centimeter line, and with the tripod magnifier count the number of spaces on the rule included between the points of the dividers. The magnifier simply makes it easy to count the spaces on the rule included between the points of the dividers — it does not, of course, increase the number of spaces or change their value. As the distance between any two lines of the image of the scale gives the size of the virtual image (Fig. 16, A S B 3 ), and as the size of the object is known, the magnification is determined by dividing the size of the image by the size of the object. Thus, suppose the distance between the two lines of the image is measured by the dividers and found on the steel scale to be 15 millimeters, and the actual size of the space between the two lines of the object is 2 millimeters, then the magnification must be i5-=-2=7/^. That is, the image is 7'j times as long or wide as the object. In this case the image is said to be magnified ■j 1 2 diameters, or 7*2 times linear. The magnification of any simple magnifier may be determined experimentally in the way described for the tripod. .MAGNIFICATION OF A COMPOUND MICROSCOPE § 158. The Magnification of a Compound Microscope is the ratio between the final or virtual image (Fig. 21, B^A 3 ), and the object magnified (A B ). The determination of the magnification of a compound microscope may be made as with a simple microscope (§ 156), but this is very fatiguing and unsatisfactory. § 159. Stage, Object or Objective Micrometer. — For deter- mining the magnification of a compound microscope and for the purpose of micrometry, it is necessary to have a finely divided scale or rule on glass or on metal. Such a finely divided scale is called a micrometer, and for ordinary work one mounted on a glass slide (1X3 in, 25 X 76 mm.) is most convenient. 106 MAGNIFICATION AND MICROMETRY [CH. IV The spaces between the lines should be y 1 -^ and ^\ Ti mm. (or if in inches, T -^ ¥ and twit m -) Micrometers are sometimes ruled on the slide, but more satisfactorily on a cover-glass of known thickness, preferably o. 15-0. 18 mm. The covers should be perfectly clean before the ruling, and afterwards simply dusted off with a camel's hair duster, and then mounted, lines downward over a shellac or other good cell. (See Ch. VII). If one rubs the lines the edges of the furrow made by the diamond are liable to be rounded and the sharp- ness of the micrometer is lost. If the lines are on the slide and un- covered one cannot use the micrometer with an oil immersion, as the oil obliterates the lines. Cleaning the slide makes the lines less sharp as stated. If the lines are very coarse, it is an advantage to fill them with plumbago. This may be done either with some very fine plum- bago on the end of a soft cork, or by using an exceedingly soft lead pencil. Lines properly filled may be covered with balsam and a cover- glass as in ordinary balsam mounting (Ch. VII). § 160. Determination of Magnification. — This is most readily accomplished by the use of some form of camera lucida (Ch. V), that of Wollaston being most convenient as it may be used for all powers, and the determination of the standard distance of 250 millimeters at which to measure the images is very readily determined (Fig. 97, S 162)- Employ the 16 mm. ( -'3 in. ) objective and a 37 mm. (or x S ocu- lar with a stage micrometer as object. For this power the yV mm. spaces of the micrometer should be used as object. Focus sharply. Fig. 96. Diagram of a stage micrometer, with a ring on the lines to facilitate finding them. It is somewhat difficult to find the mi- crometer lines. To avoid this it is well to have a small ring enclosing some of the micrometer lines (Fig. 96). The light must also be carefully regu- lated. If too much light is used, i. e., too large an aperture, the lines will be drowned in the light. In focusing with the high powers be very careful. Remember the micrometers are expensive, and one can- not afford to break them. As suggested in § 74, focus on the edge of the cement ring enclosing the lines, then in focusing down to find the lines, move the preparation very slightly, back and forth. After the lines are sharply focused, and the slide clamped in posi- tion make the tube of the microscope horizontal, by bending the flexible pillar, being careful not to bring any strain upon the fine adjustment (frontispiece ). CH. IV] MAGNIFICATION AND MICROMETRY 107 Fig. 97. Wollaston's Camera Lu- cida, showing the rays from the micro- scope and from the drawing surface, also the position of the pupil of the eye. Axis, Axis. Axial rays from the microscope and from the drawing surface (Ch. V). Camera Lucida. A section of the quadrangular prism showing the course of the rays in the prism from the microscope to the eye. As the rays are twice reflected, they have the same relation on entering the eye that they would have by looking directly into the ocular. A. B. The lateral rays from the microscope and their projection upon the drawing surface. C. D Rays from the drawing surface to ths eye. A. D. A' D' . Overlapping portions of the two fields, where both the micro- scopic image and the drawing surface, pencil, etc., can be seen. It is represented by the shaded part of the overlapping circles at the right. Ocular. The ocular of the microscope. P. The drawing pencil. Its point is shown in the overlapping; fields. Fig. 97. Put a Wollaston camera lucida (Fig. 97 and Ch. V) in position, and turn the ocular around if necessary so that the broad flat surface may face directly upward, as shown in Fig. 97. Elevate the micro- scope by putting a block under the base, so that the perpendicular dis- tance from the upper surface of the camera lucida to the table is 250 mm. (§ 162). Place some white paper on the work-table beneath the camera lucida. Close one eye, and hold the head so that the other may be very close to the camera lucida. Look directly down. The image will ap- pear to be on the table. It may be necessary to readjust the focus after the camera lucida is in position. If there is difficulty in seeing dividers and image consult Ch. V. Measure the image with dividers and obtain the power exactly as above (§156-157). Thus : Suppose two of the T \jth mm., spaces were taken as object, and the image is measured by the dividers, and the spread of the dividers is found on the steel rule to be 9+ millimeters. If now the object is fVths of a millimeter and the magnified image is gi milli- meters, the magnification (which is the ratio between size of object io8 MAGNIFICATION AND MICROMETRY [CH. IV and image) must be gf -5- -^ = 47. That is, the magnification is 47 diameters, or 47 times linear. If the fractional numbers in the above example trouble the student, both may be reduced to the same denom- ination, thus : If the size of the image is found to be 9-j mm. this number mav be reduced to tenths mm., so it will be of the same Image Fig. 98. Fig. 99. Figs, 9S-99. Figures showing that the size of object and image very directly as their distance from the center of the lens. In Fig. qq one can also see why it is necessary to focus down, 1. , Optical Co.). •5 175. Varying the Ocular Micrometer Valuation. — Any change in the objective, the ocular or the tube-length of the microscope, that is to say, any change in the size of the real image, produces a cor- responding change in the ocular micrometer valuation (§ 161, 171, 176). ij 176. Remarks on Micrometry. — In using adjustable objectives ( (-24, 103), the magnification of the objective varies with the position of the adjusting collar, being greater when the adjustment is closed as for thick cover-glasses than when open, as for thin ones. This variation in the magnification of the objective pro- duces a corresponding change in the magnification of the entire microscope, and the ocular micrometer valuation — therefore it is necessary to determine the mag- nification and ocular micrometer valuation for each position of the adjusting collar. While the principles of micrometry are simple, it is very difficult to get the exact size of microscopic objects. This is due to the lack of perfection and uni- *There are three ways of using the ocular micrometer, or of arriving at the size of the objects measured with it : (A) By finding the value of a division of the ocular micrometer for each optical combination and tube-length used, and employing this valuation as a multiplier. This is the method given in the text, and [the one most frequently employed. Thus, suppose with a given optical combination and tube-length it required five divisions on the ocular micrometer to include the image of f\ths millimeter of the stage micrometer, then obviously one space on the ocular micrometer would in- clude 1th of ,- M ths mm. or ,'th mm.; the size of any unknown object under the CH. IV] MAGNIFICATION AND MICROMETRY 119 formity of micrometers, and the difficulty of determining the exact limits of the object to be measured. Hence, all microscopic measurements are only approxi- mately correct, the error lessening with the increasing perfection of the apparatus and the skill of the observer. microscope would be obtained by multiplying the number of divisions on the ocular micrometer required to include its image by the value of one space, or in this case, i^th mm. Suppose some object, as the fly's wing, required 15 spaces of the ocular micrometer to include some part of it, then the actual size of this part of the wing would be 15 X A = fths, or 0.6 mm. (B) By finding the number of divisions on the ocular micrometer required to include the image of an entire millimeter of the stage micrometer, and using this number as a divisor. This number is also sometimes called the ocular micrometer ratio. Taking the same case as in (A), suppose five divisions of the ocular mi- crometer are required to include the image of fjthstiim., on the stage micrometer, then evidently it would require 5 h- t 2 (7 = 25 divisions on the ocular micrometer to include a whole millimeter on the stage micrometer, and the number of divisions of the ocular micrometer required to measure an object divided by 25 would give the actual size of the object in millimeters or in a fraction of a millimeter. Thus, suppose it required 15 divisions of the ocular micrometer to include the image of some part of the fly's wing, the actual size of the part included would be 15-j- 25 = ; ; or 0.6 mm. This method is really exactly like the one in (A), for dividing by 25 is the same as multiplying by -}-\\x. ( C) By having the ocular micrometer ruled in millimeters and divisions of a millimeter, and then getting the size of the real image in millimeters. In employ- ing this method a stage micrometer is-used as object and the size of the image of one or more divisions is measured by the ocular micrometer, thus : Suppose the stage micrometer is ruled in iVjth and T( Toth mm. and the ocular micrometer is ruled in millimeters and y'ljth mm. Taking i 2 , T th mm. on the stage micrometer as object, as in the other cases, suppose it requires 10 of the ^th mm. spaces or 1 mm. to measure the real image, then the real image must be magnified |I}-^t 2 o = 5 diame- ters, that is, the real image is five times as great in length as the object, and the size of an object may be determined by putting it under the microscope and getting the size of the real image in millimeters with the ocular micrometer and dividing it by the magnification of the real image, which in this case is 5 diameters. Use the fly's wing as object, as in the other cases, and measure the image of the same part. Suppose that it required 30 of the j 1 ^ mm. divisions = \% mm. or 3 mm. to include the image of the part measured, then evidently the actual size of the part measured is 3 mm, -h— 5 = ■§ mm., the same result as in the other cases. In comparing these methods it will be seen that in the first two (A and B) the ocular micrometer may be simply ruled with equidistant lines without regard to the absolute size in millimeters or inches of the spaces. In the last method the ocular micrometer must have its spaces some known division of a millimeter or inch. In the first two methods only one standard of measure is required, viz, the stage micrometer ; in the last method two standards must be used, — a stage mi- crometer and an ocular micrometer. Of course, the ocular micrometer in the first two cases must have the lines equidistant as well as in the last case, but ruling lines equidistant is quite a different matter from getting them an exact division of a millimeter or of an inch apart. MAGNIFICATION AND MICROMETRY [CH. IV A difficulty when one is using high powers is the width of the lines of the micrometer. If the micrometer is perfectly accurate half the width of each line belongs to the contiguous spaces, hence one should measure the image of the space from the centers of the lines bordering the space, or as this is somewhat difficult in using the ocular micrometer, one may measure from the inside of one border- ing line and from the outside of the other. If the lines are of equal width this is as accurate as measuring from the center of the lines. Evidently it would not be right to measure from either the inside or the outside of both lines (Fig. 10S). It is also necessary in micrometry to use an objective of sufficient power to enable one to see all the details of an object with great distinctness. The necessity of using sufficient amplification in micrometry has been especially remarked upon by Richardson, Monthly Micr. Jour., 1S74, 1S75, ; Rogers, Proc. Amer. Soc. Micro- scopists, 18S2, p. 239; Ewell, North American Pract., 1S90, pp. 97, 173. Fig. 10S. Tlic appearance of the coarse A B stage micrometer and of 'the fine ocular ■mi- crometer lines when using a high objective. (,•/). The method of measuring the spaces by putting the fine ocular mi- crometer lines opposite the center of the coarse stage micrometer lines. ( H). Method of measuring the spaces of Ike stage micrometer by pulling one line of the ocular micrometer (0. m. ) a I the inside and one at the outside of the coarse s/a^e micrometer lines (s. m.). Fig. 10S. As to the limit of accurac}- in micrometry, one who has justly earned the right to speak with authority expresses himself as follows: "/ assume that 0.211 is the limit of precision in microscopic measures beyond which it is impossible to go with certainty." W. A. Rogers Proc. Amer. Soc. Micrs., 1SS3, p. 19S. In comparing the methods of micrometry with the compound microscope given above ( \ 167, 16S, 169, 175), the one given in '',. 167 is impracticable, that given in '',, 168 is open to the objection that two standards are required, — the stage microme- ter, and the steel rule ; it is open to the further objection that several different operations are necessary, each operation adding to the probability of error. Theoret- ically the method given in ji 169 is good, but it is open to the very serious objection in practice that it requires so many operations which are especially liable to intro- duce errors. The method that experience has found most safe and expeditious, and applicable to all objects, is the method with the ocular micrometer. If the valuation of the ocular micrometer has been accurate!}' determined, then the only difficulty is in deciding on the exact limits of the objects to be measured and so arranging the ocular micrometer that these limits are inclosed by some divisions of the micrometer. Where the object is not exactly included by whole spaces on the ocular micrometer, the chance of error comes in, in estimating just how far into a space the object reaches 011 the side not in contact with one of the micrometer \_Cff. IV MAGNIFICATION AND MICROMETRY 121 lines. If the ocular micrometer has some quite narrow spaces, and others consid- erably larger, one can nearly always manage to exactly include the object by some two lines. The ocular screw micrometers (Fig. 106-107) obviates this entirely as the cross hair or lines traverse the object or its real image, and whether this distance be great or small it can be read off on the graduated wheel, and no estimation or guess work is necessary. For those especially interested in micrometry, as in its relation to medical jurisprudence, the following references are recommended. These articles consider the problem in a scientific as well as a practical spirit : The papers of Prof. Wm. A. Rogers on micrometers and micrometry, in the Amer. Quar. Micr. Jour., Vol, I. PP- 97. 2 °S ; Proceedings Amer. Soc. Microscopists, 18S2, 1SS3, 18S7. Dr. M. D. Ewell, Proc. Amer. Soc. Micrs., 1S90 ; The Microscope, 18S9, pp. 43-45 ; North Amer. Pract., 1S90, pp. 97, 173. Dr. J. J. Woodward, Amer. Jour, of the Med. Sci., 1875. M. C. White, Article "Blood-stains," Ref. Hand-Book Med. Sciences, 18S5. Medico-Legal Journal, Vol. XII. For the change in magnification due to a change in the adjustment of adjustable objectives, see Jour. Roy. Micr. Soc, 18S0, p. 702 ; Amer. Monthly Micr. Jour., 1880, p. 67. Carpenter-Dallinger, p. 270. If one consults the medico-legal journals ; the microscopical journals, the Index Medicus, and the Index Catalog of the Library of the Surgeon General's Office, under Micrometry, Blood, and Jurisprudence, he can get on track of the main work which has been and is being done. Dry objectives of id mm. {-. in. ), j mm. (4 /';;.) and homogeneous immersion objective 0/2 mm. (,'. in. ) in their mountings. (Bausch & Lonib Opt. Co.). CHAPTER V DRAWING WITH THE MICROSCOPE APPARATUS AND MATERIAL FOR THIS CHAPTER Microscope, Abbe and Wollaston's camera lucidas, drawing board, thumb tacks, pencils, paper, and microscope screen, (Fig. 59), microscopic preparations. DRAWING MICROSCOPIC OBJECTS § 177. Microscopic objects may be drawn free-hand directly from the microscope, but in this way a picture giving only the general ap- pearance and relations of parts is obtained. For pictures which shall have all the parts of the object in true proportions and relations, it is necessary to obtain an exact outline of the image of the object, and to locate in this outline all the principal details of structure. It is then possible to complete the picture free-hand from the appearance of the object under the microscope. The appliance used in obtaining out- lines, etc., of the microscope image is known as a camera htcida. § 178. Camera Lucida. — This is an optical apparatus for en- abling one to see objects in greatly different situations, as if in one field of vision, and with the same eye. In other words it is an optical device for superimposing or combining two fields of view in one eye. As applied to the microscope, it causes the magnified virtual im- age of the object under the microscope to appear as if projected upon the table or drawing board, where it is visible with the drawing paper, pencil, dividers, etc., by the same eye, and in the same field of vision. The microscopic image appears like a picture on the drawing paper (see note to § 181). This is accomplished in two distinct ways : (A) By a camera lucida reflecting the rays from the microscope so that their direction when they reach the eye coincides with that of the rays from the drawing paper, pencil, etc. In some of the camera lucidas from this group (Wollaston's, Fig. 112), the rays are reflected twice, and the image appears as when looking directly into the micro- scope. In others the rays are reflected but once, and the image has the inversion produced by a plane mirror. For drawing purposes this CH. K] DRA WING WITH TLIE MICROSCOPE 123 inversion is a great objection, as it is necessary to similarly invert all the details added free-hand. (B ) By a camera lucida reflecting the rays of light from the draw- ing paper, etc., so that their direction when they reach the eye coin- cides with the direction of the rays from the microscope (Fig. 58, 109). In all of the camera lucidas of this group, the rays from the paper are twice reflected and no inversion appears. / - 1 1 \ v_ XI J i\ -" l\ '! \ l\ « \ L\ x , a \ 1 T^-— — - Hi ll I r Hi Fig. 1 10. Fig. hi. Fig. 109. Fig. 109. Abbe Camera Lucida with the mirror at /5 , the drawing surface horizontal, and the microscope vertical. Axis, Axis. Axial ray from the mi- croscope and from the drawing surface. A, B. Marginal rays of the field on the drawing surface, a b. Sectional view of the silvered surf ace on the upper of the tri- angular prisms composing the cubical prism (P). The silvered surface is shown as incomplete in the center, thus giving passage to the rays from the microscope . Foot. Foot or base of the microscope . G. Smoked glass seen in section. It is placed between the mirror and the prism to reduce the light from the drawing surface. Mirror. The mirror of the camera lucida. A quadrant (Q) has been added to indicate the angle of inclination of the mirror, which in this case is 45 . Ocular. Ocular of the microscope over which the prism of the camera lucida is placed. P, P. Drawing pencil and the cubical prism over the ocular. Fig. iio. Geometrical figure showing the angles made by the axial ray with the drawing surface and the mirror. A,B. The drawing surface. Fig. in. Ocular showing eye-point, E. P. It is at this point both horizontally and vertically that the hole in the silvered surface should be placed {}. 182). 124 DRAWING WITH THE MICROSCOPE [CH. V The better forms of camera lucidas(Wollaston's, Grunow's, Abbe's, etc. ), may be used for drawing both with low and with high powers. Some require the microscope to be inclined (Fig. ioo) while others are designed to be used on the microscope in a vertical position. As in biological work, it is often necessary to have the microscope vertical, the form for a vertical microscope is to be preferred ; but see Figs. 115- 116. § 179. Avoidance of Distortion. — In order that the picture drawn by the aid of a camera hicida may not be distorted, it is necessary that the axial ray from the image on the drawing surface shall be at right angles to the drawing surface (Figs. 112, 114.) g 1S0. Wollaston's Camera Lucida. — This is a quadrangular prism of glass put in the path of the rays from the microscope, anil it serves to change the direction of the axial ray 90 degrees. In using it the microscope is made horizon- tal, and the rays from the microscope enter one-half of the pupil while rays from the drawing surface enter the other half of the pupil. As seen in the figure (Fig. 112), the fields partly overlap, and where they do so overlap, pencil or dividers and microscopic image can be seen together. In drawing or using the dividers with the Wollaston camera lucida it is neces- sary to have the field of the microscope and the drawing surface about equally lighted. If the drawing surface is too brilliantly lighted the pencil or dividers may be seen very clearly, but the microscopic image will be obscure. On the other hand, if the field of the microscope has too much light the microscopic image will be verv definite, but the pencil or dividers will not be visible. It is necessary, as with the Abbe camera lucida (2 1S2), to have the Wollaston prism properly arranged with reference to the axis of the microscope and the eye-point. If it is not, one will be unable to see the image well, and may be entirely unable to see the pencil and the image at the same time. Again, as rays from the micro- scope and from the drawing surface must enter independent parts of the pupil of the same eye, one must hold the eye so that the pupil is partly over the camera lucida and partly over the drawing surface. One can tell the proper position by trial. This is not a very satisfactory camera to draw with, but it is a very good form to measure the vertical distance of 250 mm. at which the drawing surface should be placed when determining magnification (I 162). §181. *Abbe Camera Lucida. — This consists of a cube of glass cut into two triangular prisms and silvered on the cut surface of the *For some persons the image and drawing surface, pencil, etc., do not appear on the drawing board as stated above, but under the microscope, according to the general principle that "objects appear in space where they could be touched along a perpendicular to the retinal surface stimulated," — that is in the line of rays entering the eye. This is always the case with the Wollaston camera lucida. The explanation of the apparent location of the image, etc., on the drawing board with the Abbe camera lucida is that the attention is concentrated upon the draw- ing surface rather than upon the object under the microscope (Dr. W. B. Pillsbury ). CH. /'] DRAWING WITH THE MICROSCOPE 125 upper one. A small oval hole is then cut out of the center of the sil- vered surface and the two prisms are cemented together in the form of the original cube with a perforated 45 degree mirror within it (Fig. 109, a b). The upper surface of the cube is covered by a perforated metal plate. This cube is placed over the ocular in such a way that the light from the microscope passes through the hole in the silvered face and thence directly to the eye. Light from the drawing surface is reflected by a mirror to the silvered surface of the prism and reflect- ed by this surface to the eye in company with the rays from the microscope, so that the two fields appear as one, and the image is seen as if on the drawing surface (Figs. 109, 114). It is designed for use with a vertical micro- scope, but see § 1S4. Fig. 112. Wollaston's Camera Lucida, shovnng the rays from the microscope and from the drawing sur- face, and the position of the pupil of the eye. For full explanation see Fig. gj. § 182. Arrangement of the Camera Lucida Prism. — In plac- ing this camera lucida over the ocular for drawing or the determination of magnification, the center of the hole in the silvered surface is placed in the optic axis of the microscope. This is done by properly arrang- ing the centering screws that clamp the camera to the microscope tube or ocular. The perforation in the silvered surface must also be at the level of the eye-point. In other words the prism must be so arranged vertically and horizontally that the hole in the silvered surface will be in the axis of the microscope and coincident with the eye-point of the ocular. If it is above or below, or to one side of the eye-point, part or all of the field of the microscope will be cut off. As stated above, the centering screws are for the proper horizontal arrangement of the prism. The prism is set at the right height by the makers for the eye-point of a medium ocular. If one desires to use an ocular with the eye-point farther away or nearer, as in using high or low oculars the position of the eye-point may be determined as directd in 126 DRAWING WITH THE MICROSCOPE \_CH. V Fig. 113. One of the latest and best forms of the Abbe Camera Lueida (Bausch & Lomb Optical Co. ). § 59 and the prism loosened and raised or lowered to the proper level ; but in doing this one should avoid setting the prism obliquely to the mirror. In the latest and best forms of this camera lueida special arrange- ments have been made for raising or lowering the prism so that it may be used with equal satisfaction on oculars with the eye-point at differ- ent levels, and the prism is hinged to turn aside without disturbing the mirror. One can determine when the camera is in a proper position by looking into the microscope through it. If the field of the microscope appears as a circle and of about the same size as without the camera lueida, then the prism is in a proper position. If one side of the field is dark, then the prism is to one side of the center ; if the field is consid- erably smaller than when the prism is turned off the ocular, it indicates that it is not at the correct level, i. e., it is above or below the eye- point. § 183. Arrangement of the Mirror and the Drawing Surface. —The Abbe camera lueida was designed for use with a vertical micro- scope (Fig. 109). On a vertical microscope if the mirror is set at an angle of 45 , the axial ray will be at right angles with the table top or a drawing board which is horizontal, and a drawing made under these conditions will be in true proportion and not distorted. The stage of most microscopes, however, extends out so far at the sides that with a 45 mirror the image appears in part on the stage of the microscope. In order to avoid this the mirror may be depressed to some point below 45 , say at 40 or 35°(Fig. 114). But as the axial ray from the mirror to the prism must still be reflected horizontally, it follows that the axial ray will no longer form an angle of 90 degrees with the drawing sur- face, but a greater angle. If the mirror is depressed to 35 , then the axial ray must take an angle of no° with a horizontal drawing surface (see the geometrical figure Fig. 1 14, A). To make the angle 90 again, so that there shall be no distortion, the drawing board must be raised toward the microscope 20 . The general rule is to raise the draw- ing board twice as many degrees toward the microscope as the mirror is depressed below 45 . Practically the field for drawing CH. V\ DRAWING WITH THE MICROSCOPE B A 127 Fig. 114. C Abbe Camera Lucida in position to avoid distortion. Fig. 114. — The Abbe Camera Lucida with the mirror at 33°. Axis, Axis. Axial ray from the microscope and from the drawing surface. A. B. Drawing surface raised toward the microscope 20° . Foot. The foot or base of the microscope. Mirror with quadrant ( 0). The mirror is seen to be at an angle of 35° . Ocular. Ocular of the Microscope. P. P. Drawing pencil and the cubical prism over the ocular. W. Wedge to support the drawing board. A. Geometrical figure of the preceding, showing the angles made by the axial ray with the mirror and the necessary elevation of the drawing board to avoid distortion. From the equality of opposite angles, the angle of the axial ray reflected at 33° must make an angle of 110° with a horizontal drawing board. The board must then be elevated toward the -microscope 20° in order that the axial ray may be perpendicular to it, and thus fulfill the requirements necessary to avoid distortion ( \ ijg, 183 ) . B. Upper view of the prism of the camera lucida. A considerable portion of the face of the prism is covered, and the opening in the silvered surface appears oval . C. Quadrant to be attached to the mirror of the Abbe Camera Lucida to in- dicate the angle of the mirror. As the angle is nearly always 43°, 40°, 01-33°, only those angles are shown. 128 D RAWING WITH THE MICROSCOPE [CH. V can always be made free of the stage of the microscope, at 45 , at 40 , or at 35 . In the first case (45° mirror) the drawing surface should be horizontal, in the second case (40 mirror; the drawing surface should be elevated io°, and in the third case (35 mirror) the drawing board should be elevated 20 toward the microscope. Furthermore it is necessary in using an elevated drawing board to have the mirror bar project directly laterally so that the edges of the mirror will be in planes parallel with the edges of the drawing board, otherwise there will be front to back distortion, although the elevation of the drawing board would avoid right to left distortion. If one has a micrometer ruled in squares (net micrometer) the distortion produced by not having the axial ray at right angles with the drawing surface may be very strikingly shown. For example, set the mirror at 35 and use a hori- zontal drawing board. With a pencil make dots at the corners of some of the squares, and then with a straight edge connect the dots. The figures will be considerably longer from right to left than from front to back. Circles in the object would appear as ellipses in the drawings, the major axis being from right to left. The angle of the mirror may be determined with a protractor, but that is troublesome. It is much more satisfactory to have a quadrant attached to the mirror and an indicator on the projecting arm of the mirror. If the quadrant is graduated throughout its entire extent, or preferably at three points, 45 , 40 and 35 , one can set the mirror at a known angle in a moment, then the drawing board can be hinged and the elevation of io° and 20 determined with a protractor. The draw- ing board is very conveniently held up by a broad wedge. By marking the position of the wedge for 10° and 20 the protractor need be used but once, then the wedge may be put into position at any time for the proper elevation. § 184. Abbe Camera and Inclined Microscope. — It is very fatiguing to draw continuously with a vertical microscope, and many mounted objects admit of an inclination of the microscope, when one can sit and work in a more comfortable position. The Abbe camera is as perfectly adapted to use with an inclined as with a vertical micro- scope. All that is requisite is to be sure that the fundamental law is observed regarding the axial ray of the image and the drawing surface, viz., that they should be at right angles. This is very easily accom- plished as follows : The drawing board is raised toward the microscope twice as many degrees as the mirror is depressed below 45° (§ 183), then it is raised exactly as man}' degrees as the microscope is inclined, cm. n DRAWING WITH THE MICROSCOPE 129 and in the same direction, that is, so the end of the drawing board shall be in a plane parallel with the stage of the microscope. The mirror must have its edges in planes parallel with the edges of the drawing board also (.Figs. 115, 116.) Fig. 115. Arrangement of the drawing board for using the microscope in an inclined position with the Abbe camera lucida [de- signed by Mrs. S. P. Gage, /SSy.) $ 1S5. Drawing with the Abbe Camera Lucida. — (A) The light from the microscope and from the drawing surface should be of nearly equal intensity, so that the image and the drawing pencil can be seen with about equal distinctness. This may be accomplished with very low powers (16 mm. and lower objectives) by covering the mirror of the microscope w r ith white paper when transparent objects are to be drawn. For high powers it is best to use a substage condenser. Often the light may be balanced by using a larger or smaller opening in the diaphragm. One can tell which field is excessively illuminated, for it is the one in which objects are most distinctly seen. If it is the microscopic, then the image of the microscopic object is very distinct and the pencil is invisible or very indistinct. If the drawing surface is too brilliantly lighted the pencil can be seen clearly, but the micro- scopic image will be obscure. When opaq'ue objects, that is objects which must be lighted with reflected light (§ 63), like dark colored insects, etc., are to be drawn the light must usually be concentrated upon the object in some way. The microscope may be placed in a very strong light and the drawing board shaded or the light may be concentrated upon the object by means of a concave mirror or a bull's eye condenser (Fig. 53). If the drawing surface is too brilliantly illuminated, it may be shaded b3^ placing a book or a ground glass screen between it and the window, also by putting one or more smoked glasses in the path of the rays from the mirror (Fig. 109 G). If the light in the microscope is too intense, it may be lessened by using white paper over the mirror, or by a ground glass screen between the microscope mirror and the source of light (Piersol, Amer. M. M. Jour., 18S8, p. 103). It is also an excellent plan to blacken the end of the drawing pencil with carbon DRAWING WITH THE MICROSCOPE [CH. V ink. Sometimes it is easier to draw on a black surface, using a white pencil or style. The carbon paper used in manifolding letters, etc. , may be used, or ordinary black paper may be lightly rubbed on one side with a moderately soft lead pencil. Place the black paper over white paper and trace the outlines w T ith a pointed style of ivory or bone. A corresponding dark line will appear on the white paper beneath. (Jour. Roy. Micr. Soc, 1883, p. 423). Fig. 116. Bernhard's Drawing Board for the Abbe Camera Lucida. This drawing board is adjustable vertically, and the board may be inclined to prevent distortion. It is also arranged for use with an inclined microscope, having the base board hinged. Microscope and drawing surface are then inclined together. (Zeit. wiss. Mikroskopie, vol. vi (/S94, p. 29S). [Zeiss Catalog). (A) It is desirable to have the drawing paper fastened with thumb tacks, or in some other way. (B) The lines made while using the camera lucida should be very light, as they are liable to be irregular. (C) Only outlines are drawn and parts located with a camera lucida. Details are put in free-hand. (D) It is sometimes desirable to draw the outline of an object with a moderate power and add the details with a higher power. If this is done it should always be clearly stated. It is advisable to do this only with objects in which the same structure is many times duplicated, as a nerve or a muscle. In such an object all CH. V~\ DRAWING WITH THE MICROSCOPE 131 the different structures could be shown, and by omitting some of the fibers the others could be made plainer without an undesirable enlarge- ment of the entire figure. (E) If a drawing of a given size is desired and it cannot be ob- tained by any combination of oculars, objectives and lengths of the tube of the microscope, the distance between the camera lucida and the table may be increased or diminished until the image is of the desired size. This distance is easily changed by the use of a book or a block, but more conveniently if one has a drawing board with adjust- able drawing surface like that shown in Fig. 116. The image of a few spaces of the micrometer will give scale of enlargement, or the power may be determined for the special case (§ 186-187). (F) It is of the greatest advantage, as suggested by Heinsius (Zeit. w t . Mikr., 1889, p. 367), to have the camera lucida hinged so that the prism may be turned off the ocular for a moment's glance at the prepa- ration, and then returned in place without the necessity of loosening screws and readjusting the camera. This form is now made by several opticians, and the quadrant is added by some. (Fig. 114.) Any skilled mechanic can add the quadrant. § 1S6. Magnification of the Microscope and size of Draw- ings with the Abbe Camera Lucida. — In determining the standard distance of 250 millimeters at which to measure the image in getting the magnification of the microscope, it is necessary to measure from the point marked P on the prism (Fig. 109) to the axis of the mirror and then vertically to the drawing board. In getting the scale to which a drawing is enlarged the best way is to remove the preparation and put in its place a stage micrometer, and to trace a few (5 or 10) of its lines upon one corner of the drawing. The value of the spaces of the micrometer being given, thus : Fig. 117. Showing the method of indicating the scale at which a drawing was made. The enlargement of the figure can then be accurately determined at any time by measuring with a steel scale the length of the image of the micrometer spaces and dividing it by their known size. Thus, suppose the 5 spaces of the scale of enlargement given with a drawing were found to measure 25 millimeters and the spaces on the 132 DRA WING WITH THE MICROSCOPE \CH. V micrometer were T > th millimeter, then the enlargement would be 25 -j- y&j = 500. That is, the image was drawn at a magnification of 500 diameters. If the micrometer scale is added to every drawing, there is no need of troubling one's self about the exact distance at which the drawing is made, convenience may settle that, as the special magnifi- cation in each case may be determined from the scale accompanying the picture. It should be remembered, however, that the conditions when the scale is drawn must be exactly as when the drawing was made. § 1S7. Drawing at Slight Magnification. — Some objects are of considerable size and for drawings should be enlarged but a few diame- ters, — 5 to 20. By using sufficiently low objectives and different ocu- lars a great range may be obtained. Frequently, however, the range must be still further increased. For a moderate increase in size the drawing surface may be put farther off, or, as one more commonly needs less rather than greater magnification, the drawing surface ma3' be brought nearer the mirror of the camera lucida by piling books or other objects on the drawing board. If one takes the precaution to draw a scale on the figure under the same conditions, its enlargement can be readily determined (§ 1S6). A very satisfactory way to draw at low magnifications is to use a simple microscope and arrange a camera lucida over it as over the ocular. In this way one may get drawings at almost any low magnification. If one has many large objects to draw at a low magnification, then some form of embryo- graph is very conven- ient. (Jour. Roy. Micr., Soc, 1899, p. 223. ) The writer has made use of a photographic camera and different photo- graphic objectives for the purpose. The object is Fig. iiS. Camera lucida for drawing objects natural size. (H. Bausch Jour. Applied Microscopy, vol. Hi (/ooo, p. Sgi). illuminated as if for a photograph and in place of the ground glass a plain glass is used and on this some tracing paper is stretched. Noth- m ©o ch. n DRA WING WITH THE MICROSCOPE 133 ing is then easier than to trace the outlines of the object. See also Ch. VIII. REFERENCES Beale, 31, 355 ; Behrens, Kossel and Schiefferdecker, 77 ; Carpenter-Dallinger, 278 ; Van Heurck, 91 ; American Naturalist, 18S6, p. 1071, 1887, pp. 1 040-1 043 ; Amer. Monthly Micr. Jour., 1SS8, p. 103, 1890, p. 94; Jour. Roy. Micr. Soc, 1881, p. S19, 1SS2, p. 402, 1883, pp. 2S3, 560, 1SS4, p. 115, 1SS6, p. 516, 18S8, pp. 113, 809, 79S; Zeit. wiss. Mikroskopie, 1S84, pp. 1-21, 1889, p. 367, 1893, pp. 289-295. Here is described an excellent apparatus made by Winkel. Consult also the latest catalogs of the opticians. 10 CENTIMETER RULE The upper edge is in millimeters, the lower in centimeters, and half centimeters. THE METRIC SYSTEM UNITS. The most commonly used divisions and multiples. THE METER for I Cenizmeter < c - ra - I. r-iooth Meter; Millimeter (ln.m.), i-ioooth Meter- Micron levgth ] lfi '' I - I000tl1 Millimeter; the Micron Is the unit in Micrometry \i 166) 1 A ilometer, 1000 Meters ; used in measuring- roads and other long- distances. the gram for j Milligram (m.g), I-ioooth Gram. weight. . . 1 Kilogram, 1000 Grams, used for ordinary masses, like groceries, etc. the liter for j Cubic Centimeter ice), i-ioooth Liter. This is more common than the correct capacity. I form, Milliliter. Divisions of the Units are indicated by the Latin prefixes : deci, I-loth ; cent! i-iooth ■ Milli i-toooth ; Micro, i-i,ooo,oooth of any unit. ' ' ' Multiples are designated by Greek prefixes ; deka, 10 times ; myria, 10,000 times ; Mega, 1,000,000 times any unit. l/ecto, 100 times ; kilo, 1000 times CHAPTER VI MICRO-SPECTROSCOPE AND POLARISCOPE, MICRO-CHEMISTRY, MICRO-MATALXOGRAPHY TEXTILE FIBERS APPARATUS AND MATERIAL REQUIRED FOR THIS CHAPTER Compound microscope ; Micro-spectroscope (2 iSS) ; Watch-glasses and small vials, slides and covers ( ',. 207) ; Various substances for examination (as blood and ammonium sulphide, permanganate of potash, chloropbj-11, some colored fruit, etc., [\ 20S-217); Alicro-polarizer (% 21S); Selenite plate (/. 26S), 2- 24 hours. 3. Saturating the tissue in ether-alcohol (| 269), 2-24 hours. 4. Infiltrating with thin collodion ('i 270), 2 hours to 2 days. 5. Infiltrating in thick collodion(5 271 ), 5 hours to several days. 6. Imbedding the tissue (§272), 15 to 20 minutes. 7. Hardening the collodion with chlo- roform (§ 273), 5-24 hours. 8. Clarifying and further hardening the collodion with castor-xylene (? 273), 10-36 hours. 9. Cutting the sections (2 274), 10 min- utes to 2 hours. 10. Transferring the sections to a slide with paper (''/. 275), 1 minute. 11. Fastening the sections to the slide with ether-alcohol (is 276), 1 or 2 minutes. 12. Removing the oil from the sections with benzin and alcohol (§ 277), 3-5 minutes, or 24 hours. 13. Staining the sections with an alco- holic dye \l 278), 2 minutes to 24 hours. 14. Staining the sections with an aque- ous dye ('i 279), 2-10 minutes. 15. Removing the superfluous dye by washing in water or alcohol (? 278- 279), 2-5 minutes. 16. Staining with a general dye ( \ 279), 15-30 seconds. 17. Washing with water or alcohol ( 'i 27S-279), 1 to 2 minutes. iS. Dehydrating the sections in 95",, al- cohol ( i;2Si ), 5 min. to 24 hours. 19. Clearing the sections ( J.2S1 ), 5 min- utes to 24 hours. 20. Draining the sections, 1-2 minutes. 21. Mounting in Canada balsam (I 281), 1-2 minutes. 22. Labeling the preparation (I 30S), 2 minutes. 23. Cataloging the preparation (\ 309), 5-10 minutes. CH. VII •] PARAFFIN SECTIONING 183 ? 2S1. Mounting in Balsam. — After the sections are stained they must be dehydrated and cleared before mounting in balsam. For the dehydration the slide is plunged into a jar of 95",, alcohol. For clearing after the dehydration the slide is drained of alcohol and put down flat and the clearer poured on, or the whole slide is immersed in a jar of clearer f§ 318). Clearing usually is sufficient in a few minutes ; a stay of an hour or even over night does not injure most sections. In mounting in balsam the clearer is drained away by standing the slide nearly vertically on some blotting paper, or by using the waste bowl and standing it up in the little funnel (Fig. 14S). Then the balsam is put on the sections or spread on the cover-glass and that placed over the sections. For cataloging and labeling, see y i. 307-310. Fig. 153. Small spirit lamp modified into a bal- sam bottle, or a glycerin or glycerin-jelly bottle, or a bottle for homogeneous immersion liquid. For all of these purposes it should contain a glass rod. See also Fig-. /6S. .§ 2S2. The Collodion Method with Alcohol.— A good method of procedure for making collodion sections is to do exactly as described, including ? 272, and then instead of hardening the collodion in chloroform and clarifier, it is hardened in S2" alcohol for a day or two before sectioning. In sectioning the knife and tissue are kept wet with 82% alcohol and the sections are dehydrated with 95% alcohol and then fastened to the slide with ether alone or with ether-alcohol. The staining and mounting {\ 27S-2S1 ) are as described. One may preserve the tissue after imbedding for a long time in the 82% alcohol before sectioning and sections may be made at any time. While this method appears somewhat simpler, the results are not so satisfactory as by the oil method given above. THE PARAFFIN METHOD § 2S3. As with the collodion method, the tissues are first properly fixed and hardened and then entirely filled with the imbedding mass, but unlike the collo- dion the mass must be entirely removed before the sections are finally mounted. The tissue thus imbedded and infiltrated is like a homogeneous mass and sections may be cut of extreme thinness. i 284. Harden perfectly fresh tissue in picric-alcohol [\ 333) from one to three days. (Any good method for fixing and hardening the elements may be used. One must observe in each case, however, the special conditions necessary 1 8 4 PARAFFIN SECTIONING [CH. VII for each method. The time might be longer or shorter than for the picric-alcohol. (See Lee, the Microtomists' Vade-Mecum. ) If picric-alcohol is used, pour it off after the proper time for fixing has elapsed, and add (>"]% alcohol. Leave this on the tissue from one to three days, and if it becomes ver}' yellow it is well to change it two or three times. After two or three days pour off the 67%' alcohol and add 82%. The tissue should remain in this one or two days, and it may remain indefinitely. In case the alcohol becomes much yellowed, it should be changed. g oTJ X = iv JE-- i< £3 w cO ! cs o Oh ! Camera and Objective CC - 5 Efl - 11 UN . oa OJ 1 1 1 l 220 PHOTO-MICROGRAPHY \_CH. V1I1 tweea two cover-glasses is needed. By combining a liltle picric acid with the solution or by the use of a thin piece of signal green glass only light between the fixed lines E and F, is allowed to pass. This is not therefore so generally useful as dichroinate. (4) Bothamley's aurantia color screen is a saturated alcoholic solution of the aurantia added to collodion of 3 to 4",,. The collodion is poured on a large cover- glass or a glass plate and allowed to dry. Pringle advises several screens of aurantia of different shades. That is easily managed by adding a greater or less amount of the solution to the collodion. This is a good screen and easily used. Petroleum light serves as a yellow color screen, and one can often get excel- lent results with such a light when daylight or the electric light without a color screen does not give a good picture. For all photography with the microscope isochromatic or orthochroniatic plates are advised. For many objects no color screen is needed if one uses a petroleum lamp. 195, 202). If this study were supplemented by a spectroscopic ex- amination of the object to be photographed, one would learn to choose with great accuracy the color screen which would give the best results, PHOTOGRAPHING WITH A MICROSCOPE* § 361. The first pictures made on white paper and white leather, sensitized by silver nitrate, were made by the aid of a solar microscope ( 1802). The pictures "^Considerable confusion exists as to the proper nomenclature of photography with the microscope. In German and French the term micro-photography is very common, while in English photo-micrography and micro-photography mean dif- ferent things. Thus : A photo-micrograph is a photograph of a small or microscopic object usually made with a microscope and of sufficient size for observation with the unaided eye ; while a micro-photograph is a small or microscopic photograph of an object, usually a large object, like a man or woman and is designed to be looked at with a microscope. Dr. A. C. Mercer, in an article in the Proc. Amer. Micr. Soc, 1886, p. 131, savs that Mr. George Sbadboltmade this distinction. See the Liverpool and Manches- ter Photographic Journal ( now British Journal of Photography), Aug. 15, 1S5S, p. 203 ; also Sutton's Photographic Notes, Vol III, 1858, pp. 205-208. On p. 208 of the last, Shadbolt's word "Photomicrography" appears. Dr. Mercer puts the case very neatly as follows : " k photo-micrograph is a macroscopic photograph of a microscopic object ; a micro-photograph is a microscopic photograph of a macro- scopic object, vSee also Medical News, Jan. 27, 1894, p. 108. CH. VIII} PHOTO-MICROGRAPHY 221 were made by Wedgewood and Davy, and Davy says : "I have found that images of small objects produced by means of the solar microscope may be copied without difficulty on prepared paper, "f Thus among the very first of the experiments in photography the microscope was called into requisition. And naturally, plants and motionless objects were photographed in the beginnings of the art when the time of exposure required was very great. At the present time photography is used to an almost inconceivable degree in all the arts and sciences and in pure art. Even astronomy finds it of the greatest assistance. It has also accomplished marvels in the production of colored plates for book illustrations, especially in natural history. For an example see Comstock's Insect Life, 2d edition. Although first in the field, Photo-Micrographv has been least successful of the branches of photography. This is due to several causes. In the first place, microscopic objectives have been naturally constructed to give the clearest image to the eye, that is the visual image as it is sometimes called, is for microscopic ob- servation, of prime importance. The actinic or photographic image, on the other hand, is of prime importance for photography. For the majority of microscopic objects transmitted light ( \ 64) must be used, not reflected light as in ordinary vis- ion. Finally, from the shortness of focus and the smallness of the lenses, the proper illumination of the object is accomplished with some difficulty, and the fact of the lack of sharpness over the whole field with any but the lower powers, have combined to make photo-micrography less successful than ordinary macro- photography. So tireless, however, have been the efforts of those who believed in the ultimate success of photo-micrography, that now the ordinary achromatic ob- jectives with ortho-chromatic or isochromatic plates and a color screen or petrol- eum light give good results, while the apochromatic objectives with projection oculars give excellent results, even in hands not especially skilled. The problem of illumination has also been solved by the construction of achromatic and apoch- romatic condensers and by the electric and other powerful lights now available. There still remains the difficulty of transmitted light and of so preparing the object that structural details stand out with sufficient clearness to make a picture which approaches in definiteness the drawing of a skilled artist. The writer would advise all who wish to undertake photo-micrography seri- ously, to study samples of the best work that has been produced. Among those who showed the possibilities of photo-micrographs was Col. Woodward of the U. tin a most interesting paper by A. C. Mercer on "The Indebtedness of Pho- tography to Microscopv," Photographic Times Almanac, 1S87, it is shown that : "To briefly recapitulate, photography is apparently somewhat indebted to microscopy for the first fleeting pictures of Wedgewood and Davy [1802], the first methods of producing permanent paper prints [Reede, 1837-1839], the first offering of prints for sale, the first plates engraved after photographs for the purpose of book illus- tration [Donne & Foucalt, 1S45], tne photographic use of collodion [Archer & Dia- mond, 1851], and finally, wholly indebted for the origin of the gelatino-bromide process, greatest achievement of them all [Dr. R. L. Maddox, 1871]. See further for the history of Photo-micrography, Neuhauss, also Bousfield. PHO TO-MICROGRAPHY \CH. VIII S. Army Medical Museum. The photo-micrographs made by him and exhibited at the Centennial Celebration at Philadelphia in 1876, serve still as models, and no one could do better than to study them and try to equal them in clearness and general excellence. According to the writer's observation no photo-micrographs of histological objects have ever exceeded those made by Woodward, and most of them are vastly inferior. It is gratifying to state, however, that at the present time many original papers are partly or wholly illustrated by photo-micrographs, and no country has produced works with photo-tnicrographic illustrations superior to those in "Wilson's Atlas of Fertilization and Karyokinesis" and "Starr's Atlas of Nerve Cells," issued by the Columbia University Press. — In passing the writer would like to pay a tribute to Mr. W. H. Walmsley who has labored in advancing photo-micrograph}' for the last twenty years. His con- venient apparatus and abundant experience have been placed freely at the com- mand of every interested worker, ;and many a beginner has been helped over difficulties by him. His last contribution in "International Clinics," vol. i. ser. 11, 12, is encouraging in the highest degree both for its matter and for the illustrations. Fig. 183. Zeiss' Vertical Photo-micro- graphic Camera. A. Set screw holding the rod [S ) in any desired position. P. Q. Set screws by which the bellows are held in place.. B. Stand with tripod base in which the sup- porting rod (S) is held. This rod is now graduated in centimeters and is a ready means of determining the length of the cam- era. M. Mirror of the microscope. L. The sleeve serving to make a light-tight connection between the camera and microscope. O. The lower end of the camera. R. The upper end of the camera where the focusing screen and plate holder are situated. (From Zeiss' Photo- micrographic Catalog ). As the difficulties of photo-micrography are so much greater than of ordinary photography, the advice is almost universal that no one should try to learn photo- graphy andjphoto-micrography at the same time, but that one should learn the CH. VI 77] PHOTO-MICROGRAPHY 223 processes of photography by making portraits, landscapes, copying drawings, etc., and then when the principles are learned one can take up the more difficult subject of photo-micrography with some hope of success. The advice of Sternberg is so pertinent and judicious that it is reproduced : "Those who have had no experience in making photo-micrographs are apt to ex- pect too much and to underestimate the technical difficulties. Objects which under the microscope give a beautiful picture, which we desire to reproduce by photography may be entirely unsuited for the purpose. In photographing with high powers it is necessary that the objects to be photographed be in a single plane and not crowded together and overlying each other. For this reason photograph- ing bacteria in sections presents special difficulties and satisfactory results can only be obtained when the sections are extremel}' thin and the bacteria well stained. Even with the best preparations of this kind much care must be taken in selecting a field for photography. It must be remembered that the expert microscopist, in examining a section with high powers, has his finger on the fine adjustment screw and focuses up and down to bring different planes into view. He is in the habit of fixing his attention on the part of the field which is in focus and discarding the rest. But in a photograph the part of the field not in focus appears in a promi- nent way which mars the beauty of the picture." APPARATUS FOR PHOTO-MICROGRAPHY \ 362. Camera. — For the best results with the least expenditure of time one of the cameras especially designed for photo-micrography is desirable but is not by any means indispensable fordoing good work. An ordinary photographic camera, especially the kind known as a copying camera, will enable one to get good results, but the trouble is increased, and the difficulties are so great at best, that one would do well to avoid as many as possible and have as good an outfit as can be afforded (Figs. 1S4, 192). The first thing to do is to test the camera for the coincidence of the plane occu- pied by the sensitive plate and the ground glass or focusing screen. Cameras even from the best makers are not always correctly adjusted. By using a straight edge of some kind, one can measure the distance from the inside or ground side of the focusing screen to the surface of the frame. This should be done all around to see if the focusing screen is equally distant at all points from the surface of the frame. If it is not it should be made so. When the focusing screen has been ex- amined, an old plate, but one that is perfectly flat, should be put into the plate holder and the slide pulled out and the distance from the surface of the plate holder determined exactly as for the focusing screen. If the distance is not the same the position of the focusing screen must be changed to correspond with that of the glass in the plate holder, for unless the sensitive surface occupies exactly the position of the focusing screen the picture will not be sharp, no matter how accurately one may focus. Indeed, so necessary is the coincidence of the plane of the focusing screen and sensitive surface that some photo-micrographers put the focusing screen in the plate holder, focus the image and then put the sensitive plate in the holder and make the exposure (Cox). This would be possible with the older forms of plate holders, but not with the double plate holders mostly used at the present day. 2^4 PHOTO-MICROGRAPHY [CH. VIII \ 362a. Size of Camera. — The majority of photo-micrographs do not exceed 8 centimeters in diameter and are made on plates 8x 11, 10 x 13 or 13 x 18 centimeters ( V4.'x4'4 in., 4x5 in., or 5x7 in.). Most of the vertical cameras are for plates not exceeding 10 x 13 centimeters (4x5 in. ) but Zeiss' new form will take plates 21x21 centimeters (S'+xS 1 / in.). 8 363. Work Room. — It is almost self-evident that the camera must be in some place free from vibration. Frequently a basement room where the camera table may rest directly on the cement floor or on a pier is an excellent situation. Such a place is almost necessary for the best work with high powers. For those living in cities, a time must also be chosen when there are no heavy vehicles moving in the streets. For less difficult work an ordinary room in a quiet part of the house or laboratory building will suffice. \ ^64. Arrangement and Position of the Camera and the Microscope. — For much of photo-micrography a vertical camera and microscope are to be preferred (Fig. 1S4). Excellent arrangements were perfected long ago, especially by the French. (See Moitessier. ) Vertical photo-micrographic cameras are now commonly made, and by some firms only vertical cameras are produced. They are exceedingly convenient, and do not require so great a disarrangement of the microscope to make the picture as do the horizontal ones. Van Heurck advises their use, then whenever a structure is shown with especial excellence it is photographed immediately. The variation in size of the picture is obtained by the objective and the projection ocular rather than by length of bellows (see below Fig. 184). It must not be forgotten, how- ever, that penetration varies inversely as the square of the power, and only in- versely as the numerical aperture (''/, 34), consequently there is a real advantage in using a low power of great aperture and a long bellows rather than an objective of higher power with a short bellows. A horizontal camera is more convenient for use with the electric light also (Fig. 192). For convenience and rapidity of work a microscope with mechanical stage is very desirable. It is also an advantage to have a tube of large diameter so that the field will not be too greatly restricted (Fig. 189). In some microscopes the tube is removable almost to the nose-piece to avoid interfering with the size of the image. The substage condenser should be movable on a rack and pinion. The microscope should have a flexible pillar for work in a horizontal position. While it is desirable in all cases to have the best and most convenient apparatus that is made, it is not by any means necessary for the production of excellent work. A simple stand with flexible pillar and good fine adjustment will answer. \ 365. Objectives and Oculars for Photo-Micrography. — The belief is almost universal that the apochromatic objectives are most satisfactory for photography. They are employed for this purpose with a special projection ocular. Two very low powers are used without any ocular (Fig. 180). Some of the best work that has ever been done, however, was done with achromatic objectives (work of Woodward and others). One need not desist from undertaking photo-micrography if he has good achromatic objectives. From a somewhat extended series of ex- periments with the objectives of many makers the good modern achromatic ob- jectives were found to give excellent results when used without an ocular. Most of them also gave good results with projection oculars, although it must be said that the best results were obtained with the apochromatic objectives and projec- Fig. 1S4. — Vertical photo- micrographic camera , screen and small table. The table is about 45 centimeters high and in the legs are large screw eyes for leveling screws. The operator can stand on the floor and per- form all the necessary opera- tions, and in adjusting the mi- croscope can sit on a low stool. The screen is of zinc and has two heavy lead feet to hold it steady. Near the lower left hand corner of the screen is an aperture for the light to shine through upon the mirror. This opening is closed by a black slide which is just balanced so that it stays in any position. In mak- ing the exposure il is raised sufficiently to admit the light to the mirror, but the stage is left in shadow. 'This screen shades the microscope and the face of the operator. ( Trans. Amer. Micr. Soc. /go/. ) 226 PHO TO-MICROGRAPH Y [ CH. VIII tion oculars. It does not seem to require so much skill to get good results with the apochromatics as with the achromatic objectives. The majority of photo- micrographers do not use the Huygenian oculars in photography, although excel- lent results have been obtained with them. An amplifier is sometimes used in place of an ocular. Considerable experience is necessary in getting the proper mutual position of objective and amplifier. The introduction of oculars especially designed for projection, has led to the discarding of ordinary oculars and of amplifiers. However the projection oculars of Zeiss restrict the field very greatly, hence the necessity of using the objective alone for large specimens.* Fig. 185. Projection Oculars tenth section removed to show the construction. Below are shown the tipper end with graduated circle to indicate the amount of rotation found necessary to focus the diaphragm on the screen. No. 2, No. 4. The numbers indicate the amount the ocular magnifies the image formed by the ob- jective as with the compensation oculars. (Zeiss' Catalog. ) (J 366. Difference of Visual and Actinic Foci. — Formerly there was much difficulty ex- perienced in photo-micrographiiig on account of the difference in actinic ami visual foci. Modern objectives are less faulty in this respect and the apochromatics are practically free from it. Since the introduction of orthochromatic or isochromatic plates and, in many cases the use of colored screens, but little trouble has arisen from differences in the foci. This is especially true when mono-chromatic light and even when petroleum light is used. In case the two foci are so unlike in an objective, it would be better to discard it for photograph}' altogether, for the estimation of the proper position of the sensitive plate after focusing is only guess work and the result is mere chance. If sharp pictures cannot be obtained with an objective when petroleum light and orthochromatic plates are used the fault may not rest with the objective but with the plate holder and focusing screen. They should be very carefully tested to see if there is coincidence in position of the focusing screen and the sensitive film as described in 'i 362. J 367. Apparatus for Lighting. — For low power work (35 mm. and longer focus) and for large objects, some form of bull's eye condenser is desirable although fairly good work may be done with diffused light or lamp-light reflected by a mirror. If a bull's eye is used it should be as nearly achromatic as possible. The engraving glass shown in Fig. 188 answers well for large objects. For smaller *A comparative study both with projection oculars, and without an ocular was made with the achromatic objective 25 mm. (1 inch), iS mm. ( j inch), 5 mm. ( ! to \ inch) and 2 mm. ( ,', inch) homogeneous immersion of the Bausch & Lomb Optical Co.; Gundlach Optical Co.; Leitz ; Reichert ; Winkel, Zeiss and the Spencer Lens Co. Good results were obtained with all of these objectives both with and without projection oculars. CH. VII 1 '] PHOTO-MICROGRAPHY 227 objects a Steinheil lens combination gives a more brilliant light and one also more nearly achromatic. For high power work all are agreed that nothing will take the place of an achromatic condenser. This may be simply an achromatic condenser, but preferably it should be an apochromatic condenser. Whatever the form of the condenser it should possess diaphragms so that the aperture of the condenser ma3' be varied depending upon the aperture of the objective. For a long time objec- tives have been used as achromatic condensers, and they are very satisfactory, although less convenient than a special condenser whose aperture is great enough for the highest powers and capable of being reduced by means of diaphragms to the capacitv of the lower objectives. It should also be capable of accurate centering. (? Si 1. Fig. 186. Arrangement for Artificial Illumination. 1. Lamp with metal chimney, easily made by rolling up some ferrotype plate and making a slit-like opening in one side. This opening should be covered by an oblong cover-glass. A glass slide, being of considerable thickness, breaks too easily. The lamp should have a wick about 40 mm. -wide, so that the thickness of the flame,- if taken edgewise, will give an intense light. A wide flame also enables one to get a larger image of the flame , and thus to illuminate a larger object than as though a small flame was used. 2. Bull's-eye condenser on a separate stand. The engraving glass shown in Fig. iSS, or the tripod magnifier {Fig. 172) answers fairly. The Steinheil lenses are still better. j. Screen showing image of the flame inverted. The lamp and bull's-eye stand are on blocks with screw-eyes as leveling screws. \ 36S. Objects Suitable for Photo-micrographs. — While almost any large ob- ject may be photographed well with the ordinary camera and photographic objec- tive, only a small part of the objects mounted for microscopic study can be photo- micrographed satisfactorily. Many objects that give beautiful images when look- ing into the microscope and constantly focusing with the fine adjustment, appear almost without detail on the screen of the photo-micrographic camera and in the photo-micrograph . 228 PHO TO-MICROGRAPHY [ CH. I' 111 Fig. 187. Adjustable lens holder. This lens holder zuill take magnifiers of various sizes, and from its adjustable mechanism is 'eery convenient for dissecting, or for holding a Stcinheil and other lenses for illumination ( The Bausch & Lomb Opt. Co.). Fig. [88. Engraving glass to serve as a condenser and for a dis- secting lens. [Bausch & Lomb Opt. Co.) ' If one examines a series of photo-micrographs the chances are that the greater number will be of diatoms, plant sections or preparations of insects. That is, they are of objects having sharp details and definite outlines, so that contrast and defi- niteness may be readily obtained. Stained microbes also furnish favorable objects when mounted as cover-glass preparations. CH. Villi PHOTO-MICROGRAPHY 229 Preparations in animal histology must approximate as nearly as possible to the conditions more easily obtained with vegetable preparations. That is, they must be made so thin and be so prepared that the cell outlines will have something of the definiteness of vegetable tissue. It is useless to expect to get a clear photo- graph of a section in which the details are seen with difficulty when studying it under the microscope in the ordinary way. Many sections which are unsatisfactory as wholes, may nevertheless have parts in which the structural details show with satisfactory clearness. In such a case the part of the section showing details satisfactorily should be surrounded by a delicate ring by means of a marker (see Figs. 61-66). If one's preparations have been carefully studied and the special points in them thus indicated, they will be found far more valuable both for ordinary demonstration and for photograph}-. The amount of time saved by marking one's specimens can hardly be overestimated. The most satisfactory material for making the rings is shellac colored with lamp- black. Ten years ago many histologic preparations could not be satisfactorily photo- graphed. But now with improved section cutters, better staining and mounting methods, and with the color screens d 356) and isochromatic plates (I355) almost any preparation which shows the elements clearly when looking into the micro- scope can be satisfactorily photographed. Good photographs cannot, however, be obtained from poor preparations. \ 369. Light. — The strongest available light is sunlight. That has the defect of not always being available, and of differing greatly in intensity from hour to hour, day to day and season to season. The sun does not shine in the evening when many workers find the only opportunity for work. Following the sunlight the electric light is the most intense of the available lights. Then come magnesium, acetylene, the lime light, the gas-glow or Wellsbach light, and lastly, petroleum light. The last is excellent for the majority of low and moderate power work. And even for 2 mm. homogeneous immersion objectives, the time of exposure is not excessive for many specimens (40 seconds to 3 minutes). This light is also cheapest and most available and has the advantage of being somewhat yellow, and therefore in many cases makes the use of a color screen unnecessary if one uses isochromatic plates. Acetylene light is excellent and may be used where the arc light is not available. A lamp with flat wick about 40 mm. ( \% in. ) wide has been found most gen- erally serviceable. For large objects and low powers the flame may be made large and the face turned toward the mirror. This will light a large field. For high powers the edge toward the mirror gives an intense light. The ordinary glass chimney answers well, especially where a metal screen is used as shown in Fig. 184. EXPERIMENTS IN PHOTO-MICROGRAPHY § 370. The following experiments are introduced to show prac- tically just how one would proceed to make photo-micrographs with various powers, and be reasonably certain of fair success. If one con- sults prints or the published figures made directly from photo-micro- graphs it will be seen that, excepting the bacteria, the magnification ranges mostly between 10 and 150 diameters. 230 PHOTO-MICROGRAPHY [CH. VIII §371. Focusing Screen for Photo-Micrography. — One cannot expect a picture sharper than the image seen on the focusing screen. Hence the greatest care must be taken in focusing. The general focus- ing may be done with the unaided eye on the ground glass, but for the final focusing a clear screen and a focusing glass must be used. (Figs. 172, 173). See § 347. With the clear focusing screen one cannot at first see the image without using a focusing glass, but with a little ex- perience the aerial image may be seen as with the microscope (§ 54 J. § 372. Photo-micrographs of 20 to 50 Diameters. — For pic- tures under 15 or 20 diameters it is better to use the camera for embryos with the objective in the end of the camera, and the special microscope stand for focusing (Fig. 175). For pictures at 25 to 50 diameters one may use the microscope with a low objective, 25 to 35 mm. equivalent focus, and no ocular (Fig. 184). The object is placed on the stage of the microscope, and focused as in ordinary observation. If a vertical microscope is used the light from the petroleum lamp or other artificial light, is reflected upward by the mirror. It may take some time to get the whole field lighted evenly. Refer back to § 95 for directions. In some cases it may be advisable to discard the condenser and use the mirror only. For some purposes one will get a better light by placing the bull's eye or other condenser between the lamp and the mirror to make the rays parallel or even to make a sharp image of the lamp flame on the mirror. Re- member also that in many cases it is necessary to have a color screen between the source of light and the object (§ 356). For a horizontal camera it is frequently better to swing the mirror entirely out of the way and allow the light to enter the condenser directly or after traversing the bull's eye ( Figs. 1S2, 186). If the ob- ject is small an achromatic combination like a Steinheil magnifier or an engraving glass is excellent (Fig. 188). When the light is satisfac- tory as seen through an ordinary ocular, remove the ocular. (A) Photographing without an Ocular. — After the removal of the ocular put in the end of the tube a lining of black velvet to avoid re- flections. Connect the microscope with the camera, making a light- tight joint and focus the image on the focusing screen. One may make a light-tight connection by the use of black velveteen or more con- veniently by the Zeiss' double metal hood which slips over the end of the tube of the microscope, and into which fits a metal cylinder on the lower end of the camera (Figs. 184, 189, 183). In the last figure the connection has been made. CH. VIII] PHOTO-MICROGRAPHY 231 Fig. 189. Zeiss' special photo-micrographic stand. It has a very large tube, a slow acting fine adjustment, mechanical stage and all appliances for the most sat- isfactory work. ( Cut loaned by Einier and Amend). It will be necessary to focus down considerably to make the image clear. Lengthen or shorten the bellows to make the image of the de- sired size, then focus with the utmost care. In case the field is too much restricted on account of the tube of the microscope, remove the draw-tube. When all is in readiness it is well to wait for three to five minutes and then to see if the image is still sharply focused. If it has got out of focus simply by standing, a sharp picture could not be ob- 232 PHOTO-MICROGRAPHY [CH VIII Figs. 190-191. Fine tint, half-tone reproductions of photo-micrographs of sec- tions made by Mrs. Gage, to show the possibilities of photo-micrography with pho- tographic objectives and with low microscopic objectives without a projection ocnlar. 1. Frontal section of the head of a large red Diemyctylus viridescens ( red newt) at the level of the portae of the brain, magnified 10 diameters. Negative made with a Gundlach perigraphic objective of about go mm. equivalent focus. 2. Frontal section of a larval Diemyctylus about 10 millimeters in length. Negative made with a Winkel objective of 22 millit>ieters equivalent focus ; no ocular. Magnified §0 diameters. ( Mrs. Susanna Phelps Gage, the Wilder Quarter Century Book). tained. If it does not remain in focus, something is faulty. When the image remains sharp after focusing make the exposure. From 20 to 60 seconds will usually be sufficient time with medium plates and the light as described. If a color screen is used it will require 40-300 seconds, i. e., 2 to 5 times as long, for a proper exposure (§ 359). B. Photographing with a Projection Ocular. — If the object is small enough to be included in the field of a projection ocular (Fig. 185) use that for making the negative as follows : Swing the camera around so that it will leave the microscope free. Use an ordinary ocular, focus and light the object, then insert a projection ocular in place of the or- dinary one, and swing the camera back over the microscope. It is not necessary to use an ordinary ocular for the first focusing, but as its field is larger it is easier to find the part to be photographed. The first step is then to focus the diaphragm of the projection ocular sharply on the focusing screen. Bring the camera up close to the mi- croscope and then screw out the eye-lens of the ocular a short distance. Observe the circle of light on the focusing screen to see if its edges are perfectly sharp. If not, continue to screw out the eye lens until it is. If it cannot be made sharp by screwing it out reverse the operation. Unless the edge of the light circle, i. e. , the diaphragm of the ocular, is sharp, the resulting picture will not be satisfactorv. It should be stated that for the X 2 projection ocular the bellows of the camera must be extended about 30 or 40 centimeters or the diaphragm cannot be satisfactorily focused on the screen. The X4 projection ocular can be focused with the bellows much shorter. For either projection ocular the screen distance can be extended almost indefinitely. When the diaphragm is sharply focused on the screen, the micro- scope is focused as though no ocular were present, that is, first with the unaided eye then with the focusing glass. The exposure is also made in the same way, although one must have regard to the greater mag- CH. VITI-] PHOTO-MICROGRAPHY 233 nifieation produced by the projection ocular and increase the time ac- cordingly ; thus when the X4 ocular is used, the time should be at least doubled over that when no ocular is employed. The time will be still further increased if a color screen is used (§ 359). Zeiss recommends that when the bellows have sufficient length the lower projection oculars be used, but with a short bellows the higher ones. It is also sometimes desirable to limit the size of the field by putting a smaller diaphragm over the eye lens. This also aids in making the field uniformly sharp. § 373. Determination of the Magnification of the Photo- Micrograph. — After a successful negative has been made, it is desirable and important to know the magnification. This is easily determined by removing the object and putting in its place a stage micrometer. If the distance between two or more of the lines of the image on the focusing screen is obtained with dividers and the distance meas- ured on one of the steel rules, the magnification is found by dividing the size of the image by the known size of the object (§ 154). If now the length of the bellows from the tube of the microscope is noted, say on a record table like that in section 360, one can get a close approxi- mation to the power at some other time by using the same optical com- bination and length of bellows. For obtaining the magnification at which negatives are made it is a great advantage to have one micrometer in half millimeters ruled with coarse lines for use with the lower powers, and one in 0.1 and 0.01 millimeter ruled with fine lines for the higher powers. § 374. Photo-Micrographs at a Magnification of 100 to 150 Diameters. — For this, the simple arrangements given in the preceding section will answer, but the objectives must be of shorter focus, 8 to 3 mm. It is better, however, to use an achromatic condenser instead of the engraving glass or the Steinheil lens. § 375. Lighting for Photo-Micrography with Moderate and High Powers. — ( 100 to 2,500 diameters). No matter how good one's apparatus, successful photo-micrographs cannot be made unless the ob- ject to be photographed is properly illuminated. The beginner can do nothing better than to go over with the greatest care the directions for centering the condenser, for centering the source of illumination, and the discussion of the proper cone of light and lighting the whole field, as given on pp. 41-52. Then for each picture the photographer must take the necessary pains to light the object properly. An achromatic condenser is almost a necessity (§ 80). Whether a color-screen should 234 PHOTO-MICROGRAPHY \_CH. VIII be used depends upon judgment and that can be attained only by ex- perience. In the beginning one may try without a screen, and with different screens and compare results. A plan used by many skilled workers is to light the object and the field around it well and then to place a metal diaphragm of the proper size in the camera very close to the plate holder. This will insure a clean, sharp margin to the picture. This metal diaphragm must be removed while focusing the diaphragm of the projection ocular, as the diaphragm opening is smaller than the image of the ocular dia- phragm. If the young photo-micrographer will be careful to select for his first trials, objects of which really good photo-micrographs have already been made, and then persists with each one until fairly good results are attained, his progress will be far more rapid than as if poor pictures of many different things were made. He should, of course, begin with low magnifications. § 376. Adjusting the Objective for Cover-Glass. — After the object is properly lighted, the objective, if adjustable, must be cor- rected for the thickness of cover. If one knows the exact thickness of the cover and the objective is marked for different thicknesses, it is easy to get the adjustment approximately correct mechanically, then the final corrections depend on the skill and judgment of the worker. It is to be noted too that if the objective is to be used without a projec- tion ocular the tube-length is practically extended to the focusing screen and as the effect of lengthening the tube is the same as thickening the cover-glass, the adjusting collar must be turned to a higher number than the actual thickness of the cover calls for (see § 103). § 377. Photographing Without an Ocular. — Proceed exactly as described for the lower power, but if the objective is adjustable make the proper adjustment for the increased tube-length (§ 103). § 378. Photographing with a Projection Ocular. — Proceed as described in § 372 B, only in this case the objective is not to be adjusted for the extra length of bellows. If it is corrected for the ordinary ocular, the projection ocular then projects this correct image upon the focusing screen. § 379. Photo-Micrographs at a Magnification of 500 to 2000 Diameters. — For this the homogeneous immersion objective is em- ployed, and as it requires a long bellows to get the higher magnifica- tion with the objective alone, it is best to use the projection oculars. CH. Villi PHOTO-MICROGRAPHY 235 For this work the' directions given in § 372 B must be followed with great exactness. The edge of the petroleum lamp flame is sufficient to fill the field in most cases. With many objects the time required with good lamp light is not excessive ; viz., 40 seconds to 3 minutes. The reason of this is that while the illumination diminishes directly as the square of the magnification, it increases with the increase in numerical aperture, so that the illuminating power of the homogeneous immersion is great in spite of the great magnification (§ 34). For work with high powers a stronger light than the petroleum lamp is employed by those doing considerable photo-micrography. Good work may be done, however, with the petroleum lamp. It may be well to recall the statement made in the beginning, that the specimen to be photographed must be of especial excellence for all powers. No one will doubt the truth of the statement who undertakes to make photo-micrographs at a magnification of 500 to 2000 diameters. If one has a complete outfit with electric arc light (Fig. 192) the time required for photographing objects is much reduced, i. e. ranging from 1 to 20 seconds even with a color screen. As the light is so in- tense with the arc light it is necessary to soften it greatly for focusing. Several thicknesses of ground glass placed between the lamp and the microscope will answer. These are removed before taking the nega- tive. It is well also to have a water bath on the optical bench to ab- sorb the heat rays. This should be in position constantly (see Ch. IX). §380. Use of Oculars in Photo-Micrography. — There is much diversity of opinion whether or not the ordinary oculars used for ob- servation should be used in photographing. Excellent results have been obtained with them and also without them. For great magnification Zeiss recommends the use of the compen- sation oculars with the apochromatics. The Zeiss projection oculars may be used with achromatic objectives of large aperture as well as with the apochromatics. PHOTOGRAPHING OPAQUE OBJECTS AND METALLIC SURFACES WITH A MICROSCOPE All of the objects cansidered in the first part of this chapter are opaque and some of them were to be photographed somewhat larger than natural size. To meet the needs of modern work, especially with metals and alloys one must be able to examine and photograph prepared surfaces at magnifications ranging from five or ten to five hundred or more diameters. \ 381. Microscope for Opaque Objects. — If one does not need to magnify more than about 100 diameters, any good microscope will answer. For the higher 236 PHO TO M I CROC R A PH Y [CH. VIII CH. VIII-}, PHO TO-MICROGRA PH ) ' 237 Fig. 192. Buxton's Photo-Micrographic outfit for use with the arc light. (Jour, Ap. Microscopy, 1901 , p. 1367). ( Cut loaned by the Bausch & Lomb. Opt. Co.) As will be seen from the figure this apparatus is for work in the horizontal position. The optical bench containing the microscope \ water bath, color screen and the electric light, swings sidewise sufficiently for the operator to arrange the speci- men exactly as desired. It then swings back into position and is joined to the camera. This is in two sections for either a short or long bellows. This seems to be the most convenient of all the expensive outfits for photo-micrography . powers it is far more convenient to employ a special microscope for metallography ( micro-metalloscope.) (German, Metallmikroskop ; French, Microscope pour l'etude des surfaces metalliques et des objets opaque ). ( Fig. T93. ) Fig. 193. Special microscope of the Boston Testing Laboratories for the study and photography of metals and alloys {\ jS/). (Cut loaned by the Boston Testing Laboratories. ) 238 PHOTO-MICROGRAPHY [CH. VIII Such a microscope has the following general characters : The stage is mova- ble up and down with rack and pinion, it is rotary and more or less mechanical by means of centering screws. With some at least the stage may be removed entirely. No substage condenser is present, and a mirror is only present for occa- sional transparent objects. A revolving nose-piece is not so good as the objective changers. \ 382. Illumination of Opaque Objects. — (A) for 25 to ico diameters. The directions of Mr. Walmsley are excellent (Trans. Amer. Micr. Soc, 1898, p. 191). "Altogether the best light for the purpose is diffused daylight. Proper lighting is more easily obtained with a vertical camera. An even illumination avoiding deep shadows is preferable in most cases and is more easily attained with the object in a horizontal position. For many objects it is better not to use a bull's eye or any form of condenser but for others the condenser may be needed, but when the con- denser is used one must avoid too much glare. The now little used parabolic re- flector and Lieberkiihn serve well in many cases, but he adds "the majority yield better results under the most simple forms of illumination," i. e., with the dif- fused light from the window. This has been the experience of the writer also. In case diffused daylight is employed the camera should be near a good sized window, and the object should be somewhat below the window ledge so that the illumination is partly from above and from the side. (This is easily attained with the small table and vertical camera shown in Figs. 175, 184). The vertical illum- inator is advantageous for these powers also. See (B. ). (B) For 100 to 500 diameters, — For the magnifications above 50 it is desirable and for those above 100 it is necessary to use some form of "vertical illuminator," that is some arrangement by which the light is reflected down through the objec- tive upon the object, the objective acting as a condenser, and from the object back through the objective and ocular to the eye of the observer. This is accom- plished in two ways : ( 1 ) By means of a small speculum-metal mirror in the tube of the micro- scope. This is set at an angle of 45 degrees and the light thrown into the tube upon it is reflected straight down through the objective upon the object. The speculum metal being opaque cuts out a part of the light. Instead of a metal mirror a circular disc of glass is now more frequently used. This allows the major part of the light reflected from the object, up through the objective to reach the eye. (2) By means of a small glass 45 degree prism inserted into the side of the objective or of a special adapter. The light is from the side of the microscope, and is reflected by the prism straight down through the objective upon the object as before.* *The idea of the vertical illuminator apparently originated with Hamilton L,. Smith. He used the metal reflector. Beck substituted a cover-glass and Powell and Zealand a disc of worked glass; i. e, glass that had been carefully polished and leveled on the two sides. Carpenter-Dallinger, pp. 336-338. The use of the prism with the objective is due to Tolles (see Jour. Roy. Micr. Soc, vol. iii, 1880, pp. 526, 574). In Zeiss' catalog the prism form is figured. In the catalog of Nachet both the glass disc and the prism forms are figured. CH. VIII] PHOTO-MICROGRAPHY 239 For both these devices uncovered objects are most successful or if the object is covered it must be in optical contact with the cover-glass. Naturally good re- flecting surfaces like the rulings on polished metal bars give most satisfactory images, hence this method of illumination is especially adapted to micro-metal- lography. Indeed, without some such adequate method of illumination the study of metals and alloys with high powers would be impossible. So successful is it that oil immersion objectives may be used. ( Carpenter- Dallinger, pp. 335-338). § 383. Light for the Vertical Illuminator. — For moderate powers one may place the microscope in front of a window, or one may use a petroleum or gas lamp. For the higher powers acetylene or preferably the electric arc light is used. In either case it may be neces- san r to soften the light somewhat either by a color screen or by some ground glass. The light should be concentrated upon the exposed end of the prism or into the hole leading to the glass disc. Both the prism and the disc should be adjustable for different objectives and different specimens. The cone of light, especially with the electric arc light, should be enclosed in a hollow metal or asbestos cone to avoid the glare in the eyes of the operator, and it may be necessary to soften the light with ground glass before attempting to focus and arrange the speci- men. This ground glass would in most cases be removed before mak- ing the exposure (§ 379). With the electric light and for long exposure or observation a water bath to absorb the heat rays will be necessary to avoid injuring the lenses. (See also under projection in the next chapter J. As it is somewhat difficult to adjust the light in a way to give the best effect, one can see the advantage of the adjustment for raising and lowering the stage. This will serve for all but the finest focusing, and thus avoid moving the tube for focusing enough to throw the lighting out of adjustment. It might be advantageous to have a fine adjust- ment on the stage also. \ 384. Mounting of Objects. — For observation only and with low powers, the objects may be mounted either in a liquid or dry as seems best. There should be a black background for most objects, then light will reach the e)'e only from the object. A light background is sometimes desirable, especially where one cares only for outlines. '// 385. Preparation of Metallic Surfaces. — In the first place a flat face is obtained by grinding or filing, and then this is polished. For polishing, finer and finer emery or other polishing powders are used, (rouge or diamantine, or specially prepared alumnina, etc). The aim is to get rid of the scratches so that the surface will be smooth and free from lines. \ 3S6. Etching. — After the surface is polished it should be etched with some substance. This etching material will corrode the less resistant material, the edges of crystals, etc., so that the structure will appear clearly. For etching, 240 PHOTO-MICROGRAPHY [CH. VIlI tincture of iodine, nitric acid in various degrees of strength, hydrochloric acid, etc., are used or one may use electricity, the metal being immersed in an indiffer- ent liquid. See numerous articles in the Metallographist for methods and micro- graphs. After the etching, the surface should be washed well with water to remove the etcher. Le Chatelier recommends that the etched surface when dry be coated with a very thin coating of collodion to avoid tarnishing. The preparation will then last for several months untarnished. I 387. Mounting the Preparations of Metal. — In order to get a satisfactory image the flat, polished and etched face should be at right angles to the optic axis. For preliminary observation one can approximate this by mounting the specimen on a piece of beeswax. (Behrens). Very elaborate arrangements of the stage have also been devised (Reichert). A simple and effective device is shown in Fig. 193 in which the specimen is held against the under side of the plane face of the stage attachment. Rubber bands answer well to support the metal, and only one side need be flat. § 388. Photographing Opaque Objects. — The general directions given in § 347 should be followed with the necessary modifications. The time of exposure is usually considerably greater with opaque objects than with transparent ones. Very few such objects can be photgraphed in less than 30 seconds, even with day- light For metallic surfaces and magnifications of 100, 150, 250 to 500, with the electric arc light as illuminant the time required for favorable objects is I, 2, 4 and 7 seconds ; with the Wellsbacb lamp the time is 5, 10, 30 and 60 minutes (Sauveur ). Fig. 194. Rack for drying negatives (Rochester Opt. Co). Fig. 194. References to Ch. VIII. See the works and journals dealing with photography. For Photo-Micrography see Pringle, Bousfield, Neuhauss, Sternberg, Francotte and the special catalogs on photo-micrography and projection issued by the great opticians. The Journal of the Royal Microscopical Society and of the Quekett Micr. Club; Zeit. wiss. Mikroskopie ; the Trans Amer. Micr. Soc; the Amer. Monthly Micr. Journal ; the Journal of Applied Microscopy. For the photography of metallic surfaces, see the various journals of engineer- ing and metallurgy, but especially Sauveur's journal, the Metallographist, begun in 1898. CH. IV/f] PHOTO-MICROGRAPHY 241 ENLARGEMENTS | LANTERN SLIDES | PHOTOGRAPHING BACTERIAL CULTURES \ 389. Enlargements. — As a low power objective has greater depth of focus or penetration than a higher power (§ 34), it is desirable in many cases to make a negative of an object with considerable depth at a low magnification, and then to enlarge this picture to the desired size. As a rule negatives will not bear an enlargement of more than five diameters. For this work the camera shown in Fig. 1S1 is excellent, and the special mi- croscope stand shown in this figure and in Fig. 175 serves to enable one to get a very exact focus. One must select an objective for the enlargement with a field of sufficient size to cover the part of the negative to be enlarged. An objective of 60 to 100 mm. focus will answer in most cases. For the illumination the camera can be elevated against the sky, or artificial light may be used. It is not easy to light so large a surface evenly by artificial light. (A) Enlargement on Bromide Paper. — For this the negative is put in place and by pulling out the bellows the proper amount, one gets the right magnifica- tion. Focus now as for any other object, using the fine adjustment and focusing glass. For great exactness one must put a clear glass in the plate holder and focus on the surface away from the objective. Then place the bromide paper on this clear glass and put another over it to hold it flat against the first plate of glass. The sensitive surface will then be in the exact plane of the focus and the picture will be sharp. For the development and subsequent treatment of the paper, follow the directions of the makers. (B) Enlargement on a Glass Plate. — One may proceed in enlarging as for making lantern slides and make a positive on a glass plate. If it is then desired to get a negative for printing, place this positive on the microscope stand and make a negative from it as if it were an object. Or one may make a contact im- pression as is frequently done in lantern slide making. By this method one must make three separate pictures, (i)the original photo-micrographic negative ; (2) the enlarged positive from this ; (3) a negative from the enlarged positive. With this negative one may print as from the original negative. \ 390. Lantern Slides from Negatives. — In preparing lantern slides from photo-micrographic or ordinary negatives one may use the contact method, or the camera. With the camera one can enlarge or reduce to suit the particular case. The camera and special microscope stand shown in Fig. 181 are admirable for the purpose. For lantern slide work a photographic objective is used and the cone for enlargement removed. One may put the objective in the front of the camera or in the middle segment, making use of the little side door. # 391. Photographing Bacterial Cultures in Petri Dishes. — For the successful photographing of these cultures dark ground illumination is employed on the principal stated in § 92. That is the preparation is illuminated with rays so oblique that none can enter the objective. These striking the culture are reflected 242 PHOTO-MICROGRAPHY [CH.VIH into the objective. The clear gelatin around the growth or colonies does not reflect the light and therefore the space between the colonies is dark. For supporting the Petri dishes a hole is made in a front board for the camera. This hole is slightly larger than the dish. Over it is then screwed or nailed a rubber ring slightly smaller than the Petri dish. This will stretch and receive the dish, and grasp it firmly so that it is in no danger of falling out when put in a verti- cal position. If the camera has two divisions like the one shown the board with the Petri dish is put in the front of the camera, and the objective in the middle division through the side door. Otherwise the board holding the Petri dish must be on a separate support. The illumination is accomplished by the use of two electric lamps with conical shades. (The cheap tin shades with white enamel paint on the inside are good). The lamps are placed at the sides so that a bright light is thrown on the culture, but at such an angle that none of it enters the objective directly. A piece of black velveteen is placed 10 to 20 cm. beyond the culture. This prevents any light from being reflected through the clear gelatin to the objective. Unless some such precaution were taken the background would be gray instead of black. One may use daylight by putting the culture in a support just outside a win- dow, leaving the camera in the room. The rays from the sky are so oblique that they do not enter the objective. One must use a black non-reflecting background some distance beyond the dish as in using artificial light (Atkinson). i* 392. Photographing Bacterial Cultures in Test-Tubes. — Here the lighting is as in the preceding section, but a great difficulty is found in getting good re- sults from the refraction and reflections of the curved surfaces. To overcome this one applies the principles discussed in \ 144, and the test-tubes are immersed in a bath of water or water and glycerin. The bath must have plane surfaces. Behind it is the black velvet screen, and the light is in front as for the Petri dishes. As suggested by Spitta it is well to employ a bath sufficiently thick in order that streak cultures may be arranged so that the sloping surface will all be in focus at once by inclining the test-tube. See the works on photo-micrography and photography for the details of lan- tern slide making. See for the Petri dishes and test-tubes, Atkinson, Botanical Gazette, xviii (1S93), p. 333 ; Spitta, Photo-Micrography (1899), p. 26. CHAPTER IX CLASS DEMONSTRATIONS IN HISTOLOGY AND EMBRYOLOGY APPARATUS AND MATERIAL FOR THIS CHAPTER Demonstration microscopes, simple and compound (Figs. 195-196); Traveling microscope (Fig. 197-198); Indicator ocular (Fig. 199-201); Marker for putting rings around the parts of specimens to be demonstrated ( Fig. 61 ); Projection mi- croscope (Fig. 207); Projection objectives (Fig. 211-212); Episcope (Fig. 214). DEMONSTRATION MICROSCOPES AND INDICATORS § 393. Simple Microscope. — The simple microscope held in one hand and the specimen in the other, has always been used for demonstration, but for class demonstration it is necessary to have mi- croscope and specimen together or the part to be observed by the class is frequently missed. Originally blocks of various kinds to hold both microscope and specimen were devised, but within the last few years excellent pieces of apparatus have been devised by several opticians for the purpose. The accompanying figure shows one of the best forms. Fig. 195. Simple Demonstra- tion Microscope of Leitz ( Win. Krafft, N. Y. ) As shown in the figure this consists of a handle, a stage and a lens holder which slides up and down for focusing. Fot observation the student holds it up to the light. 195- § 394. Compound Demonstration Microscope. — This was originally called a clinical or pocket microscope. It is thus described by Mayall in his Cantor Lectures on the history of the microscope : "A small microscope was devised by Tolles for clinical purposes which seems to me so good in every way that I must ask special attention for 244 CLASS DEMONSTRATIONS [ CH. IX it. The objective is screwed into a sliding tube, and for roughly focus- ing the sliding motion suffices ; for fine adjustment, the sheath is made to turn on a fine screw thread on a cylindrical tube, which serves also as a socket-carrier for the stage. The compound microscope is here reduced to the simplest form I have met with to be a really serviceable instrument for the purpose in view ; and the mechanism is of thor- oughly substantial character. I commend this model to the notice of our opticians. " Fig. 196. Demonstration compound microscope of Leitz. Leitz now furnishes a fine adjustment in the form of an inter- mediate piece between the objective and the tube. This has in it a screw which is turned by a milled ring. For the object- ives employed it makes an efficient fine adjustment and renders it possible for each person to adjust the microscope slightly without endangering the loss of the field. Fig. 196. Since its introduction by Tolles many opticians have produced ex- cellent demonstration microscopes of this type, but most of them have not preserved a special mechanism for fine adjustment. With it one can demonstrate with an objective of 6 mm. satisfactorily. It has a lock so that once the specimen is in the right position and the instrument fo- cused it may be passed around the class. For observation it is onl}' necessary for each student to point the microscope toward a window or a lamp. CH. AY] CLASS DEMONSTRATIONS 245 Fig. 197. Fig. 197. Traveling microscope set up for work (Lei/- ■ from Wm A'rafft, N. Y). A modification of this clinical microscope was made by Zentmayer in which the microscope was mounted on a board and a lamp for illum- inating the object was placed at the right position. 246 CLASS DEMONSTRA TIONS \_CH. IX § 395. Traveling Microscope. — For many years the French opticians have produced most excellent traveling microscopes. The opticians of other countries have also brought out serviceable instru- ments. In the one here figured Mr. Leitz has combined in an admirable way a traveling microscope and a laboratory instrument. For the needs of the pathologist and sanitan* inspector a microscope must pos- sess compactness and also the qualities which render it usable for nearly all the purposes required in a laboratory. This instrument is a type of such apparatus which has grown up with the needs of advancing knowledge. Fig. 19S. Fig. 198. Tiaveling microscope folded up and in its case {Leitz ; from H'1/1. Kraft, N. >'.). CH. AY] CLASS DEMONSTRATIONS 247 § 396. Indicator or Pointer Ocular. — This is an ocular in which a delicate pointer of some kind is placed at the level where the real image of the microscope is produced. It is placed at the same level as the ocular micrometer, and the pointer like the micrometer is magnified with the real image and appears as a part of the projected image (§ 170). By rotating the ocular or the pointer any part of the real image may be pointed out as one uses a pointer on a wall or black- board diagram. By means of the indicator eye-piece one can be cer- tain that the student sees the desired object, and is not confused by the multitude of other things present in the field. The method of its use is indicated in Fig. 201. This device has been invented many times. It illustrates well the adage : "necessity is the mother of invention," for what teacher has not been in despair many times when trying to make a student see a definite object and neglect the numerous other objects in the field. So far as the writer has been able to learn, Quekett was the first to introduce an indicator ocular with a metal pointer which was adjustable and could be turned to any part of the field or wholly out of the field. See Fig. 199, § 126. \\ \ \ 1 p \ : 'f~ ~ J — 1 1 L n p -0-ij 0( v o °db°65 199. Fig. 200. like Fig. 201. the one devised by Fig Fig. 199. Indicator ocular with metal pointer Quekett {Letts ; catalog). Fig. 200. Indicator ocular with an eyelash (cilium) on the ocular diaphragm to serve as a pointer ( P) . This projects about half way across the diaphragm open- ing. On the opposite side arc shown two rays from the microscope to indicate that the real image is formed at the level of the ocular diaphragm. Fig. 201. Field of the microscope with a mammalian blood preparation to show the use of the indicator (P) for pointing out a white blood corpuscle. 248 CLASS DEMONSTAT/ONS \_CH. IX It is not known who adopted the simple device of putting the tip of a cat's whisker or an eye-lash on the diaphragm of the ocular as shown in Fig. 200. This may be done with any ocular, positive or negative. One may use a little mucilage, Canada balsam or any other cement, and stick the eyelash on the upper face of the diaphragm so that it projects about half way across the opening. When the eye- lens is screwed back in place the hair should be in focus. If it is not screw the eye-lens out a little and look again. If it is not now sharp, the hair is a little too high and should be depressed a little. If it is less distinct on screwing out the ocular it is too low and should be elevated. One can soon get it in exact focus. Of course it may be removed at any time. § 397- — Marking the Position of Objects. — In order that one may prepare a demonstration easily and certainly in a short time the specimens to be shown must be marked in someway. A very efficient and simple method is to put rings of black or colored shellac around the part to be demonstrated. For this the Marker, Fig. 61-62, is employed as described on p. 66. ® © © ® ® ® © ©(f)® © © ® ® ® ® ® ® 1 Fig. 202. Fig. 202. Ring around one of the sections of a series for demonstrating some organ especially well. Fig. 203. Figure of a microscopical preparation with a ring around a small part to show the position of some structural feature. CH. IX] PROJECTION MICROSCOPE 249 PROJECTION MICROSCOPE One of the most useful and satisfactory means at the disposal of the teacher of Microscopic Anatomy and Embryology for class demon- strations is the Projection Microscope. With it he can show two hundred as well as one person the objects which come within the range of the instrument. It is far more satisfactory than microscopic demonstrations, for with the projection microscope the teacher can point out on the screen exacth* the structural features and organs which he wishes to demon- strate, and he can thus be certain that the students know exactly what is to be studied. Unless one employs a pointer ocular (Fig. 201), there is no certainty that the student selects from the multitude of things in the microscopic field the one which is meant by the teacher. Like all other means, however, the projection microscope is limited. With it one can show organs both adult and embryonic, and the gen- eral morphology. For the accurate demonstration of cells and cell structure the microscope itself must be used by each student personally. If no ocular is used a 3 mm. or \i inch objective is about as high a power as can be employed in a room holding two hundred. Even with an oil immersion fair demonstrations can be made, but up to the pres- ent time really successful demonstrations are usually made with powers below 3 mm. rather than above. If projection oculars are used one can hardly go bej'ond an 8 mm. objective with real satisfaction. And finally it should be remembered that the Continuous Current Arc Lamp must be employed for satisfactory results. Alternating currents are wholly unsatisfactory for this purpose. § 39S. Projection Microscope. — This is an arrangement of the microscope so that an image of the object under the microscope is thrown upon a screen of some kind. The picture on the screen is looked at precisely as one looks at the pictures thrown on the screen by an ordinary magic lantern. Indeed the projection microscope is a magic lantern with short focus objectives. One of the first uses of the microscope was to throw the images of various objects on a screen so that they could be seen by several persons at once, and the light used was sunlight. Hence those early projection microscopes were called solar or sun microscopes. If sunlight were available at all times and could be controlled, it would be universally employed ; but as it is not 250 PROJECTION MICROSCOPE [CH. IX at all times available and whenever available a heliostat is needed to keep the light fixed in a given position, sunlight is practically dis- carded and the electric light is employed for illumination. § 399. Parts of a Projection Microscope. — These are named in order, beginning with the electric lamp : See Figs. 192, 207-208. (1) An arc lamp with constant current and rheostat adjustable from about 8 to 20 amperes. (2) Lamp condenser. (3) Water bath for removing the heat rays. (4) Special achromatic condenser for high powers. (5) Large square stage with opening 6 centimeters in diameter. (6) Mechanical stage with wide range of movement. (7) A very wide tube for receiving the objectives and projection oculars. This tube to be connected with coarse and fine adjustment for focusing. All of these parts should be independent and adjustable so that any one of them cau be adjusted or removed without interfering with the others. In accordance with the suggestion of Dr. Coplin all of the appa- ratus, including the mountings of the objectives should be dead black to avoid reflections. Reflections are so dazzling that the operator can- not properly focus the image on the screen. § 400. The Arc Lamp for the Projection Microscope. — This should have the following characters: (a) The carbons should be tilted so that the crater in the positive carbon is nearly vertical. It then sends the maximum number of rays to the lamp condenser. (/>) The lower carbon should be slightly in advance of the upper one as shown in Fig. 204 so that the crater in the positive carbon is on the front of that carbon (Figs. 204-205) that is it should be in position to throw the light toward the condenser. (c) The lamp must be adjustable vertically and horizontally, and the carbon holders must also be adjustable so that the carbons may be put in line from side to side and front to back. One cannot get satis- factory results unless all these adjustments are possible. Hand-feed and automatic lamps are both used for projection. The consensus of opinion among experts is that the hand-feed lamp is bet- ter for photography and for projection. That has been the experi- ence of the writer also. S401. Starting the Lamp. — For starting the lamp with the hand-feed form it is necessary to bring the carbons in contact and then CH. IX] PROJECTION MICROSCOPE 251 when the current is established to separate them slightly (1 to 2 mm. ) in order to get a satisfactory light. The carbons may be brought in con- tact before turning on the current or afterwards. An automatic lamp will start as soon as the current is turned on, but here also the carbons must be slightly separated, or one must wait a short time for the car- bons to wear away before the best light is obtained. § 402. Angle of the Carbons. — Originally arc lamps for use with the lantern had the carbons both vertical. The "projector" lamps used at sea, had, however, the carbons inclined at an angle of 30 or 40 degrees from the vertical. Lewis Wright (Optical Projection, p. 163,) states that at his urgent request a projection arc lamp for micro-projec- tion was made with inclined carbons. Certain it is that all projection lamps have now the inclined carbons. The angle of inclination varies with different makers. The lamps furnished by Zeiss and Reichert with their apparatus has the carbons at 40 degrees from the vertical ; Behrens uses and recommends 45 degrees. Barnard and Carver (J. R. M. S., 1898, p. 170) found by a careful series of experiments that an angle of about 27 degrees gave the most satisfactory light. One firm ( A.T. Thompson & Co. of Boston) arranges the carbons at right angles, the upper or positive carbon being horizontal. This gives good results. The purpose of the inclination is to turn the crater toward the con- denser ( Fig. 205), for it is now appreciated that the arc proper gives very little light comparatively. One will appreciate this by studying the carbons projected on the screen as suggested in § 406. § 403. Adjusting the Carbons. — In many arc lamps for projec- tion there is a metal shelf to show approximately where the two car- bons should meet. If one places the carbons in their holders so that the ends are at the level of this shelf they will be nearly in the correct position. The lower carbon should be slightly in advance of the upper one (Fig. 204). This will insure the formation of the crater on the side facing the condenser. S 404. Length of the Arc. — It has been found by careful inves- tigation (Carhart, Ayrton) that the maximum brilliancy and efficiency of a continuous current arc lamp are obtained when the arc is about 1 mm. long and the current is about at its maximum for the size of car- bon used. If the carbons are too far apart the light becomes purplish. If the current is too weak the lower carbon is blunt, while with a stronger current it becomes more pointed, and hides less light ; it also contributes a share of the illumination from its white tip (Fig. 205). -'5 2 PROJECTION MICROSCOPE \_CH. IX In order to see whether the carbons are in the most favorable posi- tion, Barnard and Carver (J. R. M. S., 1898, p. 171) used a pin-hole camera at right angles to the carbons. This camera has a ground glass with cross lines to serve as guides in maintaining the proper position of the carbons. Fig. 204. Fig. 205. Figs. 204-205. Front and side views of the carbons of an arc light to give the best illumination. \ and — indicate the positive and negative poles. Eig. 204 is a side view showing the carbons in section at an angle of 30 degrees from the vertical and the negative ( — ) or lower carbon slightly in front of the pos- itive ( + ) or upper carbon. The carbons have soft cores. Fig. 205 is a front view of the carbons as seen projected on the screen with a 42 mm. objective. It is a projection of the real image of the carbons formed by the special achromatic condenser next the object (ji 599, 4). This figure shows that the source of light is the crater in the positive ( + ) or upper carbon ; it shows also that the lower carbon is slightly below the lower carbon as well as slightly in front . This avoids a shadow from the lower carbon. In the center of the crater is shown a slight shadow. This is due to the pit formed in the soft core of the carbon. § 405. Positive and Negative Carbon. — The mechanism of the lantern is arranged so that the upper carbon should be positive (+ ). In setting up the apparatus one may not be able to insert the wires correctly at first. All one has to do is to make the connections, CH. /A'] PROJECTION MICROSCOPE 253 turn on the current and observe which carbon is the more brilliant. As shown in figure 205 the brilliant carbon is at the positive pole. If now the upper carbon is brilliant the wires are properly connected ; but if the lower carbon is the brighter, the wires are inserted in the wrong binding posts and should be reversed. In observing the carbons when the current is on, one should use deeply colored glass to avoid injuring the eyes. Another excellent method is to turn the current off after a minute or two and look at the carbons directly. The one at the pos- itive pole will be red or white hot while the other will be black or very dull red. § 406. Character of the Carbons and Steadiness of the Light. — One needs a steady light for photography and for projection. To hold the crater in one position and thus render the light steady, a softer core is placed in the carbons (Fig. 204). This serves as a guide, and the crater forms symmetrically around it when the carbons are in a proper position (Fig. 205). Every one who wishes to make a success of micro-projection is urged to study the appearance of the carbons by using a low objective (35 to 65 mm.). The real image of the carbons formed by the achromatic condenser next the object (§ 399, 4) can be focused and thrown on the screen as if it were an object and one can study the crater. The image on the screen will be right side up as the achromatic condenser inverts it once and the objective reinverts it (no projection ocular being used). If the ordinary hard carbons, without soft core, are used the crater shifts its position and thus gives a wavering light. The soft cored carbons give a very steady light as the crater remains constant in position. Sometimes a small hard carbon is used for the lower or negative side and a large soft cored carbon for the upper or positive pole. This works admirably. The sizes used by Ayrton were for the positive car- bon 13 mm. diameter ; for the negative 11 mm. S 407. Rapidity of Wear in the Carbons. — If one employs two carbons equal in size and composition the positive carbon wears away twice as fast as the lower one, therefore one will find the feed mechanism in many lamps moves the upper carbon down twice as fast as the lower carbon moves up. This keeps them centered. If one uses a large carbon above and a small one below, and if the sizes of upper and lower carbon are properly selected, the two car- bons wear away equally in length and the feed mechanism of the lan- tern should move the upper carbon down and the lower one up at the sa?ne rate to insure constant centering. 254 PROJECTION MICROSCOPE \_CH. IX The rapidity of wear in the carbons, irrespective of their position, depends upon the amount of the current. It is uneconomical to use more current than necessary, both on account of the cost of the elec- tricity and the wear of the carbons. It is inconvenient to change the carbons too frequently. It is certainly inconvenient to be compelled to insert new ones during a demonstration. § 408. Size of Carbons and Amount of Current. — The size of the carbons must be proportioned to the amount of current used. For an amperage not exceeding 15, a soft cored carbon of 12 mm. (*4 inch) will answer, but if 20 amperes of current are used then the car- bon should be 16 mm. (or 4 inch) in diameter. If one uses too small a carbon for the current, the carbon partly burns, and really less avail- able light is produced for the projection.* § 409. Amount of Current for Micro-Projection. — For a lec- ture room holding 200 and a screen distance of 8 meters (26 feet J, one can demonstrate almost any suitable preparation with a current of 10 to 12 amperes, the voltage being 110. This serves for objectives as high as 3 mm. when no ocular is used. According to Behrens (Zeit. wiss. Mikr., 1S98, pp. 7-23) one cannot make available more than 20 amperes for any micro-projection. The makers of micro-projection apparatus almost invariably make a 20 ampere lamp the limit. For getting good results it is vastly more important to have all the parts of the apparatus centered and the carbons in the proper rela- tive position than to use a powerful current. The light cannot serve for projection unless it is properly used (§ 406). One will be surprised to see how excellent the results are with an amperage of 10 or 12 when one makes the most of the light. With some preparations one needs more light, and must increase the amper- age. Remember that the skill of the operator is of equal account with the amperage. Do not expect the lantern to furnish brains as well as light. § 410. Lamp Condenser. — This is a large condenser next the radiant and it serves to collect the light emitted from the crater and *For the experiments made in preparing this chapter, and for practical use during the last two years, the carbons most employed are designated : "High grade Electro., Nurnberg carbons, soft cored, 12X190 mm. Other forms were also used in the experiments, sometimes the upper carbon was soft cored and the lower one solid. With study and careful experiment one can get good results with a variety of currents and carbons. The beginner is advised, however to, start with the carbons recommended and furnished by the makers of the arc lamp which he is to employ. CH. IX] PROJECTION MICROSCOPE 255 condense it either upou the object for lantern slides and low objectives, or it narrows the light into a cone of the proper size for further con- centration by the achromatic condenser (§412). One of the most used, and also one of the best condensers for the arc lamp is composed of two plano-convex lenses with their convex sides facing each other. There is then one plane face next the radiant and one toward the mi- croscope. The lens next the radiant is somewhat smaller than the other. Both are loosely mounted to allow for expansion and the cell in which they are mounted should be freely ventilated. This con- denser should be adjustable back and forth and up and down. Fig. 206. Fig. 206. Arrangement and Centering of the Radiant [Leiss) . In (/) The radiant, i. e., the crater (Fig. 205) is too far to the right ■ (2) The crater is too far to the left ; (3) The crater is too high ; (4) The crater is too lozv ; ( 5) The crater is too far from the lamp condenser ; ( 6-7) The crater is too near the condenser. (5) The crater is in the correct position. As pointed out m the explanation of Fig. 205, there may be a slight central shadow with soft cored carbons when the lamp and condenser are in the best relative position. §411. Water Bath for Removing Heat. — This is a cell of some kind with plane glass faces. It should be approximately of a size to allow the light to pass through a stratum of water 50 mm. thick. Even this will allow something over 2% of the heat to pass. It is a great advantage to have cool or cold water circulate through this constantly. 256 PROJECTION MICROSCOPE \CH. IX The water bath should be free from the condenser mounting so that it may not be heated by conduction (see §413 for special cooler ). Fig. 207. Fig 207 Leitz Large Micro-Projection Apparatus {Leitz Catalog). In this figure the apparatus is in position for projection with a projection ocular. As here shown it consists oj :— (1) An arc lamp designed for a current up to 20 amperes ; (2) A lamp condenser of three lenses, the two inner ones being adjustable; 1 3) A large water bath for removing the heat ; (4) Bellows At one end is a space for the insertion of ordinary lantern slides (see the next figure) and at the other end are three condensers of different foci on a revolving nose-piece ; , ( y) Objective stage with a special cooler (Zoth's \ 413), and a special slide carrier serving as a kind of mechanical stage. (6) An 'objective carrier. This contains a triple, revolving nose-piece at the end of the large lube. This tube contains an iris diaphragm and receives an adapter for the use of projection oculars. For low objectives and 'when the projec- tion ocular is not to be used, this adapter is removed. Fine and coarse adjustmt nts are present for focusing. Each part is independent and capable of special adjustment. {As shown in the next figure, the microscope part may be turned aside, leaving the apparatus jor ordinary lantern slide projection. ) % 4 12 - Special Achromatic Condenser for High Powers.— For objectives of 8 mm. and higher there should be a special achro- matic condenser next the stage for holding the specimens. This should CH. AY] PROJECTION MICROSCOPE 257 be adjustable and with centering attachment. For objects which would be injured by the heat allowed to pass the large water bath, a special cooler is used next the specimen. For such cases the achromatic con- denser must be especially constructed or the light would not be focused on the object, and one would not get sufficient brilliancy for high powers. Special condensers are now made admitting the use of the special cooler, and if one has delicate objects which he desires to pro- r H , S ° ria ' ° r if hewishes t0 demonstrate the circulation of the blood or living objects he should make provision for it in ordering his apparatus. s Fig. 208. FIG. 208. Lett*' Large Projection Apparatus (cut loaned by Wm. Krafft, As here shown the microscope part is turned aside, and the lowest of the three condensers Ijoo mm. equiv. focus) is in place for projecting ordinary lantern tides. % 413. Specimen Cooler—In order to project living objects and delicate preparations the heat passing the large water bath must be still farther eliminated. This is accomplished by a device of Zoth's 25« PROJECTION MICROSCOPE [CH, IX (Zeit. wiss. Mikr., 1893, p. 152, J. R. M. S. , '894, p. 112). It consists of a metal box with glass covers. Through this box is circulated cold water, and the specimen rests directly on the cooler. With this the temperature at the focus of the special condenser (§412) rarely exceeds 27 centigrade. Fig. 209. Fig 209. Spencer Winkel Mechanical Stage. [Spencer Lens Co. | This stage is convenient for use with the projection microscope as it can be attached to any square stage and has a great range of motion. This large range is especially necessary for series of 'organs or embryos. For the projection microscope it would be better to have both milled heads for moving the stage on the side as in the next. § 414. Stage for Specimens.— This should be large and prefer- ably square or oblong. The central opening should be 50 to 60 mm. in diameter, and have a large iris to lessen this diameter at will. The specimen stage should be on an independent support and adapted for independent motion. This is necessary so that all objects capable of projection may be used on the stage and moved far enough from the CH. IX] PROJECTION MICROSCOPE 259 microscope for focusing and for the best position of the specimen in relation to the other parts of the apparatus (§ 420.) The stage should be very rigid. Fig. 210. Fig. 210. Mechanical Stage of Leitz. (Cut loaned by IVm. Krajft, N. Y.) This has the advantage of having both milled heads at the side. It has the disad- vantage of not being readily attached to the stage of a projection microscope. § 415. Mechanical Stage. — For projection work this is almost a necessity. While one is demonstrating there should be no time and no energy wasted in finding the object. Preferably the milled heads of the stage should be at the side, and the stage should be easy to remove and put back into position. It should have sufficient range of motion to enable one to demon- strate any section on a slide of serial sections. § 416. Objective Carrier. — This like the stage should be on an independent support. There should be both fine and coarse adjust- ment. The tube should either be very short, or very large to avoid restricting the field. For projection oculars there must be an adapter for using them, and the adapter must be long enough to produce the proper tube-length ( 160 mm. ). As with the stage the objective car- rier should be very substantial. § 417. Objectives to Use in Micro-Projection. — One rarely needs an objective lower than 100 mm. focus or higher than 3 mm. The majority of the work with a screen distance of 5 to 8 meters will be done with objectives of 60-75, 2 5~4°> 16, 8, 6 or 3 mm. focus. For the 260 PROJECTION MICROSCOPE [ CH. IX three kept on the nose-piece most constantly a 64, 42 and 6 mm is as good a combination as one can get. Other powers should be available, however, and for special specimens and occasions one may employ the two mm. oil immersion. For powers above 20 mm., ordinary objectives are more satisfactory than most projection objectives. S 41S. Projection Oculars (Fig. 185). — If one uses the apoch- romatic objectives of 16 and S mm. focus the projection oculars are used with them. They can also be used with wide angle achromatic objectives. With them one can get an enormous magnification even with the 16 mm. objective. (See the table below, § 421.) These oculars restrict the field greatly and in the writer's exper- ience it seemed on the whole more desirable for most objects not to use the projoction oculars. They cannot be used advantageously with objectives higher than S or 6 mm. and are most satisfactory with one of 16 mm. In using the projection ocular with a 16 mm. objective one can place the special achromatic condenser next the stage, but if the ocular is not used one must either do without a specimen condenser, or take one of lontrer focus, if he has two or three as shown in Fis;. 207. lililllllllllllL,, , Fig. 211. Fig. an. Zeiss' Micro-Planar for projection. (Cut loaned by Bausch y a converging cone of light, and it should be lighted by the entire cone of light traversing the lamp condenser. If one uses a white card it is easy to determine the position and size of the cone of light. If it is too large for the specimen, either the lamp condenser is too near the radiant or the specimen is too close to the lamp condenser. If one uses an achromatic condenser the lamp condenser and the achromatic condenser should be so arranged that the entire cone of light traversing the lamp condenser can enter the achromatic condenser. If the cone is too large they are too close together, or the lamp con- denser is too near the radiant. If the cone is too small then the lamp condenser is too far from the radiant or the achromatic condenser, or perhaps both faults are present. One must remember in all his ex- periments that a converging cone of light should be used and not a diverging one. The specimen must then not be beyond the focus of the lamp condenser. If one wishes to make micro-projection a success it will be nec- essary to give the apparatus the requisite time and thought. Tr}' to understand the conditions of success and continue experimenting until you have learned to make it possible for the machine to do its best for 3-ou. The satisfaction of showing a class real things is sufficient re- ward for all the trouble. §420. Screen and Screen Distance. — For a screen nothing is so good as a dead-white, smooth wall. A lusterless, white cloth screen answers well also. It is an advantage to have this entirety opaque, so that none of the light can pass through it. One must remember that the light passing through the minute lenses of the objective must be spread out over a great space even with low powers, and over a much greater with high powers, so that one cannot afford to have any of the light lost by transmission through the screen. All are agreed that for micro-projection a translucent screen with the projection apparatus behind it is not desirable, although for ordinary lantern slide projection it answers fairly well. The distance of the screen from the microscope depends largely on the size of one's audience. The writer has found a distance of eight meters (26 feet) good for both low and high power projection. This distance answers well for a class of 200 persons. 262 PROJECTION MICROSCOPE \_CH. IX For the minute details of a projected specimen it is recommended that the audience use opera glasses. These are also useful for the oper- ator in focusing the image on the screen. TABLE SHOWING THE SIZE OF OBJECT, THE MAGNIFICATION AND THE SIZE OF THE SCREEN IMAGE WITH VARIOUS OBJECTIVES AND PROJECTION OCULARS Distance oj the screen Jrom the stage oj the microscope, 8 meters (26 feet +). Arc light, 12 ampere current, for illumination. Objective Projection Ocular Achromatic Condenser Actual Size of Field Magnification Diameter of Screen Image 64 mm. none none 20 mm. 125— 250 cm. 42 mm. none none 10 mm. 185 185 cm. 35 mm. none none 9 mm. 230 207 cm. 24 mm. none none 7 mm. 335 235 cm. 18 mm. " mm. " mm. none none X 2 condenser X 4 1 condenser 6 mm. 1.5 mm. 1.5 mm. 440 860 1640 264 cm. 130 cm. 245 cm. 16 mm. " mm. " mm. none X 2 X 4 none condenser condenser 4 mm. 1.25 mm. 1 mm. 53° 1090 2040 200 cm. 136 cm. 204 cm. 8 mm. " mm. " mm. none X 2 X 4 condenser condenser condenser 3 i mm. 0.60 mm. 0.60 mm. 1000 2250 4500 300 cm. 140 cm. 270 cm. 6 mm. " mm. " mm. none X 2 X 4 condenser condenser condenser 2 mm. 0.50 mm. 0.50 mm. 1200 2550 5000 240 cm. 130 cm. 250 cm. 5 mm - " mm. none ■ 2 condenser condenser 1.80 mm. 0.40 mm. 1750 3500 300 cm. 140 cm. 4 mm. none | condenser j 1.50 mm. | 2150 320 cm. 3 mm. none | condenser | 1 mm. | 2350 235 cm. 2 mm. none | condenser | o.6oxmm. 4000 300 cm. §421. This table shows approximately the size of object which each objective will project upon the screen. This was determined ex- actly as described in § 50. For the magnification one simply measures the distance between two or more lines of the image of the microm- eter on the screen, and divides the size of the image by the known size of the object ( §155). Any good stage micrometer will answer. It is necessary, however, to use one with coarse lines for the low pow- ers (§ 159, 170). By comparing the magnification with and without the projection oculars, and also comparing the size of object which can be projected CM. IX] PROJECTION MICROSCOPE 263 with and without the oculars one can decide quite accurately the best combination to select. On the whole the writer has found it better to employ a sufficient variety of objectives and not use the projection oculars. It is somewhat easier to obtain a brilliant image without the oculars. §422. Darkening the Room. — It is impossible to succeed in micro- projection unless the room can be made dark, the darker the better. It is especially important that the screen should be free from all light except that projected upon it in forming the image. §423. Enclosing the Projection Apparatus. — It is desirable to have the projection apparatus closed as completely as possible to avoid diffusing light through the room and thus vitiating the most careful darkening of all windows and sky lights. It is also desirable to shut in the light from the apparatus, as it dazzles the eyes of the operator and of those near it in the audience so that the image ou the screen cannot be satisfactorily seen. Some forms of apparatus are en- closed in a metal box, others have a frame over them upon which is spread black cloth like silesia. If this is made fireproof by soaking it thoroughly in a solution of alum, borax and sodium tungstate it will not readily catch fire. The cloth should not be too thick, otherwise it will retain too much heat around the apparatus. One should remember the fundamental law of vision, viz, that other things being equal, the clearest images are obtained when no light reaches the eye except from the object. § 424. Preparations Suitable for Micro-Projection. — As a generalization it may be said that any specimen which shows clearly and sharply under the microscope with a 16 mm. objective will also give an excellent projection image. Details which are not visible with the 16 mm. objective are rarely well brought out with sufficient clearness on the screen for one or two hundred people to see. (A) The stains showing best are those which are very transpar- ent, or pure differential stains like hematoxylin. Admirable results have been obtained with hematoxylin and eosin, and the various car- mines when differentiated. Every method of staining which gives either sharply differentiated results or transparent colors produces preparations adapted to projection. A weak, or washed out appearance under the microscope is sure to be even less satisfactory on the screen. (B) The thickness of the sections may vary from l/< to 40^. But one must remember that thick sections are adapted for low powers 264 PROJECTION MICROSCOPE \_CH. IX only, while thin sections, if well stained, may be used with both high and low objectives. The size of the object which one wishes to project determines the objectives to be used. By consulting the table one can get a fair idea of the size of object which each objective will satisfactorily project. An excellent plan to follow is that for ordinary microscopic study (see p. 102), that is, use first a low power to show the object as a whole, then a higher one for details. (C) For minute objects like white blood corpuscles, etc., it is necessary to use a high power and to have a small audience which can be close to the screen, or a somewhat larger audience can see well by using opera glasses. (D) For the circulation of the blood it is necessary to eliminate the heat rays almost entirely. Nothing has proven so good as the second or specimen cooler (§413). The conditions are still more favorable if a circulation of cold water is established in the large water bath also. This is easily done by the use of two large bottles. The cold water can be siphoned or aspirated from an upper one and the warm water allowed to flow off into a lower one. For this it is of much advantage to have a tube in the bottom of the water bath in which to introduce the cold water. The warm water will then flow off through a tube in the top. One must remember that perfectly clean water must be used for the water bath especially when a circulation is established, for opaque particles in the water bath give undesirable shadows in the image. ( B ) A practical suggestion is made by Lewis Wright in his book on optical projection, and that is to warm the objective before using it for showing the circulation of the blood or in any case when a moist object is under it. If the objective is cold the vapor from the object will be condensed on the objective and make satisfactory projection impossible. §425. Masks for Projection Preparations. — The light used for projection is so brilliant that it is practically impossible to arrange the object under the objective with rapidity and certainty unless there is some kind of guide. The best one found so far is a mask on the back of the slide with an opening for the preparation L to be shown. This mask should be made of black paper. One can cut the holes in it with scissors or with ticket punches. With the specimens properly masked, and the parts of the apparatus lusterless black, as suggested by Dr. CH. IX] PROJECTION MICROSCOPE 265 Coplin, the operator can work with rapidity, certainty and also with comfort. (Fig. 213.) 79 oF 5 10 86 T 01 93 o| Homo 5 Slide 10 See's 79 20m 100 1901 Fig. 213. Slide of serial sections with a black mask , perforated over the sec- tions to be demonstrated with the projection microscope. This mask is put on the back of the slide, not on the cover-glass. Unless one has a mask something like this the light is so dazzling that it is almost impossible to find the proper sections. It is easily removed by placing the slide on wet blotting paper. S 426. How to demonstrate with the Micro-Projection Ap- paratus. — Microscopical preparations are not so easily used as are lan- tern slides. The writer has found that the most successful method is for the teacher himself to stand by the apparatus, insert the specimens, and find exacts what he wishes his pupils to see. Then to point them out a bamboo fish pole with sharp end is used. This should be 2-3 meters long and if held out in the diverging cone of light leaving the microscope, a sharp shadow will be cast upon the image. With this pointer one can indicate the part to be demonstrated even more satisfactorily than as if he pointed them out directly on the screen. While it is not possible to delegate the finding of the specimen to an assistant he is of great help in keeping the carbons of the lamp in ex- actly the right position. If the light is kept perfect the teacher has very little trouble with the rest of the manipulation. §427. Cleaning the Glass Surfaces of the Micro-projection Apparatus. — Inasmuch as it is so difficult to make the light sufficiently brilliant for micro-projection, it is of the greatest importance that all glass surfaces be kept as clean as possible. The lenses of the lamp condenser should be carefully wiped occasionally ; and the water bath should be opened and the plane glass faces thoroughly cleaned. It is desirable to soak them in the cleaning mixture for glass. There is always a certain amount of deposit on the glass even though distilled water is used. Every grade of opacity renders the image on the screen less excellent. Cleanliness is one of the most important requirements for successful micro-projection. 266 PROJECTION MICROSCOPE [CH. IX Each preparation should be wiped off before it is put in position on the stage. Any particles of dust are painfully evident in the pro- jected image. V Fig. 214. Zeiss Epidiascope for Opaque Objects, and for Transparent Objects in a Horizontal Position (Zeiss' Special Catalog.) As shown in this jigure the apparatus is set up for opaque objects. For trans- parent objects M 2 (mirror 2) is removed, when the light striking Mj is reflected to III' and thence up through the object to M' and to the screen. Commencing at the right : R. Parabolic reflector, which projects the light from the crater through ( W ) the water bath, to M 2 the mirror which is at the proper an- gle for reflecting the light down upon the opaque object. From the opaque object the light is irregularly reflected up through the objective to 31'. M' serves to reflect the rays from the objective to the screen. V. Ventilator. M ; and HI' arc mirrors for use in reflecting the light through horizontal transparent objects. CH. IX ] PROJECTION MICROSCOPE 267 This apparatus is designed to project opaque objects as large as 22 centimeters in diameter, at a magnification of 5 to 10 with a 30 ampere current. For a smaller object one may magnify as high as 25 diameters. With a 50 ampere current and a larger reflector the magnification may be from 14 tip to 37 diameters. PROJECTION OF OPAQUE OBJECTS \ 42S. Episcope. — For the projection of opaque objects like anatomical prep- arations, figures in books, coins or indeed any opaque object an apparatus on the principle of the one figured (Fig. 214) is used. That is, a powerful light is thrown upon the opaque object and the rays reflected from the object are then projected upon the screen by an objective as for a lantern slide. As the objects are mostly in a horizontal position the objective points directly upward, and the rays from it must be made horizontal by means of a 45 degree mirror or prism. This apparatus is very old. Its first name was "aphengescope" or opaque lan- tern. Now it is called an episcope, or megascope, and if for both opaque and transparent objects (Fig. 214) it is designated as an epidiascope. For its satisfactory use exceedingly powerful light must be used. Some opti- cians employ two radiants, others but one. In any case currents of 30 to 50 am- peres are used. One should wear deeply stained glasses in working with it. The apparatus works well with flat objects, and rather brilliant objects, like the movements of a watch, etc. It is also more satisfactory for objects of slight thickness. For objects like bones, etc., one must focus up and down for the dif- ferent levels. REFERENCES TO CHAPTER IX Lewis Wright, Optical Projection ; Carpenter-Dallinger ; Leiss, Die optischen Instrumente der Firtna R. Fuess ; The works on Photo-Micrography ; The latest catalogs or special catalogs on projection apparatus issued by the opticians, especial- ally Zeiss, Reichert and Leitz. The volumes of the microscopical periodicals for the last few years, especially the Journal of the Royal Microscopical Society, and the Zeitschrift fur wissentschaftliche Mikroskopie. CHAPTER X THE ABBE TEST PLATE AND APERTOMETER ; EQUIVA- LENT FOCUS OF OBJECTIVES AND OCULARS ; DRAW- INGS FOR PHOTO-ENGRAVING ; WAN MODELS 2 429. On the Method of Using Abbe's Test-Plate. — This test-plate is in- tended for the examination of objectives with reference to their corrections for spherical and chromatic aberration and for estimating the thickness of the cover- glass for which the spherical aberration is best corrected. "The test-plate consists of a series of cover-glasses ranging in thickness from 0.09 mm. to 0.24 mm., silvered on the under surface and cemented side by side on a slide. The thickness of each is written on the silver film. Groups of parallel lines are cut through the film and these are so coarsely ruled that they are easily resolved by the lowest powers, yet from the extreme thinness of the silver the}' form a very delicate test for objectives of even the highest power and widest aperture. To examine an objective of large aperture the plates are to be focused in succession observing each time the quality of the image in the center of the field and the variation produced by using alternately centra) anil very oblique illumination. When the objective is perfectly corrected for spheircal aberration for the particular thickness of cover-glass under examination, the contour of the lines in the center of the field will lie perfectly sharp by oblique illumination without any nebulous doubling or indistinctness of the minute irregularities of the edges. If after exactly adjusting the objective for oblique light, central illumination is used no alteration of the adjustment should be necessary to show the contours with equal sharpness." "If an objective fulfills these conditions with any one of the plates it is free from spherical aberration when used with cover-glasses of that thickness ; on the other hand if every plate shows nebulous doubling or an indistinct appearance of the edges of the silver lines, with oblique illumination, or if the objective requires a different adjustment to get equal sharpness with central as with oblique light, then the spherical correction is more or less imperfect." "Nebulous doubling with oblique illumination indicates overcorrection of the marginal zone, want of the edges without marked nebulosity indicates under- correction of this zone ; an alteration of the adjustment for oblique and central illumination, that is, a difference of plane between the image in the peripheral and central portions of the objective points to an absence of concurrent action of the separate zones, which may be due to either an average under or overcorrection or to irregularity in the convergence of the ravs. " "The test of chromatic correction is based on the character of the color bands, which are visible by oblique illumination. With good correction the edges of the CH. A'] TEST PLATE AND A PER TO METER 269 silver lines in the center of the field should show but narrow color bands in the complementary colors of the secondary spectrum, namely, on one side yellow- green to apple-green on the other violet to rose. The more perfect the correction of the spherical aberration the clearer this color band appears." "To obtain obliquity of illumination extending to the marginal zone of the objective and a rapid interchange from oblique to central light Abbe's illuminat- ing apparatus is very efficient, as it is only necessary to move the diaphragm in use nearer to or further from the axis by the rack and pinion provided for the purpose. For the examination of immersion objectives, whose aperture as a rule is greater than 1S0 in air and those homogeneous-immersion objectives, which considerably exceed this, it will be necessary to bring the under surface of the Test-plate into contact with the upper lens of the illluminator by means of a drop of water, glycerin or oil." "In this case the change from central to oblique light may be easily effected by the ordinary concave mirror but with immersion lenses of large aperture it is im- possible to reach the marginal zone by this method, and the best effect has to be searched for after each alteration of the direction of the mirror." "For the the examination of objectives of smaller aperture (less than 4o°-50°) we may obtain all the necessary data for the the estimation of the spherical and chromatic corrections by placing the concave mirror so far laterally, that its edge is nearly in the line of the optic axis the incident cone of rays then only filling one-half of the aperture of the objective. The sharpness of the contours and the character of the color bands can be easily estimated. Differences in the thickness of the cover-glass within the ordinary limits are scarcely noticeable with such objectives. " "It is of fundamental importance in employing the lest as above described to have brilliant illumination and to use an eye-piece of high power." "When from practice the ej^e has learnt to recognize the finer differences in the quality of the contour images this method of investigation gives very trust- worthy results. Differences in the thickness of cover glasses of 0.01 or 0.02 mm. can be recognized with objectives of 2 or 3 mm. focus." "With oblique illumination the light must always be thrown perpendicularly to the direction of the lines. Fig. 215. The Abbe Test Plate. "The quality of the image outside the axis is not dependent on spherical and chromatic correction in the strict sense of the term. Indistinctness of the con- tours towards the borders of the field of view arises as a rule, from unequal mag- nification of the different zones of the objective ; color bands in the peripheral 270 PES T PLATE A NO A PER TO ME TER \_CH. X portion (with good color correction in the middle) are caused by unequal magnifi- cation of the different colored images." "Imperfections of this kind, improperly called "curvature of the field," are shown to a greater or less extent in the best objectives, where the aperture is con- siderable." ii&*°°.. .".1615 *' -*%,>„ --Carl ZeLS^Apert^ejep Ienj^__ Fig. 216. Abbe Apertometer. \ 430. Determination of the Aperture of Objectives with an Apertometer. — Excellent directions for using the Abbe apertometer may be found in the Jour. Roy. Micr. Soc, 1S78, p. 19, and 1S80, p. 20 ; in Dippel, Zimmermann and Czapski. The following directions are but slightly modified from Carpenter-Dallinger, pp. 394-396. The Abbe apertometer involves the same principle as that of Tolles, but it is carried out in a simpler manner ; it is shown in Fig. 216. As seen by this figure it consists of a semi-circular plate of glass. Along the straight edge or chord the glass is beveled at 45 , and near this straight edge is a small, perforated circle, the perforation being in the center of the circle. To use the apertometer the micro- scope is placed in a vertical position, and the perforated circle is put under the mi- croscope and accurately focused. The circular edge of the apertometer is turned toward a window or plenty of artificial light so that the whole edge is lighted. When the objective is carefully focused on the perforated circle the draw-tube is removed and in its lower end is inserted the special objective which accompanies the apertometer. This objective and the ocular form a low power compound mi- croscope, and with it the back lens of the objective, whose aperature is to be meas- ured, is observed. The draw-tube is inserted and lowered until the back lens of the objective is in focus. "In the image of the back lens will be seen stretched across, as it were, the image of the circular part of the apertometer. It will ap- pear as a bright band, because the light which enters normally at the surface is re- flected by the beveled part of the chord in a vertical direction so that in reality a fan of 180 in air is formed. There are two sliding screens seen on either side of the apertometer ; they slide on the vertical circular portion of the instrument. The images of these screens can be seen in the image of the bright band. These screens should now be moved so that their edges just touch the periphery of the back lens. They act, as it were, as a diaphragm to cut the fan and reduce it, so that its angle just equals the aperature'of the objective and no more." "This angle is now determined by the arc of glass between the screens ; thus we get an angle in glass the exact equivalent of the aperature of the objective. As the numerical ap- ertures of these arcs are engraved on the apertometer they can be read off by inspec- tion. Nevertheless a difficulty is experienced, from the fact that it is not easy to CH. X] TEST PLATE AND APERTOMETER 271 determine the exact point at which the edge of the screen touches the periphery of the back lens, or as we prefer to designate it, the limit of aperture, for curious as the expression may appear we have found at times that the back lens of an ob- jective is larger than the aperture of the objective requires. In that case the edges of the screen refuse to touch the periphery." In determining the aperture of homogeneous immersion objectives the proper immersion fluid should be used as in ordinary observation. So, also, with glycerin or water immersion objectives. I 431. Testing Homogeneous Immersion Liquid. — In order that one shall realize the full benefit of the homogeneous immersion principle it is necessary that the homogeneous immersion liquid shall be truly homogeneous. In order that the ordinary worker may be able to test the liquid used by him, Professor Hamilton L. Smith devised a tester composed of a slip of glass in which was ground accurately a small concavity and another perfectly plain slip to act as cover. (See Proc. Amer. Micr. Soc; 1S85, p. 83). It will be readily seen that this concavity, if filled with air or any liquid of less refractive index than glass, will act as a concave or dispersing lens. If filled with a liquid of greater refractive index than glass, the concavity would act like a convex lens, but if filled with a liquid of the same refractive index as glass, that is, liquid optically homogeneous with glass, then there would be no effect whatever. In using this tester the liquid is placed in the concavity and the cover put on. This is best applied by sliding it over the glass with the concavity. A small amount of the liquid will run between the two slips, making optical contact on both surfaces. One should be careful not to include air bubbles in the concavity. The surfaces of the glass are carefully wiped so that the image will not be ob- scured. An adapter with society screw is put on the microscope and the objective is attached to its lower end. In this adapater a slot is cut out of the right width and depth to receive the tester which is just above the objective. As object it is well to employ a stage micrometer and to measure carefully the diameter of the field without the tester, then with the tester far enough inserted to permit of the passage of rays through the glass but not through the concavity, and finally the concavity is brought directly over the back lens of the objective. This can be easily determined by removing the ocular and looking down the tube. Following Professor Smith's directions it is a good plan to mark in some way the exact position of the tube of the microscope when the micrometer is in focus without the tester, then with the tester pushed in just far enough to allow the light to pass through the plane glass and finally when the light traverses the concavity. The size of the field should be noted also in the three conditions (ji 50-52. ) It will be seen by glancing at the following table that whenever the liquid in the tester is of lower index than glass, that the concavity with the liquid acts as a concave lens, or in other words like an amplifier (p. 109), and the field is smaller than when no tester is used. It will also be seen that as the liquid in the concav- ity approaches the glass in refractive index that the field approaches the size when no tester is present. It is also plainly shown by the table that the greater the difference in refractive index of the substance in the concavity and the glass, the more must the tube of the microscope be raised to restore the focus. If a substance of greater refraction than glass is used in the tester the field would be larger, i. e., the magnification less, and one would have to turn the tube down instead of up to restore the focus. 272 TEST PLATE AND APERTOMETER CH. A'] The table given below indicates the points with a tester prepared by the Gund- lach Optical Co., and used with a 16 mm. apochromatic objective of Zeiss, ,/\ compensation ocular, achromatic condenser, i.oo N. A. (Fig. 41 ) : Size of the I Elevation of the Tube Tester and Liquid in the Concavity ' ... necessary to Field Restore the Focus No tester used 1.825 mm. _. Standard position. Whole thickness of the tester at one end, not over the cavity 1.S5 mm. ___ No change of focus. Tester with water 1.075 "»"• --.Tube raised 3J2 mm. Tester with 95 % alcohol 1.15 mm. ..J . . . . 3 mm. Tester with kerosene 1.4 mm. .... 2 mm. Tester with Gundlach Opt. Co's'hom. liquid t.825 mm. _. .... ,'„"„ mm. Bausch & Lomb Opt. Co.'s hum. liquid .1.825 mm. __ . • ■ ■ $5 mm - Leitz' hom. liquid [.825 mm. _. .... ,-„"„ mm. Zeiss' hom. liquid 1.825 mm. __ .... ,-„"„ mm. \ 4^2. Equivalent Focus of Objectives and Oculars. — To work out in proper mathematical form or to ascertain experimentally the equivalent foci of these complex parts with real accuracy would require an amount of knowledge and of apparatus possessed only by an optician or a physicist. The work may be done, however, with sufficient accuracy to supply most of the needs of the working microscopist. The optical law on which the following is based is : — "The size of object and image varus directly as their distance from the center oj the lens." By referring to Figs. 14, 16, 21, it will be seen that this law holds good. When one considers compound lens-systems the problem becomes involved, as the centre of the lens systems is not easily ascertainable hence it is not attempted, and only an approximately accurate result is sought. \ 433. Determination of Equivalent Focus of Objectives. — Look into the upper end of the objective and locate the position of the back lens. Indicate the level in some way outside of the objective. This is not the center of the object- ive but serves as an arbitrary approximation. Screw the objective into the tube of the microscope. If a Huygenian ocular is used with the ocular micrometer, screw off the field lens and use the eye-lens only. If a positive ocular is used no change need be made. Pull out the draw-tube until the distance between the ocular micrometer and the back lens is 250 millimeters. Use a stage micrometer as object and focus carefullv. Make the lines of the two micrometers parallel (Fig. 10S). Note the number of spaces on the ocular micrometer required to measure one or more spaces on the stage micrometer. Suppose the two microm- eters are ruled in /,„ mm. and that it required 10 spaces on the ocular micrometer to enclose 2 spaces on the stage micrometer, evidently then 5 spaces would cover one. The image, A'B' Fig 21 in this case is five times as long as the object, A,B. — Now if the size of object and image are directly as their distance from the lens it follows that as the size of object is known ( ,-„ mm. ), that of the image directly measured ( j ]J mm.), the distance from the lens to the image also determined in the beginning, there remains to be found the distance between the objective and the object, which will represent approximately the equivalent focus. The general formula is, Object, O: Image, I : : equivalent focus, F 1250. Supplying the known CH.X] TEST PLATE AND APERTOMETER 273 values, O - ,-„, I -];| then |^m.:i 111111. ::F: 250 whence F— 50 mm. That is, the equivalent focus is approximately 50 millimeters. '{ 434. Determination of Initial or Independent Magnification of the Objec- tive. — The initial magnification means simply the magnification of the real image (A'B', Fig. 21) unaffected by the ocular. It may be determined experimentally exactly as described in \ 433. For example, the image of the object ( -j-,, mm.) measured by the ocular micrometer, at a distance of 250 mm. is \% mm., i. e., it is five times magnified, hence the initial magnification of the 50 mm. objective is approximately live. Knowing the equivalent focus of an objective, one can determine its initial magnification by dividing 250 mm. by the equivalent focus in millimeters. Thus the initial magnification of a 5 mm. objective is - T :"- = 50 ; of a 3 mm., -5" =83.3 ; of a 2 mm., -}'i = 125, etc. \ 435. Determining the Equivalent Focus of an Ocular. — If one knows the initial magnification of the objective (''/, 434) the approximate equivalent focus of the ocular can be determined as follows : The field lens must not be removed in this case. The distance between the position of the real image, a position indicated in the ocular by a diaphragm, and the back lens of the objective should be made 250 mm., as described in \ 433, 434, then bv the aid of Wollaston's camera lucida the magnification of the whole mi- croscope is obtained, as described in { 160. As the initial power of the objective is known, the power of the whole microscope must be due to that initial power multiplied bv the power of the ocular, the ocular acting like a simple microscope to magnify the real image (Fig. 21 ). Suppose one has a 50 mm. objective, its initial power will be approximately 5. If with this objective and an ocular of unknown equivalent focus the magnification of the whole microscope is 50, then the real image or initial power of the objective must have been multiplied 10 fold. Now if the ocular multiplies the real image 10 fold it has the same multiplying power as a simple lens of 25 mm. focus, for, using the same formula as before : = 5:1 = 50 : : F: 250 whence F = 25. The matter as stated above is really very much more complex than this, but this gives an approximation. For a discussion of the equivalent focus of compound lens-systems, see modern works on physics ; see also C. R. Cross, on the Focal Length of Micro- scopic Objectives, Franklin Institute Jour., 1S70, pp. 401-402; Monthly Micr. Jour., 1S70, pp. 149-159 J. J. Woodward on the Nomenclature of Achromatic Objectives, Amer. Jour. Science, 1S72, pp. 406-414 ; Monthly Micr. Jour., 1872, pp. 66-74. W. S. Franklin, method for determining focal lengths of microscope lenses. Physical Review, Vol. I, 1S93, p. 142. See pp. 1119-1131 of Carpenter- Dallinger for mathematical formulas ; also Daniell, Physics for medical students ; Czapski, Theorie der optischen Instrumente ; Dippell, Nageli und Schwendener, Zimmermann. E. M. Nelson, J. R. M. S. 1S9S, p. 362, 1900, pp. 162-169. J our - Quekett Micr. Club, vol. V. pp. 456, 462. \ 436. Drawings for Photo-Engraving. — The inexpensive processes of repro- ducing drawings bring within the reach of every writer upon scientific subjects the possibility of presenting to the eye by diagrams and drawings the facts dis- cussed in the text. Though artistic ability is necessary for perfect representation of an object, neatness and care will enable anyone to make a simple illustrative draw- ing, from which an exact copy can be obtained and a plate prepared for printing. 274 APPARATUS FOR SECTIONING {CH. X A careful study of the cuts or plates used to illustrate the same class of facts as one wishes to show will enable one to produce similar effects. Out- lines which are transferred to the drawing paper may be obtained by the camera lucida or from a photograph. The drawing should be made so that it can be reduced anywhere from one-eighth to one-half. For ordinary photo-engraving for such line drawings as are used to illustrate this book, use perfectly black carbon ink. A shaded or wash drawing can be reproduced by the half-tone process, also photographs as is illustrated by figures 1 90-191. A crayon drawing on stipple paper with shadows re-enforced by ink lines and high lights scratched out with a sharp knife give admirable results for anatomical figures by the half-tone process. (See for example the work of Max Broedel in Contributions to the Science of Medicine, (Welch Book) Baltimore, 1900). For photo-engravings of line work the letters, figures or words used to desig- nate the different parts can be put on the drawing by pasting letters, etc., of the proper size in the right position. In preparing the block the photo-engraver •eliminates all shadows and the letters look as if printed on the drawings. '4 437. Wax Models. — Large wax models of the objects which one studies ■under the microscope are helpful both to the teacher and to the investigator. These models are becoming more and more appreciated for embr3'ologic and morphologic investigations, for, as one can readily appreciate, the effort to produce a representation of the embryo or organ in three dimensions helps to overcome difficulties which are almost insurmountable if studied in the sections alone. They are made from wax plates, the principle involved being that the diame- ter of the drawing on the wax plate is as much greater than the object as the wax plate is thicker than the section. The wax plate is cut with a sharp instrument, following the outlines of the object which has been traced upon it by the aid of a camera lucida or the projec- tion microscope. The sections are piled together, some line or lines obtained from a drawing or photograph of the spceimen before it was imbedded and sec- tioned being used as a guide by which the correct form of the pile of sections can be tested. Finally the whole is welded into one by the use of hot wax or a hot instrument. Models which illustrate complex internal structures are difficult to prepare, but numerous devices will occur to the worker as the representation of blood vessels and nerves by strings or wires. A large model will need much sup- port which can be given by wire gauze, wires, pins or paper according to the special needs. A practical method for wax modeling was first published by G. Born, Arch. f. Mikr. Anat., Bd. xxii, 1S83, p. 5S4. The most detailed statements of improve- ments of the method have been published by Born (Bohru u. Oppel ) 1900, and by Dr. F. P. Mall and his assistants. See contributions to the Science of Medicine, pp. 926-1045. Proceedings of the Amer. Assoc. Anatomists, 1901, 14th session (1900) p. 193. \ 438. Some Apparatus for Imbedding and Sectioning. — As a supplement to Chapter VIII, the following figures of imbedding and sectioning apparatus are appended. It will be noticed that the microtomes are complex and consequently expensive. One is figured in which the knife is moved by the hands of the oper- ator (Fig. 217). This form of instrument is excellent, and with it one can do all kinds of work, both with collodion and paraffin. One cannot work so rapidly CH. X] APPARATUS FOR SECTIONING 275 nor with the same precision. For much of the work one ma3' section free-hand, without a microtome. Indeed the great basis of histological and embryological knowledge was gained by studying free-hand sections and dissections. At the present time there is a strong reaction against the exclusive study of sections, and a tendency to combine with the serial sections dissections such as the older anatomists and embryologists made and gained so much from. Fig. 217. A Microtome for all kinds of sectioning ; the knife is guided by the top of the microtome, but moved by the hands of the operator (Bausch & Lomb Optical Co. ) Fig. 218. The Minot microtome for ribbon sections as made by Bausch and Lomb Optical Co. It is arranged for sections from in to 2511 and any intermedi- ate thickness. 276 APPARATUS FOR SECTIONING I [ CH. X Fig 219. The Minot microtome for ribbon sections as made by the Franklin Laboratory Supply Co., Boston. This is to be made for zn, 6u, ion, 14U, 20H, and joit sections. A Fig. 220. Fig. 221. A. CH. A"] APPARATUS FOR SECTIONING 277 Fig. 221. B. Fig. 221. A B Fig. 222. Fig. 220-222. A paraffin holder clamp and a razor support for the Minot Mi- crotome. ( Trans. Amer. Micr. Soc, igoi). Fig. 220. Clamp for the paraffin block holder. In A it is shown in section, in a side view. With this clamp one can use stove bolts as well as the expensive par- affin holders furnished with the instrument. A laboratory can have as many par- affin block holders as necessary without undue expense. Fig. 22i. Razor Support and Razor. {A) Support with heavy base and -vertical piece. The base should be capable of moving endwise one or two centimeters to bring the opening in the vertical part opposite the paraffin block. (B) Front piece to the razor (see Fig. 222 A ). (C) Razor with straight back and edge. By moving this back and forth on the support nearly the entire cutting edge can be utilized. Fig. 222. The knife support of the microtome with the razor support and razor in position. (A) Front view ; (B) Back view. In the inclination of the knife toward the paraffin block is shown. 278 APPARATUS FOR SECTIONING [CH. X Fig. 223. Sliding microtome adapted especially for collodion sectioning. {The Bausch & Lomh Optical Co.). CH. X] APPARATUS FOR SECTIONING 279 A Fig. 224 Paraffin dish for infiltrating in the Lillie oven. It is made 0/ cop- per and as shown has a handle for ease in transference. A the whole dish, B the dish in section. | four. Appl. Micr. iSgg, p. 266). Fig. 225. The Lillie compartment, paraffin oven for infiltrating tissues with paraffin. Various sizes of this are made I 8, r6 and _=y compartments). Except for the largest laporalories the one with 16 compartments and trays will be found of suf- ficient capacity. ( Bausch cr Lomb Optical Co. ). 28o APPARATUS FOR SECTIONING \_CH. X Fig. 226. Circulation board, especially for Neclurus. This is prepared from a board about S x 20 centimeters. Near one edge it has a hole for a perforated cork. On the top of the cork is cemented a thick cover-glass with shellac or rubber cement. The cork can be raised or lowered in the board. The gills of Necturus or the web of a frog' s foot can be spread out on glass over the cork. (four. Appl Micr 1898, p. 131. Fig. 22; embryologic material and small aquai especially designed for collecting with a bicycle (four. Appl. Micr., iSgS,p. /j/). 227. Copper can with screw top for collecting J^ ic material and small aquatic animals. It was <±ff Fig. 22S. Egg pipette. This is made by pulling a short piece of soft rubber tubing over the cud of a i^/ass pipette with rubber bulb. Willi this one can handle the eggs both fresh and hardened without any degree of in- jury, (four. Appl. Micr. /S9S, p. 129), Fig. I syia& Fig. 229 Hashing affa, a/us for tissues fixed in osmic and chromium mi v- tures As shown in the figure the apparatus is connected with the -water fiifie by a small side cock. It is composed of a double vessel, the inner one being made of per- forated brass. There are .fecial perforated dishes to insert in the little compart- ments. J his apparatus is convenient for washing cover-glasses, for the washing out for iron hematoxylin, etc. The deeper box at the right answers for the stile baskets or holders (Fig. /^g). CH. A"] APPARATUS FOR SECTIONING 281 I-lt * i^ Fig. 229 A. — Same as the preceding with the inner, perforated box on edge. Name of Article From K.OU.WQ. A'V^tV.lA.rWV Cost,$ B.GO - °. ^kk^jJl. GXWL \G,o. ,. y itt*- Address ^ L ...,.,.. ^.^ 3 CCr clouu . .5 K (YV,^ _ Order No II 3 - Date A<\A.-^iAjL. J. £T\ I ft ^ ? Date of receipt of Articles ^r>- \^V» v x^\--| ^»„ | ^^ O — Remarks U. S^oL 4-.8.T(^Z.. U> .— Molekularphysik mit besonderer Berticksichtiguug mikroskopischer luter- suchungen und Anleituug zu solchen, sowie einem Auhang liber mikroskopische Analyse. 2 vols. Illustrated. Leipzig, 1XS.S-1SS9. Leiss, C— Die optischen Instrumente der Firnia R. b'uess, deren Beschreibung. Justierung und Anwendung. Pp. 397, illustrated. Leipzig, 1899. Lockyer, J. X. — The spectroscope and its application. Pp. 117, illustrated. London and New York, [S73 BIBLIOGRAPHY 285 Ltiquer, L. MC, I. — Minerals hi Rock Sections. Practical methods of identifying minerals in rock sections with the microscoDe. M'Kendrick, J. G. — A text-book of physiology. Vol. I, general physiology. Pp- 516, illus- trated. New York, iSSS. Mace, E. — L,es substances alimentaire Studies an microscope surtout au point de vne de lenrs alterations et de leur falsifications. Illustrated. Paris, 1891, Macdonald, J. D. — A guide to the microscopical examination of drinking water. Illustrated. London, 1875. Methods and descriptions. MacMunn, C. A. — The spectroscope in medicine. Pp. 325, illustrated. London, 1S85. Marktanner-Turneretscher, G. — Die Mikro-Photographie als Hilfsmittel naturwissenchaft- licher Forschung, Pp. 344, illustrated. Halle, a, S., 1S90. Martin, John II. — A manual of microscopic mounting with notes on the collection and exam- ination of objects. 2d ed. Illustrated. London, 1S7S. Mason, John J. — Minute structure of the central nervous system of certain reptiles and batrachians of America. Illustrated by permanent photo-micrographs. Newport, 1S79-82. Matthews, C. G., and Lott, F. E. — The microscope in the brewery and the malt house. Illustrated. London, 1SS9. Mayall, John, Jr. — Cantor lectures on the microscope, delivered before the society for the encouragement of arts, manufacturers and commerce. Nov. -Dec, 1SS5. History of the micro- scope, and figures of many of the forms used at various times. Mever, A. Die Grundlagen und die Methodeu fur die mikroskopische Untersuchuug von Pflanzenpulvern. Pp. 258, illustrated. Jena, 1901. Moeller, J. — Leitfaden zu mikroskopisch-pharmakgnostischen Ubungen. Pp. 336, illus- trated. Wien, 1901. Moitessier, A. — La photographic appliquee aux recherches micrographiques. Paris, 1866. Morel et Soulie. — Manuel de technique microscopique. Paris. 1S99. Nageli und Schwendener.— Das Mikroskop, Theorie und Anwendung desselben. 2d ed. Pp. 647, illustrated. Leipzig. 1877. Neuhauss, R.— Lehrbuch der Mikro-photographie. Pp. 266. Illustrated. 2d ed. revised. Braunschweig, 189S. Nichols, E. L-— The outlines of physics. Illustrated, N. Y., 1S97. Nichols, E. L- and Franklin Yv. S.— The Elements of Physics ; Light and Sound. Pp. 201, illustrated. New Y'ork and London, 1S97. Pappenheim, A.— Grundriss der Farbenchemie zum Gebrauch bei mikroscopischen Arbei- ten. Pp.476. Berlin, 1901. Petri, R. J. — Das Mikroskop von seinen Anfiingen bis zur jetzigen Vervollkommung flir alle Freunde dieses Instiuments. Pp. 248. Illustrated. Berlin, 1S96. Phin, J.— Practical hints on the selection and use of the microscope for beginners. 6th ed. Illustrated. New York, 1S91. Posselt, E. A.— The structure of fibres, yarns and fabrics. Illustrated. Philadelphia and London, 1891. Preyer, W.— Die Blutkrystalle. Jena, 1S71. Full bibliography to that date. Pringle. A.— Practical photo-micrography. Pp. 193, illustrated. New York, 1S90. Procter, R. A.— The spectroscope and its work. London, 18S2. Quekett, J.— A practical treatise on the use of the microscope, including the different methods of preparing and examining animal, vegetable and mineral structures. Pp. 515, 12 plates. 2d ed. London, 1*52. Rafter, G. YV— The Microscopical Examination of Potable Water. Pp. 160, New York, 1892. Ranvier, L.— Traits technique d'histologie. Pp.1109, illustrated. Paris, 1875-1SSS. Structure and methods. Also German translation, 18S8. Reeves, J. E-— A hand-book of medical microscopy, including chapters on bacteriology, neo- plasms and urinary examination. Illustrated. Philadelphia, 1894. 2S6 BIBLIOGRAPHY Reference Hand-Book of the medical sciences. Albert H. Buck, editor. S quarto vols. Illus- trated with many plates, and figures in the text. New York, 1885-1889. Supplement, 1H93. New edition, 1901 + . Richardson, J. G.— A hand-hook of medical microscopy. Pp. 333, illustrated. Philadelphia, 1871. Methods and descriptions. Robin, Ch.— Traite du microscope et des injections. 2d ed. Pp. 1 lor, illustrated. Paris, 1S77. Methods and structure. Roscoe, Sir Henry.— Lectures on spectrum analysis. 4th ed. London, 1885. Rosenbnsch, K. H. P. translated by Iddings, P.— Microscopical physiography of the rock making minerals. Illustrated. New York, 1SS9. Ross, Andrew.— The microscope. Being the article contributed to the "Penny Cyclopaedia." Republished in New York in 1877. Illustrated. Rusby, H. H. and Jeliffe. S. E-— Morphology and Histology of Plants designed especially as a guide to plant analysis and classification, and an introduction to pharmacognosy and vegetable physiol. Pp. 378, illustrated. New York, 1899. Rutherford, \V.— Outlines of practical histology. 2d. ed. Illustrated. Pp. 194. London and Philadelphia, 1S76. Methods and structure. Sayre, I,. E.— Organic Materia Medica and Pharmacognosy. An introduction to the vegetable kingdom and the vegetable and animal drugs. Chapters on insects injurious to drugs and on pharmacal botany. Pp. 220, Detroit, 1880. Schafer, E- A.— A course of practical histology, being an introduction to the use of the micro- scope. Pp. 304, 40 Fig. Philadelphia, 1S77. Methods. Scbellen, H.— Spectrum analysis, translated by Jane and Caroline Lassell. Edited with notes by W. Huggins. 13 plates, including Angstrom's and Kirchhoff's maps. London 1885. Schimper, A. F. W.— Anleitung zur mikroskopischen Untersuchungen der vegetabilischen Genussmittel. Pp. 158, illustrated. Jena, 1900. Schneider, A. — Microscopy and micro-technique. Pp. 1S9, illustrated. Chicago, 1S99. Sedgwick and Wilson. — General biology. 2d ed. Illustrated. New York, 1S95. Seiler, C— Compendium of microscopical technology. A guide to physicians and students in the preparation of histological and pathological specimens. Pp. 130, illustrated. New York, 1881. Silliman, Benj., Jr, — Principles of physics, or natural philosophy. 2d ed., rewritten. Pp. 710, 722 illustrations, New York and Chicago, i860. Spitta, E. J. — Photomicrography. 4 . Pp. 163. Illustrated by half-tone reproductions from original negatives. Text illustrations. London, 1899. .Starr, Allen M., with the cooperation of Oliver S. Strong and Edward Learning. — An atlas of nerve-cells. Columbia University Press, New York, 1896. The atlas consists of text, diagrams, and some of the best photo-micrographs that have ever been published. Sternberg, G. M. — Photo-micrographs, and how to make them. Pp. 204, 20 plates. Boston, 18S3. Stoehr, S. P. — Text-book of histology including the microscopic technique, 3d American from 8th German edition. Translated by Dr. Emma Bilstein and edited by Dr. A. Schaper. Pp. 432, illustrated. Philadelphia, 1901. Stokes, A. — Aquatic microscopy for beginners, or common objects from the ponds and ditches. Illustated, Portland, Conn., 1896. .Suffolk, W. T, — On microscopical manipulation. 2d ed. Pp. 227, Illustrated. London, 1870. Szymonowicz, L. — Lehrbuch der Histologie-einschliesslich der mikroskopischen Techuik. Illustrated. WUrzburg, 1900. Thomas, Mason B., and Win. R. Dildlev. — A laboratory manual of plant histology. Illus- trated. Crawfordsville, Ind., 1894. Trelease, Wtn. — Poulseu's botanical micro-chemistry ; an introduction to the study of veget- able histology. Pp. 118. Boston, 18S4. Mehods, Trutat, M. — La photographie appliquee ii histoire naturelle. Pp. 22^, illustrated. Paris, 18S4. BIBLIOGRAPHY 287 Valentin, G. — Die Untersuchuug der Pflauzen und der Thiergewebe in polarisirtem Liclit. Leipzig, 1861, Van Heurck, H — The microscope. Illustrated. London and New York, 1S9S. Vierordt, Die quantitative Spectralaualyse in ihrer Amvendung auf Physiologie, 1876. Vogl, A. H- — Die wichtigsten vegetablischen Nahrungs und Genussmittel. Pp. 575., illus- trated. Wien, 1899. Vogel, Conrad. — Practical pocket-book of photography. Pp. 202, Figs. London, 1893. Vogel, H. W.— Practische Spectralaualyse irdischer stoffe ; Anleitung zur Benutzung der Spectralapparate in der qualitativeu und quantitativeu chemische Analyse organischer und un- organscher Korper. 2d ed. Figs. Berlin 1889. Wall, O. A. — Notes on Pharmacognosy. — Pp. 1S0, illustrated. St Louis, 1897. Weinschenk, E. Anleitung zum Gebrauch des Polarisationsmikroskopes. Pp. 123, illus- trated. Freiburg. 1901. YVethered, M. — Medical microscopy. Pp. 406, Figs. London and Philadelphia, 1S92. Whipple, G. C— The Microscopy of Drinking Water. Pp. xii - 300. Illustrated. New York, 1S99. Whitman, C. O. — Methods of research in microscopical anatomy and embryology. Pp. 255, illustrated. Boston, 1SS5. Wilder and Gage. — Anatomical technology as applied to the domestic cat. An introduction to human, veterinary and comparative anatomy. Pp. 575, 130 Fig. 2d ed. New York and Chicago, 1SS6. Wilson., Edmund, B., with the cooperation of Edward I, earning. — An atlas of fertilization and karyokinesis. Columbia University Press, New York, 1895. This atlas marks an era in embryological study. It has admirable texts and diagrams, but the distinguishing feature is the large number of almost perfect photo-micrographs. Wood, J. G. — Common objects for the microscope. Pp. 132. London, no date. Upwards of 4.00 figures of pretty objects for the microscope, also brief descriptions and direction for prepa- rtions. Wormly, T. G. — The micro-chemistry of poisons. 2d ed. Pp. 742, illus. Philadelphia, 1SS5. Wright, Lewis, — Optical Projection, a treatise on the use of the lantern in exhibition and scientific demonstration. Pp. 425, illustrated. London, 1891. (Beginners will find this book very helpful ), Wythe, J. H. — The microscopist ; a manual of microscopy and a compendium of microscopi- cal science. 4th ed. Pp. 434, 2^2 Fig. Philadelphia, 1SS0 Zimmermann, Dr. A. — Das Mikroskop, ein Leitfaden der wissenschaftlichen Mikroskopie. Illustrated. Leipzig und Weill, 1S95. See also Watt's chemical dictionary, and the various geneial and technical encyclopedias. PERIODICALS* The American journal of anatomy (including histology, embryology and cytology). Balti- more, 1901 — . The American journal of medical research, Boston, 1901+. The American Journal of physiology. Boston, 1S96+ . The American journal of microscopy and popular science. Illustrated. New York, 1876-1881. The American monthly microscopical journal. Illustrated. Washington, D. C, 1SS0+. American naturalist. Illustrated. Salem and Philadelphia, 1S67 — . American quarterly microscopical journal, containing the transactions of the New York microscopical society. Illustrated. New York, 18784-. American microscopical society, Proceedings. 1S78-1S94 ; Transactions, 1895-f-. *Note — When a periodical is no longer published, the dates of the first and last volumes are given ; but if still being published, the date of the first volume is followed by a plus sign. See Vol. XVI of the index Catalog of the Library of the Surgeon General's office for a full list of periodicals. 288 BIBLIOGRAPHY Anatomischer Anzeiger. Centrablatt flir die gesammte wissenchaftliche Anatomie. Amt- liches Organ der anatomischen Gesellschaft. Herausgegebeii von Dr. Karl Bardeleben. Jena, 1SS6 :-. Resides articles relating to the microscope or histology, a full record of current anatomi- cal literature is given. Annales de la societe beige de microscopie. Bruxelles, 1874 f . Archives d'Anatomie microscopique. Illustrated. Paris, 1897. (Balbiani et Ranvier). Archiv fllr miroscopische Anatomie. Illustrated. Bonn, 1865+. Centrablatt flir Physiologic Unter Mitwirkung der physiologischen Gesellschaft 211 Berlin Herausgegeben von S. Exner und J. Gad. Leipzig and Wien. 18K7 + . Brief extracts of papers having a physiological bearing. Full bibliography of current literature. English mechanic. London, 1866-f-. Contains many of the papers of Mr. Nelson on light- ing, photo-micrography, etc. Index medicus. New York, 1879+. Bibliography, including histology and microscopy. International journal of micioscopy and popular science. London, 1S90 + . Journal of anatomy and physiology. Illustrated. London and Cambridge, 1867-f, Journal of Applied Microscopy and Laboratory methods. Illustrated. Rochester, N. Y., 1898+ . Journal de micrographie. Illustrated. Paris, 1877-1892. Journal of microscopy and natural science. London, 1885+ . Journal of the New York microscopical society. Illustrated. New York, 1885+. Journal of physiology. Illustrated. London and Cambridge, 1S7S + . Journal of the American chemical society. New York, 1879+. Journal of the chemical society. London, 184S-J-. Journal of the royal microscopical society. Illustrated. London, 1878 + . Bibliography of works and papers relating to the microscope, microscopical methods and histology. It also in- cludes a summary of many of the papers. Journal of the Quekett microscopical club. London, 1868 + . The Lens, a quarterly journal of microscopy, and the allied natural sciences, with the tran- sactions of the state microscopical society of Illinois. Chicago, 1872-1873. The Metallographist, a quarterly publication devoted to the study of metals with special reference to their physics, microstructure, their industrial treatment and application. Illustrated especially by photo-micrographs of metals and alloys. Boston, 1898 + . The Microscope. Illustrated. Washington, D. C, 1881-1897. Microscopical bulletin and science news. Illustrated. Philadelphia, 1S83 + . The editor, Ed- ward Pennock introduced the term " par-focal " for oculars (see vol. iii, p 31). Monthly microscopical journal. Illustrated. London, 1869-1877. Nature. Illustrated. London, 1869+. The Observer. Portland, Conn., 1890-1897. Philosophical Transactions of the Royal Society of London. Illustrated. London, 1665+. Proceedings of the American microscopical society, 1S7S+, Proceedings of the Royal Society. London, 1S54 + . Quarterly journal of microscopical science. Illustrated. London, 1S53 + . Science Record. Boston, 18S3-4. Zeitschrift f. Angewandte. Mikroskopie. 1S9S + . Zeitschrift flir Instrumentenkunde. Berlin, iSSi-f. Zeitschrift ft\r physiologiscbe Chemie. Strassburg, 1S77 + . Zeitschrift flir wisseuschaftliche Mikroskpie und fllr mikroskopische Technik. Illustrated. Braunschweig. 1S844-. Methods, bibliography and original papers. Besides the above-named periodicals, articles on the microscope or the application of the microscope appear occasionally in nearly all of the scientific journals. One is likely to get references to these articles through the Jour. Roy. Micr. Soc. or the Zeit. wiss. Mikroskopie. Excellent articles on Photo-micrography occur in the special Journals and Annuals of Photog- raphy. INDEX Abbe apertometer, 270-272. Abbe camera lucida, 123-132. Abbe condenser or illuminator, 45-50. Abbe's test-plate, method of using, 268- 270. Aberration, chromatic, 4, 5 ; by cover- glass, 55 ; spherical, 4, 5, 26S. Absorption spectra, 136-139, 145-150. Acetelyne light, 37, 51, 229. Achromatic condenser, 42-43, 256 ; objec- tives, 11, 64 ; oculars, 22. Achromatism, 12. Actinic focus, 226 ; image, 221. Adjustable objectives, II, 12, 54-57 ; ex- periments, 54 ; and micrometry, 11S ; and photo-micrography, 234. Adjusting collar, graduation of, 55. Adjustment, of analyzer, 151 ; coarse or rapid, and fine, 63, Frontispiece ; of objective, 11, 12, 54; of objective for cover-glass, specific directions, 55 ; testing, 63. Aerial image, 30, 31, 230. Air bubbles, 94, 95. Albumen fixative, Mayer's, 198. Alcohol, absolute, 198; ethylic, 198; methyl, 198, 203. i^lcoholic dve, staining sections with, 1S9. Alum solution, 19S. Amici prism, 134, 140. Amplifier, 109. Amplification of microscope, 103. Analyzer, 141, 151. Anas'tigmatic objective, 20S, 212. Angle, of aperture, 15, 16 ; of carbons for arc lamps, 251-252 ; critical, 54. Angstrom and Stokes' law of absorption spectra, 13S. Angular aperture, 15, 16. Anisotropic, 152. Apertometer, Abbe's, 270. Aperture of objective, 15, 21, 270; angu- lar, 15, 16; formula for, 16-18; of illuminating cone, 45 ; numerical, 17, 19, 270 ; numerical of condenser, 44 ; significance of, 20. Aphengescope, 267. Aplanatic cone, 46 ; objectives, 11 ; ocu- lar, 22 ; triplet, 7. Apochromatic condenser, 42 ; objectives, 12, 64, 214. Apparatus and material, 1, 34, 90, 103, 1.22, 134, 205, 243 ; for micro-projec- tion, 256, 257 ; for photography, 233 ; for photo-micrography, 205. Appearances, interpretation, 90-102. Arc lamp, 37, 229, 250-255 ; continuous current, 249. Arrangement of condenser, 46 ; of lamp, bull's eye and microscope, 51 ; mi- nute objects, 204 ; serial sections, 192; tissue for sections, 191. Artifacts, 91. Artificial illumination, 37, 48, 50 ; for photo-micrography, 229. Avoidance of diffusion currents, 180 ; of distortion, 124. Axial light, 36 ; experiments, 40 ; with Abbe illuminator, 48. Axial, point, 15 ; ray, 36. Axis, optic, 2, 3, io ; of illuminator, 49 ; secondary, 3. 6, 49. Back-ground for photographing, 209. Back combination or system of objective, 9-13. Bacterial cultures, photographing, 241- 242. Balsam, 199 ; bottle, 183 ; mounting in, 1S3, 190 ; preparation of, 199 ; re- moval from lenses, 61 ; natural, 190, 199 ; neutral, 199 ; removal from slides, 162 ; xylene, 199. Bands, absorption, 137. Base of microscope, Frontispiece. Bath, water, 255. Bed, camera, 207. Benzin, removing from sections, 189. Biaxial crystals, 154. Bibliography, 26, 33, 99, 101, 121, 133, 150. 155, 157. 158,159. T 96, 204, 220, 240, 242, 251, 258, 267, 273, 274, 282. Blocks for collodion, 178 ; for shell vials, 174. Blood, absorption spectrum of, 146 ; cir- culation of with micro-projection, 257,. 264. Board, circulation, 280. Body of microscope, Frontispiece. 290 INDEX Bottle for balsam, glycerin or shellac, 172 ; reagent, 179. Bowl, waste, 181. Box, glass, i8r ; slide, 198. Brownian movement, 99. Bubble, air, 94, 95. Bull's-eye, 51, 217, 227 ; engraving glass for, 228. Burning point, 7, 30. Buxton's photo-tnicrographic outfit, 237- 23S. Cabinet for microscopical preparations, 196,197. Calipers, micrometer, 164 ; pocket, 164. Camera, bed, 207 ; for embn'os, 211 ; for large, transparent sections, 215 ; photo-micrographic, 222, 225, 235 ; testing, 223; vertical, 205-210. Camera lucida, Abbe, 122-132; Wollas- ton's, 107, 125. Canada balsam, 199 ; mounting in, 183, 190; preparation of, 199; removal from lenses, 6r ; removal from slides, 162. Carbol-xylene, 200. Carbon-monoxide hemaglobin, spectrum of, 147. Carbons, for projection apparatus, 250- 255; adjusting, 251 ; angle, 251; kinds, 253 ; positive and negative, 252 ; size, 254 ; wear, 253. Card, catalog, 196 ; centering, 169 ; inven- tory, 281. Care of, eyes, 61 ; micro-projection appa- ratus, 265 ; microscope, mechanical parts, 59 ; optical parts, 60 ; nega- tives, 211 ; water immersion objec- tives, 58. Carmine to show currents and pedesis, 99 ; spectrum of, 14S. Carrier, objective, 259. Castor-xylene clarifier, 200. Cataloging, formula, 195 ; preparations, 194-196. Cedar-wood oil, bottle for, 199 ; for clear- ing, 184-185, 200; for oil immersion objectives, 200. Cells, deep, thin, 168 ; isolated, prepara- tion of, 175 ; mounting, 168 ; stain- ing, 174- Cement, shellac, 203. Cementing collodion, 201. Center, optical, 2, 3. Centering, and arrangement of illumina- tor, 43, 47 ; card, 169 ; image of source of illumination, 44 ; the radiant, 255. Centimeter rule, 104, 133. Central light, 36, 95. Chamber, moist, 171. Cheese-cloth for cleaning slides, 161. Chemical focus, 12 ; rays, 12. Chemistry, Micro-155. Chromatic, aberration, 4 ; correction, 11, 12 ; correction, test for, 270. Circulation of blood with micro-projec- tion, 257, 264 ; board, 226. Clarifier, 200 ; castor-xylene, 200. Class demonstrations in histology and embryology, 243. Cleaning back lens of objective, 61 ; ho- mogeneous objectives, 59 ; micro-pro- jection apparatus, 265 ; mixtures for glass, 165 ; sildes and cover-glasses, 161-163 ; water immersion objectives, 58. Clearer, 173, 183, 190, 200. Clearing mixture, preparation of, 200 ; tissues with cedar-wood oil, 184. Clinical microscope, 243-245. Cloudiness of objective and ocular, how to determine, 92 ; removal 60. Coarse adjustment of microscope, Front- ispiece ; testing, 63. Cob-web micrometer, 117. Collecting can, 280 Collodion, 200 ; for coating glass rod, 97 ; cementing, 201 ; clarifying, 178 ; cot- ton, 200; for fastening sections to slide, 18S ; hardening, 17s ; method, 176 ; thick, thin, 177, 201. Color, correct photography, 217 ; correc- tion, 12 ; images, 54, 58 ; law of, 13S ; production of, 154 ; screens, 218, 220. Colored minerals, absorption spectra of, 149 ; specimens, photography of, 21S. Comparison prism, 141, 142 ; spectrum, 142. Compensating ocular, 24, 25. Complementary spectra, 139. Compound microscope, see under micro- scope. Concave lenses, 3 ; mirror, use of, 37. Condenser, 41-50 ; Abbe, 46, 4S-49"; ach- romatic 42, 43, 227, 256 ; apochro- matic, 42, 227 ; bull's-eye, 51, 227, 228 ; centering, 43, 47 ; illuminating cone with, 45 ; lamp, for projection, 250 ; mirror for, 48 ; non-achromatic, 46 ; numerical aperture of, 44-46 ; optic axis of, 43, 47 ; for photo-micro- graphy, 42, 227, 233 ; standard size for, 26, 47 ; substage, 42; See also illuminator. Condensing lens, 36. Cone, aplanatic, 46 ; illuminating, 45. Conjugate foci, 4. Construction of images, geometrical, 6. INDEX 291 Continuous, current arc lamp, 249 ; spec- trum, 137. Contoured, doubly, 97. Converging lens, 3-5 ; lens system, 9. Convex lenses, 3-5. Cooler, specimen, 257. Correction, chromatic, or color, 5, 12, 268- 270 ; cover-glass, 55-56 ; cover, tube- length for, 56-57 ; over and under, 12. Cotton, collodion, gun, or soluble, 200. Counterstaining, [89-190. Cover-glass, or covering glass, 162-163 \ aberration by, 54 ; adjustment, spe- cific directions, 55 ; adjustment for, in photo-micrography, 234 ; adjust- ment and tube-length, 12, 13, 55 ; anchoring, 170 ; cleaning, [62-163 ! correction, 55, 56 ; effect on rays from object, 17, 55 ; gauges, 164-165 ; meas- urer, 164-165; measuringthicknessof, 163 ; non-adjustable objectives, table of thickness, [4; No. 1, variation of thickness, [64 ; putting on, 94, 167 ; sealing, [69, [70; size of, [64, [62; thickness of, 13, [4, [63~[65, [93 ; tube-length with, 13, 56, 57 ; wiping, [63 ; with, serial sections, [93. Crater of carbons, 252. Critical angle, 54. Crystals, biaxial, depolarizing, [54; from frog for pedesis, 100. Crystallization under microscope, 50, 157. Crystallography, 155 ; list of substances for, 156-157. Currents, diffusion, avoidance of, [So ; in liquids, 9S. Cutting sections, 17S, [S6, 191-192. D Dark-ground illumination, 37, 49-50; with Abbe illuminator, 50 ; with mirror, 50. Daylight, lighting with, 35. Deck-plugs for collodion blocks, [78. Dehydration, [77, [90. Demonstration, microscope, 243-245; with micro-projection apparatus, 265. Depolarizing crystals, [54. Designation, of oculars, 25 ; of w-ave iength; [43. Determination, of, field of microscope, 28 ; equivalent focus, 272-273 ; mag- nification, [03-109, 273; of working distance, 39. Diamond, writing, 196. Diaphragms and their employment, 36-50. Diffraction, grating, [37; illusory ap- pearances due to, IOI. Diffusion currents, avoidance of, [80. Direct, light, 35 ; vision spectroscope, 134- Dispersing prism, 137. Dissecting microscope, S, 33, [75, 228. Dissociator, formaldehyde, 201 ; nitric acid, 203. Distance, principal focal, 3. 30; standard at which the virtual image is meas- ured, [09 ; working d. of simple mi- croscope or objective, 39 ; working d. of compound microscope, n, 34, 39-4o. Distinctness of outline, 96. Distortion in drawing, avoidance of, 124. Diverging lens, 3. Double spectrum, [42 ; vision, 103, 105. Doubly contoured, 97 ; refracting, [52. Draw-tube, Frontispiece ; pushing in, 38. Drawing, with Abbe camera lucida, 129- [31 ; board for Abbe camera lucida, [29, 130; distortion, avoidance of, [24 ; embryograph for, [32 ; with microscope, 122; photographic cam- era for, 132 ; for photo-engraving, 273 ; scale and enlargement of, 131 ; with simple microscope, [32. Dry objectives, [o, [6-1S ; light utilized, 17 ; dry mounting, [67 ; numerical aperture, 16 ; dry plates, discovery by Maddox, 221. Dust, of living rooms, examination of, io[ ; on objectives and oculars, how to determine, 92 ; removal, 60. Dye, general, staining with, 1,89 ; aque- ous, 1S0, 1S9 ; alcoholic, [So, 1S9. Eccentric diaphragm, 44, 49-50. Egg pippett, 2S0. Electric light, 37, 229, 235, 250. Embryograph, 132 Embryos, camera for, 21 [ photograph- ing, 2U-214; records of, 213. Engraving glass for bull's-eve condenser, ' 228 Enlargements, 241. Eosin, 20 1. Epidiascope, 266-267. Episcope, 267. Equivalent focal length or focus of objec- tives and oculars, to, 25, 272. Erect image I. Ether, alcohol, 201 ; sulphuric, 201, Ethvlic alcohol, 19S. Examination of dust of the living rooms, bread crumbs, corn starch, fibres of cotton, linen, silk, human and ani- mal hairs, potatoes, rice, scales of butterflies and moths, wheat, 101. INDEX Experiments, Abbe condenser, 4S ; with adjustable and immersion objectives, 54 ; compound microscope, 26 ; ho- mogeneous immersion objective, 58 ; lighting and focusing, 37 ; in micro- chemistry, 157 ; with micro spectro- scope, 145 ; with micro-polariscope, 152 ; in mounting, 166 ; photo-micro- graphy, 229 ; simple microscope, 6. Exposure, of photographic plates, 232, 235, 240; with color-screen 220. Extraordinary ray of polarized light, 150. Eve and microscope, 6, 9, 32. Eyes, care of, 6i ; muscae volitantes of, 100. Eye-lens of the ocular, 22. Eye-piece, 22 ; micrometer, 1 14. Eye-point, 7, 22, 123 ; of ocular, demon- stration, 3:. Eye-shade, adjusting, 62 ; double, 62. Farrants' solution in mounting objects, 171 ; preparation of, 201. Fibers, examination of, 101 ; textile, 158. Field, 2S ; with camera lucida, 107 ; illu- mination of, 45, 51 ; with orthoscopic ocular, 23 ; with periscopic ocular, 24 ; of view with microscope, 28, 29, 105, 123-125; size of, with different objectives and oculars, 28, 29. Field-lens, of ocular, 22 ; action of, 32 ; dust on, 92. Filar, micrometer ocular, 23, 26 ; ocular micrometer, 117, 118. Filter, hot, 1S5. Filtering balsam, etc., 1S5, 199. Fine adjustment, Frontispiece ; testing, 63. Fir, balsam of, 199. Fixative, albumen, Mayer's, 198. Fixing, reagents for, 19S; tissue, 176. Focal distance, or point, principal, 30 ; length equivalent, 10. Focus, 6; actinic, 226; chemical, 12 ; conjugate, 4 ; equivalent, of object- ives and oculars, 10, 25, 272-273 ; op- tical, 12 ; principal, 3, 5 ; virtual, 3 ; visual, 226. Focusing, 6, 34 ; adjustments, testing, 63; with compound microscope, 34 ; em- bryos, 213; experiments, 38 ; glass, 209 ; with high objectives, 38 ; with low objectives, 38 ; objective for mi- cro-spectroscope, 144 ; for photo-mi- crography, 206, 213, 230; scale for, 206 ; screen for photo-micrography, 209 ; with simple microscope, 6, 34 ; slit of micro-spectroscope, 145. Food, detection of adulteration in, 158. Form of objects, determination of, 93. Formal, 201 . Formaldehyde, dissociator, 2or ; for isola- tion, 173. Formula, for aperture, 16, 17 ; for catalog- ing, 195 ; for refraction, 52-54. Fraunhofer lines, 137. Free, hand sections, 274 ; working dist- ance, 40. Front combination or lens of objective, 9-1 1. Frontal sections, 192. Function of objective, 29-31 ; of ocular, 31- Gauge, cover-glass, 164-165. Gelatin, liquid, preparation of, 203. Geometrical construction of images, 6. Glass, cleaning mixture for, 165 ; focus- ing, 209 ; ground, 29, 209 ; rod appear- ance under microscope, 96, 97 ; slides or slips, 161, 162. Glasses, opera, 262. Glue, liquid, preparation of, 203. Glycerin, bottle for, 1S3 ; mounting ob- jects in, order of procedure, 170, 201 ; removal, 61. Glycerin jelly, mounting objects in, or- der of procedure, 170-171 ; prepara- tion of, 201. Grating, diffraction, 137. Ground glass, focusing screen, 209 ; pre- paration of, 29. Gun cotton, 200. H Half-tones from photo-micrographs, 232, 274. Hardening collodion, 178; tissue, 176, 183. Hematein, 203. Hematoxylin, 202 ; stained preparations, photographing, 21S. Hemoglobin spectrum, 147. High school microscope, 64, 71-S9. Histology, physiological, 196. History of photo-micrography, 220. Holder, lens, 7, 217, 22S ; needle, 167 ; slide, 18S. Homogenous immersion objective, 16- 19; cleaning, 59; experiments, 58; numerical aperture, 16-21. Homogenous liquid, 11; tester for, 5S, 271 ; vessel for, 199. Horizontal camera, 230, 236-237. Huygenian ocular, 22, 24, 32. INDEX 293 Illuminating, cone (for condenser), aper- ture of, 45; objective, 13, 238; power, 21. Illumination, for Abbe camera lucida, 129; artificial, 37, 48, 50; artificial for photo-micrography, 229 ; center- ing image of source of, 44 ; with air and oil, 94, 95 ; dark ground, 49, 50 ; daylight, 35 ; of entire field, 51 ; lamp for, 50 ; methods of, 35, 49 ; for micro-polariscope, 151 ; for micro- spectroscope, 144 ; oblique with air and oil, 94, 95 ; of opaque objects, 144, 238 ; for photography, 208, 216, 229, 241 ; for photo-micrography, 2 3°> 2 33. 2 3S ; for projection, 250 ; for Wollaston's camera lucida, 124, Illuminator, 41-50; vertical, 13, 23S. See also condenser. Image, actinic, 221 ; aerial, 30, 230 ; center- ing i. of source of illumination, 44 ; color, 52, 58 ; inverted, real of ob- jective, 30; of flame, 45 ; geomet- rical construction of, 6 ; and object, size and position, 5, 9, 10S ; real, 5, 9, 30-32, 103 ; refraction, 52, 5S ; retinal, 6, 9, 32 ; swaying of, 4S ; virtual i, and standard distance at which meas- ured, 6, 109. Image-power of objectives, iS. Imbedding, 177, 185. Immersion, fluid or liquid, 58, 271 ; illu- minator, 47 ; objective, 11, 58-59. Incandescence or line spectra, 137. Incident light, 35. Index, of refraction, 53 ; of medium in front of objective, 16-19. Indicator ocular, 247. Infiltration, collodion, 177 ; paraffin, 1S4; paraffin dish for, 279. Ink for labels and catalogs, 195 ; for drawing, 274. Interpretation of appearances under the microscope, 90-102. Inventory card, 2S1. Iris diaphragm, 82, 157. Irrigating with reagents, 170. Isochromatic plates, 217. Isolation, 173 ; with formaldehyde, 173 ; nitric acid, 175. Isotropic, 152. J-K Japanese filter or tissue paper, 60. Jelly, glycerin, 170-171, 201. Jena glass, 71. Jurisprudence, micrometry in, 121. Knife support, 277. Labels and catalogs, 194-196, 203. Labeling microscopical preparations, 194; photographic negative, 211 ; serial sections. 193. Laboratory compound microscope, 64, 71-89. Lamp, acetylene, 37, 5r, 229 ; condenser, 250 ; electric arc, 37, 229, 250-255 ; petroleum, 37, 50-51, 220, 229 ; spirit, 1S3. Lantern, magic, 249 ; slides, 241. Law of color, 13S. Lens, concave, 3 ; converging, 3 ; convex, 4 ; eye, 22 ; field, 32 ; holder, 7, 175, 217, 22S ; paper, 60 ; system, 9 ; thick, 3. Lenses of micro-projection apparatus, cleaning, 265. Letters in stairs, 93 ; fur photo-engraving, 274. Lettering oculars, 26. Light, with Abbe illuminator, 4S ; ace- tylene, 37, 51, 229 ; artificial, 37, 50, 229 ; axial, 36, 40, 48 ; direct, 35 ; central, 36, 40 ; electric, 37, 229, 250 ; incident, 35 ; with mirror, 37 ; ob- lique, 36, 41, 48 ; petroleum, 37, 50, 220, 229 ; for photo-micrography, 229; polarized, 150; reflected, 35; sun, 229; transmitted, 36; utilized with different objectives, 17; for vertical illuminator, 239 ; wave length of, 142 ; Welsbach, 37, 229. Lighting, 35 ; for Abbe camera lucida, 129; artificial, 50; experiments, 37; with horizontal camera, 230; for mi- cro-polariscope, 151; for micro-spec- troscope, 144 ; with a mirror, 37, 40; with daylight, 35, 229 ; for photog- raphy, 20S, 216; for photo-micro- graphy, 229, 230; for vertical illum- inator, 229. Line spectrum, 137. Liquid, currents in, 98 ; homogenous, 1 1, 58, 271. M Magic lantern, 249. Magnification of compensating oculars, 24 ; effect of adjusting objective, 1 18 ; determination of, 103-109 ; expressed in diameters, 103 ; initial or indepen- dent, 273 : of microscope, 103 ; in micro-projection, table, 262 ; of mi- croscope with Abbe camera lucida, 131 ; of microscope, compound, 105 ; of microscope, simple, 104; of photo- 294 INDEX micrographs, determination of, 233 ; ' real images, 103 ; table of, with ocu- lar micrometer, ill : with projection microscope, 262 ; varying with com- pound microscope, 109; and velocity, 98. Magnifier, tripod, 7, 104, 209. Marker for preparations, 65-66. Marking objects, 65-66,248 ; negatives, l 211, 214; objectives, 71. Masks for preparations, 264. Material and apparatus, :, 34, 90, 103, 123, 134, 156, 19S, 205, 223, 243. Measure, unit of, in micrometry, 112 ; of wave length, 143. Measurer, cover-glass, 164-165. Measuring the thickness of cover-glass, 163. Mechanical parts of compound micro- scope, 64 ; Frontispiece, 8 ; of micro- scope, care of, 59 ; testing, 63. Mechanical stage, 65, 67-70, 25S, 259. Megascope, 267. Metallic surfaces, photography of, 235- 240 ; preparation of, 239. Metallography, microscope in, 159. Metals, examination of, 159, 235. Met-hemaglobin, spectrum of, 136, 147. Methods, collodion, 176-1S3 ; paraffin, 183-191. Metric measures and equivalents, cover 1st p., 133. Micro-chemistry, 155-157 \ slides for, 161. Micro-metallography, objects for, 238. Micrometer, 103 ; calipers, 164 ; cob-web, 117; filar m. ocular, 117, 118; filling lines of, ro6 ; lines, arrangement of ocular and stage, 120 ; lines, finding, 106; net, 128; object or objective,! 105; ocular or eye-piece, 1 14-120;' ocular, micrometry with, 116; ocular, ratio, 119; ocular, valuation of, in, 114; ocular, varying valuation of, 118 ; for photo-micrography, 233 ; screw ocular, 117; stage, 105, 106; table of magnification, 11 r. Micrometry, definition, 112, 114; with adjustable objectives, 118; compari- son of methods, 1 19-120; with com- pound microscope, 112; and juris- prudence, 121 ; limit of accuracy in, 120; with ocular micrometer, 116 ; with simple microscopic, 112; remarks on, 118; unit of measure in, 112. Micro-millimeter, 113. Micron, 113 ; for measuring wave length of light, 143. Micro-photograph, 220. Micro-photograph}', distinguished from photo-micrography, 220. Micro-planar objective, 212, 260. Micro-polariscope, 100, 150 155. Micro-polarizer, 150. Micro-projection, 249-267 ; apparatus, 256-257 ; carbons for, 250-254 ; cir- culation of blood with, 257, 264 ; con- denser, 254, 256 ; current, 254 ; dem- onstration with, 265 ; magnification, 262 ; masks for specimens, 264 ; me- chanical stages, 258-259 ; microscope for, 249 ; objectives and oculars, 259 ; pointer for, 265 ; preparations, 263 ; screen and screen distance, 26J ; specimen cooler, 257 ; stage, 258 ; stains for, 263 ; water-bath, 255. Microscope, definition, 1 ; amplification of, 103 ; clinical, 243 ; demonstration, 243 ; dissecting, 8, 33, 175, 176 228; care of, 59; eye and, 1, 6, 9; field of, 28, 29 ; focusing, 34 ; magnification, 103; for metallography, 159-160 ; for mi- crochemical analysis, 153; for opaque objects, 235-237 ; for photo-micro- graphy, 225, 231, 237 ; polarizing, 150; preparation, with erecting prism 176; projection, 249-250 ; price of, 64, 71 ; putting an object under, 27 ; sun or solar, 249 ; screen, 59 ; stand for large, transparent objects, 215, 216 ; stand, for embryos, 212 ; traveling, 245-246. Microscope compound, definition, S ; drawing with, 122 ; figures, frontis- piece, 9, 71-S9, 102, 153, 160, 237, 244-246 ; focusing, 38-39 ; for High schools, 64, 71 ; for laboratory, 64, 71-S9 ; lamp for, 50 ; magnification or magnifying power, 105 ; magnifi- cation and size of drawing with Abbe camera lucida, 131 ; mechani- cal parts, 64 ; micrometry with, 112 ; optic axis of, 9, 10 ; optical parts of, 9, 64 ; varying magnification, 109 ; working distance of, 39 ; testing, 63. Microscope, simple, definition, 1 ; exper- iments with, 6 ; figures, 6-8, 33, 104, 175, 209, 228, 243; focusing with, 34; magnification of, 104 ; micrometry with, U2; working distance of, 34; Microscopic, objective, 9 ;obj ective low, attached to camera, 214; objects, drawing, T22 ; ocular, 22 ; slides or slips, 161 ; tube-length, 13, 14. Microscopical preparations, cabinet for, 197; cataloging, 194; labeling, 194; mounting, 166-193. Microtome, hand, 274-275 ; Minot's 275- 276 ; razor support for, 276-277 ; slid- ing, 278. INDEX 295 Micro-spectroscope, 134-150; adjusting, 139 ; experiments, 145 ; focusing, 144 ; focusing the slit, 139 ; lighting for, 144 ; objectives to use with, 144 ; reversal, apparent, of colors in, 134 ; slit, mechanism of, 135, 139. Micrum, 113. Mikron, 113. Milk globules, to overcome pedesis of, 100 Minerals, absorption spectra of, 149. Minot's microtomes, 275-276. Minute objects, arrangement of 204. Mirror, 9-1 1 ; for Abbe illuminator, 4S ; of camera lucida, arrangement for drawing, 126; concave, use of, 37; dark ground illumination, 50 ; light with, central and oblique, 40, 41 ; lighting with, 37 ; plane, use of, 37. Mixture, clearing, 200. Models, wax, 274. Moist, chamber, 171. Molecular movement, 99. Monazite sand, spectrum of, 149. Monnting cells, preparation of, 168 ; me- dia and preparation of, 198 203 ; ob- jects for polariscope, 151 ; perma- nent, 167 ; temporary, 166. Mounting objects, dry in air, order of procedure, 167-16S ; in glycerin, or- der of procedure, 170 ; in glycerin jelly, order of procedure, 170; in media miscible with water, 169 ; mi- nute objects, 204 ; opaque objects, 239 ; permanent, 167 ; in resinous media, by drving or desiccation or- der of procedure, 172 ; in resinous media, by successive displacements, order of procedure, 172 ; temporarv, 166. Movement, Brownian, or molecular, 99. Muscae volitantes, 100. Muscular fibers, isolation of, 175. N Natural balsam, 199. Needle-holder, 167. Negatives, labeling, 21 r, 214; oculars, 22 ; rack for drying, 240 ; record of, 2 [4 ; storing, 211, 2 [4. Net micrometer, 12S. Neutral balsam, 199. Nicol prism, 150. Nitric acid, dissociator, 203. Nomenclature of objectives, 10. Non-achromatic condenser, 46 ; object- ives, 11. Non-adjustable objectives, 12; thickness of cover glass for, table, 14. Normal salt solution, 203. Nose-piece, 27, 3S, So; marking object- ives on, 71. Numerical aperture, of condenser, 44 ; of objectives, 16, 270 ; table of, 19. o Object, determination of form, 93 ; hav- ing plane or irregular outlines, rela- tive position in a microscopical prep- ration, 92 ; and image, size of, 10, 108 ; marking parts of, 65-66 ; mark- ing position of, 248 ; micrometer, 105; mounting, 166 ; putting under micro- scope, 27 ; shading, 59 ; suitable for photo-micrography, 227 ; transparent with curved outlines, relative posi- tion in microscopic preparations, 94. Objective, 9-13, 121; achromatic, 11; adjustable, 11, 12, 54; adjustable, micrometry with, 118; adjustable, photo-micrography with, 234 ; adjust- ment for, 54 ; aerial image of, 30 ; anastigmat, 208 ; aperture of, 15-22, 270; aplanatic, n ; apochromatic, 12, 224; back combination of, 10, 11 ; carrier, 259 ; cleaning back lens of, 61 ; collar, graduated for adjustment, 56 ; cloudiness or dust, how to deter- mine, 92; designation of, 10; dry, 10, 17-19, 121 ; equivalent focus of, 10, 25, 29, 272 ; field of, 28-29 ; focus- ing for micro-spectroscope, 144 ; front combination of, 10, 11 ; function of, 29 31 ; glass for, 11-13, 71 ; high, focusing with, 38 ; homogeneous im- mersion, 17-19, 121 ; homogeneous immersion, cleaning, 59; homogene- ous immersion, experiments, 58 ; il- luminating, 13, 238 ; image, power of, 18 ; immersion, 11, 121 ; index of refraction of medium in front of, 17, 19 ; initial or independent magnifica- tion of, 273 ; inverted, real image of, 30; for laboratory microscope, 64; lettering, 10 ; light utilized with, 17 ; low, focusing with, 38 ; magnifica- tion of, 272 ; marking, by Krauss' method, 71 ; for micrometallography, 13, 159; micro-planar, 212, 260; to use with micro-polariscope, 151 ; mi- croscopic, 9 ; to use with micro- spectroscope, 144 ; for micro-spectro- scope, focusing, 144 ; nomenclature of, 10; non-achromatic, 11; non- adjustable, 12; non-adjustable, thick- ness of cover-glass for, table, 14 ; with nose piece, 38 ; numbering, 10 ; numerical aperture, 16-22, 270 ; oil 296 INDEX immersion, 11 ; panto-chromatic, 13; para-chromatic, 13 ; for photography, 20S, 212, 214, 224; for photo-microg- raph}', 224; projection, 13, 212, 214, 259 ; putting in position and remov- ing, 26 ; semi-apochromatic, 13; table of field, 29 ; terminology of, 10 ; un- adjustable, 12; variable, 13; visual and actinic foci of, in photo-microg- raphy, 226: water immersion, 17-19, 56 ; working distance of, 39-40. Oblique light, 36, 41 ; with Abbe illumi- nator, 48; with a mirror, 41, 50. Ocular, various forms, 22-25 ; cloudiness, how to determine and remove, 60, 92; equivalent focus of, 25, 29, 273 ; eye- point of, 32 ; field-lens, 32 ; filar or screw micrometer, 26, 117; focus, equivalent of, 25, 273 ; function of, 31-33 ; indicator, 67, 247 ; iris dia- phragm for, 157 ; lettering and num- bering, 26 ; micrometer, micrometry with, 1 1 4-1 21 ; parfocal, 24, 3S ; for photo-micrography, 224, 232, 235 ; pointer, 247 ; projection, 260 ; spec- troscopic. 134 ; standard size for, 26; table, effect on field, 29. Oil, and air, appearances and distinguish- ing optically, 95 ; removal, 61 ; re- moval from sections, 179. Oil-globules, with central and oblique illuminations, 95. Oil immersion objectives, 11. Opaque objects, lighting, 144, 238 ; pho- tography of, with microscope, 235- 240; projection of, 267. Opera glasses, 262. Optic axis, 2, ; of condenser or illumi- nator, 47 ; of microscope, 10. Optical, bench, 237 ; center, 2 ; focus, 12 ; parts of compound microscope, 9, 64; parts of microscope, care of, and test- ing, 60, 63 ; section, 98. Order of procedure in mounting ob- jects dry or in air, 167 ; in glycerin, 170; in glycerin jelly, 170: in resin- nous media by desiccation, 172 ; in resinous media by successive dis- placement, 172. Ordinary ray, with polarizer, 150. Orthoch'romatic plates, 217. Orthoscopic ocular, field with, 2S. Outline distinctness of, 96. Oven paraffin, 279. Over-correction, 5. Oxy-hemoglobin, spectrum of, 138, 147. Paper, bibulous, filter, lens, or Japanese for cleaning oculars and objectives, 60, 1 So. Paraffin, 1S5, 203 ; filtering, 1S5 ; infil- trating with, 185 ; dish for infiltrat- ing, 279; imbedding in, 185, 186; method, 1S3 ; oven, 279 ; pail for melting, 184; removal from lenses, 61 ; removing from sections, '1 88. Parfocal oculars, 24, 38. Parts, optical and mechanical of micro- scope, S, 64 ; testing, 63. Pedesis, 99 ; compared with currents, 99 ; to overcome, 100; with polarizing microscope, 100 ; proof of reality of, 100. Penetrating power, 21. Penetration of objective, 21. Permanent mounting, 167 ; preparations of isolated cells, 175. Permanganate of potash, absorption spec- trum of, 136, 146. Petri dish, photographing bacterial cul- tures in, 241. Petroleum light, 37, 229 ; as color screen. 220. Pharmacological products, examination of, 15S. Photo-engraving, drawing for, 273 ; let- tering for, 274. Photographic, camera, 207 ; negatives, labeling, 211, 214; objectives, 20S ; prints, 211. Photography, back-ground for, 209 ; of bacterial cultures, 241-242 ; color- correct, 217 ; of colored objects, 218 ; compared with photo-micrography, 222; of embryos, 211-214; focusing and exposure, 206, 213 ; indebtedness to photo-micrography, 220; of large transparent objects, 214-216; lighting for, 20S, 216; metallic objects, 235- 240 ; objectives for, 20S, 212, 214 ; of objects in alcohol or water, 206 ; opaque objects, 235-240 ; plates for, 217; stage for, 211 ; with vertical camera, 205-209, 225. Photo-micrograph, 220 ; determination of magnification for, 233 ; at 5-20 di- ameters, 212; 20-50 diameters, 230; 100-2500 diameters, 233 ; of metallic surfaces, 235-240 ; objects suitable for, 227 ; of opaque objects, 235-240 ; prints of. 211; plates for, 217 ; repro- ductions of, 232 ; with and without an ocular, 230-234. Photo-micrographic, camera, 222, 225, 236; outfit, 236-237; stand, 231. INDEX 297 Photo-micrography, 220-240 ; cover-glass correction, 234 ; apparatus for, 223 ; compared with ordinary photogra- phy, 222 ; condenser for, 42, 226, 227- distinguished from micro-photogra- phy, 220 ; experiments, 229 ; expos- ure for, 213, 220, 232, 235, 240 ; focus- ing for, 213, 216 ; focusing screen for, 209 ; lighting, 229, 230, 233, 239 ; mi- crometer for, 233 ; objectives and oc- ulars for, 13, 224, 232,235; vertical camera with, 211, 222, 225; actinic foci in, 226 ; with and without ocular, 230,232, 234 ; record table for, 219. Physiological histology, 196. Picric-alcohol, 203. Picro-fuchsin, 190. Pillar of microscope, Frontispiece. Pin-hole diaphragm, 47. Pippett, 179; egg, 280. Plane mirror, use of, 37. Plates, color-correct, 217; exposure of, 213. 220, 232, 235, 240 ; isochromatic, or orthochromatic, 217 ; size of, 224. Pleochroism, 152. Pleurosigma angulatum, 41. Point, axial, 15 ; burning, 7. Pointer ocular, 247. Polariscope, 140, 150. Polarized light, extraordinary and ordi- nary ray of, 150. Polarizer and analyzer, 140, 151. Polarizing microscope, pedesis with, 100. Position of objects or parts of same ob- ject, 92 ; marking p , 24S. Positive oculars, 10, 22. Power, of microscope, 103 ; illuminating, penetrating, resolving, of objective, 19-21 ; of ocular, 25. Preparation of Canada balsam, Farrant's solution, glycerin, glycerin jelly, etc., 19S-204. Preparation, of clearing mixture, liquid gelatin and shellac cement, 19S-204 ; of ground glass, 29 ; of metallic sur- faces, 239 ; vials, 174. Preparations, cataloging, 194-196 ; cabi- net for, 196-197 ; labeling, 194 ; for microprojection, 263 ; permanent, of isolated cells, 175. Price of American and foreign micro- scope, 71. Principal, focus, 3, 5 ; focal distances, 3, 30 ; optic axis, 2, 5. Prism of Abbe camera lucida, 124, 127; Amici, 140; comparison, 141; dis- persing, 141; erecting, 176; Nicol, 150; and slit of micro-spectroscope, mutual arrangement, 139; of Wollas- ton's camera lucida, 125. Prints, photographic, 211. Projection, apparatus, 249, 263-297 ; mi- croscope, 249-266 ; see micro-projec- tion ; objective, 13, 214, 259 ; ocular, ?4i 25, 232, 260 ; opaque objects, 267 ; in photo-micrography, 232, 234. Putting, on cover-glass, ' 167 ; an object under microscope, 27 ; an objective and ocular in position, 26, 27. Pyroxylin, 200. Q-R Quadrant for camera lucida, 127, 12S. Radiant, centering, 255. Ratio, ocular micrometer, 119. Razor and support, 276-277. Reagent, bottle, 179 ; for fixing, 19S; irri- gation with, 170; for mounting, 198. Real image, 5, 8, 9, 30; magnification, 103, 262. Record, of negatives, 214; table for photo- micrograph v, 219. Reflected light. "35. Reflection, total, 54. Refraction, 52 ; images, 52, 58 ; index of, 53 ; of medium in front of objective, 1 Si- Refractive, doubly, 152 ; highly, 97 ; ■ singly, 152. Relative position of objects, 92. Resinous media, mounting objects in, order of procedure, by drying or desiccation, 172; by a series of dis- placements, 172. Resolution and numerical aperature, 20. Resolving power, 20. Retinal image, 6, 9. Revolving nose-piece, marking objectives on, 71. Ribbon sections, 186-187 ; tray for, 1S7. Sagittal sections, 192. Salicylic acid, crystallization, 50. Salt solution, normal, 203. Scale, of drawing, 131 ; of sizes for pho- tographing, 206 ; of wave lengths, 142. Screen, color, 21S-219; focusing s. for photo-micrography , 209 ; of ground glass, 29; for micro-projection, 261 ; for microscope, 59. Screw, society, 64 ; micrometer, 26, 117. Sealing cover-glass, 169, 170. Searching ocular, 24. Secondary axis, 3. Section, lifter, 1S1-182 ; optical, 98. Sections, arrangement of tissue for, 191 ; clearing, 190; cutting, 178, 186 ; de- 298 INDEX hydration of, 190 ; extending with water, 186; fastening to slide, 179, 1S7 ; frontal, 192 ; mounting, 183, 190 ; removing benzine, oil and para- ffin from, 179, 188 ; ribbon, '1S6 ; sagittal, 192 ; serial, 191-193 ; stain- ing, 1S0, 189; transferring, 179; thickness for micro-projection, 263. Selenite plate for polariscope, 154. Semi-apochromatic objective, 13. Serial sections, 191-193; arranging and labeling, 192, 193 ; stage for, 67, 69 ; thickness of cover-glass for, 193. Shell vials, 174. Shellac cement, preparation of, 203 ; re- moval from lenses, 61. Significance of aperture, 20. Simple microscope, see under microscope. Sines, table of, 3d page of cover. Slides, 161 ; box for 198; cleaning, 161 ; holder for, 188-189 ; lantern, 241 ; for micro-chemistry, 161 ; tray for, 1S7. Sliding microtome, 27S. Slips, 161. Slit mechanism of micro-spectroscope, 135, 139- Society screw, 64. Sodium, lines and spectrum, 136-137. Solar spectrum or s. of sunlight, 136-137. Soluble cotton, 200. Solution, alum, 19S; Farrants', 201. Specimen cooler, 257. Spectral, colors, 138; ocular, 134, 139. Spectroscope, direct vision, 134, 145. Spectroscopic, examination of color- screens, 220 ; ocular, 134. Spectrum, 136-150; absorption, 137; amount of material necessary and its proper manipulation, 145 ; analysis, 150 ; Angstrom and Stokes' law of, 138 ; banded, not given by all colored objects, 14S ; of blood, 146 ; of carbon monoxide hemaglobin, 147 ; of car- mine solution, 148 ; of minerals, 149 ; of colorless bodies, 14S ; comparison, 142 ; complementary, 139 ; continu- ous, 137 ; double, 142 ; incandescence; 137 ; line, 137 ; met-hemaglobin, 147, monazitesand, 149 ; oxy-hemoglobin, 135, 147; permanganate of potash, 136, 146; single-banded of hemaglobin, 1 38, 147 ; sodium, 136, T37 ; solar, 136, 137 ; two-banded of oxy-hema- globin, 147. Spherical aberration, 4, 5 ; test for, 26S. Stage, Frontispiece, 65 ; mechanical, 65, 67-70, 2,59 ; micrometer, 105 ; for mi- cro-projection, 258 ; for photograph- ing, 211. Stain, alcoholic, 180, 189; aqueous, 180, 189 ; for micro-projection, 263. Staining, cells, 174 ; dish, 190 ; sections, 180, 1 89. Stand, of microscope, 65 ; photo-micro- graphic, 231 ; special for embryos, 212 ; special for large transparent ob- jects, 215, 216. Standard, distance (250 mm.) at which the virtual image is measured, 109 ; screw, 64 ; size for condenser, 47 ; size for oculars, 26. Stokes and Angstrom's law of absorption spectra, 138. Storing negatives, 211 ; preparations, 196. Substage, S6, Frontispiece. Substances for crystallography, 156. Sulphonal with polarizer, 154. Sulphuric ether, 2or. Support for knife of microtome, 276-277. Swaying of image 48. Synthol, 19S. System, back, front, intermediate of lenses, 10, 11 ; crystal, 156; metric, cover 1st p., 133. Table, for immersion fluid, 272 ; of mag- nification and valuation of ocular mi- crometer, in ; magnification with projection microscope, 262 ; of tube- length and thickness of cover-glasses, 14 ; natural sines, third page of cover; of numerical aperture, 19 ; record, for photo-micrography, 219 ; size of fields, 29 ; testing homogeneous liq- uids, 272 ; of valuations of ocular micrometer, in ; weights and meas- ures, 2d page of cover. Temporary mounting, 166. Terminology of objectives, 10. Test of chromatic and spherical aberra- tion, 26S-270. Tester, cover-glass, 164-165 ; for homo- geneous liquids, 58, 271. Testing a camera, 223 ; a microscope and its parts, 63. Test-plate, Abbe's, method of using, 26Ss Textile fibers, examination of, 101, 158. Thickness, of cover-glass for non-adjust- able objectives, table, 14 ; of serial sections, 193. Tissues, arranging for sections, 191 ; fix- ing or hardening, 176, 1S3 ; washing apparatus for, 280. Tolles-Mavall mechanical stage, 67. Transections, 192. Transferring sections, 179. Transmitted light, 36. INDEX 299 Tray for slides, 1S7. Triplet, aplanatic, 7. Tripod, 7, 104 ; base for microscope, S6 ; as focusing glass, 209. Tube of microscope, Frontispiece. Tube-length, 13, 14,56, for cover-glass adjustment, 56, 57 ; importance of, 56 ; microscopical, 13, 14 ; of various opticians, table, 14 ; and optical com- binations, 11S. Turn-table, 16S. U— V— W— X Unadjustable objectives, 12. Under-correction, 5. Unit of measures, in micrometry, 112 ; of wave length, 143. Valuation of ocular micrometer, 114-116; table, 1 1 1. Variable objective, 13. Varying magnification of compound mi- croscope, 109. Varying ocular micrometer valuation, 118. Velocity under microscope, 98. Vertical, camera, 205-210, 225 : illumina- tor, 13, 23S-239. Vials, preparation, 174 ; blocks for, 174. Virtual image, 5, 6, 9, 32 ; standard dis- tance at which measured, 109. Visibility with objectives, 20. Vision, double, 103, 105 ; microscopic, 21. Washing apparatus for tissues, 2S0. Waste bowl, 181. Water immersion objective, 16-19, 5^ I light utilized, 17 ; numerical aper- ture, 19 Water, bath, 235, 255 ; for immersion ob- jectives and removal of, 56, 58. Wave length, designation of, 143 ; scale of, 142. Wax models, 274. Weights and measures, see 2d page of cover. Welsbach light, 37, 229. Wollaston's camera lucida, 107, 125. Work-room for photo-micrography, 224. Work-table, position, etc., 62. Working distance of microscope or ob- jective, 11, 34, 39-40- Writing diamond, 196. Xylene, 17S, 200 ; balsam, 199 ; for re- moving oil, 179 ; removing from slides, 1S9. Xylol, German form of xylene, 17S, 200. TABLE OF NATURAL SINES Compiled from Prof. G. W. Jones' Logarithmic Tables 1 Minutes. Degrees and Quarter Degrees up to 90°. i' 0.00029 l0 > 0.01745 16 , 0.27564,31°, 0.51504:46°, 0.71934 6i°, 0.87462 76°, 0.97030 2 0.00058 i°,i5 '0.02181 i6°,i5 '0.27983 31°, 15 '0.51877 46°, 15 '0.72236 6i°,i5 '0.87673 76°, 15 '0.97134 3 0.00087 1,30 0.0261S 16,30 0.28402 31,30 0.52250 46,30 0.72537 61,30 0.87882 76,30 0.97237 4 0. 001 16 1,45 0.03054 16,45 0.28820 31,45 0.52621 46,45 0.72837 6i,45 0.88089 76,45 o.97338 5 0.00145 2 0.03490 17 0.29237 32 0.52992 47 0.73*35 62 0.88295 77 0-97437 6 0.00175 2 > I 5 0.03926117,15 0.2965432,15 0.53361 47,15 0.7343262,15 0.88499 77,15 o.97534 7 0.00204 2,30 0.04362117,30 0.30071 32,30 o.5373o 47,30 o.73728'62,30 0.88701 77,30 0.97630 8 0.00233 2,45 0.04798 17,45 0.3048632,45 0.54097 47,45 0.74022 62,45 0.88902 77,45 0.97723 9 0.00262 3 0.0523418 0.3090233 0.54464 48 0.74314 63 0.89101 78 0.97815 10 0.00291 3,15 0.05669 18, 15 0-3131633,15 0.54829 48,15 0. 74606 63,15 0.89298 78,15 0.97905 11 0.00320 3,30 0.06105 18,30 0.31730 33,30 0.55194 48,30 0.74896 63,30 0.89493 78,30 o.97992 12 0.00349' 3,45 0.06540 18,45 o-32i44!33,45 0-55557 48,45 0.75184 63,45 0.89687 78,45 0.98079 13 0.00378 4 0.0697619 0.32557 34 o.559 J 9 49 0-75471 64 0.89879 79 0.98163 14 0.00407 4,15 0.0741 1 19,15 0.32969 34,15 0.562S0 49,15 0.75756 64,15 0.90070 79,15 0.9S245 15 0.00436 4,30 0.07846 19,30 o.3338i 34,30 0.56641 49,3o 0.76041 64,30 0.90259 79,3o 0.98325 16 0.00465 4,45 0.08281 19,45 o.33792 34,45 0.57000 49,45 0.7632364,45 0.90446 79,45 0. 98404 17 0.00495 5 0.0S716 20 0.34202 35 o.57358 50 0.7660465 0.90631 80 0.98481 iS 0.00524 5,15 0.09150 20,15 0.34612 35,15 o.577i5 50,15 0.7688465,15 0.90814 80,15 0.98556 19 0.00553 5.3° 0.0958520,30 0.35021 35,3° 0.58070 50,30 0.77162 65,30 0.90996 80,30 0.98629 20 0.00582 5,45 0.10019 20,45 0.35429 35,45 0.58425 5o,45 o.77439 65,45 0.91176 8o,45 0.98700 21 0.00611 6 0.10453 21 0.35S3736 0.5S77951 0.77715 66 0.9I355 81 0.9S769 22 0.00640 6,15 0.10887 21,15 0.36244136,15 0.59i3ii5i,i5 0.779S8 66,15 0.9153 1 81,15 0.98836 23 0.00669 6,30 0.11320:21,30 0.3665036,30 0.59482,51,30 0.78261 66,30 0.91706 81,30 0.98902 24 0.0069S 6,45 0.1175421,45 0.3705636,45 0.5983251,45 0.78532 66,45 0.91S79 8i,45 0.98965 25 0.00727 7 0.12187 22 o.3746i 37 0.60182.52 0.78801 67 0.92050 82 0.99027 26 0.00756 7,15 O. I262o'22,I5 0.3786537,15 0.6052952,15 0.79069 67,15 0.92220 82,15 0.99087 27 0.00785 7,30 0.1305322,30 0.3826837,30 0.60876 52,30 0-79335 67,30 0.92388^2,30 0.99144 28 0.00814 7,45 O.I3485!22,45 o.3867i'37,45 0.61222,52,45 0.79600 67,45 0.9255482,45 0.99200 29 0.00S44 S 0-I39I7P3 0.39073 38 0.61566 53 0.79864 68 0.92718 83 0.99255 30 0.00873 8,15 0.14349123,15 o.39474!38,i5 0.61909 53,15 0.80125 68,15 0.92881 83,15 0.99307 31 0.00902 8.30 O.I4781 23,30 o.39 8 753 s ,30 62251 53,30 0. 80386 68,30 0.93042 83,30 0-99357 32 0.00931 3,45 0.1521223,45 0.4027538,45 0.62592 53,45 0. 80644 68,45 0.93201 83,45 0.99406 33 0.00960 9 O.I564324 o.4o674'39 0.62932 54 0. 80902 69 0.9335S 84 0.99452 34 0.00989 9,15 O.16074 24, 15 0.4107239,15 0.6327154,15 0.S1157 69,15 0-9351484,15 0.99497 35 0.01018 9,3° 0.16505124,30 0.41469.39,30 0.6360854,30 0.81412 69,30 0.93667184,30 o.9954o 36 0.01047 9,45 0.1693524,45 0.4186639,45 0.63944 54,45 0.81664 69,45 0.93819:84,45 0.99580 37 0.01076 10 O.I7365 25 0.42262 40 0.64279 55 0.81915 70 0.9396985 0.99619 38 0.01105 10,15 O.I7794|25,I5 o.42657 ! 40,i5 0.6461255,15 0.82165 70,15 o.94ii8;85,i5 0.99657 39 0.01134 10,30 O.I8224 25,30 0.43051,40,30 0.6494555,30 0.82413 70,30 0. 94264 85,30 0.99692 40 0.0T164 io,45 0.18652 25,45 o.43445!4°,45 0.6527655,45 0.82659 70,45 0.94409 85,45 0.99725 41 0.01193 11 0. i9o8i'26 0.4383741 0.6560656 0.82904 71 o.94552 86 0.99756 42 0.01222 11,15 0.1950926,15 0.4422941,15 o.65935 56,i5 0.S3147 7i,i5 0.94693 86,15 0.99786 43 0.01251 11,30 0.19937:26,30 0.44620 41,30 0.66262 56,30 0.83389 71,30 0.94832 86,30 0.99813 44 0.01280 ii,45 0.20364:26,45 0.45010 41,45 o.6658S; 5 6,45 0.83629 71,45 0.9497086,45 0.99839 45 0.01309 12 0.20791 27 0.45399,42 0.66913 57 0.83867 72 0.9510687 0.99863 46 0.01338 12,15 0.21218 27,15 0.45787142,15 0.67237 57,15 0.84104 72,15 0.95240 87,15 0.99885 47 0.01367 12,30 0.21644 27,30 0.4617542,30 0.67559 57,30 0.84339 72,30 o.95372 87,30 0.99905 48 0.01396 12,45 0.22070)27,45 0.4656142,45 0.67880 57,45 0.84573 72,45 0.95502 87,45 0.99923 49 0.01425 13 0.2249528 0.46947 43 0.68200 58 0,84805 73 0.95630 88 o- 99939 50 0.01454 I3, J 5 0.22920 28,15 0.47332 43,15 0.68518 58,15 0.85035 73, 1 5 0-95757 88,15 o. 99953 51 0.01483 13,30 0.2334528,30 0.47716143,30 0.68835 58,30 0.85264 73,30 0.95882 88,30 0.99966 52 0.01513 13,45 0.2376928,45 0.4809943,45 0.69151 58,45 0.85491 73,45 0.96005 88,45 0.99976 53 0.01542 14 0.24192 29 0.48481 44 0.69466 59 0.85717 74 0.96126 89 0.99985 54 0.01571 14,15 o.246i5 l 29,i5 o.48862!44,i5 0.69779 59, J 5 0.85941 74,15 0.96246 89,15 0.99991 55 0.01600 14,30 0.2503829,30 0.4924244,30 0.70091 59,3° 0.86163 74,30 0.96363 89,30 0.99996 56 O.Ol62g 14,45 0.25460 29,45 0.49622144,45 0.70401 59,45 0.86384 74,45 0.96479 89,45 0.99999 57 0.01658 15 0.25882 30 0.5000045 0.70711 60 0.86603 75 0.96593 90 1. 00000 58 0.01687 15,15 0.26303 30,15 0.50377,45,15 0.71019 60,15 0.86820 75,15 0.96705 59 0.01716 15,30 0.26724 30,30 o.50754i45,30 0.71325 60,30 0.87036 75,30 0.96815 . . 60 0.0174c 15,45 0,27144 30,45 0.5112945,45 0.71630 60,45 0.87250 75,45 0.96923 IHffilifl ' II ill i!h It 111 i II i 1 ill li 1 1 ii