TABLE OF METRIC AND ENGLISH MEASURES. The measures of length, volume and weight most frequently used in micro- scopical and histological work are the following : LENGTH, THE METER = THE UNIT. METER = 100 centimeters, 1000 millimeters, 1,000,000 microns, 39.3704 inches. Centimeter (cm.) = 10 millimeters, meter. Millimeter (mm.) = 1000 microns, millimeter, yyVffth meter, 2]5th inch, ap- proximately. Micron (u) (Unit of Measure in Micrometry) mm. Ttrcro'ireoth meter, (0.000039 inch) sjVoo inch, approximately. Inch (in.) = 25.399772 mm. (25.4 mm., approx.) CAPACITY, THE LITER = THE UNIT. Liter = 1000 milliliters or 1000 cubic centimeters, i quart (approx.) Cubic centimeter (cc. or cctm.) = T-crootb of a liter. Fluid o Gram Kilogri Ounce Ounce ‘ >prox. To cha the equiv fo chs duce 50° . Farenhei = — 40° to find i, to rf- jr — 40 72° x | Addre supplies named tube-ler icopical pticians table of The Bausch & Tomb Optical Co., Rochester, N. Y. The Franklin Supply Co., 6 Hamilton Place, Boston, Mass. J. Grunow, 70 West 39th St., New York. The Gundlach Optical Co., Rochester, N. Y. The McIntosh Battery and Optical Co., . . . 141, 143 Wabash Aye., Chicago, 111. Queen & Co., 1010 Chestnut St., Philadelphia, Pa. The Spencer and Smith Optical Co., 367-373 Seventh St., Buffalo, N. Y. W. H. Walmsley (with Geneva Opt. Co.), . . 67, 69 Washington St., Chicago, 111. Williams, Brown & Earl, iotli and Chestnut Sts., Philadelphia, Pa. G. S. Woolmau, 116 Fulton St., New York. J. Zentmayer 209 South nth St.,. Philadelphia, Pa. THE MICROSCOPE AND MICROSCOPICAL METHODS, BY SIMON HENRY GAGE, Associate Professor of Anatomy, Histology aflS Embryology in Cornell University, Ithaca, N. Y., U. S. A. FIFTH EDITION, REWRITTEN, GREATLY ENLARGED, AND ILLUSTRATED BY 103 FIGURES IN THE TEXT. PART I OF THE MICROSCOPE AND HISTOLOGY. ITHACA, N. Y. Comstock Publishing Co. 1894. Copyright, 1894, By Simon Henry Gage, All Rights Reserved. Printed by Andrus & Church, Ithaca, N. Y. PREFACE. THIS edition has been enlarged nearly one-half by the elaboration of the mat- ter in the previous edition, and by the addition of a wholly new chapter on photo-micrography and on photographing natural history objects in a hori- zontal position with a vertical camera. The figures have been distributed in the text, and many new ones added. It is hoped that the book as it now appears may, while remaining strictly ele- mentary, still more fully meet the needs of those who wish to use the microscope for serious study and investigation. The aim has been to produce a book for be- ginners in microscopy, such as the author himself felt sorely the need of when he began the study. This purpose has been strengthened and furthered by noting the difficulties of the various classes that have used the work and aided in its evolution during the last fifteen years. The author wishes to acknowledge the aid rendered by the various Optical Com- panies for information freely given, and for the loan of cuts and instruments (Bausch & Tomb Optical Co., Gundlach Optical Co., Queen & Co., and all the op- ticians mentioned in the table of tube-length, p. io). I feel under special obliga- tion to my various classes for the enthusiasm and earnestness with which they have followed the instructions in the book, to my colleagues, Professor Wilder and Instructors Hopkins and Fish for suggestions, to Mrs. Gage for criticising the man- uscript, reading proof, preparing the index and the original figures, to Dr. A. C. Mercer for aid in preparing the chapter on photo-micrography, to Dr. M. D. Ewell for information and for the loan of apparatus, and finally, to many other friends who have used the previous editions, and have made suggestions whereby it is hoped the present edition is greatly improved. I would like to repeat a part of the preface to the third and to the fourth editions, and to call especial attention to the address of the Hon. J. D. Cox at the recent meeting of the American Microscopical Society : “A plea for systematic instruc- tion in the technique of the microscope at the university,” in the Proceedings for 1893- Extract from the preface of the fourth edition : “The author would feel grateful to any person who uses this book, if he would point out any errors of statement that may be discovered, and also suggest modifi- cations which would tend to increase the intelligibility, especially to beginners.” From the third edition : “It is thoroughly believed by the writer that 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 knowl- edge shall be made alive, it must be made a part of the student’s experience by ac- tual experiments carried out by the student himself. Consequently, exercises illus- trating 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 PREFACE. 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 this retinal image, whether it is made with or without the aid of a microscope, must always depend upon the character and training of the seeing and appreciating brain behind the eye. The microscope simply aids the eye in furnishing raw material, so to speak, for the brain to work upon. “The necessity for doing a vast deal of drudgery or ‘dead work,’ as it has been happily styled by Professor Leslie, before one has the training necessary for the appreciation and the production of original results, has been well stated by Beale : “ ‘The number of original observers emanating from our schools will vary as practical work is favored or discouraged. It is certain that they who are most fully conversant with elementary details and most clever at demonstration, will be most successful in the consideration of the higher and more abstruse problems, and will feel a real love for their work which no mere superficial inquirer will experience. It is only by being thoroughly grounded in first principles, and well practised in mechanical operations, that any one can hope to achieve real success in the higher branches of scientific enquiry, or to detect the fallacy of certain so-called experi- ments.’ ” In Part II, which is in preparation, will be given the application of the micro- scope to study and investigation in Vertebrate Histology. SIMON HENRY GAGE, Cornell University, Ithaca, New York, U. S. A. February 12, 1894. CONTENTS. CHAPTER L l 1-45. The Microscope and its Parts—Demonstration of the Function of each Part, 1-27 PAGE. \ 46-104. Lighting and Focusing, Manipulation of Dry, Adjustable and Immersion Objectives ; Care of the Microscope and of the Eyes, 28-51 CHAPTER II. § 105-127. Interpretation of the Appearances under the Microscope, . . 52-62 CHAPTER III. CHAPTER IV. \ 128-155. Magnification of the Microscope ; Micrometry, 63-77 CHAPTER V. § 156-166. Drawing with the Microscope, 78-87 $ 167-204. Micro-spectroscope and Micro-polariscope—Use and applica- tion, , 88-104 CHAPTER VI. CHAPTER VII. | 205-256. Slides and Cover-Glasses, Mounting, Labeling and Storing Microscopical Preparations—Experiments in Micro-chem- istry, X05-129 \ 257-280. Photo-Micrography, and the Photography of Natural History Specimens with a Vertical Camera, 130-150 Bibliography, 151-156 Index, 157-163 CHAPTER VIII. LIST OF ILLUSTRATIONS. All of the Figures, except when otherwise indicated, were drawn by Mrs. Gage. FIG. PAGE 1. Convex lens showing the formation of a real image 2 2. Convex lens showing the formation of a virtual image 2 3. Principle of the simple microscope, and the eye of the observer forming an integral part of it' 3 4. Tripod magnifier 3 5. Achromatic triplet for the pocket (Bausch & bomb Optical Co.) 4 6. Tens holder with Steinheil magnifier (Leitz) 4 7. Dissecting microscope (Bausch & Lomb Opt. Co.) 5 8. Principle of the compound microscope 6 9. Sectional view of a concave lens with its principal focus 7 10. Sectional view of a convex lens with its principal focus 7 11. Sectional view of a dry objective, showing working distance and lighting by reflected light • 7 12. Sectional view of an immersion, adjustable objective, and transmitted, ax- ial and oblique light 8 13. Figure showing the parts of the microscope included in tube-length by the various opticians 10 14. Diagram showing angular aperture u 15. Abbe’s apertometer (from Zeiss’ catalog) n 16. 17, 18. Figures showing the relative number of rays entering the microscope through a dry, a water immersion and a homogeneous immersion objective. (Modified from Ellenberger) 13 19. Sectional view of a Huygenian ocular showing eye-point 17 20. Stand of a compound microscope with names of parts 21 21. Triple nose-piece or revolver (Queen & Co.) 22 22. Specimens with ring to include parts to be observed 22 23. Figure of the actual size of the field with various objectives 23 24. Principle of the simple microscope (Fig. 3 repeated) 26 25-26. Drj' and immersion objectives (Fig. 11, 12 repeated) 29 27-30. Sectional views of the Abbe illuminator, showing different methods of illumination 37 31-32. Relative aperture of illuminator and objective (Nelson) 39 33. Acme microscope lamp (Queen &Co.) 40 34. Lamp and bull’s eye for artificial light 41 35-36. Centering the sub-stage illuminator 41 37-38. Centering the source of illumination 41 39-41. Diagrams illustrating refraction. (Slightly modified from Carpenter- Dallinger) 42 42. Effect of cover-glass (Ross) 43 43. Screen for shading the microscope and observer 46 44. Double eye-shade 49 45. Ward’s eye-shade (Bausch & Lomb Optical Co.) 49 46. Laboratory microscope (Bausch & Lomb Optical Co.) 51 LIST OF ILL USTRA TIONS. 47- Letters in stairs to show order of coming into focus 55 48. Putting on the cover-glass 55 49. Oil globule and air bubble 56 50. Sectional view of an air bubble and an oil globule in water to show the virtual focus in the air and the real focus in the oil 56 51. Solid glass rod in air and in water 58 52. Solid rod coated with collodion to show double contour 59 53. Stage micrometer with lines enclosed by a ring to facilitate finding them (Fig. 22 repeated) 65 54. Wollaston’s camera lucida 65 55. Position of the microscope for determining magnification 67 56. Mutual arrangement of stage and ocular micrometer 75 57- Abbe camera lucida with 450 mirror 79 58- Huygeniau ocular with eye-point 79 60-63. Abbe camera lucida with 350 mirror 82 64. Drawing board for inclined microscope • 84 65. Abbe camera lucida (Bauscli & Lomb Optical Co.) 85 66. Spectra : Solar, sodium, permanganate and met-hemaglobiu 89 67. Spectra of oxy-hemoglobiu and hemoglobin (Gamgee and Macmunn) . . 91 68-69. The micro-spectroscope 92 70. The micro-polarizer • 92 71. Micrometer calipers (Brown & Sharpe, cut from Wm. Wood & Co.) . . . 108 72. Bausch’s cover-glass measurer (Bausch & Lomb Opt. Co.) 109 73. Zeiss’cover-glass measurer (cut furnished by Wm. Wood & Co.) .... 109 74. Method of applying the cover-glass (Fig. 48 repeated) hi 75. Needle holder (Queen & Co.) 111 76. Turn table for sealing preparations (Queen & Co.) 112 77. Centering card IJ4 78. Anchoring the cover-glass in glycerin mounting .... 115 79. Method of irrigation ri5 80. Moist chamber ri5 81. Waste bowl (P. A. P'ish, cut furnished by Wm. Wood & Co.) 118 82. Balsam bottle • Ix9 83. Arrangement of serial sections 121 84. Writing diamond (Queen & Co.) 124 85-86. Cabinet for preparations 125 87. Walmsley’s plioto-micrographic camera (from Mr. Walmsley) 132 88. Leitz’ vertical photo-micrographic camera (Leitz) 134 89. Ground glass focusing screen, with clear center 139 90. Perigraphic objective (Gundlacli Optical Co.) 139 91. Zeiss-Anastigmat photographic objective (Bausch & Lomb Optical Co.). . 140 92. Focusing glass (Gundlach Optical Co.) • 141 93. Tripod magnifier as focusing glass (Fig. 4 repeated) 141 94. Engraving glass for bull’s eye condenser (Bausch & Lomb Optical Co.) . 142 95. Lamp for artificial illumination (Fig. 34 repeated) 143 96-99. Centering the condenser and the image of the illuminaut 144 100. Rack for drying negatives (Rochester Optical Co.) 145 101-102. Plioto-micrographs of the Newt’s head and brain 146 103. Vertical camera for photographing natural history specimens in a hori- zontal position 148 THE MICROSCOPE AND HISTOLOGY. CHAPTER I. THE MICROSCOPE AND ITS PARTS. APPARATUS AND MATERIAL FOR THIS CHAPTER. A simple microscope (§ 2, 4); A compound microscope with nose-piece (Fig. 46), eye-shade (Fig. 44, 45), achromatic (§ 12), apochromatic (§ 14), dry (g 9), immersion (§ 10), unadjustable and adjustable objectives ($ 15, 16), Huygenian or negative (§ 23» 25), positive (§ 24) and compensation oculars ($ 26), stage micrometer, homogeneous immersion liquid (§ 10, 88-93), benzine and distilled water (§ 87, 93). Mounted letters or figures ($ 39) ; Ground-glass and lens paper (§ 39) ; Aper- tometer (g 18); Tester for homogeneous immersion liquid ($ 89). A MICROSCOPE. | I. A Microscope is au 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 greatly 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 au integral part of the optical combination (Fig. 3, 8). § 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 (Fig. 3, 5), and the object must be placed within the principal focus ($ 4). The simple microscope may be held in the hand or it may be mounted in some way to facilitate its use (Fig. 6, 7). MICROSCOPE AND ACCESSORIES. Fig. i and 2.—i. Convex lens show- ing the position of the object (A-B) outside the principal focus (F), 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, i, 2, 3. Rays after traversing the lens. They are converging, and consequently form a real image. The dotted lines and the line 2 give the direction of the rays unaffected by the lens. F. The princi- pal focus. Fig. 2. Convex lens showing the po- sition 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' B' virtual image formed by tracing the rays backward. It appears on the same side of the lens as the object, and is erect (§ f). Axis. The optic axis of the lens. F. The principal focus. 1, 2, 3. Rays from the point B of the object. They are diverging after travers- ing the lens, but not so divergent as if no lens were present, as is shown by the dotted lines. Ray / traverses the center of the lens, and is therefore not deviated. g 3. A Compound Microscope.—This enables one to see an enlarged, inverted image. It always consists of two optical parts, —an objective, to produce an en- larged, inverted, real image of the object, and an ocular acting in general like a simple microscope to magnify this real image (Fig. 8). 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 (Fig. 20). SIMPLE MICROSCOPE : EXPERIMENTS. § 4- Employ a tripod or other simple microscope, and for object a printed page. Hold the eye about two centimeters from the upper sur- face 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 may be seen, is called focusing, see § 46). When a clear image is seen, note that the letters appear as with the unaided eye except that they are larger, and the letters 3 MICROSCOPE AND ACCESSORIES. appear erect or right side up, instead of being inverted, as with the compound microscope (§ 3, 39). Hold the simple microscope directly toward the sun and move it a- Fig. 3. 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. A1 B\ The object within the principal focus. A3 B3. 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. B2 A2. Retinal image of the object (A' B1). The virtual image is simply a projection of the retinal image in the field of vision. Axis. The principal optic axis of the microscope and of the eyeCr. Cornea of the eye. L. Crystal- line lens of the eye. R. Ideal refracting surface at which all the refractions of the eye may be assumed to take place. way from and toward a piece of printed pa- per 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. 9, 10). 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 cen- timeter 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 § 39. Fig. 4. Tripod Magnifier. After getting as clear an image as possible with a simple microscope, 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 position, the largest field may be seen. This position corresponds to the eye point (Fig. 19) of an ocular, and is the point at which the largest number of 4 MICROSCOPE AND ACCESSORIES. rays from the microscope enter the eye. Note that the image appears on the same side of the magnifier as the object (§ 39). Simple microscopes are very convenient when only a small magnifica- tion (Ch. IV) is desired, as for dissecting. Achro- matic triplets are excel- lent and convenient for Fig. 5. Achromatic Triplet for the pocket. As shown in the figure it is com- posed of three lenses, one of crown and two of flint glass. The whole is protected by a metal covering when not in use. (Bauscli & Lomb.) the pocket (Fig. 5). For use in conjunction with a compound micro- scope, the tripod magnifier (Fig. 4) is one of the best forms. For many purposes a special mechanical mounting like that of Fig. 6, 7 is to be preferred. Fig. 6. Adjustable lens holder with universal joint. This is especially useful for gross dissections and for supporting a bull's eye or lens-combination when lamp-light is used. Compare Fig. 34. {Leitz). 5 MICROSCOPE AND ACCESSORIES. Fig. 7. Simple Microscope with special mechanical mounting to hold and focus the magnifier and to support and light the object; used especially for dissection. (Bausch and Lomb.) COMPOUND MICROSCOPE £ 5. The Mechanical Parts of a laboratory, compound microscope are shown in Fig. 20, and are described in the explanation of that figure. The student should study the figure with a microscope before him atid become thoroughly familiar with the names of all the parts. MECHANICAL PARTS. OPTICAL PARTS- \ 6. 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 (Fig. i, 8). And as for the formation of real images generally, the ob- ject must be placed outside the principal focus, instead of within it, as for the simple microscope. (See 4, 39, Fig. 3, 8). Modern microscopic objectives usually consist of two or more systems or com- binations of lenses, the one next the object being called the front combination or lens, the one farthest from the object and nearest the ocular, the back combina- 6 MICROSCOPE AND ACCESSORIES. Fig. 8. Diagram showing the prin- ciple 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 ocular and into the eye to the retinal image (A2B2), and the projection of the retinal image into the field of vision as the virtual image (B3A*). A B. The object. A2B2. The retinal image of the inverted real image, B'A', formed by the objective. B'A*. The in- verted virtual image, a projection of the retinal image. Axis. The optic axis of the micro- scope and the eye. Cr. Cornea of the eye. L. Crystal- line lens of the eye. R. Single, ideal, refracting surface at which all the re- fractions of the eye may be supposed to take place. F.F. The principal focus of the posi- tive ocular and of the objective. ? Mirror. The mirror reflecting par- allel 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. j). tion or system. There may be also one or more intermediate systems. Each combination is, in general, composed of a convex and a concave lens. The combined action of the systems serves to produce an image free from color and from spherical distortion. In the ordinary achromatic objectives the con- vex lenses are of crown and the concave lenses of flint glass (Fig. n, 12). NOMENCLATURE OR TERMINOLOGY OF OBJECTIVES § 7. Equivalent Focus.—In America, England, and sometimes also on the Con- tinent, 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 prin- 7 MICROSCOPE AND ACCESSORIES. Fig. 9, io. Sectional views of a concave or diverging and a convex or converging lens io show that in the concave 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 lens opposite to that from which the light comes. The straight line traversing the lenses from left to right is a section of the principal plane, at which the total re- fractions of both curved surfaces are most conveniently shown. The optical center of each lens is at the intersection of the principal plane arid the axis, and the prin- cipal focal distance is the distance along the axis, from the optical center to the principal focus (F). cipal focal distance is inch or 2 millimeters (Fig. 10). An objective marked 3 in. or 75 mm., produces approximately the same sized real image as a simple converg- ing lens of 3 inches or 75 millimeters focal length. And in accordance with the law that the relative size of object and image vary directly as their distance from the center of the lens (Fig. 1, 2), 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 size of the real image. $ 8. 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 arbitrary and does not, like the one above, give direct information concerning the objective. $ 9. Dry Objectives.—These are objectives in which the space between the front of the objective and the object or cover-glass is filled with air (Fig. 11). Most ob- jectives of low and medium power (*. e., in. or 3 mm. and lower powers) are dry. Fig. ii. Section of a dry objective showing working distance and lighting by reflected light. Axis. The optic axis of the objective. B C. Back Combination, composed of a plano-concave of flint glass (F), and a double convex of crown glass (c). F C. Front Combination. C O si. The cover-glass, object and slide. Mirror. The mirror 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. IV. The Working Distance, that is the distance from the front of the objective to the object when the objective is in focus (g 4). 8 MICROSCOPE AND ACCESSORIES. I 10. 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 immeision 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 liomogeneons with the front glass of the objective. It may consist of thickened cedar-wood oil or of glycerin containing some salt, as stannous chloride, 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. 42) is almost wholly eliminated by the use of homogeneous immersion objectives. Fig. 12. Sectional view of an Immersion Ad- justable Objective, and the object lighted with axial or central and with oblique light. Axis. The optic axis of the objective. B C, M C, F C. The back, middle and front combination of the objective. In this case the front is not a combination, but a single plano-con- vex lens. A B. Parallel rays reflected by the mirror axi- ally or centrally upon the object. C. Ray reflected to the object obliquely. I. Immersion fluid between the front of the ob- jective and the cover-glass or object (O). Mirror. The mirror of the microscope. O. Object. It is represented without a cover- C glass. Ordinarily objects are covered whether ex- amined with immersion or with dry objectives. Stage. Section of the stage of the microscope. I ii. 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. $ 12. Achromatic Objectives.—In these the chromatic and the spherical aber- ration are both largely eliminated by combining concave and convex lenses of different kinds of glass “so disposed that their opposite aberrations shall correct each other.” All the better forms of objectives are achromatic and also aplanatic (§ 13). That is the various spectral colors come to the same focus. I 13. Aplanatic Objectives, etc.—These are objectives or other pieces of optical apparatus (oculars, illuminators, etc.), in which the spherical distortion is 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 appa- ratus are usually achromatic also. $ 14. 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 fluoride, 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 objectives. 9 MICROSCOPE AND ACCESSORIES. The special characteristics of these objectives, when used with the “ compen- sating oculars ” (§ 26), are as follows : (1) Three rays of different color are brought to one focus, leaving a small ter- tiary spectrum only, 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 two different colors in 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 rays 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 ozvr-corrected for the blue rays. (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. 1886, p. 300), dry apochromatic objectives give as clear images as the same, power water immersion objectives of the old form. \ 15. Non-Adjustable or Unadjustable Objectives.—Objectives in which the lenses or lens systems are permanently fixed in their mounting so that their rela- tive position always remains the same. Tow power objectives and those with homogeneous immersion are mostly non-adjustable. For beginners and those un- skilled in manipulating adjustable (§ 16) objectives, 11011-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 § 17 and table and figure of tube length and thickness of cover-glass below.) § 16. 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 by 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 $ 17. 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. 12. See also practical work with adjustable objectives). $ 17. 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), ex- cept with homogeneous immersion objectives, has a marked effect on the light passing from the object (Fig. 42). To compensate for this the relative positions of the systems-composing the objective are different from what they would be if the 10 MICROSCOPE AND ACCESSORIES. object were uncovered. Consequently, in non-adjustable objectives some standard thickness 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. Length in Millimeters and Parts included in “ Tube-Length ” by Various Opticians.* Pts. included in “ Tube- length.” See Diagram. “ Tube.length ” in Millimeters. f Grunow, New York 203 mm. I E. Leitz, Wetzlar 160 to 170 mm. | Nacliet et Fils, Paris 146 or 200 mm. a-d -j Powell and Lealand, London 254 mm. I C. Reichert. Vienna 160 to 180 mm. | Spencer and Smith, Buffalo 235 or 160 mm. [_ W. Wales, New York 254 mm. 'Bausch & Bomb Opt. Co. Rochester. . 216 or 160 mm. B£zu, Hausser et Cie, Paris 220 mm. , , Klonne und Muller, Berlin 160-180 or 254 mm. W. & H. Seibert, Wetzlar 190 mm. Swift & Son, London 165 to mm. C. Zeiss, Jena 160 or 250 mm. a-g . Gundlach Optical Co., Rochester . . . 254 mm. a-g . R. Winkel, Gottingen 220 mm. c-d . Ross & Co., London 254 mm. c-e . R. & J. Beck, London, 254 mm. c.f . J. Green, Brooklyn ... .... 254 mm. Fig. 13. Thickness of Cover-Glass for Which Non-Adjustable Objectives are Corrected by Various Opticians. J. Green, Brooklyn. J. Grunow, New York. Powell and Lealand, London. Spencer & Smith, Buffalo. W. Wales, New York. Klonne und Muller, Berlin. E. Leitz, Wetzlar (when tube 160-170 mm.). R. Winkel, Gottingen, Germany. Ross & Co., London. 1 Bausch & Lomb Optical Co., Rochester. C. Zeiss, Jena for apochromatic oil immersions). C. Reichert, Vienna. Gundlach Optical Co., Rochester. W. & H. Siebert, Wetzlar. R. &J. Beck, London. J. Zentmayer, Philadelphia. Nachet et Fils, Paris. Bdzu, Hausser et Cie, Paris. Swift and Son, London. nnymm. Ttnymm. iV—° = 0.5263. And by consulting the table of sines it will be found 1.52 that this is the sine of 310 whence 2 zz or the entire angle (balsam or oil angle) is 63° 31'. 15 MICROSCOPE AND ACCESSORIES. 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, 730 58' in water and 63° 31' in homogeneous immersion liquid. § 20. Table of a Group of Objectives with the Numeral Aper- ture (N. A.) and the method of obtaining it. Half the angular 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 = i, for water immersions n — i.jj and for homogeneous immersions n = 1.52. (For a table of natural shies, see third page of cover). Objective. Angular Aperture. (211). Naturae Sine of half the angu- lar aperture. (sin u.) Index of Refraction of ,the medium in front of the objective, (n). Numericae Aperture (N.A.) = n sin u 25 mm. (Dry.) 20° 20 . Sm —= 0.1736 n = i N.A.= 1X0.1736=0.173 25 mm. (Dry.) 0 O o- 40 Sm —=0.3420 n = 1 N.A.= 1X0.3420=0.342 12 mm. (.Dry.) 42° Sin ~= 0.3583 n = 1 N. A.= 1X0.3583=0.358 mm. (Dry.) IOO° Sin I°C>= 0.7660 n = i N.A.= 1X0.7660=0.766 6 mm. (Dry.) 75° Sin — 0.6087 n = 1 N.A.= 1X0.6087=0.608 6 mm. (Dry.) 136° Sm — = 0.9272 11 = 1 N.A.= 1X0.9272=0.927 3 mm. (Dry.) 1150 Sin 0.8434 n = 1 N.A.= 1X0.8434=0.843 3 mm. (Dry.) 163° Sin “ = 1 N.A.= 1X0.9890=0.989 2 Him. 96° 12' 96° I2/ Sin —^—=0.7443 n = 1.33 N.A.=i.33Xo. 7443=o- 99 Immersion. 2 mm. Homogeneous Immersion. no0 38'' iio°38/ S111 —- =0.8223 n = 1.52 N.A.=1.52X0.8223=1.25 2 mm. Homogeneous Immersion. 1340io' i34°io/ Sm — - = 0.921 2 11 = 1.52 N.A.=i.52Xo.92io=i.4o § 2i. 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— 16 MICROSCOPE AND ACCESSORIES. (i) Objectives vary directly as their numerical aperture in their ability to define or make clearly visible minute details (resolving power). For example an objective of 4 mm. equivalent focus and a numerical aper- ture of 0.50 N. A. 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. (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.)2. Thus if two 4 111m. objective 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.202:o.402 =0.0400:0.1600 or 1:4. That is the ob- jective of 0.20 N. A. would have but %tli the illuminating power of the one of 0 40 N. A. In considering illuminating power the equivalent focal length must also be considered. If the N. A. were the same in a 3 mm. and a 6 mm. objective their illuminating power would vary directly with the square of the foci. Thus the illuminating power of the 6 and the 3 mm. objectives would be as 62:32 or 36 to 9 or 4:1, that is 4 times as great in the 6 mm. as in the 3 mm. objective. As the magnification of an objective varies indirectly as the equivalent focus, it follows also that the illuminating power will vary indirectly as the square of the magnification of the objective. The magnification of the 6 mm. is 42 and of the 3 min. 84, whence the illuminating power of the two objec- tives are as 422 : 842 or 1764:7056 or 1:4. As the ratio is inverse in this case the result is the same as before, that is 4 times as great for the 6 mm. as for the 3 mm. objective. (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 —-1-—- so that in an objective of a given focus the greater the aperture the less the penetrating power. I11 comparing the penetrating power of objectives of different foci, the numerical aperture being the same, it is found that the penetrating power increases directly as the square of the focus. For example, two objectives of the same N. A., one of 4 mm. and the other of 2 mm. focus, the penetrating power would be as 42:22 or 16:4 or 4:1. That is, numerical aperture remaining the same, the greater the equivalent focus the greater the penetration. MICROSCOPE AND ACCESSORIES. To briefly summarize : With resolution the numerical aperture is con- cerned and the resolving power varies directly with the numerical aper- ture, thus if the N. A. is doubled, twice as many lines to the millimeter or inch can be resolved. With illuminating and penetrating power the equivalent focus of the objective must be considered, as well as the numerical aperture. With objectives of the same equivalent focus, to double the N. A. is to in- crease the illuminating power 4-fold but the penetrating power is halved. The numerical aperture remaining constant, the illuminating and penetrating power vary directly as the square of the equivalent focus, thus a 4 mm. objective would give four times the illuminating and penetrating power of a 2 mm. objective. Of course when equivalent focus and numerical aperture both differ the problem becomes more complex. For a consideration of the aperture question, its history and signifi- cance, see J. D. Cox, Proc. Amer. Micr. Soc., 1884, pp. 5-39; Jour. Roy. Micr. Soc., 1881, pp. 303, 348, 365, 388 ; 1882, pp. 300, 460 ; 1883, p. 790; 1884, p. 20. Carpenter-Dallinger, Chapters II and V. THE OCUEAR. \ 22. 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. 19. Sectional view of a Huygenian ocular {Hg. ocu- lar), 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 in looking into the ocular, i. e., in transection, it appears as a circle of light. It is at the point where 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. § 23. 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 some- what so that the real image is smaller than as if the field- lens were absent (Fig. 19). As the field-lens of the ocu- lar 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. $ 24. Positive Oculars are those in which the real, inverted image of the objec- tive is formed outside the ocular, and the entire system of ocular lenses magnifies the real image like a simple microscope (Fig. 24). 18 MICROSCOPE AND ACCESSORIES. 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 glass 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. Special names have also been applied to oculars, depending upon the designer, the construction, or the special use to which the ocular is to be applied. The following are most used.* $ 25. Huygenian Ocular.—A negative ocular designed by Huygens for the tele- scope, but adapted also to the microscope. It is the one now most commonly em- employed. It consists of a field-lens or collective (Fig. 19), aiding the objective in forming the real image, and an eye-lens which magnifies the real image. * 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 seemed 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. aplauatisches Okular (see § 13). Binocu- lar, stereoscopic Ocular; Fr. Oculaire binoculaire stereoscopique ; Ger. stereosko- pisches Doppel-Okular. A11 ocular consisting of two oculars 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 being sent to each eye. The most satisfactory form was worked out by Tolies and is constructed on true stereotomic principles, both fields being equalty illuminated. His ocular is also erecting. CampanV 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. 19). Continental Ocular. An ocular mounted in a tube of uniform diameter as in Fig. 13, 19. Deep Ocular, see high ocular. Erecting Ocular ; Fr. Oculaite redresseur ; Ger. bildumkehrendes 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 d gonio- metre ; 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 Ocu- lar, Huygens’ O., Campatii’s O., Airy’s O. ; Fr. Oculaire d’Huygens, o. de Cam- pani; Ger. Huygens’sches Okular, Campaniches Okular, see § 25. 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 satisfactorily for micrometry. Kellner's Ocular, see orthoscopic ocular. Low Ocular, also called shallow ocular. An ocular which magnifies the real image only moderately, i. e.t 2 to 8 fold. Micrometer or micrometric Ocular ; Fr. Ocu- laire micrometrique or a micrometre ; Ger. Mikrometer-Okular, Mess-Okular, Bdneche’s O., Jackson m. o., see £ 28. Microscopic Ocular ; Fr. Oculaire micro- scopic ; Ger. mikroskopisches Okular. An ocular for the microscope instead of 19 MICROSCOPE AND ACCESSORIES. While 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. § 26. 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 readily eliminated in the objective itself. Oculars of this kind, magnifying but once or twice, are made for use with high powers, for the sake of the large field in finding objects (§ 37); they are called searching oculars; those ordinarily used for observation are in contradistinction called working oculars. Part of the compensating oculars are positive and part negative. g 27. Projection Oculars.—These are oculars especially designed for projecting a microscopic image on the screen for class demonstrations, or for photographing with the microscope. While they are specially adapted for use with apochromatic objectives, they may also be used with ordinary achromatic objectives of large numerical aperture. § 28. Micrometer Ocular.—This is an ocular connected with an ocular microme- ter. 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 may be moved back and forth across the field. (See Ch. IV). § 29. Spectral or Spectroscopic Ocular.—(See Micro-Spectroscope, Ch. VI). one for a telescope. Negative Ocular, see $ 23. Nelson’s screw-micrometer ocu- lar. A modification of the Ramsden’s screw or cob-web micrometer in which positive compensating oculars may be used. Orthoscopic Oculars ; also called Kellner’s Ocular; Fr. Oculaire orthoscopique ; Ger. Kellner’sches oder orthoskop- isches Okular. An ocular with an eye-lens like one of the combinations of an objective (Fig. 11, 12) 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 re- mains 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 $ 24. Projection Ocular; Fr. Oculaire de projection ; Ger. Projections-Okular, see § 27. 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 \ 26. Shallow Ocular, see low ocular. Solid Ocular, holosteric O. ; Fr. Oculaire holostere ; Ger. holosterisches Okular, Vollglass-Okular. A nega- tive eye-piece devised by Tolies. It consists of a solid piece of glass with a mod- erate 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 or spectroscopic Ocular ; Fr. Oculaire spectroscopique ; Ger. Spectral-Okular, See Microspectroscope, Ch. VI. Stauroscopic Ocular, Fr. Ocu- laire Stauroscopique. Stauroskop-Ocular. An ocular with a Bertrand’s quartz plate for miueralogical purposes. Working Ocular ; Fr. Oc,ulaire de travail ; Ger. Arbeits-Okular, see § 26. 20 MICROSCOPE AND ACCESSORIES. § 30. Equivalent Focus.—As with objectives, some opticians designate the ocu- lars by their equivalent focus ($ 7). With this method the power of the ocular, as with objectives, other lenses or lens systems, 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 advocated by Dr. R. H. Ward for many years, and was recommended by the committee of the American Microscopical Society (Proc. Amer. Micr. Soc. 1884, p. 228). § 31. 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. \ 32. Magnification or Combined Magnification and Equivalent Focus.—The compensating oculars are marked both with their equivalent focus and the amount they magnify the real image. Thus, an ocular marked x 4, 45 mm., indicates that the equivalent focus is 45 millimeters, and that the real image of the objective is multiplied four-fold by the ocular. The projection oculars are designated simply by the amount thej7 multiply the real image of the objective. Thus for the short or 160 mm. tube-length they are, x 2, x 4; and for the long, or 250 mm. tube, they are x 3 and x 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. DESIGNATION OF OCULARS. COMPOUND MICROSCOPE. EXPERIMENTS. § 33- Putting an Obje<5tive in Position and Removing it.—Ele- vate the body of the microscope by means of the coarse adjustment (Fig. 20) 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 in the lower end of the body (Fig. 20). With two fingers of the right hand take hold of milled ring near the back or upper end of the objective, and screw7 it into the body of the microscope. Reverse this operation for removing the objective. By following this method the danger of dropping the objective will be avoided. § 34. Putting an Ocular in Position and Removing it.—Elevate the body of the microscope wdth the coarse adjustment (Fig. 20), so that the objective will be 2 cm. or more from the object—grasp the ocular by the milled ring next the eve-lens (Fig. 8), and the coarse adjustment or the tube of the microscope apd gently force the ocular into position. In removing the ocular, reverse the operation. If the above precautions are not taken, and the oculars fit snugly, there is danger in inserting them of forcing the body of the microscope downward and the objective upon the object. MICROSCOPE AND ACCESSORIES. 21 Fig. 20. Stand. That is, the mechanical parts of a simple form of oompound microscope, with the names of the parts written upon them. Arm. The part connecting the body or tube lo the pillar. Base. The part of the stand on which it rests. It should he heavy and so formed that it will give steadiness, and not be in the way of the mirror. fiody. The tube and draw-tube together. Also frequently called the tube. Coarse Adjustment. The rack and pinion for moving the tube or body rapidly up or down. Fine Adjustment. The micrometer screw arrangement for moving the body or tube of the microscope slowly up or down. Compressor. One of the pair of light springs to hold the preparation in position on the stage. Flexible .Pilla r. The pillar of the microscope, with a joint to incline the microscope. Mirror. The movable mirror with plain and concave face for lighting the ob- ject. Mirror Bar. The bar supporting the mirror. Society Screw. The screws at the lower end of the draw-tube and the main tube or body tube for receiving the objective or nose-piece. Stage. The horizontal plate for supporting the object. Substage. The cylinder below the stage for diaphragms, the illuminator and other substage accessories. 22 MICROSCOPE AND ACCESSORIES. g 35. 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 (§ 36). With low powers, it is not difficult to get an object under the micro- scope. The difficulty increases, however, with the power of the microscope and the smallness of the object. It is usually neces- sary to move the object in various directions while looking into the microscope, in order to get it into the field. Time is usually saved by getting 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.* § 36. Field or Field of View of a Microscope.—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, different 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 Fig. 2i. Triple nose piece or revolver for quickly chang- ing objectives (Queen & Co.). * As specimens are sometimes very small, or some part ©f a large specimen shows a particular structure with special excellence, it is desirable to mark the preparation so that the minute object or the part of a large object may be found quickly and with certainty. A simple way to do this is to find the object under the micro- scope, and then place a minute spot of black ink at one side. After this is done, remove the slide from the stage and surround the object with a ring of shellac cement, making the ring as small as possible and not cover the object. It will then always be known that the part to be examined is within the ring (Beale 47). The enclosure in a ring may also be very ele- gantly done by the use of a marking apparatus like that of Winkel’s (Belierens, Kossel and Schiefferdecker, p. 48), making use of either a diamond point or a delicate brush dipped in shellac or other cement. Fig. 22. Diagram showing how to enclose the lines of a mi- crometer, or of some part of a preparation by a small ring to facilitate finding it under the microscope ($ 35). MICROSCOPE AND ACCESSORIES. 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. 2mm s m m 83 m m 45mm 17 mm Fig. 23. Figures showing approximately the actual size of the field with ob- jectives of 83 mm., 43 mm., 17 mm., 5 mm., and 2 mm., equivalent focus, and ocular of 37 y2.mm., equivalent focus in each case. This figure shows graphically what is also very clearly indicated in the table (§ 37). § 37. Table showing the actual size in 7nillimeters of the field of a group of commonly used objectives and oculars. Compare with the graphic representation in Fig. 23. See also § 36. Equivalent Focus and N. A. of Objec- tive. Diameter of Field in mm. Ocular. 85 mm. 15-4 10.6 8-3 37'A mm. 25 12 yi “ Huygenian. 45 mm. 7.0 5-o 4.0 37lA mm. 25 12/4 “ Huygenian. 17 mm. N. A. = 0.25 3-o 2.0 1.6 37 lA mm. 25 12 y2 “ Huygenian. 5-7 2.8 1.4 0.97 180 mm. 45 15 10 “ Compensation. 5 mm. N. A. = 0.92 0.541 0.371 0.290 37 'A mm. 25 12/4 “ Huygenian. 0.850 0.501 0.250 0.173 180 mm. 45 “ 15 “ 10 “ Compensation. 2 mm. N. A. = 1.25 0.270 0.186 0.147 37 'A mm. 25 I2>4 “ Huygenian. 0.450 0.251 0.125 0.088 180 mm. 45 15 10 “ Compensation. MICROSCOPE AND ACCESSORIES. 24 § 38. The size of the field of the microscope as projected into the field of vision of the normal human eye (i. €., 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). FUNCTION OF AN OBJECTIVE!. § 39- Pllt a 2-in. (50 mm ) objective on the microscope, or screw off the front combination of a (18 mm.), and put the back-combina- tion 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 body of the microscope.* Rower the body of the microscope by means of the coarse adjust- ment (Fig. 20), until the objective is within 2-3 cm. of the object on the stage. Rook at the screen on the top of the body, holding the head about as far from it as for ordinary reading, and slowly elevate the body by means of the coarse adjustment until the image of the letters 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 microscope is shaded. The letters will appear as if printed on the ground glass or paper, but will be inverted (Fig. 8). 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 latter, and raise the body of the microscope until the letters do appear on the screen. 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 mi- croscope with the coarse adjustment until the burning or focal point is found (§ 4). Measure the distance from the paper object on the stage to the objective, and it will represent approximately the principal focal distance (Fig. 9,10). 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 *Grouud glass may be very easily prepared by placing some fine emery 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. MICROSCOPE AND ACCESSORIES. 25 the object and the objective is now taken, it will be found considerably greater than the principal focal distance (compare § 4). § 40. 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 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 fibers of the paper reflect the rays irregularly, so that the image may be seen at almost any angle, as if the letters were actually printed on the paper or glass. § 41. 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 ob- ject (Fig. 8). FUNCTION OF AN OCUUAR § 42- Using the same objective as for § 39, get as clear an image of the letters as possible on the lens paper screen. Look at the image with a simple microscope (Fig. 4 or 5) 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 (§ 4). Remove the screen and observe the aerial image with the tripod. Put a 50 mm., A, No. 1, or 2 in. ocular (i. e., an ocular of low magnification) in position (§ 34). Hold the eye about 10 to 20 milli- meters from the eye-lens and look into the microscope. The letters will appear as when the simple microscope was used (see above), the image will become more distinct by slightly raising the body of the microscope with the coarse adjustment. 26 MICROSCOPE AND ACCESSORIES. § 43- The function of the Ocular, as seen from the above, is that of a simple microscope, viz. : It magnifies the real image formed by the objective as if that image were an object. Compare the image formed by the ocular (Fig. 8), and that formed by a simple microscope, (Fig. 3,24). It should be borne in mind, however, that the rays from an object as usually examined with a simple microscope, extend from the object in all directions, and no matter at what angle the simple microscope is held, provided it is sufficiently near and points toward the object, an image may be seen. The rays from a real image, however, are con- Fig. 24. 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. A1 B'. The object within the principal focus. A* IP. 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. IP- A2. Retinal image of the object (A1 B'). The virtual image is simply a projection of the retinal image in the field of vision. Axis. The principal optic axis of the microscope and of the eye. Cr. Cornea of the eye. L. Crystal- line lens of the*eye R. Ideal refracting surface at which all the refractions of the eye may be assumed to take place. tinued in certain definite lines and not in all directions ; hence, in order to see this aerial image with an ocular or simple microscope, or in order to see the aerial image with the unaided eye, the simple microscope, ocular or eye must be put in the path of the rays (§ 40). § 44. 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. 19). Demonstrate this by screwing off the field-lens and using the eye-lens alone as in the ocular, refocusing if necessary. Note also that the let- ters or other image is bordered by a colored haze (§ 5). 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. § 45. The Eye-Point.—This is the point above an 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. 19), it is the smallest and brightest light circle 27 MICROSCOPE AND ACCESSORIES. 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 (§ 39). 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 seen that the eye-point is nearer the eye-lens in high than in low oculars, 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 the appendix 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. For the history of the microscope, Mayall’s Cantor Lectures on the microscope are very satisfac- tory. See also Beale, E. Bausch, Beherens, Kossel and Schiefferdecker, Dippel, Frey, Harting, Hogg, Nageli and Scliweudener, Robin, Van Heurck. The following special articles in periodicals may be examined with advantage : Apochromatic Objectives, etc. Dippel in Zeit. wiss. Mikr. 1886, p. 303 ; also in the Jour. Roy. Micr. Soc., 1886, pp. 316, 849, mo; same, 1890, p. 480; Zeit. f. Instrumentenk., 1890, pp. 1-6; Micr. Built., 1891, pp. 6-7. Tube-length, etc. "Gage, Proc. Amer. Soc. Micrs., 1887, 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, 1890, pp. 289-296. Aperture. J. D. Cox, Presidential Address, Proc. Atner. Soc. Micrs., 1884, pp. 5-39. Jour. Roy. Micr. Soc., 1881, pp. 303, 348, 365, 388 ; 1882, pp. 300, 460 ; 1883, p. 790 ; 1884. p. 20. CHAPTER II. LIGHTING AND FOCUSING, MANIPULATION OF DRY, ADJUSTABLE AND IMMERSION OBJECTIVES; CARE OF THE MICROSCOPE AND OF THE EYES. APPARATUS AND MATERIAL FOR THIS CHAPTER. Microscope supplied with plane and concave mirror, Abbe and achromatic con- densers, dry, adjustable and immersion objectives, oculars, tripple nose-piece. Microscope lamp and movable condenser (Bulks eye or other form (Fig. 34), Homogeneous immersion liquid; Benzine, alcohol, distilled water; Mounted preparation of fly’s wing 58) ; Mounted preparation of Pleurasigma. Stage or ocular micrometer with lines filled with graphite (§ 63, 64) ; Glass slides and cover glasses (Ch. VII) ; 10 per ct. solution of salicylic acid in 95 per ct. alcohol (§ 73) ; Preparation of stained microbes (§ 91) ; Vial of equal parts olive or cotton seed oil and benzine (g 95); Ward’s and double eye-shade (Fig. 44, 45); Screen for whole microscope (Fig. 43). FOCUSING. \ 46. Focusing is mutually arranging an object and the microscope so that a clear image may be seen. With a simple microscope (% 4) 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 (Fig. 20). 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 body 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 object and objective be brought. ? 47. 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 considerably less than the equivalent focal length of the objective. For example, the front-lens of a %th in., or 6 mm. objective would not be inch, or 6 milli- meters 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 very thin cover glasses over the ob- 29 LIGHTING AND FOCUSING. ject. (See § 16, 17). 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 ex- amination with high powers it is always better to use thin covers. lighting WITH daylight $ 48- 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. The light should come from the left ; but if it is necessary to face the window a vertical, adjustable screen between the face and the window is desirable (Fig. 43). It is of the greatest importance and advantage for one who is to use the micro- scope 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 re- flected, and transmitted, axial and oblique. Fig. 25. Fig. 26. Fig. 25, 26. For full explanation see Fig. 11 and 12 £ 49- 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 mi- croscope. 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 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 (Fig. 27). (See also Carpenter-Dallinger, p. 278). 30 LIGHTING AND FOCUSING. § 50. Transmitted Light.—-By this is meant light which passes through an ob- ject 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 an illuminator ($ 65), and thence transmitted to the object from below (Fig. 27-30). \ 51. 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 parallel with the optic axis of the microscope. In either case the object is symmetrically illuminated. | 52. Oblique Light.—This is light in which parallel rays from a plane mirror form an angle with the optic axis of the microscope (Fig. 12). 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. 28),. DIAPHRAGMS. | 53. 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 con- denser serve to vary its aperture to the needs of each object and each objective. g 54. 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 used. 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 with the mirror by cutting off a part of the illuminating pencil. If the diaphragm is a considerable 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. With an illuminator or condenser (Fig. 27), the diaphragm serves to narrow the pencil to be transmitted through the condenser, and thus to limit the aperture or for any special purpose to be served (see \ 61). 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 ($ 60 ; Fig. 30). 31 LIGHTING AND FOCUSING. ARTIFICIAL ILLUMINATION. \ 55. For evening work and for certain special purposes, artificial illumination is employed. No more satisfactory artificial light has been found than that from a good petroleum lamp with a flat wick. Whatever artificial source of light is employed the light should be brilliant and steady. (See \ 70 and Ch. VIII). § 56. Lighting with a Mirror.—Put a mounted fly’s wing under the microscope, put the % in. (18 mm.) or other low objective in posi- tion, also a low ocular. With the coarse adjustment (Fig. 20), lower the body of the microscope within about 1 cm. of the object. Use an opening in the diaphragm about as large as the front lens of the ob- jective ; then with the plane mirror try to reflect light up through the diaphragm upon the object. One can tell when the field (§ 36) is illu- minated, 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 concave mirror condenses the light, the field will look brighter with it than with the plane mirror. It is especially desirable to re- member that the excellence of lighting depends in part on the position of the diaphragm (§ 47). If the greatest illumination is to be obtained from the concave mirror, its position must be such that its focus will 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. § 57. Use of the Plane and of the Concave Mirror.—The mirror 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. § 58. Focusing with Low Objectives.—Place a mounted fly’s wing under the microscope ; put the three-fourths (18 mm.) objective in position, and also the lowest ocular. Select the proper opening in the diaphragm and light the object well with transmitted light (§ 50, 54)- Hold the head at about the level of the stage, look toward the win- dow, and between the object and the front of the objective ; with the coarse adjustment lower the body (Fig. 20), until the objective is within about half a cm. of the object. Then look into the microscope and slowly elevate the body with the coarse adjustment. The image will appear dimly at first, but will become very distinct by turning the body LIGHTING AND FOCUSING : EXPERIMENTS 32 LIGHTING AND FOCUSING. still higher. If the body is raised too high the image will become in- distinct, and finally disappear. It will again appear if the body 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 oc- ular in place of the low one (§ 30). If the oculars are not par-focal it will be necessary to lower the tube somewhat to get the image in focus. * Pull out the draw-tube (Fig. 20) 4-6 cm., thus lengthening the body of the microscope, and it will be found necessary to lower the tube of the microscope somewhat. § 59. 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. §60. Focusing with High Objectives.—Employ the same object as before, elevate the body of the microscope and remove the % in. (18 mm.) objective as indicated. Put the in., (5 mm.) or a higher objective in place, and use a low ocular. Eight well, and employ the proper opening in the diaphragm, etc. (§ 54). Look between the front of the objective and the object as be- fore (§ 58), and lower the body with the coarse adjustment till the ob- jective almost touches the cover-glass over the object. Look into the microscope, and with the coarse adjustment, raise the body very slowly until the image begins to appear, then turn the milled head of the fine adjustment, (Fig. 20), first one way and then the other, if necessary, until the image is sharply defined. 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 oculars are not par-focal) or lengthening the body of the microscope it will be found necessary to bring the objective still nearer the object, as with the low objective (§ 58). § 61. Always Focus Up, as directed above. If one lowers the body 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. * 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. 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. 33 LIGHTING AND FOCUSING. When the instrument is well focused, move the object around in order to bring different parts into the field of view (§ 36). 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 adjustment constantly, and the focus is continually varied. § 62. Determination of Working Distance.—As stated in § 47 this is the distance between the front lens of the objective and the ob- ject when the objective is in focus. It is always less than the equiva- 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 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 op- posite 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 mm. and the tripod to see the divisions. Or one may use a cover glass measurer, for determining the thickness of the wedge (Fig. 7l~73)- For the higher powers, if one has a microscope in which the fine adjustment is graduated, the working distance may be readily deter- mined when the thickness of the cover-glass over the specimen is known, as follows : Get the object in focus, lower the body of the mi- croscope until 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 distance. For example, a 3 mm. objective after being brought in con- tact with a cover glass was raised by the fine adjustment a distance rep- resented 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 -f- .15 = .31 mm. CENTRAL, AND OBLIQUE LIGHT WITH A MIRROR §63. Axial Light, (§ 51).-— Place a preparation containing minute air-bubbles under the micoscope. (The preparation may be easily made by beating a drop of mucilage on a slide and covering it. (See LIGHTING AND FOCUSING. 34 Ch. III). Use a inch (3 mm.) or No. 7 objective, and a medium ocular. Remove all the diaphragms and the sub-stage. 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 chang- ing the position of the mirror in the least, replace the air-bubble pre- paration by one of Pleurasigma angulatum or some other finely marked diatom. Study the appearance very carefully. § 64. Oblique Light, (§ 52).—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 different appearance with that of cen- tral light. The effect of oblique light is not so striking with histologi- cal preparations as with diatoms. It should be especially noted in §§ 63, 64, that one cannot determine the exact direction of the rays by the position of the mirror. This is especially true for axial light (§ 63). To be certain that the light is axial some such test as that given in §63 should be applied. (See also Ch. Ill, under Air-bubbles). CONDENSERS OR IEEUMINATORS § 65. These are lenses or lens-systems for the purpose of illuminating with transmitted light the object to be studied with the microscope. For the highest kind of investigation their value cannot be overesti- mated. They may be used either with natural or artificial light, and should be of sufficient numerical aperture to satisfy objectives of the widest angle.* It is of the greatest advantage to have the sub-stage condenser mounted with rack and pinion 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 conden- ser should be supplied with central stops for dark-ground illumination (§ 73) and with blue and neutral tint glasses to soften the glare when artificial light is used (§ 104). * The value of a substage condenser and the character it should possess is no new discovery, but was thoroughly understood by the older opticians as may be seen from the following by Sir David Brewster, 1831 :—“ I have no hesitation in saying that the apparatus for illumination requires to be as perfect as the appa- ratus for vision, and on this account I would recommend that the illuminating lens should be perfectly free from 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.” 35 LIGHTING AND FOCUSING. They fall into two great groups the non-achromatic and the apian - atic (§ii, 13). § 66. Abbe Illuminator.—Of the non-achromatic illuminators this is the most generally useful. It consists of two or three very large lenses which are placed in some form of mounting beneath the stage. It serves to concentrate a very wide pencil of light from the mirror upon the object (Fig. 27). § 67. Arrangement of the Illuminator.—The proper position of the illuminator for high objectives is one in which the beam of light traversing it is brought to a focus on the object. If parallel rays are reflected from the plane mirror to it, they will be focused only a few millimeters above the upper lens of the illuminator ; consequently the illuminator should be about on the level of the top of the stage and therefore almost in contact with the lower surface of the slide. For some purposes, when it is desirable to avoid the loss of light by reflec- tion or refraction, a drop of water or homogeneous immersion fluid is put between the slide and condenser, forming the so-called immersion illuminator. This is necessary only with objectives of high power and large aperture or for dark-ground illumination. § 68. Centering the Illuminator.—The illuminator should be cen- tered to the optic axis of the microscope, that is the optic axis of the condenser and of the microscope should coincide. Unfortunately there is extreme difficulty in determining when the Abbe illuminator is centered. Centering is approximated as follows : Put a pin-hole dia- phragm over the end of the condenser (Fig. 27)—that is a diaphragm with a small central hole—the central opening should appear to be in the middle of the field of the microscope. If it does not, the condenser should be moved from side to side by loosening the centering screws until it is in the center of the field. In case no pin-hole diaphragm accompanies the condenser, one may put a very small drop of ink, as from a pen-point, on the center of the upper lens and look at it with the microscope to see if it is in the center of the field. If it is not, the condenser should be adjusted until it is. When the condenser is cen- tered as nearly as possible remove the pin-hole diaphragm or the spot of ink. The microscope and illuminator axes may not be entirely co- incident even when the center of the upper lens appears in the center of the field, as there may be some lateral tilting of the condenser, but the above is the best the ordinary worker can do, and unless the me- chanical arrangements of the illuminator are very deficient, it will be very nearly centered. § 69. Mirror and Light for the Abbe Illuminator.—It is best to use light with parallel rays. The rays of daylight are practically par- 36 LIGHTING AND FOCUSING. allel, it is best, therefore, to employ the plane mirror for all but the lowest powers. If low powers are used the whole field might not be illuminated with the plane mirror when the condenser is close to the object; furthermore, the image of the window frame, objects outside the building, as trees, etc., would appear with unpleasant distinctness in the field of the microscope. To overcome these defects, one can lower the condenser and thus light the object with a diverging cone of light, or use the concave mirror and attain the same end when the con- denser is close to the object (Fig. 27). § 70. Artificial Light. —If one uses lamplight, it is recommended that a large bull’s eye be placed in such a position between the light and the mirror that parallel rays fall upon the mirror or in some cases an image of the lamp flame. If one does not have a bull’s eye the concave mirror may be used to render the rays less divergent. It ma)r be necessary to lower the illuminator somewhat in order to illuminate the object in its focus. ABBE ILLUMINATOR : EXPERIMENTS. § 7i. Abbe Illuminator, Axial and Oblique Light.—Use a dia- phragm a little larger than the front lens of the }i (3 mm.) or No. 7 objective, have the illuminator on the level or nearly on the level of the upper surface of the stage, and use the plane mirror. Be sure that the diaphragm carrier is in the notch indicating that it is central in position. Use the Pleurasigma as object. Study carefully the appearance of the diatom with this central light, then make the diaphragm eccentric so as to light with oblique light. The differences in appearance will prob- ably be even more striking than with the mirror alone (§ 63, 64) § 72. Lateral Swaying of the Image.—Frequently in studying an object, especially with a high power, it will appear to sway from side to side in focusing up or down. A glass stage micrometer or fly’s wing is an excellent object. Make the light central or axial and focus up and down and notice that the lines simply disappear or grow dim. Now make the light oblique, either by making the diaphragm opening ec- centric or if simply h mirror is used, by swinging the mirror sidewise. On focusing up and down, the lines will sway from side to side. What is the direction of apparent movement in focusing down with reference to the illuminating ray ? What in focusing up ? If one understands this experiment it may somtimes save a great deal of confusion. (See under testing the microscope for swaying with central light § 104). § 73. Dark-Ground Illumination.—When an object is lighted with rays of a greater obliquity than can get into the front-lens of the objec- tive, the field will appear dark (Fig. 30). If now the object is com- 37 LIGHTING AND FOCUSING. Fig. 27, 28, 29, 30. Sectional views of the Abbe Illuminator of 1.20 N. A. (§$ 18, 66), showing various methods of illumination {\ 65-70). Fig. 27, axial light with parallel rays. Fig. 28, oblique light. Fig. 29, axial light with con- verging beam. Fig. 30, dark-ground illumination with a central stop diaphragm. Axis. The optic axis of the illuminator and of the microscope. The illumina- tor is centered, that is its optic axis is a prolongation of the optic axis of the microscope. S. Axis. Secondary axis. In oblique light the central ray passes along a sec- ondary axis of the illuminator, and is therefore oblique to the principal axis. A represents the upper part of the illuminator. D D. Diaphragms. These are placed in sectional and in face views. The dia- phragm is placed between the mirror and the illuminator. In Fig. 27 the opening is eccentric for oblique light, and in Fig. 30 the opening is a narrow band, the central part being stopped out, and thus giving rise to dark-ground illumination (2 73)- Obj. Obj. The front of th£ objective. 27 28 29 30 posed of fine particles, or is semi-transparent, it will refract or reflect the ligljt which meets it, in such a way that a part of the very oblique rays will pass into the objective, hence as light reaches the objective only from the object, all the surrounding field will be dark and the ob- ject will appear like a self-luminous one on a dark back-ground. This form of illumination is only successful with low powers and objectives of small aperture. It is well to make the illuminator immersion for this experiment, see § 67. LIGHTING AND FOCUSING. (A) With the Mirror—Remove all the diaphragms so that very oblique light may be used, employ a stage micrometer in which the lines have been filled with graphite, use a (18 mm.) or No. 4 ob- jective, and when the light is sufficiently oblique the lines will appear something like streaks of silver on a black back-ground. A specimen like that described below in (B) may also be used. (B) With the Abbe Illuminator.—Have the illuminator so that the light would be focused on the object (see § 67) and use a diaphragm with the annular opening (Fig. 30) ; employ the same objective as in (A). For object place a drop of 10% solution of salicylic acid in 95% alcohol on the middle of a slide and allow it to dry and crystallize. The crystals will appear brilliantly lighted on a dark back-ground. Put in an ordinary diaphragm and make the light oblique by making the dia- phraghm eccentric. The same specimen may also be tried with a mir- ror and oblique light. In order to appreciate the difference between this dark-ground and ordinary transmitted light illumination, use an ordinary diaphragm and observe the crystals. A very striking and instructive experiment may be made by adding a very small drop of the solution to the dried preparation, putting it under the microscope very quickly, lighting for dark-ground illumination and then watching the crystallization. § 74. Aplanatic Condensers.—It is believed by many of the most competent microscopists, that the condenser used for lighting the object should have the same excellence as the objective used in observing it. It has therefore been the custom to use objectives for illuminators. It is more convenient, however, to have a specially constructed condenser that may be used with all powers and all numerical apertures up to the highest. § 75. Centering the Condenser.—From the arrangement of the diaphragms in the best forms of aplanatic condensers, an image of the diaphragm may be thrown into the field of view by lowering the con- denser. If it is not in the center, the condenser is easily moved by ad- justing-screws until the diaphragm opening is centered (Fig. 35, 36). § 76. Centering the Image of the Source of Illumination.—For the best results it is not only necessary that the condenser be properly centered, but that the object to be studied should be in the image of the source of illumination and that this should also be centered (Fig. 37, 38). This is easily accomplished by manipulation of the mirror or, if a lamp is used, by changing the position of the lamp or of the bull’s eye (Fig. 34). § 77. Proper Numerical Aperture of the Condenser.—As stated above, the aperture of the condenser should have a range by means of 39 LIGHTING AND FOCUSING. properly selected diaphragms to meet the requirements of all objectives from the lowest to those of the highest aperture. It is found in prac- tice that in most cases the best images are obtained when the object is lighted with a cone which shall fill about three-fourths of the diameter of the back lens of the objective with light. To determine this in any case, focus the object carefully, take out the ocular, look down the tube at the back lens. If less than three- fourths of the back lens is lighted, increase the opening in the dia- phragm—if more than three-fourths, diminish it. For some objects it is advantageous to use the entire aperture, for others, less than three- fourths. Experience will teach the best lighting for special cases. Fig. 31. Fig. 32 Fig. 31, 32. Figures showing the dependence of the objective upon the illumi- nating co?ie of the condenser. {Nelson). Fig. 31. {A) 'The illuminating cone from the condenser. {Ilium). This is seen to be just sufficient to fill the objective {Obj.). {B). The back lens of the objective entirely filled with light, showing that the numerical aperture of the illuminator is equal to that of the objective. Fig. 32. {A). In this figure the illuminating cone from the condenser {Ilium.) is seett to be insufficient to fill the objective {Obj.). {B). The back lens of the objective only partly filled with light, due to the re- stricted aperture of the illuminator. ARTIFICIAL ILLUMINATION. § 78. For evening work and for regions where daylight is not suf- ficiently brilliant, artificial Illumination must be employed. Further- more, for the most critical investigation of bodies with fine markings like diatoms, artificial light has been found superior to daylight. On the whole, a petroleum lamp gives the most satisfactory light. It is found that a metal chimney with a slit-like opening, covered with an oblong cover-glass, opposite the flame is better than the ordinary glass chimney. 40 LIGHTING AND FOCUSING. Whenever possible, the edge of the flame is turned toward the micro- scope, the advantage of this arrangement is the greater brilliancy, due to the greater thickness of the flame in this direction. § 79. Mutual Arrangement of Lamp, Bull’s Eye and Micro- scope.—To fulfill the conditions given above—namely—that the object be illuminated by the image of the source of illumination the lamp must be in such a position that the condenser projects a sharp image of the flame upon the object (Fig. 37) and only by trial can this position be determined. In some cases it is found advantageous to discard the mirror and allow the light from the bull’s eye to pass directly into the condenser. This method is especially excellent in photomicrography (see Ch. VIII). § 80. Illuminating the Entire Field.—With low objectives and large objects, the entire object might not be illuminated if the above method were strictly followed ; in this case, turn the lamp so that the flame is oblique, or if that is not sufficient, continue to turn the lamp until the full width of the flame is used. If necessary the condenser may be lowered also. § 81. Very excellent and convenient lamps for the microscope have been devised, of which Fig. 33 is a good example. Probably, however, most workers will find a good flat-wicked lamp with a metal chimney and a separate bull’s eye capable of more varied uses. Fig. 33. Acme Microscope Lamp. {Queen & Co.). LIGHTING AND FOCUSING. Fig. 34. 1. Lamp with slit-opening in metal chimney. 2. Bull's eye on sepa- rate stand. 3. Screen showing image of flame. Fig. 35. Shows that the optic axis of the condenser does not coincide with that of the microscope. (D). Dia- phragm of the condenser shown at one side of the field of the microscope. Exc.). Eccentric diaphragm. Fig. 36. Shows the diaphragm (D) in the center of the field of the mi- croscope and thus the coincidence of the axis of the condenser with that of the microscope. (C). Centered condenser. Fig. 37. Shows the image of the flame (FI.) in the center (C) of the field of the microscope and illumi- nating the object. Fig. 38. Shows the image of the flame {FI.) at one side of the center {Exc.) and not properly illumi- nating the object. REFRACTION AND COEOR IMAGES. $ 82. Refraction Images are those mostly seen in studying microscopic objects. They are the appearances produced by the refraction of the light on entering and leaving an object. They therefore depend (a) on the form of the object, (b) on the relative refractive powers of object and mounting medium. With such images the diaphragms should not be too large (see § 54). If the color and refractive index of the object were exactly like the mount- LIGHTING AND FOCUSING. 42 ing medium it could not be seen. In most cases both refractive index and color differ somewhat, there is then a combination of color and refraction images which is a great advantage. This combination is generally taken advantage of in histol- ogy. Fig. 51 is an example of a purely refractive image. Fig. 39-41. Diagrams illustrating refraction in different media and at plane and curved surfaces. In each case the denser medium is represented by line shading and the perpendicular or normal to the refracting surface is represented by the dotted line N, the refracted ray by the bent line A C. § 83. 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. 39, light passing from air into water does not continue in a straight course but is bent toward the normal, 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 to CL Conversely, if the ray of light is passing from water into air, on reaching the air it is bent from the normal, the ray C B passing to A and not in a straight line to C//. By comparing Fig. 40, 41, 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 den- ser 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. This relationship is called the index of re- fraction, and is expressed thus : Sine of the angle of incidence divided by the sine of the angle of refraction. In the figures, U = index of refrac- 0 0 Sin CBN tion. Worked out completely in Fig. 39, A B N = 40°, C B N = 28° 54' —7 = ——l—— =1.33, i. e., the index of refraction from air to water is 1.33. Sin 28° 54/ 0.48327 ’ 00 (See | 20). In Fig. 40, 41, illustrating refraction in crown glass, the angles being given, the problem is easily solved as just illustrated. (For table of natural sines see 3d page of cover). \ 84. Color Images.—These are images of objects which are strongly colored 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 specially applicable to the study of stained microbes. (See below § 91). 43 LIGHTING AND FOCUSING ADJUSTABLE, WATER AND HOMOGENEOUS OBJECTIVES. EXPERIMENTS. § 85. Adjustment for Objectives.—As stated above (§ 16), the ab- erration produced by the cover-glass (Fig. 42), is compensated for by giving the combinations 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 unad- justable objectives, one can secure the best results of which the object- ive is capable by selecting covers of the thickness for which the object- ive was corrected. (See table in § 17). Adjustment may be made also by increasing the tube-length for covers thinner than the standard, and by shorteiiing the tube-length for covers thicker than the standard (§ 17)- Fig. 42. Effect of the cover-glass on the rays from the object to the objec- tive (Ross). Axis. The projection of the optic axis of the microscope. F. Focus or axial point of the ob- jective. 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 (Cover). It is for the correction of this dis- turbance or so-called * ‘ negative aberration ” produced by the cover-glass that objec- tives are fixed in their mounting for a given thickness of cover, or that the com- binations making up the objective are made adjustable; that is so that the back combinations may be brought nearer the front lens ork combination as the cover thickens, and separated with a thinner cover or for uncovered objects. The thicker the cover, the nearer the front and back combinations ; the thiner the cover the far- ther apart are front and back combinations separated (§ 86). § 86. Adjustable Objectives.—The proper adjustment of object- ives, that is, the adjustment which gives the truest image, requires both insight and experience ; for the structure of an object does not appear the same with different adjustments of the objective. And as the opinion of different observers on the structure of objects varies, they adjust the objectives differently, and try to obtain the adjustment which will show a structure in accordance with their opinion. Byes also differ, and two observers might find it necessary to adjust the same objective differently to produce an identical appearance for each of them. LIGHTING AND FOCUSING. 44 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, shading 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 dis- tance between the systems of lenses, usually the front and the back systems (Fig. 26). In adjustable objectives the back systems should be movable, the front one remaining fixed so that there will be no danger of bringing the objective down upon the object. If the front system is movable, the body of the microscope should be raised slightly every time the adjustment is altered. General Directions.—(A) The thinner the cover-glass the further must the systems of lenses be separated, i. e., the adjusting collar is turned nearer the zero or the mark “ uncovered, ” and conversely, (B) the thicker the cover-glass, the closer together are the systems brought by turning the adjusting collar from the zero mark. This also in- creases the magnification of the objective (Ch. IV). The following specific directions for making the cover glass adjust- ment are given by Mr. Wenham (Carpenter, 166). “ Select any dark speck or opaque portion of the object, and bring the outline into perfect focus ; then lay the finger on the milled-head of the fine motion, and move it briskly backwards and forwards in both directions from the first position. Observe the expansion of the dark outline of the object, both when within and when without the focus. If the greater expansion or coma, is when the object is without the focus, or farthest from the objective- [i. e,, in focusing up], the lenses must be placed further asunder, or toward the mark uncovered [i. e., the ad- justing collar is turned toward the zero mark as the cover-glass is too thin for the present adjustment]. If the greater expansion is when the object is within the focus, or nearest the objective, [i. e., in focusing down], the lenses must be brought closer together or toward the mark covered [i. e., the adjusting collar should be turned away from the zero mark, the cover-glass being too thick for the present adjustment].” In most objectives the collar is graduated arbitrarily, the zero (O) mark representing the position for uncovered objects. Other objectives have the collar graduated to correspojid to the various thickness of cover-glasses for which the objective may be adjusted. This seems to be a?i admirable plan; then if one knows the thick?iess of the cover-glass o?i the preparation (.Fig. 71-73) the adjtisting collar may be set at a corresponding mark, and one will feel confident that the adjustment will be approximately correct. It is then only ?iecessary for the observer to make the slight adjustment to com- pensate for the mounting medium or any variation from the standard 45 LIGHTING AND FOCUSING. length of the tube of the microscope. In adjusting for variations of the length of the tube from the standard it should be remembered that : (A) If the tube of the microscope is longer than the standard for which the objective was corrected, the effect is approximately the same as thickening the cover-glass, and therefore the systems of the objective must be brought closer together, i. e., the adjusting collar must be turned away from the zero mark. (B) If the tube is shorter than the standard for which the objective is corrected, the effect is ap- proximately the same as diminishing the thickness of the cover-glass, and the systems must therefore be separated (Fig. 26). Furthermore, whatever the interpretation by different opticians of what should be included in “tube-length,” and the exact length in millimeters, its importance is very great; for each objective gives the most perfect image of which it is capable with the “ tube-length ” for which it is corrected, and the more perfect the objective the greater the ill effects on the image of varying the “ tube-length ” from this stand- ard. The plan of designating exactly what is meant by “tube- length,” and engraving on each objective the “tube-length ’’for which it is corrected, is to be commended, for it is manifestly difficult for each worker with the microscope to find out for himself for what “ tube-length ” each of his objectives was corrected. § 87. Water Immersion Objectives.—Put a water immersion ob- jective in position (§ 33) and the fly’s wing for object under the micro- scope. Place a drop of distilled water on the cover-glass and with the coarse adjustment lower the tube till the objective dips into the water, then light the field well and turn the fine adjustment one way and the other till the image is clear. Water immersions are exceedingly con- venient in studying the circulation of the blood and for many other purposes where aqueous liquids are liable to get on the cover-glass. If the objective is adjustable, follow the directions given in § 85. When one is through using a water immersion objective, remove it from the microscope and with some lens paper wipe all the water from the front-lens. Unless this is done dust collects and sooner or later the front-lens would be clouded. It is better to use distilled water to avoid the gritty substances that are liable to be present in natural waters, as these gritty particles might scratch the front-lens. HOMOGENEOUS IMMERSION OBJECTIVES : EXPERIMENTS. § 88. As stated above, these are objectives in which a liquid of the same refractive index as the front-lens of the objective is placed be- tween the front-lens and the cover glass. § 89. Tester for Homogeneous Liquid.—In order that full ad- 46 LIGHTING AND FOCUSING. vantage be derived from the homogeneous immersion principle, the liquid employed must be truly homogeneous. To be sure that such is the case, one may use a tester like that constructed by the Gundlach Optical Co., then if the liquid is too dense it may be properly diluted and vice versa. For the cedar oil immersion liquid, the density may be diminished by the addition of pure cedar-wood oil. The density may be increased by allowing it to thicken by evaporation. (See H. Iy. Smith, Proc. Amer. Soc. Micr., 1885, p. 83). § 90. Refraction Images.—Put a 2 mm. in.) homogeneous immersion objective in position, employ an illuminator. Use some histological specimen like a muscular fiber as object, make the diaphragm opening about 3 mm. in diameter, add a drop of the homogeneous immersion liquid and focus as directed in § 87, 91. The object will be clearly seen in all details by the unequal refraction of the light traversing it. The difference in color between it and the sur- rounding medium will also increase the sharpness of the outline. If an air bubble preparation (§ 63) were used, one would get pure, refrac- tion images. § 91. Color Images.—Use some stained microbes, as Bacillus tuberculosis for object. Put a drop of the immersion liquid on the cover-glass or the front-lens of the homogeneous objective. Remove the diaphragms from the illuminator or in case the iris diaphragm is used, open to its greatest extent. Focus the objective down so that the immersion fluid is in contact with both the front-lens and the cover- glass, then with the fine adjustment get the microbes in focus. They will stand out as clearly defined colored objects on a bright field. Fig. 43. Screen for shading the microscope and the face of the observer. This is very readily constructed as shown in the figure by supporting a wire in a disc of lead, iron or heavy wood. The screen is then com- pleted by hanging over the bent wire, cloth or manilla> paper 30 x 40 cm. The lower edge of the screen should be a little below the stage of the microscope and the upper edge high enough to screen the eyes of the ob- server. § 92. Shading the Object. —To get the clear- est image of an object no light should reach the eye except from the object. A handker- chief or a dark cloth wound around the objec- tive will serve the purpose. Often the proper effect may be obtained by simply shading the top of the stage with the hand or with a piece of bristol board. Unless one has a favorable light the shading of 47 LIGHTING AND FOCUSING. the object is of the greatest advantage, especially with homogene- ous immersion objectives. The screen (Fig. 43) is the most satisfac- tory means for this purpose, as the entire microscope above the illumi- nating apparatus is shaded. § 93. Cleaning Homogeneous Objectives.—After one is through with a homegeneous objective, it should be carefully cleaned as follows : Wipe off the homogeneous liquid with a piece of the lens paper (§ 97), then if the fluid is cedar oil, wet one corner of a fresh piece in benzine and wipe the front lens with it. Immediately afterward wipe with a dry part of the paper. The cover-glass of the preparation can be cleaned in the same way. If the homogeneous liquid is a glycerin mixture proceed as above, but use water instead of benzine to remove the last traces of glycerin. CARE OF THE MICROSCOPE. § 94. The microscope should be handled carefully, and kept perfectly clean. The oculars and objectives should never be allowed to fall. When not in use keep it in a place as free as possible from dust. All parts of the microscope should be kept free from liquids, espe- cially from acids, alkalies, alcohol, benzine, turpentine and chloroform. § 95. Care of the Mechanical Parts.—To clean the mechanical parts put a small quantity of some fine oil, (olive oil and benzine equal parts), on a piece of chamois leather or on the lens paper, and rub the parts well, then with a clean dry piece of the chamois or paper wipe off most of the oil. If the mechanical parts are kept clean in this way a lubricator is rarely needed. Where opposed brass surfaces “cut,” i. e., when from the introduction of some gritty material, mi- nute grooves are worn in the opposing surfaces, giving a harsh move- ment, the opposing parts should be separated, carefully cleaned as described above and any ridges or prominences scraped down with a knife. Where the tendency to “ cut ” is marked, a very slight applica- tion of equal parts of beeswax and tallow well melted together serves a good purpose. In cleaning lacquered parts, benzine alone answers well, but it should be quickly wiped off with a clean piece of the lens paper. Do not use alcohol as it dissolves the lacquer. § 96. Care of the Optical Parts.—These must be kept scrupulously clean in order that the best results may be obtained. Glass surfaces should never be touched with the fingers, for that will soil them. The glass of which the lenses are made is quite soft, consequently it is necessary that only soft, clean cloths or paper be used in wiping them. 48 LIGHTING AND FOCUSING. § 97- Lens Paper.—The so-called Japanese filter paper, which from its use with the microscope, I have designated lens paper, has been used in the author’s laboratory for the last ten years for cleaning the lenses of oculars and objectives, and especially for removing the fluid used with immersion objectives. Whenever a piece is used once it is thrown away. It has proved more satisfactory than cloth or chamois, because dust and sand are not present; and from its bibulous character it is very efficient in removing liquid or semi-liquid substances. § 98. Dust may be removed with a camel’s hair brush, or by wiping with the lens paper. Cloudiness may be removed from the glass surfaces by breathing on them, then wiping quickly with a soft cloth or the lens paper. Cloudiness on the inner surfaces of the ocular lenses may be removed by unscrewing them and wiping as directed above. A high objective should never be taken apart by an inexperienced person. If the cloudiness cannot be removed as directed above, moisten one corner of the cloth or paper with 95 per cent, alcohol, wipe the glass first with this, then with the dry cloth or the paper. Water may be removed with soft cloth or the paper. Glycerin may be removed with cloth or paper saturated with distilled water ; remove the water as above. Blood or other albuminous material may be removed while fresh with a moist cloth or paper, the same as glycerin. If the material has dried to the glass, it may be removed more readily by adding a small quan- tity of ammonia to the water in which the cloth is moistened, (water 100 cc., ammonia 1 cc). Canada Balsam, damar, paraffin, or any oily substance, may be re- moved with a cloth or paper wet with chloroform, benzine or xylol. The application of these liquids and their removal with a soft, dry cloth or paper should be as rapid as possible, so that none of the liquid will have time to soften the setting of the lenses. Shellac Cement may be removed by the paper or a cloth moistened in 95 per cent, alcohol. Brunswick Black, Gold Size, and all other substances soluble in chloroform, etc., may be removed as directed for balsam and damar. In general, use a solvent of the substance on the glass and wipe it off quickly with a fresh piece of the lens paper. It frequently happens that the upper surface of the back combination of the objective becomes dusty. This may be removed in part by a brush, but more satisfactorily by using a piece of the soft paper loosely twisted. When most of the dust is removed some of the paper may be put over the end of a pine stick (like a match stick) and the glass sur- face carefully wiped. 49 LIGHTING AND FOCUSING. Fig. 44. Double eye-shade. This is made by cut- ting a hole slightly larger than the tube near one edge. A rubber band is then used to loop around the tube and hold the screen from falling over in front. It is desirable to have the screen covered with velveteen. CARE OF THE EYES. § 99- Keep both eyes open, using the eye-screen if necessary (Fig. 44, 45) ; and divide the labor between the two eyes, i. e., use one eye for observing the image awhile and then the other. In the beginning it is not advisable to look into the microscope continuously for more than half an hour at a time. One never should work with the micro- scope after the eyes feel fatigued. After one becomes accustomed to microscopic observation he can work for several hours with the inicioscope without fatiguing the eyes. This is due to the fact that the eyes be- come inured to labor like the other organs of the body by judicious exercise. It is also due to the fact that but very slight accommodation is required of the eyes, the eyes remaining nearly in a condition of rest as for distant objects. The fatigue incident upon using the microscope at first is due partly at least to the constant effort on the part of the observer to remedy the defects of focusing of the microscope by accom- modation of the eyes. This should be avoided and the fine adjustment of the microscope used instead of the muscles of accommodation. With a microscope of the best quality, and suitable light—that is light which is steady and not so bright as to dazzle the eyes nor so dim as to strain them in determining details—microscopic work should improve rather than injure the sight. § roo. Position and Character of the Work-Table.—The work- table should be very firm and large (ixiji meter, 3x4 feet) so that the necessary apparatus and material for work may not be too crowded. The table should also be of the right height to make work by it com- fortable. An adjustable stool, something like a piano stool is conven- ient, then one may vary his height corresponding to the necessities of special cases. It is a great advantage to sit facing the window if day- light is used, then the hands do not constantly interfere with the illu- mination. To avoid the discomfort of facing the light a screen like that shown in Fig. 43 is very useful (see also under lighting, § 48). Fig. 45.— Ward's Eye-Shade. 50 LIGHTING AND FOCUSING. TESTING A MICROSCOPE. § ioi. To be of real value this must be accomplished by a person with both theoretical and practical knowledge and also with an unprejudiced mind. Such persons are not common, and when one is found, does not show an over anxiety to pass judgment. Those most ready to offer advice should as a rule be avoided, for in most cases they simply “ have an ax to grind, ” and are sure to commend only those instruments that conform to the “ fad ” of the day. From the writer’s experience it seems safe to say that the inexperienced can do no better than to trust to the judgement of one of the optical companies. The makers of micro- scopes and objectives guard with jealous care the excellence of both the mechanical and optical part of their work and send out only instruments that have been carefully tested and found to conform to the standard. This would be done by them as a matter of business prudence on their part, but it is believed by the writer that microscope makers are artists first and take an artist’s pride in their work, they therefore have a stimulus to excellence greater than business prudence alone could give. § 102. Mechanical Parts.—All of the parts should be firm, and not too easily shaken. Bearings should work smoothly. The mirror should remain in any position in which it is placed. Focusing Adjustments.—The coarse or rapid adjustment should be by rack and pinion and work so smoothly that even the highest power can be easily focused with it. In no case should it work so easily that the body of the microscope is liable to run down and plunge the objective into the object. If any of the above defects appear in a microscope that has been used for some time a person with moderate mechanical instinct will be able to tighten the proper screws, etc. The Fine Adjustment is more difficult to deal with. From the nature of its purpose, unless it is approximately perfect it would better be off the microscope entirely. It should work smoothly and be so balanced that one cannot tell by the feeling when using it whether the screw is going up or down. Then there should be ab- solutely no motion except in the direction of the optic axis, otherwise the image will appear to sway even with central light. Compare the appearance when using the coarse and when using the fine adjustments. There should be no swaying of the image with either if the light is central (§ 72). § 103. Testing the Optical Parts.—As stated in the beginning, this can be done satisfactorily only by an expert judge. It would be of very great advantage to the student if he could have the help of such a person. In no case is the con- demnation of a microscope to be made by an inexperienced person. If the be- ginner will bear in mind that his failures are due mostly to his own lack of knowledge and lack of skill; and will truly endeavor to learn and apply the principles laid down in this and in the standard works referred to he will learn after a while to estimate at their true value all the pieces of his microscope. A LABORATORY COMPOUND MICROSCOPE. \ 104. A great deal of beginning work with the microscope in biological labora- tories is done with simple and inexpensive apparatus. Indeed if one contemplates the large classes in the universities and medical schools, it can be readily under- stood that microscopes costing from $30-50 each and magnifying from 25 to 500 51 LIGHTING AND FOCUSING. diameters, are all that can be expected. But for the purpose of modern histologi- cal investigation and of advanced microscopical work in general, a microscope should have something like the following character : Its optical outfit should comprise, (a) dry objectives of 50 mm. (2 in.), 16-18 mm. in.) and 3 mm. (}i in.) equivalent focus. There should be present also a 2 mm. in) or 1.5 mm. ( in.) homogeneous immersion objective. Of oculars there should be sev- eral of different power. An illuminator or substage condenser, and an Abbe cam- era lucida are also necessities. A micro-spectroscope and a micro-polarizer are very desirable. Even in case all the optical parts cannot be obtained in the beginning, it is wise to secure a stand upon which all may be used when they are finally secured. As to the objectives. The best that can be afforded should be obtained. Cer- tainly at the present, the apochromatics stand at the head, although the best achromatic objectives approach them very closely. Mechanical Parts or Stand.—The stand should be low enough so that it can be used in a vertical position on an ordinary table without inconvenience ; it should have a jointed (flexible) pillar for inclination at any angle to the horizontal. The adjustments for focusing should be two,—a coarse adjustment or rapid movement with rack and pinion, and a fine adjustment by means of a micrometer screw. Both adjustments should move the entire body of the microscope. The body or tube should be short enough for objectives corrected for the short or 160 milli- meter tube-length, and the draw-tube should be graduated in centimeters and millimeters. The lower end of the draw-tube and of the tube should each possess a standard screw for objectives (Fig. 20). The stage should be quite large for the examination of slides with serial sections ; it is also of considerable advantage to have the stage with a circular, revolving top, and two centering screws with milled heads. In this way a mechanical stage with limited motion is secured, and this is of the highest advantage in using powerful objectives. The sub-stage fittings should be so arranged as to enable one to dispense entirely with diaphragms, to use ordinary diaphragms, or to use the illuminator. The illuminator mounting should allow up and down motion, preferably by rack and pinion. The base should be sufficiently heavy and so arranged that the microscope will be steady in all positions, and interfere the least possible amount with the manipulation of the mirror and other sub-stage accessories. Fig. 46. Represents a microscope of the continental pattern that nearly fulfills the above specifications. With the outfit named the cost would be not far from $200. If the microspectroscope and micropolarizer were omitted the cost in Eu- rope would be about $115. Fortunately some of the optical companies in America furnish their materials at duty free rates to those allowed by law to import foreign instruments duty free. For Books and Periodicals treating the subjects of this chapter, see close of Ch. I. Fig. 46. A Laboratory microscops of the Continental Pattern, (Bausch & Lomb). CHAPTER III. INTERPRETATION OF APPEARANCES. APPARATUS AND MATERIAL FOR CHAPTER III. A laboratory, compound microscope ($ 104) ; Preparation of fly’s wing ; 50 per cent, glycerin ; Slides and covers ; Preparation of letters in stairs (Fig. 47); Mu- cilage for air-bubbles and olive or clove oil for oil-globules ($ 111). Solid glass rod, and glass tube (§ 119) ; Collodion ($ 121) ; Carmine, India ink, or lamp black ($ 123) ; Frog, castor oil and micro-polariscope ($ 126). interpretation of appearances under the microscope. § 105. 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 puzzled by appearances which are constantly met with in microscopical investigation. Any one can look into a microscope, but it is quite another matter to interpret correctly the meaning of the appearances seen. It is especially important to remember that the more of the relations of any object are known, the truer is the comprehension ot 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 ?is 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 in- crease in magnification 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. INTERPRETATION OF APPEARANCES. 53 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 evident with a higher power or a more perfect objective. Indeed the problems 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 watchfulness lest the appearances may be deceptive are thus admirably stated by Dallinger (See Carpenter-Dallinger, pp. 368-369). : “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 most practised 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 useful warning against hasty conclusions drawn from a too cursory examination. 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 con- ceivable 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 waste 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 may be proved fallacious at some future time, perhaps even by our own more extended and careful researches. The suspension of the judg- ment whenever there seems room for doubt is a lesson inculcated by all 54 INTERPRETATION OF APPEARANCES. 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 practise. ’ ’ § 106. Dust or Cloudiness on the Ocular.—Employ the 18 mm. (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 (§ 34). Eight the field well and focus sharply. The image 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 mi- croscope, and rotate it. The irregular object will rotate with the ocu- lar. Cloudiness or particles of dust on any part of the ocular, may be detected in this way. § 107. 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 gly- cerin 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 can- not be made clear by focusing. Then use a clean slide, and the image may be made clear by elevating the body slightly. The obscurity pro- duced in this way is like that caused by clouding the front-lens of the objective. 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. 35). If the diaphragm is lowered or a sufficiently large one employed the entire field will be lighted. § 108. Relative Position of Objects or parts of the same ob- ject.—The general rule is that objects highest up come into focus last in focusing Jirst \n focusing down. § 109. Objects Having Plane or Irregular Outlines.—As ob- ject use three printed letters mounted in stairs in Canada balsam (Fig. 47). The first letter is placed directly upon the slide, and covered with a 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 (§ 106). 55 INTERPRETATION OF APPEARANCES. Fig. 47. Letters mounted in stairs to show the order of coming into focus. a, b, c, d. The various letters indicated by the oblique row of black marks in the 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 middle one, etc. The relative position of objects is determined ex- actly in this way in practical work. § no. Transparent Objects Having Curved Outlines.—The suc- cess of these experiments will depend entirely upon the care and skill used in preparing the objects, in lighting, and in focusing. Employ a 5 111m. in.) or higher objective and a high ocular for all the experiments. It may be necessary to shade the object (§ 92) to get satisfactory results. When a diaphragm is used the opening should be small and it should be close to the object. § in. Air Bubbles.—Prepare these by placing a drop of thin muci- lage 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. 48. Diagram showing how to place a cover-glass upon an object with jine forceps. § 112. Air Bubbles with Central Illumination.—Shade the ob- ject ; and with the plane mirror, light the field with central light (Fig. 12). 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 (§ 51). Focus both up and down, noting, that in focusing up, the central 56 INTERPRETATION OF APPEARANCES. spot becomes very clear and the black ring very sharp. On elevating the body of the microscope still more the center becomes dim, and the whole bubble loses its sharpness of outline. § 113. Air Bubbles with Oblique Illumination..—Remove the sub-stage of the microscope (Fig. 20), and all the diaphragms. Swing the mirror so that the rays may be sent very obliquely upon the object '(Fig. 12, C). The bright spot will appear no longer in the center but ■on the side away from the mirror (Fig. 49). §114. Oil Globules.—Prepare these by beating a small drop of ■clove oil with mucilage on a slide and covering as directed for air bubbles (§ hi). § 115. Oil Globules with Central Illumination.—Use the same diaphragm and light as above (§ 112). Find an oil globule appearing about 1 mm. in diameter. If the light is central the bright spot will appear 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 focusing up. A Rig. 49. Very small Globule of Oil (O) and an Air Bubble (A) seen by Oblique Light. The arrow indicates the direction of the light rays. § 116. Oil Globules with Oblique Illumination.— Remove the sub-stage, etc., as above, and swing the mirror 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. 49). § 117. Oil and Air Together.—Make a preparation exactly as described for air bubbles (§ in), and add at one edge a little of the mixture of oil and mucilage (§ 114) ; cover and examine. o Fig. 50. Section of an air bubble and oil globule in water or thin mucilage to show that the burning point or focus of the air bubble is virtual, as it is in a denser medium; it thus acts like a con- cave lens in air (Fig. 9), and the focus of the oil globule is real, as it is denser than the water surrounding it. Axis. Principal axis. F. Principal focus, virtual for the air bubble and real for the oil. Hf). Water, or mixture of water and gum arabic, serving as a mounting medium. 57 INTERPRETATION OF APPEARANCES. The sub-stage need not be used in this experiment. Search the prepa- ration until an air bubble and an oil globule, each appearing about 1 mm. in diameter, are found in the same field of view. Tight first with cen- tral 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. 49.* § 118. Air and Oil by Reflected Light.—Cover the diaphragm or mirror so that no transmitted light (§ 50) can reach the preparation, using the same preparation as in §117. The oil and air will appear like globes of silver on a dark ground. The part that was darkest in each will be lightest, and the bright central spot will be somewhat dark.f § 119. 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 out- line as each differed greatly in refractive power from the medium which surrounded them, the oil being more refractive than the mucilage and the air less. (Fig. 39, 50). Place a fragment of a cover-glass on a clean slide, and cover it (see under mounting). The outline will be very distinct with the unaided eye. Use it as object and employ the 18 mm. 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 50 % glycerin. Put a cover-glass on each. Employ * It should be remembered that the image in the compound microscope is in- verted (Fig. 8), hence the bright spot really moves toward the mirror for air, and away from it for oil. f 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 observation. If an 18 mm. in.) is used instead of a 3 mm. in.) objective, the appearances 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 ap- pearances with objectives of small and of large aperture. 58 INTERPRETATION OF APPEARANCES. the same optical arrangement as before. Examine the one in air first. There will be seen a narrow, bright band, with a wide dark band on each side. The one in glycerin will show a much wider bright central band, with the dark borders correspondingly narrow (Fig. 51) 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 (Fig. 39, 51) that they cannot enter the objective and contribute to the formation of the image. Fig. 51. Solid glass rod showing the appearance when viewed with transmitted, central light and with an objective of medium aperature. a. Mounted dry or 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 eosine 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. § 120. Highly Refractive.—This expression is often used in describ- ing microscopic objects, (inedulated nerve fibres 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 (§ 119), it would be known that the refractive power of the object, and the medium in which it was mounted must differ considerably. § 121. 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) a light band, and (3) a second dark line (Fig. 52). This may be demonstrated by coating a fine glass rod (§ 119) with *Some of the rods have air bubbles in them, and then there results a capillar}- 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. 59 INTERPRETATION OF APPEARANCES. one or more coats of collodion or celloidin and allowing it to dry, and then mounting in 50 u/o glycerin as above. Employ a 5 mm. 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. 52.) Fig. 52. Solid glass rod coated with collodion to show a double contour. Toward one end the collodion had gathered in a fusiform drop. § 122. Optical Section.—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 may be viewed in optical section. A striking example will be found in studying mammalian red blood-copuscles on edge. The experiments with the solid glass rods (Fig. 51) furnish excellent and striking examples of optical sections. § 123. Currents in Liquids.—Employ the 18 mm. in.) objective, 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 pro- duced 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 (§§ 3, 39) ? Lamp-black rubbed in water containing a little mucilage answers well for this experiment. § 124. 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. 23 it will be seen that the actual size of the field of the microscope * The collodion used is a 5 per cent, solution of gun cotton in equal parts of sulphuric ether and 95 per cent, alcohol. It is well to dip the rod two or three times in the collodion 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 mem- branous covering (Fig. 52). 60 INTERPRETATION OF APPEARANCES. with the different objectives and oculars is inversely as the magnifica- tion. 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 studying the circulation of the blood. The truth of what has just been said can be easily demonstrated in stud)Ting the circulation in the gills of Necturus or in the frog’s foot by using first a low power in which the field is actually of considerable diameter (Fig. 23, Table, § 37) and then using a high power. With the high power the apparent mo- tion will appear much more rapid. For the form of motion, spiral, serpentine, etc. see Carpenter-Dallinger, p. 375. § 125. Pedesis or Brownian Movement.—Employ the same object as above, but a 5 mm. (A in.) or higher objective in place of the 18 mm. Make the body of the microscope vertical, so that there may be no cur- rents 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, molecular 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 of soap ; sulphuric acid and various saline compounds retard or check the motion. One of the best objects is pumice stone ground finely. In this the movement is so active that it is difficult to follow the course of single particles. Pedesis is ex- hibited by all solid matter if finely enough divided and in a suitable liquid. No adequate explanation of this phenomenon has yet been offered. See Carpenter-Dallinger 373, Beale 195, Jevons in Quart. Jour. Science, new series, Vol. VIII, (1878), p. 167. Robert Brown, 1828, A brief account of microscopical observations on the particles contained in the pollen of plants, and on the general existence of active molecules in organic and inorganic bodies. C. Aug. Sigm. Schultze, Mikroskopische Untersuchuugen fiber des Herrn Robert Brown Ent- deckung lebender, selbst in Feuer unzerstoerbarer Theilchen in alien Korpern, 1828. Compare the pedetic motion with that of a current by slightly inclin- ing the body of the microscope. The small particles will continue their independent leaping movements while they are carried along by the current. 61 INTERPRE TA TION OF APPEARANCES. § 126. Demonstration of Pedesis with the Polarizing Micro- scope.—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 specimen will do if it is soaked in water for some time, but a fresh specimen is more satisfac- tory). Turn the viscera to one side and observe the small whitish masses at the emergence of the spinal nerves. With fine forceps re- move 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 y% in.). In the field will be seen multitudes of crystals of carbonate of lime, the larger crystals are mo- tionless but the smallest ones exhibit marked pedetic movement. Use the micro-polariscope, light with great care and exclude all ad- ventitious light from the microscope by shading the object (§ 92) and also by shading the eye. Focus sharply and observe the pedetic mo- tion 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 mo- tionless crystals will shine continuously and a part will remain dark, but the small crystals between the large ones will shine for an instant, then disappear, only to appear again the next instant. This demon- stration is believed to furnish absolute proof that the pedetic movement is real and not illusory. § 127. Muscae Volitantes.—These specks or filaments in the eyes due to minute shreds or opacities of the vitreus 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 by looking at the brightly illuminated snow or other white surface. By studying them carefully it will be seen that they are somewhat movable and float 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. § 128. In addition to the above experiments it is very strongly recommended that the student follow the advice of Beale, p. 248, and examine first with a low then a higher power, mounted dry, then in 62 INTERPRETATION OF APPEARANCES. water, lighted with reflected light, then with transmitted light, the fol- lowing : 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, especially cat hairs. These are very liable to be present in preparations made in this laboratory. 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 different appearances due to the illuminator see Nelson, in Jour. Roy. Micr. Soc., 1891, pp. 90-105; and for the illusory appearances due to diffraction phenomena see Carpenter-Dallinger, p. 376. 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 and 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 investiga- tion 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. CHAPTER IV. MAGNIFICATION AND MICROMETRY. APPARATUS AND MATERIAL FOR THIS CHAPTER. Simple and compound microscope; Steel scale or rule divided to millimeters and |ths ; Block for magnifier and compound microscope (§ 130, 134); Dividers (§ I3°» I3I> x34) ! Stage micrometer (g 133) ; Wollaston’s camera lucida (g 134); Ocular micrometer (\ 149); Micrometer ocular ($ 151). Abbe camera lucida (5 l6°)- § 129. The Magnification, Amplification or Magnifying Power of a microscope or any of its parts is the number obtained by dividing any linear dimension of the image by the corresponding linear dimen- sion of the object magnified. For example, if the image of some ob- ject is 40 mm. long, and the actual length of the object magnified is 2 mm. the magnification is 40-5-2=20. Magnification is expressed in diameters or times linear, that is but one dimension is considered. In giving the scale at which a microsco- pical 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. MAGNIFICATION OF A SIMPLE MICROSCOPE. § 130. The Magnification of a Simple Microscope is the ratio between the object magnified (Fig. 3, A'B'), and the virtual image (A3B3). To obtain the size of this virtual image place the tripod magnifier near the edge of a support of such a height that the distance from the upper surface of the magnifier to the table is 250 millimeters. As object, place a scale of some kind ruled in millimeters on the sup- port under the magnifier. Put some white paper on the table at the base of the support, and on the side facing the light. 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 (§ 4). Open the closed eye, and the image of the rule will appear as 64 MAGNIFICATION AND MICROMETRY. 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 binocular or double vision in which the microscopic image is seen with one eye and the dividers with the other, the two images appearing to be fused in a single visual field. § 131. Measuring the Spread of Dividers—This should be done on a steel scale divided to millimeters and |ths. As £ mm. cannot be seen plainly by the unaided eye, place one arm of the dividers at a centimeter line, and then 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 3, A3 B3), and as the size of the ob- ject is known, the magnification is determined by dividing the size of 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 be- tween the two lines of the object is 2 millimeters, then the magnifica- tion must be 15 2 — 7)4. That is, the image is 7)4 times as long or wide as the object. In this case the image is said to be magnified 7)4 diameters, or 7)4 times linear. The magnification of any simple magnifier may be determined ex- perimentally in the way described for the tripod. MAGNIFICATION OF A COMPOUND MICROSCOPE. § 132. The Magnification of a Compound Microscope is the ratio between the final or virtual image (Fig. 8, B3 A3), and the object magnified (A B). The determination of the magnification of a compound microscope may be made as with a simple microscope (§ 130), but this is very fatiguing and unsatisfactory. § 133. Stage, Object or Objective Micrometer.—For determining the magnification of a compound microscope and for the purposes 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 on glass is most convenient. The spaces between the lines should be and yj-y millimeter, and when high powers are to be used the lines should be very fine. It is of advantage to have the coarser lines filled with graphite (plumbago), especially when low powers are to be used. If one has an uncovered micrometer 65 MA GNIFICA TION AND MICRO ME TR Y. the lines may be very readily filled by rubbing some of the plumbago on the surface with the end of a cork; the super- fluous plumbago may be removed by using a clean dry cloth or a piece of lens paper. After the lines are filled and the plum- bago wiped from the surface, the slide should be examined and if it is found satisfactory, i. e., if the lines are black, a cover-glass on which is a drop of warm balsam may be put over the lines to protect them. § 134. 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 image is very readily determined (Fig. 54, 55, § 136). Employ the 18 mm. in.) objective and a 50 mm. (2 in., A or No. 1) ocular and stage micrometer as object. For this power the T\j-th mm. spaces of the micrometer should be used as object. Focus sharply, and make the body of the microscope horizontal, by bending the flexible Fig. 53. Diagram of a stage micrometer, with a ring on the lines to facilitate finding them. Fig. 54. Wollaston's Camera Luci- da, showing the rays from the micro- scope and from the drawing surface, and the position of the pupil of the eye. Axis, Axis. Axial rays from the microscope and from the drawing sur- face (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 mi- croscope and their projection on the drawing surface. CD. Rays from the drawing surface to the eye. A D, A' D'. Overlapping portion of the two fields, where both the microscopic image and the drawing surface, pencil, etc., may both be seen. It is represented by the shaded part in the overlapping circles at the right. Ocular. The occular of the microscope. P. The dra wing pencil. Its point is shown in the overlapping fields. Fig. 54. 66 MAGNIFICATION AND MICROMETRY. pillar, being careful not to bring any strain upon the fine adjustment (Fig. 20). Put a Wollaston’s camera lucida (Ch. V.) in position, and turn the ocular around if necessary so that the broad flat surface may face di- rectly upward as shown in Fig. 54. Elevate the microscope by putting a block under the base, so that the perpendicular distance from the up- per surface of the camera lucida to the table is 250 mm. (§ 136). 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. Took directly down. The image will appear 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 (§ 130, 131). Thus : Suppose two of the Tyth 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 T2ytlis of a millimeter and the magnified image is 9$ millimeters the magnifi- cation (which is the ratio between size of object and image) must be 9f-7-T27—47. That is, the magnification is 47 diameters or 47 times linear. If the fractional numbers in the above example trouble the stu- dent, both may be reduced to the same denomination, thus : If the size of the image is found to be 9% mm. this number may be reduced to tenths mm. so it will be of the same denomination as the object. In 9 mm. there are 90 tenths, and in f there are 4 tenths, then the whole length of the image is 90-1-4=94 tenths of a millimeter. The object is 2 tenths of a millimeter, then there must have been a magnification of 94 -T- 2 =47 diameters in order to produce an image 94 tenths, of a mil- limeter long. Put the 25 mm. (1 in. C or No 4) ocular in place of one of 50 mm. Focus, and then put the camera lucida in position. Measure the size of the image with dividers and a rule as before. The power will be considerably greater than when the low ocular was used. This is be- cause the virtual image (Fig. 8, B3A3), seen with the high ocular is larger than the one seen with the low one. The real image (Fig. 8, A’B1), remains nearly the same, and would be just the same if positive, par-focal oculars (§ 24,58 note), were used. Eengthen the body of the microscope 50-60 mm. by pulling out the draw-tube. Remove the camera lucida, and focus, then replace the camera, and obtain the magnification. It will be greater than with the shorter body. This is because the real image (Fig. 8, A’B1) is formed farther from the objective when the body is lengthened, and being 67 MAGNIFICATION AND MICROMETRY. formed farther from the objective it must necessarily be larger (§ 7 and Fig. 54)- * § J35- Varying the Magnification of a Compound Microscope. It will be seen from the above experiments (§ 134) that independently of the distance at which the microscopic image is measured (§ 136), there are three ways of varying the power of a compound microscope. These are named below in the order of desirability. (1) By using a higher or lower objective. (2) By using a higher or lower ocular. (3) By lengthening or shortening the tube of the microscope.* Fig. 55. Figure showing the position of the microscope, the camera lucida, and the eye, and the different sizes of the image depending upon the distance at which it is projected from the eye. {a) The size at 25 cm.; {b) at 35 cm. (| 136). § 136. Standard Distance of 250 Millimeters at which the Vir- tual Image is Measured.—For obtaining the magnification of both the simple and the compound microscope the directions were to measure the virtual image at a distance of 250 millimeters. This is not that the image could not be seen and measured at any other distance, but be- cause some standard must be selected, and this is the most common one. The necessity for the adoption of some common standard will be seen at a glance in Fig. 55, where is represented graphically the fact that the size of the virtual image depends directly on the distance at which it is projected, and this size is directly proportional to the verti- cal distance from the apex of the triangle, of which it forms a base. The distance of 250 millimeters has been chosen on the supposition that it is the distance of most distinct vision for the normal human eye. * Amplifier.—In addition to the methods of varying the magnification given in \ I35> the magnification is sometimes increased by the use of an amplifier, that is a diverging lens or combination placed between the objective and ocular and serv- ing to give the image-forming rays from the objective an increased divergence. An effective form of this accessory was made by Tolies, who made it as a small achromatic concavo-convex lens to be screwed into the lower end of the draw-tube (Fig. 20) and thus but a short distance above the objective. The divergence given the rays increases the size of the real image about two fold. 68 MA GNIFICA TION AND MICRO ME TR Y. Demonstrate the difference in magnification due to the distance at which the image is projected, by raising the microscope so that the dis- tance will be 350 millimeters, then lowering to 150 millimeters. In preparing drawings it is often of great convenience to make them at a distance somewhat lessor somewhat greater than the standard. In such a case the magnification must be determined for the special dis- tance. (See the next chapter.) For discussions of the magnification of the microscope, see : Beale, pp. 41, 355; Carpenter-Dallinger, pp 26, 238; Nageli and Schwen- dener, p. 176; Ranvier, p. 29; Robin, p. 126; Amer. Soc. Micrs., 1884, p. 183 ; 1889, p. 22 ; Amer. Jour. Arts and Sciences, 1890, p. 50 ; Jour. Roy. Micr. Soc., 1888, 1889. § 137. Table of Magnifications and of the Valuations of the Ocular Micrometer.— The following table should be filled out by each student. In using it for Micrometry and Drawing it is necessary to keep clearly in mind the exact conditions under which the determinations were made, and also the ways in which variation in magnification and the val- uation of the ocular micrometer 7nay be produced (§ 135, 136, 151, 154). OCULAR OCULAR 50 mm. 25 mm. Objective. Tube in Tube OUT MM. Tube in Tube OUT MM. Ocuear Micrometer Vaeuation. tube in. out MM. X X X X X X X X X X X X • X X X X X X X X X X X X Simpee Microscope. X 69 MAGNIFICATION AND MICROMETRY. EQUIVALENT FOCUS OF OBJECTIVES AND OCULARS. § 138. 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 optican. 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 varies directly as their distance from the centre of the lens. ” By referring to Fig. 1, 3, 8. 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. I 139. Determination of Equivalent Focus of Objectives.—Took into the upper end of the objective and locate the position of the back lens. Indicate the level in someway on the outside of the objective. This is not the center of the object- ive but serves as an arbitrary approximation. Screw the objective into the body of the microscope. Remove the field lens from a micrometer ocular, thus making a positive ocular of it (Fig. 8). Pull out the draw-tube until the distance between the ocular micrometer and the back lens is 250 millimeters. Use a stage microm- eter as object and focus carefully. Make the lines of the two micrometers parallel (Fig. 56). 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'B1 Fig. 8, 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 (r2ff mm.), that of the image directly measured (jjj 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 : 250. Supplying the known values, O = x%, I = then T25 m : 1 mm : : F : 250 whence F = 50 mm. That is, the equivalent focus is approximately 50 millimeters. § 140. Determination of Initial or Independent Magnification of the Objective. —The initial magnification means simply the magnification of the real image (A1 B1, Fig. 8) unaffected by the ocular. It may be determined experimentally exactly as described in \ 139. For example, the image of the object 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 approxi- mately five. Knowing the equivalent focus of an objective, one can determine its initial mag- nification by dividing 250 mm. by the equivalent focus in millimeters. Thus the initial magnification of a 5 mm. objective is = 50; of a 3 mm., = 83.3 ; of a 2 mm., = 125, etc. § 141. Determining the Equivalent Focus of an Ocular.—If one knows the ini- tial magnification of the objective (§ 140) 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 posi- 70 MAGNIFICATION AND MICROMETRY. tion 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 § 139, 140, then by the aid of Wollaston’s camera lucida the magnification of the whole microscope is obtained, as described in \ 134. As the initial power of the objective is known, the power of the whole microscope must be due to that initial power multiplied by the power of the ocular, the ocular acting like a simple microscope to magnify the real image (Fig. 8). Suppose one has a 50 mm. objective, its initial power will be 5. If with this ob- jective and an ocular of unknown equivalent focus, the magnification of the whole microscope is 25, then the real image or initial power of 5 must have been multi- plied by 5 to produce the final power. If the ocular multiplies the real image five fold, using the same formula as in \ 139, 0 = 5: /= 25 : : F: 250, whence 25 F — 1250 O or F= 50. Conversely, if one knows the equivalent focus of the ocular, the initial power, as with objectives, may be obtained by dividing 250 by the equiv- alent focus. Thus the initial power of an ocular of 50 mm. equivalent focus is -275ff°- = 5 ; if the ocular is of 25 mm. focus, W°- = 10, etc. g 142. Tables of Magnifications with different Oculars and Objectives Fur- nished by Opticians in their Catalogs.—These tables are prepared by multiply- ing together the initial power of objective and ocular in each case. For example, what is the power of a 2 in. or 50 mm. objective with a 2 in. or 50 111m. ocular ? In this case the initial magnification of each is 5, hence the total magnification, i. e., the virtual image (Fig. 8, A3 B3) will be 25, etc. 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 Microscopic Ob- jectives, Franklin Inst. Jour., 1870, pp. 401-402 ; Monthly Micr. Jour., 1870, pp. 149-159. J. J. Woodward, on the Nomenclature of Achromatic Objectives, Amer. Jour. Science, 1872, 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, 1893, p. 142. MICROMETRY. § 143. Micrometry is the determination of the size of objects by the aid of a microscope. MICROMETRY WITH THE SIMPLE MICROSCOPE. § 144- With a simple microscope, (A) the easiest and best way is to use dividers and then the simple microscope to see when the points of the dividers exactly include the object. The spread of the dividers is then obtained as above (§ 131). This amount will be the actual size of the object, as the microscope was only used in helping to see when the divider points exactly enclosed the object, and then for reading the di- visions on the rule in getting the spread of the dividers. (B) One may put the object under the simple microscope and then as in determining the power (§ 130), measure the image at the standard distance. If now the size of the image so measured is divided by the MAGNIFICATION AND MICROMETRY. magnification of the simple microscope, the quotient will give the actual size of the object. Use a fly’s wing or some other object of about that size and try to determine the width in the two wdys described above. If all the work is accurately done the results will agree. MICROMETRY WITH THE COMPOUND MICROSCOPE. There are several ways of varying excellence for obtaining the size of objects with the compound microscope, the method with the ocular micrometer (§ 154,155) being most accurate. § 145. Unit of Measure in Micrometry.—As most of the objects measured with the compound microscope are smaller than any of the originally named divisions of the meter, and the common or decimal fractions necessary to express the size are liable to be unnecessarily cumbersome, Harting, in his work on the microscope (1859), proposed the one thousandth of a millimeter mm- or 0.001 mm.) or one millionth of a meter (tttowot or 0.000001 meter) as the unit. He named this unit micro-millimeter and designated it mmm. In 1869, Listing (Carl’s Repetorium fiir Experimental-Pliysik, Bd, X, p. 5) favored the thousandth of a millimeter as unit and introduced the name Mikron or micrum. In English it is most often written Micron, plural micra or microns, pronunciation Mic'ron, or Mic'rdn. By universal con- sent the sign or abbreviation used to designate it is the Greek p.. Adopting this unit and sign, one would express five thousandths of a millimeter (Y7'A mm ocular. The prism turns off the ocular, thus giving an unobstructed view into the microscope. The mirror is supplied with quadrant for determining its angle, and instead of a series of smoked glasses, a smoked glass wedge is made to be inserted between mirror and prism. Any darkening effect de- sired may be obtained by sliding the wedge along. To avoid distortion, the smoked glass wedge is combined zoith a similar clear glass one, as shown in the figure. (Bausch & Lomb). (E) If a drawing of a given size is desired and it cannot be obtained by any combination of oculars, objectives and lengths of the body of the microscope, the distance between the'camera hieida and the table be increased or diminished until the image is of the desired size. The image of a few spaces of the micrometer, will give the scale of enlarge- ment, or the power may be determined for the special case (§ 165-166). (F) It is of the greatest advantage, as suggested by Heinsius (Zeit. 86 DR A WING WITH THE MICROSCOPE. w. 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. Any skilled mechanic can add the quadrant. § 165. Magnification of the Microscope and Size of Drawings with the Abbe Camera Lucida.—In determining the standard dis- tance 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. 57) to the axis of the mirror and then vertically to the drawing board. In getting the scale at 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, 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 width. 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 micrometer were millimeter, then the enlargement would be 25 tw = 5°°- That is, the image was drawn at a magnification of 500 diameters. If the micrometer scale is used with 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 magnification 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. § 166. 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 may 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 87 DR A WING WITH THE MICROSCOPE. draw a scale on the figure under the same conditions, its enlargement can be readily determined (§ 165). If one has many large objects to draw at a low magnification, then some form of embryograph is very convenient. The writer has made use of a photographic camera and different photographic objectives for the purpose. The object is 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. Nothing is then easier than to trace the outlines of the object. See also Ch. VIII. references. Beale, 3i) 355 i Behrens, Kossel and Scliiefferdecker, 77; Carpenter-Dallinger, 233; Van Heurck, 91 ; American Naturalist, 1886, p. 1071, 1887, pp. 1040-1043; Amer. Monthly Micr. Jour., 1888, p. 103, 1890, p. 94; Jour. Roy. Micr. Soc., i88ir p. 819, 1882, p. 402, 1883, pp. 283, 560, 1884, p. 115, 1886, p. 516, 1888, pp. 113, 809, 798; Zeit. wiss. Mikroskopie, 1884, pp. 1-21, 1889, p. 367, 1893, pp. 289-295. Here is described an excellent apparatus made by Winkel. CHAPTER VI. MICRO-SPECTROSCOPE AND POEARISCOPE. APPARATUS AND MATERIAL REQUIRED FOR THIS CHAPTER. Compound microscope; Micro-spectroscope ($ 167); Watch-glasses and small vials, slides and covers (§ 186) ; Various substances for examination (as blood and ammonium sulphide, permanganate of potash, chlorophyll, some colored fruit, etc. (§ 187—r8S) ; Micro-polarizer (§ 197); Selenite plate (§ 204 F.); Various doubly refracting objects, as crystals, textile fibers, starch, section of bone, etc. MICRO-SPECTROSCOPE. # 167. A Micro-Spectroscope, Spectroscopic or Spectral Ocular, is a direct vision spectroscope in connection with a microscopic ocular. The one devised by Abbe and made by Zeiss consists of a direct vision spectroscope prism of the Amici pattern, and of considerable dispersion, placed over the ocular of the micro- scope. This direct vision or Amici prism consists of a single triangular prism of heavy dint glass in the middle and one of crown glass on each side, the edge of the crown glass prisms pointing toward the base of the flint glass prism, i. e., the edges of the crown and flint glass prisms point in opposite directions. The flint glass prism serves to give the dispersion or separation into colors, while the crown glass prisms serve to make the emergent rays approximately parallel with the in- cident rays, so that one looks directly into the prism along the axis of the micro- scope. The Amici prism is in a special tube which is hinged to the ocular and held in position by a spring. It may be swung free of the ocular. In connection with the ocular is the slit mechanism and a prism for reflecting horizontal rays verti- cally for the purpose of obtaining a comparison spectrum (§ 180). Finally near the top is a lateral tube with mirror for the purpose of projecting an Angstrom scale of wave lengths upon the spectrum ($ 181). $ 168. Apparent Reversal of the Position of the Colors in a Direct Vision Spec- troscope.—In accordance with the statements in § 167 the dispersion or separation into colors is given by the flint glass prism or prisms and in accordance with the general law that the waves of shortest length, blue, etc., will be bent most, conse- quently the colors have the position indicated in the top of Fig. 68, also above Fig. 69. But if one looks into the direct vision spectroscope or holds the eye close to the single prism (Fig. 69), the colors will appear reversed as if the red were more bent. The explanation of this is shown in Fig. 69, where it can be readily seen that if the eye is placed at E, close to the prism, the different colored rays will ap- pear in the direction from which they reach the eye and consequently are crossed in being projected into the field of vision and the real position is inverted. The 89 MICRO-SPECTROSCOPE AND POLARISCOPE. same is true in looking into the micro-spectroscope. The actual position of the different colors may be determined by placing some ground glass or some of the lens-paper near the prism and observing with the eye at the distance of distinct vision.* Fig. 66. Various Spectrums.—All except that of Sodium were obtained by dif- fused day-light with the slit of such a width as gave the most distinct Fraunhofer lines. It frequently occurs that with a substance giving several absorptions bands (e. g., chlorophyll) the density or thickness of the solution must be varied to show all the different bands clearly. Solar Spectrum. — With diffused day light and a narrow slit the spectrum is not visible much beyond the fixed line B. In order to extend the visible spectrum in the red to the line A, one should use direct sunlight arid a piece of ruby glass in place of the watch glass in Fig. 68. Sodium Spectrum.—The line spectrum , (\ 170) of sodium obtained by lighting the microscope with an alcohol flame in which some salt of sodium is glowing. With the micro-spectroscope the sodium line seen in the solar spectrum and with the incandescent sodium appears single, except under very favorable circumstances ($ 181). By using a comparison spectrum of day-light with the sodium spectrum the light and dark D-line will be seen to be continuous as here shown. Permanganate of Potash.—This spectrum is characterized by the presence of five absorption bands in the middle of the spectrum and is best shown by using a y'g- per cent, solution of permanganate in water in a watch glass as in Fig. 68. Met-hemoglobin.—The absorption spectrum of met-hemoglobin is character- ized by a considerable darkening of the blue end of the spectrum and of four ab- sorptions bands, one in the red near the line C and two between D and E nearly in the place of the two bands of oxy-hemoglobin ; finally there is a somewhat faint, wide band near F. Such a met-hemoglobin spectrum is best obtained by making a solution of blood in water of such a concentration that the two oxy-hemoglobin bands run together (\ 189), and then adding three or four drops of a j-’g per cent, aqueous solution of permanganate of potash. Soon the bright red will change to a brownish color, when it may be examined. * The author wishes to acknowledge the aid rendered by Professor F. F. Nichols in the explanation offered in this section. 90 MICRO-SPECTROSCOPE AND POLARISCOPE. VARIOUS KINDS OF SPECTRA. By a spectrum is meant the colored bands appearing when light traverses a dis- persing prism or a diffraction grating, or is affected in any way to separate the different wave lengths of light into groups. When daylight or some good artificial light is thus dispersed one gets the appearance so familiar in the rainbow. $ 169. Continuous Spectrum.—In case a good artificial light or the electric light is used the various rainbow or spectral colors merge gradually into one another in passing from end to end of the spectrum. There are no breaks or gaps. \ 170. Line Spectrum.—If a gas is made incandescent the spectrum it produces consists, not of the various rainbow colors, but of sharp, narrow, bright lines, the color depending on the substance. All the rest of the spectrum is dark. These line spectra are very strikingly shown by various metals heated till they are in the form of incandescent vapor. \ 171. Absorption Spectrum.—By this is meant a spectrum in which there are dark lines or bands in the spectrum. The most striking and interesting of the absorption spectra is the Solar Spectrum, or spectrum of sun-light. If this is ex- amined carefully it will be found to be crossed by dark lines, the appearance be- ing as if one were to draw pen marks across a continuous spectrum at various levels, sometimes apparently between the colors and sometimes in the midst of a color. These dark lines are the so-called Fraunhofer Lines. Some of the princi- pal ones have been lettered with Roman capitals, A. B. C. D. E. F. G. H., com- mencing at the red end. The meaning of these lines was for a long time enig- matical, but it is now known that they correspond with the bright lines of a line spectrum (g 170). Forexample, if sodium is put in the flame of a spirit lamp it will vaporize and become luminous. If this light is examined there will be seen one or two bright yellow bands corresponding in position with D of the solar spectrum (Fig. 67). If now the spirit lamp-flame, colored by the incandescent sodium, is placed in the path of the electric light, and it is examined as before, there will be a continuous spectrum, except for dark lines in place of the bright sodium lines. That is, the comparatively cool yellow light of the spirit lamp cuts off or absorbs the intensely hot yellow light of the electric light; and although the spirit flame sends a yellow light to the spectroscope it is so faint in comparison with the electric light that the sodium lines appear dark. It is believed that in the sun’s atmosphere there are incandescent metal vapors (sodium, iron, etc.), but that they are so cool in comparison with the rays of their wave length in the sun that the cooler light of the incandescent metallic vapors absorbs the light of corresponding wrave length, and are, like the spirit lamp-flame, unable to make up the loss, and therefore the presence of the dark lines. | 172. Absorption Spectra from Colored Substances.—While the solar spectrum is an absorption spectrum,.the term is more commonly applied to the spectra ob- tained with light which has passed through or has been reflected from colored objects which are not self-luminous. It is the special purpose of the micro-spectroscope to investigate the spectra of colored objects which are not self-luminous, as blood and other liquids, various minerals, as monazite, etc. The spectra obtained by examining the light reflected from these colored bodies or transmitted through them, possess, like the solar spectrum dark lines or bands, but the bands are usually much wider and less sharply defined. Their number and position depend on the substance or its con- 91 MICRO-SPECTROSCOPE AND POLARISCOPE. stitution (Fig. 67), and their width, in part, upon the thickness of the body. With some colored bodies, no definite bands are present. The spectrum is simply restricted at one or both ends and various of the other colors are considerably lessened in intensity. This is true of many colored fruits. Fig. 67. Absorption Spectrum of Oxy-Hemoglobin or arterial blood (/) and of Hemoglobin or venous blood (2). (From Gamgee and MacMunn.) A, B, C, D, E, F, G, H. Some of the principal Fraunhofer lines of the solar spectrum ($ 171). .go, .80, .70, .60, .go, .40. Wave lengths in microns, as shown in Angstrom's scale ($181). It will be seen that the wave lengths increase toward the red and decrease toward the violet end of the spectrum. Red, Orange, Yellow, etc. Color regions of the spectrum. Indigo should come between the blue and the violet to complete the seven colors usually given. It was omitted through inadvertence. $ 173. Angstrom and Stokes Law of Absorption Spectra.—The wave lengths of light absorbed by a body when light is transmitted through some of its substance are precisely the waves radiated from it when it becomes self-luminous. For example, a piece of glass that is yellow when cool, gives out blue light when it is hot enough to be self-luminous. Sodium vapor absorbs two bands of yellow light (D lines); but when light is not sent through it, but itself is luminous and exam- ined as a source of light its spectrum gives bright sodium lines, all the rest of the spectrum being dark. \ 174. Law of Color.—The light reaching the eye from a colored, solid, liquid or gaseous body lighted with white light, will be that due to white light less the light waves that have been absorbed by the colored body. Or in other words, it will be due to the wave lengths of light that finally reach the eye from the object. For example, a thin layer of blood under the microscope will appear yellowish green, but a thick layer will appear pure red. If now these two layers are exam- ined with a micro-spectroscope, the thin layer will show all the colors, but the red end will be slightly, and the blue end considerably restricted, and some of the colors will appear of considerably lessened intensity. Finally there may appear two shadow-like bands, or if the layer is thick enough, two wrell-defined dark bands in the green (§ 189). If the thick layer is examined in the same way, the spectrum will show only red with a little orange light, all the rest being absorbed. Thus the spectroscope shows which colors remain, in part or wholly, and it is the mixture of this remain- ing or unabsorbed light that gives color to the object. \ 175. Complementary Spectra.—While it is believed that Angstrom’s law ($ 173) is correct, there are many bodies on which it cannot be tested, as they change in 92 MICRO-SPECTROSCOPE AND POLARISCOPE. chemical or molecular constitution before reaching a sufficiently high temperature to become luminous. There are compounds, however, like those of didymium, erbium and terbium, which do not change with the heat necessary to render them luminous, and with them the incandescence and absorption spectra are mutually complementary, the one presenting bright lines where the other presents dark ones (Daniell). Fig. 68. Fig. 69. Fig. 70. Fig. 68 (i). Section of the tube and stage of the microscope with the special ocu- lar or micro-spectroscope in position. Amici Prism (£ 167).—The direct vision prism of Amici in which the central shaded prism of flint glass gives the dispersion or separation into colors, while the MICRO-SPECTROSCOPE AND POLARISCOPE. end pnsms of crown glass cause the rays to emerge approximately parallel with the axis of the microscope. A single ray is represented as entering the prism and this is divided into three groups (Red, Yellow, Blue), which emerge from the prism, the red being least and the blue most bent toward the base of the flint prism (see Fig. 69). Hinge.—The hinge on which the prism tube turns when it is swung off the ocular. Ocular (\ 167).— The ocular in which the slit mechanism takes the place of the diaphragm (l 177). The eye-lens is movable as in a micrometer ocular, so that the slit may be accurately focusedfor the different colors (§ 179). 6*. Screw for setting the scale of wave lengths ($ 181). S'. Screw for regulating the width of the slit ($ 177). S". Screw for clamping the micro-spectroscope to the tube of the microscope. Scale Tube.—The tube near the upper end containing the Angstrom scale and the lenses for projecting the image upon the upper face of the Amici prism, whence it is reflected upward to the eye with the different colored rays. At the right is a special mirror for lighting the scale. For arranging and focusing the scale, see \ 181. Slit.— The linear ope7iing between the knife edges. Through the slit the light passes to the prism. It must be arranged parallel with the refracting edge of the Prism, and of such a width that the Fraunhofer or Fixed Lines ate very clearly and sharply defined when the eye-lens is properly focused ($ 177-179). Stage.—The stage of the microscope. This supports a watch-glass with sloping sides for containing the colored liquid to be examined. (3) Comparison Prism with tube for coloi'ed liquid (C. L.), and mirror. The prism reflects horizontal rays vertically, so that when the prism is made to cover part of the slit two parallel spectra may be seen, one from light sent directly through the entire microscope and one from the light reflected upward from the comparison prism. (4) View of the Slit Mechanism from below.—Slit, the linear space between the knife edges through which the light passes. P. Comparison prism beneath the slit and covering part of it at will. S. S'. Screws for regulating the width and length of the slit. Fig. 69. Flint-Glass Prism showing the separation or dispersion of white light into the three groups of colored rays (Red, Yellow, Blue), the blue rays being bent the most from the refracting edge (\ 168). Fig. 70. Sectional View of a Microscope with the Polariscope in Position (§ 197- 204). Analyzer and Polarizer.—They are represented with corresponding faces paral- lel so that the polarized beam could traverse freely the analyzer. If either nicol were rotated 90° they would be crossed and 710 light would traverse the analyzer wi- less some polarizing substance were used as object (§198). (a) Slot in the analyzer titbe so that the a7ialyzer may be raised or lowered to adjust it for differe7ice of level of the eye-p>oint in differe7it oculars (g 200). Pointer and Scale.— The pointer attached to the analyzer a7id the scale or divided circle clamped (by the screw S) to the tube of the microscope. The pointer and scale enable one to determine the exact amourit of rotation of the analyzer (\ 199). Object.—The object whose character is to be investigated by polarized light. 93 MICRO-SPECTROSCOPE AND POLARISCOPE. ADJUSTING THE MICRO-SPECTROSCOPE. § 176. The micro spectroscope or spectroscopic ocular is put in the place of the ordinary ocular of the microscope, and clamped to the top of the tube by means of a screw for the purpose. § 177. Adjustment of the Slit.—In place of the ordinary dia- phragm with circular opening, the spectral ocular has a diaphragm composed of two movable knife edges by which a slit-like opening of greater or less width and length may be obtained at will by the use of screws for the purpose. To adjust the slit depress the lever holding the prism-tube in position over the ocular, and swing the prism aside. One can then look into the ocular. The lateral screw should be used and the knife edges approached till they appear about half a millimeter apart. If now the Amici prism is put back in place and the micro- scope well lighted, one will see a spectrum by looking into the upper end of the spectroscope. If the slit is too wide, the colors will over- lap in the middle of the spectrum and be pure only at the red and blue ends ; and the Fraunhofer or other bands in the spectrum will be faint or invisible. Dust on the edges of the slit gives the appearance of longitudinal streaks on the spectrum. § 178. Mutual Arrangement of Slit and Prism.—In order that the spectrum may appear as if made up of colored bands going directly across the long axis of the spectrum, the slit must be parallel with the refracting edge of the prism. If the slit and prism are not thus mu- tually arranged, the colored bands will appear oblique and the whole spectrum may be greatly narrowed. If the colored bands are oblique, grasp the prism tube and slowly rotate it to the right or to the left until the various colored bands extend directly across the spectrum. § 179. Focusing the Slit.—In order that the lines or bands in the spectrum shall be sharply defined, the eye-lens of the ocular should be accurately focused on the slit. The eye lens is movable, and when the prism is swung aside it is very easy to focus the slit as one focused for the ocular micrometer (§151). If one now uses daylight there will be seen in the spectrum the dark Fraunhofer lines (Fig. 67 E. F., etc.). To show the necessity of focusing the slit, move the eye-lens down or up as far as possible, and the Fraunhofer lines cannot be seen. While looking into the spectroscope move the ocular lens up or down and when it is focused the Fraunhofer lines will reappear. As the different colors of the spectrum have different wave lengths, it is neces- sary to focus the slit for each color if the sharpest possible pictures are desired. It will be found that the eye-lens of the ocular must be farther from 94 MICRO-SPECTROSCOPE AND POLARISCOPE. the slit for the sharpest focus of the red end than for the sharpest focus of the lines at the blue end. This is because the wave length of red is markedly greater than for blue light. Longitudinal dark lines on the spectrum may be due to irregularity of the edge of the slit or to the presence of dust. They are most troublesome with a very narrow slit. § 180. Comparison or Double Spectrum.—In order to compare the spectra of two different substances it is desirable to be able to examine their spectra side by side. This is provided for in the better forms of micro-spectroscopes by a prism just below the slit, so placed that the light entering it from a mirror at the side of the drum shall be totally reflected in a vertical direction, and thus parallel with the rays from the microscope. The two spectra will be side by side with a narrow dark line separating them. If now the slit is well fo- cused and daylight be sent through the microscope and into the side to the reflecting or comparison prism, the colored bands and the Fraun- hofer dark lines will appear directly continuous across the two spectra. The prism for the comparison spectrum is movable and may be thrown entirety out of the field if desired. When it is to be used, it is moved about half way across the field so that the two spectrums. shall have about the same width. § 181. Scale of Wave Lengths.—In the Abbe micro-spectroscope the scale is in a separate tube near the top of the prism and at right angles to the prism-tube. A special mirror serves to light the scale, which is projected upon the spectrum by a lens in the scale-tube. This scale is of the Angstrom form, and the wave lengths of any part of the spectrum may be read off directly, after the scale is once set in the proper position, that is, when it is set so that any given wave length on the scale is opposite the part of the spectrum known by previous in- vestigation to have that particular wave length. The point most often selected for setting the scale is opposite the sodium lines where the wave length is, according to Angstrom, 0.5892 /u,. In adjusting the scale, one may focus very sharply the dark sodium line of the solar spectrum and set the scale so that the number 0.589 is opposite the sodium or D line, or a method that is frequently used and serves to illustrate § 171, is to sprinkle some salt of sodium (carbonate of sodium is good) in an alco- hol lamp flame and to examine this flame. If this is done in a dark- ened place with a spectroscope, one or two narrow bright bands will be seen in the yellow part of the spectrum. If now ordinary daylight is sent through the comparison prism, the bright line of the sodium will be seen to be directly continuous with the dark line at D in the solar spectrum (Fig. 67). Now, by reflecting light into the scale-tube 95 MICRO-SPECTROSCOPE AND POLAR/SCOPE. the image of the scale will appear on the spectrum, and by a screw just under the scale-tube, but in the prism-tube, the proper point on the scale (0.589 fx) can be brought opposite the sodium band. All the scale will then give the wave lengths directly. Sometimes the scale is ob- lique to the spectrum. This may be remedied by turning the prism- tube slightly one way or the other. It may be due to the wrong posi- tion of the scale itself. If so, grasp the milled ring at the distal end of the scale tube and, while looking into the spectroscope, rotate the tube until the lines of the scale are parallel with the Fraunhofer lines. It is necessary in adjusting the scale to be sure that the larger number 0.70 is at the red end of the spectrum. The numbers on the scale should be very clearly defined. If they do not so appear, the scale-tube must be focused by grasping the outer tube of the scale-tube and moving it toward or from the prism-tube until the scale is distinct. In focusing the scale, grasp the outer scale- tube with one hand and the prism tube with the other, and push or pull in opposite directions. In this way one will be less liable to injure the spectroscope. § 182. Designation of Wave Length.—Wave lengths of light are designated by the Greek letter X, followed by the number indicating the wave length in some fraction of a meter. With the Abbe micro- spectroscope the micron is taken as the unit as with other microscopical measurements (§ 147). Various units are in use, as the one hundred thousandth of a millimeter, millionths or ten millionths of a milli- meter. If these smaller units are taken, the wave lengths will be in- dicated either as a decimal fraction of a millimeter or as whole numbers. Thus, according to Angstrom, the wave length of sodium light is 5892 ten millionths mm. or 589.2 millionths, or 58.92 one hundred thousandths, or 0.5892 of one thousandth mm., or 0.5892 fi. The last would be indicated thus, X 0=0.5892 /a. § 183. Lighting for the Micro spectroscope.—For opaque objects a strong light should be thrown on them either with a concave mirror or a condensing lens. For transparent objects the amount of the sub- stance and the depth of color must be considered. As a general rule it is well to use plenty of light, as that from an Abbe illuminator with a large opening in the diaphragm, or with the diaphragm entirely re- moved. For very small objects and thin layers of liquids it may be better to use less light. One must tty both methods in a given case, and learn by experience. For many objects some good artificial light is better than daylight. In general, the objects giving absorption bands in the blue end of the spectrum are best shown by daylight, and those giving bands in the red end by lamp light. Furthermore, one should 96 MICRO-SPECTROSCOPE AND POLARISCOPE. be on his guard against confusing the ordinary absorption bands with the Fraunhofer lines when daylight is used. With lamp-light the Fraunhofer lines are absent and, therefore, not a source of possible con- fusion. The direct and the comparison spectrums should be about equally illuminated. One can manage this by putting the object requiring the greater amount of illumination on the stage of the microscope, and lighting it with the Abbe illuminator. § 184 Objectives to Use with the Micro-specStroscope. — If the material is of considerable bulk a low objective (18 to 50 mm.) is to be preferred. This depends on the nature of the object under examina- tion, however. In case of individual crystals one should use sufficient magnification to make the real image of the crystal entirely fill the width of the slit. The length of the slit may then be regulated by the screw on the side of the drum, and also by the comparison prism. If the object does not fill the whole slit the white light entering the spec- troscope with the light from the object might obscure the absorption bands. In using high objectives with the micro-spectroscope one must very carefully regulate the light (§ 48, 92), and sometimes shade the object. § 185. Focusing the Objective.—For focusing the objective the prism-tube is swung aside, and then the slit made wide by turning the adjusting screw at the side. When the slit is open, one can see objects when the microscope is focused as with an ordinary ocular. After an object is focused, it may be put exactly in position to fill the slit of the spectroscope, then the knife edges are brought together till the slit is of the right width ; if the slit is then too long it may be shortened by using one of the mechanism screws on the side, or if that is not suffi- cient, by bringing the comparison prism farther over the field. If one now replaces the Amici prism and looks into the microscope, the spec- trum is liable to have longitudinal shimmering lines. To get rid of these focus up or down a little so that the microscope will be slightly out of focus. § 186. Amount of Material Necessary for Absorption Spectra and its Proper Manipulation.—The amount of material necessary to give an absorption spectrum varies greatly with different substances, and can be determined only by trial. If a transparent solid is under investigation it is well to have it in the form of a wedge, then succes- sive thicknesses can be brought under the microscope. If a liquid sub- stance is being examined, a watch glass with sloping sides forms an excellent vessel to contain it, then successive thicknesses of the liquid can be brought into the field as with the wedge-shaped solid. Fre- 97 MICRO-SPECTROSCOPE AND POLAR/SCOPE. quently only a very weak solution is obtainable ; in this case it can be placed in a homoeopathic vial, or in some glass tubing sealed at the end, then one can look lengthwise through the liquid and get the effect of a more concentrated solution. For minute bodies like crystals or blood corpuscles, one may proceed as described in the previous section. MICRO-SPECTROSCOPE—EXPERIMENTS. § 187. Put the micro-spectroscope in position, arrange the slit and the Amici prism so that the spectrum shall show the various spectral colors going directly across it (§ 176-177) and carefully focus the slit. This may be done either by swinging the prism-tube aside and proceed- ing as for the ocular micrometer (§ 151), or by moving the eye-lens of the ocular up and down while looking into the micro-spectroscope until the dark lines of the solar spectrum are distinct. If they cannot be made distinct by focusing the slit, then the light is too feeble or the slit is too wide (§ 179). With the lever move the comparison prism across half the field so that the two spectra shall be of about equal width. § 188. Absorption Spectrum of Permanganate of Potash.—Make a solution of permanganate of potash in water of such a strength that a stratum 3 or 4 mm. thick is transparent. Put this solution in a watch-glass with sloping sides and put it under the microscope. Use a 50 mm. or 18 mm. objective and use the full opening of the illumina- tor. Tight strongly. Took into the spectroscope and slowly move the watch-glass into the field. Note carefully the appearance with the thin stratum of liquid at the edge and then as it gradually thickens on moving the watch-glass still farther along. Count the absorption bands and note particularly the red and blue ends. Compare carefully with the comparison spectrum (Fig. 66). § 189. Absorption Spectrum of Blood.—Obtain blood from a re- cently killed animal, or flame a needle, and after it is cool prick the finger two or three times in a small area, then wind a handkerchief or a rubber tube around the base of the finger and squeeze the finger with the other hand. Some blood will ooze out of the pricks. Rinse this off in a watch-glass partly filled with water. Continue to add the blood until the water is quite red. Place the watch-glass of diluted blood under the microscope in place of the permanganate, using the same objective, etc. Note carefully the spectrum. It would be advan- tageous to determine the wave length opposite the center of the dark bands. This may be done easily by setting the scale property as de- scribed in § 181. Make another preparation, but use a homoeopathic vial instead of a watch-glass. Cork the vial and lay it down upon the stage of the microscope. Observe the spectrum. It will be like that 98 MICRO-SPECTROSCOPE AND POLARISCOPE. in the watch-glass. Remove the cork and look through the whole length of the vial. The bands will be very much darker and if the solution is thick enough only red and a little orange will appear. Re- insert the cork and incline the vial so that the light traverses a very thin layer, then gradually elevate the vial and the effect of a thicker and thicker layer may be seen. Note especially that the two charac- teristic bands unite and form one wide band as the stratum of liquid thickens. Compare with the following : Add to the vial of diluted blood a drop or two of ammonium sul- phide, such as is used for a reducing agent in chemical laboratories. Shake the bottle gently and then allow it to stand for ten or fifteen minutes. Examine it and the two bands will have been replaced by a single less clearly defined band in about the same position. The blood will also appear somewhat purple. Shake the vial vigorously and the color will change to the bright red of fresh blood. Examine it again with the spectroscope and the two bands will be visible. After five or ten minutes another examination will show but a single band. Incline the bottle so that a very thin stratum may be examined. Note that the stratum of liquid must be considerably thicker to show the absorption band than was necessary to show the two bands in the first experiment. Furthermore, while the single band may be made quite black on thickening the stratum, it will not separate into two bands with a thinner stratum. In this experiment it is very instructive to have a second vial of fresh diluted blood, say that from the watch- glass, before the opening of the comparison prism. The two banded spectrum will then be in position to be compared with the spectrum of the blood treated with the ammonium sulphide. The two banded spectrum is of oxy hemoglobm or arterial blood, the single banded spectrum is of hemoglobin (sometimes called reduced hemoglobin) or venous blood, that is the respiratory oxygen is present in the two banded spectrum but absent from the single banded spec- trum. When the bottle was shaken the hemoglobin took up oxygen from the air and became oxy-liemoglobin, as occurs in the lungs, but soon the ammonium sulphide took away the respiratory oxygen thus reducing the oxy-hemoglobin to hemoglobin. This may be repeated many times (Fig. 67). § 190. Met-Hemoglobin.—The absorption spectrum of met-hemo- globin is characterized by a considerable darkening of the blue end of the spectrum and of four absorption bands, one in the red near the line C and two between D and E nearly in the place of the two bands of oxy-hemoglobin ; finally there is a somewhat faint, wide band near F. Such a met-hemoglobin spectrum is best obtained by making a solu- 99 MICRO-SPECTROSCOPE AND POLARISCOPE. tion of blood in water of such a concentration that the two oxy-hemo- globin bands run together (§ 146), and then adding three or four drops of a per cent, aqueous solution of permanganate of potash. Soon the bright red will change to a brownish color, when it may be ex- amined, (Fig. 66). § 191. Carbon Monoxide Hemoglobin (CO Hemoglobin).—To ob- tain this one may kill an animal, after anaesthetization, in illuminating gas, or one may allow illuminating gas to bubble through some blood already taken from the body. The gas should bubble through a minute or two. The oxygen will be displaced by carbon monoxide. This forms quite a stable compound with hemoglobin, and is of a bright cherry-red color. Its spectrum is nearly like that of oxy-hemo- globin, but the bands are farther toward the blue. Add several drops of ammonium sulphide and allow the blood to stand some time. No reduction will take place, thus forming a marked contrast to solutions of oxy-hemoglobin. By the addition of a few drops of glacial acetic acid a dark brownish red color is produced. § 192. Carmine Solution.—Make a solution of carmine by putting gram of carmine in 100 cc. of water and adding 10 drops of strong ammonia. Put some of this in a watch-glass or in a small vial and compare the spectrum with that of oxy-hemoglobin or carbon monoxide hemoglobin. It has two bands nearly in the same position, thus giv- ing the spectrum a striking similarity to blood. If now several drops, 15 or 20, of glacial acetic acid are added to the carmine, the bands re- main and the color is not very maikedly changed, while with either oxy-hemoglobin or CO-hemoglobin the color would be very markedly changed from the bright red to a dull reddish brown and the spectrum, if any could be seen, would be markedly different. Carmine and O-hemoglobin can be distinguished by the use of ammonium sulphide, the carmine remaining practically unchanged while the blood shows the single band of hemoglobin (§ 189). The acetic acid serves to dif- ferentiate the CO-hemoglobin as well as the O-hemoglobin. § 193. Colored Bodies not giving Distinctly Banded Absorp- tion Spectra.—Some quite brilliantly colored objects, like the skin of a red apple, do not give a banded spectrum. Take the skin of a red apple, mount it on a slide, put on a cover-glass and add a drop of water at the edge of the cover. Put the preparation under the microscope and observe the spectrum. Although 110 bands will appear, in some cases at least, yet the ends of the spectrum will be restricted and vari- ous regions of the spectrum will not be so bright as the comparison spectrum. Here the red color arises from the mixture of the unab- sorbed wave lengths, as occurs with other colored objects. I11 this 100 MICRO-SPECTROSCOPE AND POLARISCOPE. case, however, not all the light of a given wave length is absorbed, consequently there are no clearly defined dark bands, the light is simply less brilliant in certain regions and the red rays so predominate that they give the prevailing color. § 194. Nearly Colorless Bodies with Clearly Marked Absorp- tion Spectra.—In contradistinction to the brightly colored objects with no distinct absorption bands are those nearly colorless bodies and solu- tions which give as sharply defined absorption bands as could be de- sired. The best examples of this are afforded by solutions of the rare earths, didymium, etc. These in solutions that give hardly a trace of color to the eye give absorption bands that almost rival the Fraunhofer lines in sharpness. § 195. Absorption Spedtra of Colored Minerals.—As example take some monazite sand on a slide and either mount it in balsam (see Ch. VII), or cover and add a drop of water. The examination may be made also with the dry sand, but it is less satisfactory. Light well with transmitted light, and move the preparation slowly around. Absorp- tion bands will appear occasionally. Swing the prism-tube off the oc- ular, open the slit and focus the sand. Get the image of one or more grains directly in the slit, then narrow and shorten the slit so that no light can reach the spectroscope that has not traversed the grain of sand. The spectrum will be very satisfactory under such conditions. It is frequently of great service in determining the character of unknown mineral sands to compare their spectra with known minerals. If the absorption bands are identical, it is strong evidence in favor of the identity of the minerals. § 196. While the study of absorption spectra gives one a great deal of accurate information, great caution must be exercised in drawing conclusions as to the identity or even the close relationship of bodies giving approximately the same absorption spectra. The rule followed by the best workers is to have a known body as control and to treat the unknown body and the known body with the same reagents, and to dissolve them in the same medium. If all the reactions are identical then the presumption is very strong that the bodies are identical or very closely related. For example, while one might be in doubt between a solution of oxy- or CO-liemoglobin and carmine, the addition of ammonium sulphide would serve to change the double to a single band in the O-hemoglobin, and glacial acetic acid would enable one to dis- tinguish between the CO-blood and the carmine, although the ammonium sulphide would not enable one to make the distinction. Furthermore it is unsafe to compare objects dissolved in different media. The same objects as “ cyanine and aniline blue dissolved in 101 MICRO-SPECTROSCOPE AND POLARISCOPE. alcohol give a very similar spectrum, but in water a totally different one. ” “ Totally different bodies show absorption bands in exactly the same position (solid nitrate of uranium and permanganate of potash in the blue). ” (MacMunn). The rule given by MacMunn is a good one : * ‘ The recognition of a body becomes more certain if its spectrum consists of several absorption bands, but even the coincidence of these bands with those of another body, is not sufficient to enable us to infer chemical identity ; what enables us to do so with certainty is the fact : that the two solutions give bands of equal intensities in the same parts of the spectrum which undergo analogous changes on the addition of the same reagent. ’ ’ REFERENCES TO THE MICRO-SPECTROSCOPE AND SPECTRUM ANALYSIS. The micro-spectroscope is playing an ever increasingly important role in the spectrum analysis of animal and vegetable pigments, and of colored mineral and chemical substances, therefore a somewhat extended reference to literature will be given. Full titles of the books and periodicals will be found in the Bibli- ography at the end. * / Angstrom, Recherches sur le spectre solaire, etc. Also various papers in periodi- cals. See Royal Soc’s Cat’l Scientific Papers ; Anthony & Brackett; Beale, p. 269; Behrens, p. 139 ; Kossel und Schiefferdecker, p. 63 ; Carpenter, p. 104 : Browning, How to Work with the Spectroscope, and in Monthly Micr. Jour., II, p. 65 ; Daniell, Principles of Physics. The general principles of spectrum analysis are especially well stated in this work, pp. 435-455 ; Dippel, p. 277; Frey ; Gamgee, p. 91 ; Halliburton ; Hogg, p. 122 ; also in Monthly Micr. Jour., Vol. II, on colors of flowers; Jour. Roy. Micr. Soc., 1880, 1883 and in various other vols. ; Kraus; Lockyer; M’Kendrick ; MacMunn ; and also in Philos. Trans. R. S., 1886; Various vols. of Jour. Physiol. ; Nageli und Schwendener; Proctor; Ref. Hand- Book Med. Sciences, Vol. I, p. 577, VI, p. 516, VII, p. 426 ; Roscoe ; Schellen ; Sorby, in Beale, p. 269, also Proc. R. S., 1874, p. 31, 1867, p. 433 ; see also in the Scientific Review, Vol. V, p. 66, Vol. II, p. 419. The larger works on Chemistry and Physics may also be consulted with profit. Vogel, Spectrum analysis, also in Nature Vol. xix, p. 495 on absorption spectra. The bibliography in MacMunn is excellent and extended. MICRO-POLARISCOPE. \ 197. The micro-polariscope or polarizer is a polariscope used in connection with a microscope. The most common and typical form consists of two Nicol prisms, that is twTo somewhat elongated rhombs of Iceland spar cut obliquely and cemented together with Canada balsam. These Nicol prisms are then mounted in such a way that the light passes through them lengthwise, and in passing is divided into two rays of plane polarized light. The one of these rays obeying most nearly the ordinary law of refraction is called the ordinary ray, the one departing farthest from the law is called the extra-ordinary ray. These two rays are not only polarized, but 102 MICRO-SPECTROSCOPE AND POLARISCOPE. polarized in planes almost exactly at right angles to each other. The Nicol prism totally reflects the ordinary ray at the cemented surface so that only the extra- ordinary ray is transmitted. | 198. Polarizer and Analyzer.—The polarizer is one of the Nicol prisms. It is placed beneath the object and in this way the object is illuminated with polarized light. The analyzer is the other Nicol and is placed at some level above the ob- ject, very conveniently above the ocular. When the corresponding faces of the polarizer and analyzer are parallel, i. e., when the faces through which the oblique section passed are parallel, light passes freely through the analyzer to the eye. If these corresponding faces are at right angles, that is if the Nicols are crossed, then the light is entirely cut off and the two transparent prisms become opaque to ordinary light. There are then, in the complete revolution of the analyzer, two points, at o° aud 180°, where the corre- sponding faces are parallel and where light freely traverses the analyzer. There are also two crossing points of the Nicols, at 90° and 270°, where the light is ex- tinguished. In the intermediate points there is a sort of twilight. \ 199. Putting the Polarizer and Analyzer in Position.—Swing the diaphragm carrier of the Abbe illuminator out from under the illuminator, remove the disk diaphragm or open widely the iris diaphragm and place the analyzer in the dia- phragm carrier, then swing it back under the illuminator. Remove the ocular, put the graduated ring on the top of the tube aud then replace the ocular and put the analyzer over the ocular and ring. Arrange the graduated ring so that the in- dicator shall stand at o° when the field is lightest. This may be done by turning the tube down so that the objective is near the illuminator, then shading the stage so that none but polarized light shall enter the microscope. Rotate the analyzer until the lightest possible point is found, then rotate the graduated ring till the index stands at o°. The ring may then be clamped to the tube by the side screw for the purpose. Or more easily, one may set the index at o°, clamp the ring to microscope, then rotate the draw-tube of the microscope till the field is lightest. $ 200. Adjustment of the Analyzer.—The analyzer should be capable of moving up and down in its mounting, so that it can be adjusted to the eye-point of the ocular with which it is used. If on looking into the analyzer with parallel Nicols the edge of the field is not sharp, or if it is colored, the analyzer is not in a proper position with reference to the eye-point and should be raised or lowered till the edge of the field is perfectly sharp and as free from color as the ocular with the analyzer removed. \ 201. Objectives to Use with the Polariscope.—Objectives of the lowest power may be used and also all intermediate forms up to a 2 mm. homogeneous immer- sion. Still higher Objectives may be used if desired. In general, however, the lower powers are somewhat more satisfactory. A good rule to follow in this case is the general rule in all microscopic work, “ use the power that most clearly and satisfactorily shows the object under investigation. ” l 202. Lighting for Micro-Polariscopic Work.—Follow the general directions given in Chapter II. It is especially necessary to shade the object so that no un- polarized light can enter the objective, otherwise the field cannot be sufficiently darkened. No diaphragm is used over the polarizer for most examinations. Direct sunlight may be used to advantage with some objects, and as a rule the object would best be very transparent. That is, tissues, fibers, etc., should be mounted in balsam (Suffolk). 103 MICRO-SPECTROSCOPE AND POLARISCOPE. | 203. Purpose of a Micro-Polariscope.—The object of a micro-polariscope is to determine, in microscopic masses, one or more of the following points : (A) Whether the body is singly refractive, mono-refringent or isotropic, that is opti- cally homogeneous, as are glass and crystals belonging to the cubical system ; (B) Whether the object is doubly refractive or anisotropic, uniaxial or biaxial ; (C) Pleochroism ; (D) The rotation of the plane of polarization, as with solutions of sugar, etc. ; (E) To aid in petrology and mineralogy ; (F) To aid in the determi- nation of very minute quantities of crystallizable substances ; (G) For the pro- duction of colors. For petrological and mineralogical investigations the microscope should pos- sess a graduated, rotating stage so that the object can be rotated and the exact angle of rotation determined. It is also found of advantage in investigating objects with polarized light where colors appear, to combine a polariscopic and spectroscope (Spectro-Polariscope). MICRO—POLARISCOPE—EXPERIMENTS. § 204. Arrange the polarizer and analyzer as directed above ( § 199) and use an 18 mm. objective except when otherwise directed. (A) Isotropic or Singly Refractive Objects.—Iyight the micro- scope well and cross the Nicols, shade the stage and make the field as dark as possible ( § 198). As an isotropic substance, put an ordinary glass slide under the microscope. The field will remain dark. As an example of a crystal belonging to the cubical system and hence iso- tropic, make a strong solution of common salt (sodium chloride Na Cl.), put a drop 011 a slide and allow it to crystallize, put it under the micro- scope, remove the analyzer, focus the crystals and then replace the an- alyzer and cross the Nicols. The field and the crystals will remain dark. (B) Anisotropic or Doubly Refracting Objects.—Make a fresh preparation of carbonate of lime crystals like that described for pedesis (§ 126), or use a preparation in which the crystals have dried to the slide, use a 5 or 3 mm. objective, shade the object well, remove the an- alyzer and focus the crystals, then replace the analyzer. Cross the Nicols. In the dark field will be seen multitudes of shining crystals, and if the preparation is a fresh one in water, part of the smaller crys- tals will alternately flash and disappear. By observing carefulty, some of the larger crystals will be found to remain dark with crossed Nicols, others will shine continuously. This shows that the crystals are uniaxial. If the crystals are in such a position that the light passes through them parallel with the principal axis, the crystals are isotropic like the salt crystal and remain dark. If, however, the light traverses them in any other direction the ray from the polarizer is divided into two constitu- ents vibrating in planes at right angles to each other, and one of these 104 MICRO-SPECTROSCOPE AND POLARISCOPE. will traverse the analyzer, hence such crystals will appear as if self luminous in a dark field. The experiment with these crystals from the frog succeeds well with a 2 mm. homogeneous immersion. As further illustrations of anisotropic objects, mount some cotton fibers in balsam (Ch. VII) also some of the lens paper (§ 97). These furnish excellent examples of vegetable fibers. Striated muscular fibers are also very well adapted for polarizing ob- jects. As examples of biaxial crystals, crystallize some borax, or carbonate of lead on a slide as directed for the common salt, and use the crystals as object. As all these objects restore the light with crossed Nicols, they are sometimes called depolarizing. (C) Pleochroism.—This is the exhibition of different tints as the anal- yzer is rotated. An excellent subject for this will be found in blood crystals. (D) For the aid given by the polariscope in micro-chemistry, see (Ch. VII). (E) See works on petrology and mineralogy for the application of the micro-polarizer in those subjects. (F) For the production of gorgeous colors, a plate of selenite giving blue and yellow colors is placed between the polarizer and the object. If properly mounted, the selenite is very conveniently placed on the diaphragm carrier of the Abbe illuminator, just above the polarizer. It is very instructive and interesting to examine organic and inor- ganic substances with a micro-polarizer. If the objects enumerated in §128 were all examined with polarized light an additional means of de- tecting them would be found. references to the poeariscope and to the use of polarized EIGHT. Anthony & Brackett; Behrens, 133 ; Behrens, Kossel und Schiefferdecker; Car- noy, 61; Carpenter-Dallinger, 262, 269, 992 ; Daniell, 494 ; v. Ebener ; Gamgee ; Halliburton, 36, 272; Hogg, 133, 729; Rehmann; M’Kendrick; Nageli und Schwendener, 299; Quekett; Suffolk, 125 ; Valentin. Physical Review, I., p. 127. CHAPTER VII. SLIDES AND COVER-GLASSES, MOUNTING, LABELING AND STORING MICROSCOPICAL PREPARATIONS—EX- PERIMENTS IN MICRO-CHEMISTRY. APPARATUS AND MATERIAL FOR THIS CHAPTER. Microscope, compound and simple ( Ch. I ) ; Micro-Spectroscope and polari- scope ( Ch. VI) ; Slides and cover-glasses (§ 205, 206) ; Cleaning mixtures for glass ( \ 213 ) ; Alcohol and distilled or filtered water (§ 208) ; fine forceps for handling cover-glasses ( § 208, 222 ) ; Old handkerchiefs or lens paper ( § 97, 209 ). Paper boxes for storing cover-glasses ($ 209, 211 ) ; Cover-glass measurer; Mounting material,—Farrant’s solution, glycerin, glycerin-jelly and Canada balsam (§ 243, 246) ; Centering card and lined card for serial sections ($ 222) ; Net-micrometer for arranging minute objects like diatoms ($ 251) ; Labels, (f 250) ; Carbon ink for writing labels ($ 241) ; Writing diamond ($241) ; Shellac cement (£ 224, 249) ; Cabinet ($ 242) ; Reagents for experiments in micro-chemistry (§ 252). SLIDES AND COVER-GLASSES. § 205. Slides, Glass Slides or Slips, Microscopic Slides or Slips. —These are strips of clear flat glass upon which microscopic speci- mens are usually mounted for preservation and ready examination. The size that has been almost universally adopted for ordinary prepara- tions is 25 x 76 millimeters (1x3 inches). For rock sections, slides 25 x 45 mm. or 32 x 32 mm. are used ; for serial sections, slides 25 x 76 mm., 50x75 mm. or 37 x 87 mm. are used. For special purposes, slides of the necessary size are employed without regard to any con- ventional standard. Whatever size of slide is used, it should be made of clear glass and the edges should be ground. It is altogether false economy to mount microscopic objects on slides with uuground edges. § 206. Cleaning Slides.—For new slides a thorough rinsing in clean water with subsequent wiping with a soft towel, and then an old soft handkerchief, usually fits them for ordinary use. If they are not satis- factorily cleaned in this way, soak them a short time in 50 per cent, or 75 per cent, alcohol, let them drain for a few moments on a clean towel 106 MOUNTING AND LABELING. or on blotting paper and then wipe with a soft cloth. In handling the slides grasp them by their edges to avoid soiling the face of the slide. After the slides are cleaned, they should be stored in a place as free as possible from dust. For used slides, if only water, glycerin or glycerin jelly has been used on them, they may be cleaned with water, or preferably, warm water and then with alcohol if necessary. Where balsam or any oily or gummy substance has been used upon the slides they may be freed from the balsam, etc., by soaking them for a week or more, in one of the cleaning mixtures for glass. If they are first soaked in xylol, benzine or turpentine to dissolve the balsam, then soaked in the clean- ing mixture, the time required will be much shortened (§ 213) After all foreign matter is removed the slides should be very thoroughly rinsed in water to remove all the cleaning mixture. They may then be treated as directed for new slides. § 207. Cover-Glasses or Covering Glasses.—These are circular or quadrangular pieces of thin glass used for covering and protecting mi- croscopic objects. They should be very thin, Ty(I to T millimeter (see table, § 17). It is better never to use a cover-glass over mm. thick then the preparation may be studied with a 2 mm. oil immersion as well as with lower objectives. Except for objects wholly unsuited for high powers, it is a great mistake to use cover-glasses thicker than the working distance of a homogeneous objective (§ 47). Indeed if one wishes to employ high powers the thicker the sections the thinner should be the cover glass (see §. 235). The cover-glass should always be considerably larger than the object over which it is placed. § 208. Cleaning Cover-Glasses.— New cover-glasses should be put into a glass dish of some kind containing one of the cleaning mix tures (§ 213) and allowed to remain a day or longer. In putting them in, push one in at a time and be sure that it is entirely immersed, other- wise they adhere very closely, and the cleaning mixture is unable to act freely. Soiled covers should be left a week or more in the cleaning mixture. An indefinite sojourn in the cleaner does not seem to injure the slides or covers. After one day or longer, pour off the cleaning mixture into another glass jar, and rinse the cover-glasses, moving them around with a gentle rotary motion. Continue the rinsing until all the cleaning mixture is removed. One may rinse them occasionally,and in the meantime allow a very gentle stream of water to flow on them or they may be allowed to stand quietly and have the water renewed from time to time. When the cleaning mixture is removed rinse the covers MOUNTING AND LABELING. 107 well with distilled water, and then cover them with 50 per cent, to 75 percent, alcohol. §209. Wiping the Cover-Glasses.—When ready to wipe the cover-glasses remove several from the alcohol and put them on a soft dry cloth or on some of the lens paper to let them drain. Grasp a cover-glass by its edges, cover the thumb and index of the other hand with a soft clean cloth or some of the lens paper. Grasp the cover be- tween the thumb and index and rub the surfaces. In doing this it is necessary to keep the thumb and index well opposed on directly oppo- site faces of the cover so that no strain will come on it, otherwise the cover is liable to be broken. When a cover is well wiped, hold it up and look through it toward some dark object. The cover will be seen partly by transmitted and partly by reflected light, and any cloudiness will be easily seen. If the cover does not look clear, breath on the faces and wipe again. If it is not possible to get a cover clear in this way it should be put again into the cleaning mixture. As the covers are wiped, put them in a clean paper box. Handle them always by their edges, or use fine forceps. Do not put the fingers on the faces of the covers, for that will surely cloud them. § 210. Cleaning Large Cover-Glasses.—For serial sections and especially large sections, large quadrangular covers are used. These are to be put one by one into cleaning mixture as for the smaller covers and treated in every way the same. In wiping them one may proceed as for the small covers, but special care is necessary to avoid breaking them. A safe and good way to clean the large covers is to take two perfectly flat, smooth blocks considerably larger than the cover-glasses. These blocks are covered with soft clean cloth, or with several thick- nesses of the lens paper ; if now the cover-glass is placed on the one block and rubbed with the other the cover may be cleaned as by rubbing its faces with the cloth-covered finger and thumb. §211. Measuring the Thickness of Cover-Glasses.—It is of the greatest advantage to know the exact thickness of the cover-glass on an object; for, (a) One would not try to use objectives in studying the preparation of a shorter working distance than the thickness of the cover (§ 47) ; (b) In using adjustable objectives with the collar gradu- ated for different thicknesses of cover, the collar might be set at a favor- able point without loss of time; (c) For unadjustable objectives the thickness of cover may be selected corresponding to that for which the objective was corrected (see table § 17). Furthermore, if there is a variation from the standard, one may remedy it, in part at least, by 108 MOUNTING AND LABELING. lengthening the tube if the cover is thinner, and shortening it if the cover is thicker than the standard (§ 85). In the so-called No. 1 cover-glasses of the dealers in microscopical supplies, the writer has found covers varying from y mm. to y mm. To use cover-glasses of so wide a variation in thickness without know- ing whether one has a thick or thin one is simply to ignore the funda- mental principles on which correct microscopic images are obtained. Fig. 71. Micrometer Calipers (Brown and Sharpe). Pocket calipers, graduated either in inches or millimeters, and well adapted for measuring cover-glasses. It is then strongly recommended that every preparation shall be cov- ered with a cover-glass whose thickness is known, and that this thick- ness should be indicated in some way on the preparation. § 212. Cover-Glass Measurers.—For the purpose of measuring cover-glasses there are three very excellent pieces of apparatus. The micrometer caliper, used chiefly in the mechanic arts, is convenient, and from its size easily carried in the pocket. The two cover-glass measurers specially designed for the purpose are shown in Fig. 72-73. With either of these the covers may be more rapidly measured than with the caliper. With all of these measurers or gauges one should be certain that the index stands at zero when at rest. If the index does not stand at zero it should be adjusted to that point, otherwise the readings will not be correct. As the covers are measured the different thicknesses should be put into different boxes and properly labeled. Unless one is striving for the most accurate possible results, cover-glasses not varying more than mm. may be put in the same box. For example, if one takes y mm. as a standard, covers varying mm. on each side may be put into the same box. In this case the box would contain covers of tW tV%> ttrT) and iVir mm. 109 MOUNTING AND LABELING. Fig. 72. Cover-Glass Measurer (Edward Bausch). The cover-glass is placed in the notch between the two screws, and the drum is turned by the milled head at the right till the cover is in contact with the screws. The thickness is then indicated by the knife edge on the drum, and may be read off directly in f-fh mm. or yfffh inch. In other columns is given the proper tube- length for various unadjustable objectives, (f, J, and Tlj in.) made by the Bausch and Lomb Optical Co. Fig. 73. Zeiss Cover-Glass Meas- urer . With this the knife edge jaws are opened by means of a lever, and the cover inserted. The thickness may then be read off on the face as the pointer indicates the thickness in hundredths millimeter in the out- er circle and in hundredths inch on the inner circle. § 213. Cleaning Mixtures for Glass.—The cleaning mixtures used for cleaning slides and cover glasses are those commonly used in chem- ical laboratories : 110 MOUNTING AND LABELING. (A) Dichromate of Potash and Sulphuric Acid. Dichromate of potash (K2 Cr2 O,) 200 grams. Water, distilled or ordinary 1000 cc. Sulphuric acid (H2 S04) 1000 cc. Dissolve the dichromate in the water by the aid of heat. Pour the solution into a bottle that has been warmed and surrounded by a wet towel. Add slowly and at intervals the sulphuric acid. For making this mixture, ordinary water, commercial dichromate and strong commercial sulphuric acid should be used. It is not necessary to employ chemically pure materials. This is a very excellent cleaning mixture and is practically odorless. It is exceedingly corrosive and must be kept in glass vessels. It may be used more than once, but when the color changes markedly from that seen in the fresh mixture it should be thrown away. (B) Sulphuric and Nitric Acid Mixture. Nitric acid (H N03) 200 cc. Sulphuric acid (H2 S04) 300 cc. The acids should be strong, but they need not be chemically pure. The two acids are mixed slowly, and kept in a glass-stoppered bottle. This is a more corrosive mixture than (A), and has the undesirable feature of giving off very stifling fumes, therefore it must be carefully covered. It may be used several times It acts more rapidly than the dichromate mixture, but on account of the fumes is not so well adapted for general laboratories. For slides on which are balsam-mounted obj'ects, it is best to dissolve away the balsam first with xylol or turpentine, or other solvent of bal- sam. Then the slides and covers may be readily cleaned in either of the above. If slides with large covers, as in mounted series, are put into the cleaning mixture, the swelling of the balsam is liable to break the covers. Dissolving away the balsam with turpentine, etc., avoids this, and greatly shortens the time necessary for cleaning the old slides and covers. MOUNTING, AND PERMANENT PREPARATION OF MICROSCOPICAL, OBJECTS. § 2i4- Mounting a Microscopical Object is so arranging it upon some suitable support (glass slide) and in some suitable mounting me- dium that it may be satisfactorily studied with the microscope. The cover-glass on a permanent preparation should always be consid- erably larger than the object; and where several objects are put under one cover-glass it is false economy to crowd them too closely together. MOUNTING AND LABELING. § 215. Temporary Mounting.—For the study of living objects, like amoebae, white blood corpuscles, and many other objects both animal and vegetable, their living phenomena can best be studied by mount- ing them in the natural medium. That is, for amoebae, in the water in which they are found ; for the white blood corpuscles, a drop of blood is used and, as the blood soon coagulates, they are in the serum. Sometimes it is not easy or convenient to get the natural medium, then some liquid that has been found to serve in place of the natural medium is used. For many things, water with a little common salt (water 100 cc., common salt gram) is employed. This is the so-called nor- mal salt or saline solution. For the ciliated cells from frogs and other amphibia, nothing has been found so good as human spittle. What- ever is used, the object is put on the middle of the slide and a drop of the mounting medium added, and then the cover-glass. The cover is best put on wfith fine forceps, as shown in Fig. 74. After the cover is in place, if the preparation is to be studied for some time, it is better to avoid currents and evaporation by painting a ring of castor oil around the cover in such a way that part of the ring will be on the slide and part on the cover (Fig. 85). Fig. 74. To show the method of putting a cover-glass upon a microscopic preparation. The cover is grasped by one edge, the opposite edge is then brought down to the slide and then the cover gradually lowered upon the object. Fig. 75. Needle Holder (Queen & Co.) By means of the screw clamp or chuck at one end the needle may be quickly changed. § 2i6. Permanent Mounting. — For making per- manent microscopical preparations there are three great methods. Special methods of procedure are necessary to mount objects successfully in each of these ways. The best mounting medium and the best method of mounting in a given case can only be determined by experiment. In most cases some previous observer has already made the necessary experiments and furnished the desired information. The three methods are the following: (A) Dry or in air (§ 217) ; (B) In some medium miscible with water, as glycerin or glycerin jelly (§ 221) ; (C) In some resinous medium like dammar or Canada balsam (§ 226). Fig. 74. 112 MOUNTING AND LABELING. § 217. Mounting Dry or in Air.—The object^should be thoroughly dry. If any moisture remains it is liable to cloud the cover-glass, and the specimen is liable to deteriorate. As the specimen must be sealed, it is necessary to prepare a cell slightly deeper than the object is thick. This is to support the cover-glass, and also to prevent the running in by capillarity of the sealing mixture. ORDER OF PROCEDURE IN MOUNTING OBJECTS DRY OR IN AIR. 1. A cell of some kind is prepared. It should be slightly deeper than the object is thick (■§ 219). 2. The object is thoroughly dried (desiccated) either in dry air or by the aid of gentle heat 3. If practicable the object is mounted on the cover-glass ; if not it is placed in the bottom of the cell. 4. The slide is warmed till the cement forming the cell wall is some- what sticky, or a very thin coat of fresh cement is added ; the cover is warmed and put on the cell and pressed down all around till a shining ring indicates its adherence (§ 220). 5. The cover-glass is sealed (§ 220). 6. The slide is labeled (§ 238). 7. The preparation is cataloged and safely stored (§ 239-242). § 218. Example of Mounting Dry, or in Air.—Prepare a shal- low cell and dry it (§ 219). Select a clean cover-glass slightly larger than the cell. Pour upon the cover a drop of a 10 per cent, solution of salicylic acid in 95 per cent, alcohol. L,et it dry spontaneously. Warm the slide till the cement ring or cell is somewhat sticky, then warm the cover gently and put it on the cell, pressing down all around (§ 217). Seal the cover, label and catalog (§ 220, 238, 240). Fig. 76. Turn-Table for sealing cover-glasses and making shallow mounting tells. (Queen & Co.) A preparation of mammalian red blood corpuscles may be made very satisfactorily by spreading a very thin layer of fresh blood on a cover with the end of a slide. After it is dry, warm gently to remove the 113 MOUNTING AND LABELING. last traces of moisture and mount precisely as for the crystals. One can get the blood as directed for the Micro-spectroscopic work (§ 189). § 219. Preparation of Mounting Cells.—(A) Thin Cells. These are most conveniently made of some of the microscopical cements. Shellac is one of the best and most generally applicable (§ 249). To prepare a shellac cell, place the slide on a turn-table (Fig. 76.) and center it, that is get the center of the slide over the center of the turn-table. Select a guide ring on the turn-table which is a little smaller than the cover- glass to be used, take the brush from the shellac, being sure that there is not enough cement adhering to it to drop. Whirl the turn table and hold the brush lightly on the slide just over the guide ring selected. An even ring of the cement should result. If it is uneven, the cement is too thick or too thin or too much was on the brush. After a ring is thus prepared remove the slide and allow the cement to dry spontan- eously or heat the slide in some way. Before the slide is used for mounting, the cement should be so dry when it is cold that it does not dent when the finger nail is applied to it. A cell of considerable depth may be made with the shellac by adding successive layers as the previous one drys. (B) Deep cells are sometimes made by building up cement cells, but more frequently, paper, wax, glass, hard rubber or some metal is used for the main part of the cell. Paper rings, block tin or lead rings are easily cut out with gun punches. These rings are fastened to the slide by using some cement like the shellac. § 220. Sealing the Cover-Glass for Dry Objects Mounted in Cells.—When an object is mounted in a cell, the slide is warmed until the cement is slightly sticky, or a very thin coat of fresh cement is put on. The cover-glass is warmed slightly also, both to make it stick to the cell more easily, and to expel any remaining moisture from the ob- ject. When the cover is put on it is pressed down all around over the cell until a shining ring appears, showing that there is an intimate con- tact. I11 doing this use the convex part of the fine foceps or some other blunt, smooth object; it is also necessary to avoid pressing on the cover except immediately over the wall of the cell for fear of breaking the cover. When the cover is in contact with the wall of cement all around, the slide should be placed on the turn-table and carefully ar- ranged so that the cover-glass and cell wall will be concentric with the guide rings of the turn-table. Then the turn-table is whirled and a ring of fresh cement is painted, half on the cover and half on the cell wall (Fig. 85). If the cover-glass is not in contact with the cell wall at any point and the cell is shallow, there will be great danger of the fresh cement running into the cell and injuring or spoiling the preparation. 114 MOUNTING AND LABELING. When the cover-glass is properly sealed, the preparation is put in some safe place for the drying of the cement. It is advisable to add a fresh coat of cement occasionally. § 221. Mounting Objects in Media Miscible with Water.— Many objects are so greatly modified by drying that they must be mounted in some medium other than air. In some cases water with something in solution is used. Glycerin of various strengths, and glycerin jelly are also much employed (§ 243, 244). All these media keep the object moist and therefore in a condition resembling the natural one. The object is usually and properly treated with gradually increasing strengths of glycerin or fixed by some fixing agent (See Part II) before being permanently mounted in strong glycerin or either of the other media. In all of these different methods, unless glycerin of increasing strengths has been used to prepare the tissue, the fixing agent is washed away with water before the object is finally and permanently mounted in either of the media. For glycerin jelly no cell is necessary unless the object has a con- siderable thickness. Fig. 77. Centering Card. A card with stops for the slide and circles in the position occupied by the center of the slide. If the slide is put upon such a card it is very easy to arratige the ob- ject so that it will be approximately in the center of the slide. (From the Mi- croscope Dec., 1886.) § 222. Order of Procedure in Mounting Objects in Glycerin. 1. A cell must be prepared on the slide if the object is of considerable thickness (§219, 221). 2. A suitably prepared object (§ 221) is placed on the center of a clean slide, and if no cell is required a centering card is employed to facilitate the centering (Fig. 77). 3. A drop of pure glycerin is put upon the object, or if a cell is used, enough to fill the cell. 4. In putting on the cover-glass it is grasped with fine forceps and the under side breathed on to slightly moisten it so that the glycerin will adhere, then one edge of the cover is put on the cell or slide and the cover gradually lowered upon the object (Fig. 74). The cover is then gently pressed down. If a cell is used, a fresh coat of cement is added before mounting (§ 220). 115 MOUNTING AND LABELING. Fig. 78. Slide and cover-glass showing method of anch- oring a cover-glass with a glycerin preparation when no cell is used. A cover-glass so anchored is not liable to move when the cover is being sealed (g 224). Fig. 79. Glass slide with cover-glass, a drop of reagent and a bit of absorbent paper to show method of irriga- tion (g 253). 5. The cover-glass is sealed (§ 220). 6. The slide is labeled (§ 238). 7. The preparation is catalogued and safely stored (§ 240, 242). § 223. Order of Procedure in Mounting Objects in Glycerin Jelly. 1. Unless the object is quite thick no cell is necessary with glycerin jelly. 2. A slide is gently warmed and placed on the centering card (Fig. 77) and a drop of warmed glycerin jelly is put on its center. The suitably prepared object is then arranged in the center of the slide. 3. A drop of the warm glycerin jelly is then put on the object, or if a cell is used it is filled with the medium. 4. The cover-glass is grasped with fine forceps, the lower side breathed on and then gradually lowered upon the object (Fig. 74), and gently pressed down. A B c Fig. 8o. A—Simple form of moist chamber made with a plate and bowl. B, bowl serving as a bell-jar; P, plate containing the water and over which the bowl is inverted; S, slides on which are mounted preparations which are to be kept moist. These slides are seen endwise and rest upon a bench made by cementing short pieces of large glass tubing to a strip of glass of the desired length and width. B—Two cover-glasses (C) made eccentric, so that they may be more easily sepa- rated by grasping the projecting edge. C—Slide (S) with projecting cover-glass (C). The projection of the cover en- ables one to grasp and raise it without danger of moving it on the slide and thus folding the substance under the cover. (From Proc. Arner. Micr. Soc., 1891). 116 MOUNTING AND LABELING. 5- After mounting, the preparation is left flat in some cool place till the glycerin jelly sets, then the superfluous amount is scraped and wiped away and the cover-glass sealed with shellac (§ 220, 224). 6. The slide is labeled (§ 238). 7. The preparation is cataloged and safely stored (§ 239, 242). § 224. Sealing the Cover-Glass when no Cell is Used.—(A) For glycerin mounted specimens. The superfluous glycerin is wiped away as carefully as possible with a moist cloth, then four minute drops of cement are placed at the edge of the cover (Fig. 78), and allowed to harden for half an hour or more. These will anchor the cover-glass, then the preparation may be put on the turn-table and a ring of cement put around the edge while whirling the turn-table. (B) For objects in glycerin jelly, Farr ant's solution or a resinous me- dium. The mounting medium is first allowed to harden, then the su- perfluous medium is scraped away as much as possible with a knife, and then removed with a cloth moistened with water for the glycerin jelly and Farrant’s solution or with alcohol, chloroform or turpentine, etc., if a resinous medium is used. Then the slide is put on a turn-table and a ring of the shellac cement added. (C) Balsam preparations may be sealed with shellac as soon as they are prepared, but it is better to al- low them to dry for a few days. One should never use a cement for sealing preparations in balsam or other resinous media unless the solvent of the cement is not a solvent of the balsam, etc. Otherwise the ce- ment will soften the balsam and finally run in and mix with it, and partly or wholly ruin the preparation. Shellac is an excellent cement for sealing balsam preparations, as it never runs in, and it serves to avoid any injury to the preparation when cedar oil, etc., are used for ho- mogeneous immersion objectives. §225. Example of Mounting in Glycerin Jelly.—For this select some stained and isolated muscular fibers or other suitably prepared objects. Arrange them on the middle of a slide, using the centering card, and mount in glycerin jelly as directed in § 223. Air bubbles are not easily removed from glycerin jelly preparations, so care should be taken to avoid them. § 226. Mounting Objects in Resinous Media.—While the media miscible with water offer many advantages for mounting animal and vegetable tissues the preparations so made are liable to deteriorate. In many cases, also, they do not produce sufficient transparency to en- able one to use sufficiently high powers for the demonstration of minute details. By using sufficient care almost any tissue may be mounted in a resin- ous medium and retain all its details of structure. 117 MOUNTING AND LABELING. For the successful mounting of an object in a resinous medium it must iu some way be deprived of all water and all liquids not miscible with the resinous mounting medium. There are two methods of bring- ing this about : (A) By drying or desiccation (§ 227,) and (B)by suc- cessive displacements (§ 229). § 227. Order of Procedure in Mounting Objects in Resinous Media by Desiccation: 1. The object suitable for the purpose (fly’s wings, etc.) is thoroughly dried in dry air or by gentle heat. 2. The object is arranged as desired in the center of a clean slide on the centering card (Fig. 77). 3. A drop of the mounting medium (§ 246) is put directly upon the object or spread on a cover-glass. 4. The cover-glass is put on the specimen with fine forceps (Fig. 74), but in no case does 011c breathe on the cover as when media miscible with water are used. 5. The cover glass is pressed down gently. 6. The slide is labeled (§ 238). 7. The preparation is cataloged and safely stored (§ 239-242 . 8. Although it is not absolutely necessary, it is better to seal the cover with shellac after the medium has hardened round the edge of the cover (§ 224 C). § 228. Example of Mounting in Balsam by Desiccation.—Find a fresh fly, or if in winter procure a dead one from a window sill or a spider’s web. Carefully remove the fly’s wings, being especially care- ful to keep them the dorsal side up. With a camel’s hair brush remove any dirt that may be clinging to them. Place a clean slide on the cen- tering card, then with fine forceps put the two wings within one of the guide rings. Beave one dorsal side up, turn the other ventral side up. Spread some Canada balsam on the face of the cover-glass and with the fine forceps place the cover upon the wings (Fig. 74). Probably some air-bubbles will appear in the preparation, but if the slide is put in a warm place these will soon disappear. Babel, catalog, etc., (§227, 238, 239)- § 229. Mounting in Resinous Media by a Series of Displace- ments.—The first step in the series is Dehydration, that is the water is displaced by some liquid which is miscible both with the water and the next liquid to be used. Strong alcohol (95 per cent, or stronger) is usually employed for this. Plenty of it must be used to displace the last trace of water. The tissue may be soaked in a dish of the alcohol, or alcohol from a pipette may be poured upon it. Dehydration usually occurs iu the thin objects to be mouuted in balsam in 5 to 15 minutes. 118 MOUNTING AND LABELING. If a dish of alcohol is used it must not be used too many times, as it loses in strength. The second step is clearing. That is some liquid which is miscible with the alcohol and also with the resinous medium is used. This liquid is highly refractive in most cases and consequently this step is called clearing and the liquid a Clearer. The clearer displaces the alcohol, and renders the object more or less translucent. In case the water was not all removed a cloudiness will appear in parts or over the whole of the preparation. In this case the preparation must be re- turned to alcohol to complete the dehydration. One can tell when a specimen is properly cleared by holding it over some dark object. If it is cleared it can be seen only with difficulty, as but little light is reflected from it. If it is held toward the window, however, it will appear translucent. The third and final step is the displacement of the clearer by the resinous mounting medium. The specimen is drained of clearer and allowed to stand for a short time till there appears the first sign of dullness from evaporation of the clearer from the surface. Then a drop of the resinous medium is put on the object and finally a cover glass is placed over it, or a drop of the mounting medium is spread on the cover and it is then put on the object. It is in many ways more convenient to perform the series of displace- ments on the slide. This must be done with serial sections. If the Fig. 81. Waste Bowl {from the Reference Hand-Book of the Medical Sciences, Supple- ment'). “ The apparatus is easily constructed and exceed- ingly useful. The ends of two glass rods of the proper length are fastened into two strips of lead, slightly curved to fit around the margin of an ordinary bowl. Between the glass rods is a tin funnel, held in position by flanges of tin which may be bent around the rods and permit the fun- nel to slide back and forth. The rods are a convenient resting place for the slide while in process of prepara- tion. The various solutions used in treating the sections may be readily drained from the slide by placing it for a few moments in the funnel" (P. A. Fish ) 119 MOUNTING AND LABELING. preparations are not fastened to the slide, some workers perform the dehydration and clearing in separate dishes. § 230 Order of Procedure in Mounting Objects in Resinous Media by successive Displacements: 1. A suitable object is selected, for example a section of animal tis- sue, and is centered on a clean slide. 2. The slide is held in the hand and the object is dehydrated by dropping upon it strong alcohol (§ 229). 3. The alcohol is drained from the specimen and removed by blotting paper held at the edge of the object. 4. Two or three drops of the clearer (§ 247) are put on the object to displace the alcohol (§ 229). 5. When the object appears translucent the clearer is drained off and blotted from the edge of the specimen (§ 229). 6. A drop of the resinous medium is put directly on the object or spread upon a cover-glass. 7. The cover-glass is put upon the object and pressed down. It may then be heated gently. 8. The slide is labeled (§ 238J. 9. The preparation is catalogued and safely stored (§ 239-242). 10. After the resin has hardened round the edge of the cover the superfluous material may be cleaned away and the cover-glass sealed with shellac. This is not absolutely neces- sary, but is desirable (§ 224). Fig. 82. Small spirit lamp modified into a balsam 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 as shown in the figure. By adding a small brush, it answers well for a shellac bottle also. § 231. Example of Mounting in Balsam by Displacement.—For this experiment select a stained section of any organ or tissue, as the skin, or myel, then proceed exactly as described in § 229, 230. § 232. In histological studies it is frequently of the greatest advan- tage to have the sections in serial order, then an obscure feature in one section is frequently made clear by the following or preceding section. While serial sections may be very desirable in histological studies, they serial sections. 120 MOUNTING AND LABELING. are absolutely necessary for the solution of morphological problems presented in complex organs like the brain, in embryos and in minute animals where gross dissection is impossible. § 233. Arrangement of Serial Sections.—The numerical order may be very conveniently like the words on a printed page, from the upper left hand corner and extending from left to right, top to bottom (Fig. 83). The position of the various aspects of the sections should be in gen- eral such that when they are under the compound microscope the rights and lefts will correspond with those of the observer. This may be ac- complished as follows for sections made in the three cardinal sectional planes, Transections, Frontal Sections, Sagittal Sections : (A) Transections, i e., sections across the long axis of the embryo or animal dividing it into equal or unequal cephalic and caudal parts. (a) In accordance with the generally approved method of numbering serial parts in anatomy, the most cephalic section should be first (No. 1 of Fig. 83). (b) The caudal aspect of the section should face upward toward the cover-glass, the cephalic aspect being next the slide. (c) The ventral aspect should face toward the upper edge of the slide (Fig. 83). This arrangement may be easily accomplished in transections in two ways : (1) The embryo or animal is imbedded in such a way that the sectioning shall begin at the cephalic end. In this case the first section is placed in the upper left hand corner of the slide (No. 1 of Fig. 83), but it must be turned over so that the caudal aspect shall face up. The ventral aspect must be made to look toward the upper edge of the slide, then under the compound microscope the dorsal side will appear toward the upper edge of the slide and the right and left correspond with the observer. (2) The embryo or animal is imbedded so that the sectioning begins at the caudal end, then the sections are not turned over, as they are al- ready caudal face up, but they must be put on the slide in reverse order, i. e.y the first section made is put in the lower right hand corner (No. 10 of Fig. 83). In this way the most cephalic section will be number one as before. As in the previous case the ventral side of the section should be toward the upper edge of the slide (Fig. 83). (B) Frontal Sections, i. I4° ! putting in position and removing, 20 ; single lens, 13 ; termi- nology of, 6 ; unadjustable, 9 ; visual and actinic foci of in photo-microgra- phy, 135 ; water immersion, 13, 14, 15 ; experiments, 43, 45 ; working distance of, 28, 33. Oblique light, 30, 36 ; with Abbe illu- minator, 36 ; experiments, 34 ; with a mirror, 34. Ocular, 2 ; achromatic, 18 ; Airy’s, 18 ; aplanatic, 18 ; binocular, 18 ; cloud- iness, how to determine and remove, 48, 54; Campani’s, and cobweb mi- crometer, 18 ; compensating, 9, 19; compound, 18; continental, 18; deep, 18 ; designation by magnification or combined magnification and equiva- lent focus, 20 ; dust, how to deter- mine, 54 ; equivalent focus of, 20, 69; erecting, 18; eve-point of, dem- onstration, 26 ; field-lens, 17, 26 ; filar micrometer, 18 ; focus, equiva- lent of, 20 ; function of, 25, 26 ; gon- iometer, 18 ; high, 9,18 ; holosteric, INDEX. 163 19 ; Huygenian, 18 ; index, 18 ; Jack- son micrometer, 18 ; Kellner’s, 18 ; lettering of, 20; low, 18 ; magnifi- cation, table, 70; micrometer, 18, 19, 72, 73 ; micrometer, micrometry with, 74; micrometer, putting in position, 73 ; micrometer ratio, 75 ; micrometer, valuation of, 73 ; mi- crometer, varying valuation, 74 ; mi- crometer, ways of using, 75 ; micro- metric, 18 ; microscopic, 17, 18; neg- ative, 17, 19 ; numbering, 20 ; ortho- scopic, 19, 22 ; par-focal, 12, 19, 32 ; periscopic, 19, 22 ; for photo-microg- raphy, 135; photo-micrography with and without o., 142, 145 ; positive, 6, 17 ; projection, 19, 135 ; projection, designation of, 20 ; projection, use of in photo-micrography, 142, 145 ; putting in position and removing, 20 ; Ramsden’s, 19 ; screw microm- eter, 18, 19 ; searching, 19 ; shallow, 19 ; solid, 19 ; spectral, 19, 88 ; spec- troscopic, 9, 88, 92 ; stauroscopic, 19 ; stereoscopic, 18 ; working, 19. Oil and air, appearances and distinguish- ing optically, 56, 57. Oil-globules, with central illumination, 56 ; with oblique illumination, 56. Oil-immersion objectives, 8. Optic axis, 2, 3, 6 ; of condenser, or illu- minator, 35 ; of microscope, 6. Optical center, 7 ; combination, 1; focus, 9; parts of compound microscope, 5 ; parts of microscope, care of, and testing, 47, 51 ; section, 59. Order of procedure in mounting objects, dry or in air, 112 ; in glyceriu, 114 ; in glycerin jelly, 115: in resinous media by desiccation, 117 ; in resin- ous media by successive displace- ments, 119. Ordinary ray with polarizer, 101. Orthochromatic plates, 135. Orthoscopic ocular, field with, 22, Outline, distinctness of, 57. Oxy-hemoglobin, spectrum of, 98, P Paper, aristotype, 150 ; bibulous, filter, lens, or Japanese, 24, 25 ; bromide, 150 ; for cleaning oculars and ob- jectives, 48. Paraffin, removal from lenses, 48. Parfocal oculars, 12, 19, 32. Parts, optical and mechanical of micro- scope, 1, 5, 21, 47, 51 ; testing, 51. Pedesis, 60; compared with currents, 60 ; with polarizing microscope, 61 ; proof of reality of, 61. Penetrating power, 16, 17. Penetration, 134. Perigraphic objective, 139. Periscopic ocular, field with, 22. Permanent mounting, m. Permanganate of potash, absorption spec- trum of, 97, 101. Pin-hole diaphragm, 35, 144. Photographic objectives, 148 ; for photo- micrography, 137 ; figures of, 139, 140. Photography, 9 ; basis for figures, 147 ; compared with photo-micrography, 131 ; exposure for, 138 ; indebted- ness to microscopy, 130; lighting large objects for, 138 ; of objects in alcohol or water, 147 ; with a verti- cal camera, 146. Photogravures from photo-micrographs, I5°- . Photo-micrograph, 130 ; and drawing, 147 ; exposure for, 138; at 5-20 di- ameters, 137 ; 20-50 diameters, 142 ; 100-150 diameters, 143 ; 500-2000 di- ameters, 145 ; objects suitable for, 136 ; prints of, 150 ; reproductions of, 146, 150; with and without an ocular, 142, 145. Photo-micrographic camera, 132-134. Photo-micrography, 130 ; apparatus for, 133 ; arrangement of camera for, 133 ; compared with ordinary pho- tography, 131 ; distinguished from micro - photography, 130; experi- ments, 137 ; exposure for, 138 ; fo- cusing for, 133 ; focusing screen for, 138 ; lighting, 40, 136-138, 143, 146; vertical camera with, 146 ; visual and actinic foci in, 135 ; with long and short bellows, 143 ; with and with- out ocular, 142, 145. Plane mirror, use of, 31. Plates, exposure of, 138, 142, 143 ; gela- tino-bromide, 130; isochromatic, 135, 138, 140 ; orthochromatic, 135, 138. Pleochroism, 103, 104. Pleurosigma angulatum, 34, 36. Point, axial, 11. Polariscope, 92, 101. Polarized light, 101 ; extraordinary and ordinary ray of, 101. Polarizer, 92, 101, 102 ; and analyzer, putting in position, 102. Polarizing microscope, pedesis with, 61. Position of objects or parts of same ob- ject, 54. Positive oculars, 6, 17. Potato, examination of, 62. Power of microscope, 63 ; illuminating, penetrating, resolving, 16, T7. Preparation of Canada balsam, Farrant’s solution, glycerin, glycerin jelly, 126. 164 INDEX. Preparation of clearing mixture, liquid gelatin and shellac cement, 127. Price of American and foreign micro- scope, 51. Principal focus, 1-7 ; for oil and air bub- bles, 56 ; focal distance, 6, 7. Prism of Abbe camera lucida, 80, 83 ; Amici, 88, 92 ; comparison, 92, 94 ; dispersing, 90 ; Nicol, 101 ; reflect- j ing, 94; and slit of micro-spec- troscope, mutual arrangement, 93 ; of Wollaston’s camera lucida, 65. Prints and mechanical printing of photo- micrographs, 150. Projection ocular, 19 ; designation of, 20 ; in photo-micrography, 142, 145 ; of Zeiss, 135. Pumice stone for pedesis, 60. Pushing in draw-tube, 32. Putting on cover-glass, 111, 114; an ob- ject under microscope, 22. Q-R Quadrant for camera lucida, 82, 83, 86. Quinine, Herapath’s method of deter- mining minute quantities of, 129. Rack for drying negatives, 145. Ratio, ocular micrometer, 75. Ray, chemical, 9 ; ordinary of polarized light, 101 ; extraordinary, 101 ; re- fracted, 42. Real image, 1, 7, 24 ; inverted, 5. Reflected light, 29. Reflecting prism, 94. Refracted ray. 42. Refracting surface, 26, 42. Refraction, 42 ; images, 41, 46 ; index of, 42 ; of medium in front of objective, I3-. Refractive, doubly, 103 ; highly, 58 ; singly, 103. Resinous media, mounting objects in, order of procedure, by drying or desiccation, 117 ; by a series of dis- placements, 117-119. Resolution and numerical aperture, 17. Resolving power, r6. Retinal image, 26. Revolver, 22. Rice, examination of, 62. Rule or scale for magnification and mi- crometry, 64. s Sagittal sections, 120. Salicylic acid, crystallization, 38. Scale, of magnification and micrometry, 64 ; of wave lengths, 94. Scales of butterflies and moths, examin- ation of, 62. Screen, focusing s. for photo-microgra- pliy, 138 ; of ground glass, 24, 25 ; for microscope, 46. Screw, society, 21 ; micrometer, 73. Sealing cover-glass, 113, 117. Secondary axis, 37. Section, frontal, 120; optical, 59; sagit- tal, 120; serial, 119, 122. Sediment in water, determination of character, 128. Selenite plate for polariscope, 104. Serial sections, 119 ; arranging and label- ing, 120, 121 ; determining thick- ness of, 121 ; stage for, 51 ; thick- ness of cover-glass for, 121. Shading object, 46 ; for micro-polari- scope, 61. Shellac cement, preparation of, 127 ; re- moval from lenses, 48. Sight, injury or improvement in micro- scopic work, 49. Significance of aperture, 15. Silk, examination of, 62. Simple microscope, see under micro- scope. Slides, 105 ; cleaning, 105. Slips, 105. Slit mechanism of micro-spectroscope, 88, 92 ; adjusting and focusing, 93 ; slit and prism, mutual arrangement, 93- Society screw, 21. Solar spectrum or s. of sunlight, 89, 90. Spectral, colors, 8; ocular, 19, 88. Spectroscope, direct vision, 88. Spectroscopic ocular, 19, 88. Spectrum, 9, 82-92 ; absorption, 90, 100; amount of material necessary and its proper manipulation, 96 ; analysis, 101 ; Angstrom and Stokes’ law of, 91 ; banded, not given by all colored objects, 99 ; of blood, 91, 97 ; of car- bon monoxide hemaglobin, 99, 100 ; of carmine solution, 99, 100 ; of col- ored minerals, 100 ; of colorless bod- ies, 100 ; comparison, 92, 94 ; com- plementary, 91 ; continuous, 90; double, 94 ; incandescence, 92 ; line, 90 ; met-hemaglobin, 89, 98 ; mona- zite sand, 100 ; nitrate of uranium, 101 ; oxy-hemaglobin, 91, 98 ; per- manganate of potash, 89, 96, 101 ; single-handed of hemaglobin, 91, 98 ; sodium, 89 ; solar, 89, 90 ; two-band- ed of oxy-hemaglobin. 98, 100. Spherical aberration, 8 ; distortion, 6, 8. Stage, 21 ; mechanical, 51 ; micrometer, 64, 72, 75 ; for mineralogical studj% 103 ; for serial sections, 51. Stand, of microscope, 1,21; for labora- tory microscope, 51. INDEX. Standard distance (250 mm.) at which the virtual image is measured, 65, 67. Starch, examination of, 62. Steinheil lens, 136, 143. Stokes and Angstrom’s law of absorption spectra, 91. Structure, microscopic, 53. Substage, 21. Surface, refracting, 26, 42. Swaying of image, 36. System, back, front, intermediate, of lenses, 5-8. T Table, of magnification and valuation of ocular micrometer, 68 ; of tube- length and thickness of cover-glass- es, 10 ; natural sines, third page of cover; of weights and measures, second page of cover; size of field, 23 ; of numerical aperture, 15. Temporary mounting, 111. Terminology of objectives, 6. Tester, cover-glass, 108 ; for homogen- eous liquids, 45. Testing a camera, 133 ; a microscope and its parts, 50. Textile fibres, examination of, 62. Thickness of cover-glass for nou-adjust- able objectives, table, 10. Transections, 120. Transmitted light, 29. Transparent objects having curved out- lines, relative position in microscop- ic preparations, 55. Triplet, achromatic, 4. Tripod, 3 ; as focusing glass, 141. Tube of microscope, 21. Tube-length, 9, 10, n* for cover-glass adjustment, 45 ; importance of, 45 ; microscopical, 9; of various opti- cians, table, 10. Turn-table, 112. u-v—w Unadjustable objectives, 9. Unit of measures, in micrometry, 71 ; of wave length, 95. Uranium nitrate spectrum of, 101. Valuation of ocular micrometer, 73, 74. Varying ocular micrometer valuation, 74. Velocity under microscope, 59. Vertical camera, 134, 146, 148. Virtual image, 26, 63 ; standard distance at which measured, 67. Ward’s eye-shade, 49. Waste bowl, 118. Water immersion objective, 45; light utilized, 13 ; numerical aperture, 14. Water, for immersion objectives, 45 ; re- moval, 48; solid sediment in, 128. Wave length, designation of, 95 ; scale of, 94. Weights and measures, see 2d p. of cover. Wheat, examination, 62. Wollaston’s camera lucida, 65, 66, 80. Work-room for photo-micrography, 133. Work-table, position, etc., 49. Working distance of microscope or ob- jective, 7, 28; determination of, 33. Writing diamond, 124. TABLE OF NATURAE SINES. Compiled, from Prof. G. IV. fones' Logarithmic Tables. Minutes. Degrees AND Quarter Degrees up to 90°. i/0.00029 i°, 0.01745' 160, 0.27564 31°, 0.51504 46°, o.7i934 6i°, 0.87462 76°, 0.97030 2 0.00058 i°, 15 0.02181 160, i5/ 0.27983 31°, 15 ' 0.51877! 46°, 15" 0,72236 6i°, 15 ' 0.87673 76°,15 '0.97134 3 0.00087 i,3° 0.02618' 16,30 0.28402 31,30 0.5225046,30 0.72537 61,30 0.87882 76,30 0.97237 4 0.00116 i,45 0.03054 i6,45 0.28820 31,45 0.52621 46,45 0.72837 6i,45 0.88089 76,45 0.97338 5 0.00145 2 0.03490 17 0.29237 32 0.52992 47 o.73i35 62 0.88295 77 0-97437 6 0.00175 2,I5 0.03926 I7D5 0.29654 32,15 .0.53361 47,15 o.73432 62,15 0.88499 77D5 0-97534 7 0.00204 2,30 0.04362 17,30 0.30071 32,30 0.5373047,30 0.73728 62,30 0.88701 77,30 0.97630 8 0.00233 2,45 0.04798 17,45 0.30486 32,45 0.54097 47,45 0.74022 62,45 0.88902 77,45 0.97723 9 0.00262 3 0.05234 18 0.30902 33 0.54464 48 0.7431463 0.89101 78 0.97815 10 0.00291 3,i5 0.05669 18,15 0.31316 33,15 0.54829 48,15 0.7460663,15 0.89298 78,15 0.97905 11 0.00320 3,30 0.06105 18,30 0.31730 33,30 0.5519448,30 0.74896 63,30 0.89493 78,30 0.97992 12 0.00349 3,45 0.06540 i8,45 0.32144 33-45 o.55557i48,45 0.75184163.45 0.89687 78,45 0.9807Q 13 0.00378 4 0.06976 19 0.32557 34 0-5591949 0.75471 64 0.89879 79 0.98163 14 0.00407 4,15 0.07411 19D5 0.32969 34D5 0.5628049,15 0.7575664,15 0.90070 79,i5 0.98245 15 0.00436 4,30 0.07846 19,30 o.3338i 34,30 0.56641:49,30 0.76041 64,30 0.90259 79,30 0-98325 16 0.00465 4,45 0.082S1 19,45 0.33792 34,45 0.5 7000'49,45 0.76323:64,45 0.90446 79,45 0.08404 17 0.00495 5 0.08716 20 0.34202 35 o.57358;5o 0.76604:65 0.90631 80 0.98481 18 0.00524 5,15 0.09150 20,15 0.34612 35,i5 0.57715550,15 0.76884165,15 0.90814 80,15 0.98556 19 0.00553 5,30 0.09585 20,30 0.35021 35,30 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 50,45 0.7743965.45 0.91176 80,45 0.98700 21 0.00611 6 0.10453 21 0.35837 36 0.58779:51 °.777i5 66 o-9i355 81 0.98769 22 0.00640 6,15 0.10887 21,15 0.36244 36,15 0.59131 5i,i5 0.77988 66,15 o-9i53i 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 0.00698 6,45 0.11754 2i,45 0.37056 36,45 0.5983251,45 0.78532 66,45 0.91879 8i,45 0.98965 25 0.00727 7 0.121S7 22 0.37461 37 0.60182^52 0.78801 67 0.92050 82 0.99027 26 0.00756 7-i5 0.12620 22,15 0.37865 37D5 0.60529 52,15 0.79069 67,15 0.92220 82,15 0.99087 27 0.00785 7,30 0.13053 22,30 0.38268 37,30 0.60876 52,30 0-79335 67,30 0.92388 82,30 0.99144 28 0.00814 7,45 0.13485 22,45 0.38671 37,45 0.61222 52,45 0.79600 67,45 0.92554 82,45 0.99200 29 0.00844 8 0.13917 23 0.39073 38 0.61566 53 0.79864 68 0.92718 83 0.99255 30 0.00873 8,15 0.14349 23.15 0.39474 38,15 0.61909 53D5 0.80125 68,15 0.92881 83,15 0.99307 31 0.00902 8,30 0.14781 23,30 0.39875 38,30 0.62251 53,30 0.80386 68,30 0.93042 83,30 0-99357 32 0.00931 8,45 0.15212 23,45 0.40275 38,45 0.62592 53,45 0.80644 68,45 0.93201 83,45 0.99406 33 0.00960 9 0.15643 24 0.40674 39 0.62932 54 0.80902 69 0.93358 84 0.99452 34 0.00989 9J5 0.16074 24D5 0.41072 39D5 0.63271 54D5 0.81157 69,15 o.935i4 84,15 0.99497 35 0.01018 9,30 0.16505 24,30 0.41469 39,30 0.63608 54,30 0.81412 69,30 0.93667 84,30 o.9954o 36 0.01047 9,45 0.16935 24.45 0.41866 39,45 0.63944 54,45 0.81664 69,45 0.93819 84,45 0.99580 37 0.01076 10 0.17365 25 0.42262 40 0.64279 55 0.81915 70 0.93969 85 0.99619 38 0.01105 10,15 0.17794 25,15 0.42657 40,15 0.64612 55D5 0.82165 70,15 0.94118 85,15 0.99657 39 0.01134 10.30 0.18224 25,30 0.43051 40,30 0.64945 55-30 0.82413 70,30 0.94264 85,30 0.99692 40 0.01164 io,45 0.18652 25,45 0.43445 40,45 0.65276 55,45 0.82659 70,45 0.94409 85,45 0.99725 41 0.01193 11 0.19081 26 0.43837 4i 0.65606 56 0.82904 7i 0.94552 86 0.99756 42 0,01222 n,i5 0.19509 26,15 0.44229 4GI5 0.65935 56,15 0.83147 7G15 0.94693 86,15 0.99786 43 0.01251 ”,30 0.19937 26,30 0.44620 41,30 0.66262 56,30 0.83389 7i,3o 0.94832 86,30 0.99813 44 0.01280 ii,45 0.20364 26,45 0.45010 4G45 0.66588 56,45 0.83629 7i,45 0.94970 86,45 0.99839 45 0.01309 12 0.20791 27 0-45399 42 0.66913 57 0.83867 72 0.95106 87 0.99863 46 0.01338 12,15 0.21218 27D5 0.45787 42,15 0.67237 57,15 0.84104 72D5 0.95240 87D5 0.99885 47 0.01367 12,30 0.21644 27,30 0.46175 42,30 0.67559 57,30 0.84339 72,30 o.95372 87,30 0.99905 48 0.01396 12,45 0.22070 27,45 0.46561 42,45 0.67880 57,45 0.84573 72,45 0.95502 87,45 0.99923 49 0.01425 13 0.22495 28 0.46947 43 0.68200 58 0.84805 73 0.95630 88 0-99939 50 0.01454 I3,i5 0.22920 28,15 0.47332 43D5 0.68518 58,15 0.85035 73D5 0-95757 88,15 0-99953 51 0.01483 J3,30 0.23345 28,30 0.47716 43,30 0.68835 58,3° 0.85264 73,3° 0.95882 [88,3° 0.99966 52 0.01513 13,45 0.23769 28,45 0.48099 43,45 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 I4,i5 0.24615 29D5 0.48862 44D5 0.69779 59D5 0.85941 74D5 0.96246 89,15 0.99991 55 0.01600 14,30 0.25038 29,30 0.49242 44,30 0.70091 59-30 0.86163 74,30 0.96363 89,30 0.99996 56 0.01629 54,45 0.25460 29,45 0.49622 44,45 0.70401 59,45 0.86384 74,45 0.96479 89,45 0-99999 57 0.01658 15 0.25882 30 0.50000 45 0.70711 60 0.86603 75 0.96593 90 1.00000 58 0.01687 15,15 0.26303 30,15 0.50377 45,i5 0.71019 60,15 0.86820 75D5 0.96705 59 0.01716 55,30 0.26724 30,30 0.50754 45,30 0.71325 60,30 0.87036 75,30 0.96815 60 0.01745 55,45 0.27144 30,45 0.51129 45,45 0.7163060,45 0.87250 75,45 0.96923