Popular Science Monthly/Volume 61/June 1902/Educational Value of Photomicrography

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Popular Science Monthly Volume 61 June 1902  (1902) 
Educational Value of Photomicrography by Arthur Curtis Scott




THE part of the universe which the penetrating power of the microscope reveals to the student of nature, though concerned with the infinitesimal, equals the macroscopic portion in magnitude and significance.

Modern scientific consideration recognizes the fact that no more accurate method of research can be concentrated on the question of origin, cyclic changes in development and existing structure of various forms of matter, both organic and inorganic, than that of their minute examination under the microscope. It is a familiar fact that early investigators with this instrument, as well as many at the present time, exhibit as results of their work drawings of the objects examined. While these pictures made with the camera lucida may be reasonably exact when drawn by a careful investigator under the best conditions, it is true that they are frequently inaccurate under ordinary circumstances, and when numerous reproductions are desired the photomicrograph is largely superseding the laborious work of the draftsman.

It is probable that the application of photography to the reproduction of microscopic structure has largely been due to the demand for unmodified and unprejudiced exactness of detail. Again the photographic plate is more sensitive and more efficient than the retina, for not only is the human eye easily fatigued, but it is quite unable to regard slight differences of illumination, or to differentiate the most minute characteristics of specimens. While it is a fact that the plate is most sensitive to light of a certain color and intensity, it is also true that the requirements can be readily obtained, and that the silver salt is able to indicate the action of light that fails to stimulate the sense of vision. The existence of funiculi in the coma-bacilli of Asiatic cholera, for example, was proved by the aid of photography after repeated failures to discover them by other means.

The middle of the nineteenth century marks the beginning of attention to photomicrography when Mayer of Frankfort devised apparatus for this work. Since that time wonderful advance has been made both in photography and microscopy, and we now may obtain not only extremely rapid and color sensitive plates, with readily modified developers that allow a considerable latitude of exposure, but scopes of the highest perfection mechanically, and closely approaching that standard, optically. It may be said in this connection also that though apparatus of excellent quality is thus readily obtained, yet a reasonably accurate knowledge of the optical principles involved and of photographic manipulation is quite necessary to insure satisfactory results. In fact it is the writer's conclusion, after some years' experience connected with the various branches of scientific photography, that no more difficult problem has presented itself than the production of a thoroughly satisfactory photomicrograph of a specimen magnified to 2,000 diameters.

Probably many failures in the work are directly traceable to the use of inferior microscopes, and a few points noticed here may be interesting and possibly instructive.

It would seem hardly necessary to mention the elementary principle in optics, that the spectrum resulting from the resolution of white light is divided into three more or less distinct parts, according to wave length and effect upon matter. Beginning with the longest wave length, we have the infra red and red, or heat portion; then the yellow and green, or light (visual) portion; and finally the blue and violet, or chemical portion. Simple lenses, being of prismatic origin in manufacture, refract white light in such a way as to resolve it into its component colors, and the wave length of the red being greater, it suffers less refraction than the yellow, which in turn is refracted less than the blue; and thus the converging rays do not focus at the same point as a whole, and chromatic aberration results. Further, the fact that for any given lens there is a decrease in thickness from center to circumference, results in unequal refraction of the light as a whole and spherical aberration results, which practically means distortion of the image of an object. Both these defects, chromatic and spherical aberration, are reduced to a minimum by the combination of crown and flint glass to correct the former, and a reduction of aperture or multiplicity of lenses to overcome the latter.

It is a well-known fact, however, that the ordinary microscope objectives have what may be called residual chromatic aberration which focuses the light rays a trifle nearer the lens than the chemical rays, and thus when the image is perfectly sharp upon the ground glass of the camera, the chemical rays, which alone are active upon the photographic plate, do not accurately delineate the object. Fraunhofer has shown, that when the portion of the spectrum of greatest intensity upon the retina, that between the yellow and green is expressed by 1,000, the part between the blue and violet is only 31, so the difficulty in focusing the chemical rays is obvious. It actually amounts to focusing carefully the image, and then moving the objective toward the object an indefinite distance, in order to obtain the desired result. Of course with low power objectives one may do this after repeated trials with some accuracy, but the chief difficulty is found in determining whether an indistinct negative is due to improper manipulation, or imperfect resolving power in the objective. For this reason, even for elementary work, such apparatus is not economical and is quite unsatisfactory.

A consideration of the apochromatic objectives of Professor Abbe and Professor Hastings, which are readily obtained with either the Zeiss or Bausch & Lomb Optical Co.'s instruments, is therefore important. These objectives having a uniform correction for spherical aberration, correct also for three colors, resulting in a better concentration of image rays and greater resolving power; this improvement in objectives brings the photographer's work nearer perfection, though there is, with high power objectives, an error which thus far remains uncorrected. This defect is balanced, however, by over-corrected eyepieces known as compensating oculars, which are designed for use with these objectives. Thus for photographic work neither an apochromatic objective with the ordinary Huyghenian eyepiece, nor the achromatic objective with compensating eyepiece is satisfactory.

The bulk of literature on photomicrography advises the use of the objective without any eyepiece; such advice is good with ordinary achromatic objectives, as the addition of the eyepiece would only introduce more absorbing and reflecting parts without correcting any of the defects in the objective. Where fine work is desired, however, and apochromats are used, the compensating ocular is not only necessary for the highest degree of correction of the system, but is useful in the regulation of magnifying power.

From the fact that so many different makes of objectives and oculars are in the market, accompanying their respective microscopes, the simplest method of rating in photographic work seems to be a statement of the number of diameters which the object is magnified. The magnifying power is most readily and accurately obtained by using a stage micrometer, one millimeter divided into hundredths, and measuring directly the magnification of the image upon the ground glass with an engine-divided steel rule.

The illumination of the object to be photographed is of so much importance that a few points may be briefly considered. An almost indispensable adjunct of the microscope in this connection is the Abbe sub-stage condenser, which not only condenses the light, but illuminates the object with a cone of light having an angular aperture equal to that of the objective employed. It is arranged to move with rack and pinion, thus providing a means for controlling the illumination to an important extent, Experience shows that the best illumination upon the object in general obtains when the Abbe condenser is as far away from the microscopic slide on one side as is the objective on the other. Thus for oil immersion work a drop of oil must be placed between the condenser and slide as well as upon the objective. It frequently happens that when the proper position of the condenser is located for low power objectives the detail is somewhat obliterated because of too intense illumination, and the manipulation of the iris diaphragm connected with the condenser is necessary in order to obtain the best results.

From whatever source the light falls upon the Abbe condenser, an auxiliary condenser before it is advantageous, except when daylight is used upon the mirror beneath the stage. The use of daylight has disadvantages, however, which do not recommend it for photographic work. With diffused light falling upon the mirror it is impossible to use even moderately high power objectives and obtain sufficient light for focusing,

Fig. 1. Convenient Arrangement of Apparatus for Ordinary Photomicrographic Work.

while with the direct rays of the sun, unless a heliostat is used to maintain the position of the sun constant upon the mirror, one cannot obtain a photograph before the sun has changed enough to throw the image of the object off the plate. In addition to this inconvenience in general, the optical imperfections of the mirror are such as to make the production of sharp photographs difficult, and much better results are obtained with high magnifying power, by dispensing with its use and transmitting the light directly from the source through the optical system.

Photomicrographic work of low magnification has been successfully done with the various forms of artificial illuminants of low candle power. The writer, in the production of first photographs used very successfully a series of such lights, beginning with a two-wick oil lamp behind a large Florence flask filled with water, the flask serving at once as a condenser and heat filter. Afterward, in succession, was used an argand gas lamp; a Welsbach burner; a three-wick projection lantern, burning camphorated oil; a 50-candle power incandescent lamp; and a four-burner acetylene lamp with stereopticonFig. 2. Apparatus Complete for making Instantaneous Pictures of Living Microscopic Animals. double condenser, to the final and most efficient light of all, an arc light which can be regulated between 1,000 and 5,000 candle power. Of course the arc light gives out a great amount of heat as well as a satisfactory quantity of light, and were the rays to fall directly upon the microscope lenses the temperature would rise high enough to endanger their mountings. To obviate this difficulty a distilled water cell two inches thick is placed in front of the stereopticon double condenser, which is directly before the arc at such a distance as to parallelize the divergent beam. It is a common idea that an alum solution is the best for absorbing heat while transmitting light. That such is not the case is proved by experiment. Melloni and others have shown that distilled water will intercept more heat rays than a solution of alum. The writer has verified that conclusion and is also able to show that distilled water transmits more light, has practically the same heat-absorbing power through a considerable range of temperature and also has the advantage of no formation of crystals in the cell. Distilled water is necessary, as with common water the air bubbles collect on the parallel faces of the cell as the temperature rises, and obstruct the passage of light.

The form of apparatus shown complete in Fig. 1, using a Zeiss microscope and Bausch & Lomb adjustable camera with automatic regulating arc light, gives excellent results for ordinary dry mounted slides that may be readily photographed with the microscope in a horizontal position. A mechanical stage is of great convenience in this work as it saves much time in accurately placing a slide, especially where high power objectives are used and when focusing must be done at arm's length. In the making of photographs of rock sections, it is frequently necessary to use Nicol prisms in order to properly differentiate particular portions. The one is placed over the ocular, or between it and the objective, and the other beneath the sub-stage condenser. As the latter is free to be rotated, it is an easy matter to bring out clearly the special features desired. Fig. 2 illustrates the apparatus in a vertical position. It must be operated in this position when freshly mounted slides are used and where photo-micrographs are to be made of living organisms. A special shutter devised by the writer for the photographing of living specimens, a detailed description of which will be found in the Scientific American of March 24, 1900, is shown in place on the draw tube of the microscope. In place of the mirror a hole is drilled through the base of the camera stand in order to make transmitted light available. With this arrangement, as illustrated, photographs have been made in 1/50 of a second, (see Figs. 3 and 4).

Fig. 3. Phyllopod. Line Specimen. 250. Exposure 1/40 Sec. Fig. 4. Daphina. Line Specimen. 150. Exposure 1/50 sec.

The character of the plates used in connection with the work and a word as to their manipulation is worthy of notice, because ultimate results are not dependent upon first quality optical apparatus alone. With ordinary light and low power objectives, a slow plate is to be preferred, and with such conditions the Carbutt's B brand has given excellent results. Where polarized light is employed a color sensitive plate is preferable. Such light often gives the most beautiful colors with rock sections and crystal specimens, and the Cramer isochromatic plate renders the color values admirably.

The question of exposure must be decided principally by experience. The candle power of the illuminant; the character of the light, whether ordinary or polarized; the kind of stain used upon the slide, if any; the magnifying power employed, and the rapidity of plate, all combine to determine the limiting values for exposure. There is scarcely opportunity in this paper to enter into discussion of these

Fig. 5. Crystal of Chloritoid in Quartz, 110 Diameters. Polarized Light. Fig. 6. Band of Quartz Crossed by Mica, 100 Diameters. Polarized Light.

factors in detail. In general, however, it may be said that the blues transmit more actinic light than they appear to do; and so there is danger of over-exposure, while with the dark yellows and reds it is quite the reverse. Other conditions remaining the same the exposure is directly proportional to magnifying power, and since frequent changes in objectives and oculars are necessary to obtain the desired magnification

Fig. 7 Section of Iron. 60 Diameters.

of different objects, the calculation of the exposure in terms of magnification expressed in diameters simplifies the work considerably. With living organisms that are given plenty of space to move about, in order to be photographed under conditions favorable to them, the exposure must necessarily be very short, and of course only moderately high powers can be used, and the intensity of the light must be very high. While the subject of this article can be expected to admit of only a limited discussion of photomicrographic apparatus and its manipulation, it has seemed best to thus briefly mention a few points on the modus operandi of the work that one may the better comprehend its value.

To convey an idea of the scope of photomicrography in its application to the study and subsequent presentation of the details of objects

Fig. 8. Fig. 9.
Fig. 10. Fig. 11.
Fig. 8. Cross Section of Juniper Stem showing Three Resin Ducts. 50 Diameters.
Fig. 9. Longitudinal Section of Linden. 90 Diameters.
Fig. 10. Cross Section of Sugar Cane. 50 Diameters. Polarized Light.
Fig. 11. Wing of Seed of Ecoremo-carpus. 80 Diameters. Polarized Light.

in nature we may well begin with the inanimate substance and finish with the living animal organism. Time was when men referred to the 'everlasting hills' as though such portions of nature were unchangeable, but modern geologic science has shown that the face of nature is ever changing, that rocks are constantly being formed, metamorphosed and disintegrated; that the earth has undergone radical changes, and that the geography of the world in many particulars may be for one generation very different from that for the following one. Much information on the details of these changes is gained from a minute study of rocks and earth materials.

By the aid of the microscope one is able to study the fine points in the relation of different rock materials to one another, and by the aid of the camera exhibit the result to others. As an illustration of this fact Fig. 5 shows the position of a crystal of chloritoid in quartz, and Fig. 6, quartz, crossed by laminæ of mica, taken with crossed Nicols to properly differentiate the materials. These photographs are some of a number made for the Geological Survey to be used in connection with reports. The U. S. Geol. Report for 1899 on the geology of Yellowstone Park shows many photomicrographs, admirably illustrating special geologic features.

Fig. 12. Cotton Fiber Injured in the Process of Ginning. 40 Diameters.

Much valuable information is also gained in this way of the structure and properties of metals both in the ore and after smelting and refining. Fig. 7 illustrates the appearance of iron whose tensile strength has been exceeded, and it is easily possible to show also differences in composition or quality of iron or steel by microscopic methods.

The photography of microscopic sections of wood aid very materially in a detailed consideration of forestry. It shows the character of the small tubes or cells of which wood is made up, indicating the definite way in which the cells of the new wood formed each year at the inner surface of the cambium layer are arranged, depending upon the climate where the tree grows. It shows the characteristic differences in cellular structure of different woods, together with the lines separating the growth of successive seasons in the trunk and bark; the medullary rays, which make the silver grain in quartered oak; and a host of other inter

Fig. 13. Plumose Quinidine. 70 Diameters. Polarized Light. Fig. 16. Cinchonidine. 50 Diameters. Polarized Light.
Fig. 14. Platino-cyanide of Magnesia. 70 Diameters. Polarized Light. Fig. 17. Bichromate of Potash Crystals. 70 Diameters. Polarized Light.
Fig. 15. Crystal of CACO3 in India Rubber Plant, 120 Diameters. Fig. 18. Brucine Crystal. 70 Diameters. Polarized Light.

esting details about seeds, leaf structure, etc. It is quite possible that the microscope may yet serve to answer the present disputed question as to how the water can rise into the tops of the great trees of California, some of which are over 300 feet from the ground. It may also be mentioned Fig. 19. Snow Crystals. that photomicrographs are of much importance in studying insects and parasites that infest and destroy forest and fruit trees. In fact, the preservation or annihilation, as seems best to serve man's purpose, of certain organisms is determined very largely by the revelations of the microscope. When such a thing as the lack of assimilating power of a leaf due to insufficient amount of light may be shown by a photograph, the far-reaching value of this science can be understood.

Its intimate connection with botany and plant structure in general is in fact so well recognized as to need little comment here. Some work in connection with cotton fibers, however, is interesting as it shows how it is possible to detect injury to the fiber in the process of ginning; such photographs in connection with others of like character, render valuable information concerning textile materials (Fig. 12).

In crystallography the photomicrograph is useful. Figs. 13-18 illustrate the appearance in polarized light of a few crystals of both organic and inorganic substances. Perhaps one of the richest of all substances in variety of form of crystals is snow. While the geometrical form is invariably hexagonal, it occurs in countless combinations and many of the crystals are very beautiful. For procuring pictures of snow crystals it seems necessary to use the apparatus in a temperature below the freezing point. The snow crystals are collected as they fall upon a surface too cold to tend to melt them, and if the work is done quickly, excellent results are obtained. It is a singular fact that almost every snow crystal differs in some particular from all others collected. Mr. Bentley, of Nashville, Vermont, has doubtless given more attention to snow crystal photography than any one else, and the number of different forms which he has already obtained is remarkably large, and many of them are exquisite. Much has also been done on questions relative to the crystalline structure of various minerals by the aid of the microscope, as for example, 'Inclusions of Petroleum in Quartz' (Jour. Am. Chem. Soc.) 20: 795, and also concerning the 'Solution Vein Theory' for the origin of gold. In the same way it is possible that in the future a unanimity of opinion may be produced concerning the crystallization of iron, which is worthy of serious attention as the enormous amount of that metal used in construction increases year by year.

Fig. 20. Cow Hair, 2,200 Diameters Fig. 21. Cross Section of Horn of African Rhinoceros, x 60 Diameters. Polarized Light.

The value of the part which the microscope plays in determining facts about the minute structure of the animal organisms can scarcely be overestimated. The bacteriological analysis of water in connection with zymotic diseases; the determination of disease germs in impure air; the examination of useful and injurious bacteria in food; all this and more must be credited to the microscope in the development of medical science and sanitation.

A detailed description of all the applications of photomicrography to nature study can not well be given here, and so a few of the important ones only have been briefly noticed. The chief value of it all seems to be the aid rendered in the dissemination of knowledge. To attempt to look beneath the superficial and discover the ulterior is a fundamental desire in the active, civilized mind. Since the beginning of the seventeenth century, when Galileo was imprisoned for stating his belief in the motion of the earth, we look with pride at the development of science through the minds of such men as Newton, Kepler, Lyell, Huxley, Ohm, Faraday, Joule, Helmholtz, Le Conte, Darwin and scores of their contemporaries.

The beginning of the twentieth century finds the major part of the civilized world bound to recognize the power of scientific thought and investigation in its bearing upon the prosperity of nations, and the equality of man. More and more attention is wisely being given to methods of teaching in our primary and secondary schools as well as to college and university work. Such attention is even now beginning to manifest its results, as we note men having completed their university training for the Doctor's degree under twenty-five years of age. No doubt it may truly be said that such men are brilliant, or have

Fig. 22. Palate of a Whelk, X 90 Diameters. Polarized Light. Fig. 23. Proboscis of Fly. x 80 Diameters.

exceptional advantages or both combined for the result attained, yet the fact remains that elementary knowledge gained from secondary instruction molds to a great extent the future of the individual. This phase of study is all the more important since it is beyond the control of the pupil. Beyond his control, for though volumes may be written, and instructors may be at hand, the average student does not early in life appreciate the necessity or value of application to study.

Interest in a subject is the prime factor in its mastery, and the method of presenting the subject will tend either to stimulate or allay the interest of the pupil.

The time devoted to the study of a subject in the secondary school is frequently very limited, and the teacher may profitably consider the needs of the pupils and seek to originate such methods as will accomplish the most for the time allotted. One important saving of time in the class room, both for elementary and advanced work along most scientific lines, is the use of pictures in making certain points clearly understood, and it seems that either the stereopticon, or wall pictures large enough to be seen by the class as a whole, are most desirable. The instructor may thus explain to a class in a few minutes what would under other conditions require hours to make clear to them. This mode of procedure is as useful with pictures of the infinitesimal organisms and material in nature as with views of miles of landscape concerned with the geography of the globe.

Take for example the little daphina, shown in Fig. 4, magnified 150 diameters. This when thrown from a slide on an eightfoot screen makes the original magnified to nearly six thousand diameters. A class of students may look first at a tiny speck in a glass of clear water, which is perhaps one third the size of a pin-head, apparently without definite form, and of no consequence, but which, when seen enlarged, is shown to possess all the organs of a living animal. And further to illustrate how very tiny a particle of matter may become and yet be a mass as distinguished from molecules and atoms, the student has only to note such an illustration; for he sees that this microscopic animal is a mass as truly as is the elephant. In the study of rocks, of plants, of animals, even in a most elementary way, some very instructive lessons concerning their minute structure and how it concerns their outward forms and functions may be learned by such pictures.

Of course, all schools are not so situated as to be able to use a lantern in this way, though many have felt the importance of its use and of making a place for it. With the cheap forms of apparatus on the market at present and the readiness with which electricity or acetylene gas is obtained for an illuminant, the lantern should find its proper place in the class-room. Various forms of apparatus are also accessible for the projection of the microscopic slides directly, but in general, they give more trouble in handling to the average person than they are worth. The lantern slide is infinitely more satisfactory for general illustrative teaching. Another and in some eases better method for illustrating a subject is by the use of large pictures which can be hung up before a class and readily explained. Where classes are not too large, a photograph 20″ x 24″ in size is large enough, and there is then no necessity for a darkened room as with the lantern, and the picture may be considered in just the proper place in the course of a talk on the subject. These pictures are simple enlargements on bromide or velox paper from the original microscopic negative and cost but little more than lantern slides.

Since the scientific knowledge structure is continually building upon the foundation of accepted results of earlier investigators, it may be said in conclusion that photomicrography serves a double purpose. First, itenables the scientific investigator to determine accurately a knowledge of the minute physical structure of matter, and secondly, it provides a means of placing such information before others in a comprehensive manner.