PHOTO-MICROGRAPHY THE BEST MEANS OF ILLUSTRATING AND TEACHING PATHOLOGY BY MOSES GREELEY PARKER, M.D. Lowell, Mass. PHOTO-MICROGRAPHY THE BEST MEANS OF ILLUSTRATING AND TEACHING PATHOLOGY BY MOSES GREELEY PARKER, M.D. Lowell, Mass. READ BEFORE THE NINTH INTERNATIONAL MEDICAL CONGRESS [Reprint from Annals of Gynaecology, Boston, August, 188S] B O S T O N Press of Rockwell and Churchill, 39 Arch Street 1888 PHOTO-MICROGRAPHY THE BEST MEANS OF ILLUSTRATING AND TEACHING PATHOLOGY. PHOTO-MICROGRAPHY is the art of producing enlarged pictures of microscopical objects, by projecting the image of the object through the microscope, or combination of lenses, and catching this image, many times enlarged, on the sensitive film. Micro-photography is a term sometimes used to express the same thing, but erroneously, as it is the art of producing miniature pictures of objects so minute as to require a magnifying glass for their examination. Thus we see that photo-micrography is the reverse of micro-photography ; the one being the art of obtaining a large picture of a minute object, the other the art of obtaining a minute picture of a large object; while photography is the art of fixing the image produced by the camera, or combination of lenses, on the sensitive film. Photo-micrography dates back to the discovery of Daguerre, published in 1839. After Daguerre came Reade, Donne, Hodgson, Kingsley, and Talbot, early workers in this attractive field. After these, many in France, Germany, England, and America took up this most interesting study ; among the latter Woodward stands pre- eminent. His advantages in Washington enabled him to obtain photo- graphs of diatomes which have never been surpassed. Koch first used photo-micrography to delineate bacteria ; his first publication was in 1877, in which he speaks in the highest terms of the method. By photo-micrography we can demonstrate more clearly, and teach more accurately, the minute changes constantly taking place in disease, than by any other means. Photo-micrography is entirely void of any mental impression conveyed by the observer, through the engraver, to the picture. A photo-micrograph is the true picture engraved by the constant, faithful, all-observing artist "Light," as the lens sees it; and we all know that the lens sees vastly more than the human eye, therefore is the instru- ment used to record scientific investigations. 4 PHOTO-MICROGRAPHY. The oculist tells us that all eyes are not alike, and consequently do not see alike. The result is sometimes difference of opinion, and here the photo-micrograph comes in to settle the question. It is of the greatest aid in microscopic research, as we have seen, when used for illustration on the screen in the section of gynaecology. The gentlemen used also the most beautiful and attractive colored microscopical specimens in their illustrations, but all depended on photo- micrographs to illustrate the detail of disease, for in no other way can this be brought out as well. I shall not, in this paper, describe the process of making photo-micro- graphs, as this has been so well done in this country by Woodward,1 Sternberg,2 Walmsley,3 Piersoil,4 Y. May King,5 and others, but say to the beginner, much time can be saved, fewer plates spoiled, and more satis- faction obtained, by taking a few practical lessons from a good instructor at first, rather than work the art out from books alone, although good results will be obtained, if one follows the directions given by the above writers. In focusing an obj'ect accurately, one will find some difficulty, owing to the great difference in eyes. It is true that blue glass, or a solution of ammonia sulphate of copper, will greatly assist in getting a sharp chemical focus. The sharpness of this focus is what gives brilliancy and detail to the picture. In my opinion it is incorrect focusing rather than jarring that gives a blurred picture. What else can it be when the image is caught in the small fraction of a second? This is further illustrated by the individual who possesses an eye color-blind as to greens and reds. For such an eye the blue glass is of little assistance, as it sees the chemical focus unaided, and in this branch of science possesses great advantages over the normal eye, which sees the many variations of color. For this work the color-blind eye has a great advantage over the normal eye, a fact I have not seen mentioned before. There are many difficulties in photo-micrography yet unsolved, but good work can be done up to several hundred diameters, depending greatly on the specimen. The thinnest specimens, finely colored with alum cochineal, give admirable results. Thickness of specimen or diffuse cnlnrincr are fatal to clearness and brilliancv of detail. 1 Woodward. - " American Journal of Science and Arts." Vol. 42, September, 1866. Report to Surg.-Gen. U.S.A., 1S70-71. 2 Sternberg. - " Photo-micrographs and how to make them." Published by Osgood & Co. 1883. "Walmsley. - "Anthony's Photographic Bulletin," December 25, 1886, January 8-22 and Feb- ruary 12, 1887. 4 Piersol 1. - " Medical News," Philadelphia, June 19,1886; "New York Medical Journal," June 26, 1S86. BY. May King. - "New York Medical Journal," July 2, 1887. PHOTO-MICR O GRAPHY. 5 Having obtained a good negative of any valuable microscopical speci- men, it is a satisfaction to know that the picture from this negative can be reproduced, by various processes, and at a price within the reach of all. Everything considered, the stippled electrotype plate, prepared from the negative for press-work, is probably the best. This is used in illus- trating many of our best magazines. It would be an invaluable addition to our medical journals, and would have a far-reaching effect on the study and demonstration of pathological questions. In studying microscopical objects it is impossible for several to ex- amine the same object at the same time, unless the image is projected on a screen. Even then it is interesting to know that the image would be found out of focus for any one who happened to be color-blind ; for the color- blind a different adjustment must be made. All microscopists know that with high powers the slightest turn of the fine adjustment causes the image to present different lights and shades, or disappear entirely' ; consequently the hand is on the fine adjustment screw, and almost intuitively turns it to accommodate the eye to the dif- ferent parts of the field. The same instrument is not used by all micro- scopists, consequently they do not all have the same illumination and the same amplification. One may work with a large diaphragm, the other with a small ; one with a condenser, the other without. These differences, and many others, occur, even when the same specimen is passed around for examination. Should each person prepare his own specimens, a greater difference will occur. The method of preparing will not be the same, the cutting and staining will vary, and the mounting medium may not have the same refractive power. Under all these varying conditions, how can we expect all to agree as to what they see, and more especially when the objects have to be magni- fied from 100 to 200 diameters before they are seen at all? With these conditions, does any one wonder that varied opinions prevail, or that even the existence of some things is denied? Under the existing circumstances it is impossible that it should be otherwise, and to settle the disputed points, without the aid of something more, is absolutely impossible. All this unrecorded microscopic study is lost to science. Fortunately we have in photography a method of recording that is most accurate and valuable ; for, no matter how carefid one may be, his own drawings carry his own personal impressions, and when one attempts to record through an engraver, as we have before mentioned, he is no better off. 6 PHO TO-MICR O GRAPHY. A good photo-micrograph is often more valuable than the original specimen, as it places the object before the observer in such a way that he can compare, measure, and make his own observations, and draw his own conclusions. This is not possible with drawings. Again, the photograph gives the picture with its lights and shadows. If these are not good in the specimen, one cannot improve them by pho- tography. This, however, is not true with drawings. One can make a very good drawing from a very poor specimen, and, by so doing, give no idea of the value of the specimen. Not so with photography. Photography reproduces the image so faithfully, that in this repro- duction it criticises the preparation of the specimen itself, and thereby claims its own superiority ; a claim most valid, and one, I think, all will concede. To illustrate this difference of representation by engravings and photo- micrography I would call attention to the illustrations on the opposite page. Fig. lisa copy of an engraving, photographed from a book illustration, with the characteristic diagrammatic appearance of the arteries and co- lumnar epithelium. Fig. 13 is a copy direct from the photo-micrographic negative, not so much enlarged, but representing the same pathological change.1 We add, to further illustrate the value of photo-micrography, sixteen photo-micrographs, with explanations on opposite pages (Figs. 1 to 16, inclusive), used to illustrate Dr. Cushing's valuable paper on "Cancerous Degeneration of Hyperplastic Glands of the Cervix Uteri," read before the Ninth International Congress, and reprinted in the Annals of Gynaecology, April, May, and June, 18SS. 1 Since the above was written, Hydrochinone has appeared on the market as a developer, and un- like pyro does not stain everything it comes in contact with, while it develops equally as well as pyro or iron, a little slower perhaps, but much stronger, and does not blacken the fingers while holding the plate in the solution. It retains its developing power for a long time after being mixed and used over and over again. I have used it with good results two months after first mixing. I have developed the bromide paper in the solution with good results, the picture coming out clear, and the paper remain- ing perfectly white. PHOTO-MICR O GRAPHY. 7 Fig. 1. Fig. 13. 8 PHO TO-MICR O GRAPH V CANCEROUS DEGENERATIONS. EXPLANATION OF FIGURES. Fig. i shows at a the normal epithelial covering of the mucous mem- brane ; at b, this is broken through rather abruptly, and the rest of the surface is not covered by any mucous membrane, nor is it divided from the subjacent tissues by any regular layer of cells, as is the case in the " erosions" caused by papillary thickening or glandular hypertrophy ; at c is seen a tortuous blood-vessel running up into the cancerous nodule, of which the processes or pegs are seen in cross section. The rapid increase of the cells raises the cancerous surface at d ; above the level of the sur- rounding parts at c the mucous membrane reappears. On each side of the cancerous nodule is seen a gland. In that on the left the duct is preserved, while in that on the right it is known from other sections that a duct existed leading up to surface. These glands are seen enlarged in Figs. 2, 3, and 4. Enveloping the glands, and reaching beyond them in every direction, is seen a dark stain covering about half the field. This repre- sents a very active proliferation or immigration of small round cells. For want of any accurate knowledge this is called an inflammatory reaction of the tissues ; " its resemblance to other " inflammatory" accumulations of cells around points of bacterial infection is very striking. Fig. 2. Above is one of the glands seen in Fig. 1. At a and b the gland is normal and lined with cylindrical epithelium. To the left of b the contour of the gland is lost, and at c it is seen again, but it now is represented by a solid epithelial process. This, however, I do not con- sider to be a transition to a cancerous peg, but a process of rapid growth of the gland in which a lumen will afterwards appear ; d shows the duct of the gland; c,y, z, cross sections of cancer-pegs; g, k, a longitudinal section. The centre of the field is occupied by a cancerous mass, which, by shrinking in alcohol, has left a narrow open space. Between this and the gland above the tissue is packed with small cells entirely obscuring the proper muscular uterine cells; k is a shadow, caused by improper adjustment of the ray of light in photographing. PHO TO-MICR O GRAPH Y. 9 Fig. 1. Fig. 2. 10 PHO 7 O-MICR O GRAPH Y. Fig. 3 shows the larger gland shown in Fig. i ; towards a the cylin- drical epithelium lining the gland is still to he seen intact; everywhere else it has disappeared, and is replaced hy a thick layer of large cells, forming a ring around the lumen of the gland, seen in the figure, about half an inch wide. At b and c were apparently processes of the gland, already now destroyed ; at d, and running up to a, there is a thicker mass of larger cells, already seemingly cancerous. In the lumen of the gland is seen a mass of cells which represent the contents of the gland, shrunken by the alcohol. Fig. 4 shows with a higher power the part of Fig, 3, opposite a. In the centre is seen a row of cylindrical epithelium, abruptly terminated at either end by a growth of large cells, b, which, although not arranged in distinct pegs, or processes, are distinctly larger than the small cells with which the tissues adjacent to the gland are crowded. It will be noticed that there is no transition between the cylindrical epithelium and the flat cells. The boundary of each kind is sharp, and gives the idea of a malignant invasion of the lining of the gland. As far as I know, this appearance is pathognomonic of cancer. Above c are seen the nuclei of the cells contained in the lumen of the gland. These cells, also, are large and flat, and it may well be that a cancerous process has invaded the gland at some point, and, growing in its cavity, has secondarily invaded the walls. This explanation is implied more strongly by other sections of the same gland, which are filled cpiite full of a cancerous mass. The shadow at d is a photographic error. Below the cylindrical cells, between e and f, the tissues are comparatively normal. PHOTO-MICR O GRAPH Y. 11 Fig. 3. Fig. 4. 12 PHOTO-MICR O GRAPHY. Fig. 5 shows at a, a, a, a a cancerous mass, occupying nearly one- fourth of the field. Most of the processes, or pegs, happen to be cut lon- gitudinally. In the right upper corner are the remains of two glands, showing remains of cylindrical epithelium on a level with e. Above this, on a level with d^ a growth of cells has replaced the cylindrical epithelium on both sides of the inner gland and on the left side of the outer gland. Here also there is no transition, but the glandular epithelium is, as it were, eaten up by the invading cells. A dense infiltration of small cells precedes the growth of the cancer, reaching about to a line drawn from e to g. At c is a normal gland. Above b is a gland not yet invaded by the cancer, but showing in a high degree the changes mentioned by Ruge and Veit, and described in the next figure. At and neary is normal uterine tissue. Fig. 6 shows the last-mentioned gland with a higher power. At a the lining cylindrical epithelium is seen to be growing rapidly, one layer being superimposed on another, forming brush-like projections into the lumen. Only the nuclei of the very thin and transparent cells are seen in the figure. At b the shrinking of the preparation has separated from the walls of the gland the lining of cells, which here show coherent masses. There is no infiltration of the periphery of the gland with a new growth, as in Fig. 3 and Fig. 5, d. I hardly think that the changes here ought to be considered as a transitional stage in the process of cancerous degenera- tion, for similar changes occur in glands, under irritation, when there is no cancer present or impending, as I shall hereafter show. The rapid growth of the epithelium in this case, like the sprouting of solid buds from the gland, as seen in Figs. 2 and 8, seems to be a reaction of the glandular structures, liable to occur from various stimuli, one of which is the prox- imity of cancer. PHOTO-MICR O GRAPH Y. 13 Fig. 3. Fig. 6. 14 PHOTO-MICR O GRAPH Y. Fig. 7, from the same specimen, shows at <?, a. a cancerous mass ex- tending two-thirds across the field, broken by a cleft under c, where part of the new growth has ulcerated away. The cancer-pegs are seen on both sides of this cleft, and between it and a, - some cut longitudinally and some cut across. On the left, at b, are normal glands ; between e, e below is normal uterine tissue, darkened somewhat by a photographic shadow. Over y is a cotton fibre, accidentally imbedded in the Canada balsam. Over d is a gland which is quite close to the cancer, and surrounded by a growth of small cells. Fig. 8 shows this gland much enlarged. It seems nearly normal, but on each side is a solid sprout. While this might be due to a simple bud- ding growth, or an accidental section, near the side of an acinus, giving a tessellated appearance from cross section of the cylindrica lepithelia, yet there are to be seen signs of incipient degeneration, not by transitional stages or changes of the epithelial lining of the glands, but by invasion from ozitside of it. All around the gland are seen the nuclei of the infil- trating growth of new cells, taking the color strongly in comparison with the nuclei of the cylindrical epithelial cells. Over b the former are massed very strongly, and these new highly-stained nuclei can be seen all along the lower edge of the gland wedged in between the cylindrical cells. This is very well marked in the sprout near a. It is readily seen that a pro- liferation of these new and active cells would soon give a picture like that seen in Figs. 3 and 4. PHO TO-MICR O GRAPHY. 15 Fig. 7. Fig. 8. 16 PHO TO- MICR O GRAPHY. Fig. 9 (50 x) shows a part of the specimen where the cancerous development has supplanted the normal tissues almost entirely. There are no glands visible. To the left, at A, is relatively normal uterine tissue, thickly sprinkled with the nuclei of new cells, the "inflammatory reaction" preceding the advent of the cancer. At the level of B B are lymph spaces. All the middle of the field is filled with characteristic processes, or pegs, of cancer. These are composed of cells, not very large nor flat, crowded together with no connective tissue between them. The different processes are cut longitudinally, obliquely, or transversely in different places. Their outline is regularly irregular, very different from bundles of uterine muscle, or solid new-formed glands. At the right the free edge represents the surface of the cancerous ulcer, ragged, necrotic, bare of epithelium, - a condition very different from the so-called erosion (yide Annals of Gynecology, Oct., '87), and characteristic of cancer, tuber- culosis, syphilis, or similar grave " constitutional" disease. Between the pegs of cancer the tissues are infarcted with new cells, somewhat smaller than the cancer-cells, in such abundance that the uterine muscular tissue cannot be distinguished. The next three figures are from a case of cancer of the cervix, which apparently commenced among the glands of the cervical canal, or in the parenchyma of the cervix, spreading downward and outward, but involving the flat epithelium of the portio vaginalis very little. A large cone of cervical tissue was removed, the uterine canal thoroughly cauterized, and the angles of the wound in the cervix brought together with sutures. There has been no recurrence in nine months from the time of operation. Fig. 10 (50 x) shows at A' A pegs of cancer, forming part of the mass which fills all the left part of the field, sharply defined even to the naked eye. At B is a large gland surrounded by irritated tissue. Below this the field shows normal uterine tissue between the cancer at the left and the glands at C. The latter are growing rapidly. Microscopic examination shows them to be budding with solid sprouts, and to be surrounded by a tissue filled with small cells, as at B. Between these glands and a little cystic dilatation opposite D the free surface of the cervix is seen to have its flat epithelium thickened, the papillae greatly elongated, and the surface, as at E, "eroded" in places, or rather undermined by glandular pockets, as described by Ruge and Veit. PHO TO-MICR 0 GRAPHY. 17 Fig. 9. Fig. IO. 18 PHO TO-MICR O GRAPHY. Fig. ii (150 x) shows the gland seen at B in the last figure. The infiltration with small cells of the tissues around the gland is plainly visible. Various sprouts or branches of the gland are shown by this section, of which the youngest, to the right of A and B and to the left of C, appear solid, being cut near the end and across the lining cylindrical epithelium. It is plain that such glandular processes have nothing to do with the solid cancerous pegs shown in the next figure. Where a number of glandular solid sprouts are shown together in a cross section, there is a certain similarity of appearance, but in such cases some of the sprouts will show a lumen, or the arrangement of the outer layer of cells in a ring of elongated epithelium, while all the sprouts will be nearly round or oval, very different from the irregular shapes of the cancer-pegs as seen in Fig. 9. The lining of this gland is seen to be an unbroken layer of epithelium, and although so near the cancerous mass and evidently soon to be attacked by it, there is no indication of any transition of its epithelia into cancerous cells, such as can be shown in adenomata of the fundus. Fig. 12 (250 x) shows with a higher power the part of Fig. 10 to the right of A1. At A B C are partitions of relatively healthy tissue between the cancerous masses, dividing the field into alveoli. DEF show aggregations of cells, neither large nor flat, forming cancerous masses lying in alveoli, as seen in Fig. 10. These masses are not penetrated by any septa of connective tissue, have no lumina, and show the cells lying close together. The whole tissue is, however, infiltrated with similar cells not collected into alveoli, and of various sizes, from ordinary wander- cells upward. All these new cells take the color strongly. The whole specimen corresponds to the description of Ruge and Veit, who have in cancer of the portio vag., " here precisely in the spot where cancroid most com- monly occurs, most frequently observed the origin of the latter from connective tissue."' PHO TO-MICR O GRA PHY. 19 Fig. H. Fig. 12. 20 PHO TO-MICR O GRAPH Y Fig. 13 (25 x) is from case N. H., mentioned in the foregoing paper. Here the age, clinical history, and gross appearances all indicated an incipient cancer; and the diseased tissue, with a wide margin of healthy vaginal portion, was freely excised. There has been no return now in nearly a year. The figure shows on the right the free edge of the section, which was made per- pendicular to the margin of the os externum. The epithelial covering is gone; the ragged surface is covered with papillary outgrowths. These, with the tissue under them, are so thickly crowded with small cells that they appear black and opaque, since the nuclei of the newly-formed cells take the color strongly and prevent the light from passing through. The loss of the epithelial layer is here not post-mortem, as the portion excised was put immediately into Muller's fluid. To the left of the densely infiltrated border, that Is, below the abraded surface, the tissue is seen to be studded with glands lined with cylindrical epithelium. These are in a state of great activity, and the heavy dark lines bounding them indicate the rapid cell- formation on their surfaces. At A is a deep cleft bordered by these newly-formed glands. This is seen enlarged in the next figure. At B and all along the left of the field is normal uterine tissue. No cancer pegs anywhere. Fig. 14 (150 x) shows the cleft in the tissues, seen in the last figure at A. It is lined in its upper half with ciliated epithelium, beneath, and in some places re- placing, which is a dense infiltration of small cells, making the picture look thick and black. There is no such lining of the lower half of the cleft here ; the open space prob- ably represents a rent in the tissues, the sides of which are sundered by shrinking in alcohol. The same shrinking will account for most of the free space in the upper half of the cleft. This, then, would represent one great gland with numerous branches which are growing rapidly in either direction from the main trunk. These glandular branches are lined with cylindrical epithelium, and in some the lumen is indicated, as in those opposite A, B, and C, and the upper half of D. In most of the sprouts, however, no lumen is visible ; and this may be explained in two ways, either by the section running in one wall of a hollow sprout, or by the fact that the sprout buds out into the tissues as a solid mass of cells, and becomes hollow as it gets older and larger. I am convinced that although the first explanation may account for the appearances in some sections, as in the gland opposite C, and in Fig. 15, yet the second explanation is true as concerning the mode of growth of rapidly- forming new glands, as seen at D, E, E, and in Fig. 16. The subjacent uterine tissue of the vaginal portion is seen in the upper part of the picture to be darker and infiltrated with round-cell nuclei, while in the lower part of the field it is normal. PHOTO-MICR O GRAPHY. 21 Fig. 13. Fig. 14. 22 PHO TO-MICR O GRA PH Y. Fig. 15 (450 x) shows the glandular branch seen at C, in Fig. 14. Below the open lumen the ciliated columnar lining is shown very clearly. Over A, B can be seen the dark nuclei occupying the lower half of each cell. To the right, as far as the space over C, the nuclei are shorter and more irregular, corresponding to an in- filtration of. cells of another and rounder form, apparently due to a rapid prolifera- tion of the cylindrical epithelium, or to a change similar to that seen in Fig. 8. Higher up, opposite D, the section runs obliquely through the cylindrical epithelium lining the wall of the gland, which here makes a turn, and correspond- ingly the cross-sections of the cells are seen in various degrees of foreshortening. In the middle, on the level of D and above B, the cells have an appearance like pave- ment epithelium, which they clearly are not; and it is also of importance not to confound them with cancer cells, nor to attribute to them any particular significance which they do not possess. The border of this gland on the right is darker, from intense staining of the cell nuclei. By comparing the different parts of this figure with the corresponding places in Figs. 14 and 13, a correct idea may be formed of the significance of the dark lines and patches in the latter figures, which represent, in general, dense aggregations of highly-stained nuclei of newly-formed cells. Fig. 16 (300 x), from another section of the same specimen, shows the above- mentioned mode of growth of the glandular sprouts as solid processes, at A and B, less clearly at C and D. The clubbed end of the large gland opposite E shows divisions, and in these an apparent filling up with cells, which I understand, how- ever, as a stage of the process of becoming hollow in sprouts previously solid, and not the reverse. The uterine tissue in which these sprouts lie is normal. B FiQ. 15. Fig- 16-