MICROSCOPIC rechniques FOR THE S T U D Y OF COLLAGEN1

Transcription

1 183. MICROSCOPIC rechniques FOR THE S T U D Y OF COLLAGEN1 ALFRED L. E V E R E T T Interest in all phases of collagen and connective tissue research has greatly increased during the past two decades. Undoubtedly the medical field has led all othersin ktensifying its efforts in this area in order to make some headway against aging effects and the poorly understood collagen diseases. M s o, those of US in the leather field have been actively seeking new information about the composition, structural organization and chemical reactivity of collagen, especially as it occurs in animal skins. But the study of collagen from any and all sources, and from every conceivable viewpoint, should be of mutual benefit to us all. Leather chemists are justifiably concerned with collagen since it is the essential raw material for their product. Processing methods are designed, in simplest terms, for purifying the collagen and converting it to a stable and durable form while adding certain other desirable properties. This viewpoint is obviously very different from yours in meat research, for you are more concerned with minimizing the effects of connective tissue. In fact I am sure you would be happy to be rid of it entirely! Recently it has become more apparent than ever that the microscope is an invaluable tool for revealing the structural origins of many of our problems. After looking over the Proceedings of your recent meetings I can see that you have given much attention to the approaches of histology and histochemistry, and also to some excellent biochemical studies. I intend to add a few observations from our different viewpoint that may, hopef'ully, stimulate further investigation. It might be informative to start by showing you our light microscope, on the first slide, since it is rather unique and highly versatile. Two intermediate lenses and five objectives allow rapid shifting between Slide 1 camera and automatic exposure device. 'Presented at the 18th Annual Reciprocal Meat Conference of the American Meat Science Association, Eaanhattan, Kiinsas, June 15-17, Eastern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture.

2 184. film in a built-in camera at the back, while the device on the left, after suitable calibration, automatically sets the correct exposure with few exceptions. Slide 2 shows Slide 2 Electron Microscope our electron microscope and its operator. This work is performed in another division, utilizing most of the modern techniques for sectioning, shadowing, etc. Non-collagenous components Before concentrating on collagen I would like to touch briefly on the principal non-collagenous components of connective tissue. This area has been well discussed in previous meetings but a few points deserve further comment. Elastic tissue is somewhat less important to leather than it is to meat properties. A number of good staining methods are available; perhaps the best two of these are acid orcein and Verhoeffrs iron-hematoxylin (9, 11, 12) for their intensity of staining. Where a m i n i m of nonspecific background staining is desirable we prefer Fullmer &Lillie's orcinol-new fuchsin (3, 12) or Gomori1s aldehyde-fuchsin (4, 6, 9, 11, 12). The identity and significance of reticular tissue, or reticulum (13) have long been controversial, although histologists still recognize such a fiber. Many staining methods hsve been described but all are notoriously fickle. Slide 3 shows the only one we have tried a variant of the Bielschowsky- - ~ Reticular fibers, stained black by silver impregnation, in a horizontal (parallel to surface) section of cowhide, X l O O Maresch method (9, 11, 12). This is a horizontal section of cowhide through the sebaceous glands, where a number of black reticular fibers can be seen in several locations. Mucopolysaccharides are c o m n constitutents of connective tissue and are relatively abundant in skin. Metachromatic stains (5, 6, 9, 11, 12) are very useful for demonstrating the acidic types because a single solution can stain several different materials at once in a variety of colors. The presence of strongly acidic groups results in a chemical conversion of the dye to a different color form, the final effect being dependent on ph. Slide 4 shows a vertical section of cowhide stained with toluidine blue Slide 4 Nktachromatic staining of acid mucopolysaccharides in cowhide, using toluidine blue at ph 6, X50 ~~~ ~~ - ~ buffered at ph 6. The upper, dark layer of epidermis and the central hair surround two areas of unstained dermal collagen, in which are many bluishpurple cell nuclei and several reddish-violet regions. This violet color is

3 185. indicative of the acidic material, and a closer look in Slide 5 reveals that it is diffusely spread among the fibers, and not associated with any distinct physical structure. "he minehart-hale dialyzed iron reagent Slide 5 1 Similar area at higher magnification, XlOO I (5, 6, 9, 11, 12) and Stedman's Alcian blue stain (6, 9, 11, 12) are also useful for this purpose. Mucoproteins of the ground substance are also abundant in skin and can best be visualized by a periodic acid-schiff (PAS) sequence such as that of Hotchkiss (5, 6, 8, 9, 11, 12), as shown in Slide 6. I n addition, notice that this stain a l s o demonstrates a wide band of intensely stained material along basement membranes. This PAS-positive material is Slide 6 Ground substance and basement membrane in cowhide; periodic acid-schiff, XlOO, solubilized to varying degrees by treatment with certain salt solutions and enzymes, and is apparently involved in binding the epidermis of skin to the underlying dermis (2) Attractive combination stains have been developed with this procedure, such as following with acetic alum-hematoxylin to stain nuclei (9), or preceding with Alcian blue to show mucopolysaccharides (11). Finally it should be noted that fat staining is of considerable value in the study of both leather and meat. In our experience the best general purpose stains have been Oil Red 0 and Sudan Black B (5, 6, 9, 11,12). Polarization Microscopy I n our studies of collagen fiber morphology and organization, the most valuable single technique has been the use of polarized light (7, 12). The pronounced birefringence of collagen makes all fiber bundles stand out clearly when they are in or near the plane of the section, while those near the perpendicular plane are very dark. Unfortunately, the intensity of this effect is related to fiber diameter. Little or nothing can be seen at thicknesses less than 1 micron, so its application has definite limits. Likewise the effect is masked, even in heavy hides, by strongly colored dyes, pigments and vegetable tannins. Let me illustrate with a problem we are now studying. Certain Hereford hides have an inherent defect, commonly described as pulpy, mushy, or vertical fiber (1,14), consisting of an abnormal, vertical alignment of their fibers. When leather from these hides is split for making shoe uppers, it is usually so weak as to be practically worthless. Slide 7 is a composite of full-depth cross-sections of two hides, one with normal fiber Slide 7 Vertical cross-sections of 2 hides comparing normal structure vs. pulpy fiber (vertical), X5

4 186. pattern on the left compared with the abnormal, vertical fiber pattern on the right. The angular, interwoven, normal arrangement is of course responsible for high tensile strength, whereas the abnormally vertical arrangement lead6 to much lower strength. This is mre apparent in Slide 8, which shows a vertical cross-section of the upper half of a normal hide in polarized light. Slide 8 i The angular fiber pattern can now be recognized more clearly. Slide 9 shows Vertical section, upper half of normal hide, polarized light, X10 Slide 9 I Similar section of pulpy fiber hide, XlO 1 a similar section from an abnormal, pulpy fiber hide and there is no mistaking the vertical arrangement of the collagen fibers in the lower (corium) area. We have found that the distinction between horizontal and vertical fibers can be demonstrated more dramatically by cutting sections parallel to the surface through the center of the corium, and viewing them in polarized light. Slide 10 shows such an unstained section of normal Angus hide, and Slide 10 - I Horizontal s ec tion through mid- corium I of normal Angus hide, unstained, X10 most of the heavy fibers can be recognized fairly well. But notice how Slide 11, in polarized light, greatly clarifies the same field and permits Slide 11 1 Same field as 10, polarized light, XlO 1 much more detailed analysis of the intricate architecture. Note also that the vast majority of fibers are bright, proving that they are predominantly horizontal, and are fairly wide in diameter. Then we found that by adding a special filter to the light path, called a first-order red compensator or retardation plate (15), placed at 450 to the plane of polarization, we could create the beautiful effect shown in Slide 12. This adds a whole new Slide 12 Same field as 10, polarized light with red compensator, XlO dimension to the interpretation. The bright, brizontal fibers now appear in two interference colors: yellow in one direction and blue at right angles to this. Vertical fibers are still dark but no longer opaque as in -

5 187. t h e previous slide, so t h a t a d d i t i o n a l d e t a i l s are apparent i n t h e s e regions. The background i s c l e a r red, i n d i c a t i n g where holes o r f a t c e l l s a r e located. L e t u s compare t h i s normal p i c t u r e w i t h what we see i n t h e abnormal Hereford hides, shown i n S l i d e 13. Here it is evident t h a t the yellow and blue h o r i z o n t a l fibers are narrower and fewer i n number, with many more dark a r e a s S l i d e 13 Horizontal s e c t i o n through corium of abnormal Hereford hide, polarized l i g h t with red compensator, X l O of v e r t i c a l fibers. Ivbst of these are a c t u a l l y compound groups of s e v e r a l fibers c l o s e l y spaced. I hope you l i k e our new o p t i c a l a r t form! This i s a purely o p t i c a l e f f e c t ; there i s no s t a i n i n g involved. Phase Contrast Microscopy I would l i k e t o d i s c u s s b r i e f l y some recent work involving p a r a l l e l study of u l t r a t h i n hide s e c t i o n s i n both l i g h t and e l e c t r o n microscopes. Phase c o n t r a s t is e s p e c i a l l y applicable t o such t h i n specimens, b u t becomes less e f f e c t i v e as thickness increases. This l i m i t a t i o n is i n t h e opposite d i r e c t i o n t o t h a t described f o r p o l a r i z a t i o n, b u t t h e r e is an intermediate area where both can be u s e f u l. Slide 14 shows a resin-embedded (10) sect i o n of hide corium, about 1 micron t h i c k, i n r e g u l a r light; f i b e r o u t l i n e s Slide 14 Thin s e c t i o n of high corium, approx. 1 micron, regular l i g h t, X 5 1 ~ are f a i r l y c l e a r b u t n o t much d e t a i l i s v i s i b l e. S l i d e 15 shows t h e same s e c t i o n i n polarized l i g h t with a l i t t l e more detail, b u t t h e g r e a t e r Slide 15 Same section, polarized l i g h t, X 5 1 advantage here is t h a t t h e r e l a t i v e brightness i n d i c a t e s d i r e c t i o n of o r i e n t a t i o n. Then S l i d e 16 shows w h a t t h e same s e c t i o n looks l i k e i n phase c o n t r a s t. I c h more of the s u r f a c e d e t a i l i s now apparent, a g a i n s t the b l u e background produced by a d a y l i g h t f i l t e r. As t h e magnification is Slide 16 Same section, phase c o n t r a s t, X 5 1 I increased, i n S l i d e 17, f r o m about 50 t o over 200, more and more of t h e surface f e a t u r e s show up w i t h three-dimensional c l a r i t y. S l i g h t l y t h i n n e r Slide 1 7 Portion of same s e c t i o n, phase c o n t r a s t, X204 I s e c t i o n s a r e more s a t i s f a c t o r y f o r higher magnification b u t r e s o l u t i o n i s extremely poor with t h i c k e r s e c t i o n s.

6 ~~~ ~ 188 Electron Microscow Using alternate sections of the same material, cut at about 0.1 micron, or less, considerably more detail and better resolution can naturally be achieved with the electron microscope. At present we are attempting to overlap the two ranges of magnification for a Eore comprehensive picture of fiber organization. Slide 18 shows a section from the grain layer of hide magnified about 400 times on the slide. The fiber bundles Slide 18 Ultrathin section of hide grain layer, electron micrograph, X395 look quite similar to what we have seen in the light microscope. There is one group cut longitudinally and several others in cross-section. As the magnification is gradually increased it is found that the large fiber bundles are in turn made up of smaller and smaller bundles, and each of these composed of tiny fibrils. Slide 19, magnified 6,500 times, shows 1 Slide 19 Ultrathin section of hide grain layer, electron micrograph, X6,500 clearly some of these fibrils lengthwise and also portions of two fibril bundles in cross-section; and finally Slide 20, at about 36,OOOX, shows much of the fine structural detail to be found in a collagen fibril, with Slide 20 Electron micrograph of collagen fibril, X35,946 its typical periodicity. There are also a number of fibrils in crosssection. We are still in the preliminary stages of this work, but hope to gain useful information as we compare samples from different sources. Application to lviuscular Tissue In conclusion I will try to indicate briefly how these techniques can be applied to meat or muscular tissue, using two routine hematoxylineosin slides, prepared from a normal Eolstein cow, from the files of our Meat Inspection Division in Beltsville, Md. Slide 21 shows some connective Slide 21 Cross-section of Holstein skeletal muscle, hematoxylin-eosin, X256! tissue fibers in the space between two large bands of skeletal muscle. Switching to polarized light, as shown in Slide 22, immediately indicates that at least some of these fibers are collagen because of their brightness. To make sure how many are collagen it is necessary to rotate

7 ~ 189. Slide 22 Same Field, polarized light, X256 the slide about 45' and watch for additional bright objects. The birefringence of muscle is also apparent here, but it is much less intense and the striations at low magnification make its identity obvious. Slide 23 then shows the same field in phase contrast, merely to demonstrate how Slide 23 Same Field, phase contrast, X256 distinctly this of no value for picks up even the smallest fiber fragments. However this is actually identifying collagen. Slide 24 shows another area Slide 24 Cross-section of skeletal muscle, hematoxylin-eosin, X64 similar to the previous one at one-fourth its magnif'ication, but this time the fibers are running perpendicular to the original direction. Converting to polarized light in Slide 25, the muscle is seen to be in the dark position and again the collagen can easily be identified. But the bright spots in Slide 25 I Same field, polarized light, X64 I the thin, dark fiber suggest that rotation will prove this to be collagen also, Finally in Slide 26 we show a section of tongue for comparison. Here the heavy fibers in the center, between two portions of muscle, appear more Slide 26 Cross-section of cow's tongue, hematoxylin-eosin, X64 typically collagenous. Polarization, as shown in the last slide (27), Slide 27 1 Same field, polarized light, X64 I makes it quite obvious that this is true. Scanning such slides with polarized light is a good routine method for sptting collagen rapidly, keeping in mind that the slide must be rotated frequently. Admittedly some of these techniques are more effective with material rich in collagen, but it has been shown that the principles of polarization and phase contrast microscopy have wide application for investigating both the composition and the structural organization of fibrous tissues.

8 190. REPERENCES Amos, G. L. V e r t i c a l f i b r e i n r e l a t i o n t o t h e p r o p e r t i e s of chrome s i d e l e a t h e r. J. SOC. Leather Trades' Chem. 42, - 79 (1958). Everett, A. L. and Cordon, T.C. The use of s a l t t o a s s i s t enzymatic unhairing of f r e s h hides, with histochemical observations. J. Amer. Leather Chem. Assoc. 53, 548 (1958). 3. Fullmer, H. M. and L i l l i e, R. D. S t a i n Techn. 31, 27 (1956). A selective s t a i n f o r elastic tissue. 4. Gomori, G. Aldehyde-fuchsin; a new s t a i n f o r e l a s t i c t i s s u e. J. Clin. Path. 20, 665 (1950). 5. Gomori, G. Microscopic Histochemistry. Chicago. (1952). 6. Gurr, E. Methods of Analytical Histology and Histo-Chemistry. W i l l i a m s and Wilkins Co., Baltimore. (1960). 7. Holmstrand, K., Longacre, J. J., and de Stefano, G. A. s t r u c t u r e of collagen i n skin, s c a r s and keloids. Reconstructive Surgery 27, 597 (1961). 0. Hotchkiss, R. D. A microchemical reaction r e s u l t i n g i n t h e s t a i n i n g of polysaccharide s t r u c t u r e s i n f i x e d t i s s u e preparations. Arch. 16, 131 (1948). Biochem L i l l i e, R. D. Histopathologic Technic and P r a c t i c a l Histochemistry. The Blakiston Co., Inc., New York. (1954). Amer. University of Chicago Press, The The u l t r a P l a s t i c and 10. Luft, J. H. Improvements i n epoxy r e s i n embedding methods. Biochem. Cytol. 9, 409 (1961). J. Biophys. 11. McYTnus, J. F. A. and Mowry, R. W. Staining Methods, Histologic and Histochemical. Paul B. Hoeber, Inc., New York. (1960). 12. Pearse, A. G. E. Histochemistry, Theoretical and Applied. Brown and Co, Boston. Second ed. (1960).. Little, 13. F'uchtler, H. On t h e o r i g i n a l d e f i n i t i o n of t h e term " r e t i c u l i n. " J. Histochem. Cytochem. 12, 552 (1964) Roddy, W. T. Testing of pulpy hides. Oct. 31 (1964). Leather and Shoes, 148, Tonna, E. A. An evaluation of birefringence of s k e l e t a l t i s s u e s s t a i n e d with metachromatic dyes. J. Royal Microscopical SOC. 83, 307 (1964).

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10 192. Mgure 5 Figure 6 Electron micrograph of hide Cross-section of Holstein skeletal muscle showing section. Portions of two connective tissue fibers adjacent to strands of fibril bundles in cross-see- striated muscle. Negative print, hematoxylin bion; C O l h P fibrils length- eosin, regular light. x 940. wise at upper left. x 17,230. Figure 7 x Same field as Fig. 6 in polarized light to distinguish collagen Negative print.

11 193. R. G. CASSENS: Thank you Mr. Everett for the informative presentation. Our next speaker is Dr. M. Judge. Dr. Judge received his B.S. from F'urdue University, his M.S. from the Ohio State University and his Ph.D. from Purdue University. He is now in charge of &at Science work at Purdue University. During the past year he has been working as a Post-doctoral fellow with Dr. E. J. Briskey at the University of Wisconsin. Dr. Judge's presentaticn is concerned with adrenal gland histology as related to post-mortem muscle properties. ############if