BioEngineering Unit, University of Strathclyde, Glasgow

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Loren Lester
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1 SCANNING ELECTRON MICROSCOPE STUDIES OF HUMAN SKIN By K. E. CARR, Ph.D. BioEngineering Unit, University of Strathclyde, Glasgow TrIE mechanical properties of human skin have been examined using engineering techniques to explore its load-bearing characteristics (Kenedi, Gibson and Daly, 1965; Kenedi, Gibson, Daly and Abrahams, 1966). These results have been linked with changes in the histological structure of the dermis by Craik and McNeil (I965) who found that the collagen fibres of the dermis become orientated in the plane of stress, develop an affinity for the red dye oftrichrome staining methods and finally are disrupted completely. An attempt is now being made to explore further the three-dimensional relationships of the different components of the dermis and the alterations induced in these relationships by loading. This paper describes the structure of human skin as seen by the scanning electron microscope, an instrument which has already proved of use in the study of wound healing in the rat (Forrester et al, 1969). Preliminary results of experiments using the scanning electron microscope to study loaded and control human skin are described elsewhere (Finlay, 1969). Materials and Methods.--Human skin samples, either full thickness or split skin, were obtained at operation and were fixed in formol saline, pinned out on card. The tissue was then processed in the usual way for light microscopy and embedded in wax. Sections 25 to 35 # thick were cut and picked up on a metal stub suitable for use in the scanning electron microscope. After dewaxing in xylol, the specimens were coated on a rotating stagewith either 5ooA of gold/palladiumor 5o to iooa of carbon/platinum. They were then examined in a Cambridge Instrument Company Stereoscan Mark 2A operated at accelerating voltages of between io and 3o kv. The results were recorded with a Zenza Bronica-S camera with Ilford FP4 12o film. I2 # thick stained sections were also prepared for parallel histological studies. Results.--The low-power views of skin sections examined by scanning electron microscopy are readily comparable with the familiar histological picture (Fig. I). The epidermis, the dermis and in some cases the subcutaneous adipose tissue can all be seen. Each layer will be described in turn. The keratinised cells of the superficial stratum corneum are seen as layers of angular material (Fig. I). At higher magnification (Fig. 2) the boundaries of the remains of individual flattened cells can be seen. In most cases neighbouring cells lie closely apposed to each other within different layers which lie parallel to the surface of the skin, but periodically a flattened cell spans the gap between one layer and the next. The variations in shape of the cells comprising the different layers of the epidermis can be seen. The cells of the stratum basale are polygonal in shape while those more superficially placed are often quite flattened. The" intercellular bridges "of the stratum spinosum are particularly prominent, but similar features are also seen elsewhere. These areas of cell contact can be seen in greater detail at higher power (Fig. 3) and bundles of cytoplasmic fibrils, presumably tonofilaments, can sometimes be observed. At medium power the connective tissue of the dermis can be seen to consist of a multi-directional network of fibres (Gibson et al., 1965 ; Forrester et al., 1969). The fibres (Fig. 4) are grouped in large and small bundles which are often linked by networks of fine, thread-like fibrils, which can also be seen crossing the fibre bundles (Fig. 5). 66

Fig. r.--low-power scanning micrograph of section of h u m a n skin, normal to the surface. keratinised layer and the epidermis and dermis can be made out. 3oo. The Fig. 2.

2 Fig. r.--low-power scanning micrograph of section of h u m a n skin, normal to the surface. keratinised layer and the epidermis and dermis can be made out. 3oo. The Fig. 2.--High-power view of keratinised layer. T h e remains of several flattened cells can be seen, lying in layers. At one point there is a link between one layer and its neighbour, x3,o0o. Fig. 3.--High-power view of cells in the stratum basale of the epidermis. T h e "intercellular b r i d g e s " can be seen. Considerable shrinkage of the cells has taken place during preparation, x 6,9o0.

Fig. 4. - - M e d i u m - p o w e r view of dermis. Large fibre bundles can be seen~ the majority being cut in cross-section.

3 Fig M e d i u m - p o w e r view of dermis. Large fibre bundles can be seen~ the majority being cut in cross-section. Networks of fibrils joining these bundles can be made out. I~75o. Fig. 5.--Higher-power view o f an area from Fig. 4. T h e fibrillar sub-units making up the bundles can be seen and a clearer view of the networks of fibrils is shown. 8~75o.

Fig. 6.--High-power view of fibril network. Both straight and curled fibrils are seen. T h e fibrils link round each other, sometimes forming quite complicated knots, x 2o,0oo. Fig. 7.

4 Fig. 6.--High-power view of fibril network. Both straight and curled fibrils are seen. T h e fibrils link round each other, sometimes forming quite complicated knots, x 2o,0oo. Fig M e d i u m - p o w e r view of fibre bundle, with an extensively coiled fibril running beside it. x-4,75o. Fig. 8.--High-power view of the collagen fibrillar sub-units from a bundle cut in cross-section. T h e crossbanding can be made out, although the contrast is poor. x 25,000.

Fig. I o. - - M e d i u m - p o w e r view of hair follicle.

5 Fig. 9.--Low-power View of adipose tissue deep to the dermis. T h e outline of the cells is seen, although little cellular material is left. 4oo. Fig. I o. - - M e d i u m - p o w e r view of hair follicle. T h e hair with its cuticle can be seen. Fig. I I.--Low-power view of hair follicle, with accompanying sebaceous gland. 3oo. x 56o.

SCANNING ELECTRON MICROSCOPE STUDIES OF HUMAN SKIN 71 Examination of these networks of fibrils at higher magnification shows that they are interwoven and in some cases appear to be anchored between

6 SCANNING ELECTRON MICROSCOPE STUDIES OF HUMAN SKIN 71 Examination of these networks of fibrils at higher magnification shows that they are interwoven and in some cases appear to be anchored between the components of the large fibre bundles (Fig. 6). Some of the connecting fibrils take up a coiled configuration (Fig. 7). The major fibre bundles are composed of finer, parallel, dose-packed fibrillar subunits, seen in longitudinal section (Fig. 5) and more clearly in transverse section (Fig. 8). They are approximately 7ooA in diameter and show a faint but consistent banding, with a periodicity of about 6ooA. This banding suggests that they are collagen bundles. In some cases where the bundles have been transversely sectioned there is a suggestion of an external coating surrounding the whole bundle of fihrillar sub-units. Skin appendages can be dearly identified. At low power in suitable sections, hair follicles can be seen with accompanying sebaceous glands (Fig. IX) and on occasion the arrector pili muscle is seen. At higher power, the outer and inner root sheaths and the hair itself can all be seen (Fig. io). The scales of the cuticle are prominent and in cases where the hair has been pulled out during sectioning, the inner aspects of the sheath can be seen to have a matching pattern, forming what is essentially a replica of the cuticle. At low accelerating voltages it is possible to pick out structures which are roughly spherical and probably represent cells of the root sheath (Fig. Io). At low magnification, the layer of subcutaneous fatty tissue can be seen clearly in suitable sections. At higher magnifications a clearer image of the cells can be obtained but since the fat has been extracted during processing, only the thin surrounding rim of cytoplasm remains (Fig. 9). DISCUSSION The lower range of magnifications available with the Stereoscan, using dewaxed sections as described above, allows comparison with conventional histology. This permits easy orientation of the section and allows identification of the images formed in the scanning electron microscope. In skin, for example, the layers of the epidermis, hair follicles and fatty tissue can all be identified in the scanning microscope by direct comparison with an adjacent section under the light microscope. The higher magnification ranges enable detail to be seen which is normally only resolved with the transmission electron microscope, although the latter instrument does not present an image with such great depth of focus. For example, it is possible using the scanning microscope to resolve some of the substructure of the connective tissue fibres of the dermis, while still displaying in three dimensions the winding appearance of the large fibre bundles, since the thick sections allow full use of the greater depth of focus of the Stereoscan. One of the problems of scanning electron microscopy lies in the mechanism of contrast production, perhaps best illustrated by the differences between the image of collagen fibres obtained with the transmission and with the scanning electron microscope. The transmission picture shows the 64oA periodicity in sharp contrast. The scanning image however, shows only a faint banding which can barely be seen on the video screen, since contrast effects in the scanning electron microscope are due to differences in secondary electron emission or to contour effects on the surface of the specimen. Transmission electron microscopy, on the other hand, produces an image of high contrast in which dark areas represent parts of the specimen containing "electron dense" material. The image in this case depends on differential scattering of the electron beam as it passes through the specimen. Such contrast effects in transmission electron microscopy depend mainly on the adsorption of electron dense stains on the specimen, whereas suitable differential "staining" methods to enhance secondary emission contrast have not yet been devised for use with the scanning microscope.

7 2 BRITISH JOURNAL OF PLASTIC SURGERY Another problem is set by the interpretation of medium and high magnification scanning electron images.

7 7 2 BRITISH JOURNAL OF PLASTIC SURGERY Another problem is set by the interpretation of medium and high magnification scanning electron images. When examining histological preparations it is possible to identify different parts of the specimen by their observed reactions to chcmical stains. The interpretation of the image in scanning electron microscopy however, dcpends cssentiauy on the recognition, in this case using crude tissue sections, of surface contours which reflect boundarics present within cells and tissues. These are unsatisfactory criteria for structural studies and many problems of interpretation arc raised. It is possible, for example, only to guess at the nature of the different fibre types in the dermis. Althoughthc general appearance of the large bundles and their ~ 6o0 A periodicity suggests that they are collagen, while the thin coiled fibres are probably elastin, it is not possible even tentatively to identify the straight fibres of medium to small diameter. Extensive comparison based on the use of transmission electron microscopy and histological preparations will be necessary in order to identify the relatively non-specific images formed by the scanning electron microscope. At the present time, however, the instrument is well suited for the examination of the interrelationships of the dermal fibres as a whole and their variations under different forms of stress. SUMMARY The scanning electron microscope has been used to examine dewaxed thick sections of human skin. The features of the epidermis and dermis are described, particular attention being paid to the fibres of the dermis. The advantages and disadvantages of the technique are pointed out and the difficulties of interpretation of the scanning electron micrograph are discussed. The author is grateful to Professor R. M. Kenedi and to Professor T. Gibson for research facilities and encouragement in this project. The work was supported by Science Research grant No. B/sR/4322. REFERENCES CRAm, J. E. and McNEIL, I. R. R. (1965). In " Biomechanics and Related Engineering Topics ", ed. Kenedi, R. M. (I966). Nature, Lond. 2o9, 931. FINLAY, J. B. (1969). Bio-med. Engng., 4, 322. FO~STER, J. C., HAYES, T. L., PEASE, R. F. W. and HUNT, T. K. (I969). Nature, Lond. 22x, 373- GIBSON, T, KENEm, R. M. and CRAm, J. E. (I965). Br. J. Surg. 52, 764. KE~DX, 12.. M. GIBSON, T. and DALY, C. H. (1965). Digest 6th int. Conf. Med. Elect. Biol. Engng, Tokyo. KENEDI, R. M., GIBSON, T., DALY, C. H. and ABRAHAMS, M, (1966). Fed. Proc. 25, May/ June, Pt. x.

Light Microscopy. Upon completion of this lecture, the student should be able to:

Light Microscopy. Upon completion of this lecture, the student should be able to: Light Light microscopy is based on the interaction of light and tissue components and can be used to study tissue features. Upon completion of this lecture, the student should be able to: 1- Explain the