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Scanning Electron Microscopy
Vol. 59, No.3 Printed in U.S.A.
GASTROENTEROLOGY
Copyright © 1970 by The Williams & Wilkins Co.
SCANNING ELECTRON MICROSCOPY A new method in the study of rectal mucosa HYMIE KAVIN, M .B. , B.Ch., DIP. MED ., D. G. HAMILTON, M . B ., B.Ch., F.F.(PATH.), R. E. GREASLEY , J. D . EcKERT, AND G . ZuiDEMA
Departments of Medicin e and Pathology, University of Witwatersrand Medical School and the Johannesburg Hospital; Electron Microscopy Unit , University of the Witwatersrand and Adamant Research Laboratory, Johannesburg, South Africa
Preliminary observations indicate that the scanning electron microscope is useful for studying the surface structure of the rectum in health and disease. The surface of normal rectal mucosa presents a remarkably neat and geometrical pattern comprising numerous polygonal units, each with a central crypt surrounded by epithelial cells and goblet cells. The units are delineated by shallow furrows . On scanning electron microscopy, the rectal mucosal surface of patients with chronic ulcerative colitis in remission is distorted by irregular corrugations. Scanning electron microscopy may be a valuable additional tool in the investigation of the surface pathology of the rectal mucous membrane. The scanning electron microscope is designed primarily for visualization of the surface structure of an object. The basic principles of this technique were well known in the 1930's, but the first commercial scanning electron microscope became available in 1965 (Stereoscan, Cambridge Instrument Company, Cambridge, England) . Nixon 1 has reviewed the deReceived September 23, 1969. Accepted March 4, 1970. This paper was presented in part at the 3rd Annual Scanning Electron Microscopy Symposium, in Chicago, lllinois on April 30, 1970. Address requests for reprints to : Dr. H. Kavin , Gastrointestinal Unit, Depa rtment of Medicine, University of Witwatersrand Medical School, Hospital Street, Johannesburg, South Africa. This work was supported by a grant from the South African Medical Research Council. The authors wish to thank Dr. C. Abrahams for valuable advice. They are indebted to Mr. G. S. James, Research Director, Adamant Research Laboratory, for facilities granted. The authors also wish to thank the Photo Unit, Department of Medicine, and Mr. A. Veenstra and Mrs. V. Cohen of the Departments of Surgery and Radiology, University of Witwatersrand for the photographs. 426
velopment of the instrument and its wide application. Recently, the ultramicroscopic structure of the surface of normal and abnormal human small intestine has been described.2' a The Stereoscan also has been used to study the surface of rat intestinal villi. 4 The purpose of this report is to describe observations on the three-dimensional appearances of the surface of the rectum in normal subjects and in patients with chronic ulcerative colitis as viewed through the scanning electron microscope. Materials and Methods The Cambridge Scanning Electron Microscope Mk II A (Stereoscan) . In the conventional transmission electron microscope the electron beam passes through the specimen and it is the scattering of these electrons as they pass through the tissue that produces the image. The scanning electron microscope is based on a different principle: an electron beam, emitted from a hot tungsten filament in a conventional electron gun, is reduced in size and focused by a series of electromagnetic lenses into a very fine probe 100 A in diameter. As the beam scans the surface of the speci-
September /970
SCANNING ELECTRON MICROSCOPY
men, secondary electrons are emitted and accelerated into a collector. The signal produced by the secondary electrons is amplified by a photomultiplier. The final output is then displayed on a television type cathode ray tube and 1000-line screen. The instrument is designed so that the electron beam scanning each point of the surface of the specimen is in exact synchrony with the spot on the cathode ray tube as it traverses the screen. Since the depth of focus of the Stereoscan is at least 300 times that of the optical microscope, accurate focusing can be obtained upon all parts of the specimen at the same time. The limit of resolution of the instrument for biological material is approximately 200 A at magnifications ranging from 20 to 10,000 times. Specimens as large as 12 mm in diameter can be examined. Preparation of tissue. Rectal biopsies were obtained with punch biopsy forceps approximately 10 em from the anal verge. The surface
427
was gently swabbed with a cotton wool pledget soaked in normal saline before the biopsy was taken. The biopsy specimens, varying between 0.3 to 1.0 em in diameter, were carefully oriented on muslin gauze and washed in normal saline to eliminate surface debris. They were fixed in 10% formol-saline solution for at least 16 hr. This simple method of fixation also was used in the preparation of small bowel biopsies and proved just as good when compared with the techniques described by Marsh and Swift"·" and Carr and Toner.' Two biopsies were obtained from adjacent areas of the rectum in each patient. Histological sections of one biopsy were stained with hematoxylin and eosin. The tissue from the other biopsy was mounted in the desired orientation on 12 mm diameter aluminium stubs for scanning electron microscopy. The specimens were glued to the stubs with colloidal graphite alcohol (Dag 580) to reduce charg-
FIG. 1. Scanning electron micrograph of normal rectal mucosa showing subdivision of the surface into polygonal units each with a central crypt lumen (C) . Each unit is delineated by a shallow furrow (F). P, goblet cell partially filled with mucus; G, empty goblet cell; M, strand of mucus. X 240.
428
KAVIN ET AL.
ing. The specimens were dried directly from the 10% formol-saline fixative and coated in a modified Edwards Coating Unit Model 12 E 6/487 fitted with a rotatory shaft seal in order to transmit a rotatory motion within the evaporation chamber. The stubs were attached to the edges of a disc fixed to this rotatory shaft. After drying at a pressure of 0.2 to 0.3 JJ. Hg, the specimens were coated with the thinnest layer of silver necessary to render the surface conducting by evaporating the silver from a basket type tungsten filament. If charging occurred, additional silver was applied. During the evaporation process the specimens were raised, lowered, and rotated continuously through 360°. The coated tissue then was examined directly in the scanning electron microscope operated at an accelerating voltage of 20 kv. Photographs were recorded on llford 35-mm HP3 or HP4 film.
Vol. 59, No.3
Results Appearances of normal rectal mucous membrane. The surface of normal rectal mucosa viewed through the Stereoscan (fig. 1) presents a neat and geometrical pattern. A striking feature is the division of the surface of the tissue into multiple polygonal units each comprising a central hole approximately 18 J.l. in diameter, which is the mouth of a crypt. Each polygonal unit is delineated by a sharp but shallow depression or furrow. The mouth of the crypt slopes gently, like a funnel. Small oval pits on the surface of the specimens, approximately 5 tJ. in diameter, may represent the surface of partially filled goblet cells. The surfaces
FIG. 2. Polygonal units (see text). C, crypt lumen; P, goblet cell partially filled with mucus; G, empty goblet cell; H , hemispherical projections of the surfaces of columnar epithelial cells or goblet cells bulging with mucus. X 590.
September 1970
SCANNING ELECTRON MICROSCOPY
429
...
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FIG. 3. Scanning electron micrograph of a control rectal biopsy. The mouths of the crypts (C) are surrounded by numerous goblet cells which have discharged their contents (G). H, hemispherical projections; F, furrow. X 500.
of goblet cells which have presumably discharged their contents appear as holes also approximately 5 /J. in diameter. Hemispherical projections on the surface may represent columnar epithelial cells or goblet cells bulging with mucus (fig. 2) . The surface appearances of the goblet cells and epithelial cells of small intestinal villi are similar. 2 The exact nature of the furrows which appear to divide the surface into polygonal units is not clear. There is no obvious histological counterpart. It is possible that the furrows are formed by localized contraction of the muscularis mucosa during the process of fixation. In a light microscopic study of the surface of colonic mucosa Fabbrini et al. have described similar
polygonal areas containing the openings of the crypts. They suggest that capillary networks delineate the polygonal units_r.. 1; Whatever the mechanism of the furrows, their appearances clearly suggest the existence of a structural unit of the surface of the rectum. It is conceivable that, like the villus of the small intestine, this may be a functional unit. This concept merits further exploration. The surface structure of a rectal biopsy from a 2nd control subject is shown in figure 3. The mouths of the crypts of Lieberkuhn are surrounded by numerous goblet cells which have discharged their mucus content. Light microscopy of the biopsy taken from an immediately adjacent area confirmed that the predomi-
430
KAVIN ET AL.
Vol. 59, No. 3
FIG. 4. Scanning electron micrograph of rectum from patient with chronic ulcerative colitis in remission. The surface is distorted by corrugations (CR) adjacent to the furrows (F). C, crypt; G, empty goblet cells. The inset illustrates the light microscopy appearance of an immediately adjacent a rea. Hematoxylin and eoson stained. X 310; inset X 240.
FIG . 5. Low power scanning electron micrograph of a rectal biopsy from the 2nd patient with chronic ulcerative colitis in remission. There is marked variability in the diameters of the rectal units and crypts (C). D, surface debris. At higher magnification (inset), the surface corrugations (CR) are prominent. M, mucus; G, empty goblet cell. X 80; inset, X 320.
September 1970
SCANNING ELECTRON MICROSCOPY
nant cell of the surface epithelium is the goblet cell. Chronic ulcerative colitis. Rectal biopsies were examined from 2 patients with classical chronic ulcerative colitis during a clinical remission. The 1st patient, a 36-year-old Caucasian male, had a 15-year history of ulcerative colitis. During this period he experienced only three relapses. At the time of examination he complained of mild diarrhea and sigmoidoscopically, the only findings were a lustreless rectal mucosa, absence of the normal vascular pattern, and mild edema. The barium enema showed a contracted, "hosepipe" colon and the rectum was narrow and rigid. Light microscopy of the rectal biopsy from this patient (fig. 4, inset) reveals an atrophic rectal mucosa. The crypts of Lieberkiihn are short and broad. The surface is lined by flattened epithelial cells. The lamina propria is mildly infiltrated with chronic inflammatory cells. The scanning electron microscopic appearances of the rectal surface is shown in figure 4. The uniform pattern of the hypothetical units is preserved. However, the surface is strikingly distorted by irregular corrugations adjacent to the furrows. In addition, there are very few goblet cells. The corrugations probably represent the surfaces of bands of irregularly regenerating epithelial cells. The hemispherical projections corresponding to the surfaces of columnar epithelial cells or goblet cells filled with mucus in normal biopsies are not evident. The surface, instead, appears irregular and flattened. The 2nd patient, 20-year-old Caucasian woman, had frequent attacks of diarrhea and rectal bleeding for 4 years. The sigmoidoscopic and barium enema findings were similar to those of the first patient. Rectal biopsies were taken during a 9-month remission. The scanning electron microscopic appearances of the mucosal surface are strikingly . abnormal. At low magnification (fig. 5) the morphology is grossly irregular. In general there is marked variability in the surface structure between adjacent areas. No particular area is representative. The pattern
431
of the hypothetical units is variable. Some appear circular, others ovoid, and in some areas the pattern is completely lost. In addition, the diameters of the mouths of the crypts vary considerably. Goblet cells are very sparse. A considerable amount of debris and mucus is present. At higher magnification (fig. 5, inset) the surface is markedly corrugated. Discussion Surface scanning electron microscopy has been used to study the surface properties of many types of inorganic materials such as metals. 1 The external structure of hard biological specimens like bone 7 and teeth 8 may also be readily inspected. In order to examine soft biological material with the Stereoscan, specimens require fixation, complete drying, and coating with a thin layer of evaporated metal to render the surface conducting before insertion into the vacuum chamber of the instrument. The technique has been applied successfully to a variety of tissues, such as blood cells!' and small intestinal villi." - 4 The structure of the surface of large bowel has been a neglected field of study. Fabbrini and his colleagues, using low power conventional light microscopy, have described the surface morphology of normal human colon. ''· 1; Normal mucosa presented either a f1at or papillary surface pattern on which the openings of the mucous glands were uniformly spread . A single case of ulcerative colitis showed depressed areas and a rough surface. With the Stereoscan the mucosal surface of human large intestine can be examined at low and high magnification. The high resolutions obtained and the great depth of focus of the instrument provides a three-dimensional picture of the surface of the specimen, all parts of which may be brought into focus for detailed inspection and recording. In the present communication the appearances of the surface of human rectal tissue obtained by biopsy are described. The openings of the crypts of Lieberkuhn are neatly and uniformly scattered on the surface.
432
KAVIN ET AL.
The surfaces of goblet cells and columnar epithelial cells are readily identifiable. In addition, shallow furrows appear to divide the surface of the rectum into polygonal units. It is likely that these furrows are produced by contraction of the muscularis mucosa under the conditions of fixation. Fabbrini and colleagues believe that the polygonal areas are delineated by submucosal capillary plexuses. 5 ' 6 The surfaces of rectal biopsies from 2 patients with chronic ulcerative colitis in remission are strikingly abnormal. In addition to the distortion of the surface architecture, corrugations and micropolyps are evident. In the present study it was not possible to identify microvilli on the surface of the epithelial cells of normal and abnormal rectum. This may be due to the presence of a prominent and adherent filamentous coat or fuzzy layer which covers the surface of intestinal epithelial cells. 10 By combining ion beam etching of the surface of the epithelial cell with scanning electron microscopy, it may be possible to peel away successive layers of the specimen for inspection of the subsurface structure. This technique has been applied successfully to the red blood cell. 11
Vol . 59, No. 3
REFERENCES 1. Nixon WC : Scanning electron microscopy. Contemp Phys 10:71-96, 1969 2. Marsh MN, Swift JA: Studies of small intestinal mucosa with the scanning electron microscope. Brit Med J 2:95-96, 1968 3. Marsh MN, Swift JA: A study of the small intestinal mucosa using the scanning electron microscope. Gut 10:940-949, 1969 4. Carr KE, Toner, PE: Scanning electron microscopy of rat intestinal villi . Lancet 2:570-571, 1968 5. Fabbrini A, Torsoli A, Allessandrini A, et al: Surface microscopy of the large bowel. Experientia 22:408-410, 1966 6. Onori L, Greco V, Fabbrini A, et al: Microscopic patterns of the colonic mucosal surface. Investigati on in human subjects and experimental animals. Experientia 24:1252, 1968 7. McCall JG: Scanning electron microscopy of articular surfaces. Lancet 2:1194, 1968 8. Boyde A: The development of enamel structure. Proc Roy Soc Med 60:923-928, 1967 9. Clarke JA, Salisbury AJ: Surface ultra microscopy of human blood cells. Nature (London) 215:402-404, 1967 10. Trier JS: The surface coat of gastrointestinal epithelial cells. Gastroenterology 56:618-622, 1969 11. Lewis SM, Osborn JS, Stuart PR: Demonstration of an internal structure within the red blood cell by ion etching and scanning electron microscopy. Nature (London) 220:614-616, 1968
GASTROENTEROLOGY
Copyright © 1970 by The Williams & Wilkins Co.
SCANNING ELECTRON MICROSCOPY A new method in the study of rectal mucosa HYMIE KAVIN, M .B. , B.Ch., DIP. MED ., D. G. HAMILTON, M . B ., B.Ch., F.F.(PATH.), R. E. GREASLEY , J. D . EcKERT, AND G . ZuiDEMA
Departments of Medicin e and Pathology, University of Witwatersrand Medical School and the Johannesburg Hospital; Electron Microscopy Unit , University of the Witwatersrand and Adamant Research Laboratory, Johannesburg, South Africa
Preliminary observations indicate that the scanning electron microscope is useful for studying the surface structure of the rectum in health and disease. The surface of normal rectal mucosa presents a remarkably neat and geometrical pattern comprising numerous polygonal units, each with a central crypt surrounded by epithelial cells and goblet cells. The units are delineated by shallow furrows . On scanning electron microscopy, the rectal mucosal surface of patients with chronic ulcerative colitis in remission is distorted by irregular corrugations. Scanning electron microscopy may be a valuable additional tool in the investigation of the surface pathology of the rectal mucous membrane. The scanning electron microscope is designed primarily for visualization of the surface structure of an object. The basic principles of this technique were well known in the 1930's, but the first commercial scanning electron microscope became available in 1965 (Stereoscan, Cambridge Instrument Company, Cambridge, England) . Nixon 1 has reviewed the deReceived September 23, 1969. Accepted March 4, 1970. This paper was presented in part at the 3rd Annual Scanning Electron Microscopy Symposium, in Chicago, lllinois on April 30, 1970. Address requests for reprints to : Dr. H. Kavin , Gastrointestinal Unit, Depa rtment of Medicine, University of Witwatersrand Medical School, Hospital Street, Johannesburg, South Africa. This work was supported by a grant from the South African Medical Research Council. The authors wish to thank Dr. C. Abrahams for valuable advice. They are indebted to Mr. G. S. James, Research Director, Adamant Research Laboratory, for facilities granted. The authors also wish to thank the Photo Unit, Department of Medicine, and Mr. A. Veenstra and Mrs. V. Cohen of the Departments of Surgery and Radiology, University of Witwatersrand for the photographs. 426
velopment of the instrument and its wide application. Recently, the ultramicroscopic structure of the surface of normal and abnormal human small intestine has been described.2' a The Stereoscan also has been used to study the surface of rat intestinal villi. 4 The purpose of this report is to describe observations on the three-dimensional appearances of the surface of the rectum in normal subjects and in patients with chronic ulcerative colitis as viewed through the scanning electron microscope. Materials and Methods The Cambridge Scanning Electron Microscope Mk II A (Stereoscan) . In the conventional transmission electron microscope the electron beam passes through the specimen and it is the scattering of these electrons as they pass through the tissue that produces the image. The scanning electron microscope is based on a different principle: an electron beam, emitted from a hot tungsten filament in a conventional electron gun, is reduced in size and focused by a series of electromagnetic lenses into a very fine probe 100 A in diameter. As the beam scans the surface of the speci-
September /970
SCANNING ELECTRON MICROSCOPY
men, secondary electrons are emitted and accelerated into a collector. The signal produced by the secondary electrons is amplified by a photomultiplier. The final output is then displayed on a television type cathode ray tube and 1000-line screen. The instrument is designed so that the electron beam scanning each point of the surface of the specimen is in exact synchrony with the spot on the cathode ray tube as it traverses the screen. Since the depth of focus of the Stereoscan is at least 300 times that of the optical microscope, accurate focusing can be obtained upon all parts of the specimen at the same time. The limit of resolution of the instrument for biological material is approximately 200 A at magnifications ranging from 20 to 10,000 times. Specimens as large as 12 mm in diameter can be examined. Preparation of tissue. Rectal biopsies were obtained with punch biopsy forceps approximately 10 em from the anal verge. The surface
427
was gently swabbed with a cotton wool pledget soaked in normal saline before the biopsy was taken. The biopsy specimens, varying between 0.3 to 1.0 em in diameter, were carefully oriented on muslin gauze and washed in normal saline to eliminate surface debris. They were fixed in 10% formol-saline solution for at least 16 hr. This simple method of fixation also was used in the preparation of small bowel biopsies and proved just as good when compared with the techniques described by Marsh and Swift"·" and Carr and Toner.' Two biopsies were obtained from adjacent areas of the rectum in each patient. Histological sections of one biopsy were stained with hematoxylin and eosin. The tissue from the other biopsy was mounted in the desired orientation on 12 mm diameter aluminium stubs for scanning electron microscopy. The specimens were glued to the stubs with colloidal graphite alcohol (Dag 580) to reduce charg-
FIG. 1. Scanning electron micrograph of normal rectal mucosa showing subdivision of the surface into polygonal units each with a central crypt lumen (C) . Each unit is delineated by a shallow furrow (F). P, goblet cell partially filled with mucus; G, empty goblet cell; M, strand of mucus. X 240.
428
KAVIN ET AL.
ing. The specimens were dried directly from the 10% formol-saline fixative and coated in a modified Edwards Coating Unit Model 12 E 6/487 fitted with a rotatory shaft seal in order to transmit a rotatory motion within the evaporation chamber. The stubs were attached to the edges of a disc fixed to this rotatory shaft. After drying at a pressure of 0.2 to 0.3 JJ. Hg, the specimens were coated with the thinnest layer of silver necessary to render the surface conducting by evaporating the silver from a basket type tungsten filament. If charging occurred, additional silver was applied. During the evaporation process the specimens were raised, lowered, and rotated continuously through 360°. The coated tissue then was examined directly in the scanning electron microscope operated at an accelerating voltage of 20 kv. Photographs were recorded on llford 35-mm HP3 or HP4 film.
Vol. 59, No.3
Results Appearances of normal rectal mucous membrane. The surface of normal rectal mucosa viewed through the Stereoscan (fig. 1) presents a neat and geometrical pattern. A striking feature is the division of the surface of the tissue into multiple polygonal units each comprising a central hole approximately 18 J.l. in diameter, which is the mouth of a crypt. Each polygonal unit is delineated by a sharp but shallow depression or furrow. The mouth of the crypt slopes gently, like a funnel. Small oval pits on the surface of the specimens, approximately 5 tJ. in diameter, may represent the surface of partially filled goblet cells. The surfaces
FIG. 2. Polygonal units (see text). C, crypt lumen; P, goblet cell partially filled with mucus; G, empty goblet cell; H , hemispherical projections of the surfaces of columnar epithelial cells or goblet cells bulging with mucus. X 590.
September 1970
SCANNING ELECTRON MICROSCOPY
429
...
-
FIG. 3. Scanning electron micrograph of a control rectal biopsy. The mouths of the crypts (C) are surrounded by numerous goblet cells which have discharged their contents (G). H, hemispherical projections; F, furrow. X 500.
of goblet cells which have presumably discharged their contents appear as holes also approximately 5 /J. in diameter. Hemispherical projections on the surface may represent columnar epithelial cells or goblet cells bulging with mucus (fig. 2) . The surface appearances of the goblet cells and epithelial cells of small intestinal villi are similar. 2 The exact nature of the furrows which appear to divide the surface into polygonal units is not clear. There is no obvious histological counterpart. It is possible that the furrows are formed by localized contraction of the muscularis mucosa during the process of fixation. In a light microscopic study of the surface of colonic mucosa Fabbrini et al. have described similar
polygonal areas containing the openings of the crypts. They suggest that capillary networks delineate the polygonal units_r.. 1; Whatever the mechanism of the furrows, their appearances clearly suggest the existence of a structural unit of the surface of the rectum. It is conceivable that, like the villus of the small intestine, this may be a functional unit. This concept merits further exploration. The surface structure of a rectal biopsy from a 2nd control subject is shown in figure 3. The mouths of the crypts of Lieberkuhn are surrounded by numerous goblet cells which have discharged their mucus content. Light microscopy of the biopsy taken from an immediately adjacent area confirmed that the predomi-
430
KAVIN ET AL.
Vol. 59, No. 3
FIG. 4. Scanning electron micrograph of rectum from patient with chronic ulcerative colitis in remission. The surface is distorted by corrugations (CR) adjacent to the furrows (F). C, crypt; G, empty goblet cells. The inset illustrates the light microscopy appearance of an immediately adjacent a rea. Hematoxylin and eoson stained. X 310; inset X 240.
FIG . 5. Low power scanning electron micrograph of a rectal biopsy from the 2nd patient with chronic ulcerative colitis in remission. There is marked variability in the diameters of the rectal units and crypts (C). D, surface debris. At higher magnification (inset), the surface corrugations (CR) are prominent. M, mucus; G, empty goblet cell. X 80; inset, X 320.
September 1970
SCANNING ELECTRON MICROSCOPY
nant cell of the surface epithelium is the goblet cell. Chronic ulcerative colitis. Rectal biopsies were examined from 2 patients with classical chronic ulcerative colitis during a clinical remission. The 1st patient, a 36-year-old Caucasian male, had a 15-year history of ulcerative colitis. During this period he experienced only three relapses. At the time of examination he complained of mild diarrhea and sigmoidoscopically, the only findings were a lustreless rectal mucosa, absence of the normal vascular pattern, and mild edema. The barium enema showed a contracted, "hosepipe" colon and the rectum was narrow and rigid. Light microscopy of the rectal biopsy from this patient (fig. 4, inset) reveals an atrophic rectal mucosa. The crypts of Lieberkiihn are short and broad. The surface is lined by flattened epithelial cells. The lamina propria is mildly infiltrated with chronic inflammatory cells. The scanning electron microscopic appearances of the rectal surface is shown in figure 4. The uniform pattern of the hypothetical units is preserved. However, the surface is strikingly distorted by irregular corrugations adjacent to the furrows. In addition, there are very few goblet cells. The corrugations probably represent the surfaces of bands of irregularly regenerating epithelial cells. The hemispherical projections corresponding to the surfaces of columnar epithelial cells or goblet cells filled with mucus in normal biopsies are not evident. The surface, instead, appears irregular and flattened. The 2nd patient, 20-year-old Caucasian woman, had frequent attacks of diarrhea and rectal bleeding for 4 years. The sigmoidoscopic and barium enema findings were similar to those of the first patient. Rectal biopsies were taken during a 9-month remission. The scanning electron microscopic appearances of the mucosal surface are strikingly . abnormal. At low magnification (fig. 5) the morphology is grossly irregular. In general there is marked variability in the surface structure between adjacent areas. No particular area is representative. The pattern
431
of the hypothetical units is variable. Some appear circular, others ovoid, and in some areas the pattern is completely lost. In addition, the diameters of the mouths of the crypts vary considerably. Goblet cells are very sparse. A considerable amount of debris and mucus is present. At higher magnification (fig. 5, inset) the surface is markedly corrugated. Discussion Surface scanning electron microscopy has been used to study the surface properties of many types of inorganic materials such as metals. 1 The external structure of hard biological specimens like bone 7 and teeth 8 may also be readily inspected. In order to examine soft biological material with the Stereoscan, specimens require fixation, complete drying, and coating with a thin layer of evaporated metal to render the surface conducting before insertion into the vacuum chamber of the instrument. The technique has been applied successfully to a variety of tissues, such as blood cells!' and small intestinal villi." - 4 The structure of the surface of large bowel has been a neglected field of study. Fabbrini and his colleagues, using low power conventional light microscopy, have described the surface morphology of normal human colon. ''· 1; Normal mucosa presented either a f1at or papillary surface pattern on which the openings of the mucous glands were uniformly spread . A single case of ulcerative colitis showed depressed areas and a rough surface. With the Stereoscan the mucosal surface of human large intestine can be examined at low and high magnification. The high resolutions obtained and the great depth of focus of the instrument provides a three-dimensional picture of the surface of the specimen, all parts of which may be brought into focus for detailed inspection and recording. In the present communication the appearances of the surface of human rectal tissue obtained by biopsy are described. The openings of the crypts of Lieberkuhn are neatly and uniformly scattered on the surface.
432
KAVIN ET AL.
The surfaces of goblet cells and columnar epithelial cells are readily identifiable. In addition, shallow furrows appear to divide the surface of the rectum into polygonal units. It is likely that these furrows are produced by contraction of the muscularis mucosa under the conditions of fixation. Fabbrini and colleagues believe that the polygonal areas are delineated by submucosal capillary plexuses. 5 ' 6 The surfaces of rectal biopsies from 2 patients with chronic ulcerative colitis in remission are strikingly abnormal. In addition to the distortion of the surface architecture, corrugations and micropolyps are evident. In the present study it was not possible to identify microvilli on the surface of the epithelial cells of normal and abnormal rectum. This may be due to the presence of a prominent and adherent filamentous coat or fuzzy layer which covers the surface of intestinal epithelial cells. 10 By combining ion beam etching of the surface of the epithelial cell with scanning electron microscopy, it may be possible to peel away successive layers of the specimen for inspection of the subsurface structure. This technique has been applied successfully to the red blood cell. 11
Vol . 59, No. 3
REFERENCES 1. Nixon WC : Scanning electron microscopy. Contemp Phys 10:71-96, 1969 2. Marsh MN, Swift JA: Studies of small intestinal mucosa with the scanning electron microscope. Brit Med J 2:95-96, 1968 3. Marsh MN, Swift JA: A study of the small intestinal mucosa using the scanning electron microscope. Gut 10:940-949, 1969 4. Carr KE, Toner, PE: Scanning electron microscopy of rat intestinal villi . Lancet 2:570-571, 1968 5. Fabbrini A, Torsoli A, Allessandrini A, et al: Surface microscopy of the large bowel. Experientia 22:408-410, 1966 6. Onori L, Greco V, Fabbrini A, et al: Microscopic patterns of the colonic mucosal surface. Investigati on in human subjects and experimental animals. Experientia 24:1252, 1968 7. McCall JG: Scanning electron microscopy of articular surfaces. Lancet 2:1194, 1968 8. Boyde A: The development of enamel structure. Proc Roy Soc Med 60:923-928, 1967 9. Clarke JA, Salisbury AJ: Surface ultra microscopy of human blood cells. Nature (London) 215:402-404, 1967 10. Trier JS: The surface coat of gastrointestinal epithelial cells. Gastroenterology 56:618-622, 1969 11. Lewis SM, Osborn JS, Stuart PR: Demonstration of an internal structure within the red blood cell by ion etching and scanning electron microscopy. Nature (London) 220:614-616, 1968