8Journal of Microscopy, Vol. 116, Pt 2, July 1979, pp. 255-264. Revised paper accepted 19 January 1979
Ultrastructure of cell organelles by scanning electron microscopy of thick sections surface-etched by an oxygen plasma
by WALTERJ. HUMPHREYS and W I L L I A MG. HENK,Central Electron Microscopy Laboratory, Barrow Hall, University of Georgia, Athens, Georgia 30602, U.S.A. SUMMARY
Kidney tissue double fixed in glutaraldehyde and osmium tetroxide and embedded in epoxy resin by standard techniques used for transmission electron microscopy was cut into sections 1 pm or more thick and surface-etched by an oxygen plasma. Etching caused ash residues (possibly composed partly or organo-metallic complexes) of membranes and other etch resistant cell components to emerge as recognizable structures projecting upward from the surrounding embedment which was combusted and removed as volatile products. Using the secondary electron mode for image formation, structural features of cells which could be imaged with clarity with the scanning electron microscope included: profiles of peripheral and infolded plasma membranes, the nuclear envelope and profiles of cut mitochondria1 matrix granules, cristae and the outer limiting membranes. Resolution was better than that obtainable from most other methods of specimen preparation currently being used in scanning electron microscopy for viewing the internal structures of cells and organelles in bulk samples of tissue. INTRODUCTION
Attempts to visualize cell organelles and their internal structural details with the scanning electron microscope, using the standard secondary electron mode, have been only partially successful. The use of fracturing techniques, advocated by Boyde & Wood (1969) for opening up tissues and cells for examination with the scanning electron microscope, has permitted limited observation of some cellular constituents. Humphreys & Wodzicki (1972) were able to see cytoplasmic organelles in plant cells which were freeze-fractured and subsequently freeze-dried. Tanaka & Iino (1972) and Woods & Ledbetter (1974) could identify organelles inside cells that had been infiltrated with epoxy resin, frozen, fractured and after removal of the resin by solvents, critical point dried. Humphreys et al. (1973a) identified some cell organelles with the scanning electron microscope by fracturing tissues embedded in epoxy resin, originally intended for ultramicrotomy. The technique of freezing tissues in certain non-polar intermediate fluids that are used for critical point drying (e.g. ethanol, acetone, Freons) followed by cryofracturing, thawing the fractured tissue in the intermediate fluid, and critical point drying (Sybers & 0022-2720/79/0700-0255 S02.00
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WalterJ. Humphreys and William G. Henk Ashraf, 1973; Humphreys et al., 1973b, 1974; Miyai et al., 1976; Munger &Mumaw, 1976) has become a valuable technique that has provided a new and very useful way of looking inside tissues with the scanning electron microscope. Myklebust et al. (1975) studied the ultrastructure of heart muscle by scanning electron microscopy of deparaffinized, critical point dried thick sections of paraffin-embedded tissue. In none of these studies was the internal structure of organelles such as the cristae of mitochondria clearly visible. Haggis et al. (1976) were able to visualize mitochondrial cristae and nuclear chromatin using the scanning electron microscope. Their specimens were freeze-fractured from an aqueous solution containing a cryoprotectant, thawed and fixed in a solution of glutaraldehyde containing a cryoprotectant, and then critical point dried. T o make identification of cytoplasmic organelles unmistakable at fractured or cut surfaces of tissues, a method is required which would etch away enough cytoplasmic matrix, mitochondrial matrix, or other matrices both inside and surrounding organellar structures so that stabilized etch-resistant membranes (and other etchresistant constituents) would project above the surface far enough to be imaged by the scanning electron microscope. Then, as a consequence of the etching, positive identification of many cytoplasmic organelles could be made by virtue of the membrane patterns or other swucrural patterns that would em-rge. Sections 1 pm thick cut from blocks of tissue embedded in epoxy have been etched chemically with epoxy solvents to cause cell organelles such as mitochondria to appear as rounded elevations (Winborn & Guerrero, 1974). Erlandsen et al. (1973) obtained similar results with this technique by etching the smooth surface of a block from which sections had been cut. However, this method did not reveal the internal structure of organelles. Another chemical agent that can be used for surface-etching thick sections of plastic-embedded tissue is an oxygen plasma. If radio-frequency electrical discharges are passed through a gentle stream of pure oxygen gas while it is being drawn past the specimens by a mechanical vacuum pump connected to the reaction chamber and if the oxygen is admitted at a controlled flow rate to attain an operating pressure of 6-130PaY the gas becomes highly reactive chemically. It forms a plasma that destroys organic bonds and organic material is removed from the specimen as volatile products that are swept out by the oxygen stream. Inorganic residues remain on the surface very near their position in the unreacted sample. The resulting ash patterns remaining on the etched surface can then be examined by scanning electron microscopy. This was suggested to us as a method for preparing specimens for scanning electron microscopy from previous studies in which excited oxygen was used for low temperature ultra-microincineration of biological specimens that were to be studied by transmission electron microscopy. Thomas (1962) used high temperature ashing of ultra-thin sections of bacterial spores for transmission electron microscopy studies. Following this, Thomas (1964) pioneered the use of low temperature microincineration of thin-sectioned biological materials for transmission electron microscopy. Thomas & Greenawalt (1968) demonstrated by transmission electron microscopy of metal-shadowed thin sections that an oxygen plasma could be used at a low temperature (specimen temperature less than 373 K) to differentially surface-etch thin sections of Epon-embedded isolated mitochondria. This incomplete ashing caused the membrane remnants of the sectioned cristae to emerge as recognizable structures projecting upward as ridges from the surrounding embedded material which was volatilized and removed by the excited oxygen. An excellent review on the status of spodography (the technique of producing ash patterns) for light and electron microscopy has been published by Thomas (1974).
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SEM of plasma-etched sections Hohman (1967, 1974) and Hohman & Schraer (1972) carried out the first extensive studies which utilized the technique of low temperature ultramicroincineration of thin sectioned tissue. Sections of hen shell gland 100-500 nm thick were completely ashed onto silicon monoxide support films and the resulting spodograms were examined by transmission electron microscopy. Resolution was remarkably good and it was easily possible to identify from the ash patterns such cell components as mitochondria, plasma membranes, cytoplasmic membranes and chromatin. Frazier (1971) using essentially the same method carried out transmission electron microscopy of shadowed sections of odontoblasts which were etched in an oxygen plasma for varying lengths of time at a low power level. After only 30 s of etching, enough of the epoxy was preferentially removed to reveal ash patterns of plasma membranes, mitochondria, and endoplasmic reticulum with a fine definition better than in most of the preparations shown by Hohman & Schraer (1972). Frazier's improved resolution was probably due to the use of thinner sections (about 60-100 nm). The above results suggested to us the use of an oxygen plasma for surfaceetching fractured or cut surfaces of bulk samples of tissue embedded for transmission electron microscopy. It was anticipated that a gentle, and differential surface-etching would cause the emergence of structural features within cells and organelles that could be imaged with considerable clarity with the scanning electron microscope operated in the secondary electron mode. '
M A T E R I A L S AND M E T H O D S
Mouse kidney was fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer at p H 7.2 for 1 h and washed in 0.1 M cacodylate buffer with 50,; sucrose added. It was post-fixed in 1% Os04 in cacodylate buffer for 1 h, washed in cacodylate buffer, dehydrated in a graded series of ethanols and embedded in Araldite-Epon (Mollenhauer, 1964). Ultrathin sections were cut, stained with aqueous uranyl acetate (Watson, 1958), rinsed, stained with lead citrate (Reynolds, 1963), and examined in a transmission electron microscope in order to make sure that fixation was good. For scanning electron microscopy, 1 pm thick sections were cut with a diamond knife, placed on a fragment of coverslip 10 x 10 mm or smaller and allowed to dry flat against the glass. The coverslip fragments bearing 1 pm sections were placed on a microscope slide which was placed in the reaction chamber of a Tegal Plasmod" plasma generator. The sections were etched by an oxygen plasma following the method described by Thomas & Hollahan (1974). Briefly, the reaction chamber was evacuated to a mild vacuum by a mechanical pump. Oxygen gas obtained from standard-grade commercial oxygen bottles was admitted at a controlled flow rate 13.8 kPa (2 psi) to obtain an operating pressure in the range of 6-130 Pa. Radio-frequency power was applied around the chamber at 13.56 MHz. This excited the oxygen molecules and changed some of them into other species such as atoms, radicals, ions and free electrons. The highly reactive gaseous plasma caused a gentle, low-temperature combustion of the organic materials in the sample. The combustion products were carried away in the gas stream leaving behind the thick sections with the exposed surfaces etched. An R F power setting of 50 W was arbitrarily chosen and the sections were etched for a period of 60 s. The etched sections were shadowed by vacuum evaporation of platinum-palladium wire from an angle of 30" at a distance of 100mm. After shadowing, the specimens were lightly sputter-coated with gold using a Hummer I1 sputter coater (Technics, Inc.). The etched sections were photographed with a Philips 501 scan-
* Available from the Tegal Corporation, 860 Wharf Street, Richmond, CA 94804, U.S.A. 257
WalterJ. Humphreys and William G. Henk
Figs. 1 and 2
258
SEM of plasma-etched sections ning electron microscope at an operating voltage of 15 kV with the specimen tilted to an angle of about 25 '. OBSERVATIONS
A low magnification scanning electron micrograph showing the typical appearance of a 1 pm thick epoxy section of mouse kidney surface-etched by an oxygen plasma is shown in Fig. 1. The nuclear, cytoplasmic, mitochondrial, and plasma membranes show up as light lines against a grey background. The overall appearance of the etched surface resembles a transmission electron micrograph of a thin section printed in reverse contrast. In general, the structures that would appear electron dense in typical transmission electron micrographs of thin sections (Fig. 3) appear light in scanning electron micrographs of this type of preparation. The lumen (L) of the distal convoluted tubule and the vesicles (V) in the cells of a proximal convoluted tubule appear as the darkest areas in the specimen. Both structures are spaces essentially filled with only the epoxy embedment. The inset of Fig. 1 is an enlargement of an area containing the brush border of a proximal convoluted tubule cut at an oblique angle. The limiting plasma membranes of microvilli appear as bright lines surrounded by the darker lumen into which the microvilli project. An enlargement of a portion of Fig. 1 is shown as Fig. 2. Differential etching and metal shadowing has caused the nuclear envelope to project upward like a ridge rising above the background of etched nucleoplasm on the one side and etched cytoplasmic matrix on the other. Similarly, profiles of mitochondrial membranes appear as ridges rising above the etched mitochondrial matrix and the cytoplasmic matrix. Thus, cristae are clearly recognizable. A few mitochondrial matrix granules (G), which seem to be refractory to etching by oxygen plasma as used here, are found lying over the cristae of some mitochondria. A typical transmission electron micrograph of a thin section of the embedded kidney tissue used for the 1 pm thick sections shown in all the other figures is shown in Fig. 3. Figure 4 is a scanning electron micrograph of the plasma-etched surface of a 1 pm thick section cut from the same block of embedded tissue as that shown in Fig. 3 and at about the same magnification. Comparing Fig. 4 with Fig. 3 it appears that, in general, the structures that are most electron dense in Fig. 3 are the ones that were most resistant to the plasma-etching as shown in Fig. 4. After etching with the oxygen plasma the electron-dense structures such as heterochromatin (H), and cytoplasmic and mitochondrial membranes leave ash residues Fig. 1. Scanning electron micrograph of a 1 pm thick Epon-Araldite section of mouse kidney tissue. The section was surface-etched by an oxygenplasma and shadowed at a 30" angle with Pt.-Pd. At the upper left and upper right corners structural features of cells of distal convoluted tubules are recognizable. Ash patterns of the membranes of the nucleus (N), plasma membranes and cytoplasmic membranes appear light against a grey background. Erythrocytes (E), in a capillary lumen are at top centre. L, lumen of tubule; V, vesicles. x 3870. Inset: Brush border of a distal convoluted tubule at a higher magnification, enlarged from the area designated by the arrow. x 7000. Fig. 2. Higher magnification of the area outlined in Fig. 1. Ash patterns of membranes of the nucleus (N), plasma membranes (PL) including lateral cell membranes (CM), and cytoplasmic membranes can be identified. Mitochondria (M) are identifiable by the ash patterns of their cristae and outer limiting membranes. Mitochondria matrix granules (G) lie on top of the cut profiles of cristae of several mitochondria. x 12,000.
259
S-& 'SsTd
092
SEM of plasma-etched sections that are sufficiently elevated above the background to permit their imaging by the scanning electron microscope. The ash patterns remain on the surface of the section in undisturbed configurations that can be used to identify the structures from which the ash patterns originated during the limited surface-incineration of the section. In Fig. 4 the ash patterns that originated at the site of cut profiles of numerous mitochondrial membranes that were surface-ashed by the oxygen plasma are unmistakably recognizable. The embedding epoxy and the mitochondrial matrices were partially etched away leaving the ash pattern of the mitochondrial walls and cristae. Residues of the outer and inner mitochondrial membranes cannot be distinguished from one another even at higher magnifications (Fig. 5). Residues of the two membranes and the membrane space between them appear as a single structure in these preparations, probably because the metal shadowing and the subsequent metal coating caused the space between the two membranes to be bridged by the metal deposit. For the same reason, the double membrane structure of the cristae is not discernible from the residues of the surface-ashed cristae. The ash patterns of mitochondrial walls and cristae are much thicker (Fig. 4) than the profiles of the same structures as seen in thin sections (Fig. 3) because of the metal coating on the etched sections. DISCUSSION
In order to allow a direct comparison of our etched preparations as seen by scanning electron microscopy with the characteristic ultrastructural appearance of the same tissue in thin section, and thus ensure the adequacy of the fixation, the tissue used in this study was fixed and processed in the conventional manner. That is, all tissue samples were fixed in glutaraldehyde, post-fixed in osmium tetroxide and embedded in epoxy using the standard techniques employed for ultramicrotomy and transmission electron microscopy. Although the etch-resistance of cytoplasmic membranes might have been increased by en bloc staining, or staining of the thick sections with solutions containing non-volatile metal salts, the cytoplasmic membranes were sufficiently etch-resistant to the oxygen plasma without further treatment. A consistent observation was that those cell constituents that were most electron dense in thin sections were also the most etch-resistant structures in the plasmaetched preparations. As is well known, much of the electron density of structures in thin sections is due to the presence of reduced osmium. But this would not seem to be a significant factor in the etch-resistance since there is the distinct possibility Fig. 3. Transmission electron micrograph of a thin section cut from the same block as the surface-etched 1 pm thick sections shown in the other figures. x 10,500. Fig. 4. Scanning electron micrograph of a surface-etched 1 pm thick section prepared the same as in Fig. 1. Cells of a convoluted tubule of a mouse kidney are shown. Ash patterns of numerous mitochondrial profiles remain after the low temperature surface-incineration. The heterochromatin (H) was more resistant to the oxygen-plasma than the other nuclear material. Compare with Fig. 3. x 11,400. Fig. 5. Scanning electron micrograph of a surface-etched 1 pm thick sections prepared the same as in Fig. 1. Mitochondria1 profiles in cells of mouse kidney convoluted tubules are shown. The low temperature plasma-etching appears to cause very little lateral migration of the ash from the incinerated membranes. Ashed cristae remain discretely separated from one another. x 21,800.
26 1
WalterJ. Humphreys and William G. Henk pointed out by Thomas (1974) that in the presence of the oxygen plasma osmium is a volatile stain and in his words, ‘evidently the metallic osmium is reoxidized by the plasma to volatile osmium tetroxide which leaves no trace’. Hohman (1974) found in spodograms of totally low-temperature ashed thin sections of avian shell gland tissue that most cellular structures appeared to be similar, with or without osmium fixation. However, certain membranes such as membranes between cells appeared only when glutaraldehyde fixation was followed by osmium post-fixation. The ash from these membranes was not apparent in spodograms from tissue fixed by glutaraldehyde alone. Whether the ash residue in these membranes consisted in part of complexed osmium that was not volatilized or whether the residue resulted indirectly from the action of osmium cannot be determined until further studies designed to answer this question have been made. Most spodography of embedded tissue that has previously been described (Thomas, 1964; Thomas & Greenawalt, 1968; Hohman, 1967; Hohman & Schraer, 1972; Hohman, 1974) has involved the use of excited oxygen for total ashing of thin sections onto silicon monoxide films for transmission electron microscopy. The method reported here describes an alternative method for spodography in which thick sections are partially ashed or surface-etched by the use of excited oxygen and then examined in the scanning electron microscope as bulk samples using the secondary electron mode. The depth of etching can be controlled by varying the length of time used for etching and by the selection of different wattages used for producing the oxygen plasma, or by an appropriate combination of these two variables. Thus spodograms can be produced by etching thick sections to depths equal to the thicknesses of thin sections that have been totally ashed. The etched thick sections can be examined by the scanning electron microscope and the totally ashed sections can be examined by transmission electron microscopy and the resulting spodograms can be compared. If such a comparison is made, the main advantage of etching compared to complete ashing is a distinct reduction in ash displacement and an increased resolution. Having an intact ‘base’ under the ashed material may help to maintain the stability of the ash. The totally ashed sections studied by Hohman & Schraer (1972) showed evidence of considerable ash migration that, for example, resulted in clear zones around nuclei. This artefact was found in all sections thicker than about 0.1 pm, the clear zones becoming wider as thicker sections were used. Such artefacts were not found in our preparations of thicker sections etched for scanning electron microscopy. The nuclei in Figs. 4 and 5, for example, show no such artefacts. Recently Tanaka et al. (1976) used ion-etching on critical point dried tissue that had been freeze-cracked by various methods. Such specimens when examined by scanning electron microscopy appeared similar to our etched tissue sections. The specimens were etched by high-energy sputter etching and differential etching was achieved because membraneous structures in the cell are generally more resistant to sputter-etching and the cytoplasmic matrix is easily etched. Structures revealed this method of surface etching were nuclear pores, endoplasmic reticulum with ribosomes, Golgi apparatus, and mitochondria1 cristae. We attempted to use oxygen to surface-etch freeze-fractured critical point dried tissue prepared by the method of Humphreys et al. (1974). We were unsuccessful because the critical point dried tissue is freely permeable to the gaseous oxygen plasma which reacted chemically with the interior of the tissue at the same time as with the surface. As a consequence organic material was volatilized from the interior of the tissue at the same rate as from the tissue’s surface and no differential surface-etching could be achieved. In these preliminary studies we found that more satisfactory results were obtained when the plasma-etched sections were metal-shadowed before they were coated
262
SEM of plasma-etched sections with metal prior to their examination in the scanning electron microscope. Theoretically this step is not necessary and higher resolution would be possible if it were dispensed with, since less metal would be present on the specimen to obscure surface detail. Our attempts to omit this step, however, usually, but not invariably, resulted in specimens that lacked adequate contrast when observed in the scanning electron microscope. Better resolution could also be obtained if the etching were restricted to a depth just sufficient to generate enough variability in surface topography to permit imaging the surface after a conducting metal coat of minimal thickness was deposited on to it. Optimization of these parameters will require further study. With careful attention to such details, plasma-etched sections could conceivably be capable of showing cellular fine structure that is not now resolvable by most state-of-the-art scanning electron microscopes using the secondary electron mode. Unanswered questions regarding the technique of etching with an oxygen plasma such as the role and the fate of osmium tetroxide during etching, the question of how best to obtain the optimal amount of etching and how to render the etched specimens electrically conductive without obscuring surface detail must be determined by further experience. Because of these questions the use of an oxygen plasma for etching embedded tissue for observation with the scanning electron microscope must be considered still in the state of technical development and exploration. Even so certain advantages are obvious. Etching thick sections with excited oxygen, for scanning electron microscopy, is considerably less arduous than total ashing of thin sections for transmission electron microscopy. The need for preparing silicon monoxide films is eliminated, as well as the need to cut ultra thin sections and the concern for mounting the sections onto the film with smooth intimate contact. Resolution of the spodograms produced by etching is at least as good as in spodograms produced by total ashing of thin sections. Finally, as a general technique for scanning electron microscopy etching thick sections of embedded tissue by the use of an oxygen plasma yields specimens that show a resolution that is considerably better than that obtainable by most other methods of scanning electron microscopy currently being used for viewing the internal structure of cells and organelles in bulk samples. ACKNOWLEDGMENTS
We are grateful to Dr Richard S. Thomas of the U.S. Department of Agriculture, Western Regional Research Laboratory, Berkeley, California, for very helpful discussions and correspondence that provided us with a substantial amount of technical advice and guidance that made this investigation much simpler to undertake. References Boyde, A. & Wood, C. (1969) Preparation of animal tissue for surface-scanning electron microscopy. 3. Microsc. 90, 221. Erlandsen, S.L., Thomas, A. & Wendelschafer, G. (1973) A simple technique for correlating SEM with T E M on biological tissue originally embedded in epoxy resin for TEM. In: Proc. Sixth Ann. SEM Symp. (Ed. by 0. Johari and I. Corvin), p. 349. I I T Res. Inst., Chicago. Frazizr, P.D. (1971) An electron microscopic investigation of mineralized tissues. Ph.D. thesis, Washington University, Seattle, 1971. University Microfilms, Ann Arbor, Mich. Haggis, G.H., Bond, E.F. & Phipps, B. (1976) Visualization of mitochondria1 cristae and nuclear chromatin by SEM. In: Proc. Ninth Ann. S E M Symp. Part I (Ed. by 0. Johari), p. 281. I I T Res. Inst., Chicago. Hohman, W.R. (1967) A study of low temperature ultramicroincineration of avain shell gland mucosa by electron microscopy. Ph.D. dissertation, Pennsylvania State University, University Park, Pennsylvania.
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WalterJ. Humphreys and William G . Henk Hohman, W.R. (1974) Ultramicroincineration of thin-sectioned tissue. In : Principles and Techniques of Electron Microscopy-Biological Applications, Vol. 4 (Ed. by M. A. Hayat), p. 129. van Nostrand Reinhold, New York. Hohman, W. & Schraer, H. (1972) Low temperature ultramicroincineration of thin-sectioned tissue. 3. Cell Biol. 55, 328. Humphreys, W.J. & Wodzicki, T.J. (1972) Methods for viewing by scanning electron microscopy the interior organization of protoplasts of plant cells. In: Proc. 30th Annual Meeting Electron Microscopy Society of America (Ed. by C. J. Arceneaux), p. 238. Claitor’s Publishing Division, Baton Rouge. Humphreys, W. J., Wodzicki, T.J. & Paulin, J. J. (1973a) Fractographic studies of plasticembedded cells by scanning electron microscopy. J . Cell Biol. 56, 876. Humphreys, W.J., Spurlock, B.O. & Johnson, J.S. (1973b) Critical point drying of freezefractured tissue for scanning electron microscopy. In : Proc. 31st Annual Meeting Electron Microscopy Society of America (Ed. by C. J. Arceneaux), p. 452. Claitor’s Publishing Division, Baton Rouge. Humphreys, W.J., Spurlock, B.O. & Johnson, J.S. (1974) Critical point drying of ethanolinfiltrated, cryofractured biological specimens for scanning electron microscopy. In : Proc. Seventh Ann. SEM Symp. (Ed. by 0 . Johari and I. Corvin), p. 275. IIT Res. Inst., Chicago. Miyai, K., Abraham, J.L., Lithicum, D.S. & Wagner, R.M. (1976) Scanning electron microscopy of hepatic ultrastructure-secondary, backscattered, and transmitted electron imaging. Lab. Invest. 35, 369. Mollenhauer, H.H. (1964) Plastic embedding mixtures for use in electron microscopy. 3. Stain Technol. 39, 111. Munger, B.L. & Mumaw, V.R. (1976) Specimen preparation for SEM study of cells and cell organelles in uncoated preparations. In: Proc. Ninth Ann. SEM Symp. Part I (Ed. by 0. Johari), p. 275. IIT Res. Inst., Chicago. Myklebust, R., Dalen, H. & Saetersdal, T.S. (1975) A comparative study in the transmission electron microscope of intracellular structures in sheep heart muscle cells. 3. Microsc. 105,57. Reynolds, E.S. (1963) The use of lead citrate at high p H as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 208. Sybers, H.D. & Ashraf, M. (1973) Preparation of cardiac muscle for SEM. In: Proc. Sixth Ann. SEM Symp. (Ed. by 0.Johari and I. Corvin), p. 341. IIT Res. Inst., Chicago. Tanaka, K. & Iino, A. (1972) Frozen resin cracking method for scanning electron microscopy and its application to cytology. In: Proc. 30th Annual Meeting Electron Microscopy Society of America (Ed. by C. J. Arceneaux), p. 408. Claitor’s Publishing Division, Baton Rouge. Tanaka, K., Iino, A. & Naguro, T. (1976) Scanning electron microscopic observation on intracellular structures of ion-etched materials. Arch. Hist. 3ap. 39, 1976. Thomas, R.S. (1962) Demonstration of structure-bound mineral constituents in thinsectioned bacterial spores by microincineration. In : Electron Microscopy (Ed. by s. s. Breese), Vol. 2, RR-11. Academic Press, New York. Thomas, R.S. (1964) Ultrastructural localization of mineral matter in bacterial spores by microincineration. 3. Cell BioZ. 23, 113. Thomas, R.S. (1974) Use of chemically reactive gaseous plasmas in preparation of specimens for microscopy. In: Techniques and Applications of Plasma Chemistry (Ed. by J. R. Hollahan and A. T. Bell), p. 255. John Wiley, New York. Thomas, R.S. & Greenawalt, J.W. (1968) Microincineration, electron microscopy, and electron diffraction of calcium phosphate-loaded mitochondria. 3. Cell Biol. 39, 55. Thomas, R.S. & Hollahan, J.R. (1974) Use of chemically-reactive gas plasmas in preparing specimens for scanning electron microscopy and electron probe microanalysis. In : Proc. Seventh Ann. SEM Symp. (Ed. by 0. Johari and I. Corvin), p. 83. I I T Res. Inst., Chicago. Watson, M.L. (1958) Staining of tissue sections for electron microscopy with heavy metals. 3. biophys. biochem. Cytol. 4, 475. Winborn, W.B. & Guerrero, D.L. (1974) The use of a single tissue specimen for both transmission and scanning electron microscopy. Cytobios, 10, 83. Woods, P.S. & Ledbetter, M.C. (1974) A method of direct visualization of plant cell organelles for scanning electron microscopy. In : Proc. 32nd AnnuaZ Meeting Electron Microscopy Society of America (Ed. by C. J. Arceneaux), p. 122. Claitor’s Publishing Division, Baton Rouge.
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Ultrastructure of cell organelles by scanning electron microscopy of thick sections surface-etched by an oxygen plasma
by WALTERJ. HUMPHREYS and W I L L I A MG. HENK,Central Electron Microscopy Laboratory, Barrow Hall, University of Georgia, Athens, Georgia 30602, U.S.A. SUMMARY
Kidney tissue double fixed in glutaraldehyde and osmium tetroxide and embedded in epoxy resin by standard techniques used for transmission electron microscopy was cut into sections 1 pm or more thick and surface-etched by an oxygen plasma. Etching caused ash residues (possibly composed partly or organo-metallic complexes) of membranes and other etch resistant cell components to emerge as recognizable structures projecting upward from the surrounding embedment which was combusted and removed as volatile products. Using the secondary electron mode for image formation, structural features of cells which could be imaged with clarity with the scanning electron microscope included: profiles of peripheral and infolded plasma membranes, the nuclear envelope and profiles of cut mitochondria1 matrix granules, cristae and the outer limiting membranes. Resolution was better than that obtainable from most other methods of specimen preparation currently being used in scanning electron microscopy for viewing the internal structures of cells and organelles in bulk samples of tissue. INTRODUCTION
Attempts to visualize cell organelles and their internal structural details with the scanning electron microscope, using the standard secondary electron mode, have been only partially successful. The use of fracturing techniques, advocated by Boyde & Wood (1969) for opening up tissues and cells for examination with the scanning electron microscope, has permitted limited observation of some cellular constituents. Humphreys & Wodzicki (1972) were able to see cytoplasmic organelles in plant cells which were freeze-fractured and subsequently freeze-dried. Tanaka & Iino (1972) and Woods & Ledbetter (1974) could identify organelles inside cells that had been infiltrated with epoxy resin, frozen, fractured and after removal of the resin by solvents, critical point dried. Humphreys et al. (1973a) identified some cell organelles with the scanning electron microscope by fracturing tissues embedded in epoxy resin, originally intended for ultramicrotomy. The technique of freezing tissues in certain non-polar intermediate fluids that are used for critical point drying (e.g. ethanol, acetone, Freons) followed by cryofracturing, thawing the fractured tissue in the intermediate fluid, and critical point drying (Sybers & 0022-2720/79/0700-0255 S02.00
ri"
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WalterJ. Humphreys and William G. Henk Ashraf, 1973; Humphreys et al., 1973b, 1974; Miyai et al., 1976; Munger &Mumaw, 1976) has become a valuable technique that has provided a new and very useful way of looking inside tissues with the scanning electron microscope. Myklebust et al. (1975) studied the ultrastructure of heart muscle by scanning electron microscopy of deparaffinized, critical point dried thick sections of paraffin-embedded tissue. In none of these studies was the internal structure of organelles such as the cristae of mitochondria clearly visible. Haggis et al. (1976) were able to visualize mitochondrial cristae and nuclear chromatin using the scanning electron microscope. Their specimens were freeze-fractured from an aqueous solution containing a cryoprotectant, thawed and fixed in a solution of glutaraldehyde containing a cryoprotectant, and then critical point dried. T o make identification of cytoplasmic organelles unmistakable at fractured or cut surfaces of tissues, a method is required which would etch away enough cytoplasmic matrix, mitochondrial matrix, or other matrices both inside and surrounding organellar structures so that stabilized etch-resistant membranes (and other etchresistant constituents) would project above the surface far enough to be imaged by the scanning electron microscope. Then, as a consequence of the etching, positive identification of many cytoplasmic organelles could be made by virtue of the membrane patterns or other swucrural patterns that would em-rge. Sections 1 pm thick cut from blocks of tissue embedded in epoxy have been etched chemically with epoxy solvents to cause cell organelles such as mitochondria to appear as rounded elevations (Winborn & Guerrero, 1974). Erlandsen et al. (1973) obtained similar results with this technique by etching the smooth surface of a block from which sections had been cut. However, this method did not reveal the internal structure of organelles. Another chemical agent that can be used for surface-etching thick sections of plastic-embedded tissue is an oxygen plasma. If radio-frequency electrical discharges are passed through a gentle stream of pure oxygen gas while it is being drawn past the specimens by a mechanical vacuum pump connected to the reaction chamber and if the oxygen is admitted at a controlled flow rate to attain an operating pressure of 6-130PaY the gas becomes highly reactive chemically. It forms a plasma that destroys organic bonds and organic material is removed from the specimen as volatile products that are swept out by the oxygen stream. Inorganic residues remain on the surface very near their position in the unreacted sample. The resulting ash patterns remaining on the etched surface can then be examined by scanning electron microscopy. This was suggested to us as a method for preparing specimens for scanning electron microscopy from previous studies in which excited oxygen was used for low temperature ultra-microincineration of biological specimens that were to be studied by transmission electron microscopy. Thomas (1962) used high temperature ashing of ultra-thin sections of bacterial spores for transmission electron microscopy studies. Following this, Thomas (1964) pioneered the use of low temperature microincineration of thin-sectioned biological materials for transmission electron microscopy. Thomas & Greenawalt (1968) demonstrated by transmission electron microscopy of metal-shadowed thin sections that an oxygen plasma could be used at a low temperature (specimen temperature less than 373 K) to differentially surface-etch thin sections of Epon-embedded isolated mitochondria. This incomplete ashing caused the membrane remnants of the sectioned cristae to emerge as recognizable structures projecting upward as ridges from the surrounding embedded material which was volatilized and removed by the excited oxygen. An excellent review on the status of spodography (the technique of producing ash patterns) for light and electron microscopy has been published by Thomas (1974).
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SEM of plasma-etched sections Hohman (1967, 1974) and Hohman & Schraer (1972) carried out the first extensive studies which utilized the technique of low temperature ultramicroincineration of thin sectioned tissue. Sections of hen shell gland 100-500 nm thick were completely ashed onto silicon monoxide support films and the resulting spodograms were examined by transmission electron microscopy. Resolution was remarkably good and it was easily possible to identify from the ash patterns such cell components as mitochondria, plasma membranes, cytoplasmic membranes and chromatin. Frazier (1971) using essentially the same method carried out transmission electron microscopy of shadowed sections of odontoblasts which were etched in an oxygen plasma for varying lengths of time at a low power level. After only 30 s of etching, enough of the epoxy was preferentially removed to reveal ash patterns of plasma membranes, mitochondria, and endoplasmic reticulum with a fine definition better than in most of the preparations shown by Hohman & Schraer (1972). Frazier's improved resolution was probably due to the use of thinner sections (about 60-100 nm). The above results suggested to us the use of an oxygen plasma for surfaceetching fractured or cut surfaces of bulk samples of tissue embedded for transmission electron microscopy. It was anticipated that a gentle, and differential surface-etching would cause the emergence of structural features within cells and organelles that could be imaged with considerable clarity with the scanning electron microscope operated in the secondary electron mode. '
M A T E R I A L S AND M E T H O D S
Mouse kidney was fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer at p H 7.2 for 1 h and washed in 0.1 M cacodylate buffer with 50,; sucrose added. It was post-fixed in 1% Os04 in cacodylate buffer for 1 h, washed in cacodylate buffer, dehydrated in a graded series of ethanols and embedded in Araldite-Epon (Mollenhauer, 1964). Ultrathin sections were cut, stained with aqueous uranyl acetate (Watson, 1958), rinsed, stained with lead citrate (Reynolds, 1963), and examined in a transmission electron microscope in order to make sure that fixation was good. For scanning electron microscopy, 1 pm thick sections were cut with a diamond knife, placed on a fragment of coverslip 10 x 10 mm or smaller and allowed to dry flat against the glass. The coverslip fragments bearing 1 pm sections were placed on a microscope slide which was placed in the reaction chamber of a Tegal Plasmod" plasma generator. The sections were etched by an oxygen plasma following the method described by Thomas & Hollahan (1974). Briefly, the reaction chamber was evacuated to a mild vacuum by a mechanical pump. Oxygen gas obtained from standard-grade commercial oxygen bottles was admitted at a controlled flow rate 13.8 kPa (2 psi) to obtain an operating pressure in the range of 6-130 Pa. Radio-frequency power was applied around the chamber at 13.56 MHz. This excited the oxygen molecules and changed some of them into other species such as atoms, radicals, ions and free electrons. The highly reactive gaseous plasma caused a gentle, low-temperature combustion of the organic materials in the sample. The combustion products were carried away in the gas stream leaving behind the thick sections with the exposed surfaces etched. An R F power setting of 50 W was arbitrarily chosen and the sections were etched for a period of 60 s. The etched sections were shadowed by vacuum evaporation of platinum-palladium wire from an angle of 30" at a distance of 100mm. After shadowing, the specimens were lightly sputter-coated with gold using a Hummer I1 sputter coater (Technics, Inc.). The etched sections were photographed with a Philips 501 scan-
* Available from the Tegal Corporation, 860 Wharf Street, Richmond, CA 94804, U.S.A. 257
WalterJ. Humphreys and William G. Henk
Figs. 1 and 2
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SEM of plasma-etched sections ning electron microscope at an operating voltage of 15 kV with the specimen tilted to an angle of about 25 '. OBSERVATIONS
A low magnification scanning electron micrograph showing the typical appearance of a 1 pm thick epoxy section of mouse kidney surface-etched by an oxygen plasma is shown in Fig. 1. The nuclear, cytoplasmic, mitochondrial, and plasma membranes show up as light lines against a grey background. The overall appearance of the etched surface resembles a transmission electron micrograph of a thin section printed in reverse contrast. In general, the structures that would appear electron dense in typical transmission electron micrographs of thin sections (Fig. 3) appear light in scanning electron micrographs of this type of preparation. The lumen (L) of the distal convoluted tubule and the vesicles (V) in the cells of a proximal convoluted tubule appear as the darkest areas in the specimen. Both structures are spaces essentially filled with only the epoxy embedment. The inset of Fig. 1 is an enlargement of an area containing the brush border of a proximal convoluted tubule cut at an oblique angle. The limiting plasma membranes of microvilli appear as bright lines surrounded by the darker lumen into which the microvilli project. An enlargement of a portion of Fig. 1 is shown as Fig. 2. Differential etching and metal shadowing has caused the nuclear envelope to project upward like a ridge rising above the background of etched nucleoplasm on the one side and etched cytoplasmic matrix on the other. Similarly, profiles of mitochondrial membranes appear as ridges rising above the etched mitochondrial matrix and the cytoplasmic matrix. Thus, cristae are clearly recognizable. A few mitochondrial matrix granules (G), which seem to be refractory to etching by oxygen plasma as used here, are found lying over the cristae of some mitochondria. A typical transmission electron micrograph of a thin section of the embedded kidney tissue used for the 1 pm thick sections shown in all the other figures is shown in Fig. 3. Figure 4 is a scanning electron micrograph of the plasma-etched surface of a 1 pm thick section cut from the same block of embedded tissue as that shown in Fig. 3 and at about the same magnification. Comparing Fig. 4 with Fig. 3 it appears that, in general, the structures that are most electron dense in Fig. 3 are the ones that were most resistant to the plasma-etching as shown in Fig. 4. After etching with the oxygen plasma the electron-dense structures such as heterochromatin (H), and cytoplasmic and mitochondrial membranes leave ash residues Fig. 1. Scanning electron micrograph of a 1 pm thick Epon-Araldite section of mouse kidney tissue. The section was surface-etched by an oxygenplasma and shadowed at a 30" angle with Pt.-Pd. At the upper left and upper right corners structural features of cells of distal convoluted tubules are recognizable. Ash patterns of the membranes of the nucleus (N), plasma membranes and cytoplasmic membranes appear light against a grey background. Erythrocytes (E), in a capillary lumen are at top centre. L, lumen of tubule; V, vesicles. x 3870. Inset: Brush border of a distal convoluted tubule at a higher magnification, enlarged from the area designated by the arrow. x 7000. Fig. 2. Higher magnification of the area outlined in Fig. 1. Ash patterns of membranes of the nucleus (N), plasma membranes (PL) including lateral cell membranes (CM), and cytoplasmic membranes can be identified. Mitochondria (M) are identifiable by the ash patterns of their cristae and outer limiting membranes. Mitochondria matrix granules (G) lie on top of the cut profiles of cristae of several mitochondria. x 12,000.
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S-& 'SsTd
092
SEM of plasma-etched sections that are sufficiently elevated above the background to permit their imaging by the scanning electron microscope. The ash patterns remain on the surface of the section in undisturbed configurations that can be used to identify the structures from which the ash patterns originated during the limited surface-incineration of the section. In Fig. 4 the ash patterns that originated at the site of cut profiles of numerous mitochondrial membranes that were surface-ashed by the oxygen plasma are unmistakably recognizable. The embedding epoxy and the mitochondrial matrices were partially etched away leaving the ash pattern of the mitochondrial walls and cristae. Residues of the outer and inner mitochondrial membranes cannot be distinguished from one another even at higher magnifications (Fig. 5). Residues of the two membranes and the membrane space between them appear as a single structure in these preparations, probably because the metal shadowing and the subsequent metal coating caused the space between the two membranes to be bridged by the metal deposit. For the same reason, the double membrane structure of the cristae is not discernible from the residues of the surface-ashed cristae. The ash patterns of mitochondrial walls and cristae are much thicker (Fig. 4) than the profiles of the same structures as seen in thin sections (Fig. 3) because of the metal coating on the etched sections. DISCUSSION
In order to allow a direct comparison of our etched preparations as seen by scanning electron microscopy with the characteristic ultrastructural appearance of the same tissue in thin section, and thus ensure the adequacy of the fixation, the tissue used in this study was fixed and processed in the conventional manner. That is, all tissue samples were fixed in glutaraldehyde, post-fixed in osmium tetroxide and embedded in epoxy using the standard techniques employed for ultramicrotomy and transmission electron microscopy. Although the etch-resistance of cytoplasmic membranes might have been increased by en bloc staining, or staining of the thick sections with solutions containing non-volatile metal salts, the cytoplasmic membranes were sufficiently etch-resistant to the oxygen plasma without further treatment. A consistent observation was that those cell constituents that were most electron dense in thin sections were also the most etch-resistant structures in the plasmaetched preparations. As is well known, much of the electron density of structures in thin sections is due to the presence of reduced osmium. But this would not seem to be a significant factor in the etch-resistance since there is the distinct possibility Fig. 3. Transmission electron micrograph of a thin section cut from the same block as the surface-etched 1 pm thick sections shown in the other figures. x 10,500. Fig. 4. Scanning electron micrograph of a surface-etched 1 pm thick section prepared the same as in Fig. 1. Cells of a convoluted tubule of a mouse kidney are shown. Ash patterns of numerous mitochondrial profiles remain after the low temperature surface-incineration. The heterochromatin (H) was more resistant to the oxygen-plasma than the other nuclear material. Compare with Fig. 3. x 11,400. Fig. 5. Scanning electron micrograph of a surface-etched 1 pm thick sections prepared the same as in Fig. 1. Mitochondria1 profiles in cells of mouse kidney convoluted tubules are shown. The low temperature plasma-etching appears to cause very little lateral migration of the ash from the incinerated membranes. Ashed cristae remain discretely separated from one another. x 21,800.
26 1
WalterJ. Humphreys and William G. Henk pointed out by Thomas (1974) that in the presence of the oxygen plasma osmium is a volatile stain and in his words, ‘evidently the metallic osmium is reoxidized by the plasma to volatile osmium tetroxide which leaves no trace’. Hohman (1974) found in spodograms of totally low-temperature ashed thin sections of avian shell gland tissue that most cellular structures appeared to be similar, with or without osmium fixation. However, certain membranes such as membranes between cells appeared only when glutaraldehyde fixation was followed by osmium post-fixation. The ash from these membranes was not apparent in spodograms from tissue fixed by glutaraldehyde alone. Whether the ash residue in these membranes consisted in part of complexed osmium that was not volatilized or whether the residue resulted indirectly from the action of osmium cannot be determined until further studies designed to answer this question have been made. Most spodography of embedded tissue that has previously been described (Thomas, 1964; Thomas & Greenawalt, 1968; Hohman, 1967; Hohman & Schraer, 1972; Hohman, 1974) has involved the use of excited oxygen for total ashing of thin sections onto silicon monoxide films for transmission electron microscopy. The method reported here describes an alternative method for spodography in which thick sections are partially ashed or surface-etched by the use of excited oxygen and then examined in the scanning electron microscope as bulk samples using the secondary electron mode. The depth of etching can be controlled by varying the length of time used for etching and by the selection of different wattages used for producing the oxygen plasma, or by an appropriate combination of these two variables. Thus spodograms can be produced by etching thick sections to depths equal to the thicknesses of thin sections that have been totally ashed. The etched thick sections can be examined by the scanning electron microscope and the totally ashed sections can be examined by transmission electron microscopy and the resulting spodograms can be compared. If such a comparison is made, the main advantage of etching compared to complete ashing is a distinct reduction in ash displacement and an increased resolution. Having an intact ‘base’ under the ashed material may help to maintain the stability of the ash. The totally ashed sections studied by Hohman & Schraer (1972) showed evidence of considerable ash migration that, for example, resulted in clear zones around nuclei. This artefact was found in all sections thicker than about 0.1 pm, the clear zones becoming wider as thicker sections were used. Such artefacts were not found in our preparations of thicker sections etched for scanning electron microscopy. The nuclei in Figs. 4 and 5, for example, show no such artefacts. Recently Tanaka et al. (1976) used ion-etching on critical point dried tissue that had been freeze-cracked by various methods. Such specimens when examined by scanning electron microscopy appeared similar to our etched tissue sections. The specimens were etched by high-energy sputter etching and differential etching was achieved because membraneous structures in the cell are generally more resistant to sputter-etching and the cytoplasmic matrix is easily etched. Structures revealed this method of surface etching were nuclear pores, endoplasmic reticulum with ribosomes, Golgi apparatus, and mitochondria1 cristae. We attempted to use oxygen to surface-etch freeze-fractured critical point dried tissue prepared by the method of Humphreys et al. (1974). We were unsuccessful because the critical point dried tissue is freely permeable to the gaseous oxygen plasma which reacted chemically with the interior of the tissue at the same time as with the surface. As a consequence organic material was volatilized from the interior of the tissue at the same rate as from the tissue’s surface and no differential surface-etching could be achieved. In these preliminary studies we found that more satisfactory results were obtained when the plasma-etched sections were metal-shadowed before they were coated
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SEM of plasma-etched sections with metal prior to their examination in the scanning electron microscope. Theoretically this step is not necessary and higher resolution would be possible if it were dispensed with, since less metal would be present on the specimen to obscure surface detail. Our attempts to omit this step, however, usually, but not invariably, resulted in specimens that lacked adequate contrast when observed in the scanning electron microscope. Better resolution could also be obtained if the etching were restricted to a depth just sufficient to generate enough variability in surface topography to permit imaging the surface after a conducting metal coat of minimal thickness was deposited on to it. Optimization of these parameters will require further study. With careful attention to such details, plasma-etched sections could conceivably be capable of showing cellular fine structure that is not now resolvable by most state-of-the-art scanning electron microscopes using the secondary electron mode. Unanswered questions regarding the technique of etching with an oxygen plasma such as the role and the fate of osmium tetroxide during etching, the question of how best to obtain the optimal amount of etching and how to render the etched specimens electrically conductive without obscuring surface detail must be determined by further experience. Because of these questions the use of an oxygen plasma for etching embedded tissue for observation with the scanning electron microscope must be considered still in the state of technical development and exploration. Even so certain advantages are obvious. Etching thick sections with excited oxygen, for scanning electron microscopy, is considerably less arduous than total ashing of thin sections for transmission electron microscopy. The need for preparing silicon monoxide films is eliminated, as well as the need to cut ultra thin sections and the concern for mounting the sections onto the film with smooth intimate contact. Resolution of the spodograms produced by etching is at least as good as in spodograms produced by total ashing of thin sections. Finally, as a general technique for scanning electron microscopy etching thick sections of embedded tissue by the use of an oxygen plasma yields specimens that show a resolution that is considerably better than that obtainable by most other methods of scanning electron microscopy currently being used for viewing the internal structure of cells and organelles in bulk samples. ACKNOWLEDGMENTS
We are grateful to Dr Richard S. Thomas of the U.S. Department of Agriculture, Western Regional Research Laboratory, Berkeley, California, for very helpful discussions and correspondence that provided us with a substantial amount of technical advice and guidance that made this investigation much simpler to undertake. References Boyde, A. & Wood, C. (1969) Preparation of animal tissue for surface-scanning electron microscopy. 3. Microsc. 90, 221. Erlandsen, S.L., Thomas, A. & Wendelschafer, G. (1973) A simple technique for correlating SEM with T E M on biological tissue originally embedded in epoxy resin for TEM. In: Proc. Sixth Ann. SEM Symp. (Ed. by 0. Johari and I. Corvin), p. 349. I I T Res. Inst., Chicago. Frazizr, P.D. (1971) An electron microscopic investigation of mineralized tissues. Ph.D. thesis, Washington University, Seattle, 1971. University Microfilms, Ann Arbor, Mich. Haggis, G.H., Bond, E.F. & Phipps, B. (1976) Visualization of mitochondria1 cristae and nuclear chromatin by SEM. In: Proc. Ninth Ann. S E M Symp. Part I (Ed. by 0. Johari), p. 281. I I T Res. Inst., Chicago. Hohman, W.R. (1967) A study of low temperature ultramicroincineration of avain shell gland mucosa by electron microscopy. Ph.D. dissertation, Pennsylvania State University, University Park, Pennsylvania.
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WalterJ. Humphreys and William G . Henk Hohman, W.R. (1974) Ultramicroincineration of thin-sectioned tissue. In : Principles and Techniques of Electron Microscopy-Biological Applications, Vol. 4 (Ed. by M. A. Hayat), p. 129. van Nostrand Reinhold, New York. Hohman, W. & Schraer, H. (1972) Low temperature ultramicroincineration of thin-sectioned tissue. 3. Cell Biol. 55, 328. Humphreys, W.J. & Wodzicki, T.J. (1972) Methods for viewing by scanning electron microscopy the interior organization of protoplasts of plant cells. In: Proc. 30th Annual Meeting Electron Microscopy Society of America (Ed. by C. J. Arceneaux), p. 238. Claitor’s Publishing Division, Baton Rouge. Humphreys, W. J., Wodzicki, T.J. & Paulin, J. J. (1973a) Fractographic studies of plasticembedded cells by scanning electron microscopy. J . Cell Biol. 56, 876. Humphreys, W.J., Spurlock, B.O. & Johnson, J.S. (1973b) Critical point drying of freezefractured tissue for scanning electron microscopy. In : Proc. 31st Annual Meeting Electron Microscopy Society of America (Ed. by C. J. Arceneaux), p. 452. Claitor’s Publishing Division, Baton Rouge. Humphreys, W.J., Spurlock, B.O. & Johnson, J.S. (1974) Critical point drying of ethanolinfiltrated, cryofractured biological specimens for scanning electron microscopy. In : Proc. Seventh Ann. SEM Symp. (Ed. by 0 . Johari and I. Corvin), p. 275. IIT Res. Inst., Chicago. Miyai, K., Abraham, J.L., Lithicum, D.S. & Wagner, R.M. (1976) Scanning electron microscopy of hepatic ultrastructure-secondary, backscattered, and transmitted electron imaging. Lab. Invest. 35, 369. Mollenhauer, H.H. (1964) Plastic embedding mixtures for use in electron microscopy. 3. Stain Technol. 39, 111. Munger, B.L. & Mumaw, V.R. (1976) Specimen preparation for SEM study of cells and cell organelles in uncoated preparations. In: Proc. Ninth Ann. SEM Symp. Part I (Ed. by 0. Johari), p. 275. IIT Res. Inst., Chicago. Myklebust, R., Dalen, H. & Saetersdal, T.S. (1975) A comparative study in the transmission electron microscope of intracellular structures in sheep heart muscle cells. 3. Microsc. 105,57. Reynolds, E.S. (1963) The use of lead citrate at high p H as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 208. Sybers, H.D. & Ashraf, M. (1973) Preparation of cardiac muscle for SEM. In: Proc. Sixth Ann. SEM Symp. (Ed. by 0.Johari and I. Corvin), p. 341. IIT Res. Inst., Chicago. Tanaka, K. & Iino, A. (1972) Frozen resin cracking method for scanning electron microscopy and its application to cytology. In: Proc. 30th Annual Meeting Electron Microscopy Society of America (Ed. by C. J. Arceneaux), p. 408. Claitor’s Publishing Division, Baton Rouge. Tanaka, K., Iino, A. & Naguro, T. (1976) Scanning electron microscopic observation on intracellular structures of ion-etched materials. Arch. Hist. 3ap. 39, 1976. Thomas, R.S. (1962) Demonstration of structure-bound mineral constituents in thinsectioned bacterial spores by microincineration. In : Electron Microscopy (Ed. by s. s. Breese), Vol. 2, RR-11. Academic Press, New York. Thomas, R.S. (1964) Ultrastructural localization of mineral matter in bacterial spores by microincineration. 3. Cell BioZ. 23, 113. Thomas, R.S. (1974) Use of chemically reactive gaseous plasmas in preparation of specimens for microscopy. In: Techniques and Applications of Plasma Chemistry (Ed. by J. R. Hollahan and A. T. Bell), p. 255. John Wiley, New York. Thomas, R.S. & Greenawalt, J.W. (1968) Microincineration, electron microscopy, and electron diffraction of calcium phosphate-loaded mitochondria. 3. Cell Biol. 39, 55. Thomas, R.S. & Hollahan, J.R. (1974) Use of chemically-reactive gas plasmas in preparing specimens for scanning electron microscopy and electron probe microanalysis. In : Proc. Seventh Ann. SEM Symp. (Ed. by 0. Johari and I. Corvin), p. 83. I I T Res. Inst., Chicago. Watson, M.L. (1958) Staining of tissue sections for electron microscopy with heavy metals. 3. biophys. biochem. Cytol. 4, 475. Winborn, W.B. & Guerrero, D.L. (1974) The use of a single tissue specimen for both transmission and scanning electron microscopy. Cytobios, 10, 83. Woods, P.S. & Ledbetter, M.C. (1974) A method of direct visualization of plant cell organelles for scanning electron microscopy. In : Proc. 32nd AnnuaZ Meeting Electron Microscopy Society of America (Ed. by C. J. Arceneaux), p. 122. Claitor’s Publishing Division, Baton Rouge.
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