C Journal of Microscopy, Vol. 105, P t 1, September 1975, pp. 57-65. Received 10 March 1975; revision received 28 M a y 1975
A comparative study in the transmission electron microscope and scanning electron microscope of intracellular structures in sheep heart muscle cells
by R E I D A RMYKLEBUST, H E L G ED A L E N *and T H v . S E L M E R S A ETERSDAL, Institute of Anatomy, University of Bergen, 5000 Bergen, Norway and Department of Pathology, Karolinska Sjukhuset, 10401 Stockholm 60, Sweden SUMMARY
The internal cellular structures of the sheep ventricular myocardium have been comparatively studied in the transmission electron microscope (TEM) and in the scanning electron microscope (SEM). For T E M studies the tissue was prepared according to standard methods. Thick sections (10 pm) of paraffin embedded material were, after they had been deparaffinized in toluene, critical point dried, coated with gold and examined in the SEM. The comparative T E M and SEM investigations revealed very good correspondence, and it is evident that the described preparation procedure for SEM has preserved the fine structures of myofibrils, mitochondria, T-Tubules and sarcoplasmic reticulum in an excellent life-like pattern. Of special interest was the three-dimensional demonstration of triads and of circumferentially arranged T-tubules. INTRODUCTION
Three-dimensional studies of soft biological material in the scanning electron microscope (SEM) have mainly been limited to the outer surfaces of cells and tissues. Some methods have, however, been developed for direct observations of internal cellular structures in the SEM. The first attempt in this direction was described by McDonald et al. (1967), who used thick paraffin sections. After removing the embedding material with xylene, the sections were air-dried and coated with a thin conductive film of gold for examination in the SEM. Several authors have since applied paraffin sections for studying the internal architecture of cells and tissues (Dalen et al., 1970; Geissinger, 1971; Herbst et al., 1972; Poh et al., 1971). As an alternative method Pachter et al. (1973) have used thick sections of Epon-embedded muscle cells. I n this case the embedding material was etched away by means of an iodine acetone solution. On the other hand Makita & Sandborn (1970) successfully omitted the embedding step by cutting thick sections of fixed material on a Smith-Farquar tissue sectioner. Common for all procedures mentioned above is the dehydration of the thick
* Present address : Laboratory of Clinical Electron Microscopy, The Gade Institute, Department of Pathology, University of Bergen, 5016 Bergen, Norway. Correspondence : Reidar Myklebust, Institute of Anatomy, University of Bergen, Arstadveien 19, 5000 Bergen, Norway 57
Reidar Myklebust, Helge Dalen and Thv.Selmer Saetersdal sections by air-drying. In this connection it should be kept in mind, that airdrying is associated with a considerable shrinkage of soft tissues (Boyde & Wood, 1969; Porter et al., 1972). However, this technical problem was overcome by employing the cryofracturing technique (Haggis, 1970; Lim, 1971). The fractured tissues were dehydrated by freeze-drying. Even though by this method a life-like spatial distribution of internal cellular structures is retained, minor fractures due to ice-crystallization can often be a problem (Boyde & Wood, 1969; Porter et al., 1972). The cryofracture technique developed by Tanaka (1972) utilized frozen blocks of freshly epoxy-embedded material. After the resin had been removed from the fractured tissue by propylene oxide, the pieces were transferred to amyl acetate and critical point dried according to the method of Anderson (1951). Today this dehydration procedure is considered to be superior to air-drying and freezedrying (Boyde & Wood, 1969; Porter et al., 1972). The works of Sybers & Ashraf (1973) and McAllister et al. (1974) are also in favour of Anderson's procedure, as very good preservation of internal cellular structures was obtained after critical point drying of deparaffinized thick sections and of cryofractured tissue. The purpose of the present investigation was to conduct a comparative study in the TEM and SEM of the internal structures of the sheep myocardial cell. The ultrastructure of this tissue has previously been examined in the TEM by Simpson & Oertalis (1962), but these authors did not deal with the cell three-dimensional architecture. It should also be pointed out that the preservation of the cell fine structure in their work did not appear satisfactory. MATERIAL AND METHODS
Tissue preparation for TEA4 Freshly dissected pieces of sheep ventricular myocardium were fixed under ice-cold conditions for 1.5 h in Karnowsky's fixative (Karnowsky, 1965) followed by 1 h in 29b OsO,. Both fixatives were made up in 0.1 M cacodylate buffer at p H 7.2. The tissues were stained en bloc for 1 h in 2% aqueous uranyl acetate. After dehydration at room temperature in increasing concentrations of ethanol, the tissues were embedded in Epon 812 (Luft, 1961) and polymerized for 24 h at 40"C, followed by 48 h at 60°C. The cured blocks were cut with glass knives on a Reichert ultramicrotome. Thick sections (1 pm) of the Epon-embedded material were stained with toluidine blue (Trump et al., 1961) for examination in the light microscope, while the ultrathin sections were stained with lead citrate (Reyholds, 1963) for examination in a Philips 300 electron microscope, operated at 80 kV.
Tissue preparations for SEA4 The myocardial tissue selected for SEM studies, was fixed in the same manner as described above. However, after dehydration in ethanol the material was transferred to toluene and embedded in paraffin. Thick sections (10 pm) were cut on a microtome and mounted on glass cover-slips. The first section in each series was stained according to standard histological methods in haematoxylin and eosin for examination in the light microscope. The rest of the sections were deparafinized in toluene and transferred through a series of absolute ethanol baths to amyl acetate. The critical point drying was carried out in a Polaron apparatus according to the method described by Anderson (1951). At this stage some specimens were directly embedded in Epon and sectioned for the transmission electron microscope. Prior to the examination in a Cambridge Stereoscan S4,
58
TEM and SEM of heart muscle ceIIs operated at 10-20 kV, the specimens were coated with a thin conductive film of gold in an Edwards vacuum coater. RESULTS
The contractile material of the sheep myocardial cell is arranged in separated myofibrils running parallel to each other (Fig. 1). The TEM micrograph (Fig. 5) reveals the striated nature of the fibrils. Sarcomeres of different fibrils are almost exactly in register. The myofibrils are separated by rows of mitochondria (Figs. 1, 2 and 5). It is apparent, both from SEM and T E M micrographs (Figs. 2 and 5),
Fig. 1. A SEM micrograph showing parallel myofibrils separated by rows of mitochondria. Some of the fibrils have been torn up by the knife. Sheep myocardial cell. x 4560. Fig. 2. The same as Fig. 1 at higher magnification. MF = myofibril. M = mitochondrion. Arrow = T-tubule. x 11,360.
59
Reidar Myklebust, Helge Dalen and Thv. Selmer Saetersdal that the distribution of mitochondria is closely related to the sarcomere pattern of the myofibrils. T h e mitochondria are consistently found between two adjacent Z-line levels, which seems to be the case whether the sarcomeres are contracted or relaxed. This indicates that the mitochondria change their form during the contraction-relaxation cycle.
Fig. 3. (a) SEM micrograph of circumferentially arranged T-tubules (arrows). x2280. (b) The same at higher magnification. The tubules (arrows) are covered by sarcolemma except in the lower right of the micrograph. Note the connections of adjacent tubules (at the middle of micrograph). x 11,360. Fig. 4. A SEM micrograph showing the course of the T-tubules (arrows) into the cell. Note how closely they invest the myofibrils (MF). x 11,360.
60
TEA4 and SEM of heart muscle cells The transverse tubules (T-tubules) are prominent structures in this kind of myocardial cell, showing a regular pattern related to the pattern of sarcomeres and mitochondria. Fig. 4 shows the spatial relationship between the myofibrils and the T-tubules. The T-tubules invest the fibrils very closely, passing from one myofibril to the next in a predominantly transverse direction. The TEM-micrograph shows that they are situated at the Z-band levels of sarcomere (Fig. 5).
Fig. 5. A TEM micrograph showing the structure and position of the coupling (C). MF = myofibril. Arrows = T-tubules. SR = sarcoplasmic reticulum. x 21,600. Fig. 6.A SEM micrograph of the coupling (circles). Note the dilatation of the junctional SR. MF = myofibril. Arrows = T-tubules. x 11,360.
61
Reidar Myklebust, Helge Dalen and Thv. Selmer Saetersdal Occasionally, short longitudinal branches may be seen connecting the T-tubules at two adjacent Z-line levels. Where the cytoplasmic side of the sarcolemma was exposed, a special T-tubule arrangement was observed. The tubules could be seen running circumferentially around the muscle fibres, just beneath the sarcolemma (Fig. 3a and b). The sarcoplasmic reticulum (SR) is rather poorly developed, forming a loose and irregular network around the myofibrils. The SR is somewhat expanded at the H-bands. From here it sends branches to the Z-line levels on both sides of the H-bands, where it makes contacts (couplings) with the T-tubules (Figs. 5 and 6). Figure 6 shows triadic junctions consisting of one T-tubule and of two components of the SR. At the junctional area, the SR forms knoblike dilatations (Fig. 6). These dilatations probably correspond to the junctional SR described by Sommer & Johnson (1969). Figure 5 shows that the junctional part of the SR is filled with electron dense material, this in contrast to the nonjunctional part of the SR. This electron dense material is called junctional granules by Sommer & Johnson (1969), and it may represent calcium binding sites (Philpott & Goldstein, 1967). Figure 7 shows a T E M micrograph of SEM prepared tissue. No damage of intracellular structures due to SEM preparation can be observed.
Fig. 7. A TEM micrograph of SEM prepared tissue. The different structures have retained a life-like pattern and no damage due to the SEM preparation seems to have occurred. Arrows = T-tubules, M = mitochondrion, SR = sarcoplasmic reticulum. x 30,150.
62
TEM and SEM of heart muscle cells DISCUSSION
It is evident from the present study that the internal cellular structures have retained a life-like pattern after critical point drying. This is a great improvement compared with the air-drying of sectioned material which always leads to a considerable shrinkage of the soft material (Boyde & Wood, 1969). Also, freezedrying is superior to air-drying, as the former method minimizes the surface tension (Arenberg et al., 1970). Accordingly, freeze-fractured material reveals a good spatial preservation of the internal cellular structures. However, minor disruptions of the tissue due to ice-crystallization are common artefacts associated with this type of dehydration (Boyde & Wood, 1969). In his studies of mouse mammary gland, Nemanic (1972) embedded the tissue in agar, cut it on a SmithFarquar tissue sectioner and applied critical point drying to minimize dehydration artefacts. This technique revealed a very good preservation of internal cellular structures. However, by using paraffin sections it is possible to investigate corresponding areas in succeeding planes by both the light microscope and SEM. Besides, sectioned material reveals the cell architecture very clearly. The good preservation of very fine cell structures after the critical point drying, makes this method very useful in getting a more spatial and precise picture of the cell organization. Intracellular structures of muscle cells have previously been studied in sectioned and air-dried tissue of normal (Buss et al., 1971; Pachter et al., 1973; Poh et al., 1971) pathological material (Geissinger, 1971) and of cryofractured and freezedried muscles (Haggis, 1970). The information these works give about the cell organization, are limited due to the preparation techniques. Some distortions seem to have occurred, and the preservation of fine structures like myofibrils, mitochondria, T-tubules and sarcoplasmic reticulum is not satisfactory. The fine and fragile tubules of the sarcoplasmic reticulum are especially difficult to preserve. The tubules easily collapse, making them difficult to distinguish from the underlying myofibrils. In critical point dried material the tubules retain their tubular structure and the network of these tubules is therefore more easily followed (Sybers & Ashraf, 1973). An important consequence of this is the preservation of the couplings. The size and form of the junctional sarcoplasmic reticulum and the frequency of the couplings are more easily told. The interesting observation of the circumferentially arranged T-tubules just beneath the sarcolemma are in good agreement with findings of McAllister et al. (1974). Although T E M micrographs revealed the ultrastructure of intracellular structures very clearly, serial sectioning is necessary in order to reveal their spatial orientation. The direct spatial observation of sectioned material made possible by the SEM, has to some extent made the serial sectioning unnecessary. The difficulty has been artefacts caused by unsatisfactory preparation techniques. However, comparative studies of SEM micrographs of critical point dried material with T E M micrographs show very good correspondence. I n this connection it should be mentioned that the T E M micrographs were taken both from conventionally prepared tissue and from tissue originally prepared for SEM and subsequently embedded in Epon and sectioned for TEM. This makes it safe to form three-dimensional pictures of intracellular structures from SEM micrographs. Moreover, the high resolution in the T E M micrographs, combined with the three-dimensional picture from the SEM micrographs, make up a very complete picture of the cell organization. ACKNOWLEDGMENTS
The authors wish to express their thanks to Professor Bo Thorell for his support
63
Reidar Myklebust, Helge Dalen and Thv.Selmer Saetersdal and interest in the present investigation. The skilful1 technical assistance by Mrs Maud Edenholm and Miss Ingeborg Kranz is gratefully acknowledged. This work was supported by grants from the Norwegian Research Council for Science and the Humanities and from the Swedish Medical Research Council (B67- 12x-215601).
References Anderson, T.F. (1951) Techniques for the preservation of three-dimensional structure in preparing specimens for the electron microscope. Trans. N. Y . Acad. Sci. 13, 130. Arenberg, J.K., Marovitz, W.F. & MacKenzie, A.P. (1970) Preparative techniques for the study of soft biological tissues in the scanning electron microscope: A comparison of air drying, low temperature evaporation, and freeze drying. Proc. Cambridge Stereoscan Colloquim, 1970, Morton Grove, Ill. p. 121. Buss, H., Hanrath, P., Kronert, W. & Schoenmackers. (1971) Uber raster-elektronenmikroskopische Untersuchungen am Myokard der Ratte. Arch. Kreislaufforsch.65, 58. Boyde, A. & Wood, C. (1969) Preparation of animal tissues for surface-scanning electron microscopy. 3. Microscopy, 90, 221. Dalen, H., Forte, J.G. & Forte, G.M. (1970) The application of the scanning electron microscope for some anatomical studies of the frog stomach. Proc. Seventh Int. Congr. Electron Microsc. I, 475. Geissinger, H.D. (1971) Correlated light optical and scanning electron microscopy of Gram smears of bacteria and paraffin sections of cardiac muscle.3. Microsc. 93, 109. Haggis, G.H. (1970) Cryofracture of biological material. Proc. Third Ann. SEM Symp., ZIT Res. Znst., Chicago, Ill, 97. Herbst, R., Multier-Lajous, A. M. & Kassner, G. (1972) Methode fur Direktabbildung von Zellkernen im Raster-Elektronenmikroskop. Beitr. Pathol. 145, 395. Karnowsky, M. J. (1965) A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J . Cell Biol. 27, 137A. Lim, D. J. (1971) Scanning electron microscopic observation on non-mechanically cryofractured biological tissue. Proc. Fourth Ann. SEM Symp., ZIT Res. Znst., Chicago, Ill. I, 257. Luft, J.H. (1961) Improvements in epoxy resin embedding methods. J . biophys. biochem. Cytol. 9, 409. McAllister, L.P., Murnaw, V.R. & Munger, B.L. (1974) Stereo ultrastructure of cardiac membrane systems in the rat heart. Proc. Seventh Ann. SEM Symp., ZIT Res. Inst. Chicago, Ill. 111, 712. Makita, T. & Sandborn, E.B. (1970) Scanning electron microscopy of secretory granules in albumen gland cells of the laying hen oviduct. Expl. Cell Res. 60, 477. McDonald, L.W., Pease, R.F.W. & Hayes, T.L. (1967) Scanning electron microscopy of sectioned tissue. Lab. Invest., 16, 532. Nemanic, M.K. (1972) Critical point drying, cryofracture, and serial sectioning. Proc. Fifth Ann. SEM Symp., ZZT Res. Inst., Chicago, Ill. 11, 295. Pachter, B.R., Davidowitz, J., Zimmer, B. & Breinin, G.M. (1973) Scanning electron microscopy of etched Epon extraocular muscle sections. J. Microsc. 99, 85. Philpott, C.W. & Goldstein, M.A. (1967) Sarcoplasmic reticulum of striated muscle. Localization of potential calcium binding sites. Science 155, 1019. Poh, R.L., Altenhoff, J., Abraham, S . & Hayes, T. (1971) Scanning electron microscopy of myocardial sections originally prepared for the light microscopy. Exp. MoZ. Pathol. 14, 404. Porter, K.R., Kelley, D. & Andrews, P.M. (1972) The preparation of cultured cells and soft tissues for scanning electron microscopy. Proc. Cambridge Stereoscan Colloquim, 1972, Morton Grove, Ill., p. 1. 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. Simpson, F.O. & Oertalis, S.J. (1962) The fine structure of sheep myocardial cells, sarcolemma1 invaginations and the transverse tubular system. 3. Cell BioZ. 12, 91. Sommer, R.J. & Johnson, E.A. (1969) Cardial muscle. A comparative ultrastructural study with special reference to frog and chicken hearts. 2. Zellforsch. 98,437.
64
TEM and SEM of heart muscle cells Sybers, H.D. & Ashraf, M. (1973) Preparation of cardiac muscle for SEM. Proc. Sixth A n n . SEM Symp., I I T Res. Inst., Chicago, Ill. 111, 341. Tanaka, K . (1972) Freezed resin cracking method for scanning electron microscopy of biological materials. Naturwissenschaften, 59, 77. Trump, B.F., Smuckler, E.A. & Benditt, E.P. (1961) A method for staining epoxy sections for light microscopy. J . Ultrastruct. Res. 5, 343.
65
A comparative study in the transmission electron microscope and scanning electron microscope of intracellular structures in sheep heart muscle cells
by R E I D A RMYKLEBUST, H E L G ED A L E N *and T H v . S E L M E R S A ETERSDAL, Institute of Anatomy, University of Bergen, 5000 Bergen, Norway and Department of Pathology, Karolinska Sjukhuset, 10401 Stockholm 60, Sweden SUMMARY
The internal cellular structures of the sheep ventricular myocardium have been comparatively studied in the transmission electron microscope (TEM) and in the scanning electron microscope (SEM). For T E M studies the tissue was prepared according to standard methods. Thick sections (10 pm) of paraffin embedded material were, after they had been deparaffinized in toluene, critical point dried, coated with gold and examined in the SEM. The comparative T E M and SEM investigations revealed very good correspondence, and it is evident that the described preparation procedure for SEM has preserved the fine structures of myofibrils, mitochondria, T-Tubules and sarcoplasmic reticulum in an excellent life-like pattern. Of special interest was the three-dimensional demonstration of triads and of circumferentially arranged T-tubules. INTRODUCTION
Three-dimensional studies of soft biological material in the scanning electron microscope (SEM) have mainly been limited to the outer surfaces of cells and tissues. Some methods have, however, been developed for direct observations of internal cellular structures in the SEM. The first attempt in this direction was described by McDonald et al. (1967), who used thick paraffin sections. After removing the embedding material with xylene, the sections were air-dried and coated with a thin conductive film of gold for examination in the SEM. Several authors have since applied paraffin sections for studying the internal architecture of cells and tissues (Dalen et al., 1970; Geissinger, 1971; Herbst et al., 1972; Poh et al., 1971). As an alternative method Pachter et al. (1973) have used thick sections of Epon-embedded muscle cells. I n this case the embedding material was etched away by means of an iodine acetone solution. On the other hand Makita & Sandborn (1970) successfully omitted the embedding step by cutting thick sections of fixed material on a Smith-Farquar tissue sectioner. Common for all procedures mentioned above is the dehydration of the thick
* Present address : Laboratory of Clinical Electron Microscopy, The Gade Institute, Department of Pathology, University of Bergen, 5016 Bergen, Norway. Correspondence : Reidar Myklebust, Institute of Anatomy, University of Bergen, Arstadveien 19, 5000 Bergen, Norway 57
Reidar Myklebust, Helge Dalen and Thv.Selmer Saetersdal sections by air-drying. In this connection it should be kept in mind, that airdrying is associated with a considerable shrinkage of soft tissues (Boyde & Wood, 1969; Porter et al., 1972). However, this technical problem was overcome by employing the cryofracturing technique (Haggis, 1970; Lim, 1971). The fractured tissues were dehydrated by freeze-drying. Even though by this method a life-like spatial distribution of internal cellular structures is retained, minor fractures due to ice-crystallization can often be a problem (Boyde & Wood, 1969; Porter et al., 1972). The cryofracture technique developed by Tanaka (1972) utilized frozen blocks of freshly epoxy-embedded material. After the resin had been removed from the fractured tissue by propylene oxide, the pieces were transferred to amyl acetate and critical point dried according to the method of Anderson (1951). Today this dehydration procedure is considered to be superior to air-drying and freezedrying (Boyde & Wood, 1969; Porter et al., 1972). The works of Sybers & Ashraf (1973) and McAllister et al. (1974) are also in favour of Anderson's procedure, as very good preservation of internal cellular structures was obtained after critical point drying of deparaffinized thick sections and of cryofractured tissue. The purpose of the present investigation was to conduct a comparative study in the TEM and SEM of the internal structures of the sheep myocardial cell. The ultrastructure of this tissue has previously been examined in the TEM by Simpson & Oertalis (1962), but these authors did not deal with the cell three-dimensional architecture. It should also be pointed out that the preservation of the cell fine structure in their work did not appear satisfactory. MATERIAL AND METHODS
Tissue preparation for TEA4 Freshly dissected pieces of sheep ventricular myocardium were fixed under ice-cold conditions for 1.5 h in Karnowsky's fixative (Karnowsky, 1965) followed by 1 h in 29b OsO,. Both fixatives were made up in 0.1 M cacodylate buffer at p H 7.2. The tissues were stained en bloc for 1 h in 2% aqueous uranyl acetate. After dehydration at room temperature in increasing concentrations of ethanol, the tissues were embedded in Epon 812 (Luft, 1961) and polymerized for 24 h at 40"C, followed by 48 h at 60°C. The cured blocks were cut with glass knives on a Reichert ultramicrotome. Thick sections (1 pm) of the Epon-embedded material were stained with toluidine blue (Trump et al., 1961) for examination in the light microscope, while the ultrathin sections were stained with lead citrate (Reyholds, 1963) for examination in a Philips 300 electron microscope, operated at 80 kV.
Tissue preparations for SEA4 The myocardial tissue selected for SEM studies, was fixed in the same manner as described above. However, after dehydration in ethanol the material was transferred to toluene and embedded in paraffin. Thick sections (10 pm) were cut on a microtome and mounted on glass cover-slips. The first section in each series was stained according to standard histological methods in haematoxylin and eosin for examination in the light microscope. The rest of the sections were deparafinized in toluene and transferred through a series of absolute ethanol baths to amyl acetate. The critical point drying was carried out in a Polaron apparatus according to the method described by Anderson (1951). At this stage some specimens were directly embedded in Epon and sectioned for the transmission electron microscope. Prior to the examination in a Cambridge Stereoscan S4,
58
TEM and SEM of heart muscle ceIIs operated at 10-20 kV, the specimens were coated with a thin conductive film of gold in an Edwards vacuum coater. RESULTS
The contractile material of the sheep myocardial cell is arranged in separated myofibrils running parallel to each other (Fig. 1). The TEM micrograph (Fig. 5) reveals the striated nature of the fibrils. Sarcomeres of different fibrils are almost exactly in register. The myofibrils are separated by rows of mitochondria (Figs. 1, 2 and 5). It is apparent, both from SEM and T E M micrographs (Figs. 2 and 5),
Fig. 1. A SEM micrograph showing parallel myofibrils separated by rows of mitochondria. Some of the fibrils have been torn up by the knife. Sheep myocardial cell. x 4560. Fig. 2. The same as Fig. 1 at higher magnification. MF = myofibril. M = mitochondrion. Arrow = T-tubule. x 11,360.
59
Reidar Myklebust, Helge Dalen and Thv. Selmer Saetersdal that the distribution of mitochondria is closely related to the sarcomere pattern of the myofibrils. T h e mitochondria are consistently found between two adjacent Z-line levels, which seems to be the case whether the sarcomeres are contracted or relaxed. This indicates that the mitochondria change their form during the contraction-relaxation cycle.
Fig. 3. (a) SEM micrograph of circumferentially arranged T-tubules (arrows). x2280. (b) The same at higher magnification. The tubules (arrows) are covered by sarcolemma except in the lower right of the micrograph. Note the connections of adjacent tubules (at the middle of micrograph). x 11,360. Fig. 4. A SEM micrograph showing the course of the T-tubules (arrows) into the cell. Note how closely they invest the myofibrils (MF). x 11,360.
60
TEA4 and SEM of heart muscle cells The transverse tubules (T-tubules) are prominent structures in this kind of myocardial cell, showing a regular pattern related to the pattern of sarcomeres and mitochondria. Fig. 4 shows the spatial relationship between the myofibrils and the T-tubules. The T-tubules invest the fibrils very closely, passing from one myofibril to the next in a predominantly transverse direction. The TEM-micrograph shows that they are situated at the Z-band levels of sarcomere (Fig. 5).
Fig. 5. A TEM micrograph showing the structure and position of the coupling (C). MF = myofibril. Arrows = T-tubules. SR = sarcoplasmic reticulum. x 21,600. Fig. 6.A SEM micrograph of the coupling (circles). Note the dilatation of the junctional SR. MF = myofibril. Arrows = T-tubules. x 11,360.
61
Reidar Myklebust, Helge Dalen and Thv. Selmer Saetersdal Occasionally, short longitudinal branches may be seen connecting the T-tubules at two adjacent Z-line levels. Where the cytoplasmic side of the sarcolemma was exposed, a special T-tubule arrangement was observed. The tubules could be seen running circumferentially around the muscle fibres, just beneath the sarcolemma (Fig. 3a and b). The sarcoplasmic reticulum (SR) is rather poorly developed, forming a loose and irregular network around the myofibrils. The SR is somewhat expanded at the H-bands. From here it sends branches to the Z-line levels on both sides of the H-bands, where it makes contacts (couplings) with the T-tubules (Figs. 5 and 6). Figure 6 shows triadic junctions consisting of one T-tubule and of two components of the SR. At the junctional area, the SR forms knoblike dilatations (Fig. 6). These dilatations probably correspond to the junctional SR described by Sommer & Johnson (1969). Figure 5 shows that the junctional part of the SR is filled with electron dense material, this in contrast to the nonjunctional part of the SR. This electron dense material is called junctional granules by Sommer & Johnson (1969), and it may represent calcium binding sites (Philpott & Goldstein, 1967). Figure 7 shows a T E M micrograph of SEM prepared tissue. No damage of intracellular structures due to SEM preparation can be observed.
Fig. 7. A TEM micrograph of SEM prepared tissue. The different structures have retained a life-like pattern and no damage due to the SEM preparation seems to have occurred. Arrows = T-tubules, M = mitochondrion, SR = sarcoplasmic reticulum. x 30,150.
62
TEM and SEM of heart muscle cells DISCUSSION
It is evident from the present study that the internal cellular structures have retained a life-like pattern after critical point drying. This is a great improvement compared with the air-drying of sectioned material which always leads to a considerable shrinkage of the soft material (Boyde & Wood, 1969). Also, freezedrying is superior to air-drying, as the former method minimizes the surface tension (Arenberg et al., 1970). Accordingly, freeze-fractured material reveals a good spatial preservation of the internal cellular structures. However, minor disruptions of the tissue due to ice-crystallization are common artefacts associated with this type of dehydration (Boyde & Wood, 1969). In his studies of mouse mammary gland, Nemanic (1972) embedded the tissue in agar, cut it on a SmithFarquar tissue sectioner and applied critical point drying to minimize dehydration artefacts. This technique revealed a very good preservation of internal cellular structures. However, by using paraffin sections it is possible to investigate corresponding areas in succeeding planes by both the light microscope and SEM. Besides, sectioned material reveals the cell architecture very clearly. The good preservation of very fine cell structures after the critical point drying, makes this method very useful in getting a more spatial and precise picture of the cell organization. Intracellular structures of muscle cells have previously been studied in sectioned and air-dried tissue of normal (Buss et al., 1971; Pachter et al., 1973; Poh et al., 1971) pathological material (Geissinger, 1971) and of cryofractured and freezedried muscles (Haggis, 1970). The information these works give about the cell organization, are limited due to the preparation techniques. Some distortions seem to have occurred, and the preservation of fine structures like myofibrils, mitochondria, T-tubules and sarcoplasmic reticulum is not satisfactory. The fine and fragile tubules of the sarcoplasmic reticulum are especially difficult to preserve. The tubules easily collapse, making them difficult to distinguish from the underlying myofibrils. In critical point dried material the tubules retain their tubular structure and the network of these tubules is therefore more easily followed (Sybers & Ashraf, 1973). An important consequence of this is the preservation of the couplings. The size and form of the junctional sarcoplasmic reticulum and the frequency of the couplings are more easily told. The interesting observation of the circumferentially arranged T-tubules just beneath the sarcolemma are in good agreement with findings of McAllister et al. (1974). Although T E M micrographs revealed the ultrastructure of intracellular structures very clearly, serial sectioning is necessary in order to reveal their spatial orientation. The direct spatial observation of sectioned material made possible by the SEM, has to some extent made the serial sectioning unnecessary. The difficulty has been artefacts caused by unsatisfactory preparation techniques. However, comparative studies of SEM micrographs of critical point dried material with T E M micrographs show very good correspondence. I n this connection it should be mentioned that the T E M micrographs were taken both from conventionally prepared tissue and from tissue originally prepared for SEM and subsequently embedded in Epon and sectioned for TEM. This makes it safe to form three-dimensional pictures of intracellular structures from SEM micrographs. Moreover, the high resolution in the T E M micrographs, combined with the three-dimensional picture from the SEM micrographs, make up a very complete picture of the cell organization. ACKNOWLEDGMENTS
The authors wish to express their thanks to Professor Bo Thorell for his support
63
Reidar Myklebust, Helge Dalen and Thv.Selmer Saetersdal and interest in the present investigation. The skilful1 technical assistance by Mrs Maud Edenholm and Miss Ingeborg Kranz is gratefully acknowledged. This work was supported by grants from the Norwegian Research Council for Science and the Humanities and from the Swedish Medical Research Council (B67- 12x-215601).
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