- Email: [email protected]
Preparation of biological cryosections for analytical electron microscopy
Ultramicroscopy North-Holland
45
10 (1982) 45-54 Publishing Company
PREPARATION MICROSCOPY
OF BIOLOGICAL
CRYOSECTIONS
FOR ANALYTICAL
ELECTRON
Karl ZIEROLD Max-Planck-Institut Received
1 March
Jiir Systemphysrologie,
Rheinlanddomm
201. D-4600 Dortmund
1. Fed. Rep. OJ Germ~n.r
1982
A cooling chain for studies of ultrastructure and elemental composition of cryofixed biological cells and tissues is described. The technique is demonstrated for yeast cells. The preparation steps such as cryofixation, cryosectioning. transfer into the electron microscope as well as imaging and X-ray microanalysis in the STEM are discussed with respect to the present possibilities and problems. Whereas freeze-dried cryosections can be studied routinely, imaging of the ultrastructure and measurement of the elemental distribution in frozen-hydrated sections turn out to be limited.
1. Introduction The purpose of this contribution is to demonstrate the special interest of cell biologists and physiologists in low temperature electron microscopy and related cryopreparation techniques, and to show the state of the art as well as actual problems from this particular point of view. Classical electron microscopy of biological sections is based on chemical fixation, dehydration, staining and embedding of the specimen. For resteps view see, e.g., ref. [l]. These preparation more or less risk denaturation of biological molecules [2], deformation of tissue compartments [3], and the loss or redistribution of diffusible substances, in particular of water and ions [4,5]. The reason for the use of cryotechniques is to avoid these disadvantages and to study ultrastructure and composition of the biological object in the most defined physiological state possible [6]. This concept is not yet completely realized, but there are some encouraging results and developments in cryotechniques and analytical microscopy which will be described in the following. First the cooling chain technique as constructed at the Max-Planck-Institut in Dortmund [7,8] is presented with results obtained on yeast cells, and then the main cryopreparation steps - cryofixa0304-3991/82/0000-0000/$02.75
0 1982 North-Holland
tion, cryosectioning, transfer of the specimen, electron microscopy and elemental analysis - are discussed in detail.
2. Cryopreparation microscopy
and low temperature
electron
2.1. The cooling chain technique The preparation starts with mounting the biological sample on a gold-coated Balzers freeze-etch specimen support. A tissue block of about 1 mm in diameter or a droplet of cell suspension of the same size are simply placed on the specimen support and glued together by quench freezing. Tissue culture cells grown on these supports can be cryofixed as well. The preparation steps are illustrated in fig. 1: Cryofixation is preferably done by freezing with the propane jet method [9]. Liquid propane cooled to about 90 K is shot quickly from both sides onto the specimen support holding the specimen (fig. la). For cryosectioning a Reichert FC4 Ultracut cryoultramicrotome is used [lo]. 100 nm thick sections are cut dry by means of a glass knife at a temperature below 173 K. They are picked up by an eyelash probe [ 1l] and moved on a pioloform-
46
K. Zierold / Preparatron
of brological crvosections for ana&rccrlEM
coated [ 121 and carbon-evaporated electron microscopical grid (fig. lb). After inserting it into the transfer specimen holder the section is sandwiched T=300K
cl
V’T:gl,K’Y
1<173K
F, a scanning transmission electron microscope (STEM) with a field emission gun, operated at 100 kV accelerating voltage. The usually low contrast of unstained cryosections can be greatly enhanced by combination of the brightfield signal from the central detector and the darkfield signal obtained by the annular detector (fig. Id). For X-ray microanalysis an energy dispersive SiLi detector (USC nuclear semiconductor) and a multichannel analyzer (Link Systems) are used. Sections are studied in the frozen-hydrated and freeze-dried state. Freeze-drying of the section is performed in the transfer chamber connected to the microscope, by loosening the cold contact between the liquid nitrogen Dewar and the transfer specimen holder. 2.2. Results obtuined from freeze-dried cryosections
Fig. 1. Schematic drawing of the cooling chain technique. (a) Cryofixation by the propane jet method. Liquid propane is shot onto the specimen support and the biological sample. (b) Cutting of ultrathin cryosections by means of a glass knife. Sections are moved onto the electron microscopical grid by means of an eye-lash probe. (c) Transfer of the frozen-hydrated sections in a transfer chamber under cold nitrogen atmosphere. (d) Imaging and X-ray microanalysis of the cryosection in the cold stage of a STEM. Imaging is improved by combining the brightfield signal from the central detector with the darkfield signal from the annular detector.
by a second grid and fixed together with a snap ring. The transfer specimen holder is then taken into a transfer chamber which can be connected to the cryoultramicrotome chamber. Sectioning, mounting the section and transferring to the electron microscope is performed in cold nitrogen gas atmosphere below 173 K (fig. lc). The transfer chamber is connected to the electron microscope, and after evacuating the transfer chamber, the specimen holder can be locked into the cold stage of the microscope. Grid temperature in the cold stage is 165 K. The microscope is a Siemens Elmiskop ST 100
frozen-hydruted
md
For the sake of simplicity and better comparability the presented results are confined to yeast cells saccharomyces cerevisiae. Fig. 2 shows a frozen-hydrated section of a yeast cell suspension immediately after transfer into the microscope. Except for the cell walls, all intracellular structures are hidden in the ice. Sectioning traces from the glass knife and few ice crystals deposited during the transfer can be recognized.
Fig. 2. Frozen-hydrated c=cell, i=contaminated knife.
section of a yeast cell suspension; ice crystals, k= traces of the glass
K. Zierold / Preparation
Fig. 3. Frozen-hydrated beginning freeze-drying;
section of a yeast cell suspension c = cell, o = organelles.
of biological cryosections for analytical EM
after
The cells appear compressed perpendicular to the sectioning direction. Careful freeze-drying for some minutes removes the superficial ice crystals and the sectioning tracks. Some intracellular structures become slightly visible (fig. 3). Further freeze-drying causes sublimation of the extracellular ice, whereas the intracellular matrix remains still frozen (fig. 4). After complete freeze-drying of the section, intracellular structures - for example nucleus and mitochondria - appear (fig. 5).
Fig. 4. Cryosection dried extracellular
of frozen-hydrated space.
47
yeast cells and freeze-
Fig. 5. Cryosection of freeze-dried yeast cells; c = cytoplasm, g= granules. m = mitochondria, n = nucleus.
wB_
m-m-1
Fig. 6. X-ray spectrum
Fig. 7. X-ray spectrum freeze-dried yeast cell.
of a frozen-hydrated
of the cytoplasm
yeast cell.
(c in fig. 5) in a
K. Zterold / Preparution
48
Fig. 8. X-ray spectrum yeast cell.
of a granule
of biological cryosections for ma!vtical
(g in fig. 5) in a freeze-dried
The X-ray spectrum of a frozen-hydrated cell shows relatively small peaks above the background (fig. 6). The peak-to-background ratio is greatly enhanced after freeze-drying, as can be seen from figs. 7 and 8 which show X-ray spectra from the cytoplasm and a black granule in a yeast cell, respectively. Fig. 9 shows the extracellular spectrum as control. The X-ray spectra prove the inhomogeneous element distribution in the cells, in particular calcium accumulating structures are identified. The copper peaks in the X-ray spectra are caused by the grid, the origin of silicon is unknown. The importance of good cryofixation for imaging the ultrastructure is demonstrated by figs. 10 and 11. Yeast cell specimens plunged into liquid propane by hand (which means that the freezing rate is relatively low) show a rather smooth surface (fig. 10). Quick freezing as done by the propane jet
F ILII--
EM
Fig. IO. Freeze-dried cryosections of yeast cells. cryofixed plunging the sample by hand into liquid propane. Note smooth surface (arrows).
by the
method preserves the glycocalyx layer on the cell surface (fig. 11). Frozen-hydrated cryosections are very sensitive to radiation damage as illustrated by fig. 12. Scanning a small area of the section, which means a high electron dose, leads to an enormous mass loss, particularly of water. Freeze-drying stabilizes cryosections, which means that mass loss is re-
.._ _(uIIIII_
Fig. 9. X-ray spectrum of the extracellular dried cryosection of yeast cells.
space of a freeze-
Fig. 11. Freeze-dried cryosection of yeast cells cryofixed by the propane-jet method. Note the glycocalyx layer on the cell surface (arrows).
K. Zierold / Preparation
of biological cryoseciions for analytical EM
Fig. 12. Frozen-hydrated section of yeast cells after scanning a smaller area with high electron irradiation. Large mass loss, particularly of water, results in bright rectangular area; c= yeast cell.
duced considerably also at high electron doses. After long irradiation contamination may occur, but this effect depends to a great extent on the quality of the vacuum.
3. Discussion 3.1. Cryofixation Biological material consists of about 80% water. The idea of cryofixation is to transform the wet biological specimen into a solid state which can be sectioned and which is resistant to vacuum. Simply freezing by lowering the temperature causes segregation phenomena of the water with great distortions of the ultrastructure and the composition of the specimen. Therefore cryofixation intends to freeze the specimen in such a way that ice crystals remain as small as possible. In practice this means cooling very quickly to a temperature of at least 190 K. Below that temperature intracellular ice crystal growth does not occur [ 131. In principle cryoprotectants - for example glycerol, some PVP (polysugars, DMSO (dimethylsulfoxide), vinylpyrrolidone) or HES (hydroxyethylstarch) can reduce ice crystal damage, but they might affect the ultrastructure and the element distribu-
49
tion of the native state [14] and should therefore be avoided. Freezing without ice crystal formation, which means that water is fixed in a vitreous state, is only achieved in some particular cases, as reported recently [ 151. Vitrification of water needs a freezing rate above lo4 K/s [16]. Because of the low thermal conductivity of water and ice of about 2-4 W/m . K [ 171, vitrification can only be realized for very small objects, for example single cells or cell organelles. As a consequence the best cryofixation results are obtained from single cells, as demonstrated by the spray-freeze technique [ 181. However, not all biological material can be sprayed into a cooling medium without severe damage, and therefore different freezing techniques have been developed in order to quenchfreeze a great variety of biological objects. Much experimental work has been done to find out the most adequate cooling media. Today liquid propane cooled by liquid nitrogen is widely accepted to be the quenching liquid with the fastest freezing rate [19-211. Since dipping the specimen by hand into the quenching liquid is relatively slow, it is recommended either to shoot the specimen into the quenching fluid [22] or to spray the liquid propane onto the specimen using the so-called propane jet method, as published by several authors [9,23-251. Cold metal surfaces are successfully used for cryofixation [ 11,26-291 because of their high thermal conductivity. This method needs a very pure and well polished copper or mercury surface without contamination by ice in order to get a good contact with the specimen. As an example, the thermal conductivity of copper is about 480 W/m. K at liquid nitrogen temperature, more than 100 times higher than that of ice. Finally the high pressure freezing method [30] should be mentioned. This method means freezing under a pressure of 2 kbar which reduces the temperature range for ice crystal growth. This results in only minute ice crystals in the specimens [31]. The disadvantage of this method is that a high pressure freezing machine is expensive and not easily available at present. Not considering the particular freezing method, it can be stated that the quality of cryofixation
50
K. Zierold / Preparation
of biological cryosections for anuiytical EM
simply depends on the freezing velocity. The smaller the specimen and the specimen support are, the higher is the freezing velocity. Due to the low thermal conductivity of water only thin objects or tissue layers thinner than about 30 pm can be well preserved without visible ice crystal damage. 3.2. Cryosectioning Sectioning cryofixed specimens below the recrystallization temperature of about 193 K is often considered to be difficult, since frozen biological objects become brittle at lower temperature, and as a consequence many small chips are obtained rather than clear sections without cracks. Therefore many efforts were made to overcome this problem. Smooth sections can be obtained by infusion of sucrose or other sugar solutions into the object [32]. However, this might influence the internal composition of the biological specimen. For the same reason any trough liquid has to be avoided in cryosectioning. Only dry cutting assures the preservation of the native structure and composition of the sample [33,34]. Brittleness of cryofixed biological objects obviously depends on water content and size of the ice crystals. The smaller the ice crystals are the easier is sectioning at a temperature below 190 K. Often a high cutting speed of 30-50 mm/s seems favourable with respect to the prevention of cracking and curling of the section. This could induce melting for a short time which might cause recrystallization and displacement of diffusible elements. Therefore, most authors work at a cutting speed below 2 mm/s [35-371. Dry cut sections are mostly electrically charged, which impedes picking up and placing them onto the electron microscopic grid. Sometimes a commercially available discharge pistol helps to overcome this problem [38]. Flattening and picking up cryosections by means of a vacuum pipette, as reported some years ago [39], is not known to be reproduced. Cryosectioning presently works better than it is understood physically. Phenomena such as brittleness of frozen tissue as well as the questions of thawing during cutting and electrical charging are unclear and await a concise theoretical explanation.
3.3. Transfer to the eLectron microscope drying
andjieeze-
Besides the cooling chain described in this paper, in the literature there are many attempts and technical descriptions of transfer devices and cold stages for studies of frozen-hydrated material in an electron microscope [40-481. All presented versions work at a transfer temperature below 145 K which is sufficient for the preservation of the frozen-hydrated state of biological cryosections. In the past the lack of appropriate specimen preparation techniques and the disappointing contrast obtained in the electron microscope have prevented the establishment of this method as a routine technique. Up to now, frozen-hydrated sections have been hard to study, as demonstrated by figs. 2-4. Nonetheless cryotransfer systems also become important for studies of freeze-dried cryosections. Up to now most cryosections were freeze-dried and then brought into an electron microscope through the air. Unfortunately, cryosections are very sensitive to the moisture of the air [36,49,50]. Some authors therefore have used closed transfer devices at room temperature to avoid the contact of the freeze-dried section with the air atmosphere [5 1.521. This is an acceptable technique insofar as an alteration of ultrastructural details, molecular architecture or distribution of elements caused by warming up are unimportant or can be excluded. The cooling chain technique including freeze-drying of the section keeps the temperature below the intracelluar recrystallization point of about 193 K. In comparison with the warm transfer this would at least reduce the mobility of diffusible substances. As demonstrated in figs. 4 and 5 extracellular freeze-drying starts before the intracellular ice sublimes. This result can be explained by the assumption that the intracellular bonding of water molecules is stronger than in ordinary extracellular ice. The freeze-drying method used in experiments described in this paper leads to shrinkage of cells as concluded from comparison between cell diameters in the frozen-hydrated and freeze-dried state. It is not known if this artefact can be avoided by a particular drying method.
K. Zierold / Preparation of biological cryosections for ana(ytrcal EM
3.4. STEM
and X-ray microanalysis
No contrast is found in frozen-hydrated sections of cryofixed cells by scanning transmission electron microscopy (STEM), either in brightfield or in darkfield mode. This observation is presumably caused by the fact that the electron scattering coefficient of ice lies very close to that of biological structures. After sublimation of the ice by freeze-drying the different electron scattering between remaining biological molecules and vacuum yields the electron optical contrast. For practical purposes an image preferably obtained by division of brightfield over darkfield signal is recorded photographically. Improvement of the contrast in frozen-hydrated sections, e.g. by more precise discrimination between elastically and inelastically scattered electrons, remains a challenge for future work in electron detection and signal processing. The great mass loss by electron irradiation as observed in frozen-hydrated sections (fig. 12) is presumably caused by a chemical reaction of electrons with water molecules [53]. In contrast to the ice, freeze-dried sections are relatively stable and only exhibit a small mass loss at low temperature. Very high electron doses may result in a contamination layer built up in the exposed area. Usually this phenomenon is prevented by a cold trap with a temperature lower than that of the section situated near the grid. The cold stage of the cooling chain presented in this paper achieves the relatively high grid temperature of 165 K which has to be improved. A reduction of radiation damage can be expected by further lowering the specimen temperature [54,55]. The low peak-to-background ratios in X-ray spectra of frozen-hydrated sections which are much enhanced after freeze-drying (see figs. 6-9) are consistent with similar reports by other authors [38,56]. Perhaps the element concentration in frozen-hydrated cell compartments can be computed from those spectra with adequate calibration methods [57,58]. So far X-ray microanalysis is not a routine method to measure the element content in liquid compartments of cells and tissues, e.g. in vessels, extracellular spaces or vacuoles. Quantitative X-ray microanalysis of biological cryosections is developed only for freeze-dried material [59-611.
51
At present, only preliminary information is available about possibilities of electron energy loss spectroscopy in studying the distribution of particular substances in cells and tissues [62-651. This technique seems quite promising in order to extend studies of the distribution of biologically relevant elements as obtained by X-ray microanalysis to measurements of the molecular composition. However, even the best analytical techniques applied to biological cells or tissues cannot substitute for an adequate cryopreparation to preserve their native state.
4. Conclusion The described cooling chain technique combined with a scanning transmission electron microscope proves that ultrastructure and elemental distribution can be studied in cryofixed freeze-dried cryosections of biological cells and tissues. Whereas cryosectioning and transfer techniques can be performed routinely, cryofixation and imaging of frozen-hydrated sections turn out to be the present limitations. In particular, freezing techniques must be developed which allow a good cryofixation of tissues in a defined physiological state. Imaging of frozen-hydrated sections, which is not yet possible, would extend electron beam analysis to studies of the structure of extracellular compartments, vessels and vacuoles. In spite of these limitations the cooling chain technique is one of the few available methods to study the elemental composition of cells in defined functional states at an ultrastructural level. It can help to elucidate processes of storage and transport of substances in cellular biological systems.
Acknowledgement I want to thank Miss S. Dongard technical assistance.
for her careful
References [l] A.M. Glauert, Fixation, Dehydration Biological Specimens (North-Holland,
and Embedding of Amsterdam, 1975).
52
K. Zierold / Preparation of biologrcal cryosectwns for ancr!vtzcalEM
[2] E. Kellenberger and J. Kistler, The Physics of Specimen Preparation, in: Unconventional Electron Microscopy for Molecular Structure Determination, Eds. W. Hoppe and R. Mason (Vieweg, Braunschweig, 1979) p, 49. [3] B. Cragg, Tissue and Cell 12 (1980) 63. 14) A.J. Morgan, T.W. Davies and D.A. Erasmus. Specimen Preparation, in: Electron Probe Microanalysis in Biology, Ed. D.A. Erasmus (Chapman and Hall, London, 1978) p. 94. [5] K. Zierold, D. Schafer and J. Gullasch, Microsc. Acta, Suppl. 2 (1978) 92. [6] D. Schafer, K. Zierold, D.W. Lubbers. Eds.. Arzneim.Forsch./Drug Res. 29 (11) (1979) 1809. [7] K. Zierold. R. Konig, K.-H. Olech. D. Schafer, D.W. Lubbers, K.-H. Miiller and H. Winter. Ultramicroscopy 6 (1981) 181. [8] K. Zierold, J. Microscopy 125 (1982) 149. [9] M. Miiller, N. Meister and H. Moor, Mikroskopie 36 (1980) 129. [IO] H. Sitte, K. Neumann, H. Hlssig, H. Kleber and G. Kappl, in: Electron Microscopy 1980, Eds. P. Brederoo and W. de Priester (7th European Congr. on Electron Microscopy Foundation, Leiden, 1980) Vol. I, p. 540. [I l] A.K. Christensen. J. Cell Biol. 51 (1971) 772. [ 121 W. Stockem, Mikroskopie 26 (1970) 185. [13] H. Moor, Cryotechnology for the Structural Analysis of Biological Material, in: Freeze-Etching, Techniques and Applications, Eds. E.L. Benedetti and P. Favard (Societi Franqaise de Microscopic Electronique, Paris, 1973) p. Il. [ 141 J. Farrant, CA. Walter, H. Lee, G.J. Morris and K.J. Clarke, Structural and Functional Aspects of Biological Freezing Techniques, in: Low Temperature Biological Microscopy and Microanalysis, Eds. P. Echlin, B. Ralph and E.R. Weibel (Blackwell, Oxford, 1978) p. 17. (151 J. Dubochet and A.W. McDowall, J. Microscopy 124 (1981) RP 3. [ 161 H. Moor, Z. Zellforsch. 62 (1964) 546. [ 171 P. Hobbs, Ice Physics (Clarendon, Oxford, 1974). [ 18) L. Bachmann and W.W. Schmitt-Fumian, Spray-Freezing and Freeze-Etching, in: Freeze-Etching, Techniques and Applications, Eds. E.L. Benedetti and P. Favard (Societe Franqaise de Microscopic Electronique, Paris, 1973) p. 73. [19] J.L. Stephenson, J. Biophys. Biochem. Cytol. 2 (1956) 45. [20] M.J. Costello and J.M. Corless, J. Microscopy 121 (1978) 17. [21] K. Zierold, Microsc. Acta 83 (1980) 25. [22] A.W. Robards and N.J. Severs, Cryo-Letters 2 (1981) 135. [23] N.L. Burstein and D.M. Maurice, Micron 9 (1978) 191. [24] P. Pscheid, C. Schudt and H. Plattner, J. Microscopy 121 (1981) 149. [25] T. Espevik and A. Elgsaeter, J. Microscopy 123 (1981) 105. [26] A. van Harreveld and J. Crowell, Anat. Record 149 (1964) 381. (271 G.P. Dempsey and S. Bullivant, J. Microscopy 106 (1976) 251. [28] J.E. Heuser, T.S. Reese, M.J. Dennis, Y. Jan, L. Jan and L. Evans, J. Cell Biol. 81 (1979) 275.
[29] K.G. Schwabe and L. Terracio, Cryobiology 17 (1980) 571. [30] U. Riehle and M. Hoechli, The Theory and Technique of High Pressure Freezing, in: Freeze-Etching, Techniques and Applications. Eds. E.L. Benedetti and P. Favard (Societe Francaise de Microscopic Electronique, Paris, 1973) p. 31. [31] K.-V. Wolf, W. Stockem and K.-E. Wohlfarth-Bottermann, Cell Tissue Res. 217 (1981) 479. [32] K.T. Tokuyasu. J. Cell Biol. 57 (1973) 551. [33] M. Sjostrom and L.E. Thornell. J. Microscopy 103 (1975) 101. [34] T.C. Appleton, The Contribution of Cryoultramicrotomy to X-Ray Microanalysis in Biology, in: Electron Probe Microanalysis in Biology, Ed. D.A. Erasmus (Chapman and Hall, London, 1978) p. 148. 135) L. Seveus, J. Microscopy 112 (1978) 269. [36] P.M. Frederik and W.M. Busing, J. Microscopy 121 (1981) 191. [37] A.J. Saubermann, P. Echlin, P.D. Peters and R. Beeuwkes III, J. Cell Biol. 88 (1981) 257. [38] B.L. Gupta, T.A. Hall and R.B. Moreton, Electron Probe X-Ray Microanalysis, in: Transport of Ions and Water in Animals, Eds. B.L. Gupta, R.B. Moreton. J.L. Oschman and B.J. Wall (Academic Press, London, 1977) p. 83. [39] T.C. Appleton, J. Microscopy 100 (1974) 49. [40] H.G. Heide and S. Grund, J. Ultrastruct. Res. 48 (1974) 259. [41] Th.E. Hutchinson, M. Bacaner, J. Broadhurst and J. Lilley, Rev. Sci. Instr. 45 (1974) 252. [42] R.B. Moreton, P. Echlin, B.L. Gupta, T.A. Hall and T. Weis-Fogh, Nature 247 (1974) 113. [43] U. Valdre and R.W. Horne, J. Microscopy 103 (1975) 305. [44] T.E. Hutchinson, D.E. Johnson and A.P. MacKenzie, UItramicroscopy 3 ( 1978) 3 15. [45] Y. Talmon, H.T. Davis, L.E. Striven and E.L. Thomas, Rev. Sci. Instr. 50 (1979) 698. [46] R. Freeman, F. Booy and K. Leonard, in: Electron Microscopy 1980, Eds. P. Brederoo an W. de Priester (7th European Congr. on Electron Microscopy Foundation, Leiden, 1980) Vol. 2, p. 650. [47] S. Lichtenegger and W.M.A. Hax, in: Electron Microacopy 1980, Eds. P. Brederoo and W. de Priester (7th European Congr. on Electron Microscopy Foundation, Leiden, 1980) Vol. 2, p. 652. [48] H.G. Heide, Ultramicroscopy 6 (1981) 115. [49] G.M. Roomans and L.A. Seveus, J. Cell Sci. 21 (1976) 119. [50] T. Barnard and L. Se&us, J. Microscopy 112 (1978) 281. 1511 T.L.B. Spriggs and D. Wynne-Evans, J. Microscopy 107 (1976) 35. [52] S. Hodson and L. Williams, J. Cell Sci. 20 (1976) 687. [53] Y. Talmon, H.T. Davis, L.E. Striven and E.L. Thomas, J. Microscopy 117 (1979) 321. [54] R.M. Glaeser and K.A. Taylor, J. Microscopy 112 (1978) 127. (551 V.E. Cosslett, Radiation Damage: Experimental Work. in: Unconventional Electron Microscopy for Molecular Struc-
K. Zierold / Preparation
[56] [57] [58] [59] [60] [61]
of brologrcal cryosections for analytical EM
ture Determination, Eds. W. Hoppe and R. Mason (Vieweg, Braunschweig, 1979) p. 8 1. A. Ross, A.T. Sumner and A.R. Ross, J. Microscopy 121 (1981) 261. A.J. Saubermann, R. Beeuwkes III an P.D. Peters, J. Cell Biol. 88 (1981) 268. B.L. Gupta and T.A. Hall, Tissue and Cell 13 (1981) 623. T.A. Hall, J. Microscopy 117 (1979) 145. H. Shuman, A.V. Somlyo and A.P. Somlyo, Ultramicroscopy 1 (1976) 317. A. DGrge, R. Rick, K. Gehring and K. Thurau, Pfliigers Arch. 373 (1978) 85.
53
[62] M. Isaacson and D. Johnson, Ultramicroscopy 1 (1975) 33. [63] C. Colliex and P. Trebbia, Quantitation and Detection Limits in Electron Energy Loss Spectroscopy (EELS) of Thin Biological Sections, in: Microbeam Analysis in Biology, Eds. C.P. Lechene and R.R. Warner (Academic Press, New York, 1979) p. 65. [64] F.P. Ottensmeyer and J.W. Andrew, J. Ultrastruct. Res. 72 (1980) 336. [65] A.P. Somlyo and H. Shuman, Ultramicroscopy 8 (1982) 219.
45
10 (1982) 45-54 Publishing Company
PREPARATION MICROSCOPY
OF BIOLOGICAL
CRYOSECTIONS
FOR ANALYTICAL
ELECTRON
Karl ZIEROLD Max-Planck-Institut Received
1 March
Jiir Systemphysrologie,
Rheinlanddomm
201. D-4600 Dortmund
1. Fed. Rep. OJ Germ~n.r
1982
A cooling chain for studies of ultrastructure and elemental composition of cryofixed biological cells and tissues is described. The technique is demonstrated for yeast cells. The preparation steps such as cryofixation, cryosectioning. transfer into the electron microscope as well as imaging and X-ray microanalysis in the STEM are discussed with respect to the present possibilities and problems. Whereas freeze-dried cryosections can be studied routinely, imaging of the ultrastructure and measurement of the elemental distribution in frozen-hydrated sections turn out to be limited.
1. Introduction The purpose of this contribution is to demonstrate the special interest of cell biologists and physiologists in low temperature electron microscopy and related cryopreparation techniques, and to show the state of the art as well as actual problems from this particular point of view. Classical electron microscopy of biological sections is based on chemical fixation, dehydration, staining and embedding of the specimen. For resteps view see, e.g., ref. [l]. These preparation more or less risk denaturation of biological molecules [2], deformation of tissue compartments [3], and the loss or redistribution of diffusible substances, in particular of water and ions [4,5]. The reason for the use of cryotechniques is to avoid these disadvantages and to study ultrastructure and composition of the biological object in the most defined physiological state possible [6]. This concept is not yet completely realized, but there are some encouraging results and developments in cryotechniques and analytical microscopy which will be described in the following. First the cooling chain technique as constructed at the Max-Planck-Institut in Dortmund [7,8] is presented with results obtained on yeast cells, and then the main cryopreparation steps - cryofixa0304-3991/82/0000-0000/$02.75
0 1982 North-Holland
tion, cryosectioning, transfer of the specimen, electron microscopy and elemental analysis - are discussed in detail.
2. Cryopreparation microscopy
and low temperature
electron
2.1. The cooling chain technique The preparation starts with mounting the biological sample on a gold-coated Balzers freeze-etch specimen support. A tissue block of about 1 mm in diameter or a droplet of cell suspension of the same size are simply placed on the specimen support and glued together by quench freezing. Tissue culture cells grown on these supports can be cryofixed as well. The preparation steps are illustrated in fig. 1: Cryofixation is preferably done by freezing with the propane jet method [9]. Liquid propane cooled to about 90 K is shot quickly from both sides onto the specimen support holding the specimen (fig. la). For cryosectioning a Reichert FC4 Ultracut cryoultramicrotome is used [lo]. 100 nm thick sections are cut dry by means of a glass knife at a temperature below 173 K. They are picked up by an eyelash probe [ 1l] and moved on a pioloform-
46
K. Zierold / Preparatron
of brological crvosections for ana&rccrlEM
coated [ 121 and carbon-evaporated electron microscopical grid (fig. lb). After inserting it into the transfer specimen holder the section is sandwiched T=300K
cl
V’T:gl,K’Y
1<173K
F, a scanning transmission electron microscope (STEM) with a field emission gun, operated at 100 kV accelerating voltage. The usually low contrast of unstained cryosections can be greatly enhanced by combination of the brightfield signal from the central detector and the darkfield signal obtained by the annular detector (fig. Id). For X-ray microanalysis an energy dispersive SiLi detector (USC nuclear semiconductor) and a multichannel analyzer (Link Systems) are used. Sections are studied in the frozen-hydrated and freeze-dried state. Freeze-drying of the section is performed in the transfer chamber connected to the microscope, by loosening the cold contact between the liquid nitrogen Dewar and the transfer specimen holder. 2.2. Results obtuined from freeze-dried cryosections
Fig. 1. Schematic drawing of the cooling chain technique. (a) Cryofixation by the propane jet method. Liquid propane is shot onto the specimen support and the biological sample. (b) Cutting of ultrathin cryosections by means of a glass knife. Sections are moved onto the electron microscopical grid by means of an eye-lash probe. (c) Transfer of the frozen-hydrated sections in a transfer chamber under cold nitrogen atmosphere. (d) Imaging and X-ray microanalysis of the cryosection in the cold stage of a STEM. Imaging is improved by combining the brightfield signal from the central detector with the darkfield signal from the annular detector.
by a second grid and fixed together with a snap ring. The transfer specimen holder is then taken into a transfer chamber which can be connected to the cryoultramicrotome chamber. Sectioning, mounting the section and transferring to the electron microscope is performed in cold nitrogen gas atmosphere below 173 K (fig. lc). The transfer chamber is connected to the electron microscope, and after evacuating the transfer chamber, the specimen holder can be locked into the cold stage of the microscope. Grid temperature in the cold stage is 165 K. The microscope is a Siemens Elmiskop ST 100
frozen-hydruted
md
For the sake of simplicity and better comparability the presented results are confined to yeast cells saccharomyces cerevisiae. Fig. 2 shows a frozen-hydrated section of a yeast cell suspension immediately after transfer into the microscope. Except for the cell walls, all intracellular structures are hidden in the ice. Sectioning traces from the glass knife and few ice crystals deposited during the transfer can be recognized.
Fig. 2. Frozen-hydrated c=cell, i=contaminated knife.
section of a yeast cell suspension; ice crystals, k= traces of the glass
K. Zierold / Preparation
Fig. 3. Frozen-hydrated beginning freeze-drying;
section of a yeast cell suspension c = cell, o = organelles.
of biological cryosections for analytical EM
after
The cells appear compressed perpendicular to the sectioning direction. Careful freeze-drying for some minutes removes the superficial ice crystals and the sectioning tracks. Some intracellular structures become slightly visible (fig. 3). Further freeze-drying causes sublimation of the extracellular ice, whereas the intracellular matrix remains still frozen (fig. 4). After complete freeze-drying of the section, intracellular structures - for example nucleus and mitochondria - appear (fig. 5).
Fig. 4. Cryosection dried extracellular
of frozen-hydrated space.
47
yeast cells and freeze-
Fig. 5. Cryosection of freeze-dried yeast cells; c = cytoplasm, g= granules. m = mitochondria, n = nucleus.
wB_
m-m-1
Fig. 6. X-ray spectrum
Fig. 7. X-ray spectrum freeze-dried yeast cell.
of a frozen-hydrated
of the cytoplasm
yeast cell.
(c in fig. 5) in a
K. Zterold / Preparution
48
Fig. 8. X-ray spectrum yeast cell.
of a granule
of biological cryosections for ma!vtical
(g in fig. 5) in a freeze-dried
The X-ray spectrum of a frozen-hydrated cell shows relatively small peaks above the background (fig. 6). The peak-to-background ratio is greatly enhanced after freeze-drying, as can be seen from figs. 7 and 8 which show X-ray spectra from the cytoplasm and a black granule in a yeast cell, respectively. Fig. 9 shows the extracellular spectrum as control. The X-ray spectra prove the inhomogeneous element distribution in the cells, in particular calcium accumulating structures are identified. The copper peaks in the X-ray spectra are caused by the grid, the origin of silicon is unknown. The importance of good cryofixation for imaging the ultrastructure is demonstrated by figs. 10 and 11. Yeast cell specimens plunged into liquid propane by hand (which means that the freezing rate is relatively low) show a rather smooth surface (fig. 10). Quick freezing as done by the propane jet
F ILII--
EM
Fig. IO. Freeze-dried cryosections of yeast cells. cryofixed plunging the sample by hand into liquid propane. Note smooth surface (arrows).
by the
method preserves the glycocalyx layer on the cell surface (fig. 11). Frozen-hydrated cryosections are very sensitive to radiation damage as illustrated by fig. 12. Scanning a small area of the section, which means a high electron dose, leads to an enormous mass loss, particularly of water. Freeze-drying stabilizes cryosections, which means that mass loss is re-
.._ _(uIIIII_
Fig. 9. X-ray spectrum of the extracellular dried cryosection of yeast cells.
space of a freeze-
Fig. 11. Freeze-dried cryosection of yeast cells cryofixed by the propane-jet method. Note the glycocalyx layer on the cell surface (arrows).
K. Zierold / Preparation
of biological cryoseciions for analytical EM
Fig. 12. Frozen-hydrated section of yeast cells after scanning a smaller area with high electron irradiation. Large mass loss, particularly of water, results in bright rectangular area; c= yeast cell.
duced considerably also at high electron doses. After long irradiation contamination may occur, but this effect depends to a great extent on the quality of the vacuum.
3. Discussion 3.1. Cryofixation Biological material consists of about 80% water. The idea of cryofixation is to transform the wet biological specimen into a solid state which can be sectioned and which is resistant to vacuum. Simply freezing by lowering the temperature causes segregation phenomena of the water with great distortions of the ultrastructure and the composition of the specimen. Therefore cryofixation intends to freeze the specimen in such a way that ice crystals remain as small as possible. In practice this means cooling very quickly to a temperature of at least 190 K. Below that temperature intracellular ice crystal growth does not occur [ 131. In principle cryoprotectants - for example glycerol, some PVP (polysugars, DMSO (dimethylsulfoxide), vinylpyrrolidone) or HES (hydroxyethylstarch) can reduce ice crystal damage, but they might affect the ultrastructure and the element distribu-
49
tion of the native state [14] and should therefore be avoided. Freezing without ice crystal formation, which means that water is fixed in a vitreous state, is only achieved in some particular cases, as reported recently [ 151. Vitrification of water needs a freezing rate above lo4 K/s [16]. Because of the low thermal conductivity of water and ice of about 2-4 W/m . K [ 171, vitrification can only be realized for very small objects, for example single cells or cell organelles. As a consequence the best cryofixation results are obtained from single cells, as demonstrated by the spray-freeze technique [ 181. However, not all biological material can be sprayed into a cooling medium without severe damage, and therefore different freezing techniques have been developed in order to quenchfreeze a great variety of biological objects. Much experimental work has been done to find out the most adequate cooling media. Today liquid propane cooled by liquid nitrogen is widely accepted to be the quenching liquid with the fastest freezing rate [19-211. Since dipping the specimen by hand into the quenching liquid is relatively slow, it is recommended either to shoot the specimen into the quenching fluid [22] or to spray the liquid propane onto the specimen using the so-called propane jet method, as published by several authors [9,23-251. Cold metal surfaces are successfully used for cryofixation [ 11,26-291 because of their high thermal conductivity. This method needs a very pure and well polished copper or mercury surface without contamination by ice in order to get a good contact with the specimen. As an example, the thermal conductivity of copper is about 480 W/m. K at liquid nitrogen temperature, more than 100 times higher than that of ice. Finally the high pressure freezing method [30] should be mentioned. This method means freezing under a pressure of 2 kbar which reduces the temperature range for ice crystal growth. This results in only minute ice crystals in the specimens [31]. The disadvantage of this method is that a high pressure freezing machine is expensive and not easily available at present. Not considering the particular freezing method, it can be stated that the quality of cryofixation
50
K. Zierold / Preparation
of biological cryosections for anuiytical EM
simply depends on the freezing velocity. The smaller the specimen and the specimen support are, the higher is the freezing velocity. Due to the low thermal conductivity of water only thin objects or tissue layers thinner than about 30 pm can be well preserved without visible ice crystal damage. 3.2. Cryosectioning Sectioning cryofixed specimens below the recrystallization temperature of about 193 K is often considered to be difficult, since frozen biological objects become brittle at lower temperature, and as a consequence many small chips are obtained rather than clear sections without cracks. Therefore many efforts were made to overcome this problem. Smooth sections can be obtained by infusion of sucrose or other sugar solutions into the object [32]. However, this might influence the internal composition of the biological specimen. For the same reason any trough liquid has to be avoided in cryosectioning. Only dry cutting assures the preservation of the native structure and composition of the sample [33,34]. Brittleness of cryofixed biological objects obviously depends on water content and size of the ice crystals. The smaller the ice crystals are the easier is sectioning at a temperature below 190 K. Often a high cutting speed of 30-50 mm/s seems favourable with respect to the prevention of cracking and curling of the section. This could induce melting for a short time which might cause recrystallization and displacement of diffusible elements. Therefore, most authors work at a cutting speed below 2 mm/s [35-371. Dry cut sections are mostly electrically charged, which impedes picking up and placing them onto the electron microscopic grid. Sometimes a commercially available discharge pistol helps to overcome this problem [38]. Flattening and picking up cryosections by means of a vacuum pipette, as reported some years ago [39], is not known to be reproduced. Cryosectioning presently works better than it is understood physically. Phenomena such as brittleness of frozen tissue as well as the questions of thawing during cutting and electrical charging are unclear and await a concise theoretical explanation.
3.3. Transfer to the eLectron microscope drying
andjieeze-
Besides the cooling chain described in this paper, in the literature there are many attempts and technical descriptions of transfer devices and cold stages for studies of frozen-hydrated material in an electron microscope [40-481. All presented versions work at a transfer temperature below 145 K which is sufficient for the preservation of the frozen-hydrated state of biological cryosections. In the past the lack of appropriate specimen preparation techniques and the disappointing contrast obtained in the electron microscope have prevented the establishment of this method as a routine technique. Up to now, frozen-hydrated sections have been hard to study, as demonstrated by figs. 2-4. Nonetheless cryotransfer systems also become important for studies of freeze-dried cryosections. Up to now most cryosections were freeze-dried and then brought into an electron microscope through the air. Unfortunately, cryosections are very sensitive to the moisture of the air [36,49,50]. Some authors therefore have used closed transfer devices at room temperature to avoid the contact of the freeze-dried section with the air atmosphere [5 1.521. This is an acceptable technique insofar as an alteration of ultrastructural details, molecular architecture or distribution of elements caused by warming up are unimportant or can be excluded. The cooling chain technique including freeze-drying of the section keeps the temperature below the intracelluar recrystallization point of about 193 K. In comparison with the warm transfer this would at least reduce the mobility of diffusible substances. As demonstrated in figs. 4 and 5 extracellular freeze-drying starts before the intracellular ice sublimes. This result can be explained by the assumption that the intracellular bonding of water molecules is stronger than in ordinary extracellular ice. The freeze-drying method used in experiments described in this paper leads to shrinkage of cells as concluded from comparison between cell diameters in the frozen-hydrated and freeze-dried state. It is not known if this artefact can be avoided by a particular drying method.
K. Zierold / Preparation of biological cryosections for ana(ytrcal EM
3.4. STEM
and X-ray microanalysis
No contrast is found in frozen-hydrated sections of cryofixed cells by scanning transmission electron microscopy (STEM), either in brightfield or in darkfield mode. This observation is presumably caused by the fact that the electron scattering coefficient of ice lies very close to that of biological structures. After sublimation of the ice by freeze-drying the different electron scattering between remaining biological molecules and vacuum yields the electron optical contrast. For practical purposes an image preferably obtained by division of brightfield over darkfield signal is recorded photographically. Improvement of the contrast in frozen-hydrated sections, e.g. by more precise discrimination between elastically and inelastically scattered electrons, remains a challenge for future work in electron detection and signal processing. The great mass loss by electron irradiation as observed in frozen-hydrated sections (fig. 12) is presumably caused by a chemical reaction of electrons with water molecules [53]. In contrast to the ice, freeze-dried sections are relatively stable and only exhibit a small mass loss at low temperature. Very high electron doses may result in a contamination layer built up in the exposed area. Usually this phenomenon is prevented by a cold trap with a temperature lower than that of the section situated near the grid. The cold stage of the cooling chain presented in this paper achieves the relatively high grid temperature of 165 K which has to be improved. A reduction of radiation damage can be expected by further lowering the specimen temperature [54,55]. The low peak-to-background ratios in X-ray spectra of frozen-hydrated sections which are much enhanced after freeze-drying (see figs. 6-9) are consistent with similar reports by other authors [38,56]. Perhaps the element concentration in frozen-hydrated cell compartments can be computed from those spectra with adequate calibration methods [57,58]. So far X-ray microanalysis is not a routine method to measure the element content in liquid compartments of cells and tissues, e.g. in vessels, extracellular spaces or vacuoles. Quantitative X-ray microanalysis of biological cryosections is developed only for freeze-dried material [59-611.
51
At present, only preliminary information is available about possibilities of electron energy loss spectroscopy in studying the distribution of particular substances in cells and tissues [62-651. This technique seems quite promising in order to extend studies of the distribution of biologically relevant elements as obtained by X-ray microanalysis to measurements of the molecular composition. However, even the best analytical techniques applied to biological cells or tissues cannot substitute for an adequate cryopreparation to preserve their native state.
4. Conclusion The described cooling chain technique combined with a scanning transmission electron microscope proves that ultrastructure and elemental distribution can be studied in cryofixed freeze-dried cryosections of biological cells and tissues. Whereas cryosectioning and transfer techniques can be performed routinely, cryofixation and imaging of frozen-hydrated sections turn out to be the present limitations. In particular, freezing techniques must be developed which allow a good cryofixation of tissues in a defined physiological state. Imaging of frozen-hydrated sections, which is not yet possible, would extend electron beam analysis to studies of the structure of extracellular compartments, vessels and vacuoles. In spite of these limitations the cooling chain technique is one of the few available methods to study the elemental composition of cells in defined functional states at an ultrastructural level. It can help to elucidate processes of storage and transport of substances in cellular biological systems.
Acknowledgement I want to thank Miss S. Dongard technical assistance.
for her careful
References [l] A.M. Glauert, Fixation, Dehydration Biological Specimens (North-Holland,
and Embedding of Amsterdam, 1975).
52
K. Zierold / Preparation of biologrcal cryosectwns for ancr!vtzcalEM
[2] E. Kellenberger and J. Kistler, The Physics of Specimen Preparation, in: Unconventional Electron Microscopy for Molecular Structure Determination, Eds. W. Hoppe and R. Mason (Vieweg, Braunschweig, 1979) p, 49. [3] B. Cragg, Tissue and Cell 12 (1980) 63. 14) A.J. Morgan, T.W. Davies and D.A. Erasmus. Specimen Preparation, in: Electron Probe Microanalysis in Biology, Ed. D.A. Erasmus (Chapman and Hall, London, 1978) p. 94. [5] K. Zierold, D. Schafer and J. Gullasch, Microsc. Acta, Suppl. 2 (1978) 92. [6] D. Schafer, K. Zierold, D.W. Lubbers. Eds.. Arzneim.Forsch./Drug Res. 29 (11) (1979) 1809. [7] K. Zierold. R. Konig, K.-H. Olech. D. Schafer, D.W. Lubbers, K.-H. Miiller and H. Winter. Ultramicroscopy 6 (1981) 181. [8] K. Zierold, J. Microscopy 125 (1982) 149. [9] M. Miiller, N. Meister and H. Moor, Mikroskopie 36 (1980) 129. [IO] H. Sitte, K. Neumann, H. Hlssig, H. Kleber and G. Kappl, in: Electron Microscopy 1980, Eds. P. Brederoo and W. de Priester (7th European Congr. on Electron Microscopy Foundation, Leiden, 1980) Vol. I, p. 540. [I l] A.K. Christensen. J. Cell Biol. 51 (1971) 772. [ 121 W. Stockem, Mikroskopie 26 (1970) 185. [13] H. Moor, Cryotechnology for the Structural Analysis of Biological Material, in: Freeze-Etching, Techniques and Applications, Eds. E.L. Benedetti and P. Favard (Societi Franqaise de Microscopic Electronique, Paris, 1973) p. Il. [ 141 J. Farrant, CA. Walter, H. Lee, G.J. Morris and K.J. Clarke, Structural and Functional Aspects of Biological Freezing Techniques, in: Low Temperature Biological Microscopy and Microanalysis, Eds. P. Echlin, B. Ralph and E.R. Weibel (Blackwell, Oxford, 1978) p. 17. (151 J. Dubochet and A.W. McDowall, J. Microscopy 124 (1981) RP 3. [ 161 H. Moor, Z. Zellforsch. 62 (1964) 546. [ 171 P. Hobbs, Ice Physics (Clarendon, Oxford, 1974). [ 18) L. Bachmann and W.W. Schmitt-Fumian, Spray-Freezing and Freeze-Etching, in: Freeze-Etching, Techniques and Applications, Eds. E.L. Benedetti and P. Favard (Societe Franqaise de Microscopic Electronique, Paris, 1973) p. 73. [19] J.L. Stephenson, J. Biophys. Biochem. Cytol. 2 (1956) 45. [20] M.J. Costello and J.M. Corless, J. Microscopy 121 (1978) 17. [21] K. Zierold, Microsc. Acta 83 (1980) 25. [22] A.W. Robards and N.J. Severs, Cryo-Letters 2 (1981) 135. [23] N.L. Burstein and D.M. Maurice, Micron 9 (1978) 191. [24] P. Pscheid, C. Schudt and H. Plattner, J. Microscopy 121 (1981) 149. [25] T. Espevik and A. Elgsaeter, J. Microscopy 123 (1981) 105. [26] A. van Harreveld and J. Crowell, Anat. Record 149 (1964) 381. (271 G.P. Dempsey and S. Bullivant, J. Microscopy 106 (1976) 251. [28] J.E. Heuser, T.S. Reese, M.J. Dennis, Y. Jan, L. Jan and L. Evans, J. Cell Biol. 81 (1979) 275.
[29] K.G. Schwabe and L. Terracio, Cryobiology 17 (1980) 571. [30] U. Riehle and M. Hoechli, The Theory and Technique of High Pressure Freezing, in: Freeze-Etching, Techniques and Applications. Eds. E.L. Benedetti and P. Favard (Societe Francaise de Microscopic Electronique, Paris, 1973) p. 31. [31] K.-V. Wolf, W. Stockem and K.-E. Wohlfarth-Bottermann, Cell Tissue Res. 217 (1981) 479. [32] K.T. Tokuyasu. J. Cell Biol. 57 (1973) 551. [33] M. Sjostrom and L.E. Thornell. J. Microscopy 103 (1975) 101. [34] T.C. Appleton, The Contribution of Cryoultramicrotomy to X-Ray Microanalysis in Biology, in: Electron Probe Microanalysis in Biology, Ed. D.A. Erasmus (Chapman and Hall, London, 1978) p. 148. 135) L. Seveus, J. Microscopy 112 (1978) 269. [36] P.M. Frederik and W.M. Busing, J. Microscopy 121 (1981) 191. [37] A.J. Saubermann, P. Echlin, P.D. Peters and R. Beeuwkes III, J. Cell Biol. 88 (1981) 257. [38] B.L. Gupta, T.A. Hall and R.B. Moreton, Electron Probe X-Ray Microanalysis, in: Transport of Ions and Water in Animals, Eds. B.L. Gupta, R.B. Moreton. J.L. Oschman and B.J. Wall (Academic Press, London, 1977) p. 83. [39] T.C. Appleton, J. Microscopy 100 (1974) 49. [40] H.G. Heide and S. Grund, J. Ultrastruct. Res. 48 (1974) 259. [41] Th.E. Hutchinson, M. Bacaner, J. Broadhurst and J. Lilley, Rev. Sci. Instr. 45 (1974) 252. [42] R.B. Moreton, P. Echlin, B.L. Gupta, T.A. Hall and T. Weis-Fogh, Nature 247 (1974) 113. [43] U. Valdre and R.W. Horne, J. Microscopy 103 (1975) 305. [44] T.E. Hutchinson, D.E. Johnson and A.P. MacKenzie, UItramicroscopy 3 ( 1978) 3 15. [45] Y. Talmon, H.T. Davis, L.E. Striven and E.L. Thomas, Rev. Sci. Instr. 50 (1979) 698. [46] R. Freeman, F. Booy and K. Leonard, in: Electron Microscopy 1980, Eds. P. Brederoo an W. de Priester (7th European Congr. on Electron Microscopy Foundation, Leiden, 1980) Vol. 2, p. 650. [47] S. Lichtenegger and W.M.A. Hax, in: Electron Microacopy 1980, Eds. P. Brederoo and W. de Priester (7th European Congr. on Electron Microscopy Foundation, Leiden, 1980) Vol. 2, p. 652. [48] H.G. Heide, Ultramicroscopy 6 (1981) 115. [49] G.M. Roomans and L.A. Seveus, J. Cell Sci. 21 (1976) 119. [50] T. Barnard and L. Se&us, J. Microscopy 112 (1978) 281. 1511 T.L.B. Spriggs and D. Wynne-Evans, J. Microscopy 107 (1976) 35. [52] S. Hodson and L. Williams, J. Cell Sci. 20 (1976) 687. [53] Y. Talmon, H.T. Davis, L.E. Striven and E.L. Thomas, J. Microscopy 117 (1979) 321. [54] R.M. Glaeser and K.A. Taylor, J. Microscopy 112 (1978) 127. (551 V.E. Cosslett, Radiation Damage: Experimental Work. in: Unconventional Electron Microscopy for Molecular Struc-
K. Zierold / Preparation
[56] [57] [58] [59] [60] [61]
of brologrcal cryosections for analytical EM
ture Determination, Eds. W. Hoppe and R. Mason (Vieweg, Braunschweig, 1979) p. 8 1. A. Ross, A.T. Sumner and A.R. Ross, J. Microscopy 121 (1981) 261. A.J. Saubermann, R. Beeuwkes III an P.D. Peters, J. Cell Biol. 88 (1981) 268. B.L. Gupta and T.A. Hall, Tissue and Cell 13 (1981) 623. T.A. Hall, J. Microscopy 117 (1979) 145. H. Shuman, A.V. Somlyo and A.P. Somlyo, Ultramicroscopy 1 (1976) 317. A. DGrge, R. Rick, K. Gehring and K. Thurau, Pfliigers Arch. 373 (1978) 85.
53
[62] M. Isaacson and D. Johnson, Ultramicroscopy 1 (1975) 33. [63] C. Colliex and P. Trebbia, Quantitation and Detection Limits in Electron Energy Loss Spectroscopy (EELS) of Thin Biological Sections, in: Microbeam Analysis in Biology, Eds. C.P. Lechene and R.R. Warner (Academic Press, New York, 1979) p. 65. [64] F.P. Ottensmeyer and J.W. Andrew, J. Ultrastruct. Res. 72 (1980) 336. [65] A.P. Somlyo and H. Shuman, Ultramicroscopy 8 (1982) 219.