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Collapse phenomena in freeze-drying
JOURNAL OF ULTRASTRUCTURE RESEARCH
59, 7 0 - 7 5
(1977)
Collapse Phenomena in Freeze-Drying J. KISTLER AND E . KELLENBERGER Biozentrum der Universit6t Basel, Klingelberstrasse 70, 4056 Basel, Switzerland Received September 16, 1976 On freeze-dried fibrous, membranous, and sheet-like structures we demonstrate t h a t collapse p h e n o m e n a occur aider drying. They are of the same sort as were described much earlier by Anderson for specimens prepared by critical-point drying.
The preservation of the three-dimensional shape of specimens prepared for electron microscopy is strongly dependent on the dehydration procedure involved. In normal air-drying a liquid-gas interphase passes through the specimen, and phenomena associated with surface energy or surface tension might lead to a flattening of the structures, depending on their rigidity (2, 4). Therefore, to study the original three-dimensional shape of a biological specimen, different dehydration methods must be used combined with shadow-casting. Critical-point drying (1) and freezedrying (10, 14) were both successfully applied for ultrastructural investigations in the electron microscope. However, it was recognized early that critical-point-dried specimens show a characteristic type of dehydration artifact. On critical-point-dried preparations of bacteria, Anderson (3) observed the flagella tightly stretched in space from the cells to the supporting film on which the remainder of the flagella lay flat. He proposed that structures which are free under vacu u m vibrate as a result of their thermal energy and collapse irreversibly upon the support. In this report we show that the same type of artifact is also observed on different-sized specimens which are dehydrated by freeze-drying.
drying and shadowing procedure is described in detail in the following paper.' An alternative is t h e microdroplet method whereby the suspension is sprayed as small droplets (about 25 tLm in diameter) onto a cooled surface covered with a supporting m e m b r a n e . The description of this technique is given by Williams (14). Shadowing was carried out at a 30° elevation angle for all examples given; the shadowing materials are given in the figure legends. (b) Electron microscopy. Specimens were examined in a Phillips EM 300 which was operated a t 80 kV using a 30-t~m objective aperture and a liquid nitrogen a n t i c o n t a m i n a t i o n device. Micrographs were recorded on 70-mm Kodalith LR 2572 film and developed in Kodak 60a developer. (c) Photography. All micrographs are reversed in contrast, the heavy metal deposits appearing white and the shadows black. RESULTS
(a) The Eutectic Freezing of an aqueous solution leads first to the formation of pure ice crystals surrounded by a '~eutectic mixture" of the dissolved material. Factors like cooling rate, pressure, presence of antifreeze agents, and the final temperature determine the ice crystal size and the concentration of the eutectic mixture (9, 11, 12). For freeze-drying, the structures under investigation are adsorbed from pure water and frozen in a thin layer. Therefore, only a thin eutectic from residual salts, which reaches equilibrium concentration due to recrystallization at the sublimation tomperature (-35°C), is formed. Two aspects of the eutectic can be visualised on elec-
MATERIALS AND METHODS
(a) Specimen preparation. Unless otherwise stated, we adsorbed t h e structures in suspension onto a supporting film prior to freezing. The freeze-
' Kistler, J., Aebi, U., and Kellenberger, E., J .
Ultrastruct. Res. 59, 76, (1977). 7O
Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0022-5320
COLLAPSE PHENOMENA IN FREEZE-DRYING tron micrographs. (1) On completely dehydrated specimens we observed a network of strands usually joining in triangular connection points (Fig. la). Most of the underlying supporting film is visible, showing the shadow cast of the strands and adsorbed material. Sets of stereoscopic electron micrographs (pictures not shown) indicate that most of these strands are concentrated about a plane approximately 0.5 t~m above the support. (2) We found another appearance of the eutectic on specimens which are likely to be incompletely dehydrated. Figure lb represents this aspect, which shows a network of thin perforated sheets. The supporting membrane is visible through the holes.
( b ) Bacterial Flagella We reinvestigated the problem of collapse of bacterial flagella upon dehydration (3) by freeze-drying the specimens. Figures lc and td clearly confirm the characteristic stretched appearance of the Escherichia coli flagella (as indicated by the straight shadows) and the remainder collapsed onto the supporting film. Two thrther aspects are revealed: First, the movement, hypothetically proposed by Anderson, is visualized in the form of blurred shadow casts. We measured displacements of up to 1000/~ for these structures, approximately 200/~ thick; the largest displacement is always found near the bacterial body. Second, the flagella often ~ost their stretched appearance and showed a wavy shape instead; sometimes we found them broken. This modification must have happened after shadowing, probably due to a secondary phenomenon. (c) The Preservation of T4 Bacteriophage A striking example of thermal collapse is shown by the tail fibers ofT-even phages (20 to 30 A in diameter). Early electron microscopy showed either fibers, when carefully air-dried (6), or distal blobs, in freeze-drying (15). It was not possible to decide whether the fibers were real or an
71
artifact. From indirect experiments it was then concluded that the fibers are responsible for adsorption and that they are extended when attaching the phage to the bacteria (7). We confirmed these facts by the following experiments. When we first adsorbed the phages to a supporting film prior to freezing, we observed extended fibers (Fig. 2a) as they are also visualized on micrographs of negatively stained preparations. However, tails with a distal blob instead of fibers are seen in freeze-dried preparations carried out according to the microdroplet method of Williams and Frazer (14, 15). Figure 2b illustrates this artifact on T4 bacteriophage ghosts which are prepared by the application of the microdroplet freeze-drying procedure. Three more features of interest are contained in these pictures. First, the phage head collapses to a degree which is dependent upon whether or not it is empty. Phage ghosts clearly show a strong collapse, as indicated by the shadow cast of the empty heads (Fig. 2b); heads which still contain their DNA show smaller distortions (Fig. 2a). Second, although both freeze-drying procedures indicate the icosahedral shape of the phage head, neither of them preserves the corners of the head as revealed on micrographs of freeze-fractured preparations (5). Third, the shadow cast of the tail suggests that the latter sticks to the support over its entire length. It is not stretched free between the head and the support, as we would have expected for an artifact-free preservation.
(d) The Collapse of Empty Cylindrical Structures Although it is believed that freezedrying retains much of the specimen's three-dimensionality, we always found empty cylindrical structures to be collapsed either onto themselves or onto the supporting film. We illustrate this on two structures of different sizes. Figure 2c represents a low magnification micrograph of a freeze-dried preparation ofE. coli saccu-
FIG. 1. (a) Typical appearance of eutectic strands on completely dehydrated specimens, where most of the supporting film is visible. (b) Aspect of eutectic on specimens which are likely to be not yet completely dehydrated. The support is visible only on limited areas. (c, d) Freeze-dried preparation of E. coli and bacteriophage T2. The shadow casts indicate the vibration (V) of the free part of the flagella and the remainder is collapsed onto the support (C). (a-d, Tungsten shadowing.) 72
FIG. 2. (a) Freeze-dried bacteriophage T4 prepared by the preadsorption method. The fibers remained extended. (Tungsten shadowing.) (b) Bacteriophage T4 ghosts freeze-dried according to the microdroplet method. The arrows indicate the distal blob on the tails. (Uranium dioxide shadowing.) (c) Low magnification micrograph of a freeze-dried preparation ofE. coli sacculus. Both the collapsed structures collected on the eutectic and those flattened on the support are visible. Arrows indicate intermediates. (Tungsten shadowing.) (d) Freeze-dried E. coli sacculus collapsed fiat onto the support showing regular arrays of the matrix protein. (Tungsten shadowing.) (e) ~ polyhead type C, freeze-dried. (Platinum/carbon shadowing.) 73
74
KISTLER AND KELLENBERGER
lus (Steven et al., submitted for publication), showing the two types of collapse and intermediate states. Most of the structures which are collected on the eutectic network are found to be collapsed onto themselves; on an average, these show about 60% reduction in diameter, as compared to the original diameter calculated from the width of structures which were collapsed flat onto the supporting film. Figure 2d illustrates a typically flattened sacculus o f E . coli cell envelope with preserved patches of regularly arranged matrix protein. Arrows in Fig. 2c indicate two intermediates, one end of which is collapsed flat onto the support while the other stands upright collapsed onto itself. For h and T-even polyheads (head-related aberrant forms of mutant bacteriophage h or T-even), thin sectioning (~; T. Hohn, personal communication) and freeze-fracturing (13) confirmed the tubular shape of these structures. However, when looking at freeze-dried preparations, we always found them flattened on the supporting film. As an example we illustrate this on a preparation of ~ polyheads (Fig. 2e) (16). DISCUSSION AND CONCLUSIONS
Anderson has theoretically proposed the existence of thermal movement and pointed out that dehydrated structures collapse onto the supporting film upon contact. It is likely that Van der Waals forces are responsible for this irreversible collapse. Our observations indicate that structures may collapse not only upon contact with the supporting membrane but also upon contact with themselves. At this stage we are not yet able to quantify these phenomena on the molecular level, but consider it worthwhile to discuss the qualitative aspects in view of practical electron microscopy. Although the eutectic is not a biological structure, we think that thermal collapse is the bridging phenomenon between the two aspects shown in Figs. la and lb. The
thin eutectic layers are likely to be very unstable as soon as they are liberated from the ice; rupture would then lead to the perforated sheet appearance in Fig. lb. In the course of further dehydration these layers become free to thermal movement which makes them role up into strands and then collapse onto themselves as soon as they hit each other (Fig. la). In the case of bacterial flagella we can clearly demonstrate the existence of movements; however, we are not able to attribute them to (1) the simple thermal energy contained in a structure at a given temperature or (2) to the energy introduced during evaporation of the heavy metal or (3) to thermally induced movements due to intramolecular rearrangements in the absence of the aqueous environment. Without quantifying these contributions precisely, we consider the existence of this phenomenon important for specimen preparation. The wavy aspect of the flagella after shadowing is probably due to a release of tension either because of flagellar breakage or because of a later swelling after exposure to atmospheric water vapor. The different preservation of T-even phage tail fibers when comparing specimens prepared by the two freeze-drying methods is probably the strongest evidence for thermal collapse. Preadsorbed tail fibers have limited freedom to move and therefore remain extended. The microdroplet method essentially prevents this preadsorption but retains the particles in the ice matrix. As they are liberated from the ice during sublimation they undergo thermal movement and the fibers irreversibly collapse onto themselves upon contact, leading to the formation of the distal blob on the tail (Fig. 2b). As in the previous case, we found a different preservation of freeze-dried empty cylindrical structures depending on whether or not they had been adsorbed to the support prior to freezing. The cylindrical structures are likely to be stiff in the axial direction but to be rather flexible
COLLAPSE PHENOMENA IN FREEZE-DRYING
radially, probably as a function of the radius/wall thickness ratio. For nonadsorbed particles, thermal movement might bring the walls into contact, leading to irreversible collapse onto themselves. Since structures found on the support are mostly flattened and not randomly collapsed onto themselves, we assume a step by step collapse due to thermal movement and the interactions between the cylinder wall and the support. In order to rule out the possibility that this already happens in the liquid phase after adsorption prior to freezing Cadsorption collapse"), we performed the following experiment: T2 giant phage were first adsorbed to the grid and nonadsorbed particles were carefully washed off. The grids were then floated on a drop of Fab fragments prepared from antibodies directed against the outside of the capsid (Aebi et al., in preparation). Micrographs of negatively stained giant phages were optically diffracted and clearly showed that the Fab fragments were adsorbed all around the giant phage. In the case of adsorption collapse the Fab fragments could not have adsorbed to the giant phage side which is in contact with the support. Knowing that thermal collapse phenomena act on structures of all different sizes, a question arises regarding fine structural preservation at the molecular level after fl~eze-drying. One way to investigate this is to choose periodic structures as test objects and make use of their redundant inibrmation content. The flat preservation of such specimens allows the application of image processing techniques. A preliminary note (8) and the following paper 1 report a detailed comparison of filtrations from negatively stained preparations with those from freeze-dried and shadowed preparations of the same periodic struc-
75
ture. In both cases we obtained resolution to 25-30/~ and did not observe systematic collapse artifacts of the fine structures down to this resolution. We are grateful to Drs. A. Steven, T. Frey, and U. Aebi for critically reading the manuscript, to Marianne Schaefer for typing the manuscript, and to Regina Oetterli for the photographic work. This work was supported by a grant from the Swiss National Foundation for Scientific Research. REFERENCES 1. ANDERSON,T. F., Comptes Rendues du Premier Congr~s International de Microscopie Electronique, Paris, 1950 (Rev. d'Optique ed.), pp. 567-576, 1952. 2. ANDERSON,T. F.,Amer. Naturalist 86, 91 (1952). 3. ANDERSON, W. F., Trans. N.Y. Acad. Sci. 16, 242, (1954). 4. ARBER, W., KELLENBERGER,E., AND LASZT, L., Kolloid-Z. 150, 123, (1957). 5. BRANTON, D., AND KLUG, A., J. Mol. Biol. 92, 559, (1974). 6. KELLENBERGER,E., AND ARBER, W., Z. Naturforsch. 106, 698, (1955). 7. KELLENBERGER,E., BOLLE, A., BOY DE LA TOUR, E., EPSTEIN, R. M., FRANKLIN,N. C., JERNE, N. K., REALE-SCAFATI,A., SECHAUD,J., BURDEW, I., GOLDSTEIN,D., AND LAUFFER, M. A., Virology 26, 419, (1965). 8. KISTLER, J., AEBI, U., AND KELLENBERGER,E., Proceedings of the 6th European Congress on Electronmicroscopy, Israel (1976). 9. MOOR,H., Z. Zellforsch. 62, 546, (1964). 10. NERMUT, M., FRANKLIN,H., AND SCHAEFER,W., Virology 49, 345, (1972). 11. LUYET,B. J., Cryobiology 2, 198, (1966). 12. RIEHLE, U., AND HOCHLI, M., in BENEDETTI, E., AND FAVARD,P. (Eds.), Freeze Etching: Techniques and Application, pp. 31-61. Paris, (1973). 13. SCRABA, D. G., RASKA, I., KELLENBERGER,E., AND MOOR, S., J. Ultrastruct. Res. 44, 27, (1973). 14. WILLIAMS,n. C.,Exp. CellRes. 4, 188, (1952). 15. WILLIAMS, R. C., AND FRAZER, D., J. Bact. 66, 458, (1953). 16. WURTZ,M., KISTLER, J., AND HOHN, T., J. Mol. Biol. 101, 39, (1976).
59, 7 0 - 7 5
(1977)
Collapse Phenomena in Freeze-Drying J. KISTLER AND E . KELLENBERGER Biozentrum der Universit6t Basel, Klingelberstrasse 70, 4056 Basel, Switzerland Received September 16, 1976 On freeze-dried fibrous, membranous, and sheet-like structures we demonstrate t h a t collapse p h e n o m e n a occur aider drying. They are of the same sort as were described much earlier by Anderson for specimens prepared by critical-point drying.
The preservation of the three-dimensional shape of specimens prepared for electron microscopy is strongly dependent on the dehydration procedure involved. In normal air-drying a liquid-gas interphase passes through the specimen, and phenomena associated with surface energy or surface tension might lead to a flattening of the structures, depending on their rigidity (2, 4). Therefore, to study the original three-dimensional shape of a biological specimen, different dehydration methods must be used combined with shadow-casting. Critical-point drying (1) and freezedrying (10, 14) were both successfully applied for ultrastructural investigations in the electron microscope. However, it was recognized early that critical-point-dried specimens show a characteristic type of dehydration artifact. On critical-point-dried preparations of bacteria, Anderson (3) observed the flagella tightly stretched in space from the cells to the supporting film on which the remainder of the flagella lay flat. He proposed that structures which are free under vacu u m vibrate as a result of their thermal energy and collapse irreversibly upon the support. In this report we show that the same type of artifact is also observed on different-sized specimens which are dehydrated by freeze-drying.
drying and shadowing procedure is described in detail in the following paper.' An alternative is t h e microdroplet method whereby the suspension is sprayed as small droplets (about 25 tLm in diameter) onto a cooled surface covered with a supporting m e m b r a n e . The description of this technique is given by Williams (14). Shadowing was carried out at a 30° elevation angle for all examples given; the shadowing materials are given in the figure legends. (b) Electron microscopy. Specimens were examined in a Phillips EM 300 which was operated a t 80 kV using a 30-t~m objective aperture and a liquid nitrogen a n t i c o n t a m i n a t i o n device. Micrographs were recorded on 70-mm Kodalith LR 2572 film and developed in Kodak 60a developer. (c) Photography. All micrographs are reversed in contrast, the heavy metal deposits appearing white and the shadows black. RESULTS
(a) The Eutectic Freezing of an aqueous solution leads first to the formation of pure ice crystals surrounded by a '~eutectic mixture" of the dissolved material. Factors like cooling rate, pressure, presence of antifreeze agents, and the final temperature determine the ice crystal size and the concentration of the eutectic mixture (9, 11, 12). For freeze-drying, the structures under investigation are adsorbed from pure water and frozen in a thin layer. Therefore, only a thin eutectic from residual salts, which reaches equilibrium concentration due to recrystallization at the sublimation tomperature (-35°C), is formed. Two aspects of the eutectic can be visualised on elec-
MATERIALS AND METHODS
(a) Specimen preparation. Unless otherwise stated, we adsorbed t h e structures in suspension onto a supporting film prior to freezing. The freeze-
' Kistler, J., Aebi, U., and Kellenberger, E., J .
Ultrastruct. Res. 59, 76, (1977). 7O
Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0022-5320
COLLAPSE PHENOMENA IN FREEZE-DRYING tron micrographs. (1) On completely dehydrated specimens we observed a network of strands usually joining in triangular connection points (Fig. la). Most of the underlying supporting film is visible, showing the shadow cast of the strands and adsorbed material. Sets of stereoscopic electron micrographs (pictures not shown) indicate that most of these strands are concentrated about a plane approximately 0.5 t~m above the support. (2) We found another appearance of the eutectic on specimens which are likely to be incompletely dehydrated. Figure lb represents this aspect, which shows a network of thin perforated sheets. The supporting membrane is visible through the holes.
( b ) Bacterial Flagella We reinvestigated the problem of collapse of bacterial flagella upon dehydration (3) by freeze-drying the specimens. Figures lc and td clearly confirm the characteristic stretched appearance of the Escherichia coli flagella (as indicated by the straight shadows) and the remainder collapsed onto the supporting film. Two thrther aspects are revealed: First, the movement, hypothetically proposed by Anderson, is visualized in the form of blurred shadow casts. We measured displacements of up to 1000/~ for these structures, approximately 200/~ thick; the largest displacement is always found near the bacterial body. Second, the flagella often ~ost their stretched appearance and showed a wavy shape instead; sometimes we found them broken. This modification must have happened after shadowing, probably due to a secondary phenomenon. (c) The Preservation of T4 Bacteriophage A striking example of thermal collapse is shown by the tail fibers ofT-even phages (20 to 30 A in diameter). Early electron microscopy showed either fibers, when carefully air-dried (6), or distal blobs, in freeze-drying (15). It was not possible to decide whether the fibers were real or an
71
artifact. From indirect experiments it was then concluded that the fibers are responsible for adsorption and that they are extended when attaching the phage to the bacteria (7). We confirmed these facts by the following experiments. When we first adsorbed the phages to a supporting film prior to freezing, we observed extended fibers (Fig. 2a) as they are also visualized on micrographs of negatively stained preparations. However, tails with a distal blob instead of fibers are seen in freeze-dried preparations carried out according to the microdroplet method of Williams and Frazer (14, 15). Figure 2b illustrates this artifact on T4 bacteriophage ghosts which are prepared by the application of the microdroplet freeze-drying procedure. Three more features of interest are contained in these pictures. First, the phage head collapses to a degree which is dependent upon whether or not it is empty. Phage ghosts clearly show a strong collapse, as indicated by the shadow cast of the empty heads (Fig. 2b); heads which still contain their DNA show smaller distortions (Fig. 2a). Second, although both freeze-drying procedures indicate the icosahedral shape of the phage head, neither of them preserves the corners of the head as revealed on micrographs of freeze-fractured preparations (5). Third, the shadow cast of the tail suggests that the latter sticks to the support over its entire length. It is not stretched free between the head and the support, as we would have expected for an artifact-free preservation.
(d) The Collapse of Empty Cylindrical Structures Although it is believed that freezedrying retains much of the specimen's three-dimensionality, we always found empty cylindrical structures to be collapsed either onto themselves or onto the supporting film. We illustrate this on two structures of different sizes. Figure 2c represents a low magnification micrograph of a freeze-dried preparation ofE. coli saccu-
FIG. 1. (a) Typical appearance of eutectic strands on completely dehydrated specimens, where most of the supporting film is visible. (b) Aspect of eutectic on specimens which are likely to be not yet completely dehydrated. The support is visible only on limited areas. (c, d) Freeze-dried preparation of E. coli and bacteriophage T2. The shadow casts indicate the vibration (V) of the free part of the flagella and the remainder is collapsed onto the support (C). (a-d, Tungsten shadowing.) 72
FIG. 2. (a) Freeze-dried bacteriophage T4 prepared by the preadsorption method. The fibers remained extended. (Tungsten shadowing.) (b) Bacteriophage T4 ghosts freeze-dried according to the microdroplet method. The arrows indicate the distal blob on the tails. (Uranium dioxide shadowing.) (c) Low magnification micrograph of a freeze-dried preparation ofE. coli sacculus. Both the collapsed structures collected on the eutectic and those flattened on the support are visible. Arrows indicate intermediates. (Tungsten shadowing.) (d) Freeze-dried E. coli sacculus collapsed fiat onto the support showing regular arrays of the matrix protein. (Tungsten shadowing.) (e) ~ polyhead type C, freeze-dried. (Platinum/carbon shadowing.) 73
74
KISTLER AND KELLENBERGER
lus (Steven et al., submitted for publication), showing the two types of collapse and intermediate states. Most of the structures which are collected on the eutectic network are found to be collapsed onto themselves; on an average, these show about 60% reduction in diameter, as compared to the original diameter calculated from the width of structures which were collapsed flat onto the supporting film. Figure 2d illustrates a typically flattened sacculus o f E . coli cell envelope with preserved patches of regularly arranged matrix protein. Arrows in Fig. 2c indicate two intermediates, one end of which is collapsed flat onto the support while the other stands upright collapsed onto itself. For h and T-even polyheads (head-related aberrant forms of mutant bacteriophage h or T-even), thin sectioning (~; T. Hohn, personal communication) and freeze-fracturing (13) confirmed the tubular shape of these structures. However, when looking at freeze-dried preparations, we always found them flattened on the supporting film. As an example we illustrate this on a preparation of ~ polyheads (Fig. 2e) (16). DISCUSSION AND CONCLUSIONS
Anderson has theoretically proposed the existence of thermal movement and pointed out that dehydrated structures collapse onto the supporting film upon contact. It is likely that Van der Waals forces are responsible for this irreversible collapse. Our observations indicate that structures may collapse not only upon contact with the supporting membrane but also upon contact with themselves. At this stage we are not yet able to quantify these phenomena on the molecular level, but consider it worthwhile to discuss the qualitative aspects in view of practical electron microscopy. Although the eutectic is not a biological structure, we think that thermal collapse is the bridging phenomenon between the two aspects shown in Figs. la and lb. The
thin eutectic layers are likely to be very unstable as soon as they are liberated from the ice; rupture would then lead to the perforated sheet appearance in Fig. lb. In the course of further dehydration these layers become free to thermal movement which makes them role up into strands and then collapse onto themselves as soon as they hit each other (Fig. la). In the case of bacterial flagella we can clearly demonstrate the existence of movements; however, we are not able to attribute them to (1) the simple thermal energy contained in a structure at a given temperature or (2) to the energy introduced during evaporation of the heavy metal or (3) to thermally induced movements due to intramolecular rearrangements in the absence of the aqueous environment. Without quantifying these contributions precisely, we consider the existence of this phenomenon important for specimen preparation. The wavy aspect of the flagella after shadowing is probably due to a release of tension either because of flagellar breakage or because of a later swelling after exposure to atmospheric water vapor. The different preservation of T-even phage tail fibers when comparing specimens prepared by the two freeze-drying methods is probably the strongest evidence for thermal collapse. Preadsorbed tail fibers have limited freedom to move and therefore remain extended. The microdroplet method essentially prevents this preadsorption but retains the particles in the ice matrix. As they are liberated from the ice during sublimation they undergo thermal movement and the fibers irreversibly collapse onto themselves upon contact, leading to the formation of the distal blob on the tail (Fig. 2b). As in the previous case, we found a different preservation of freeze-dried empty cylindrical structures depending on whether or not they had been adsorbed to the support prior to freezing. The cylindrical structures are likely to be stiff in the axial direction but to be rather flexible
COLLAPSE PHENOMENA IN FREEZE-DRYING
radially, probably as a function of the radius/wall thickness ratio. For nonadsorbed particles, thermal movement might bring the walls into contact, leading to irreversible collapse onto themselves. Since structures found on the support are mostly flattened and not randomly collapsed onto themselves, we assume a step by step collapse due to thermal movement and the interactions between the cylinder wall and the support. In order to rule out the possibility that this already happens in the liquid phase after adsorption prior to freezing Cadsorption collapse"), we performed the following experiment: T2 giant phage were first adsorbed to the grid and nonadsorbed particles were carefully washed off. The grids were then floated on a drop of Fab fragments prepared from antibodies directed against the outside of the capsid (Aebi et al., in preparation). Micrographs of negatively stained giant phages were optically diffracted and clearly showed that the Fab fragments were adsorbed all around the giant phage. In the case of adsorption collapse the Fab fragments could not have adsorbed to the giant phage side which is in contact with the support. Knowing that thermal collapse phenomena act on structures of all different sizes, a question arises regarding fine structural preservation at the molecular level after fl~eze-drying. One way to investigate this is to choose periodic structures as test objects and make use of their redundant inibrmation content. The flat preservation of such specimens allows the application of image processing techniques. A preliminary note (8) and the following paper 1 report a detailed comparison of filtrations from negatively stained preparations with those from freeze-dried and shadowed preparations of the same periodic struc-
75
ture. In both cases we obtained resolution to 25-30/~ and did not observe systematic collapse artifacts of the fine structures down to this resolution. We are grateful to Drs. A. Steven, T. Frey, and U. Aebi for critically reading the manuscript, to Marianne Schaefer for typing the manuscript, and to Regina Oetterli for the photographic work. This work was supported by a grant from the Swiss National Foundation for Scientific Research. REFERENCES 1. ANDERSON,T. F., Comptes Rendues du Premier Congr~s International de Microscopie Electronique, Paris, 1950 (Rev. d'Optique ed.), pp. 567-576, 1952. 2. ANDERSON,T. F.,Amer. Naturalist 86, 91 (1952). 3. ANDERSON, W. F., Trans. N.Y. Acad. Sci. 16, 242, (1954). 4. ARBER, W., KELLENBERGER,E., AND LASZT, L., Kolloid-Z. 150, 123, (1957). 5. BRANTON, D., AND KLUG, A., J. Mol. Biol. 92, 559, (1974). 6. KELLENBERGER,E., AND ARBER, W., Z. Naturforsch. 106, 698, (1955). 7. KELLENBERGER,E., BOLLE, A., BOY DE LA TOUR, E., EPSTEIN, R. M., FRANKLIN,N. C., JERNE, N. K., REALE-SCAFATI,A., SECHAUD,J., BURDEW, I., GOLDSTEIN,D., AND LAUFFER, M. A., Virology 26, 419, (1965). 8. KISTLER, J., AEBI, U., AND KELLENBERGER,E., Proceedings of the 6th European Congress on Electronmicroscopy, Israel (1976). 9. MOOR,H., Z. Zellforsch. 62, 546, (1964). 10. NERMUT, M., FRANKLIN,H., AND SCHAEFER,W., Virology 49, 345, (1972). 11. LUYET,B. J., Cryobiology 2, 198, (1966). 12. RIEHLE, U., AND HOCHLI, M., in BENEDETTI, E., AND FAVARD,P. (Eds.), Freeze Etching: Techniques and Application, pp. 31-61. Paris, (1973). 13. SCRABA, D. G., RASKA, I., KELLENBERGER,E., AND MOOR, S., J. Ultrastruct. Res. 44, 27, (1973). 14. WILLIAMS,n. C.,Exp. CellRes. 4, 188, (1952). 15. WILLIAMS, R. C., AND FRAZER, D., J. Bact. 66, 458, (1953). 16. WURTZ,M., KISTLER, J., AND HOHN, T., J. Mol. Biol. 101, 39, (1976).