8 Journal of Microscopy, Vol. 103, Pt 1,January 1975, pp. 71-77. Received 21 M a y 1974; revision received 8 October 1974
A wet stage modification to a scanning electron microscope
by V. N. E. R O B I N S O NFaculty , of Applied Science, University of New South Wales, P.O. Box 1, Kensington, N . S .W. 2033, Australia SUMMARY
A modification to the vacuum system of a JSM2 scanning electron microscope has enabled hydrated specimens to be placed inside the specimen chamber of the instrument and to be surrounded by water vapour at a pressure up to approximately 1.3 kPa (10 Torr). The surface topography was observed by detecting the backscattered electrons using a wide angle backscattered electron detector placed close to the specimen. The microscope was operated in the normal scanning mode which allowed the examination of the surface topography of the specimens, whilst still retaining the depth of focus which is a feature of the SEM. This modification has enabled a resolution of approximately 0.2 pm to be obtained from biological specimens partially immersed in water at temperatures just above 0°C. INTRODUCTION
The ability to examine wet specimens in a controlled atmosphere in electron microscopes can greatly increase the versatility of these instruments and the information that can be obtained through their use. The operation of an electron microscope requires the electrons to travel through a high vacuum. The introduction of water vapour, or any other vapour or gas molecules, must be done in such a way as to preserve the high vacuum in the electron optics column. To achieve this, environmental control cells have been constructed for electron microscopes operated in the transmission mode, closed cells (Abrams & McBain, 1944 ; Fullam, 1972) and differentially pumped chambers (Matricardi, Hausner & Parsons, 1970; Moretz, Hausner & Parsons, 1970; Fukami et al., 1970; Parsons et al., 1972; Parsons, Uydess & Matricardi, 1974), scanning transmission mode (Swift & Brown, 1970; Morgan, Lebiedzik & White, 1973) and the conventional scanning mode (Lane, 1970). The cells for transmission and scanning transmission microscopes only provide information about the bulk structure of thin specimens. T o obtain information about the surface structure of thick specimens, Lane (1970) constructed an environmental cell whereby a flux of vapour molecules through a small aperture established a vapour cloud round the specimen. This design can accommodate only small specimens, has an unknown and variable water vapour pressure and can produce vacuum deterioration (Morgan et al., 1973). A design which allows a larger specimen size, currently about 10 mm diameter, in a static, known and controllable vapour pressure up to about 1-3 kPa has been developed for a
71
V.N . E. Robinson JSM2 scanning electron microscope. This design which incorporated a backscattered or rediffused electron detector (Robinson, 1974), allowed normal scanning electron microscope operation to obtain information about the surface structure of large specimens. DESIGN AND OPERATION
The principle employed was that at temperatures in the vicinity of O'C, the water vapour pressure was sufficiently low to enable 15-25 keV electrons to travel a few millimetres through the vapour. This distance was sufficiently long to enable these electrons to travel through the vapour, impinge upon the specimen and give off a signal which could be detected. The electron beam was formed, in the usual manner, in the high vacuum of the electron microscope column. The whole of the specimen chamber contained the water vapour in static equilibrium. The specimen was positioned as close as practical to the final aperture and the backscattered electron detector to minimize the electron path length in the vapour. The practicabilities of how this has been achieved in one instrument, a JSM2, are outlined below. At all stages of the modification, emphasis was placed upon minimum alteration to the microscope. The electron microscope column was isolated from the specimen chamber, the only connection being the 100 pm diameter final aperture. In the JSM2, this was achieved by blocking the diffusion pump from pumping the specimen chamber and placing O-rings around the final lens polepiece and the final aperture holder, the polepiece O-ring being permanently incorporated into the microscope. A vapour pressure of 1.3 kPa in the specimen chamber resulted in a slow leak across the aperture which required a pumping speed of less than 10 dm3per sec to Pa maintain a pressure of 1.3 x Torr) in the column. The loss of vapour from the specimen chamber was negligible. To situate the specimen as close as practical to the final aperture, repositioning of the final aperture to be at the bottom of the final lens polepiece was necessary. Experience has shown that this repositioning did not interfere with the double deflection scan system or alter the magnification calibration of the instrument. The emerging backscattered or rediffused electrons were detected by a wide angle detector placed as close as possible to the specimen and the final aperture. The detector was designed to collect as much of the signal as possible consistent with good specimen movement. It was constructed on the scintillator-light guide principle because this required no electrical connections likely to be fouled by the presence of water vapour and required no microscope alteration. Figure 1 illustrates the detector-specimen arrangement, embodying the above principles, currently used in a JSM2 scanning electron microscope. A block of plastic scintillator appropriately machined and positioned under the final aperture, was used for both the detector and light guide. Other scintillator light guide geometries have also been employed with similar results. To reduce beam scattering by the residual vapour, it was found necessary to reduce the residual vapour pressure by cooling the specimen and as much of the specimen chamber as practical, to a temperature just above 0°C. Partial cooling of the specimen chamber was achieved by a cold finger attached to the specimen stage and cooled externally with liquid nitrogen. Complete cooling of all the specimen chamber surfaces to 0°C has not been achieved in this modification, and this results in the vapour pressure being somewhat higher than would be expected from the temperature of the water. The specimen was mounted on a stub 10 mm diameter and 20 mm long. The
72
Wet stage mod$cation to the SEM
Fig. 1. Schematic illustration of the detector and specimen configuration employed for wet specimen microscopy in a JSMZ SEM.
specimen, stub and stub holder were cooled to 0°C prior to insertion into the chamber. A reservoir of about 5 g of ice and water was placed in the chamber when the specimen was inserted. The specimen was inserted so as to place it inside the detector. Excess vapour was pumped out through the pre-evacuation pump with the air lock valve open. When the pressure had reached equilibrium, the air lock valve was closed and the microscope was operated in the normal manner. Specimen insertion and removal was performed in the usual manner with both the column and chamber maintained under vacuum. The vapour pressure in the chamber was measured using a simple U tube manometer, one end of which was connected to a vacuum pump and maintained at a pressure of approximately 10 Pa (10-1Torr). The pressure in the chamber was measured against this, using diffusion pump oil as the manometer liquid. The beam accelerating voltage used depended upon the vapour pressure. At low vapour pressures, below approximately 100 Pa (1 Torr), very little beam scattering occurred and an accelerating voltage in the vicinity of 10 kV could be used to resolve detail of less than 0.2 pm on uncoated biological materials. At these low chamber pressures, the resolution achieved from this system, when examining gold coated specimens, appears to be the same as that achieved with the conventional secondary electron detector system, compare Fig. 2a and b. The lower resolution achieved from the low density biological materials appeared to be due to the increased penetration of the primary beam into these low density specimens. This was reduced by operating at a lower accelerating voltage and experience has shown that greater topographic contrast is obtained from the examination of low density specimens at lower accelerating voltages. On the other hand, high density specimens or specimens coated with a dense metal (Au) gave best contrast and resolution at the higher accelerating voltages (25 kV). Vapour pressures above approximately 100 Pa (1 Torr) resulted in increased beam scattering and required a higher beam voltage to obtain useful topographic information. An accelerating voltage in the range of 15-20 kV, resolving detail in the vicinity of 0.2 pm to 0.3 pm on biological material, gave optimum performance at pressures corresponding to saturated water vapour at temperatures in the vicinity of 0°C.At higher water temperatures optimum performance was achieved with accelerating voltages above 20 kV, with resolution in the vicinity of 0.5 pm. The beam currents employed depended upon vapour pressure, accelerating voltage and specimen density. With gold coated specimens at low vapour pressures, operating conditions were typically 25 kV accelerating voltage with a beam
73
V.N.E. Robinson
74
Wet stage modiJication to the SEM
Fig. 3. The condensation of water droplets onto the surface of chlorinated wool fibres partially immersed in water, no coating, 18 kV accelerating voltage. Magnification marker is 20 pm. A. Low density specimens and a vapour pressure in the current of 5 x vicinity of 0.6 kPa (6 Torr) required a beam current of approximately 200 x A with an accelerating voltage of 15 kV for optimum performance. The beam current could not be accurately measured during wet stage operation because of the scattering and conductivity influence of the water vapour and approximate beam currents were measured subsequently during dry microscopy. It takes only a few minutes to change the microscope from normal operation to wet stage capability. Additional time is required to precool the specimen chamber. This modification produces no vacuum deterioration in the column at chamber pressures less than 1.3 kPa (10 Torr), and does not result in an increase in the rate of column contamination or decrease in filament life. When the microscope is subsequently reverted to normal operation, there is no change in microscope performance. -_
Fig. 2. The secondary electron image (a) and wide angle backscattered electron image (b) of a gold coated germinating rust spore (MeZunosporu medusae). 25 kV accelerating voltage 5 x 10-la A beam current. In (b) the specimen was in a residual vapour pressure of approximately 20 Pa.
Magnification marker is 2 pm,
75
V . N.E. Robinson USES
Some of the uses to which a microscope wet stage can be applied have been previously outlined (Lane, 1970; Morgan et al., 1973; Parsons, 1974). This modification has been used for a variety of different studies, the complete results of which will be reported at a later date. T o demonstrate the versatility of this modification, this section contains a brief report of some of the results obtained to date. Figure 3 shows a micrograph of chlorinated wool fibres partially longitudinally immersed in water. What appears to be condensation droplets can be observed on the surface of the fibres. These droplets have been produced on many surfaces by cooling the specimen. They subsequently disappear, presumably by evaporation as they diminish in size, after a few minutes. Figure 4 shows formaldehyde cross linked fibres partially immersed in water. Figures 3 and 4 illustrate the image clarity attainable with this modification. This is sufficient to provide much information about the wet appearance of biological material and to make comparisons of the surface appearance, wet and dry, of these materials. The examination of wet hydrophilic specimens presents some problems because of the tendency of the water to spread over the specimen surface and obscure topographic detail. This modification has been used for evaporation and sublimation studies, with the vapourization being produced by vacuum pumping. The condensation and growth of water droplets onto different surfaces has been observed. The
Fig. 4. HCHO crosslinked wool fibres partially immersed in water, no coating, 17 kV accelerating voltage. Magnification marker is 20 pm.
76
Wet stage modification to the SEM shape and contact angle of water droplets of sizes from approximately 2 pm up have been observed. The large volume available inside the detector could enable small instruments to be placed inside the detector to perform some in situ experiments. The detector design may be modified to accommodate different types of specimen holders for Werent experiments. CONCLUSIONS
The design features of a wet stage modification to a scanning electron microscope have been discussed and the performance of a unit constructed for a JSM2 SEM has been presented. In principle this design can be modified to suit most instruments. The whole of the specimen chamber contains the water vapour at a pressure which is static, known and controllable. Specimens of size up to 1Omm diameter can be accommodated and have been examined in the SEM whilst they were partially immersed in water at temperatures close to 0°C.
References Abrams, I.M. & McBain, J.W. (1944) A closed cell for electron microscopy. J . uppl. Phys. 15, 607. Fukami, A., Etho, T., Ishihara, W., Katch, M. & Fujiwara, K. (1970) Pressurised specimen chamber for the electron microscope. Proc. 28th Ann. Meeting E.M.S.A. (Ed. by C. J. Arceneaux), p. 546. Claitor’s, Baton Rouge. Fullam, E.F. (1972) A closed wet cell for the electron microscope. Rev. scient. Instrum. 43,245. Lane, W.C. (1970) The environmental control stage. In: Scanning Electron Microscopy, 1970 (Ed. by 0. Johari), p. 41. IITRI, Chicago. Matricardi, V.R., Hausner, G.G. 81Parsons, D.F. (1970) Hydration chamber for JEOLCO 200 kV microscope. Proc. 28th Ann. Meeting, E.M.S.A. (Ed. by C. J. Arceneaux). p. 542. Claitor’s, Baton Rouge. Moretz, R.C., Hausner, G.C. Jr. & Parsons, D.F. (1970) Studies on water in the hydration chamber of a modified electron microscope. Proc. 28th Ann. Meeting, E.M.S.A. (Ed. by C . J. Arceneaux), p. 544. Claitor’s, Baton Rouge. Morgan, R.S., Lebiedzik, J. & White, E.W. (1973) Compact, controlled atmosphere cell for TSEM of hydrated biological materials. In: Scanning Electron Microscopy, 1973 (Ed. by 0. Johari and I. Corvin), p. 205. IITRI, Chicago. Parsons, D.F. (1974) Electron microscopy and electron diffraction of wet biological material. In: Electron Microscopy, 1974, Eighth Znternational Congress (Ed. by J. V. Sanders and D. J. Goodchild), Vol. 2, p. 32. Australian Academy of Science, Canberra. Parsons, D.F., Matricardi, V.R., Subject, J., Uydess, I. & Wray, G. (1972) High voltage microscopy of wet whole cancer and normal cells: Visualisation of cytoplasmic structures and surface projections. Biochim. biophys. Acta, 290, 110. Parsons, D.F., Uydess, I. & Matricardi, V.R. (1974) High voltage electron microscopy of wet whole cells: effect of different cell preparation methods on visibility of structures. 3. Microsc. 100, 153. Robinson, V.N.E. (1974) The construction and uses of an efficient backscattered electron detector for scanning electron microscopy. J. Phys. E: Scient. Instrum. 7 , 650. Swift, J.A. & Brown, A.C. (1970) An environmental cell for the examination of wet biological specimens at atmospheric pressure by transmission scanning electron microscopy. J . Phys. E: Scient. Instrum. 3, 924.
77
A wet stage modification to a scanning electron microscope
by V. N. E. R O B I N S O NFaculty , of Applied Science, University of New South Wales, P.O. Box 1, Kensington, N . S .W. 2033, Australia SUMMARY
A modification to the vacuum system of a JSM2 scanning electron microscope has enabled hydrated specimens to be placed inside the specimen chamber of the instrument and to be surrounded by water vapour at a pressure up to approximately 1.3 kPa (10 Torr). The surface topography was observed by detecting the backscattered electrons using a wide angle backscattered electron detector placed close to the specimen. The microscope was operated in the normal scanning mode which allowed the examination of the surface topography of the specimens, whilst still retaining the depth of focus which is a feature of the SEM. This modification has enabled a resolution of approximately 0.2 pm to be obtained from biological specimens partially immersed in water at temperatures just above 0°C. INTRODUCTION
The ability to examine wet specimens in a controlled atmosphere in electron microscopes can greatly increase the versatility of these instruments and the information that can be obtained through their use. The operation of an electron microscope requires the electrons to travel through a high vacuum. The introduction of water vapour, or any other vapour or gas molecules, must be done in such a way as to preserve the high vacuum in the electron optics column. To achieve this, environmental control cells have been constructed for electron microscopes operated in the transmission mode, closed cells (Abrams & McBain, 1944 ; Fullam, 1972) and differentially pumped chambers (Matricardi, Hausner & Parsons, 1970; Moretz, Hausner & Parsons, 1970; Fukami et al., 1970; Parsons et al., 1972; Parsons, Uydess & Matricardi, 1974), scanning transmission mode (Swift & Brown, 1970; Morgan, Lebiedzik & White, 1973) and the conventional scanning mode (Lane, 1970). The cells for transmission and scanning transmission microscopes only provide information about the bulk structure of thin specimens. T o obtain information about the surface structure of thick specimens, Lane (1970) constructed an environmental cell whereby a flux of vapour molecules through a small aperture established a vapour cloud round the specimen. This design can accommodate only small specimens, has an unknown and variable water vapour pressure and can produce vacuum deterioration (Morgan et al., 1973). A design which allows a larger specimen size, currently about 10 mm diameter, in a static, known and controllable vapour pressure up to about 1-3 kPa has been developed for a
71
V.N . E. Robinson JSM2 scanning electron microscope. This design which incorporated a backscattered or rediffused electron detector (Robinson, 1974), allowed normal scanning electron microscope operation to obtain information about the surface structure of large specimens. DESIGN AND OPERATION
The principle employed was that at temperatures in the vicinity of O'C, the water vapour pressure was sufficiently low to enable 15-25 keV electrons to travel a few millimetres through the vapour. This distance was sufficiently long to enable these electrons to travel through the vapour, impinge upon the specimen and give off a signal which could be detected. The electron beam was formed, in the usual manner, in the high vacuum of the electron microscope column. The whole of the specimen chamber contained the water vapour in static equilibrium. The specimen was positioned as close as practical to the final aperture and the backscattered electron detector to minimize the electron path length in the vapour. The practicabilities of how this has been achieved in one instrument, a JSM2, are outlined below. At all stages of the modification, emphasis was placed upon minimum alteration to the microscope. The electron microscope column was isolated from the specimen chamber, the only connection being the 100 pm diameter final aperture. In the JSM2, this was achieved by blocking the diffusion pump from pumping the specimen chamber and placing O-rings around the final lens polepiece and the final aperture holder, the polepiece O-ring being permanently incorporated into the microscope. A vapour pressure of 1.3 kPa in the specimen chamber resulted in a slow leak across the aperture which required a pumping speed of less than 10 dm3per sec to Pa maintain a pressure of 1.3 x Torr) in the column. The loss of vapour from the specimen chamber was negligible. To situate the specimen as close as practical to the final aperture, repositioning of the final aperture to be at the bottom of the final lens polepiece was necessary. Experience has shown that this repositioning did not interfere with the double deflection scan system or alter the magnification calibration of the instrument. The emerging backscattered or rediffused electrons were detected by a wide angle detector placed as close as possible to the specimen and the final aperture. The detector was designed to collect as much of the signal as possible consistent with good specimen movement. It was constructed on the scintillator-light guide principle because this required no electrical connections likely to be fouled by the presence of water vapour and required no microscope alteration. Figure 1 illustrates the detector-specimen arrangement, embodying the above principles, currently used in a JSM2 scanning electron microscope. A block of plastic scintillator appropriately machined and positioned under the final aperture, was used for both the detector and light guide. Other scintillator light guide geometries have also been employed with similar results. To reduce beam scattering by the residual vapour, it was found necessary to reduce the residual vapour pressure by cooling the specimen and as much of the specimen chamber as practical, to a temperature just above 0°C. Partial cooling of the specimen chamber was achieved by a cold finger attached to the specimen stage and cooled externally with liquid nitrogen. Complete cooling of all the specimen chamber surfaces to 0°C has not been achieved in this modification, and this results in the vapour pressure being somewhat higher than would be expected from the temperature of the water. The specimen was mounted on a stub 10 mm diameter and 20 mm long. The
72
Wet stage mod$cation to the SEM
Fig. 1. Schematic illustration of the detector and specimen configuration employed for wet specimen microscopy in a JSMZ SEM.
specimen, stub and stub holder were cooled to 0°C prior to insertion into the chamber. A reservoir of about 5 g of ice and water was placed in the chamber when the specimen was inserted. The specimen was inserted so as to place it inside the detector. Excess vapour was pumped out through the pre-evacuation pump with the air lock valve open. When the pressure had reached equilibrium, the air lock valve was closed and the microscope was operated in the normal manner. Specimen insertion and removal was performed in the usual manner with both the column and chamber maintained under vacuum. The vapour pressure in the chamber was measured using a simple U tube manometer, one end of which was connected to a vacuum pump and maintained at a pressure of approximately 10 Pa (10-1Torr). The pressure in the chamber was measured against this, using diffusion pump oil as the manometer liquid. The beam accelerating voltage used depended upon the vapour pressure. At low vapour pressures, below approximately 100 Pa (1 Torr), very little beam scattering occurred and an accelerating voltage in the vicinity of 10 kV could be used to resolve detail of less than 0.2 pm on uncoated biological materials. At these low chamber pressures, the resolution achieved from this system, when examining gold coated specimens, appears to be the same as that achieved with the conventional secondary electron detector system, compare Fig. 2a and b. The lower resolution achieved from the low density biological materials appeared to be due to the increased penetration of the primary beam into these low density specimens. This was reduced by operating at a lower accelerating voltage and experience has shown that greater topographic contrast is obtained from the examination of low density specimens at lower accelerating voltages. On the other hand, high density specimens or specimens coated with a dense metal (Au) gave best contrast and resolution at the higher accelerating voltages (25 kV). Vapour pressures above approximately 100 Pa (1 Torr) resulted in increased beam scattering and required a higher beam voltage to obtain useful topographic information. An accelerating voltage in the range of 15-20 kV, resolving detail in the vicinity of 0.2 pm to 0.3 pm on biological material, gave optimum performance at pressures corresponding to saturated water vapour at temperatures in the vicinity of 0°C.At higher water temperatures optimum performance was achieved with accelerating voltages above 20 kV, with resolution in the vicinity of 0.5 pm. The beam currents employed depended upon vapour pressure, accelerating voltage and specimen density. With gold coated specimens at low vapour pressures, operating conditions were typically 25 kV accelerating voltage with a beam
73
V.N.E. Robinson
74
Wet stage modiJication to the SEM
Fig. 3. The condensation of water droplets onto the surface of chlorinated wool fibres partially immersed in water, no coating, 18 kV accelerating voltage. Magnification marker is 20 pm. A. Low density specimens and a vapour pressure in the current of 5 x vicinity of 0.6 kPa (6 Torr) required a beam current of approximately 200 x A with an accelerating voltage of 15 kV for optimum performance. The beam current could not be accurately measured during wet stage operation because of the scattering and conductivity influence of the water vapour and approximate beam currents were measured subsequently during dry microscopy. It takes only a few minutes to change the microscope from normal operation to wet stage capability. Additional time is required to precool the specimen chamber. This modification produces no vacuum deterioration in the column at chamber pressures less than 1.3 kPa (10 Torr), and does not result in an increase in the rate of column contamination or decrease in filament life. When the microscope is subsequently reverted to normal operation, there is no change in microscope performance. -_
Fig. 2. The secondary electron image (a) and wide angle backscattered electron image (b) of a gold coated germinating rust spore (MeZunosporu medusae). 25 kV accelerating voltage 5 x 10-la A beam current. In (b) the specimen was in a residual vapour pressure of approximately 20 Pa.
Magnification marker is 2 pm,
75
V . N.E. Robinson USES
Some of the uses to which a microscope wet stage can be applied have been previously outlined (Lane, 1970; Morgan et al., 1973; Parsons, 1974). This modification has been used for a variety of different studies, the complete results of which will be reported at a later date. T o demonstrate the versatility of this modification, this section contains a brief report of some of the results obtained to date. Figure 3 shows a micrograph of chlorinated wool fibres partially longitudinally immersed in water. What appears to be condensation droplets can be observed on the surface of the fibres. These droplets have been produced on many surfaces by cooling the specimen. They subsequently disappear, presumably by evaporation as they diminish in size, after a few minutes. Figure 4 shows formaldehyde cross linked fibres partially immersed in water. Figures 3 and 4 illustrate the image clarity attainable with this modification. This is sufficient to provide much information about the wet appearance of biological material and to make comparisons of the surface appearance, wet and dry, of these materials. The examination of wet hydrophilic specimens presents some problems because of the tendency of the water to spread over the specimen surface and obscure topographic detail. This modification has been used for evaporation and sublimation studies, with the vapourization being produced by vacuum pumping. The condensation and growth of water droplets onto different surfaces has been observed. The
Fig. 4. HCHO crosslinked wool fibres partially immersed in water, no coating, 17 kV accelerating voltage. Magnification marker is 20 pm.
76
Wet stage modification to the SEM shape and contact angle of water droplets of sizes from approximately 2 pm up have been observed. The large volume available inside the detector could enable small instruments to be placed inside the detector to perform some in situ experiments. The detector design may be modified to accommodate different types of specimen holders for Werent experiments. CONCLUSIONS
The design features of a wet stage modification to a scanning electron microscope have been discussed and the performance of a unit constructed for a JSM2 SEM has been presented. In principle this design can be modified to suit most instruments. The whole of the specimen chamber contains the water vapour at a pressure which is static, known and controllable. Specimens of size up to 1Omm diameter can be accommodated and have been examined in the SEM whilst they were partially immersed in water at temperatures close to 0°C.
References Abrams, I.M. & McBain, J.W. (1944) A closed cell for electron microscopy. J . uppl. Phys. 15, 607. Fukami, A., Etho, T., Ishihara, W., Katch, M. & Fujiwara, K. (1970) Pressurised specimen chamber for the electron microscope. Proc. 28th Ann. Meeting E.M.S.A. (Ed. by C. J. Arceneaux), p. 546. Claitor’s, Baton Rouge. Fullam, E.F. (1972) A closed wet cell for the electron microscope. Rev. scient. Instrum. 43,245. Lane, W.C. (1970) The environmental control stage. In: Scanning Electron Microscopy, 1970 (Ed. by 0. Johari), p. 41. IITRI, Chicago. Matricardi, V.R., Hausner, G.G. 81Parsons, D.F. (1970) Hydration chamber for JEOLCO 200 kV microscope. Proc. 28th Ann. Meeting, E.M.S.A. (Ed. by C. J. Arceneaux). p. 542. Claitor’s, Baton Rouge. Moretz, R.C., Hausner, G.C. Jr. & Parsons, D.F. (1970) Studies on water in the hydration chamber of a modified electron microscope. Proc. 28th Ann. Meeting, E.M.S.A. (Ed. by C . J. Arceneaux), p. 544. Claitor’s, Baton Rouge. Morgan, R.S., Lebiedzik, J. & White, E.W. (1973) Compact, controlled atmosphere cell for TSEM of hydrated biological materials. In: Scanning Electron Microscopy, 1973 (Ed. by 0. Johari and I. Corvin), p. 205. IITRI, Chicago. Parsons, D.F. (1974) Electron microscopy and electron diffraction of wet biological material. In: Electron Microscopy, 1974, Eighth Znternational Congress (Ed. by J. V. Sanders and D. J. Goodchild), Vol. 2, p. 32. Australian Academy of Science, Canberra. Parsons, D.F., Matricardi, V.R., Subject, J., Uydess, I. & Wray, G. (1972) High voltage microscopy of wet whole cancer and normal cells: Visualisation of cytoplasmic structures and surface projections. Biochim. biophys. Acta, 290, 110. Parsons, D.F., Uydess, I. & Matricardi, V.R. (1974) High voltage electron microscopy of wet whole cells: effect of different cell preparation methods on visibility of structures. 3. Microsc. 100, 153. Robinson, V.N.E. (1974) The construction and uses of an efficient backscattered electron detector for scanning electron microscopy. J. Phys. E: Scient. Instrum. 7 , 650. Swift, J.A. & Brown, A.C. (1970) An environmental cell for the examination of wet biological specimens at atmospheric pressure by transmission scanning electron microscopy. J . Phys. E: Scient. Instrum. 3, 924.
77
