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Preparation of microgrids as specimen supports for high resolution electron microscopy
Micron, 1977, Vol. 8: 29-31. Pergamon Press. Printed in Great Britain,
SHORT COMMUNICATION
Preparation of microgrids as specimen supports for high resolution electron microscopy R. REICHELT,
T. KONIG
and
G. WANGERMANN
A k a d e m i e der Wissenschaften der D D R , Z e n t r a l i n s t i t u t f u r Molekularbiologie, A b t . Elektronenmikroskopie, 1 1 15 Berlin, G . D . R .
Manuscript
r e c e i v e d A u g u s t 25, 1976
A simple methodfor preparing microgrids is described which gives reproducible results. The hole size can be controlled up to a certain maximum value which makes up to 93-95% of the useful grid area availablefor study. The microgrids are prepared by immersing glass slides into a 0.25% solution of ceUulose acetobutyrate ( Triafol) containing 1% glycerol. The liquid film is then dried, floated off on distilled water, transferred to copper grids and exposed to an atmosphere of ethyl acetate to enlarge the hole size. Finally the meshes of the microgrids are strengthened by a coating of carbon. Microgrids prepared in this way have beenfound suitable for a variety of specimens and provide good mechanical and thermal stability.
Microgrids have been used for about a decade in high resolution electron microscopy as supports for various specimens including extremely thin foils. Their functions are to provide mechanical and thermal stability and at the same time avoiding or reducing to a minimum any interference with the amount of information about the specimen that can be obtained from the electron optical image. While a number of methods for preparing microgrids have been developed (Fukami et al., 1965, 1972 ; Kleinschmidt, 1970; Gordon, 1972 ; Hoelke, 1975) all have certain disadvantages. Some are not only time-consuming but in practice do not yield consistent results so that microgrids of variable quality are produced. The method described by Fukami et al. (1965), for example, is relatively complicated and the results influenced by changes in room temperature and humidity. Other procedures, such as that developed by Hoelke (1975), are simple but the holey area is only about 10-20% of the whole grid area so that the probability of finding suitable parts of the specimen over the holes is correspondingly low. In the present paper a simple method for producing microgrids is described which largely overcomes the disadvantages of those referred to above. The basic procedure for preparing the microgrids is shown schematically in Fig. 1. A micro29
scope glass slide is cleaned with a surface acting agent (of hydrophilic character), thoroughly rinsed with tap water and dried with a piece of cloth (Fig. la). The material we used for the microgrids was cellulose acetobutyrate (Triafol). This was prepared as a 0.25~}/O solution in ethyl acetate to which was added about 1% glycerol. The cleaned microscope glass slide was then immersed in the emulsion for about 5-10 sec. Upon removal, a thin film of Triafol interspersed with droplets of glycerol covered the glass surface (Fig. lb). The film was then air-dried for about 10min with the slide mounted vertically (Fig. 1c). In order to dissolve the droplets of glycerol, the slide was held in a stream of water vapour for about 2rain. Subsequently the film was floated off on the surface of double-distilled water (Figs. ld,e), transferred to copper grids (200, 300 or 400 mesh) and dried with infra-red light. The diameter of the holes was then enlarged by exposing the holey Triafol film on its copper grid to an atmosphere saturated with ethyl acetate. The size of the holes can be increased by prolonging the exposure time and in our case was controlled by observing the effect by phase contrast (light) microscopy. At a room temperature of 295 K, maximum enlargement of the holes are obtained between 20-30sec exposure. This was found to be applicable to all films prepared from the same batch.
R. I,t+<'ichelhT. K6ni~ a~ld ( ;. Waneevil]aim
'31)
(]
b
| ........
J
layer of Triafo|
of Trla fo!
f
.i,
.
,~ '"
~'
"-~
ml.-- microgrid
~t
Preparation of Microgrids In order to strengthen the holey films they were coated with a layer (about 30nm) of carbon in an evaporator in the usual way. T h e y can then be stored until required for use (Fig. lf). For certain types of work it may be desirable to use the microgrids to support a very thin film of carbon (i.e. one of the order of about 2 - 4 n m thick), as for example, when examining specimens of the kind shown in Fig. 4. To do this the microgrids were prepared as described above including the use of a relatively thick coating of carbon to strengthen the meshes. They were then coated with a layer of Triafol, again using a 0.25% solution in ethyl acetate, and then allowed to dry. The surface of the Triafol film was then coated with the very thin film of carbon by evaporation in the usual way. Finally the Triafol film was carefully dissolved by exposing it to an atmosphere of ethyl acetate fbr several hours using the apparatus described by Studer (see Oliver, 1973). In this process certain residues remain which act as an adhesive and closely bind the very thin film of carbon to the microgrid. It may also be remarked that during exposure to the ethyl acetate, the Triafol of the microgrid is also dissolved but the integrity of the structure is preserved by the coating of thick carbon so that the main supporting structure is essentially a microgrid of carbon. Figure 2 is a micrograph at low magnification of a microgrid mounted on a copper grid. With maximal hole size, the relative proportion of holes amounts to 93-95% of the whole grid so that the area for study is not essentially reduced by the use of such microgrids. The most frequent hole size is between l~tm and 7~tm. Figure 3 is a micrograph at higher magnification illustrating the typical appearance of the microgrids. In assessing the quality of the microgrids, two factors are especially important, their behaviour
31
when loaded with different specimens and when exposed to the electron beam, particularly when working at high magnifications when the intensity increases. In the former case, microgrids prepared as described in this paper were found to be sufficiently stable mechanically to support a range of specimens including isolated macromolecules, ribosomes and stains in solution. As a criterion for thermal stability, the amount of specimen drift was measured (Fukami et al., 1972) which, at a given magnification must be less than the attainable resolution. At electron optical magnifications of between 200,000 to 500,000 times and a beam density of about 10-°A/cm 2, the latter requirement was met. Figure 4 illustrates results obtained at such magnifications of negatively stained protein crystals mottnted on a very thin carbon film supported by a microgrid.
REFERENCES Fukami, A., Adachi, K. and Katoh, M., 1965. A new method of preparation of a self-perforated micro plastic grid and its application (I). 07. Electron. Microsc., 14:112-118. Fukami, A., Adachi, K. and Katoh, M., 1972. Micro grid techniques (continued) and their contribution to specimen preparation techniques for high resolution work. a7. Electron. Microsc., 21: 99-108. Gordon, C. N., 1972. The use of octadecanol monolayers as wetting agents in the negative staining technique, a7. Ultrastruct. Res., 39: 173-185. Hoelke, C. W., 1975. Preparation and use of holey carbon microgrids in high resolution electron microscopy. Micron, 5:307-311. Kleinschmidt, A. K., 1970. Bright field and dark field electron microscopy of biomacromolecules mounted on thin support films. Berichte Bunsengesellschaft, 74:1190-1196. Oliver, R. M., 1973. Negative stain electron microscopy of protein macromolecules. In: Methods in Enzymology, 27: 616-672. Academic Press, New York and London.
Fig. 1. Schematic diagram showing the main stages (a-f) in preparing the microgrids. Fig. 2. Electron micrograph of a Triafol microgrid reinforced with carbon and mounted on a copper grid. x 100. Fig. 3. Part of the microgrid shown in Fig. 2 as seen at higher magnification, x 4600. Fig. 4. Electron micrographs of protein crystals negatively stained with ammonium molybdalL and mounted on a very thin film of carbon supported by a microgrid, x 300,000.
SHORT COMMUNICATION
Preparation of microgrids as specimen supports for high resolution electron microscopy R. REICHELT,
T. KONIG
and
G. WANGERMANN
A k a d e m i e der Wissenschaften der D D R , Z e n t r a l i n s t i t u t f u r Molekularbiologie, A b t . Elektronenmikroskopie, 1 1 15 Berlin, G . D . R .
Manuscript
r e c e i v e d A u g u s t 25, 1976
A simple methodfor preparing microgrids is described which gives reproducible results. The hole size can be controlled up to a certain maximum value which makes up to 93-95% of the useful grid area availablefor study. The microgrids are prepared by immersing glass slides into a 0.25% solution of ceUulose acetobutyrate ( Triafol) containing 1% glycerol. The liquid film is then dried, floated off on distilled water, transferred to copper grids and exposed to an atmosphere of ethyl acetate to enlarge the hole size. Finally the meshes of the microgrids are strengthened by a coating of carbon. Microgrids prepared in this way have beenfound suitable for a variety of specimens and provide good mechanical and thermal stability.
Microgrids have been used for about a decade in high resolution electron microscopy as supports for various specimens including extremely thin foils. Their functions are to provide mechanical and thermal stability and at the same time avoiding or reducing to a minimum any interference with the amount of information about the specimen that can be obtained from the electron optical image. While a number of methods for preparing microgrids have been developed (Fukami et al., 1965, 1972 ; Kleinschmidt, 1970; Gordon, 1972 ; Hoelke, 1975) all have certain disadvantages. Some are not only time-consuming but in practice do not yield consistent results so that microgrids of variable quality are produced. The method described by Fukami et al. (1965), for example, is relatively complicated and the results influenced by changes in room temperature and humidity. Other procedures, such as that developed by Hoelke (1975), are simple but the holey area is only about 10-20% of the whole grid area so that the probability of finding suitable parts of the specimen over the holes is correspondingly low. In the present paper a simple method for producing microgrids is described which largely overcomes the disadvantages of those referred to above. The basic procedure for preparing the microgrids is shown schematically in Fig. 1. A micro29
scope glass slide is cleaned with a surface acting agent (of hydrophilic character), thoroughly rinsed with tap water and dried with a piece of cloth (Fig. la). The material we used for the microgrids was cellulose acetobutyrate (Triafol). This was prepared as a 0.25~}/O solution in ethyl acetate to which was added about 1% glycerol. The cleaned microscope glass slide was then immersed in the emulsion for about 5-10 sec. Upon removal, a thin film of Triafol interspersed with droplets of glycerol covered the glass surface (Fig. lb). The film was then air-dried for about 10min with the slide mounted vertically (Fig. 1c). In order to dissolve the droplets of glycerol, the slide was held in a stream of water vapour for about 2rain. Subsequently the film was floated off on the surface of double-distilled water (Figs. ld,e), transferred to copper grids (200, 300 or 400 mesh) and dried with infra-red light. The diameter of the holes was then enlarged by exposing the holey Triafol film on its copper grid to an atmosphere saturated with ethyl acetate. The size of the holes can be increased by prolonging the exposure time and in our case was controlled by observing the effect by phase contrast (light) microscopy. At a room temperature of 295 K, maximum enlargement of the holes are obtained between 20-30sec exposure. This was found to be applicable to all films prepared from the same batch.
R. I,t+<'ichelhT. K6ni~ a~ld ( ;. Waneevil]aim
'31)
(]
b
| ........
J
layer of Triafo|
of Trla fo!
f
.i,
.
,~ '"
~'
"-~
ml.-- microgrid
~t
Preparation of Microgrids In order to strengthen the holey films they were coated with a layer (about 30nm) of carbon in an evaporator in the usual way. T h e y can then be stored until required for use (Fig. lf). For certain types of work it may be desirable to use the microgrids to support a very thin film of carbon (i.e. one of the order of about 2 - 4 n m thick), as for example, when examining specimens of the kind shown in Fig. 4. To do this the microgrids were prepared as described above including the use of a relatively thick coating of carbon to strengthen the meshes. They were then coated with a layer of Triafol, again using a 0.25% solution in ethyl acetate, and then allowed to dry. The surface of the Triafol film was then coated with the very thin film of carbon by evaporation in the usual way. Finally the Triafol film was carefully dissolved by exposing it to an atmosphere of ethyl acetate fbr several hours using the apparatus described by Studer (see Oliver, 1973). In this process certain residues remain which act as an adhesive and closely bind the very thin film of carbon to the microgrid. It may also be remarked that during exposure to the ethyl acetate, the Triafol of the microgrid is also dissolved but the integrity of the structure is preserved by the coating of thick carbon so that the main supporting structure is essentially a microgrid of carbon. Figure 2 is a micrograph at low magnification of a microgrid mounted on a copper grid. With maximal hole size, the relative proportion of holes amounts to 93-95% of the whole grid so that the area for study is not essentially reduced by the use of such microgrids. The most frequent hole size is between l~tm and 7~tm. Figure 3 is a micrograph at higher magnification illustrating the typical appearance of the microgrids. In assessing the quality of the microgrids, two factors are especially important, their behaviour
31
when loaded with different specimens and when exposed to the electron beam, particularly when working at high magnifications when the intensity increases. In the former case, microgrids prepared as described in this paper were found to be sufficiently stable mechanically to support a range of specimens including isolated macromolecules, ribosomes and stains in solution. As a criterion for thermal stability, the amount of specimen drift was measured (Fukami et al., 1972) which, at a given magnification must be less than the attainable resolution. At electron optical magnifications of between 200,000 to 500,000 times and a beam density of about 10-°A/cm 2, the latter requirement was met. Figure 4 illustrates results obtained at such magnifications of negatively stained protein crystals mottnted on a very thin carbon film supported by a microgrid.
REFERENCES Fukami, A., Adachi, K. and Katoh, M., 1965. A new method of preparation of a self-perforated micro plastic grid and its application (I). 07. Electron. Microsc., 14:112-118. Fukami, A., Adachi, K. and Katoh, M., 1972. Micro grid techniques (continued) and their contribution to specimen preparation techniques for high resolution work. a7. Electron. Microsc., 21: 99-108. Gordon, C. N., 1972. The use of octadecanol monolayers as wetting agents in the negative staining technique, a7. Ultrastruct. Res., 39: 173-185. Hoelke, C. W., 1975. Preparation and use of holey carbon microgrids in high resolution electron microscopy. Micron, 5:307-311. Kleinschmidt, A. K., 1970. Bright field and dark field electron microscopy of biomacromolecules mounted on thin support films. Berichte Bunsengesellschaft, 74:1190-1196. Oliver, R. M., 1973. Negative stain electron microscopy of protein macromolecules. In: Methods in Enzymology, 27: 616-672. Academic Press, New York and London.
Fig. 1. Schematic diagram showing the main stages (a-f) in preparing the microgrids. Fig. 2. Electron micrograph of a Triafol microgrid reinforced with carbon and mounted on a copper grid. x 100. Fig. 3. Part of the microgrid shown in Fig. 2 as seen at higher magnification, x 4600. Fig. 4. Electron micrographs of protein crystals negatively stained with ammonium molybdalL and mounted on a very thin film of carbon supported by a microgrid, x 300,000.