The following paper appeared in Proceedings of the XIIth International Congress for Electron Microscopy; San Francisco Press, San Francisco, CA, 1990, pp. 290-291.
CONDUCTING POLYMER FILMS AS EM SUBSTRATES
M. N. Simon, B. Y. Lin, H. S. Lee*, T. A. Skotheim*, and J. S. Wall
Department of Biology, Department of Applied Science*, Brookhaven National Laboratory, Upton, NY 11973
The developing field of electroactive polymers, when the polymers are manipulated into thin film forms, offers potentially ideal materials for EM substrates. Machenically, they are strong, flat, thin, and stable in the electron beam. For biological specimens, they are hydrophilic and their chemistry can be adapted to the particular specimen so the attachment can be gentle with no denaturation. They have excellent cal properties of low Z, little grain, good electrical conductivity and no observable radiation damage in the electron beam. Such films can be made fresh just prior to use to avoid contamination.
Up to now, thin C films have been the best EM substrates for direct deposition of biological materials. With high vacuum evaporation of C, 20A thick films with >90% coverage of 4u windows in a holey film canm be obtained. However, these films rapidly become hydrophobic by absorbing contaminants fro the air and so need something like the wet-film technique or glow discharge to keep them hydrophilic.1 Even so, there are often hot spots of irreversible attachment of biological molecules causing distortion and even denaturation. Also, C films show a significant phase grain, presumably from C60 microcrystals.
Cast films such as formvar and parlodion are often used for biological specimens. They are easy to make, need little equipment and, being about 100A thick or more, are strong enough to cover a grid hole. However,m they are insulating and suffer significant damage in the electron beam. Since they are not very hydrophilic, they are usually shadowed lightly with C before depositing a biological specimen. The exceptions are basic protein-nucleic acid films where the denatured protein film adheres well to parlodion and the later staining or shadowing makes it less insulating.
The Langmuir-Blodgett (L-B) technique can be used to make monolayer films at an air-water interface. This is not a cast plastic film but a controlled aggregation where the thickness is determined by the number of monolayers. These films are very flat, can be quite strong, but are usually insulators and sensitive to beam damage. However, it has been shown recently that L-B polypyrrole films are excellent conductors (with conductivities equivalent to metals).2
Polypyrrole films are formed by casting a hydrocarbon pyrrole (in this case, 3-hexadecylpyrrole) in an organic solvent on an air-water interface where the water subphase contains an oxidant (FeCl3). Free pyrrole as the vapor is added to cause polymerization. Alternatively, the C16-pyrrole and pyrrole can be mixed before casting the film. Grids are dropped on the film, picked up and washed as with thin C films, and the sample applied. They can then be freeze-dried overnight or air-dried for visualization in the STEM. An electron micrograph of an air-dried tobacco mosaic virus (TMV) specimen is shown in Fig. 1.
The thickness of the film is determined by the number of monolayers plus surface pyrrole. Films of thickness t<30A are not mechanically strong but if t>30A the films can get lumpy from added pyrrole. However, they are all excellent conductors and extremely resistant to electron beam damage. Fig. 2 shows a plot of the measured film thickness versus the time of exposure to pyrrole vapor. Since these films are of low Z material, they are good for visualizing biological molecules and most samples seem to adhere very well to them. When the thickness is just right, they are extremely uniform with little graininess. Further work is needed to eliminate lumpiness without sacrificing mechanical strength. Other possibilities are to chemically tailor the surface to the specific biological application, e.g. to use bilayers for membrane proteins.
Figure 1 (On L). STEM image of TMV which was unstained but air-dried. The full width of the micrograph is 0.512 microns, the diameter of TMV is 18 nm, and the thickness of the film is 3.9 nm.
Figure 2 (On R). The thickness of the film plotted in A versus the length of time the C16-pyrrole film, cast on 0.02M FeCl3 for 5 min, was exposed to pyrrole vapor.
- J. S. Wall, J. F. Hainfeld and K. D. Chung. 1985. Proc. 43rd Annu. EMSA Meet., ed. G. W. Bailey, p. 318. San Francisco: San Francisco Press.
- K. Hong, R. B. Rosner and M. F. Rubner. 1990. Chemistry of Materials, 2, 82-88.
- This work was supported by USDOE-OHER and NIH Grant #RR01777.
Thanks to the San Francisco Press for allowing us to reproduce this online.