Films as substrates
The developing field of electroactive polymers, when the polymers are manipulated into thin film forms, offers potentially ideal materials for substrates. Machenically, they are strong, flat, thin, and stable in the electron beam. For biological specimens, they are hydrophilic and their chemistry could be adapted to the particular specimen so the attachment could 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 could be made fresh just prior to use to evaide contamination. Up to now, thin C films have been the best 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 could be gained. But 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. But 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. But 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 could 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.
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