Both quality and quantity of STEM results depend on the care and attention to detail of the specimen preparation. We continue to use t~e """"""""wet film"""""""" methodology developed previously and an array of test specimens,, each particularly sensitive to some aspect of specimen preparation. Quality is monitored by an automatic mass analysis program which recognizes particles, aligns them and compares their profiles to ideal models. Yield of high quality specimens has gradually improved to roughly 25%., Other specimens tend to have some acceptable areas which can be found by extensive searching. Therefore we place a high priority on improving yield by careful control of all preparation parameters. Higher resolution may be obtained with specimens embedded in vitreous """"""""ice"""""""", glucose or various negative stains. Since the STEM dark field signal is not sensitive to phase contrast, detailed interpretation then depends on differences in scattering power per unit volume. Of particular interest as an embedding medium for heavy atom staining is vanadatelborate, which gives excellent detail in protein areas but does not obscure 1.4 nm gold clusters specifically bound. Variation in the ratio of vanadate to borate permits contrast matching. Low dose, low temperature and freedom from phase contrast make images of fully embedded particles relatively straightforward to interpret, especially in conjunction with a parallel freeze dried specimen.'
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