Scanning Electron Microscopy


With this introductory chapter we attempt a synthesis of old and new knowledge of the physical principles that govern cryomethods. Interface phenomena determine the increase or decrease of the number of particles observed in frozen-hydrated suspensions because they occupy the air-liquid interface according to their specific balance of hydrophobic versus hydrophilic properties. Biological macromolecules are surrounded by organised water, the hydration shell, that prevents them from sticking to each other. Partial or complete removal of these hydration shells by freeze-drying or freeze-substitution leads to collapses or aggregations. The solvent-induced aggregation is usually decreased by prior cross-linking with adequate chemical fixatives. A new finding is that aggregations are also decreased with lower temperatures. This al lows us, for example, to preserve DNA-containing plasma from coarse aggregation even in cases where it has not been previously crosslinked.

When rapidly freezing a physiologically homogeneous population of bacterial cells without added cryoprotectants we find a 10-20 μm thick layer of cells without ice crystal formation. In deeper layers an increasing proportion of cells exhibits crystallization damage, although some cells are still well preserved.

Treatment of cells with aqueous solutions of OsO4 and/or uranyl acetate leads to 10-20 % (w/w) heavy metal deposit. Direct staining of sections of resin embedded material results in 10 times more deposit. The location of these deposited metals is at first unknown and is best visualized through a comparison with totally unstained material. Sufficient contrast is achieved with the dark field, or the ratio-contrast mode of imaging. The latter has the advantage of giving less weight to thickness variations than does the former. When observing fully unstained thin sections by ratio contrast the influence of the surface reliefs is thereby virtually eliminated. With CTEM the dark field mode requires too great a dose to use it successfully on frozen-hydrated material. With STEM this is possible for both dark field and ratio contrast.

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