Progress in scanning electron microscopy of frozen-hydrated biological specimens.

Modern scanning electron microscopy yields structural information down to 2 to 5 nm from thin, beam transparent biological specimens. This paper examines the possibilities of garnering this level of structural information from bulk, frozen-hydrated samples. Freeze-fractured, frozen-hydrated yeast cells, frequently taken as a yardstick to monitor progress in low-temperature scanning electron microscopy, have been used to optimize both metal shadowing methods and observation parameters (e.g. accelerating voltage, electron beam irradiation of the specimen). Uncoated frozen-hydrated yeast cells do not change electrically at an accelerating voltage of 30 kV. Increasing charging effects are however observed with decreasing accelerating voltages. Very thin metal films are therefore used for specimen coating to localize and enhance the specific secondary electron signal. Planar-magnetron sputtering of a 1 nm metal layer provides high resolution secondary electron images, at 30 kV, of freeze-fractured, frozen-hydrated yeast cells in an in-lens field-emission scanning electron microscope. Structural information comparable to that of transmission electron microscopy of freeze-fractures is attained. Planar-magnetron sputtering of either chromium, tungsten or platinum results in essentially the same information density (smallest visible significant structural detail). Frozen-hydrated samples are very beam sensitive and have to be observed under minimum dose conditions.