Abstract
Low-dose, dark-field imaging in the field-emission STEM is a well documented method for mass mapping isolated protein molecules and macromolecular assemblies. Now, technical advances can provide truly thin, minimally compressed cryosections of unfixed, direcdy frozen tissues. These sections are thin enough-and have enough ultrastructural detail-to make mass mapping useful for directly and quantitatively measuring the in situ mass of cell constituents without beam-induced mass loss or other damage. In this report, we illustrate how dark-field STEM of cryosections can be used to measure the mass of single organelles in Purkinje cell dendrites, which is of interest because certain of these organelles participate in ion and water flow following synaptic activity-induced calcium mobilization.Cryosections, prepared in a Reichert FC-4E cryoultramicrotome at ca. -160 C, were cut to a nominal thickness of 100 nm on a 35° Diatome diamond knife with the aid of an antistatic device. Sections mounted on carbon/Formvar-coated grids were transferred (frozen-hydrated) into a modified VG HB501 field-emission STEM prior to freeze-drying at ca. -110 C.
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