MICROANALYSIS OF RAPIDLY-FROZEN TISSUE IN THE FIELD EMISSION STEM

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Freeze-dried, ultrathin cryosections of directly frozen mouse liver and brain have been prepared and characterized by low-dose dark-field scanning transmission electron microscopy (STEM). These improved cryosections gave images comparable to those from conventional plastic sections. They were thin enough to use established dark-field techniques (modified for thickness-dependent nonlinearities) in order to measure the dry mass fraction of individual organelles, and hence to deduce their water content. Digital STEM imaging, in combination with electron and x-ray spectroscopy, has important biological applications, as illustrated by studies on calcium regulation in Purkinje neurons. Calcium concentrations per unit dry weight of dendritic compartments were determined by the peak/continuum method of energy-dispersive x-ray spectroscopy (EDXS), which necessarily overstates elemental concentrations because of beam-induced mass loss. The dry-mass content of organelles at low dose and the percent of dry mass retained after analysis at high dose were used to correct elemental concentrations for mass loss. Results indicated that the major calcium storage organelle in Purkinje cell dendrites is the endoplasmic reticulum, of which there are two types, distinguished by their levels of calcium. These techniques are being applied to characterize elemental and ionic distributions in other tissues, including hepatocytes and the protozoan parasite Giardia.