Quantitative Scanning Transmission Electron Microscopy of Ultrathin Cryosections: Subcellular Organelles in Rapidly Frozen Liver and Cerebellar Cortex

Abstract

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 (⪡1.0 elastic mean free path) to use established dark-field techniques, modified for thickness-dependent nonlinearities, 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 percentage of dry mass retained after analysis at high dose were as follows: mitochondria (46.0 g dry mass/100 g hydrated mass, 67% mass retained); endoplasmic reticulum (27.9 g/100 g, 57%); and cytoplasm (16.3 g/100 g, 41%). These values 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. Parallel electron energy loss spectroscopy of dendritic organelles corroborated EDXS measurements, with an improved sensitivity that indicates the feasibility of quantitative calcium mapping.