MICROANALYSIS OF RAPIDLY-FROZEN TISSUE IN THE FIELD EMISSION STEM

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Scanning transmission electron microscopy (STEM), electron energy-loss spectroscopy (EELS), and energy-dispersive X-ray spectroscopy (EDXS) have been developed and applied to the measurement of calcium and other ions in neurons. The developments include: capability to vitrify cultured brain slices, improved cryosectioning technique, ability to characterize frozen-hydrated sections by EELS, low-dose digital dark-field STEM imaging of the freeze-dried sections, and high-efficiency X-ray microanalysis. These methods have been applied to identify calcium buffering organelles in dendrites of CA3 hippocampal pyramidal cells in the minutes immediately following afferent synaptic activity. The results reveal a subset of endoplasmic reticulum as a major, high-capacity Ca++ buffer in these dendrites. The sequestration activity of these organelles was graded and reversible. It was also extremely robust. Following strong tetanic stimulation, the total Ca++ concentration within these organelles increased twentyfold, on average. Analytical electron microscopy has also been used to measure calcium and other elemental concentrations in rapidly frozen neurohypophyseal nerve endings, in order to determine the role of calcium in the secretion of posterior pituitary hormones.