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. Improvements in cryosectioning technique have been made, including a new accurate method for measuring the thickness of hydrated cryosections at low electron dose. These methods have been applied to the microanalysis of mouse hippocampus. Organotypic cultures of mouse hippocampus grown under well-defined physiological conditions (resting state or electrical stimulation) were rapidly frozen, cryosectioned to a thickness of 100 nanometers, and analyzed by x-ray microanalysis in a high-resolution scanning transmission electron microscope. Ionic concentrations were measured in dendrites of the CA1 region of the hippocampus. Preliminary results indicate that calcium concentrations are elevated in membrane-bound cisterns of dendritic endoplasmic reticulum in stimulated cells, whereas calcium is present at lower levels in the controls.
