Immuno Correlative Microscopy in Solution Using Atmospheric Scanning Electron Microscope (ASEM): Observation of Dynamic Rearrangements of Molecular Complexes

Tatsuhiko Ebihara, Toshiyuki Murai, Hidetoshi Nishiyama, Mari Sato, Mitsuo Suga and Chikara Sato

Abstract: High-resolution observation of cells and tissues is principally carried out by electron microscopy (EM), although standard EM requires the sample be in vacuum. In the ASEM, an inverted SEM observes the wet sample from beneath an open dish while an optical microscope (OM) observes it from above. The disposable dish with a silicon nitride (SiN) film window can hold a few milliliters of culture medium, and allows various types of cells to be cultured in a stable environment. The use of this system for in situ correlative OM/SEM immuno-microscopy is explored, the efficiency of the required dual-tagged labeling assessed and the imaging capabilities of the ASEM documented. We have visualized a dynamic string-like gathering of STIM1 on the ER in Jurkat T cells in response to Ca²⁺ store depletion. We have also visualized filamentous-actin (F-actin) and tubulin in the growth cones of primary-culture neurons as well as in synapses. Further, radially running actin fibers were shown to partly colocalize with concentric bands of the Ca²⁺ signaling component Homer1c in the lamellipodia of neuron primary culture growth cones. After synapse formation, neurite configurations were drastically rearranged; a button structure with a fine F-actin frame faces a spine with a different F-actin framework.

Key words: immuno-EM, CLEM, ClairScope, STIM, CD44