Abstract
Protein complexes in cells and tissues play critical roles in various physiological functions, including embryogenesis and signal processing.To observe the dynamics of protein complexes, high-resolution and high-throughput electron microscopy (EM) in aqueous solution is required. However, standard EM requires the sample to be in a vacuum. With ASEM, an inverted scanning electron microscope (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 film window can hold a few milliliters of culture medium, allowing various types of cells to be cultured in a stable environment. This system was used to develop in situ correlative OM/SEM immuno-microscopy in liquid. Using this method, we have observed a dynamic string-like gathering of STIM1 on the endoplasmic reticulum in Jurkat T cells in response to Ca2+ store depletion. We have also observed filamentous-actin (F-actin) and tubulin in the growth cones of primary-culture neurons, as well as in synapses. Further,we have monitored in-situ electrochemical reactions in electrolytes, and the melting and solidification of solder using ASEM.
