Development of an in-solution observation method using atmospheric scanning electron microscopy (ASEM)

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.