Simultaneous Imaging of Cryo-Bright Field, Dark Field STEM and SEM Using Unroofed Living Cells with Special Reference to Membrane Cytoskeletons

Cryo-electron microscopy has been used exclusively so far for single particle analysis or crystallography of purified proteins. Recently, technical improvements on rapid freezing and cryo-microtome permitted structural analysis of organelle or macromolecules in native state using whole cells (1,2) and vitreous cryo-sections (3). However, cryo-sections don’t scatter electrons so much and therefore show quite low contrast in addition to weakness against electron irradiation. They complicate a searching and a focusing of samples. In order to overcome these difficulties, we are improving unroofing techniques for preparation of cryo-electron microscopy of cells together with development of SEM based cryo-STEM. We succeeded simultaneous imaging of cryo-dark field and bright field STEM and secondary electron with extremely high contrast and accuracy for untreated cells.