Development of an Easy-to-use Cryo-electron Microscope for Simultaneous Observation of SEM and Transmission Images

New low voltage cryo-scanning transmission electron microscope (STEM) has been developed based on conventional high-resolution SEM, which enables to observe a transmitted image and a secondary electron (SEM) image simultaneously in a fresh frozen state. Simultaneous observation is advantageous to distinguish structures on surface or inside. New cryo-transfer holder enables to observe specimens at -190°C beneficial for low beam damage. Specimen chamber is kept clean with anti-contamination trap reached at -210°C using slash nitrogen. Frozen specimens are mounted easily on the holder in liquid nitrogen by using a cryo-station. It is possible to observe biological specimens without any reduction of resolution because extremely high contrast is retained on focusing. High-resolution single particle analysis is easily obtained even in small number of specimens because correction of contrast transfer function is not necessary due to STEM optics. In case of cell observation, cells cultured on carbon-coated grids are unroofed. Since some soluble components are eluted on unroofing, spatial architecture of cytoskeletal actin filaments and microtubules are observed at extremely high contrast.