Development of a New cryo-S(T)EM Technique Allowing Simultaneous STEM and SEM Imaging and its Application to Biological Samples

Abstract A new type of cryo-electron microscopy (cryo-S(T)EM) technique made possible by installing a new cryo-transfer holder and an anti-contamination trap on a scanning electron microscope (Hitachi SU9000) allowed simultaneous collection of both transmission (transmission electron microscopy, TEM) images and surface (scanning electron microscopy, SEM) images at -180°C. The ultimate temperatures of the cryo-transfer holder and the anti-contamination trap reached − 190°C and − 210°C, respectively, by applying a liquid nitrogen slush. The TEM images obtained by the new cryo-S(T)EM method showed quality equal or superior to that of images obtained by conventional 100 kV TEM, although the resolution did not improve at -180°C due to slight drifting of the sample stage. Cryo-S(T)EM also had the unexpected advantage of enabling observations of intracellular structures in thick frozen cells by accelerating the sublimation of ice surrounding the specimens. The spatial architecture of the cytoskeleton, poly-ribosome-chains, endoplasmic reticulum (ER), mitochondria, etc., became visible in thick frozen cells via sufficient (deep) sublimation of ice in combination with the unroofing method. In particular, it should be noted that the ER appeared as a wide and flat structure beneath the cell membrane while forming a large spatial network together with tubular ER.