Observation of Frozen-hydrated or Frozen-liquid Specimens by Cryo-SEM

Abstract

Cryo-SEM can visualize ultra-structures of hydrated or liquid specimens such as tissues, cells, gels, emulsions, and slurries without dehydration. General cryo-SEM is equipped with a cryo-stage and a cold trap on a room temperature SEM. For cryo-SEM observation it is important to freeze hydrated specimens in an amorphous state. After amorphous freezing, the specimen should be cross-sectioned to exposure the internal structure. Depending on the observation targets, we should choose a cross-sectioning method between freeze-fracturing, cryo-ultra-thin-sectioning, or ion milling. In many cases we can observe the cross-sectioned specimens without any coating even if the specimen is non-conductive. If composite images are not observed on the flat sectioned surface, the specimen should be heated to at around –100°C to sublimate the surface amorphous ice and exposure the observation targets above the cross-sectioned surface. If observation is difficult by the sample charging, conductive metal or carbon coating could help clear imaging. Cryo-SEM is a powerful tool to analyze sub-micrometer structures and their distribution or localization in hydrated or liquid specimens, since SEM allows observation extending from low magnification to high magnification.