Direct Observation of t-Butyl Alcohol Frozen and Sublimated Samples Using Low-Vacuum Scanning Electron Microscopy

Abstract

Frozen biological specimens in t-butyl alcohol were examined under a low-vacuum environment in a “wet SEM” or “variable pressure SEM (scanning electron microscope)” equipped with a cooling stage and highly sensitive backscattered detector of the YAG type. After fixation with glutaraldehyde and osmium tetroxide, rat tissue blocks (tracheae and kidneys), and cultured human carcinoma cells were dehydrated with a graded series of t-butyl alcohol. The specimens were directly frozen on the cooling stage at -10°C, evacuated to 20Pa in the specimen chamber, and observed by detecting backscattered electrons at accelerating voltages of 5-6kV. The images became clearer 20min after the vacuum reached 20Pa and revealed had good quality by 30min, probably because t-butyl alcohol was sublimated during the time. The cilia of tracheal ciliated cells, end-feet of the podocytes of the renal glomerulus, and processes of cultured cells were clearly observed without any serious preparation artifacts. Since the low-vacuum SEM of t-butyl alcohol frozen samples is both simple and provides high imaging quality, it is expected to be useful in a variety of biological fields such as the rapid pathological diagnosis.