A new drying method of biological specimens for scanning electron microscopy: the t-butyl alcohol freeze-drying method.

A simple drying method for biological materials for scanning electron microscopy was developed. Fixed specimens were immersed in t-butyl alcohol after dehydration through a graded series of ethanol. When the room temperature fell below the melting point of t-butyl alcohol (25.5 degrees C), liquidized alcohol obtained by warming was used. Specimens in the alcohol were then frozen in a refrigerator. They were placed in the bell jar of a vacuum evaporator and simply evacuated with a rotary pump. The samples were completely dried within 1 h after the frozen alcohol was sublimated in the vacuum. When examined by scanning electron microscopy (SEM), both surface and intracellular structures were demonstrated in three-dimension without any significant drying artifacts. Careful comparison of the results indicated that the SEM images obtained by this method were either superior or equal to those obtained by the critical point drying method.