TECHNIQUES FOR THE PRESERVAATION OF THREE-DIMENSIONAL STRUCTURE IN PREPARING SPECIMENS FOR THE ELECTRON MICROSCOPE†

Before examination of biological specimens can take place all volatiles must be removed. This presented paper details a method of sample preparation that reduces distortion and maintains 3-dimensional structure. Freeze drying disrupts cell structures as phase boundaries move through the specimen twice, first a solid boundary in the freezing process then a solid-vapor boundary during sublimation. To eliminate this the temperature of the ambient liquid is raised above its critical point. When it reaches the region where two phases cannot exist surface tension vanishes and fluid can escape without disrupting structures. Specimen preparation is given. Place droplet on a formvar-coated screen and fix with osmic acid vapor. Replace water with alcohol and alcohol with amyl acetate. Place specimen in a bomb and flood this with liquid carbon dioxide to replace the amyl acetate. Raise the temperature in the bomb to 35 Centigrade. Previous determinations show this to be above the critical point for carbon dioxide. Stereoscopic views of electron micrographs show three prepared specimens. There remains some minor distortion and flattening against the formvar screen. Outlines a method to retain cell structure in samples destined for the scanning electron microscope.