Improved preservation of freeze-dried biological specimens

Abstract

Specimens prepared by the wet-film technique (injecting unstained biological specimens into a drop of buffer on a thin carbon substrate which has never seen air, washing extensively, blotting to a thin layer of liquid, plunging the grid into nitrogen slush, and freeze-drying overnight) then visualized in the scanning transmission electron microscope (STEM) usually have reasonably wel1-preserved structures. However, there is a certain variability from day to day and sometimes even from one area to another on a given grid. This can occur for different reasons which may be inextricably related. The thin carbon film can be non-uniform at the molecular level with hot spots for strong attachment of some specimens, a part of a biological specimen may attach strongly while the rest of it thrashes about in Brownian motion ruining any perceivable structure, and the final thickness of liquid before freeze-drying may vary slightly which may affect the preservation of the structure.