Fixation for Electron Microscopy

Abstract

IT is a common practice to use buffers for making up fixing solutions for study of biological materials by electron microscopy, and such buffered fixatives are considered advantageous. This belief seems to have resulted from researches in days when the development of preparative techniques was in its infancy; and embedding media, like epoxy resins and polyester resins, were unknown in electron microscopy. It was experienced¹ that fixation by simple distilled water solutions of osmium tetroxide followed by embedding in monomer methacrylates caused gross vacuolization of the ground cytoplasm, which resulted in disorganization of the cellular membranous system; the nucleoplasm also precipitated into a coarse reticulum. Further experiments suggested that these defects in preservation were caused by a wave of acidity produced by the reaction of osmium tetroxide with tissues; this acidity preceded fixation of tissues by osmium tetroxide. It was, however, discovered by Palade¹ that preservation of cell structures was greatly improved if the fixative were buffered (pH. 7.3–7.5); and he recommended the use of Michaelis's² sodium acetate/ sodium veronal buffer for this purpose. This buffer seems to have been more often used than any other for making up solutions of osmium tetroxide. It has also been adopted for buffering potassium permanganate³ and formaldehyde for use in electron microscopy, though formaldehyde reacts with veronal to produce a substance that has no buffering value in the range of pH. of physiological importance (7.2–7.5)⁴.