Conductive staining of biological specimens for scanning electron microscopy with special reference to ligand-mediated osmium impregnation.

Biological specimens for scanning electron microscopy are made electron-conductive and non-charging by intense osmication with tannic acid, thiocarbohydrazide, hydrazine hydrate or other bifunctional ligands (or mordants). Such osmication also minimizes tissue shrinkage during dehydration and drying, reduces tissue damage during metal coating and scanning observations, and circumvents the use of a thick metal coating which hinders high resolution. Intense osmication when repeated eliminates the need for a metal coating and produces a well-contrasted scanning image with good resolution. Moreover, such osmication reduces tissue damage during ion implanting, enhances the depth of imaging, allows x-ray microanalysis without coated metals, and permits resin-embedded observations, as well as continuous or repeated microdissections in the scanning electron microscope without charging. Intense osmication forms a surface coat or thickens the cell membrane. This surface coat consists of electron dense deposits of 4-20 nm thickness. The hydrazine hydrate-osmium method can be used in the vapor phase. The vapor phase application has the advantage of staining even plastic samples for non-metal-coated scanning electron microscopy.