Planing frozen hydrated plant specimens for SEM observation and EDX microanalysis

Abstract

A procedure is described for forming a flat face on a frozen piece of plant tissue, which may then be observed fully-hydrated or lightly etched, and coated or uncoated with a metal film, in scanning electron microscopy (SEM). The frozen sample was planed with a glass knife at -80 degrees C in a cryo-ultramicrotome. The sections were discarded, and the planed block face placed on the cold stage in the microscope column, either for observation uncoated at low kV, or for light etching (-90 degrees C) to reveal the cell outlines. If a higher accelerating voltage was needed, the face was given an evaporative coating of Al in the cryo-preparation chamber and returned to the column. The advantages of the planed face over the usual fracture face are illustrated: imaging at a chosen rather than a chance position; clearer cellular and subcellular detail; preservation of hydrated gels like mucilage and swollen cell walls; the possibility of making serial parallel sections through the same piece of tissue; opportunities for accurate morphometric analyses on the planed face; capacity to produce longitudinal sections; preservation of very delicate structures that are destroyed by fixation and drying. A major advantage of the Al-coated planed face is the increased accuracy of energy-dispersive X-ray (EDX) microanalyses on a smooth rather than a rough surface. Tests are included which show that neither the light etching employed, nor successive planing, interferes with the analyses of elements in the frozen face.