Development of Methods to Observe a Living Cell and Macromolecules in Aqueous Solution with Scanning Electron Microscope at Nanometer Resolution

Abstract

In electron microscopy, transparency of specimens against a beam of electrons in TEM or intensity of secondary electrons and so on induced by an incident electron beam in SEM is translated into contrast. Any material surrounding a specimen, which prevents electron beam passing or detection of secondary electrons, obstructs to create an image. Hence, electron microscopy intrinsically requires high voltage electron beam irradiation of specimens and high vacuum under 10-4 Pa in the cell for specimens. Water in samples must be replaced with some resins or completely dried up. These conditions make it difficult to observe wet or living samples like enzymes retaining catalytic activities or living cells in aqueous solution. To image wet and living samples using electron microscopy at nanometer resolution, we are developing a new wet cell for SEM whereby living cells and enzymes can be maintained in aqueous solution. A carbon thin layer with thickness of 20 nm was made by vacuum evaporation. We applied it as a diaphragm withstanding a pressure gap for separating a specimen in solution at atmospheric pressure from high vacuum environment. Cells and enzymes were placed on its surface of the atmospheric side. They were imaged using SEM. The EM photographs show detailed structures of the cell membrane and the enzymes.