High voltage electron microscopy of wet whole cells

Abstract

Factors affecting the visibility of cell cytoplasmic structures and cell surface projections have been evaluated for examination of whole wet, unstained and unfixed cell preparations in the hydration chamber of a high voltage microscope.It was found that the thickness of the cells and that of the surrounding water layer were the most important factors affecting contrast and resolution of structures. One technique (surface spreading) flattened nuclei enough to see internal nuclear structure. Some washing and fixing (glutaraldehyde) methods caused regions of the grid of cultured cells to become hydrophobic with consequent improvement in contrast and resolution in these areas. Optimization of these factors appears feasible.The effects of cell freshness, blurring due to cell structure, vibration and Brownian movement, electron radiation and heat damage, microscope image mode, and photographic emulsion resolution are also discussed.Cell movements (e.g. cytoplasmic streaming) have not been observed to date, the possible reasons being radiation damage, cell bursting at lowered pressure, oxygen lack and concentration of metabolites on the grid surface. I N T R O D U C T I O NA method of examining whole cell mounts of wet cells using a hydration chamber in a 1 MV electron microscope has been previously described . Some internal vesicular cytoplasmic structures and fine surface processes of cells were visualized but other sub-cellular structure could not be clearly seen. It was shown that the thickness of the overlying water layer has a large effect in deteriorating contrast and resolution.The essential question with respect to our new technique is whether cytoplasmic structures of wet whole cell mounts can be visualized with better resolution than the light, phase or interference microscope. Even if it proves possible to examine living cells in the hydration chamber, an improvement over the light microscope images will be required to make the new technique significant.In this report we show further improvement in visualizing cytoplasmic organelles and surface processes and give an estimate of what factors control contrast and resolution. 153

Structure of Wet Specimens in Electron Microscopy

“…Quantitative comparisons between SSTEM and HVEM micrographs of whole cells (9) are not yet complete. Reported HVEM results on whole wet cells (10,11) indicate that conversion of present-day 1-and 3-Mv HVSM's to the scanning mode is required to penetrate the nucleus and cell cytoplasm of cells. Converted scanning HVEM's will have sufficient resolution for the initial phases of the wet cell electron microscope work, but eventually the application of field emission scanning HVEM's (12) will be necessary to give higher resolution.…”