Light microscopy of living cells correlative to high-voltage EM and low-voltage SEM of cell cryo-whole-mounts

Abstract

Analysis of motility phenomena in a living cell observed with light microscopy can be significantly enriched by preparing a whole-mount of this cell for high voltage electron microscopy (to reveal the intracellular organization) and for low voltage scanning electron microscopy (to reveal the surface topography). In earlier studies, cell whole-mount prepration by chemical fixation and drying was adequate for studies of slow cellular motions at the subcellular level (e.g. receptor movements). Fast cellular motions analysed at the supramolecular level (e.g. transmitter release, cytoskeleton reorganization) required development of much faster cryo-immobilization methods. However, in studies of cells grown on grids, these freezing methods involved time consuming transfer of these cells , from an incubator to a freezer, making impossible fine correlations between images of a living cell and its cryo-whole-mount. To overcome this constraint for correlative microscopical studies of neoplastic cell motility, I designed an instrument consisting of a freezer attached to a light microscope and allowing cryoimmobilization within miliseconds after recording. The main objective of the current project was refinement of an instrument and improvement of appropriate specimen cryo-preparation techniques.