Capturing Developmental Events of the C. Elegans Embryo by High Pressure Freezing After Monitoring by a Multi-Photon Imaging System.

Extract

High pressure freezing enables the rapid arrest of developmental events without prefixation. Standard chemical fixation is a time dependent event and may cause artifacts in sensitive cytoskeletal components. We are studying two developmental events in embryonic Caenorhabditis elegans: that involve changes in the cytoskeleton: spindle alignment and membrane fusion. The mitotic spindle undergoes rapid rotational alignment prior to certain differentiative divisions. We are trying to capture these events by anticipation their timing and rapid freezing. Precursor hypodermal cells of embryonic C. elegans undergo a transition from individual cells to a syncytium at the onset of morphogenesis. In an effort to visualize the fusion events, embryos were stained with the vital probe FM4-64 to highlight cell membranes. Development was monitored by fluorescent microscopy using multiple-photon excitation imaging to minimize photobleaching while providing clear images of deep sections. Small cellulose capillary tubes, as described by Hohenberg for isolation and high pressure freezing of individual cells, were not of sufficient optical quality for monitoring by a laser-scanning microscope.