[
International Worm Meeting,
2011]
Although marking the endocytic route with gold tracer has been successfully demonstrated in many mammalian cell types, its application to the intestinal cells of C. elegans has yet to be realized. In this study we fed the worm with 6 nm cationic gold particles in conjunction with OP50 for 30 minutes to two hours. Ultrathin sections of resin embedded as well as cryoprotected samples were prepared and examined with electron microscopy. The majority of gold tracer remained in the lumen of the gut in association with degrading bacteria. Rarely were gold particles found among the microvilli and much less in membrane-bound vesicles inside the cell. The apparent inaccessibility of gold tracer to endosomes from the apical pole of the intestinal cells may be inherent to this particular cell type, or our tracer may be too large. We are redoing the experiments with a smaller tracer. However, to rule out the possibility of an initial aldehyde fixation artifact, high pressure freeze fixation is also being tested. Here we report two advances in the handling of C. elegans for high pressure freezing: 1. Single worm confinement in cellulose capillary tubes for oriented frozen hydrated sectioning. 2. Rehydration of high pressure frozen/freeze substituted sample amenable for subsequent immunolocalization studies. In addition, we are testing standard post-embedding immunolocalizations on plastic-embedded tissues to identify the precise positions of actors in endocytosis.
[
International Worm Meeting,
2019]
The Tokuyasu method is room-temperature immuno-electron microscopy of cryosections. This technique is based on using sucrose as a cryoprotectant during both freezing and thawing steps [Tokuyasu 1973]. This method is optimized for preserving the antigenicity of the sample. Thus, the cocktails of heavy metals, organic solvents, and plastics that are used to stabilize, infiltrate, and stain the fine ultrastructure in plastic-section microscopy are not used. As a result, the inherent nature of the sample is minimally altered. However, compared to plastic sections, preservation of fine ultrastructure in tokuyasu sections remains inadequate and inconsistent. Despite attempts to optimize by several groups, the morphology of neurons obtained with this method remains poor still. Here we show that this method could be modified to preserve the C. elegans ventral nerve cord (VNC) at an unprecedented level of structural detail while preserving antigenicity. Osmolality driven structural collapse is a significant drawback of using sucrose in the vitrification step. When a sample block is immersed in sucrose during the process of infusion, sucrose-driven collapse of cellular turgor pressure result in deformation of cellular structures that are prone to collapse. Such tissue is the ventral nerve cord (VNC) of C. elegans. When sucrose is used, neuronal profiles of the VNC collapse inwardly, and their plasma membranes appear jagged. The critical difference between our method and previously published ones [Tokuyasu 1973, Liou et al. 1996, Bos et al. 2004, Nicolle et al. 2015], is the lack of sucrose. Removing sucrose from the sample vitrification step prevented the sucrose driven collapse of cellular turgor pressure and thus preserved the cellular structural form. This modification also necessitated compensatory changes in sample embedding, orienting and freezing steps to maintain ease of cryosectioning and sample vitrification due to lack of sucrose. To address these challenges we used high-pressure freezing to cryo-immobilize C. elegans in an enhanced copper tube. This sample carrier enables robust loading, grouping, orienting, freezing, and downstream processing to generate ribbons of hydrated cryosections of gently fixed or unfixed worms. We plan to use the Tokuyasu method of thawing cryosections as a screening step before correlative cryo fluorescence microscopy and cryo-electron tomography of C. elegans ventral nerve cord.