High-Pressure Freeze and Freeze Substitution Electron Microscopy in C. elegans.

While traditional chemical fixation methods for C. elegans electron microscopy (EM) have provided invaluable anatomical and structural information, the development of high-pressure freeze (HPF) and freeze substitution (FS) protocols offers advantages for high-resolution imaging. Specimens prepared using HPF methodology exhibit fewer distortion artifacts due to fixation and dehydration, have improved antigenicity, and result in a more physiologically accurate structural representation of the worm. In the HPF technique, freely moving worms are frozen at high-pressure (2100 bar) and low temperature (-180 °C) within milliseconds. These conditions prevent the formation of ice crystals that can damage cellular structures. Samples then undergo FS, during which worms are slowly brought to room temperature while substituting amorphous ice with organic solvents to preserve tissue in its near native state and provide contrast for imaging. FS can be performed in an automatic freeze substitution (AFS) machine or in makeshift, temperature controlled chambers. Fixed worms can be embedded in plastic resin and further processed for a variety of imaging techniques. Samples then viewed using scanning (SEM) or transmission electron microscopy (TEM) will show enhanced preservation of organelles, cell morphology, and antigenicity for immunocytochemistry.