Electron microscopy of fast intracellular membrane processes and localization of smalle easily diffusable substances like lipids using the leica high pressure freezer

Immunogold cytochemistry on ~ 100 nm thick cryosections of chemically fixed cells and tissues has revealed a wealth of information on the precise intracellular distribution of proteins. However the intracellular localization of lipids and small proteins has remained elusive, because of insufficient retention of such substances by this approach. Furthermore chemical fixation is rather slow (seconds to minutes) so that fast intracellular processes like vesicle fusion and budding can be finished off rather than being arrested. We aim to design novel methods, which will overcome these problems by cutting ultrathin cryosections from directly frozen cells without prior chemical fixation. The freezing process should be instantaneous to catch fast intracellular events and to avoid the formation of ice crystals, which would cause severe ultra structural damage. Subsequent handling of the cryosections with our recently developed technique, including an 'on section' fixation step is very promising with regard to the preservation of ultra structure and undisturbed distribution of lipids and small proteins. High pressure freezing ensures such preservation of biological specimens in an extremely short period of time, typically in about 8 ms. The high pressure of 2300 bar allows freezing of relatively thick specimens (200-300 mm) without (ultra)structural damage due to ice crystal formation The new Leica High Pressure Freezer is the apparatus of choice to cryo-preserve biological specimens adequately, and, in combination with cryo sectioning, will allow us to uncover hitherto unknown in situ distributions of molecules such as membrane lipids and will give us the opportunity to visualise membrane budding and fusion events.