High Pressure Freezing Of Cultured Neurons

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Preservation of cellular structures and molecules in their native state is important for scientists who use microscopy to probe the mechanisms of cellular function. Most chemical fixation techniques for cells and biological samples significantly alter the distribution of elements in the preparation. To date the only technique that retains the elemental distribution in the native state is cryo preservation. This technique involves rapidly freezing the sample at a cooling rate approaching one million degrees centigrade per second. Slower freezing rates permit the formation of ice crystals that disrupt cellular structures. Unfortunately, because of the relatively slow heat transfer through biological samples, the depth of acceptable freezing by either plunge freezing or slam freezing is limited to a few microns. A new technique has been recently introduced that permits freezing of aqueous samples to depths of several hundred microns. This technique referred to as high pressure freezing, utilizes significantly elevated pressure, approximately 2300 bar, to retard crystal formation within the sample while heat is removed via liquid nitrogen jets. The purpose of this project is to use high pressure freezing to cryo-preserve cultured neurons. Some samples will subsequently be freeze-substituted, embedded, and thin sectioned and evaluated tomographically in the electron microscope. Other samples will be cryo sectioned and undergo x-ray microanalysis or possibly electron energy loss spectroscopy.