An apparatus has been designed that allows light microscopic observation of living cell phenomena immediately followed by fast immobilization of these phenomena with the aid of rapid freezing. Then the ultrastructure of this cell cryo-whole-mount can be observed in an electron microscope using a cryo-stage. This apparatus consists of phase contrast, epifluorescence, and polarized light optical systems assembled onto an optical bench. These optical systems are integrated into one unit with a specimen carrier assembled onto a sliding mechanism delivering the specimen into melting ethane. The unique feature of this apparatus is that it allow us to accomplish the fundamental goal of ultrastructural studies in life sciences, ie. direct correlation between images of a living cell with images of this cell's ultrastructure, thus direct interpretation of molecular mechanisms in a living cell. The goal of this project is to build a permanent instrument, to refine its operation, and to prepare its expansion toward new imaging capabilities by making six improvements: (1) addition of an ICCD (2) addition of a filter-wheel containing fluorochrome excitation and infrared filters, as well as a multi-band barrier filter (3) addition of a dedicated computer (4) addition of Nomarski optics and motorized specimen stage (5) preparation for the attachment of a real-time CLSM (6) preparation for the attachment of an AFM.
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