It is proposed to redirect somewhat the focus of the Integrated Microscopy Resource as a national center with the instrumentation, skills and knowledge for the application of light and electron microscope techniques to the study of live cells and tissues. There will be two main areas of core research. The first is a new thrust focusing on the development of optical instrumentation for the study of live cells and tissues. The second is the extension of previous work focusing on the development of sample preparation techniques for correlative electron microscopy. The optical workstation will have facilities for recording time-lapse sequences of events in multiple planes. It will also have facilities for the manipulation of organelles by optical trapping, photobleaching, photoactivation, laser ablation and patterned irradiation. A major design goal will be to develop an economical system that is easy to use. The design detail of the workstation will be driven by biological applications. One of the main objectives for the work on sample preparation techniques for electron microscopy would be the development of correlative light microscopy and EM analysis of the same specimen. In addition to the deembedding technique developed by the IMR, protocols will be developed for rapidly demounting individual samples from the light microscope for cryofixation. Also, new metal-coating techniques of frozen-hydrated or controlled freeze-dried specimens will be developed for high resolution FESEM. It is proposed to acquire a JEOL 4000FX Intermediate Voltage Electron Microscope with electronic image capture in place of the current High Voltage Electron Microscope that is to be decommissioned. Digitally captured electron micrographs will obviate the need for the time-consuming production of a large number of photographic negatives and prints for serial-section reconstructions. A software system for manipulating and archiving images will be developed for both light and electron microscopy. In addition, the IVEM would have allowed relatively thick sections (up to 0.5 micrometer) to be used for serial reconstructions.
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