DEVELOPMENT OF PROBES FOR MULTIPHOTON & EFTEM MICROSCOPIES

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I am developing probes that may be used with both multiphoton excitation fluorescence microscopy and energy-filtered transmission electron microscopy. Integrating these two microscopies can provide us with a powerful tool for studying intracellular processes. Multiphoton excitation fluorescence microscopy provides considerable advantages for studying live specimens because of reduced out-of-focus excitation, enabling the thi-ee-dimensional organization of live tissue to be better studied because of reduced phototoxicity. In energy filtering transmission electron microscopy much smaller immuno-gold labels can be used compared with standard, transmission electron microscopy. I have recently demonstrated that 1.4 mn nahogold can be clearly visualized within the nucleus of a labeled specimen, compared with 5mn which is generally the minimum size for conventional transmission electron microscopy. The use of smaller probes reduces problems of steric hindrance, allowing the label to infiltrate better into the matrix of the cytoskeleton. The combination of featurts of both microscopies may have much to offer the cell biologist, allowing high-resolution, correlative visualization of cytological structure following analysis of in vivo dynamics of the living specimen. This integrated approach is particularly powerful, when combined with cryotechniques. The goal of these project will be to explore various combinations of markers to be used for integrated microscopy.