MICROBEAM ABLATION & CELLULAR MECHANISMS

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The renaissance of optical microscopy brings with it new capabilities to visualize molecular events at the subcellular level. No longer does microscopy provide the tools only to see the morphology of the cell and its subcomponents, it now serves as a platform for imaging discrete molecular activities with tools to perturb and manipulate these events. Utilization of laser microbeams to inactivate specific cellular components in a living cell and the development of single cell assays to monitor gene expression and other cellular activities to discern function will be highlighted in this section. Our specific aims are to a) Develop in vivo labeling of subcellular structures with green fluorescent protein (GFP) to facilitate microbeam-mediated ablation of an in vivo target and to subsequently assess the resulting structural alteration in the cell, b) Couple laser inactivation with specific cellular/molecular assays in order to elucidate the function of the ablated targets, c) Develop Fluorescence Resonance Energy Transfer (FRET) utilizing green fluorescent protein (GFP)- and blue fluorescent protein (BFP)- fusion proteins and employ FRET as an assay to access the effects of microbeam ablation of discrete subcellular structures or domains of interaction within a cell, d) Combine laser tweezers with sub-ablative microbeams to introduce biological reagents into cells via Ropto-porationS. This will expand the repertoire of microbeam technologies to previously unexplored biological areas i.e. suspension cell models.