NEW ENERGY TRANSFER PROBES FOR SINGLE CELL IMAGING

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Single molecule studies of protein interactions with artificial and living cell membranes have progressed significantly since the commencement of the grant period. Interactions of proteins with the cell membrane are essential for efficient uptake of macromolecules into living cells. Single molecule fluorescence approaches have allowed us to study primary events such as the binding of macromolecules to membranes, lateral motion in the membrane plane and internalization for individual molecules, with all measurements free from ensemble averaging. Real-time single molecule imaging is the ability to record movies of molecular motions and to follow the spatial pathways and chemical reactions of individual macromolecules or their complexes. Rhodamine based fluorescent cassettes are being synthesized in the research group of Kevin Burgess and will be investigated in our laboratories using single molecule spectroscopic tools. These cassettes were shown to employ large stokes shifts making them suitable to be used in potential applications of single molecule experiments in solution or in single cells, where the weak fluorescence has to be distinguished from scattered excitation light and other background sources. Other possible applications include their use for two-photon imaging of biological samples using a confocal microscope setup.