This application requests a Zeiss LSM510 laser scanning confocal microscope, outfitted with dedicated equipment permitting patch-clamp electrophysiological measurements to be made concurrently with confocal image acquisition. This particular instrument will feature two crucial advantages absent in all core-facility confocal microscopes at Johns Hopkins University. First, the microscope will possess the requisite flexibility in selective excitation and emission wavelengths to permit implementation of a novel 3(3)-FRET algorithm that rapidly and nondestructively specifies the FRET efficiency (E) between donor and acceptor fluorophores. (Conveniently done with GFP-color mutants like CFP and YFP), determination of E can specify interfluorophore distances between 10-100 Angstroms, thereby providing a powerful and quantitative measure of protein-protein interaction in living cells. The 3(3)-FRET algorithm overcomes a major obstacle to this approach, allowing E to be determined despite commonly observed variability in the expression levels of labeled proteins. The proposed confocal instrument will thus furnish spatially resolved maps of protein-protein interaction. Second, no core-facility confocal microscope currently accommodates simultaneous imaging and patch-clamp recording; the attachment of electrophysiological apparatus would prove disruptive to conventional users, and such apparatus would be easily damaged by a high-volume of non-electrophysiological participants. Yet, numerous scientific questions hinge critically upon simultaneous image acquisition and measurement/control of membrane voltage. By drawing from a user base that values this dual capability, the proposed confocal instrument with dedicated patch clamp would address this void. The Zeiss LSM510 platform accommodates optimal implementation of 3(3)-FRET and electrophysiology. The microscope will anchor a departmental facility supported by 3 major and 3 minor users, all of whose research would be fundamentally advanced by the platform's unique capabilities.
| Takahashi, Shoji X; Miriyala, Jayalakshmi; Tay, Lai Hock et al. (2005) A CaVbeta SH3/guanylate kinase domain interaction regulates multiple properties of voltage-gated Ca2+ channels. J Gen Physiol 126:365-77 |
