Spatially Complex Diffraction-Limited Photolysis of Caged Calcium and IP3 Combined with High-Speed Confocal Imaging

Abstract

The novel Mosaic digital illumination system (Photonic Instruments/Andor Group) integrated into a Nikon A1R confocal microscope was used to uncage Ca (DM-nitrophen, NP-EGTA) or IP3 from multiple geometrically complex (Fig.) diffraction-limited subcellular regions and simultaneously measure [Ca]i with high-speed 2-dimensional confocal imaging (430 fps). The Mosaic System uses a computer controlled spatial light modulator to map a diffraction limited mask onto the specimen plane. A Digital Micromirror Device, consisting of a high speed array of hinge-mounted individually addressable, tiltable microscopic mirrors, directs continuous wave laser light (405 nm) onto the image plane according the user-defined diffraction limited mask. Local uncaging of Ca from multiple small regions of interest (0.63 micrometer diameter) generated artificial Ca sparks outside the cell and produced CICR inside permeabilized cardiac myocytes. Uncaging Ca from a 0.63x10 micrometer region triggered CICR and propagating Ca waves. Subsarcolemmal uncaging of IP3 initiated propagating Ca waves that originated within the region of uncaging, and caused increased peak amplitude of electrically evoked Ca transients and Ca alternans, suggesting that in cardiac myocytes Ca release from IP3 recpetors primes ryanodine receptor Ca release channels and enhances CICR.