Visualizing Ca2+ sparks and substructure of Ca2+ waves by Total Internal Reflection Fluorescence Microscopy (TIRFM)

Based on near-field optical theory, total internal reflection fluorescence microscope shows a novel character that its picture has great signal-to-noise ratio and high temporal resolution achieved by high quality CCD camera. This allows us to analyze the spatiotemporal details of local Ca2+ dynamics within the nanoscale microdomain surrounding different Ca2+ channels. We have recently constructed a versatile objective TIRFM equipped with a high numerical aperture (NA = 1.45) objective. Using fluo-4 as the Ca 2+ indicator, we visualized the near-membrane profiles of Ca 2+ waves and elementary Ca2+ sparks generated by Ca 2+ release channels in rat ventricular myocytes. Different from those detected using conventional or confocal microscopy, Ca2+ waves observed with TIRFM exhibited fine inhomogenous substructures. The propagation of Ca2+ waves with anfractuous routes of spark recruitment is much more complicated than previously imagined. We believe that TIRFM will provide a unique tool for dissecting the microscopic mechanisms of intracellular Ca 2+ signaling. ? World Scientific Publishing Company.EI6709-714