Visualizing substructure of Ca<sup>2+</sup> waves by total internal reflection fluorescence microscopy

Yongqiang Bai; Aihui Tang; Shiqiang Wang; Xing Zhu

doi:10.1117/12.580734

Abstract

Total internal reflection fluorescence microscope is a new optical microscopic system based on near-field optical theory. Its character of illumination by evanescent wave, together with the great signal-to-noise ratio and temporal resolution achieved by high quality CCD, 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 Ca²⁺ indicator, we visualized the near-membrane profiles of Ca²⁺ waves and elementary Ca²⁺ sparks generated by Ca²⁺ release channels in rat ventricular myocytes. Different from those detected using conventional and confocal microscopy, Ca²⁺ waves observed with TIRFM exhibited fine inhomogenous substructures composed of fluctuating Ca²⁺ sparks. The anfractuous routes of spark recruitment suggested that the propagation of Ca²⁺ waves is much more complicated than previously imagined. We believe that TIRFM will provide a unique tool for dissecting the microscopic mechanisms of intracellular Ca²⁺ signaling.

Citation Download Citation

Yongqiang Bai, Yongqiang Bai, Aihui Tang, Aihui Tang, Shiqiang Wang, Shiqiang Wang, Xing Zhu, Xing Zhu, } "Visualizing substructure of Ca²⁺ waves by total internal reflection fluorescence microscopy", Proc. SPIE 5635, Nanophotonics, Nanostructure, and Nanometrology, (9 February 2005); doi: 10.1117/12.580734; https://doi.org/10.1117/12.580734

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