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Abstract
Recent developments in optoelectronics permit real-time Ca(2+) imaging of thin planes within cells utilizing laser scanning confocal microscopy (LSCM). However, a major complication associated with this imaging system involves increased phototoxicity with improved spatiotemporal resolution. Two-photon excitation microscopy (TPEM) helps to minimize phototoxicity due to the restriction of this technique to the volume proximal to the geometric focus of the light. In this study, the capability of Ca(2+) imaging was investigated employing recently developed real-time TPEM, RTS2000MP (Bio-Rad, Tokyo) with a mode-locked Ti-sapphire laser. Z-axis resolution of RTS2000MP with high NA objectives defined as full-width at half maximum (FWHM) with a 0.5-microm fluorescent bead provided values nearly identical to those obtained with LSCM at a small pinhole (0.2 mm) (approximately 0.6 microm). When serial sectioning of 21 sequential images at 0.3-microm intervals in cultured endothelial cells loaded with calcein and tetramethyl-rhodamine methylester were performed with TPEM, the z-axis resolution was higher than that observed with LSCM; moreover, the photobleaching rate was significantly lower than that obtained with LSCM. Maximum fluorescence intensities were detected at 780 nm in excitation spectra of fluo-3 and fluo-4 Ca(2+)-sensitive probes with TPEM. Fluorescence images in mouse arterial endothelial cells loaded with fluo-4 could be clearly visualized by TPEM in situ. Application of acetylcholine caused oscillatory increase in [Ca(2+)](i) of endothelial cells; subsequently, relaxation along the major axis of smooth muscle cells was evident. Furthermore, consecutive long-lasting experiments could be repeated with identical response in the same microscopic field. In conclusion, fluorescence imaging employing TPEM is useful for Ca(2+) imaging in blood vessels in situ.
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