Optical Bioimaging : From Living Tissue to a Single Molecule : Calcium Imaging in Blood Vessel In Situ Employing Two-Photon Excitation Fluorescence Microscopy

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Recent developments in optoelectronics permit real-time Ca<sup>2+</sup> 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<sup>2+</sup> 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-μm fluorescent bead provided values nearly identical to those obtained with LSCM at a small pinhole (0.2 mm) (approximately 0.6 μm). When serial sectioning of 21 sequential images at 0.3-μm 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<sup>2+</sup>-sensitive probes with TPEM. Fluorescence images in mouse arterial endothelial cells loaded with fluo-4 could be clearly visualized by TPEM <i>in situ</i>. Application of acetylcholine caused oscillatory increase in [Ca<sup>2+</sup>]<sub>i</sub> 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<sup>2+</sup> imaging in blood vessels <i>in situ</i>.<br>

収録刊行物

  • Journal of pharmacological sciences

    Journal of pharmacological sciences 93(3), 242-247, 2003-11-01

    The Japanese Pharmacological Society

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各種コード

  • NII論文ID(NAID)
    10012544804
  • NII書誌ID(NCID)
    AA11806667
  • 本文言語コード
    ENG
  • 資料種別
    REV
  • ISSN
    13478613
  • NDL 記事登録ID
    6757750
  • NDL 雑誌分類
    ZS51(科学技術--薬学)
  • NDL 請求記号
    Z53-D199
  • データ提供元
    CJP書誌  CJP引用  NDL  J-STAGE 
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