Jöbsis P D, Combs C A, Balaban R S
Laboratory of Cardiac Energetics, National Heart Lung and Blaood Institute, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA.
J Microsc. 2005 Mar;217(Pt 3):260-4. doi: 10.1111/j.1365-2818.2005.01442.x.
The use of 2,3-dicyanohydroquinone (DCHQ) as an emission ratiometric probe of pH in vitro and in fibroblast cells was evaluated using two-photon excitation fluorescence microscopy (TPEFM). In addition, methods for spectrally calibrating the Zeiss LSM510 META spectroscopy system for TPEFM were also developed. The emissions of both the acid and base forms of DCHQ were detectable when using an 800-nm excitation in TPEFM, thereby allowing ratiometric determination of pH. These data suggest that, in contrast to most other emission ratiometric probes, both acid and base forms of DCHQ have similar two-photon cross-sectional areas at 800 nm. Acid (maximum at approximately 457 nm) and base (maximum at approximately 489 nm) DCHQ TPEFM emission spectra were similar to previously reported one-photon excitation emission spectra. Calibration curves for pH were successfully constructed using the ratio of DCHQ emission difference maxima at 460 nm and 512 nm in vitro and in cells. To our knowledge, DCHQ is currently the only effective emission ratiometric pH indicator for two-photon microscopy and may serve as a useful starting point for the development of other TPEFM ratiometric dyes for quantitative measurement of other cell parameters such as Ca2+, Mg2+ or Na+.
使用双光子激发荧光显微镜（TPEFM）评估了2,3 - 二氰基对苯二酚（DCHQ）作为体外和在成纤维细胞中pH发射比率探针的用途。此外，还开发了用于TPEFM的蔡司LSM510 META光谱系统的光谱校准方法。在TPEFM中使用800 nm激发光时，DCHQ的酸形式和碱形式的发射均可检测到，从而可以进行pH的比率测定。这些数据表明，与大多数其他发射比率探针不同，DCHQ的酸形式和碱形式在800 nm处具有相似的双光子截面积。酸（最大波长约为457 nm）和碱（最大波长约为489 nm）的DCHQ TPEFM发射光谱与先前报道的单光子激发发射光谱相似。利用体外和细胞中DCHQ在460 nm和512 nm处发射差异最大值的比率成功构建了pH校准曲线。据我们所知，DCHQ是目前唯一用于双光子显微镜的有效发射比率pH指示剂，并且可能作为开发其他用于定量测量其他细胞参数（如Ca2 +、Mg2 +或Na +）的TPEFM比率染料的有用起点。
