Premium
Two‐photon excitation fluorescence pH detection using 2,3‐dicyanohydroquinone: a spectral ratiometric approach
Author(s) -
JÖBSIS P. D.,
COMBS C. A.,
BALABAN R. S.
Publication year - 2005
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.2005.01442.x
Subject(s) - fluorescence , two photon excitation microscopy , excitation , emission spectrum , analytical chemistry (journal) , chemistry , spectroscopy , microscopy , fluorescence spectroscopy , calibration curve , calibration , spectral line , detection limit , materials science , optics , physics , chromatography , quantum mechanics , astronomy
Summary 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 ∼457 nm) and base (maximum at ∼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 Ca 2+ , Mg 2+ or Na + .