© 2005 The Royal Microscopical Society Journal of Microscopy, Vol. 217, Pt 3 March 2005, pp. 260–264 Received 16 July 2004; accepted 7 November 2004 Blackwell Publishing, Ltd. SHORT TECHNICAL NOTE Two-photon excitation fluorescence pH detection using 2,3-dicyanohydroquinone: a spectral ratiometric approach P. D. JÖBSIS, C. A. COMBS* & R. S. BALABAN Laboratory of Cardiac Energetics, *Light Microscopy Core, National Heart Lung and Blood Institute, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, U.S.A. Key words. Cellular pH, dicyanohydroquinone, dihydroxyphthalonitrile, spectral calibration, spectral imaging, two-photon. Received 16 July 2004; accepted 7 November 2004 Summary The use of 2,3-dicyanohydroquinone (DCHQ) as an emission ratiometric probe of pH in vitro and in fibroblast cells was eval- uated 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 + . Introduction The use of spectral ratiometric approaches for monitoring the cellular ionic milieu is highly desirable because effects of bleaching, variations in excitation power or fluorophore con- centration can be minimized. Ratiometric probes rely on the spectral differences in the excitation or emission characteristics of the probe as the ionic milieu shifts, providing an internal reference. However, in two-photon excitation fluorescence microscopy (TPEFM) (Denk et al., 1990) many of the ratiometric probes that depend on either emission or excitation spectral differences have not been found to be useful. The so-called excitation ratiometric probes, such as Fura-2, do not have ade- quate spectral excitation differences to differentiate the forms of the dye due to the rather broad spectral density of TPEFM excitation. The broad TPEFM excitation profile initially would appear to be an advantage with emission ratio dyes because both forms of the probes should be excited concomitantly. However, with most probes the two-photon cross-sectional area of one form of the dye dominates the emission properties, severely limiting the ratiometric approach. The lack of adequate emission signal from one form of a ratiometric probe has been demonstrated by Baker et al. (2002) for the common pH probe SNARF and by Szmacinski et al. (1996) for the calcium indicator INDO. In the case of SNARF the base form dominates the emission charac- teristics over the entire pH range. To our knowledge, no emission ratiometric probes for pH are currently available for TPEFM. The purpose of this study was to evaluate the two-photon excitation characteristics of the ratiometric probe 2,3- dicyanohydroquinone (DCHQ) (also known as 1,4 dihydroxy- phthalonitrile; Kurtz & Balaban, 1986) as a TPEFM probe of pH. Materials and methods Calibration of the Zeiss META spectroscopy system in TPEFM The META spectral imaging system (maximum resolution 10.7 nm) incorporated in the Zeiss LSM510 Axiovert 200 m (Thornwood, NY, U.S.A.) microscope scan-head was used to estimate the emission spectrum of DCHQ in TPEFM. Several aspects of the manufacturer’s suggested methods for spectral calibration of the system were found to be suboptimal, so a customized method was adopted as outlined below. Because no visible excitation light is present in TPEFM, we removed the visible laser blocking pins in front of the META’s 32-channel Correspondence to: Dr Paul Jobsis. Fax: +1 301 402 2389; e-mail: Jobsisp@nhlbi.nih.gov