Characterization of dual-wavelength seminaphthofluorescein and seminapthorhodafluor dyes for pH sensing under high hydrostatic pressures

Hydrostatic pressure is an important physical parameter in biology, with pressures in the few-hundred-atm range having significant effects on cellular morphology, metabolism, and viability. To ensure valid results when studying pressure effects using fluorescence spectroscopy and imaging methods, metabolic probes need to be characterized for high-pressure use. Of interest is the sensing of pH at high pressures due to the key role that pH plays in cellular function. Despite the availability of pH-sensitive dyes, only a few have been characterized for high-pressure use. Here we present the effects of pressure on the acid–base equilibria of four dual-wavelength seminaphthorhodafluor and seminaphthofluorescein dyes (pKa = 6.6–7.8). Using phosphate buffers as high-pressure pH references, we investigate the pressure dependence of pKa for these dyes and determine the volume change associated with the acid-dissociation reaction. We find that if pressure-induced pKa changes are not accounted for during interpretation of emission spectra, systematic errors of up to 0.02 pH units per 100 atm would result, comparable to previously measured pressure-induced pH changes in vivo. Results are validated by correctly sensing pH changes in Tris and acetate solutions. Methods presented here are applicable to other metabolic probes utilizing dual-wavelength ratiometric sensing modes.