Characterization of probe binding and comparison of its influence on fluorescence lifetime of two pH-sensitive benzo[c]xanthene dyes using intensity-modulated multiple-wavelength scanning technique

Quantitative pH imaging using the carboxy semi-naphthofluorescein dyes SNAF L-1 and SNAFL-2 can be performed by measurement of intensity ratios or fluo rescence lifetimes. However, there is a controversy as to whether the latte r method has the practical advantage of a straightforward pH calibration in buffers compared to a cumbersome and time-consuming procedure in cells. In this study we have undertaken a systematic study of the potential factors influencing the fluorescence lifetime of the probes at different pH using c onfocal microscopy. In vitro results demonstrate that factors such as lipid and protein concentrations have a substantial influence on pH measurements based on fluorescence lifetime. The pH could be overestimated by more than 2 pH units. Studies in permeabilized COS-7 cells demonstrate the same tren ds as observed in the in vitro studies. (C) 2000 Academic Press.