QUANTITATIVE PH IMAGING IN CELLS USING CONFOCAL FLUORESCENCE LIFETIMEIMAGING MICROSCOPY

The pH-sensitive probe carboxy SNAFL-1 can be used for imaging using r atiometric and fluorescence lifetime techniques. The former method suf fers from the drawback that quantitative pH imaging in cells requires a time-consuming and cumbersome calibration procedure. In contrast, st raightforward calibrations in buffer suffice for fluorescence lifetime imaging. This is illustrated here by a comparative study of the two t echniques under different controlled conditions. The effect of probe c oncentration, protein concentration, and hydrophobicity, the contents of damaged cells and living cells on the emission ratio, and the fluor escence lifetime of carboxy SNAFL-1 were studied. The results clearly demonstrate that the fluorescence lifetime imaging technique is more c onvenient than the ratiometric method for pH determination. (C) 1995 A cademic Press, Inc.