R. Sanders et al., QUANTITATIVE PH IMAGING IN CELLS USING CONFOCAL FLUORESCENCE LIFETIMEIMAGING MICROSCOPY, Analytical biochemistry, 227(2), 1995, pp. 302-308
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.