Confocal pH imaging of microscopic specimens using fluorescence lifetimes and phase fluorometry: influence of parameter choice on system performance

We investigate the performance of confocal pH imaging when using phase fluo rometry and fluorophores with pH-dependent lifetimes. In these experiments, the specimen is illuminated by a laser beam, whose intensity is sinusoidal ly modulated. The lifetime-dependent phase shift in the fluorescent signal is detected by a lock-in amplifier, and converted into a pH value through a calibration procedure. A theoretical investigation is made of how the diff erent system parameters will influence the results concerning sensitivity a nd noise. Experiments carried out with the fluorophore SNAFL-2 support thes e theoretical predictions. It is found that, under realistic experimental c onditions, we can expect a pH change of 0.1 units to be easily detected in an 8-bit digital image. However, the pixel-to-pixel root mean square noise is often of the order of one pH unit. This comparatively high level of nois e has its origin in photon quantum noise. pH measurements on living cells s how a systematic deviation from expected values. This discrepancy appears t o be the result of fluorophore interaction with various cell constituents, and is the subject of further investigation.