Confocal pH imaging of microscopic specimens using fluorescence lifetimes and phase fluorometry: influence of parameter choice on system performance
2000 (English)In: Journal of Microscopy, ISSN 0022-2720, E-ISSN 1365-2818, Vol. 199, 106-114 p.Article in journal (Refereed) Published
We investigate the performance of confocal pH imaging when using phase fluorometry and fluorophores with pH-dependent lifetimes. In these experiments, the specimen is illuminated by a laser beam, whose intensity is sinusoidally 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 different system parameters will influence the results concerning sensitivity and noise. Experiments carried out with the fluorophore SNAFL-2 support these theoretical predictions. It is found that, under realistic experimental conditions, 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 noise has its origin in photon quantum noise. pH measurements on living cells show a systematic deviation from expected values. This discrepancy appears to be the result of fluorophore interaction with various cell constituents, and is the subject of further investigation.
Place, publisher, year, edition, pages
2000. Vol. 199, 106-114 p.
confocal microscopy, fluorescence lifetime imaging, pH measurements, time-resolved identification, scanning ims technique, multiple fluorophores, laser, cells, indicators, molecules, neurons
IdentifiersURN: urn:nbn:se:kth:diva-19976ISI: 000088807300004OAI: oai:DiVA.org:kth-19976DiVA: diva2:338669
QC 201005252010-08-102010-08-10Bibliographically approved