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
2000 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 283, no 1, 104-110 p.Article in journal (Refereed) Published
Quantitative pH imaging using the carboxy semi-naphthofluorescein dyes SNAFL-1 and SNAFL-2 can be performed by measurement of intensity ratios or fluorescence lifetimes. However, there is a controversy as to whether the latter 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 confocal 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 trends as observed in the in vitro studies.
Place, publisher, year, edition, pages
2000. Vol. 283, no 1, 104-110 p.
confocal microscopy, fluorescence lifetime imaging, pH measurement, COS-7, SNAFL-1, SNAFL-2, intracellular ph, ims technique, microscopy, fluorophores
IdentifiersURN: urn:nbn:se:kth:diva-19927DOI: 10.1006/abio.2000.4652ISI: 000088376000016OAI: oai:DiVA.org:kth-19927DiVA: diva2:338619
QC 201005252010-08-102010-08-10Bibliographically approved