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

Quantitative pH imaging using the carboxy seminaphthofluorescein 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...

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Bibliographic Details
Published in:Analytical biochemistry 2000-07, Vol.283 (1), p.104-110
Main Authors: Andersson, Ronnie M, Carlsson, Kjell, Liljeborg, Anders, Brismar, Hjalmar
Format: Article
Language:English
Subjects:
Animals
Brain - metabolism
Calibration
Cell Line
confocal microscopy
COS Cells
COS-7
Fluoresceins - metabolism
Fluoresceins - pharmacology
fluorescence lifetime imaging
Fluorescent Dyes - pharmacology
Hydrogen-Ion Concentration
Medicin och hälsovetenskap
Microscopy, Confocal - methods
pH measurement
Sheep
SNAFL-1
SNAFL-2
Spectrometry, Fluorescence - methods
Time Factors
Xanthenes - metabolism
Xanthenes - pharmacology
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Summary:Quantitative pH imaging using the carboxy seminaphthofluorescein 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.
ISSN:0003-2697
1096-0309
DOI:10.1006/abio.2000.4652