ABSTRACT
The pH-sensitive probe carboxy SNAFL-1 can be used for imaging using ratiometric and fluorescence lifetime techniques. The former method suffers from the drawback that quantitative pH imaging in cells requires a time-consuming and cumbersome calibration procedure. In contrast, straightforward calibrations in buffer suffice for fluorescence lifetime imaging. This is illustrated here by a comparative study of the two techniques under different controlled conditions. The effect of probe concentration, protein concentration, and hydrophobicity, the contents of damaged cells and living cells on the emission ratio, and the fluorescence lifetime of carboxy SNAFL-1 were studied. The results clearly demonstrate that the fluorescence lifetime imaging technique is more convenient than the ratiometric method for pH determination.
Subject(s)
Cells/chemistry , Microscopy, Confocal , Microscopy, Fluorescence/methods , Animals , Buffers , CHO Cells , Calibration , Cricetinae , Hydrogen-Ion Concentration , Proteins/chemistry , Sensitivity and Specificity , Solubility , Tumor Cells, Cultured , Water/chemistryABSTRACT
Ca(2+) concentrations in biological cells are widely studied with fluorescent probes. The probes have a high selectivity for free calcium and exhibit marked changes in their photophysical properties upon binding. The differences in the fluorescent lifetime of the probes can now be used as a contrast mechanism for imaging purposes. This technique can be further exploited for the quantitative determination of ion concentrations within the cells. We describe the use of a fast fluorescence lifetime imaging method in combination with a standard confocal laser scanning microscope for the determination of Ca(2+) concentrations in single rat cardiac myocytes using the intensity probe Calcium Green.
