We have constructed a time-resolved fluorescence lifetime imaging (FLIM) system to perform quantitative observation of microenvironments and physiological parameters of a single cell. Fluorescence intensity depends on a variety of biophysical and experimental factors such as concentration or optical condition, whereas fluorescence lifetime is an inherent property of a chromophore, and is therefore independent of photobleaching, excitation power, and other factors that limit intensity measurements. FLIM techniques therefore provide detailed information on the environment in a cell. In the present study, we have used FLIM to examine stress-induced changes in a cellular microenvironment. We expressed the enhanced green fluorescent protein (EGFP)-fusion protein in HeLa cells and examined the time course of its fluorescence lifetime under cell stress. Cell stress was induced by the treatment with medium lacking nutrition and exposure to normal air. The cells have been found to exhibit a decrease in fluorescence lifetime with the cell stress. The observed decrease in the fluorescence lifetime has been interpreted in terms of a change in local electric field produced by the protein matrix surrounding the chromophore of EGFP. The fluorescence lifetime image of EGFP in HeLa cells has also been measured with varying intracellular pH. The pH dependence of the fluorescence lifetime could be measured using monensin that equalizes intracellular pH to extracellular one. It has been found that the fluorescence lifetime of EGFP decreases with decreasing intracellular pH after photoexcitation of its neutral chromophore, which can be explained by the pH-dependent ionic equilibrium of the p-hydroxybenzylidene-imidazolidinone structure of the chromophore of EGFP. These results indicate that the intracellular pH of a single cell can be evaluated using FLIM of EGFP. From these results, we have proposed that FLIM can be used for noninvasive determination of the status of individual cells.
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