pH dependence of the fluorescence lifetime of enhanced yellow fluorescent protein in solution and cells

pH dependence of the fluorescence decay profile of enhanced yellow fluorescent protein (EYFP) depends on the excitation wavelength. The correlation between the fluorescence lifetime and pH in solution is discussed in terms of the acid–base equilibrium of the chromophore of EYFP. Fluorescence lifetime images of EYFP in HeLa cells have also been measured at various values of intracellular pH. A remarkable pH dependence of the fluorescence lifetime image is observed in pH 4.5–6.0, indicating that pH in a single cell can be evaluated using the fluorescence lifetime image of EYFP especially in the acidic condition.