Fluorescence lifetime imaging spectroscopy in living cells with particular regards to pH dependence and electric field effect

Abstract

Intracellular pH of a single cell can be imaged using FLIM of enhanced green fluorescent protein (EGFP). The correlation between the intracellular pH and the fluorescence lifetime of EGFP in HeLa cells is explained by considering the pH-dependent acid-base equilibrium of the p-hydroxybenzylidene-imidazolidinone structure of the chromophore of EGFP. The equilibrium between different forms of chromophore depends on pH of the medium. The equilibrium constant between the neutral and anionic EGFP chromophores in HeLa cells is obtained by analyzing the fluorescence lifetimes observed with different values of intracellular pH. The intracellular pH dependence has been also observed in HeLa cells where enhanced yellow fluorescent protein (EYFP) is expressed. The pH dependence of the fluorescence lifetime of EYFP may result from the pH dependence of the molecular structure of the protein bound ionic form of EYFP or the conformational change of the EYFP chromophore induced by lowering pH. The fluorescence lifetimes both of EGFP and of EYFP are not uniform in the cell. At each pH, for example, the fluorescence lifetime of EGFP located near the outer cell membrane is shorter than those located inside cell, whereas the lifetime of EYFP located near the outer cell membrane is longer than those located inside the cell. These differences are ascribed to the different distribution of the electric field surrounding the fluorescent chromophore in the cells, implying that the chromophores of EGFP and EYFP show the opposite electric field effects of the fluorescence lifetime to each other. The fact that the fluorescence lifetime of BCECF in solution is different from the one observed at the same pH in intact cells of Halobacterium salinarum has been also ascribed to the local field produced by membranes in vivo.