Intracellular pH sensing using autofluorescence lifetime microscopy.

Fluorescence lifetime images of reduced nicotinamide adenine dinucleotide (NADH) that is a key cofactor in cellular metabolism were obtained in a cell at various values of intracellular pH. The average fluorescence lifetime of NADH is found to become shorter monotonically with increasing pH, indicating that pH in a single cell can be determined by fluorescence lifetime imaging of NADH without adding exogenous fluorescent probes. The magnitude of the pH-induced lifetime change is higher in cells than that in buffer solution. The fluorescence lifetime of NADH is not uniform inside a cell; the fluorescence lifetime of nuclear NADH is shorter than that of mitochondrial NADH at each pH, and the magnitude of the pH-induced change is larger in nuclei than in other areas. The local electric field effect on the fluorescence lifetime is discussed since this effect may be one of the strong possibilities for the nonuniformity of the autofluorescence lifetime of NADH in cells.