<title>Confocal fluorescence lifetime imaging of pH in single cells</title>

Abstract

We show that the confocal fluorescence lifetime imaging method is a powerful tool for the quantitative determination of pH on a microscopic scale. This method is easily implemented using a conventional confocal microscope and moreover, utilizes currently available fluorescent probes. It is shown that both the intensity probe DM-NERF and the ratio probe BCECF are suitable for this purpose, albeit with different useful pH ranges. In addition it is shown that the fluorescence decay of both probes are independent of the probe-concentration. Furthermore, the fluorescence lifetime behavior of BCECF is found to be insensitive to changes in the hydrophobicity and protein content of the buffer solution. The intracellular pH was imaged using BCECF since this probe is sensitive in the physiological pH range. A realistic pH value of about 7.3 was found throughout CHO cells.