Fluorescence lifetime imaging of pH in cells: investigation of factors influencing the pH calibration lifetime

Intracellular ion concentrations can be measured using ratiometric fluorophores. These fluorophores often show, in addition to the change in fluorescence spectrum, a change in fluorescence lifetime for different ion strengths. Accordingly, it has been suggested that using the fluorescence lifetime would make it possible to replace the intracellular calibration with a simplified extracellular calibration step. However, there are contradictory results on whether probe binding to membranes and macromolecules has influence on lifetime measurement. We have addressed this problem by systematically investigating three different probes used for intracellular pH determination, (SNAFL-1, SNAFL-2, and SNARF-1). All probes were studied in vivo (in cells) and in vitro (in capillaries). Influence of different factors such as protein and lipid concentration on the lifetime of the probes was measured. Our results give a possible explanation as to why earlier investigators have recorded constant pH throughout the whole cell.