The real-time monitoring of the intracellular pH in live cells with high precision represents an important methodological challenge. Although genetically encoded fluorescent indicators can be considered as a probe of choice for such measurements, they are hindered mostly by the inability to determine an absolute pH value and/or a narrow dynamic range of the signal, making them inefficient for recording the small pH changes that typically occur within cellular organelles. Here, we study the pH sensitivity of a green-fluorescence-protein (GFP)-based emitter (EGFP-Y145L/S205V) with the alkaline-shifted chromophore's pKa and demonstrate that, in the pH range of 7.5-9.0, its fluorescence lifetime changes by a factor of ~3.5 in a quasi-linear manner in mammalian cells. Considering the relatively strong lifetime response in a narrow pH range, we proposed the mitochondria, which are known to have a weakly alkaline milieu, as a target for live-cell pH measurements. Using fluorescence lifetime imaging microscopy (FLIM) to visualize the HEK293T cells expressing mitochondrially targeted EGFP-Y145L/S205V, we succeeded in determining the absolute pH value of the mitochondria and recorded the ETC-uncoupler-stimulated pH shift with a precision of 0.1 unit. We thus show that a single GFP with alkaline-shifted pKa can act as a high-precision indicator that can be used in a specific pH range.
Keywords: FLIM; fluorescence lifetime; fluorescent indicators; pH; time-resolved spectroscopy.