Intracellular pH is a key component of cell metabolism. In healthy cells, intracellular pH is lower (more acidic) than extracellular pH; this gradient helps maintain a normal balance between metabolic reactions inside cells. In tumor cells, intracellular pH increases and becomes more alkaline than extracellular pH; this reverse gradient drives changes in cell metabolism that favor cancer progression. In line with this, tumor cells often switch from oxidative phosphorylation to glycolysis as the preferential metabolic pathway used to generate ATP for energy. Glycolysis favors cancer growth: generation of ATP is much quicker than during oxidative phosphorylation, intermediates of glycolysis can be utsed for growth and proliferation, and the lactate produced via glycolysis acidifies the extracellular environment to support cancer invasion.

The metabolic abnormalities seen in cancer cells make intracellular pH a valuable readout for cancer progression, but most current techniques used to measure pH in vivo are invasive or expensive or require complex data processing methods. In their work “Fluorescence lifetime-based pH mapping of tumors in vivo using genetically encoded sensor SypHerRed,” Shimolina and colleagues set out to develop a noninvasive optical method that can be used to measure intracellular pH in vitro and in vivo. This article is part of the April 5 Issue of Biophysical Journal.

This study builds upon existing methods that measure intracellular pH by using genetically encoded pH sensors. Rather than measuring fluorescence intensity, Shimolina et al. use fluorescence lifetime imaging (FLIM). Fluorescence lifetime measures the time a fluorophore spends in the excited state before it emits a photon, which makes it an intrinsic property of the fluorophore. It is thus an absolute measurement, whereas fluorescence intensity can report only relative differences between cells. Importantly, lifetime is unaffected by properties like sample thickness or fluorophore concentration, which affect intensity measurements.

The study focuses on SypHerRed, a pH sensor that shows both intensity and lifetime response to pH in cells. Once a calibration curve is generated on the basis of the relationship between SypHerRed fluorescence lifetime and pH values (in the range of 6.0–8.0 pH units), intracellular pH can be determined from microscopy images. This study demonstrates the use of SypHerRed fluorescence lifetime measurements to quantify absolute pH values for cultured cells and, for the first time, for tissues in vivo.

Importantly, another advantage of SypHerRed is that it is a single (red)-color pH sensor, so it can be combined with other fluorophores for simultaneous measurement of different cellular parameters. Accordingly, Shimolina and colleagues combine FLIM of SypHerRed and NADH to assess pH and cell metabolic states concurrently. By exposing cells to different metabolic perturbations, the authors demonstrate that this technique can also be used to measure intracellular pH and cell metabolic state, live and at the same time.