Acquisition of simultaneous dynamic changes in pH and temperature at the cellular level is essential for studying cellular physiological activities. Herein, we developed an organic small-molecule fluorescent probe, RH-Co, that simultaneously images pH and temperature in two fluorescent channels to investigate the dynamic changes of both in cellular metabolism. The fluorescence emission intensity of RH-Co at 450 nm responded to the change in pH with a pKa value of 7.14 ± 0.72, which was suitable for detecting cell pH (∼7.2). In terms of temperature response, the fluorescence emission intensity of RH-Co at 590 nm decreased linearly with increasing temperature (25-45 °C), and the temperature relative sensitivity of RH-Co was calculated to be 3.12%/°C (37 °C). Simultaneous dynamic monitoring of the pH and temperature in living cells was achieved using RH-Co. With the increase in temperature, the intracellular pH decreased significantly after 37 °C. After adding glucose to improve energy metabolism, a large amount of ATP was synthesized, which made the intracellular temperature rise rapidly. At the same time, the accompanying production of acidic metabolites caused the pH to decrease. After 5 min of FCCP stimulation, the cell temperature began to rise and the pH decreased slightly due to the blockage of the oxidative phosphorylation process. Intracellular copper metabolism triggered temperature increases, but the efficient copper transport and distribution of ATOX1 led to little change in pH. A drop in cellular pH occurred during autophagy, and a marked increase in temperature was observed due to the enhancement of energy expenditure and ATP production.
Huang et al. (2026) studied this question.