Real-time monitoring of mitochondrial temperature and calcium spikes using fluorescent organic probes

Astrocytes, the abundant glial cells, maintain cerebral homeostasis and cognitive functions through calcium signalling - a regulatory pathway that is frequently altered in brain disease. Mitochondria serve as thermal hubs in living systems, generating metabolic heat during respiratory substrate oxidation and ATP synthesis. Crucially, mitochondrial temperature variations directly reflect metabolic status, as impaired ATP production induces thermodynamic shifts. Here, we utilized a fluorescent thermometer probe MTY for <i>in vitro</i> determination and visualization of intracellular mitochondrial temperatures at the single-cell level. Through precisely controlled thermal modulation of fixed, living, and laser-stimulated astrocytes, we established a platform extendable to MCF-7 and Panc02 cell lines. The methodology enabled real-time tracking of near-infrared-induced thermal perturbations and FCCP-mediated uncoupling effects. Spinning-disk confocal microscopy revealed synchronized mitochondrial thermogenesis and calcium transients, with thermal/laser stimulation inducing 2-4-fold greater calcium spiking <i>versus</i> controls. Mechanistic analysis suggested this response was likely mediated through TRPV4 channel-mediated extracellular Ca²⁺ influx and/or intracellular calcium release from mitochondrial and endoplasmic reticulum stores.