FRET-amplified mitochondrial thermometry with an AIE–rhodamine conjugate reveals elevated temperatures in metastatic cancer cells

Mitochondria are essential organelles for cellular energy production and also serve as key sources of heat, linking cellular metabolism to temperature homeostasis. However, measuring mitochondrial temperature dynamics with high fidelity remains challenging, as traditional single-dye thermometers often lack sensitivity and quantitative accuracy. Here we show that integrating two thermoresponsive fluorophores into a single Förster resonance energy transfer (FRET)-coupled probe dramatically amplifies temperature-induced fluorescence changes, enabling more sensitive and quantitative mitochondrial thermometry. The new probe, “Mito-TPE”, covalently links a flexible aggregation-induced emission donor to a rhodamine B acceptor, so that minor temperature-dependent molecular motions produce a large fluorescence response under single-wavelength excitation. Mito-TPE exhibits a linear fluorescence decrease with rising temperature, with a relative sensitivity (Sr) of 13.82%/°C at 37 °C, which is much higher than that of conventional mitochondrial probes. It localizes specifically to mitochondria and allows estimation of organelle temperatures in living cells. Using Mito-TPE, we quantitatively detect subtle mitochondrial temperature differences induced by chemical uncouplers and inhibitors, as well as higher basal mitochondrial temperatures in aggressively metastatic cancer cells compared to less invasive or normal cells. These findings suggest a general strategy to boost the performance of molecular thermometers by dual-unit FRET coupling. By enabling more precise imaging of intracellular heat, this approach may open new opportunities to investigate fundamental bioenergetics and to identify thermal signatures of disease.