Chemically Anchored Diamond with H3 Centers for Ratiometric Measurement of Isolated Mitochondria Temperature

Precise measurement of mitochondrial temperature at different metabolic states remains one of the key challenges in cellular biophysics due to the lack of thermometers that combine nanoscale sensitivity with stable thermal contact with the organelle. Here, we present a hybrid sensing platform based on chemically functionalized diamond microparticles containing H3 luminescent centers, covalently bound to the outer membrane of isolated rat liver mitochondria. Surface activation via oxidation and EDC/HOBt chemistry provides a robust and reproducible thermal link between the thermometric probe and the organelle, minimizing heat dissipation through the surrounding medium. The local temperature is monitored ratiometrically from the emission ratio of H3 centers at 515-525 nm and 585-610 nm, showing a linear dependence on temperature with a relative sensitivity of 1.15%⋅°C^-1 in aqueous environments. Upon the uncoupling of oxidative phosphorylation and the inhibition of electron transport, the diamond thermometers reproducibly recorded the local thermal changes in the range of 0.5-10 °C, depending on the degree of coverage by anchored mitochondria. The observed response reflects efficient local heat confinement within the diamond-mitochondrion assembly, suggesting that structural organization and thermal insulation at the subcellular level are critical modulators of mitochondrial thermogenesis.