Some fluorescent chemicals exhibit significant fluorescent quenching when exposed to increasing temperatures. This effect has been used to measure and examine the thermogenic properties of mitochondria. This involves placing mitochondria-targeting thermosensitive fluorophores inside cells, which naturally localise inside the mitochondria due to the inner mitochondrial membrane matrix-faces negative charge (as the fluorophores are cationic). The temperature of these fluorophores is inversely proportional to their fluorescence emission, and thus by measuring the fluorescent output, the temperature of actively-respiring mitochondria can be deduced. The fluorophores used are typically lipophilic cations derived from Rhodamine-B, such as ThermoFishers MitoTracker probes. This technique has contributed significantly to the general scientific consensus that mitochondria are physiologically maintained at close to 50 ˚C, more than 10˚C above the rest of the cell.
The inverse relationship between fluorescence and temperature can be explained by the change in the number of atomic collisions in the fluorophore's environment, depending on the kinetic energy. Collisions promote radiationless decay and loss of extra energy as heat, so more collisions or more forceful collisions will promote radiationless decay and reduce fluorescence emission.
This temperature-measurement technique is, however, limited. These cationic fluorophores are heavily influenced by the charge of the inner mitochondrial membrane matrix-face, dependent on the cell type. For example, the thermosensitive fluorophore MTY (MitoTracker Yellow) shows a sudden and drastic drop in fluorescence after the addition of oligomycin (an ATP synthase inhibitor) to the mitochondria of human primary fibroblasts. This would suggest a sharp increase in mitochondrial temperature but is, in reality, explained by the hyperpolarisation of the mitochondrial inner membrane by oligomycin - leading to the breakdown of the positively-charged MTY fluorophore.