Fixable Molecular Thermometer for Real-Time Visualization and Quantification of Mitochondrial Temperature

A change of mitochondrial temperature can be an important indicator of mitochondrial metabolism that generates considerable heat. For this reason, development of fluorescent probes to detect mitochondrial temperature has become an attractive topic. Previous efforts have successfully addressed the major issues, such as temperature sensitivity and mitochondrial targetability. However, there remains a key obstacle to practical applications. Considering the highly dynamic features of mitochondria, especially the variation of the inner-membrane potential, it is quite necessary to permanently immobilize a temperature probe in mitochondria in order to avoid unstable intracellular localization along with the changes of mitochondrial status. Herein, we report Mito-TEM, the first fixable, fluorescent molecular thermometer. Mito-TEM is based on a positively charged rhodamine B fluorophore that has the tendency of being attracted to mitochondria, which have negative potential. This fluorophore containing rotatable substituents also contributes to the temperature-responsive fluorescence property. Most importantly, a benzaldehyde is introduced in Mito-TEM as an anchoring unit that condenses with aminos of the protein and thus immobilizes the probe in mitochondria. The specific immobilization of Mito-TEM in mitochondria is unambiguously demonstrated in colocalization imaging. By using Mito-TEM, a method of visualizing and quantifying a temperature distribution through grayscale imaging of mitochondria is established and further applied to monitor the temperature changes of live cells under light heating and PMA stimulation.