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

Abstract

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.

Cyclooctatetraene-conjugated cyanine mitochondrial probes minimize phototoxicity in fluorescence and nanoscopic imaging

“…A future extension of this strategy would be the upgrade of the dyes to immobilizable versions for permanent labelling of mitochondria. 49 Additionally, the ndings in this work could potentially inspire the development of advanced probes for various organelles and protein/nucleic acid targets, as well as next generation TSQs. A recent perspective conveys several opinions on self-healing dyes as a strategy for reducing photobleaching.…”

Latest Trends in Temperature Sensing by Molecular Probes

“…6 °C (Figure h). Cells treated with phorbol 12‐myristate‐13‐acetate also showed a decrease in fluorescent intensity over a 30 min period, which translates to an increase in temperature of about 3 °C . Both of these experiments show real‐time temperature measurements of mitochondria in reaction to external stimuli.…”

“…On top of all of that, there is a current debate on the spatial resolution of fluorescent thermometers due to the rapid heat transfer in small scale aqueous environments . Some groups have used fluorescent thermal probes to measure several degree changes in temperature due to endogenous heat generation . Nonetheless, Baffou et al .…”

“…Huang et al . attempted this and synthesized Mito‐TEM (Figure , structure 4 ), a rhodamine‐based dye that has a linker and terminal aldehyde that can react with amines in proteins . 4 was characterized with a spectrometer showing a fluorescence emission peak at 590 nm and a decrease in emission intensity as temperature increases, with a sensitivity of 6.65 %/°C in buffer.…”

Pitfalls in Monitoring Mitochondrial Temperature Using Charged Thermosensitive Fluorophores

“…At present, accurate real-time monitoring of mitochondrial temperature in living cells remains challenging (reviewed in [14][15][16]). Among the few molecular tools available, nano-sized temperature-sensitive fluorescent dyes represent a valuable option [7,[17][18][19]. These lipophilic cations, derived from rhodamine, preferentially localize into mitochondria, driven by the negative charges on the matrix face of the inner mitochondrial membrane.…”