Fluorescence polymer thermometer for the real-time mitochondrial temperature imaging of zebrafish embryos and larvae

As the bioenergetic organelles in eukaryotic cells, mitochondria exhibit thermoregulatory fluctuations, making them a critical biosensor for evaluating the functional integrity and metabolic states of eukaryotic cells. However, mitochondria’s subcellular compartmentalization and nanoscale dimensions impose substantial technical constraints on spatiotemporally resolved thermometric analyses, particularly in embryos and in vivo microenvironments. Here, we have developed a fluorescence polymer thermometer, poly(N-isopropylacrylamide)-1-[4-(7-nitrobenzo [1,2,5] oxadiazol-4-yl)-piperazin-1-yl]-propenone (PNIPAM-AANBD). It is designed with a piperazine ring capable of establishing hydrogen bonding interactions with particular mitochondrial molecules and an alkyl side chain that enhances lipophilicity. This dual-functional design achieves precise targeting of mitochondria. Notably, PNIPAM-AANBD displays pronounced thermoresponsive fluorescence enhancement across the physiologically relevant temperature range of 24.0–44.0 °C. Given the probe’s exceptional spatiotemporal resolution and mitochondrial thermal specificity, we established zebrafish embryo and larval systems to test the feasibility and efficacy of the fluorescence polymer thermometer. In the hyperthermia induction assay, compared to the control group, the average fluorescence intensity of zebrafish embryos increased by 25 %, and that of larvae increased by 49 %. In contrast, during the antipyretic assays, the average fluorescence intensity of zebrafish embryos decreased in a gradient manner (46 %–69 %) with the increase in concentration of antipyretic reagents, while that of larvae decreased by 30 %–36 % due to different types of antipyretic reagents (genipin and antondine). This fluorescence polymer thermometer offers a simple, effective method for longitudinal monitoring of mitochondrial temperature both in vitro and in vivo to elucidate fundamental physiological and pathological processes related to mitochondrial function.