Abstract
Nanothermometry methods with intracellular sensitivities have the potential to make important contributions to fundamental cell biology and medical fields, as temperature is a relevant physical parameter for molecular reactions to occur inside the cells and changes of local temperature are well identified therapeutic strategies. Here we show how the GFP can be used to assess temperature-based on a novel fluorescence peak fraction method. Further, we use standard GFP transfection reagents to assess temperature intracellularly in HeLa cells expressing GFP in the mitochondria. High thermal resolution and sensitivity of around 0.26% °C−1 and 2.5% °C−1, were achieved for wt-GFP in solution and emGFP-Mito within the cell, respectively. We demonstrate that the GFP-based nanothermometer is suited to directly follow the temperature changes induced by a chemical uncoupler reagent that acts on the mitochondria. The spatial resolution allows distinguishing local heating variations within the different cellular compartments. Our discovery may lead to establishing intracellular nanothermometry as a standard method applicable to the wide range of live cells able to express GFP.
Highlights
Cells are the basic units of life that can be used to understand various physical and biological processes in the body
In this work we demonstrate the existence of intracellular temperature variations between mitochondria regions, highlighting the suitability of emGFP-Mito as a Peak Fractions (PF)-based molecular nanothermometer
We demonstrate the capability of wt-Green Fluorescent Protein (GFP) for solution-based and emGFP-Mito for intracellular temperature measurements
Summary
Cells are the basic units of life that can be used to understand various physical and biological processes in the body. Despite the very high thermal sensitivity of this sensor, it requires a very complicated fabrication of the micro-thermocouple Since these kinds of thermal probes require physical contact with the system under study, the coupling and inefficiency of the heat transfer from the system to the probe can lead to errors in the temperature measurements. Paulik et al have achieved a very high thermal resolution of 0.002 °C using infrared thermometry in thermogenesis studies of isolated cells after exposing them to the mitochondrial uncoupler, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), or transforming the cells with the uncoupling protein-29 This method has some drawbacks such as low spatial resolution and the limitation to surface measurements only. Most of them require complicated synthesis procedures, and most of them are not commercially available
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