GFP-based Nanothermometry at the Mitochondria

Temperature plays a crucial role for the biological functioning of all living organisms. Thus, accurate and spatially resolved information on internal temperature can make an important contributions to cell biology and... [ view full abstract ]

Temperature plays a crucial role for the biological functioning of all living organisms. Thus, accurate and spatially resolved information on internal temperature can make an important contributions to cell biology and biomedical field [1]. In this context, luminescence nanothermometry that uses small luminescent thermometers to measure temperature has attracted a lot of attention due to its non-invasive nature, high spatial and thermal resolution [2]. However, it is challenging to control intracellular specific locations of nanothermometers, since some are taken up by endocytosis, and many others require microinjection. Another important drawback of existing luminescent nanothermometers is a need of complicated synthesis procedure and potential toxicity of the luminescent material or unwanted aggregation.

Here, we show how intracellular temperature can be measured using commercial available green fluorescence protein (GFP) attached to specific proteins in the cell. We show that a ratiometric approach applied to the analysis of the fluorescence emission spectrum of GFP can be used to determine temperature. First, we demonstrate the capability of wt-GFP for solution-based nanothermometry. Then, in vitro studies were performed with genetically transfected HeLa cells using a GFP agent associated to the mitochondria of the cells. Thermal images were recorded by means of confocal microscopy, where fluorescence emission is separated into two spectral wavelength ranges using a diffractive grating and selected subarrays of the 32 channel GaAsP detector.

High intracellular thermal resolution and sensitivity of around 0.26 ºC and 2.5 ºC^-1, respectively were achieved [3]. Moreover, the specific organelle association of the protein based nanothermometer at the mitochondria allowed observing temperature increase induced by a chemical uncoupling reagent. We believe that our method will facilitate basic research on cellular biological processes and furthermore may find practical application in hyperthermia therapy allowing temperature feedback during treatments.

[1]         K. Okabe, R. Sakaguchi, B. Shi, and S. Kiyonaka. Pflugers Arch. Eur. J. Physiol., vol. 470, no. 5, pp. 717–731, 2018.

[2]         H. Zhou, M. Sharma, O. Berezin, D. Zuckerman, and M. Y. Berezin. ChemPhysChem, vol. 17, no. 1, pp. 27–36, 2016.

[3]         O.A. Savchuk, O. F. Silvestre, R. M. R. Adão, J. B. Nieder*, submitted (2018).