Temperature Measurement in Single Living Cells using a Hydrophilic Fluorescent Nanogel Thermometer

Abstract

The temperature of a living cell changes with every cellular event. Thus, measuring intracellular temperature will contribute to the explanation of intricate biological processes and the development of novel diagnoses. Here we demonstrate the first intracellular thermometry with a newly developed fluorescent nanogel thermometer (FNT). The temperature-sensing function of FNT is undertaken by the thermo-responsive polyNIPAM unit combined with a water-sensitive fluorophore. Furthermore, two structural modifications were made taking into consideration its functions in intracellular environments. The first was gelation at a nanometer scale using a crosslinker, allowing the nanogel to remove undesirable responses originating from interactions between cellular components and the fluorophores. The second was the enrichment of ionic sulfate groups on the surface of FNT to avoid precipitation in living cells. In a spectroscopic study, the fluorescence enhancement of FNT with increasing temperature was independent of KCl concentration (100 to 200 mM), environmental pH (4 to 10), or surrounding proteins. Then FNT was microinjected into living COS7 cells, followed by imaging with an epi-fluorescence microscope. The total fluorescence intensity of FNT in single COS7 cells showed the temperature-dependent enhancement upon heating, which provides the calibration curve for intracellular thermometry using FNT. The temperature resolution was evaluated to be 0.29-0.50 °C (27-33 °C). Next, intracellular temperature variations induced by FCCP (mitochondria uncoupler) was investigated. FCCP provoked the fluorescence enhancement of FNT, indicating intracellular heating by 0.45 °C for 30 min. This result suggests that our thermometer FNT successfully detected the intrinsic and significant intracellular temperature change in response to stimulation. In conclusion, novel thermometer FNT is superior to other candidate thermometers in terms of biocompatibility (i.e., size, sensitivity, and solubility) and functional independence (i.e., negligible interactions with cellular components), enabling intracellular temperature measurement in single living cells.