Intracellular Thermometry with Fluorescent Polymer Sensors and Nanodiamonds

Intracellular temperature fluctuations are thought to be closely related to higher order cellular phenomena. We have developed a method for imaging the temperature distribution in single living cells using a fluorescent polymer thermometer and fluorescence lifetime imaging microscopy to study the effects of intracellular temperature on cellular physiological activity. Previous experiments using the developed method have shown that during the G1 phase, the temperature of the nucleus is approximately 1°C higher than the temperature of the cytoplasm, as well as some mitochondria and centrosomes. The mechanisms that maintain this intracellular temperature heterogeneity and its physiological significance are not well understood. Recently, however, the mechanisms of physiological phenomena caused by fluctuations in intracellular temperature have begun to be elucidated. We hypothesized that intracellularly generated heat is not only a product of reactions, but also drives cellular responses. To test this hypothesis, we focused on cell differentiation, in which cells dramatically change their protein expression patterns. Using the neuronal model cell PC12, we investigated the relationship between neuronal differentiation and intracellular temperature. The results showed that intracellular temperature is involved in neuronal differentiation and associated neurite outgrowth. In addition to the aforementioned fluorescent polymer temperature sensor and fluorescence lifetime imaging microscopy, we also measured temperature using fluorescent nanodiamond particles containing negatively charged nitrogen vacancy centers as temperature sensors. We have previously shown that fluorescent nanodiamonds are able to measure temperature independent of external environmental influences such as pH, salt, and viscosity. To measure intracellular thermal conductivity, we also fabricated nanoparticles coated with polydopamine, which generates heat when exposed to light, around fluorescent nanodiamonds. Although the intracellular thermal conductivity has been assumed to be the same as the thermal conductivity of water, whether this is true or not was verified by actually measuring the intracellular thermal conductivity using the fabricated hybrid particles of polydopamine and fluorescent nanodiamonds. The results showed that, although the data were very uneven, on average the thermal conductivity inside the cell was several times smaller than the conductivity of water. In the present study, it is clear from microscopic observations that the hybrid particles are present inside the cell, but it is not known where in the cell they are localized. The variability of the data suggests variations in thermal conductivity depending on the location within the cell. Future work will use techniques to chemically modify the surface of the polydopamine-fluorescent nanodiamond hybrid particles, such as localizing the particles in mitochondria and nuclei, to measure thermal conductivity at different local positions in the cell to clarify what exactly produces such low thermal conductivity.