Thermal engineering at the microscale, such as the regulation and precise evaluation of the temperature within cellular environments, is a major challenge for basic biological research and biomaterials development. We engineered a polymeric nanoparticle having a fluorescent temperature sensory dye and a photothermal dye embedded in the polymer matrix, named nanoheater-thermometer ( nanoHT ). When nanoHT is illuminated with a near-infrared laser at 808 nm, a subcellular-sized heat spot is generated in a live cell. Fluorescence thermometry allows the temperature increment to be read out concurrently at individual heat spots. Within a few seconds of an increase in temperature by approximately 11.4 °C from the base temperature (37 °C), we observed the death of HeLa cells. The cell death was observed to be triggered from the exact local heat spot at the subcellular level under the fluorescence microscope. Furthermore, we demonstrate the application of nanoHT for the induction of muscle contraction in C2C12 myotubes by heat release. We successfully showed heat-induced contraction to occur in a limited area of a single myotube based on the alteration of protein–protein interactions related to the contraction event. These results demonstrate that even a single heat spot provided by a photothermal material can be extremely effective in altering cellular functions.

Despite improved sensitivity of nanothermometers, direct observation of heat transport inside single cells has remained challenging for the lack of high-speed temperature imaging techniques. Here, we identified insufficient temperature resolution under short signal integration time and slow sensor kinetics as two major bottlenecks. To overcome the limitations, we developed B-gTEMP, a nanothermometer based on the tandem fusion of mNeonGreen and tdTomato fluorescent proteins. We visualized the propagation of heat inside intracellular space by tracking the temporal variation of local temperature at a time resolution of 155 μs and a temperature resolution 0.042 °C. By comparing the fast in situ temperature dynamics with computer-simulated heat diffusion, we estimated the thermal diffusivity of live HeLa cells. The present thermal diffusivity in cells was about 1/5.3 of that of water and much smaller than the values reported for bulk tissues, which may account for observations of heterogeneous intracellular temperature distributions.

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Super-resolution light microscopy (SRM) offers a unique opportunity for diffraction-unlimited imaging of biomolecular activities in living cells. To realize such potential, genetically encoded indicators were developed recently from fluorescent proteins (FPs) that exhibit phototransformation behaviors including photoactivation, photoconversion, and photoswitching, etc. Super-resolution observations of biomolecule interactions and biochemical activities have been demonstrated by exploiting the principles of bimolecular fluorescence complementation (BiFC), points accumulation for imaging nanoscale topography (PAINT), and fluorescence fluctuation increase by contact (FLINC), etc. To improve functional nanoscopy with the technology of genetically encoded indicators, it is essential to fully decipher the photo-induced chemistry of FPs and opt for innovative indicator designs that utilize not only fluorescence intensity but also multi-parametric readouts such as phototransformation kinetics. In parallel, technical improvements to both the microscopy optics and image analysis pipeline are promising avenues to increase the sensitivity and versatility of functional SRM.