Color-changing fluorescent dyes allow for accurate temperature measurements within living cells

Temperature is an important variable that affects countless biological processes at the cellular level. However, accurate measurement of temperature within living cells remains difficult. Traditional temperature measurement techniques often lack the spatial resolution required to detect subtle temperature fluctuations in complex microscopic environments. Furthermore, many existing molecular thermometers have significant limitations in terms of sensitivity, resolution, and applicable targets that highlight the need for innovative approaches and versatile tools.

Contrary to this background, a research team led by Associate Professor Konishi of the Tokyo Institute of Science in Japan has developed a molecular thermometer using a novel solvent dye fluorescent dye. Their findings published online in the Journal of the American Chemical Society on March 5, 2025 show that this new compound allows for high-precision temperature measurements through changes in fluorescence properties.

The researchers designed a series of donor-π–accptor(d-π–a) fluorophores based on the π-extended fluorene structure. These molecules are specially designed to alter their fluorescent properties depending on the polarity of their surrounding environment. As temperature rises, the polarity of the solvent decreases slightly, and these dyes emit light at different wavelengths and intensities.

By measuring the ratio of fluorescence intensities at two specific wavelengths, researchers can accurately calculate temperature changes. This “ratiometric” approach is highly reliable for eliminating variables such as dye concentration and excitation light intensity and detecting microtemperature fluctuations within microscopic environments such as cell organelles.

The newly developed dyes exhibited exceptional solvent staining properties with a shift above 200 nm between different solvents and emission wavelengths exceeding the red region (701-828 nm). In particular, researchers can perform temperature measurements with a pronounced relative sensitivity of up to 3.0%/°C and a resolution of less than 0.1°C.

“These results represent the highest sensitivity and resolution reported for small organic single fluorescence-like ratiometric fluorescence thermometers dispersed in solution, which is ideal for bioimaging.” Through further mechanical analysis, the team determined the underlying principles that contribute to the design of future molecular thermometers and lead to the extraordinary solvent staining properties of the proposed dye.

The team has successfully demonstrated the practical application of molecular thermometers by introducing one of the dyes into living human cell cultures. Ratio confocal microscopy was used to confirm that the dyes function effectively as temperature sensors in cellular environments, especially cell droplets.

“This molecular thermometer based on a solvent-dye fluorescent dye is expected to significantly expand the range of fluorescent temperature measurements and contribute to revealing unknown biological phenomena due to its excellent spatial resolution, non-invasiveness, and ease of molecular design,” Konisi explains.

Beyond biological research applications, this innovative molecular thermometer demonstrates the potential for analyzing the temperature-dependent properties of polymeric materials and other material systems. Researchers plan to develop a library of fluorescent thermometers based on this strategy to cover a variety of environments of interest.

By providing unprecedented insight into microscopic temperature fluctuations, these novel dyes could help scientists to unravel temperature-dependent biological phenomena and contribute to significant advances in fields ranging from cell biology to chemistry and materials science.