D−π–A Fluorophores with Strong Solvatochromism for Single-Molecule Ratiometric Thermometers

Fluorescence thermometry has emerged as a significant area of research in the field of remote temperature sensing with high accuracy. However, the development of noninvasive and reliable small organic fluorescence thermometers (FTs) remains challenging. In this study, we developed a high-sensitivity, high-resolution small organic ratiometric FT with a solvatochromic dye and analyzed its temperature response mechanism. π-Extended fluorene-based D-π-A-type small solvatochromic dyes (<b>FπAc</b>, <b>FπF</b>, and <b>FπVC</b>) were synthesized and characterized. A significant solvatochromic shift of >200 nm was observed between <i>n</i>-hexane and tetrahydrofuran (THF) for both <b>FπF</b> and <b>FπVC</b>, with emission in THF reaching the red region (701 nm, 828 nm). This substantial solvatochromic shift was accompanied by a concurrent decrease in the fluorescence quantum yield and is attributable to accelerated internal conversion. The fluorescence spectra of <b>FπF</b> in THF and <b>FπVC</b> in diethyl ether exhibited blue shifts and increased fluorescence intensities with a decrease in polarity at increased temperatures. Our observations revealed an absolute sensitivity of 21%/°C and a relative sensitivity of 3.0%/°C at the maximum. These results represent the highest sensitivity and resolution reported for single fluorophore small organic ratiometric FTs dispersed in solution. The positive temperature coefficient of the fluorescence intensity was attributed to control of the nonradiative decay pathway by solvatochromism. The temperature responsiveness of <b>FπF</b> could be detected in living HeLa cells by ratiometric confocal microscopy. Using the proposed strategy to develop FTs, we plan to build a library of FTs that will cover various environments of interest in both simple and complex systems.