Cellular processes are shaped by the intracellular microenvironment, which includes biomolecular, chemical, and physical factors. Among them, viscosity and temperature influence cellular activity by regulating diffusion, transport, and enzymatic kinetics. Their local fluctuations also accompany events such as cytoskeletal remodeling and stress responses. Accordingly, site-specific visualization of these two physical parameters is crucial for elucidating how local physicochemical states regulate vital cellular functions. BODIPY-rotors are fluorescent probes whose fluorescence lifetime reflects local viscosity or temperature by modulating radiative and non-radiative decay. Because fluorescence lifetime is independent of probe concentration or excitation, fluorescence lifetime imaging (FLIM)-based measurements enable quantitative, site-specific mapping of these physical parameters in cells. Although local viscosity and temperature would dynamically change, reflecting or triggering cellular functions, site-specific mapping of them at cell organelle scale is still unexplored due to the lack of tools especially for the temperature analysis. Thus, we designed and synthesized dual-color pallet of BODIPY-rotor based green- and red-fluorescent probes which selectively visualize intracellular viscosity or temperature though their fluorescence lifetime, enabling quantitative analysis of organelle viscosity and temperature via FLIM. We evaluated the viscosity and temperature sensitivity of fluorescent probes by fluorescence spectroscopy. Green BODIPY-rotors showed high sensitivity to viscosity changes, whereas red BODIPY-rotors were highly responsive to temperature with minimal interference from viscosity. Fluorescence lifetime analysis by FLIM confirmed that the green and red BODIPY derivatives function as viscosity and temperature sensors, respectively. We further demonstrated their applicability by visualizing chromatin condensation in the nucleus and excessive thermogenesis in the sarcoplasmic reticulum. In this session, we will an additional application based on multiplexing of fluorescence lifetime.
Yamazaki et al. (Sun,) studied this question.