Super-Resolution FLIM Imaging of Intracellular Lipid Microenvironments with a Dual-Responsive Polarity- and pH-Sensitive Fluorescent Probe

The microenvironment is essential for the proper function of organelles and biological systems. In particular, its physical properties─such as polarity and pH profoundly influence both physiological and pathological processes. Therefore, directly visualizing and quantitatively measuring changes in the cellular microenvironment is crucial for advancing our understanding of these fundamental processes. Fluorescent probes capable of enabling direct visualization and quantitative analysis of the cellular microenvironment using STED/FLIM (stimulated emission depletion fluorescence and lifetime imaging microscopy) offer powerful tools for exploring intracellular biophysical properties with nanoscale resolution. However, such probes remain largely unexplored. Here, we present TPA-BT-CA, a highly photostable fluorescent probe that is both polarity- and pH-sensitive, enabling real-time super-resolution imaging and quantitative mapping of intracellular lipid distributions via STED/FLIM microscopy. The donor–acceptor–acceptor (D–A–A) structural design of TPA-BT-CA facilitates efficient intramolecular charge transfer, leading to strong fluorescence signals and prolonged fluorescence lifetimes in nonpolar environments, thereby allowing precise differentiation of lipid regions with varying polarity. Furthermore, protonation and deprotonation processes under acidic or alkaline conditions induce fluorescence lifetime shifts, enabling quantitative assessment of lipid distribution in living cells. This work establishes a new approach for high-resolution visualization and quantitative measurement of the cellular microenvironment, offering new insights into lipid organization and microenvironmental dynamics.