Hyperspectral Fluorescence Imaging with a New Polarity‐Ultrasensitive Fluorescent Probe

Abstract Hyperspectral fluorescence imaging (HSFI) could simultaneously offer morphological visualization and microenvironmental information through fluorescence wavelength‐shifting of microenvironmental‐sensitive fluorescent probes, yet its advancement is directly hindered by the scarcity of probes with sufficient spectral sensitivity. Herein, this study focuses on the development of an innovative fluorescent probe that exhibits heightened spectral sensitivity to microenvironmental changes and its advanced HSFI application. First, the comprehensive investigation of various donor‐π‐acceptor molecules uncovers the critical role of π‐spacer on the wavelength sensitivity to microenvironment polarity. Based on this insight, a new fluorescent probe Lipi‐PS of whose wavelength sensitivity represents the highest level so far is rationally developed. Further combining with the features of lipid droplets (LDs) targeting and high photostability of Lipi‐PS , as well as the newly established wavelength and polarity encoding method, HSFI is successfully realized in cells, tissues, and zebrafishes. Accordingly, the structural morphologies, fluorescence wavelengths, and polarities are intuitively visualized in the HSFI images. Notably, a special HSFI of single LD is achieved using Lipi‐PS in a custom‐built 3D‐SpecDIM system, allowing 5D tracking ( xyzλt ) of individual LDs for the first time. This enables unprecedented correlation of polarity‐dynamic behavior at the single‐LD level.