A BODIPY/Coumarin Hybrid Probe for Dual‐Mode Fluorescent/FLIM Imaging of Endoplasmic Reticulum Micro‐Polarity

ABSTRACT Endoplasmic reticulum (ER) micro‐polarity, with its spatial heterogeneity and dynamic changes, regulates key physiological processes like protein folding and lipid metabolism. Its abnormal alterations under pathological conditions, such as oxidative stress are closely linked to metabolic diseases and tumors. Capturing these subtle dynamics to reveal connections with organelle dysfunction remains a critical challenge. Existing traditional physical approaches are complex and cannot achieve real‐time spatial imaging. Fluorescent probes for polarity sensing could provide spatiotemporal resolution, nevertheless, suffer from unstable signals due to nonfixed dual channels, narrow response ranges, interference from viscosity, and nonlinear responses, hindering quantitative monitoring. We designed an ER‐targeted probe MBDP via hybridizing coumarin and BODIPY fluorophores, by introducing a benzene to reduce dihedral angles and a methyl group to inhibit intramolecular rotation, enabling specific polarity response. It shows a broad linear response, strong anti‐interference ability, which quantifies ER micro‐polarity via dual‐channel and lambda mode imaging. It captures ER micro‐polarity changes under stress and hippocampal regional differences. Moreover, the remarkable lifetime variations upon polarity change endow MBDP capability under fluorescence lifetime imaging microscope (FLIM). This probe provides a powerful tool with ratiometric imaging and FLIM dual‐modal monitoring for ER micro‐polarity, aiding in revealing ER‐related physiological/pathological mechanisms and disease research.