Far-red-emitting Nile red with boosted solvatochromism enables multidimensional imaging of membrane heterogeneity and dynamics.

The heterogeneity of lipid membranes plays an essential role in regulating the biological functions of organelles. As a solvatochromic probe, Nile red exhibits optical responses to varying membrane environments; however, its limited solvatochromism restricts its ability to detect subtle changes in membrane polarity and lipid order. In this work, we rationally design the auxochrome of Nile red by incorporating an oxygenated julolidine moiety and develop oxygenated julolidine-based Nile red (JONR), which exhibits significantly enhanced sensitivity to environmental polarity, along with strong fluorogenicity from protic to aprotic solvents, large Stokes shifts, and far-red emission in polar environments. Compared to Nile red, JONR demonstrates 3-fold greater accuracy in identifying various organelles in live cells and enables real-time tracking of their dynamic interactions through ratiometric imaging. Furthermore, JONR can distinguish lipid environments through polarization modulation depth and fluorescence lifetime variations. By integrating these properties with its superior spectral response, we present the first comprehensive mapping of subtle lipid heterogeneity within migration-associated structures in L929 cells. The high sensitivity to subtle differences in lipid environments of JONR enables fine resolution of subcellular membrane structures in live cells and provides a new approach for high-throughput analysis of intracellular microenvironments.