Engineering a Viscosity-Gated Dual-Rotor Probe for Comprehensive Mapping of Lipid Droplet Heterogeneity

Small, metabolically active lipid droplets (LDs) with elevated core viscosity play pivotal roles in lipid metabolism and disease yet remain underexplored due to their transient nature and the limitations of conventional probes. Here, we report YTT-Z, a viscosity-gated dual-rotor fluorophore optimized for fixed-cell imaging that enables high-fidelity, ratiometric imaging of LD heterogeneity across diverse cell types. YTT-Z integrates a red-emissive intramolecular charge transfer (ICT) scaffold with a sterically tuned pyrrole rotor system, conferring enhanced brightness, photostability, and pronounced sensitivity to local microviscosity. Its zwitterionic structure promotes selective accumulation within LD cores, revealing small, high-viscosity LD subpopulations typically missed by standard dyes such as BODIPY. Importantly, YTT-Z uncovers mitochondria-associated LDs and reflects spatial variations in LD metabolic status, highlighting its broad utility for organelle mapping and lipid metabolism studies. This platform provides a powerful new tool to dissect LD substructures, metabolic gradients, and disease-linked lipid remodeling at subcellular resolution.