AbstractWe report a crystallizationâinduced emission fluorophore to quantitatively interrogate the polarity of aggregated proteins. This solvatochromic probe, namely âAggRetinaâ probe, inherently binds to aggregated proteins and exhibits both a polarityâdependent fluorescence emission wavelength shift and a viscosityâdependent fluorescence intensity increase. Regulation of its polarity sensitivity was achieved by extending the conjugation length. Different proteins bear diverse polarity upon aggregation, leading to different resistance to proteolysis. Polarity primarily decreases during protein misfolding but viscosity mainly increases upon the formation of insoluble aggregates. We quantified the polarity of aggregated proteinâofâinterest in live cells via HaloTag bioorthogonal labeling, revealing polarity heterogeneity within cellular aggregates. The enriched microâenvironment details inside misfolded and aggregated proteins may correlate to their bioâchemical properties and pathogenicity.