A Bright Ratiometric Dipyrene Probe for Functional Imaging of Condensate Microenvironments in Living Cells

Biomolecular condensates exhibit spatially heterogeneous microenvironments shaped by intertwined physicochemical factors, yet practical small‐molecule tools for mapping these states in living cells remain limited. Here, we report PyLUMI, a bright dipyrene ratiometric probe, optimized by linker engineering to improve fluorescence output under aqueous conditions while preserving excimer‐to‐monomer (E/M) readout capability. Solution‐phase characterization identifies a scaffold with enhanced fluorescence efficiency and an E/M response to coupled polarity–viscosity microenvironmental changes. In recombinant protein condensates, PyLUMI provides substantially increased signal and enables pixel‐resolved visualization of intra‐condensate heterogeneity. In living cells, the probe enables low‐exposure ratiometric imaging of centrosome‐associated condensate compartments and reveals microenvironmental signatures linked to cell‐cycle progression and pathology‐associated centrosome amplification beyond morphological size differences. These results establish PyLUMI as a functional chemical reporter for spatially resolved profiling of integrated physicochemical fingerprints in condensate‐associated compartments.