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.