Organelle-targeting large-Stokes-shift fluorescent probes for confocal, fluorescence-lifetime and 2-photon excitation microscopy applications

Subcellular multiplex imaging is fundamentally limited by spectral crowding, restricted photostability, and the modality-specific performance of conventional fluorophores, which constrain the simultaneous visualization of multiple organelles in living cells. Here, we report a modular class of organelle-targeting, coumarin-based fluorescent probes that overcome these limitations through a large (up to 150 nm) Stokes shift, high brightness, good photostability, and efficient twophoton excitation. Central to this platform is a synthetically accessible, sulfonamide-functionalized coumarin core that enables convenient late-stage attachment of diverse organelle-targeting motifs. This modular strategy allows selective labeling of mitochondria, endoplasmic reticulum, lysosomes, plasma membrane, and whole cells at nanomolar concentrations with high signal-to-background ratios. The large Stokes shift minimizes spectral crosstalk and enables straightforward multiplex imaging in combination with green-and red-emitting fluorophores using simplified excitation schemes. These probes perform robustly across multiple imaging modalities, including confocal microscopy, two-photon excitation microscopy, fluorescence lifetime imaging microscopy, and flow cytometry. These new dyes provide a versatile toolkit for dynamic subcellular studies in basic research and disease modelling.