Developing Quinacridone-Based Long Fluorescent Lifetime Probes for Cell Membrane Heterogeneity Imaging

The nonuniform distribution of lipids and proteins in phospholipid bilayers forms distinct microdomains, leading to the structural and functional heterogeneity of cell membranes. Fluorescence lifetime imaging (FLIM) provides microenvironment-sensitive readouts that are independent of local fluorophore concentration. Conventional fluorescent probes for membrane microdomain imaging are limited by short fluorescence lifetimes (<6 ns), which may overlap with endogenous signals and hinder precise microenvironment discrimination. To overcome this limitation, we have designed and synthesized a series of polarity-sensitive fluorescent probes with exceptionally long lifetimes to resolve membrane heterogeneity. The optimized probe CMFL-QASAG (including CQSC6, CQSC8, and CQSC10) exhibits an unprecedented lifetime shift (Δτ ≈ 18 ns) linearly responding to membrane polarity changes and effectively decoupling from cellular autofluorescence. Cholesterol depletion experiments demonstrated a robust correlation between CMFL-QASAG's extended lifetime and membrane polarity variations in glioma cells. Furthermore, we observed dynamic changes in membrane polarity during cell migration, enabling imaging analysis and tracking of heterogeneous functional microregions on the cell membrane surface.