Fluorescence Lifetime Imaging Provides Enhanced Contrast when Imaging the Phase-Sensitive Dye di-4-ANEPPDHQ in Model Membranes and Live Cells

We apply fluorescence lifetime imaging to the membrane phase-sensing dye di-4-ANEPPDHQ in model membranes and live cells. We show that the 1700 ps lifetime shift between liquid-disordered and liquid-ordered phases offers greater contrast than the 60 nm spectral shift previously reported. Detection of cholesterol-rich membrane microdomains is confirmed by observation of the temperature dependence of membrane order and by cholesterol depletion using methyl-β-cyclodextrin.