Optimized time‐gated generalized polarization imaging of Laurdan and di‐4‐ANEPPDHQ for membrane order image contrast enhancement

The membrane dyes Laurdan and di-4-ANEPPDHQ can be used to image membrane order due to a spectral blue-shift in the fluorescence emission between the liquid-ordered and liquid-disordered phases. These images typically take the form of a normalized intensity ratio image known as a generalized polarization (GP) plot. Here, we exploit the known excited state photophysics and time-resolved data acquisition via time-correlated single-photon counting (TCSPC) to demonstrate GP contrast enhancement for these two probes of 7 and 31%, respectively. This improvement in image contrast enhancement will be invaluable when studying the role of lipid rafts in fixed and live cell systems.