Link between Fluorescent Probe Partitioning and Molecular Order of Liquid Ordered-Liquid Disordered Membranes.

Fluorescence microscopy is an important technique for studying lipid membranes and is increasingly being used for examining liquid ordered-liquid disordered phase coexistence. Liquid-liquid phase coexistence is a phenomenon of biological interest because it led to the lipid raft hypothesis, which postulates the existence of lateral heterogeneities in cell membranes. Observation of membrane heterogeneity relies on differential distribution of fluorescent membrane markers, but this can also modify the phase behavior, complicating the observation. We have used H NMR to measure the physical changes to 35:35:30 (mol/mol) DOPC/DPPC-D62/chol membranes introduced by fluorescent probes Laurdan and naphthopyrene. We measured miscibility transition temperature (T) and DPPC-D62 chain order for a range of probe concentrations. We found that up to 0.5 mol% of the equipartitioning probe Laurdan does not influence DPPC-D62 acyl chain order or phase behavior. In contrast, 2.0 mol% Laurdan slightly increases the fraction of DPPC-D62 in the liquid disordered phase below the T and increases T by 1 °C. Conversely, the nominally liquid ordered phase preferring probe naphthopyrene slightly perturbs the membrane even at concentrations as low as 0.3 mol%. This suggests that the strength of fluorescent probe partitioning between liquid ordered and liquid disordered phases correlates with the degree of perturbation to membrane phase behavior.