Liquid-crystalline, liquid-ordered, rippled and gel lipid bilayer phases as observed with nile red fluorescence

The basic matrix of cellular membranes consists of a double layer (bilayer) of phospholipids. Semisynthetic lipid bilayers are commonly used in biophysical studies of membranes. According to tem-perature and composition, lipid bilayers can exist in liquid-crystalline (or liquid-disordered), liquid -ordered, rippled, and gel phases. In the present study, the hydrophobic, solvatochromic molecule Nile red has been used as a fluorescent probe to examine the physical state of bilayers of different composi-tions in the 15-60 degrees C range. Phospholipids with saturated or unsaturated acyl chains, in the presence or absence of cholesterol have been studied. Nile red shows absorption maxima at 520-550 nm and emis-sion maxima at 580-640 nm, single photon excitation not being damaging to the system. A red/orange intensity ratio (ROIR) index has been used to normalize the results. ROIR varies clearly and reproducibly with the lipid phase, increasing in the order: liquid-ordered < gel < rippled < liquid-crystalline. It increases with temperature and decreases with cholesterol contents in the bilayers. Nile red allows an unusually clear observation of the rippled-to-liquid crystalline phase transition in saturated phospho-lipids. FLIM studies with Nile red also show differences between lamellar phases. Rotational relaxation times have been determined for Nile red in liquid-disordered (0.72 +/- 0.010 ns), gel (1.16 +/- 0.070 ns), and liquid-ordered (1.79 +/- 0.14 ns) phases, the large value of the liquid-ordered phase being an indication of the sterol hindering probe tumbling in the hydrophobic matrix.(c) 2022 Published by Elsevier B.V.

Automated summary

The basic structure of cellular membranes is a double layer of phospholipids. In this study, the fluorescent probe Nile red was used to examine the physical state of lipid bilayers with different compositions at different temperatures. Nile red allows clear observation of the rippled-to-liquid crystalline phase transition in saturated phospholipids.