Phospholipid packing and hydration in pulmonary surfactant membranes and films as sensed by LAURDAN

► Packing in surfactant films are monitored by the fluorescence of the probe LAURDAN. ► Compression-driven packing of surfactant at 25 °C is similar to that of pure DPPC films. ► At 37 °C, maximal packing required the presence of cholesterol in surfactant films. ► Compressed films are more hydrated than bilayers of same composition, specially at 37 °C.