Bidirectional surface analysis of monomolecular membrane harboring nanoscale reversible collapse structures

Determination of orientation of nanoscale collapse structures formed within a Langmuir film at the air-aqueous interface has not been possible by existing experimental techniques. This is however of special importance for pulmonary surfactant films, which form reversible surface-associated reservoirs (SARs) under dynamic lateral compression and expansion. The direction of these SARs with respect to the interface has hitherto remained uncertain. We designed a methodological approach to investigate the directionality of SARs formed in a functional analogue of the pulmonary surfactant lining, where we transferred the compressed film on a holey substrate and performed bidirectional surface imaging of the hole spanning monomolecular membrane harboring SARs. This unambiguously showed association of SARs with the membrane toward the air-side, in contrast to the up to now commonly accepted view of an orientation toward the aqueous phase.