Effects of cholesterol (CHO) at various concentrations on the molecular organization and lateral structure of Curosurf films. The first column shows typical AFM topographic images of pure Curosurf at five characteristic surface pressures (π): 20, 30, 40, 50, and 60 mN/m. Columns 2–4 show AFM topographical images of Curosurf mixed with 0.1%, 1.0%, and 10% CHO, respectively. Curosurf + CHO films were compared with pure Curosurf at identical π in each row. Surface pressures of 50 and 60 mN/m cannot be reached with the addition of 10% CHO due to early film collapse. The AFM scan area was 50 × 50 µm for all images (the bar equal to 10 µm). The full z-range was set to be 5 nm for monolayers (i.e., π ≤ 40 mN/m) and 20 nm for multilayers (i.e., π > 40 mN/m). Two phospholipid domains, i.e., the tilted-condensed phase (TC) and the liquid-expanded (LE) phase, are indicated by arrows. Characteristic relative heights of multilayers are pointed by arrows. High-resolution images (5 × 5 µm at 20 and 30 mN/m, and 2 × 2 µm at 40 mN/m) of critical lateral structures are indicated by rectangular boxes and shown as inserts. The bottom-right corner shows the height profile along the line tracing indicated in the insert at 40 mN/m. AFM, atomic force microscopy.