Spm data processing

Two microliters of thylakoid membrane samples were adsorbed onto freshly cleaved mica surface with 38 μL of adsorption buffer (10 mM Tris-HCl, pH 7.5, 150 mM KCl, 25 mM MgCl 2 ) at room temperature for 1.5 h. After adsorption, the sample was carefully rinsed with 800 μL imaging buffer (10 mM Tris-HCl, pH 7.5, 150 mM KCl) 71 (link) . High-resolution imaging was performed in AC imaging mode in liquid at room temperature using a NanoWizard 3 AFM (JPK) equipped with an ULTRA S scanner and Ultra-Short Cantilever probe (0.3 N•m -1 , Nano World) at scan frequencies of 5 Hz using optimised feedback parameters and a resolution of 512 × 512 pixels. The tip spring constant was routinely calibrated. Images were processed with JPK SPM Data Processing (JPK) and ImageJ.

AFM (NanoWizard 3 AFM, JPK Instruments AG, Berlin, Germany) colloidal probe measurements were performed to investigate the surface forces between the multilayer films. The accurate spring constant of the unloaded cantilevers (CSC37/Cr-Au, Mikromasch) was determined by the Sader method. 47 (link) The sensitivity of the cantilevers (loaded, before coating) was obtained by performing force measurements against a bare silica wafer (Supporting Information, Figure S1). A similar procedure as that of the ellipsometry experiment was used herein (40 min stabilization time). A constant approach and retraction speed of 400 nm s -1 was used to avoid hydrodynamic effects. 48 (link) For each condition, three randomly chosen areas (10 μm × 10 μm, 36 points on each area) of the sample were chosen to perform the force measurements. The standard software of the instrument (JPK SPM Data Processing) was utilized to analyze the force curves.