Ultra s scanner

Freshly cleaved mica surface was immersed in 38 μL of adsorption buffer (10-mM Tris–HCl, pH 7.5, 150-mM KCl, 25-mM MgCl₂) and 2 μL of thylakoid membrane samples were immediately injected into the buffer drop and then incubated for 1.5 h in a humidor at room temperature. After adsorption, the sample was carefully rinsed with 800-μL imaging buffer to remove the free membranes (10-mM Tris–HCl, pH 7.5, 150-mM KCl) (Liu et al., 2009 (link), 2011 (link); Zhao et al., 2016 , 2020 (link); Miller et al., 2020 (link)). Then, the membranes on mica were imaged using JPK NanoWizard 3 AFM in AC imaging mode in the imaging buffer at room temperature. AFM was equipped with an ULTRA S scanner and Ultra-Short Cantilever probes (0.3 N·m⁻¹, Nano World). The tip spring constant was routinely calibrated. High-resolution imaging was performed at a scan rate of 5 Hz and a resolution of 512 × 512 pixels. Images were processed with JPK SPM Data Processing (JPK). The construction of models was carried out by Chimera and Adobe Illustrator. Simulations of AFM images were carried out as previously described (Zhao et al., 2020 (link)).

Blc. viridis photosynthetic membranes were immobilized on the mica substrate in 40 μl absorption buffer (10 mM Tris-HCl pH 7.2, 150 mM KCl, 25 mM MgCl₂) for 1 h at room temperature. The sample was rinsed with imaging buffer (10 mM Tris-HCl pH 7.2, 150 mM KCl). AFM imaging was performed using a Bruker Multimode 8.0 equipped with a 97 μm J-scanner and OTR4-10 probe (spring constant = 0.08 N m⁻¹) in PeakForce Quantitative Nanoscale Mechanical (PeakForce QNM) mode. Minimal loading forces of ~120 pN were used at scan frequencies of 3 Hz using optimized feedback parameters. High-speed AFM was carried out using a NanoWizard 3 AFM (JPK) equipped with an ULTRA S scanner and Ultra-Short Cantilever probe (0.3 N·m⁻¹, Nano World) in AC mode with the scan frequency of 20–30 Hz. Image analysis was carried out using Gwyddion and ImageJ. Statistical data are presented as mean ± standard error of the mean (SEM) unless stated otherwise.