Adsorption and Atomic Force Microscopy Imaging of Thylakoid Membranes

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)).