Microscopic Analysis of Transfected Cells

Focused-ion beam scanning electron microscopy (FIB-SEM) was performed on a Helios NanoLabG3 microscope (FEI, The Netherlands) as described previously (Nunes-Hasler et al., 2017 (link)). Briefly, transfected cells were seeded on 35-mm Ibidi polymer dishes with gridded bottom (catalog number 81166). Following incubation with the target and fixation with 4% PFA for 20 min at room temperature, brightfield and high-resolution confocal images were captured. Next, samples were fixed for EM with 2.5% glutaraldehyde and 2% PFA in Ca-Caco buffer (2 mM CaCl₂, 0.15 M sodium cacodylate, pH 7.4) for 3 h on ice and washed five times in ice-cold Ca-Caco buffer. Following dehydration, samples were embedded on Epon and prepared for FIB-SEM imaging as previously described. Samples were sputter-coated with gold for 30 s by a Q150T ES coater (Quorum Technologies, UK). Cellular footprints obtained from FIB-SEM and fluorescence and brightfield images were compared to locate the cell of interest. Images were acquired at the highest resolution setting (5×5×10 nm) using the Autoslice and View software (FEI). Drift correction and alignment of FIB-SEM images were done using Amira Software and overlay with the fluorescence image using ImageJ.