Q150t es coater

CLEM was performed according to protocols established in prior studies as follows^{7 (link),69 (link)}. Transfected cells seeded on Grid-500 polymer dishes (Ibidi) and exposed to phagocytic targets were fixed in 4% PFA for 30 min prior to high-resolution confocal and brightfield imaging. After light image acquisition dish was re-fixed in 2.5% glutaraldehyde/2% PFA/2 mM CaCl₂/0.15 M sodium cacodylate buffer (pH 7.4) for 3 h, dehydrated and embedded in Epon and stained with osmium, ferrous cyanide, lead acetate and uranyl acetate according to standard Autoslice and View protocol provided by FEI^{7 (link)}. After sectioning, the polymer grided coverslip was dissolved in xylol for 1 h, and samples were mounted and sputter-coated with gold for 30 s using a Q150T ES coater (Quorum Technologies). Focused ion beam scanning electron microscopy (FIB-SEM) imaging was performed on a Helios NanoLabG3 microscope (FEI). Images were acquired at the highest resolution setting, resulting in 5×5×10 nm pixels using the Autoslice and View software (FEI). Drift correction and alignment were performed using Amira software. Confocal stacks were deconvolved using Imaris (Oxford Instruments). Orthogonal slices and overlay were generated from the aligned stack using ImageJ.

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.