Multimodal Correlative Light and Electron Microscopy

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.