3D CLEM: Confocal and FIB-SEM imaging of endocytosed nanoparticles

Hela cells were grown on gridded glass coverslips, prepared as described by Fermie et al.³⁷. Cells were incubated with fiducial markers at a concentration of 1 μg/ml dissolved in complete DMEM and incubated for 3 hours, and fixed overnight in 1x PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl₂, pH = 6.9) containing 4% paraformaldehyde (Sigma) and 0.1% glutaraldehyde (Merck) at 4 °C. Following fixation, coverslips with cells were washed in 1x PHEM buffer and mounted in live-cell coverslip holders filled with 1x PHEM buffer to prevent dehydration of the samples. Fluorescence imaging was performed using a Zeiss LSM700 CLSM equipped with 63x/1.4 NA oil immersion objective. Nanoparticles were excited using the 555 nm laser line at 2% power. Z-stacks were collected with 200 nm step size. The position of cells relative to the grid of the coverslips was recorded using polarized light. Cells were prepared for electron microscopy according to a protocol described earlier³⁸, with minor modifications. Briefly, samples were postfixed using 1% osmium tetroxide (w/v) with 1.5% potassium ferrocyanide (w/v) for 1 h on ice, incubated with 1% thiocarbohydrazide in dH₂O (w/v) for 15 min, followed by 1% osmium tetroxide in dH₂O for 30 min. Samples were en-bloc stained with 2% uranyl acetate in dH₂O for 30 minutes and stained with Walton’s lead aspartate for 30 min at 60 °C. Dehydration was performed using a graded ethanol series. Samples were embedded in Epon resin and polymerized for 48–60 h at 65 °C. Polymerized resin blocks were removed from the glass coverslips using liquid nitrogen, mounted on aluminum stubs and rendered conductive using conductive carbon paint and a sputter coated layer of 5 nm Pt. Following sample preparation, automated serial imaging was performed using a Scios FIB-SEM (Thermo Scientific), according to a previously described workflow³⁷. Briefly, trenches were prepared surrounding the region of interest using the FIB, after which automated serial imaging was performed using 5 nm isotropic voxels. Electron microscopy images were collected at an acceleration voltage of 2 kV and a current of 0.2 nA, using the T1 backscattered electron detector. Following imaging, correlation of fluorescence and FIB-SEM data was achieved by manual registration using Fiji and ec-CLEM³⁶. FIB-SEM images are presented with inverted contrast, to resemble TEM contrast.