Fig. 1

Imaging of P. berghei liver stages using GFP-CLEM. A Illustration outlining the protocol to perform CLEM of P. berghei liver stages expressing GFP. Huh7 cells were seeded on gridded coverslips presenting alphanumerical coordinates and infected with P. berghei sporozoites. Maps of cells infected with 2-day old liver stages were acquired using GFP (green) and Hoechst (a nucleic acid stain, blue) and light and fluorescence microscopy (LM). Samples were then processed for TEM imaging, which includes embedding samples in resin, trimming resin blocks around ROIs, preparing ultra-thin sections using the microtome and staining with contrasting reagents. Samples were then imaged using TEM and ROIs were located by correlating patterns of host nuclei and liver stages. An example shows a LM map (bottom left) and low-magnification TEM micrographs (bottom right) for 4 GFP⁺ liver stages (i, ii, iii and iv). Scale bars are 200 μm and 10 μm for the LM and TEM micrographs, respectively. Drawings were created with BioRender.com. B The overlay of micrographs exemplifies how data from LM and TEM are correlated and used to re-localize ROIs. Scale bar is 10 μm. C Higher-magnification TEM micrographs showing the hepatocyte-parasite interface, and a host cell mitochondrion (M_H), a P. berghei mitochondrion (M_Pb), the parasitophorous vacuole membrane (PVM), the parasite plasma membrane (PPM), P. berghei vacuoles (V_Pb) and a P. berghei nucleus (N_Pb). Scale bar is 1 μm. The inset in (B) defined by a box with a white dotted border shows the area selected for the zoom-in micrograph presented in (C)