HEK-293 cells stably expressing GFP-ATG13 or GFP-DFCP1 were grown on poly-L-lysisne coated cover slips or were transfected with dsRED MITO for 24 hr and were then replated onto poly lysine-coated cover slip (MatTek). Before imaging with LM, the cells were fixed with 2% PFA in 0.1 M Hepes, pH 7.4. After LM imaging the cells were fixed with 2% GA in 0.2 M Hepes buffer for 30 min and post-fixed with reduced 1% OsO4 in 0.1 M NaCac, pH 7.4. Cells were then sequentially washed with 0.1 M NaCac buffer and water before UA-en-bloc staining for 1 h, +4°C. After extensive washes in water, samples were dehydrated stepwise in ethanol. Finally the cells were flat embedded, infiltrated in epoxy resin (TAAB 812 ref. T030) for 2 h and baked at +60°C overnight. Semi thick 230 nm sections were prepared and picked up on single slot grids. 10 nm colloidal gold particles were added to the sections to serve as fiducial markers in the tomogram alignment. Dual axis tilt series were acquired using SerialEM software on a Technai FEG20 microscope at 200 kV with 11,500X magnification, over a tilt range of ±62 degrees. Image correlation for correlative light-electron microscopy was done using the TrakEM2 (Cardona et al., 2012) module of Fiji by transforming the fluorescent image and superimposing it on top of a low-magnification EM image of the cell of interest, using mitochondria as the main fiducial markers. Tilt series were aligned and reconstructed into tomograms using IMOD, segmented using Microscope image browser and the models visualized using 3D Slicer. Zachari M, Gudmundsson SR, Li Z, Manifava M, Cugliandolo F, Shah R, Smith M, Stronge J, Karanasios E, Piunti C, Kishi-Itakura C, Vihinen H, Jokitalo E, Guan JL, Buss F, Smith AM, Walker SA, Eskelinen EL, Ktistakis NT. Selective Autophagy of Mitochondria on a Ubiquitin-Endoplasmic-Reticulum Platform. Dev Cell. 2019 Sep 9;50(5):627-643.e5. doi: 10.1016/j.devcel.2019.06.016. Epub 2019 Jul 25. Erratum in: Dev Cell. 2020 Oct 26;55(2):251. doi: 10.1016/j.devcel.2020.10.002. PMID: 31353311; PMCID: PMC6739445. Gudmundsson SR, Kallio KA, Vihinen H, Jokitalo E, Ktistakis N, Eskelinen EL. Morphology of Phagophore Precursors by Correlative Light-Electron Microscopy. Cells. 2022 Sep 30;11(19):3080. doi: 10.3390/cells11193080. PMID: 36231043; PMCID: PMC9562894.

2019