Tecnai feg 20

Samples were prepared as described for TEM. Dual axis tilt series (Mastronarde, 1997 (link)) were recorded from one or consecutive semi-thick (250 nm) sections using Tecnai FEG 20 (FEI Company) microscope operating at 200 kV. The sections were tilted at one-degree intervals using a high-tilt specimen holder (model 2020; E.A. Fischione Instruments) between ±62°. Images were acquired with SerialEM software (Mastronarde, 2005 (link)) using a 4 k × 4 k Ultrascan 4000 CCD camera (Gatan Corporation) at nominal magnification of 9,600× or 11,500×. The alignment of the tilt series and reconstructions were done with IMOD software package (Kremer et al, 1996 (link)) using 10-nm colloidal gold particles as fiducial markers. Tomographic reconstructions were segmented with MIB software (Belevich et al, 2016 (link)) and visualization was performed using Amira (VSG, FEI Company).

The sections stuck on EM grids were contrasted using uranyl acetate (4% (w/v) in 70% methanol) for 2 min, then after rinsing with 50% methanol in water, they were further stained with Reynolds lead citrate for 1 min^{39 (link)}. Tomography data were acquired on a FEI Tecnai FEG30, operating at 300 kV with a CMOS camera (One View, Gatan 4 Kx4K, Gatan Inc. USA) or on a FEI Tecnai FEG20, operating at 200 kV equipped with a CCD Ultrascan camera (Gatan 2 Kx2K, Gatan Inc. USA). In both cases, the tilt series were acquired with 1° increment from −60° to +60°, single axis^{38 (link)}, using SerialEM^{40 (link)} acquisition software, and reconstructed using etomo^{41 (link)}. To find the lesion site in the tomography microscope, we calculated the affine transfer matrix between the MIM and the TEM coordinate system, including scaling, rotation and translation factors (Fig. S3a,d). To find these parameters we tracked the three reference points in both LM and EM (Fig. S3b). The LM coordinates were recorded using Fiji or ImageJ software. The matrix was then used to calculate the TEM coordinates of the ROI (Fig. S3c,d). A coordinate converter GUI (Fig. S3e) allowed the users to enter the coordinates of the selected points and performed automatically the required calculation in a “user-friendly” way (Fig. S3e). The motorised stage then moved the grid to the right place (ROI) for tilt series acquisition.

ET was done on serial 250-nm thick sections as previously described52 (link), except that the tilt series images between ±62° were acquired with an UltraScan 4000 CCD camera, 4 k × 4 k (Gatan Inc.) at nominal magnification of 6.500×, 7.800× or 9.600×. Dual axis tilt series were acquired using SerialEM software running on a Tecnai FEG 20 microscope (FEI Company, Hillsboro, OR) operating at 200 kV. Gold particles on tomograms were quantified by manually tagging on separately modelled ER structures. Reconstructions were done using IMOD software53 (link) followed by visualization and modelling using MIB and Amira (VSG, FEI Company). The presented 3D models of ER are shown in perspective view, where the bars apply to the centre point of the image except in Fig. 6Cf.