Atherosclerotic plaques from human external or common carotid arteries were acquired from patients undergoing carotid endarterectomy. Intimal pieces were fixed with 2% formaldehyde and 2.5% glutaraldehyde, in 0.1 mol/L Na-cacodylate buffer (pH 7.4) supplemented with 2 mmol/L CaCl2 for 2 to 3 hours. The fixed samples were stored in 0.1 mol/L Na-cacodylate–2 mmol/L CaCl2 buffer containing 2% of paraformaldehyde for stability and were further processed for TEM using a standard TEM protocol. 250 nm thick sections were cut from a trimmed pyramid, and 10-nm colloidal gold particles were placed on top/below the section to serve as fiducial markers for the alignment. The dual-axis tilt series was recorded using a Tecnai FEG 20 transmission electron microscope operating at 200 kV. Images were acquired using SerialEM software version 3.2.243 and a 4K × 4K Ultrascan 4000 charge-coupled device camera at a nominal magnification of ×9600, providing a 2× binned pixel size of 1.94 nm. For tilt series, the specimens were tilted at one-degree intervals using a high-tilt specimen holder between ±62 degrees. The Tilt series was aligned and reconstructed with the IMOD software package version 4.7.13,44 and the tomographic reconstruction was analyzed, modeled, and visualized with MIB software version 0.999 and Amira software version 6.0.1.

2017