Microscopy and quantification of NCs taken up by HAECs and aorta in ApoE−/− mice

For HAEC samples, images were captured with a Nikon widefield deconvolution epifluorescence microscope equipped with ×20 and ×40 objectives and filters optimized for Texas Red and DAPI. For en face aorta samples, microscope slides were fluorescence scanned using a Panoramic MIDI II (3D Histech, iHisto service https://www.ihisto.io/). A Leica SP5 confocal microscope (BU imaging core) was used to perform high-resolution imaging of en face endothelium and aortic lesions.

NP images were quantified with FIJI (NIH ImageJ) by first setting a threshold value that reflected the fluorescence value of the nanoparticle. The binary threshold images were then quantified using the FIJI function by setting resolution at 2–200 pixel² and circularity at 0.10–1.00.