Examining Atherosclerotic Lesions in Three Dimensions at the Nanometer Scale with cryo-FIB-SEM
2Department of Chemical Research Support, Weizmann Institute of Science, Israel
Cholesterol crystallization is central in atherogenesis. Cholesterol crystals lead to plaque instability and rupture, which can result in myocardial infarction and stroke. Cholesterol crystals are associated with intra-lesional macrophages, however, crystallization as well as plaque regression following cholesterol crystal dissolution are still obscure.
MicroCT, cathodoluminescence combined with cryo-SEM and cryo-FIB/SEM were employed to examine rabbit and human atherosclerotic lesions in two and three dimensions, maintained in close to native conditions, with a resolution of tens of nanometers (1). Cryo-FIB/SEM data from lesions were processed and analyzed, following segmentation and volume or surface representation.
In both rabbit and human tissues, early stages of crystalline cholesterol appear to be associated with intra- or extracellular lipid droplets and the outer membrane of multi-lamellar bodies. However, in humans, many of the mature crystals displayed intriguing curved morphologies, fragmented crystals, and foamy lysosome-like structures comprising parts of mature, partially dissolved cholesterol crystals, indicative of possible plaque regression through cholesterol crystal dissolution.
Lipid droplets/multi-lamellar bodies play a role in cholesterol crystal nucleation, which may partially occur intracellularly (1). In humans, strains during cholesterol crystallization may lead to crystal deformation and fragmentation. Plaque regression may occur through active dissolution by lysosome-like structures within macrophages. Combination of cathodoluminescence, cryo-SEM and Cryo-FIB/SEM allows direct, high-resolution, three-dimensional examination of cholesterol deposits in atherosclerotic lesions.
1. Capua-Shenkar, J., Varsano, N., Itzhak, N. R., Kaplan-Ashiri, I., Rechav K. Jin, X., Niimi, M., Fan, J., Kruth, H., Addadi, L. Examining atherosclerotic lesions in three dimensions at the nanometer scale with cryo-FIB-SEM. PNAS, 2022.