Multiplex Coherent Anti-Stokes Raman Spectroscopy Images Intact Atheromatous Lesions and Concomitantly Identifies Distinct Chemical Profiles of Atherosclerotic Lipids

Abstract

Lipids are a key component of atherogenesis. However, their physiological role on the progression of atherosclerosis including plaque vulnerability has not been clearly understood, because of the lack of appropriate tools for chemical assessment. We aimed to develop a label-free chemical imaging platform based on multiplex coherent anti-Stokes Raman scattering (CARS) for the correlative study of the morphology and chemical profile of atherosclerotic lipids. Whole aortas from atherosclerotic apolipoprotein E knock-out mice were en face examined by multiplex CARS imaging and 4 distinctive morphologies of the lipids (intra/extracellular lipid droplets and needle-/plate-shaped lipid crystals) were classified. The chemical profiles of atherosclerotic lipids depending on morphologies were firstly identified from intact atheromatous tissue by multiplex CARS. We demonstrated that needle-/plate-shaped lipid crystals in advanced plaques had undergone a phase shift to the solid state with increased protein contents, implying that lipid modification had occurred beforehand. The validity of lipid-selective multiplex CARS imaging was supported by comparative results from oil red O staining and whole-mount immunohistochemistry. By spatial CARS analysis of atherosclerosis progression, we found greater accumulation of lipid crystals in both the lesser curvature of the aortic arch and the innominate artery. Furthermore, multiplex CARS measurement successfully demonstrated the effect of a drug, statin, on atherosclerotic lipids by showing the change of their chemical profiles. Multiplex CARS imaging directly provides intact morphologies of atherosclerotic lipids with correlative chemical information, thereby suggesting its potential applications in the investigation of lipid-associated disorders and the preclinical drug screening.