Imaging and Quantitative Analysis of Atherosclerotic Lesions by CARS-Based Multimodal Nonlinear Optical Microscopy

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Abstract
Objective— The purpose of this study was to assess the ability of label-free multimodal nonlinear optical (NLO) microscopy to characterize, and thus enable quantitative in situ analyses of, different atherosclerotic lesion types, according to the original scheme suggested by the AHA Committee. Methods and Results— Iliac arteries were taken from 24 male Ossabaw pigs divided into lean control and metabolic syndrome groups and were imaged by multimodal NLO microscopy where sum-frequency generation (SFG) and 2-photon excitation fluorescence (TPEF) were integrated on a coherent anti-Stokes Raman scattering (CARS) microscope platform. Foam cells, lipid deposits, matrices, and fibrous caps were visualized with submicron 3D resolution. Starting from the adaptive intimal thickening in the initial stage to the fibrous atheroma or mineralization in the advanced stages, lesions were visualized without labels. Histological staining of each lesion confirmed the lesion stages. Lipid and collagen contents were quantitati... A multimodal nonlinear optical (NLO) microscope, which integrated CARS, TPEF, and SFG on the same platform, was applied to interrogate atherosclerotic lesions without labels. Early and advanced lesions were distinguished with subcellular resolution and with compositional specificity such that in situ quantitative analyses of lipid and collagen contents could be performed.

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