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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2009;29:1342.)
© 2009 American Heart Association, Inc.

Integrative Physiology/Experimental Medicine

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

Han-Wei Wang; Ingeborg M. Langohr; Michael Sturek; Ji-Xin Cheng

From the Weldon School of Biomedical Engineering (H.-W.W., J.-X.C.), the Department of Comparative Pathobiology (I.M.B.), and the Department of Chemistry (J.-X.C.), Purdue University, West Lafayette, Ind; and the Department of Cellular & Integrative Physiology (M.S.), Indiana University School of Medicine, Indianapolis.

Correspondence to Ji-Xin Cheng, 206 S Martin Jischke Drive, West Lafayette, IN 47907-2032. E-mail jcheng{at}purdue.edu

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 quantitatively analyzed based on the CARS and SFG signals. Lipid accumulation in thickened intima culminated in type IV whereas the highest collagen deposition was found in Type V lesions. Luminal CARS imaging showed the capability of viewing the location of superficial foam cells that indicate relatively active locus in a lesion artery.

Conclusions— We have demonstrated the capability of CARS-based multimodal NLO microscopy to interrogate different stages of lesion development with subcellular detail to permit quantitative analysis of lipid and collagen contents.

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.

Key Words: coherent antistokes Raman scattering • multimodal nonlinear optical microscopy • Ossabaw miniature swine • atherosclerosis • histopathology