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

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 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. Key Words: coherent antistokes Raman scattering Ⅲ multimodal nonlinear optical microscopy Ⅲ Ossabaw miniature swine Ⅲ atherosclerosis Ⅲ histopathology A therosclerosis, the major cause of cardiovascular diseases, has been a leading contributor to morbidity and mortality in the United States, 1 and it has been on the rise globally. 2 The statistics that account for the rise in incidence consequently call for new imaging techniques to advance the research and diagnosis of atherosclerosis. Current imaging methods, such as x-ray angiography, MRI, intravascular ultrasound (IVUS), computed tomography, and optical coherence tomography (OCT) allow exquisite delineation of advanced lesions. 3 Notably, MRI can achieve molecular imaging using contrast agents. 4,5 Also, by using spectral analysis to identify lesion components, IVUS can perform virtual histology. 6 -8 Nonetheless, these techniques have not yet reached submicron resolution. As the gold standard, histology provides high performance in biopsy studies, but it is not feasible for live tissue imaging. In addition, with technical advances, fluorescence microscopy with 1-and 2-photon excitation has been applied to vascular and atherosclerosis studies 5,9 with the capability of identifying cellular and molecular compositions in vivo with labeling. 10 However, these techniques are subject to possible compromises to labeling, such as photobleaching, the requirement for extra incubation, or limited circulation lifetime, all of which could be less optimal for arterial studies. Thus, it is intriguing to explore label-free imaging methods which can also provide chemical selectivity and submicron resolution. Conclusions-WeNonlinear optical (NLO) m...

Nonlinear optical microscopy in decoding arterial diseases

“…6a). The morphology of this IEL layer resembles that in swine artery observed by Wang et al (2009). At a deeper layer (approximately 20 μm), a different fibril structure emerged with an orientation nearly perpendicular to that of IEL membrane (Fig.…”

“…Lesions, however, showed a disoriented collagen fibril morphology with the collagen fibrils in the atheroma running perpendicular to those within the artery wall. A study by Wang et al (2009) focused on imaging arterial cells in relation to the extracellular matrix using a similar multimodal CARS microscope with ps laser pulses, not fs pulses, for TPEF and SHG generation. Using two mode-locked ps Ti:sapphire lasers (one master and one slave), Wang et al (2009) obtained high-resolution images of the normal artery wall at various depths from the lumen.…”

“…A study by Wang et al (2009) focused on imaging arterial cells in relation to the extracellular matrix using a similar multimodal CARS microscope with ps laser pulses, not fs pulses, for TPEF and SHG generation. Using two mode-locked ps Ti:sapphire lasers (one master and one slave), Wang et al (2009) obtained high-resolution images of the normal artery wall at various depths from the lumen. CARS imaging was able to resolve the luminal endothelium monolayer based on the signal originating from the endothelial cell membrane.…”

“…In contrast to the typical CARS signal originating from an ordered orientation of CH 2 groups in lipid bilayers, the CARS signal from IEL was found to be polarization independent and thought to come from disordered orientation of CH 2 -rich residues in the cross-linking region. Without seeing visible thermal damage to artery tissue, Wang et al (2009) concluded that ps laser was a feasible light source to use for SHG/TPEF generation in arterial tissue despite the requirement of a higher average power compared to femtosecond sources.…”

“…In another study, Doras et al (2011) performed multiphoton image reconstruction and also analyzed the change in optical polarization that occurs in atherosclerotic plaque development. Using multimodal CARS imaging, Wang et al (2009) reported area percentage calculations of collagen and lipid content in the thickened initima of different types of atherosclerosis lesions. These authors were the first to demonstrate that this type of quantification correlates well with histology studies.…”