Laser Microbeam and Medical Program (LAMMP), Beckman Laser Institute and Medical Clinic, Department of Physiology and Biophysics, University of California, Irvine, CA, USA.
J Lipid Res. 2010 Jul;51(7):1729-37. doi: 10.1194/jlr.M003616. Epub 2010 Mar 5.
We characterized several cellular and structural features of early stage Type II/III atherosclerotic plaques in an established model of atherosclerosis-the ApoE-deficient mouse-by using a multimodal, coregistered imaging system that integrates three nonlinear optical microscopy (NLOM) contrast mechanisms: coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), and two-photon excitation fluorescence (TPEF). Specifically, the infiltration of lipid-rich macrophages and the structural organization of collagen and elastin fibers were visualized by CARS, SHG, and TPEF, respectively, in thick tissue specimens without the use of exogenous labels or dyes. Label-free CARS imaging of macrophage accumulation was confirmed by histopathology using CD68 staining. A high-fat, high-cholesterol Western diet resulted in an approximate 2-fold increase in intimal plaque area, defined by CARS signals of lipid-rich macrophages. Additionally, analysis of collagen distribution within lipid-rich plaque regions revealed nearly a 4-fold decrease in the Western diet-fed mice, suggesting NLOM sensitivity to increased matrix metalloproteinase (MMP) activity and decreased smooth muscle cell (SMC) accumulation. These imaging results provide significant insight into the structure and composition of early stage Type II/III plaque during formation and allow for quantitative measurements of the impact of diet and other factors on critical plaque and arterial wall features.
我们使用一种多模态、配准的成像系统，整合了三种非线性光学显微镜（NLOM）对比机制：相干反斯托克斯拉曼散射（CARS）、二次谐波产生（SHG）和双光子激发荧光（TPEF），对动脉粥样硬化模型（载脂蛋白 E 缺陷小鼠）中早期 II/III 型动脉粥样硬化斑块的几种细胞和结构特征进行了描述。具体而言，我们分别使用 CARS、SHG 和 TPEF 可视化了富含脂质的巨噬细胞的浸润以及胶原蛋白和弹性纤维的结构组织，而无需使用外源性标记物或染料。使用 CD68 染色进行组织病理学检查，证实了 CARS 对巨噬细胞积累的无标记成像。高脂肪、高胆固醇的西方饮食导致富含脂质的巨噬细胞的 CARS 信号所定义的内膜斑块区域增加了约 2 倍。此外，对富含脂质斑块区域内胶原分布的分析表明，在西方饮食喂养的小鼠中，胶原分布减少了近 4 倍，这表明 NLOM 对基质金属蛋白酶（MMP）活性增加和平滑肌细胞（SMC）积累减少敏感。这些成像结果深入了解了早期 II/III 型斑块形成过程中的结构和组成，并允许对饮食和其他因素对关键斑块和动脉壁特征的影响进行定量测量。
