Imaging coronary artery microstructure using second-harmonic and two-photon fluorescence microscopy.
Imaging coronary artery microstructure using second-harmonic and two-photon fluorescence microscopy.
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DOI：
10.1529/biophysj.104.042887
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发表时间：
2004-10
影响因子：
3.4
通讯作者：
Aikaterini Zoumi;Xiao Lu;G. Kassab;B. Tromberg
中科院分区：
文献类型：
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作者：
Aikaterini Zoumi;Xiao Lu;G. Kassab;B. Tromberg
由于缺乏产生这些血管壁成分的三维视图的无损方法，血管机械性能的微观结构基础尚未直接确定。在这里，我们证明，通过将切除的猪冠状动脉的机械测试（扩张）与同步双光子激发荧光和二次谐波产生显微镜相结合，多光子显微镜可用于可视化血管机械特性的微观结构基础。我们的结果表明，源自胶原蛋白的二次谐波生成信号可以与弹性蛋白和平滑肌细胞双光子荧光进行光谱分离。双光子荧光信号可以通过 495 nm 和 520 nm 处的发射最大值进一步表征，分别对应于弹性蛋白和细胞贡献。光谱融合图像的二维重建允许胶原蛋白和弹性蛋白原纤维以及从内膜到外膜的平滑肌细胞的高分辨率可视化。这些结构特征通过多光子显微镜图像与传统组织学的共配准得到证实。当将无负载（零跨壁压力）和零应力条件与 30 和 180 mmHg 膨胀压力进行比较时，观察到平均原纤维厚度和总体壁尺寸的显着变化。总的来说，这些数据表明多光子显微镜是研究血管壁微观结构形态测量特性的一种高度敏感且有前途的技术。
The microstructural basis for the mechanical properties of blood vessels has not been directly determined because of the lack of a nondestructive method that yields a three-dimensional view of these vascular wall constituents. Here, we demonstrate that multiphoton microscopy can be used to visualize the microstructural basis of blood vessel mechanical properties, by combining mechanical testing (distension) of excised porcine coronary arteries with simultaneous two-photon excited fluorescence and second-harmonic generation microscopy. Our results show that second-harmonic generation signals derived from collagen can be spectrally isolated from elastin and smooth muscle cell two-photon fluorescence. Two-photon fluorescence signals can be further characterized by emission maxima at 495 nm and 520 nm, corresponding to elastin and cellular contributions, respectively. Two-dimensional reconstructions of spectrally fused images permit high-resolution visualization of collagen and elastin fibrils and smooth muscle cells from intima to adventitia. These structural features are confirmed by coregistration of multiphoton microscopy images with conventional histology. Significant changes in mean fibril thickness and overall wall dimension were observed when comparing no load (zero transmural pressure) and zero-stress conditions to 30 and 180 mmHg distension pressures. Overall, these data suggest that multiphoton microscopy is a highly sensitive and promising technique for studying the morphometric properties of the microstructure of the blood vessel wall.