Oregon Health and Science University, School of Medicine, Department of Biomedical Engineering, 3303 SW Bond Avenue, Portland, Oregon 97239, USA.
J Biomed Opt. 2013 Jan;18(1):16014. doi: 10.1117/1.JBO.18.1.016014.
Flow chamber assays, in which blood is perfused over surfaces of immobilized extracellular matrix proteins, are used to investigate the formation of platelet thrombi and aggregates under shear flow conditions. Elucidating the dynamic response of thrombi/aggregate formation to different coagulation pathway perturbations in vitro has been used to develop an understanding of normal and pathological cardiovascular states. Current microscopy techniques, such as differential interference contrast (DIC) or fluorescent confocal imaging, respectively, do not provide a simple, quantitative understanding of the basic physical features (volume, mass, and density) of platelet thrombi/aggregate structures. The use of two label-free imaging techniques applied, for the first time, to platelet aggregate and thrombus formation are introduced: noninterferometric quantitative phase microscopy, to determine mass, and Hilbert transform DIC microscopy, to perform volume measurements. Together these techniques enable a quantitative biophysical characterization of platelet aggregates and thrombi formed on three surfaces: fibrillar collagen, fibrillar collagen +0.1 nM tissue factor (TF), and fibrillar collagen +1 nM TF. It is demonstrated that label-free imaging techniques provide quantitative insight into the mechanisms by which thrombi and aggregates are formed in response to exposure to different combinations of procoagulant agonists under shear flow.
流室分析中，血液在固定化细胞外基质蛋白表面流过，用于在切变流条件下研究血小板血栓和聚集体的形成。阐明血栓/聚集体形成对体外不同凝血途径扰动的动态响应，有助于了解正常和病理心血管状态。目前的显微镜技术，如微分干涉对比（DIC）或荧光共聚焦成像，分别不能简单、定量地了解血小板血栓/聚集体结构的基本物理特征（体积、质量和密度）。本文首次介绍了两种无标记成像技术在血小板聚集和血栓形成中的应用：非干涉定量相显微镜用于测定质量，希尔伯特变换 DIC 显微镜用于进行体积测量。这些技术一起实现了在三种表面（纤维胶原、纤维胶原+0.1 nM 组织因子（TF）和纤维胶原+1 nM TF）上形成的血小板聚集体和血栓的定量生物物理特征分析。实验表明，无标记成像技术为研究在切变流条件下，血小板血栓和聚集体形成的机制提供了定量的见解，这些机制是对不同促凝剂组合暴露的响应。
