ACRF Department of Cancer Biology and Therapeutics, John Curtin School of Medical Research.
ACRF Department of Cancer Biology and Therapeutics, John Curtin School of Medical Research; ACRF Centre for Intravital Imaging of Niches for Cancer Immune Therapy.
Biophys J. 2021 Mar 2;120(5):791-804. doi: 10.1016/j.bpj.2021.01.015. Epub 2021 Jan 26.
Microfluidics in vitro assays recapitulate a blood vessel microenvironment using surface-immobilized agonists under biofluidic flows. However, these assays do not quantify intrathrombus mass and activities of adhesive platelets at the agonist margin and use fluorescence labeling, therefore limiting clinical translation potential. Here, we describe a label-free multimodal quantitative imaging flow assay that combines rotating optical coherent scattering microscopy and quantitative phase microscopy. The combined imaging platform enables real-time evaluation of membrane fluctuations of adhesive-only platelets and total intrathrombus mass under physiological flow rates in vitro. We call this multimodal quantitative imaging flow assay coherent optical scattering and phase interferometry (COSI). COSI records intrathrombus mass to picogram accuracy and shape changes to a platelet membrane with high spatial-temporal resolution (0.4 μm/s) under physiological and pathophysiological fluid shear stress (1800 and 7500 s). With COSI, we generate an axial slice of 4 μm from the coverslip surface, approximately equivalent to the thickness of a single platelet, which permits nanoscale quantification of membrane fluctuation (activity) of adhesive platelets during initial adhesion, spreading, and recruitment into a developing thrombus (mass). Under fluid shear, pretreatment with a broad range metalloproteinase inhibitor (250 μM GM6001) blocked shedding of platelet adhesion receptors that shown elevated adhesive platelet activity at average of 42.1 μm/s and minimal change in intrathrombus mass.
在体外，微流控芯片在生物流体流动下使用表面固定的激动剂重现血管微环境。然而，这些测定方法无法在激动剂边缘定量测量血栓内的质量和黏附血小板的活性，且使用荧光标记，因此限制了其临床转化的潜力。在此，我们描述了一种无标记的多模态定量成像流分析，结合了旋转光学相干散射显微镜和定量相位显微镜。该联合成像平台能够实时评估在生理流速下仅黏附血小板的膜波动和血栓内总质量。我们将这种多模态定量成像流分析称为相干光学散射和相位干涉（COSI）。COSI 以皮克级的精度记录血栓内质量，并以高时空分辨率（0.4 μm/s）记录血小板膜的形状变化，在生理和病理生理流体切应力（1800 和 7500 s）下。通过 COSI，我们从盖玻片表面生成 4 μm 的轴向切片，大约相当于单个血小板的厚度，这允许在初始黏附、扩展和募集到正在形成的血栓时（质量）对黏附血小板的膜波动（活性）进行纳米级定量。在流体切应力下，用广泛的金属蛋白酶抑制剂（250 μM GM6001）预处理可阻止血小板黏附受体的脱落，从而显示出黏附血小板活性的升高，平均为 42.1 μm/s，血栓内质量的变化最小。
