Label-free multimodal quantitative imaging flow assay for intra-thrombus formation in vitro.

Abstract Microfluidics in vitro assays recapitulate a blood vessel microenvironment using surface-immobilized agonists under biofluidic flows. However, these assays do not quantify intra-thrombus 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 (ROCS) microscopy and quantitative phase microscopy (QPM). The combined imaging platform enables real-time evaluation of membrane fluctuations of adhesive-only platelets and total intra-thrombus mass under physiological flow rates in vitro. We call this multi-modal quantitative imaging flow assay- Coherent Optical Scattering and phase Interferometry (COSI). COSI records intra-thrombus 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-1). With COSI, we generate a thrombus axial slice 4 μm from the coverslip surface, approximately equivalent to the thickness of a single platelet, that permits nanoscale quantification of membrane fluctuation (activity) of adhesive platelets during initial adhesion, spreading and recruitment into a developing thrombus (mass). This activity was inhibited when actin polymerization was prevented. Under fluid shear, pre-treatment 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 intra-thrombus mass.