Coherent Optical Scattering and Interferometry (COSI) for label-free multiparameter quantitative imaging of intra-thrombus stability in vitro

Abstract

ABSTRACTAlthough existing microfluidics in vitro assays recapitulate blood vessel microenvironment using surface-immobilized agonists under biofluidic flows, these assays do not quantify intra-thrombus mass and activities of adhesive platelets at agonist margin and uses fluorescence labeling, therefore limiting clinical translation potential. Here, we describe a real time label-free in vitro quantitative imaging flow assay called Coherent Optical Scattering and phase Interferometry (COSI) that evaluates both intra-thrombus and adhesive-only platelet dynamics using only changes in refractive index. By combining coherent optical scattering and optical interferometry, we evaluated and quantified both intra-thrombus mass with picogram accuracy and adhesive platelet-only events/dynamics with high spatial-temporal resolution (400 nm/s) under fluid shear stress using only changes in refractive index. Using oblique illumination, COSI provide a ∼ 4 µm thin axial slice that quantifies the magnitude of physical of surface adhesive platelets (spreading, adhesion and consolidation) in a developing thrombus without labelling under fluid shear stress. We achieve real time visualization of recruitment of single platelet into thrombus and further correlate it to the developing mass of a thrombus. The adhesive platelet activity exhibit stabilized surface activity of around 2 µm/s and intra-thrombus mass exchange were balanced at around 1 picogram after treatment of a broad range metalloproteinase inhibitor (250 µM GM6001).SignificanceThe combination of phase imaging with transmitted light and backscattering imaging via oblique illumination in COSI unpicked intra-thrombus mass and adhesive platelet-only activity events at picogram and sub-micrometer precision with millisecond time resolution under fluid shear stress. COSI maps the longitudinal time dynamics of adhesive platelets along changing thrombus mass under metalloproteinase inhibition, and demonstrates potential for real-time correlative microfluidic label-free imaging for flow-dependent biological adhesive events.