Label-Free Functional Imaging Of Platelets At Nanoscale
Creators: name: Zheng, Yujie
Publisher: name: The Australian National University
Year: 2022

description: Platelet is the key cellular machinery of a blood clot/thrombus. Platelet morphodynamics under fluid shear plays an essential role in the quantitative analysis of platelet dysfunction. The mechanical properties of platelets inform the formation of haemostatic plugs that have wide application in bleeding disorders and cardiovascular diseases. During clotting, individual platelets sense physical and biochemical agonists on their surface receptors that trigger the formation of membrane protrusions. Platelets would reorganize their cytoskeletal structure upon sensing agonists to manipulate the extracellular matrix (collagen and fibrin fibres) and form a stable blood thrombus. However, platelet dysfunction would lead to an imbalance between haemostasis and thrombosis that causes life-threatening diseases, including pulmonary embolism and ischemic stroke. In the last few decades, the development of advanced imaging tools (optical, electrons, conductance, and atomic force) have revealed a highly heterogeneous distribution of platelet morphologies at the microscopic scale that spreads across a thrombus. Under platelet spreading assays, the membrane protrusions of platelets have been observed to manipulate fibrin fibres and contribute to the mechanical contraction of a blood clot. Current optical imaging tools in platelet are generally performed with specific fluorescence markers. Since the human platelets sample is unsuitable for genetic manipulation, platelet labelling is often conducted using membrane markers or with fixed samples which could restrict nanoscopic imaging of the platelet dynamics at video rate. Rapid assessment of platelet dysfunction without markers will accelerate many aspects of platelet studies. Therefore, it is imperative to develop a label-free imaging flow assay (video rate and diffraction-limited) that could measure the morphodynamics of single platelet in a thrombus. Existing label-free imaging tools such as differential interference contrast, darkfield, phase contrast, and reflectance interference contrast are regularly applied to platelets imaging but lack imaging contrast for quantifying platelet motility. Nanoscopic label-free imaging techniques based on light scattering and interferometry have emerged as a rapid high-resolution live-cell imaging technique. Applying nanoscopic label-free imaging permits real-time imaging of platelet membrane protrusion (lamellipodium, filopodia) and morphological profiling. The capability to quantify platelet morphology during thrombus formation and embolization, along with platelets detachment, enables in-depth assessment of functional responses of platelets. In the first part of the thesis (Chapter 1-3), I shall describe the role of scattering in forming a microscopic image of a cell using numerical models and review current imaging tools tailored for platelet and thrombus studies. The second part of the thesis (Chapter 4) covers the steps to design, calibrate, and characterize a series of label-free tools (COSI) for platelet imaging under flow. Using the COSI system, I demonstrate the detection sensitivity down to 20 nm gold nanoparticles without post-processing. In the third part of the thesis (Chapter 5-6), I shall elaborate on platelet spreading assays and migration studies to quantify single platelet adhesion and characterize platelet membrane protrusions with ECM (collagen, fibrin, fibrinogen, CRP). In the final chapter, I demonstrate label-free imaging on thrombus formation and study the effects of a metalloproteinase inhibitor in embolization with piconewton (pg) and high spatial-temporal resolution (0.4 um/s). In summary, this thesis paves the way for label-free nanoscale imaging of quantitative study of platelets and blood thrombus under flow.