(The) study of morphology and dynamics of platelets using quantitative phase imaging

Abstract

Platelets, or thrombocytes, are anucleated tiny blood cells and have an indispensable role in hemostatic properties of whole blood by detecting injured sites at the surface of blood vessels and forming blood clots. In particular, in the routine surgical operations, a lack of or functional loss of platelets may lead to excessive bleeding in a patient and, in contrast, excessive activation of platelets can cause an embolism. Therefore, there has been a great demand for a technique capable of quickly and objectively measuring the platelet functionalities. The most direct method for examining the platelet function is to observe the activability of the platelets, and each platelet undergoes a large morphological alteration such as the exocytosis of intraplatelet granules or formation of pseudopods at the surface during the activation process. However, due to the small size of the platelets and the irregularity of its shape, most current clinical tests mainly focus on the observation of the macroscopic aggregations among platelets, rather than the activation of individual platelets. Accordingly, imaging-based platelet studies still remain at the basic level, and, in particular, the optical observation of the whole activation process of platelets at the individual cell level has not been thoroughly investigated yet.

In this thesis, we quantitatively and non-invasively investigate the morphological and biochemical alterations of individual platelets in its activation process without using any exogenous agent by employing 3-D quantitative phase imaging (QPI) technique which can reconstruct 3-D refractive index (RI) tomograms of microscopic cells. In the first chapter of this thesis, we briefly deal with the overall morphology and intraplatelet structure of platelets and the internal mechanism of platelet activation. In the following chapter, we introduce general principles of QPI. In the third chapter, we address the changes in various characteristics of platelets depending on the extent of platelet activation by optically measuring individual platelets before and after administration of platelet agonists. Here, collagen, thrombin, and thrombin receptor activating peptides are used to selectively stimulate the activation signalling routes of platelets. In the fourth chapter, we introduce a modified QPI setup for high-speed and long-time measurements of platelet tomogram. Then, resting platelets in the plasma are activated using thrombin, and 3-D RI tomograms of the corresponding platelets are simultaneously measured at both long and short time-scales to access the continuous morphological and biochemical alterations of individual platelets under activation. In Chapter 5, in the extension of the concerns about utilization of the holographic imaging system for a clinical platelet study, we quantitatively address how the morphological and intraplatelet properties of the stored platelets change with the storage period and method. Finally, in the last chapter, we summarize the contents covered in this thesis as a whole, and present a future research direction that QPI should go ahead in order to be widely utilized in the clinical field in the future.