Three-dimensional Flow-induced Dynamics

Endothelial cells play a central role in maintaining vascular integrity. Lesions in the endothelial layer may allow the invasion of leukocytes into the artery wall and promote inflammatory responses that could possibly lead to atherosclerosis. Over the past decade, a series of studies have demonstrated the presence of a delicate surface layer, named the glycocalyx, decorating the endothelial membrane and associated with a number of physiological implications in the vasculature. This thesis studies the flow-induced dynamics of the endothelial glycocalyx layer and reports the first direct measurement of the in vitro mechanical properties as well as the thickness of the glycocalyx through a novel optical imaging approach. Here, we have developed an optical imaging system that tracks the position of single particles in three-dimensional (3D) space with high precision, typically 1 ~ 4 nm in the XY plane and 15 ~ 40nm in the Z-direction along the optical axis of the microscope. With the use of quantum dots (QDs), semiconducting nanoparticles of superior optical characteristics, this method provides the opportunity to investigate biological subjects in living cells that can be neither spatially resolved nor continuously traced over minutes by conventional optical microscopy techniques. Therefore, by 3D mapping of both the glycocalyx layer and the membrane using quantum dots of different colors, the in vitro thickness of the endothelial glycocalyx layer is found to be ~ 350nm. With QDs attached to the glycocalyx layer, we track the flow-induced motion of this thin structure at multiple levels of oscillating shear stresses from 5 to 20 dynes/cm2 . The displacement of each QD ranges from 80 400 nrm and varies from cell to cell and point to point on a single cell. The estimated Young's modulus is thus -6.7Pa, for a 3D matrix with a measured thickness of~ 350nm. The QD motion, staying in phase with the applied shear stress up to the 1Hz limit of the present study, confirms the highly elastic gel nature for the glycocalyx layer in which major deformations occur at physiological level of fluid shear stress. Thesis supervisor: C. Forbes Dewey, Jr. Title: Professor of Mechanical and Biological Engineering Acknowledgements I sincerely thank Prof. C. Forbes Dewey, the best advisor and teacher I could ever wish for. Without his wisdom, support, enthusiasm and inspiration, I would have never made even a small step of progress in my thesis. It has been a great journey to work with him all these years, where I learned so much about science and life. And most importantly, I am grateful to an invaluable friendship that will continue for the rest of my life. I wish to thank my committee members, Prof. Roger Kamm, Prof. Peter So, and Prof. Ram Sasisekharan, for providing continuous guidance and sharing with me their insightful knowledge throughout the course of the project. I am extremely fortunate to have worked with two best collaborators I could ever imagine, Maxine Jonas and Hao Huang. This thesis could not have been completed without their numerous efforts devoted into the collaboration. I am thankful to Alex Rabodzy, who prepared me for the first in vitro experiments in my life, and David Berry, Eric Osborn, Maryelise Cieslewicz and many people who have helped me through the completion of this thesis. My special thanks to Donna Wilker, Leslie Regan, and my collegues in the Hatsopolous Microfluids Lab who have made all these years such an enjoyable experience. I am forever indebted to my parents for their love, understanding and endless patience and encouragement ever since they gave birth to a little baby girl, 26 years ago. Finally, I thank my husband, Jian, for the love and support that have been around me all along. Always a true believer in me, enduring my late hours, my spoiled weekends, my bad temper, and even my computer bugs. To him, I dedicate this thesis.