24. IN VIVO TWO-PHOTON MICROSCOPY FOR IMAGING LEUKOCYTES IN ATHEROSCLEROTIC PLAQUES

Department: Bioengineering
Faculty Advisor(s): Klaus Ley

Primary Student
Name: Sara Ashley McArdle
Email: smcardle@ucsd.edu
Phone: 908-723-4157
Grad Year: 2015

Abstract
Atherosclerotic plaques form when inflammatory leukocytes (mostly lymphocytes, macrophages, and dendritic cells) migrate into the arterial wall, take up lipids, interact with other cells of the same or different subtypes and further promote inflammation. Currently, leukocyte activity within atherosclerotic plaques is often studied in vitro or ex vivo; however, mouse in vivo studies are necessary to accurately understand these cells' role in disease progression in their complex native microenvironment. Two-photon microscopy is emerging as a valuable tool to image live, fluorescent cells in explants or certain organs in vivo. However, new technical challenges are introduced when imaging tissues that move within the body, such as arteries, because in-frame and between-frame motion cause still images to be unclear and movies to appear unsteady. This work focuses on engineering an integrated hardware and software system for in vivo two-photon imaging of large arteries which expand with every heart beat and shift with every breath. A custom stage was built to precisely position the mouse under the microscope objective. To ensure that the artery is in the same position for every picture, a trigger mechanism was constructed which links image acquisition to the mouse's heartbeat measured via pulse oximetry. To minimize blur in each frame, a system was developed to acquire only a fraction of a frame during each heart beat and then stitch the resulting small strips together to form a full frame using image processing software. This method will enable us to obtain more accurate and useful information about leukocyte movement in mouse models of atherosclerosis.

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