In vivo two‐photon microscopy for imaging leukocytes in atherosclerotic plaques (547.16)

Abstract

Atherosclerosis is characterized by chronic inflammation of the walls of large and medium arteries that have been infiltrated by macrophages, dendritic cells (DCs), T cells, and other leukocytes. Disease progression is determined in part by a variety of cell functions that are regulated by the cell’s motility, including recruitment, transmigration, egress, phagocytosis, and interaction between multiple cell types. In vivo imaging is necessary to better understand leukocyte activity during atherosclerosis within the native plaque 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, which expand with every heart beat. In‐frame and between‐frame motion cause still images to be distorted and movies to appear unsteady, making cell tracking infeasible.We engineered an integrated hardware and software system for in vivo two‐photon imaging of large arteries. 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 via pulse oximetry. To minimize distortions, a method 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 larger frame. A custom stage was built to precisely position the mouse under the microscope objective in 3 dimensions in coordination with the trigger mechanism. The resulting 3D movies will be used to track cell motion. This system can be used to study multiple questions involving cell motility in vivo, including leukocyte recruitment from blood, antigen presentation, phagocytosis, chemokine function, and the role of cell subsets in disease progression.Grant Funding Source: Supported By the AHA (11PRE7580009), HHMI (56005681), and NHLBI (T32HL007089‐35)