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Atherosclerosis is a leading cause of mortality in industrialized countries. In addition to "traditional" systemic risk factors for atherosclerosis, the geometry and motion of coronary arteries, which vary substantially among individuals, may contribute to individual susceptibility to the development and progression of disease in these vessels. We have developed a high-speed microscope-based stereo-imaging system to quantify the motion of epicardial coronary arteries of wild-type and apo-E deficient mice for correlation against the degree of atherosclerotic change in these vessels. Using near-infrared non-targeted quantum dots as a contrast agent, we synchronously acquire paired images of the surgically exposed murine heart, from which the 3-D geometry of the coronary arteries is reconstructed. The reconstructed geometry is tracked frame by frame through the cardiac cycle to quantify the in vivo motion of the vessel, and displacements, curvature, and torsion parameters are derived. Illustrative results for C57BL/6J mice will be presented.
The imaging system shown in Figure 1, which also serves as the surgical microscope, consists of a stereomicroscope (SMZ-1000) with a 0.5X objective, fluorescent filter attachment, and dual beam splitter (Nikon Instruments, Melville, NY), an excitation light source, a computer, a calibration device, and two high-performance synchronized digital CCD cameras with 2 GB of internal memory (pco. 1600, The Cooke Corporation, Romulus, MI). Using IEEE 1394 cables, both cameras are connected to the computer. During image acquisition, the arterial images are stored on the camera's internal memory, and then after each experiment downloaded to a computer for image processing. The near-infrared emission light of the contrast agent is filtered using two 18-mm diameter 690--730-nm bandpass emission filters (710AF40, Omega Optical, Brattleboro, VT). To excite the contrast gent, a 150-W fiber-optic illuminator (Schott-Fostec, LLC, Auburn, NY) outfitted with a EKE High-Output halogen bulb (USHIO, Cypress, CA) and a hot mirror (0\deg angle of incidence, Edmund Optics, Barrington, NJ) is used. Attached to the illuminator is a liquid light guide (8-mm core diameter, 1-m long, Newport, Irvine, CA) and a fiber-optic focusing lens unit (Edmund Optics, Barrington, NJ), which was modified to hold the 25-mm diameter Qdot excitation filter (shortpass 650-nm cutoff, Edmund Optics, Barrington, NJ); the focusing unit is attached to an articulating arm mounting system to position the focusing unit during image acquisition. The calibration device (see Figure1 B) used in this study is analogous to that used in previous biplane angiographic studies of human coronary arterial motion; it is used to define the imaging. The device is a black Delrin "pyramid" embedded with 88 0.5-mm diameter stainless steel 440 spheres (Salem Specialty Ball, Canton, CT).
Figure 1: (Left) The high-speed microscope-based imaging system developed to quantify the motion of the murine epicardial coronary arteries consists of the following: (a) stereomicroscope, (b) dual beam splitter, (c) fluorescent filter attachment, (d) two high-performance synchronized CCD cameras, (e) computer monitor to observe the image acquisition, (f) an excitation light source, (g) a calibration device, and (h) a computer (not shown) to control the cameras. (Top Right) The transmission spectra versus wavelength for the excitation filter (dashed line) and emission filter (solid line). (Bottom Right) Photomicrographs of the top view (left image) and side view (right image) of the calibration pyramid used to calibrate the acquired images. Horizontal distance between scale bars is 1 mm.
The image processing method used here to track the murine coronary arteries using a sequence of paired images is based on the technique developed for tracking human coronary arteries in biplane angiograms (Cataloguing the Geometry of the Human Coronary Arteries). It involves three main steps: (1) calibrating the imaging system; (2) reconstructing the 3-D vessel axis in the first frame; and (3) tracking the 3-D vessel axis through the entire sequence of images.
Figure 2: (A) Paired fluorescent images of the main branch of the left epicardial coronary artery in vivo (denoted by arrow) on the beating heart of a 29.2-g C57BL/6J mouse imaged, at a frame rate of ~ 40-fps, with the stereo-imaging system. The mouse was intravenously injected with ~ 8.5-pmol/g of non-targeted, near-infrared quantum dots imaging contrast agent. Superimposed on each image in (A) is the 3-D reconstructed vessel axis segment (dotted white line). (B) For the paired images in (A), the ~4.3-mm long reconstructed segment of the vessel axis is calculated from 22 locations spaced ~ 0.2-mm along the vessel and displayed in approximately the same orientation of the heart in (A). Note: The distance between each tick mark is ~ 0.36-mm.
Figure 3: Paired fluorescent image sequence of the main branch of the left epicardial coronary artery in vivo (denoted by arrow) on the beating heart of a 29.2-g C57BL/6J mouse imaged, at a frame rate of ~ 40-fps (click QuickTime icon to see movie). The mouse was intravenously injected with ~ 8.5-pmol/g of non-targeted, near-infrared quantum dots imaging contrast agent. Superimposed on each image is the 3-D reconstructed vessel axis segment (dotted white line).
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