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1 Cardiology Division of the Department of Medicine, Emory University School of Medicine, Atlanta, GA, USA
2 Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, USA
3 Division of Cardiothoracic Surgery. Department of Surgery, Emory University School of Medicine, Atlanta, GA, USA
4 Departments of Biomedical Engineering and Chemistry, Emory University and Georgia Institute of Technology, Atlanta, GA, USA
5 Cardiology Division of the Department of Medicine, Emory University School of Medicine, Atlanta, GA, USA; Cardiology Division of the Department of Medicine, Atlanta VA Medical Center, Atlanta, GA, USA
* To whom correspondence should be addressed. E-mail: wtaylor{at}emory.edu.
Background: Traditional imaging with single-photon confocal microscopy and organic fluorophores poses several challenges for the visualization of vascular tissue, including autofluorescence, fluorophore crosstalk and photobleaching. Methods: We studied human coronary arteries (HCAs) and mouse aortas with a modified immunohistochemical (IHC) en face method using quantum dot (Qdot) bioconjugates and two-photon excitation laser scanning microscopy (TPELSM). Results: We demonstrated the feasibility of multilabeling intimal structures by exciting multicolored Qdots with only one laser wavelength (750nm). Detailed cell structures, such as the granular appearance of VWF and the subcellular distribution of eNOS, were visualized using green dots (525 nm), even when the emission maximum of these Qdots overlapped that of tissue autofluorescence (510-520 nm). In addition, sensitive fluorescence quantification of VCAM-1 expression at areas of varying hemodynamics (intercostal branches vs. nonbranching areas) was performed in normal C57Bl/6 mice. Finally, we took advantage of the photostability of Qdots and the inherent 3D resolution of TPELSM to obtain large z-stack series without photobleaching. Conclusion: This innovative en face method allowed simple multicolor profiling, highly-sensitive fluorescence quantitation and 3D visualization of the vascular endothelium with excellent spatial resolution. This is a promising technique to define the spatial and temporal interactions of endothelial inflammatory markers and quantify the effects of different interventions on the endothelium.
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 J. Suo, D. E. Ferrara, D. Sorescu, R. E. Guldberg, W. R. Taylor, and D. P. Giddens Hemodynamic Shear Stresses in Mouse Aortas: Implications for Atherogenesis Arterioscler Thromb Vasc Biol, February 1, 2007; 27(2): 346 - 351. [Abstract] [Full Text] [PDF]
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