Quantitative 3D fluorescence technique for the analysis of en face preparations of arterial walls using quantum dot nanocrystals and two-photon excitation laser scanning microscopy.
Traditional imaging with one-photon confocal microscopy and organic fluorophores poses several challenges for the visualization of vascular tissue, including autofluorescence, fluorophore crosstalk, and photobleaching. 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). We demonstrated the feasibility of multilabeling intimal structures by exciting multicolored Qdots with only one laser wavelength (750 nm). Detailed cell structures, such as the granular appearance of von Willebrand factor (VWF) and the subcellular distribution of endothelial nitric oxide synthase, 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 vascular cell adhesion molecule 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 three-dimensional (3D) resolution of TPELSM to obtain large z-stack series without photobleaching. 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.
Ferrara
Dardo E
DE
Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.
Weiss
Daiana
D
Carnell
Peter H
PH
Vito
Ray P
RP
Vega
David
D
Gao
Xiaohu
X
Nie
Shuming
S
Taylor
W Robert
WR
D000818:Animals; D001011:Aorta / Q000166:cytology*; D001057:Apolipoproteins E / Q000235:genetics; D003324:Coronary Artery Disease; D003331:Coronary Vessels / Q000166:cytology*; D042783:Endothelial Cells / Q000166:cytology*; D005453:Fluorescence; D005786:Gene Expression Regulation; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018613:Microscopy, Confocal / Q000379:methods*; D045663:Quantum Dots; D019010:Vascular Cell Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism
D001057:Apolipoproteins E; D019010:Vascular Cell Adhesion Molecule-1