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L-A Zhao, Y Song, AM Laties, R Wen; Vessel Painting, a Novel Technique to Stain and Visualize Blood Vessels in 3D . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3699.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Although there has been rapid progress in angiogenesis research in recent years, it is still difficult with available techniques either to fully appreciate the 3D nature of the blood vessel network or to perform quantitative analysis. We have developed a novel technique, dubbed Vessel Painting, to directly stain blood vessels. This technique, combined with confocal microscopy, makes it possible to obtain digitized images from whole mount retinas or thick tissue sections. The digitized images can be used for 3D reconstruction of blood vessel network or quantitative analysis. Methods: Vessel Painting is achieved by intravascular perfusion with Vessel Paint, a solution contains DiI (0.1-0.2 mg/ml). Adult rats were sacrificed by CO2 inhalation and perfused with 10 ml of PBS, followed by 10 ml of Vessel Paint and 20 ml of 4% paraformaldehyde solution. For retinal whole mount preparation, retinas were dissected and mounted with 50% glycerol on a glass slide, ganglion cell side up. For tissue sections, the anterior portion of the eye, including the cornea and the lens, was removed and the eyecup was embedded in 5% agarose. Thick sections (100-150 µm) of an eye were cut on a vibratome and mounted on glass slides. Results: The retinal blood vessel network was clearly visible by regular fluorescence microscopy using a 10X objective. High quality digital images of retinal whole mount or thick sections were obtained with a confocal microscope (Bio-Rad MRC 1024) using a 20X dry objective lens at the smallest pinhole size. Stacks of images were taken at 1-µm steps along the z-axis. No significant photobleaching was observed. Three-dimensional images were reconstructed from a stack of images using Auto Visualize-3D (version 5.5, AutoQuant Imaging, Inc). Total volume of blood vessels in a given volume of tissue can be calculated by counting the voxels above a given threshold. Conclusion: Vessel Painting enhances visualization of blood vessels in tissues. Once digitized, data can be processed and analyzed with a variety of software, including 3D reconstruction and quantitative analysis. This technique is likely to have application in ocular neovascularization research.
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