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. 2011 Oct 20;6(10):e26253. doi: 10.1371/journal.pone.0026253

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Steven et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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A) Intravital image of a lymphatic vessel in an experimentally vascularized murine cornea. The fluorochrome labeled lymphatic vessel (open arrow) is shown in green and autofluorescence of epithelium, blood vessel (white arrow) and tissue cells is shown in red, surrounding collagen fibrils in blue. B) Intravital image of unstained vascularized cornea. Tissue autofluorescence reveals individual cells (red) and blood vessels (red, white arrows). Some cells feature autofluorescence in all detection channels (yellow-white, arrowheads). Lymphatic vessels cannot be visualized by autofluorescence excitation. C) Intravital image of isotype control injected cornea. Individual cells (red) and blood vessels (white arrows) are visible. Large cells feature green fluorescence deriving from intracellular Alexa488 fluorochrome. Lymphatic vessels are not labeled by isotype controls. D) Intravital autofluorescence image of normal cornea at the limbus. A limbal blood vessel is visible (white arrows) and few individual cells (red). E) Ex vivo confocal image of specimen B) following immunohistochemistry (IHC). CD31+ blood vessels (red; white arrows) and Lyve-1+/CD31− lymphatic vessels (green, open arrows) are visible. F) Ex vivo confocal image of specimen C) following IHC. CD31+ blood vessels (red; white arrows) and Lyve-1+/CD31− lymphatic vessels (green, open arrows) are now visible. G) 3D-reconstruction of a 2-photon image stack through the vascularized cornea. The signal of the fluorochrome labeled lymphatic vessels is virtually reconstructed and depicted by surface rendering (green). Surrounding collagen fibrils are displayed in blue, epithelial cells and the blood vessel (dashed line and arrow) in red. H) Higher magnification and different angle of G). Within the lymphatic vessel an Alexa488⁺ presumed macrophage (arrow) is depicted in yellow as an overlay of Alexa488 fluorophore (green) and autofluorescence signal (red, diameter: 16.7 µm). Images recorded with acquisition times of 13.4s/image.