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Jose Gonzalez, James Tan; Multimodal Microscopy of Aqueous Drainage Channels in Live Mice. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3550.
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To non-invasively analyze the aqueous humor drainage channels of live mice by multimodal 2-photon excitation fluorescence (TPEF) imaging.
Anesthetized mice were carefully positioned so that the eye and corneoscleral limbus were accessible to a microscope objective (63X) on an objective inverter for upright TPEF microscopy. TPEF (850nm) was used to generate collagen-associated second harmonic generation (SHG) that was captured through a narrowband filter (425nm (+/- 10nm)). Wild-type C57BL/6 and BALB/c, and transgenic TIE-2-GFP (Tunica interna endothelial cell kinase, or Angiopoietin-1 receptor; BALB/c) and Prox1-GFP (lymphatic endothelial nuclear transcription factor; C57BL/6) mice were studied. RFP-conjugated dextran (70kDa) was injected intravenously prior to enucleation. For F-actin labeling, eyes were enucleated, fixed, permeabilized and incubated with Alexa-568-conjugated phalloidin overnight. Some eyes were frozen for histology.
SHG revealed loosely arranged fine collagen fibrils in the conjunctival stroma. Just deep to the conjunctiva, sclera was identifiable by coarse collagen bands. In the intervening space were episcleral vessels (diameter<5um) with phalloidin-labeled walls. Deep (5-10um) to this, lay a network of parallel interconnecting channels resembling an intrascleral plexus. The plexus was cell-lined, labeled with phalloidin and created scleral SHG signal voids. Yet deeper to the external surface (extending 15-40um), collector channels that were perpendicular and connected to the intrascleral plexuses opened via discrete ostia into a large channel (>100um in diameter) of Schlemm’s Canal. Walls of collector channels and ostia labeled with phalloidin. These findings were confirmed by 3D reconstructions and histology. Dextran-RFP and Prox1-GFP were associated with intrascleral aqueous veins and Tie-2-GFP was associated with episcleral vessels.
Multimodal TPEF that exploited transgenic fluorescence was used to analyze the distal aqueous drainage channels with reference to collagen SHG in mice. 3D reconstruction provided stunning views of the in situ drainage tissue. Combined, these approaches could yield unique insights into mouse aqueous humor outflow biology.
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