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. 2017 Dec 6;7:17071. doi: 10.1038/s41598-017-16897-y

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Filamentous actin in distal aqueous drainage tract walls. Orthogonal reconstruction (a) of serial optical sections (b-i) captured during Balb/c 2-photon trans-scleral imaging ex vivo. Eyes were labeled with Alexa-568-conjugated phalloidin for filamentous actin (F-actin; red). (a) Orthogonal image showing structures of ciliary muscle (CM; ~125 μm deep) and trabecular meshwork (TM) adjacent to Schlemm’s canal (SC). SC was continuous with a collector channel (CC) within scleral collagen (second harmonic generation (SHG); cyan). The CC connected with an intrascleral plexus (ISP). Optical sections captured progressively deeper in the sclera (b-i) showed the following: (b) prominent F-actin-positive labeling bordered the ISP lumen (SHG signal void); White crosshairs: location of CC ostium 70–80 μm deeper where ISP eventually connected to SC; (c) ISPs interconnect; Yellow crosshairs: bridging vessel connects with collector channel; (d) Relationship between CC and ISP at a deeper location; (e) Evidence of this relationship persisted; (f-g) CC course deeper into sclera (yellow crosshairs; relative depth 40–60 μm); (h): CC opening (CCO) into SC (relative depth 70 μm); (i) Deeper, SC was a fuller void in the collagen SHG signal, seen with reference to the CCO. The SHG signal (cyan) reveals collagenous bundles in sclera; (j-k): High magnification images of CC (j) and ISP (k) showing cytosolic F-actin outlining nuclei (dark ovals) and revealing a multilayered cellular wall organization surrounding lumen (dark central region) within SHG signal voids. The lumen comprise but a portion of the cross-sectional area of each intrascleral channel, with cells filling the rest of the area as part of the channel wall.