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James C. Tan, Jose M. Gonzalez, Jr., Sarah F. Hamm-Alvarez, Martin Heur, Jonathan Song; Visualization of Trabecular Meshwork Ultrastructure by In Situ Optical Sectioning. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4626.
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To image and describe the 3-dimensional (3D) ultrastructure and cells of the trabecular meshwork (TM) by in situ confocal optical sectioning without conventional histologic embedding and sectioning.
Viable ex vivo explants of the anterior chamber angle containing the aqueous drainage tissue in situ were maintained in transport medium and imaged by 1- and 2-photon laser confocal microscopy. Multimodal imaging of intravital dye in cells, autofluorescence (AF) and epifluorescence within the intact drainage tract was performed. Celltracker Red and Hoechst, intravital fluorescent dyes, aided visualization of cells. Some tissue was permeablized, fixed and incubated with phalloidin-rhodamine allowing filamentous actin to be seen. The TM was viewed by tangential, orthogonal and 3D views.
In the innermost uveal TM, a 3D network of extracellular matrix (ECM) branching fiber-like structures was seen by AF. In the adjacent region of the corneoscleral TM, fibers took on a denser organization and featured intermittent pore-like openings. Deeper, this organization gave way to arrays of fine fibers orientated in the tissue’s longitudinal axis reminiscent of the cribriform plexus of the juxtacanalicular TM (JCT). In the outermost portion of the TM, consistent with subendothelial JCT, AF of fine fibers was sparse, then undetectable as optical sections approached the presumed inner wall of Schlemm’s canal. Cells were seen in all depths of the tissue and closely associated with the fiber-like ECM. No nuclear fragmentation was observed on Hoechst staining. F-actin was present in all depths of the TM and its distribution in cells was primarily cortical.
We have applied multimodal fluorescence microscopy to analyze the aqueous drainage pathway in situ. The structural ECM, cells and subcellular elements of the TM, and their relationships can be localized in the tissue. Intravital dye, AF and epifluorescence features combined with optical sectioning reveal a unique 3D perspective of TM ultrastructure and cells. Much of this analysis can be performed in viable tissue and could lead to interesting research possibilities.
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