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James C. H. Tan, Jose M. Gonzalez, Sarah Hamm-Alvarez, Jonathan Song; In Situ Autofluorescence Visualization of Human Trabecular Meshwork Structure. Invest. Ophthalmol. Vis. Sci. 2012;53(4):2080-2088. doi: 10.1167/iovs.11-8141.
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To characterize the three-dimensional (3-D) structure of the human trabecular meshwork (TM) by two-photon excited (TPEF) autofluorescence (AF) and optical sectioning without conventional histologic embedding and sectioning.
Viable human ex vivo explants of the anterior chamber angle containing the aqueous humor drainage tissue in situ were imaged by TPEF to localize AF and Hoechst 33342 nuclear fluorescence. An autofluorescent marker in Schlemm's Canal (SC) aided SC situ visualization. En face and orthogonal views of the TM were generated.
In the innermost uveal TM, AF signals outlined an intricate 3-D network of fine branching beams with large openings between the beams. In the adjacent corneoscleral TM, beams were thicker and coalesced as plate-like structures with pore-like openings. Linear and coiled AF fibers were visible on the background AF of beams. Deeper, in the external TM, this organization changed to fine fiber arrays orientated in the tissue's longitudinal axis, reminiscent of the cribriform plexus of the juxtacanalicular TM (JCT). In the outermost JCT, AF of fine fibers was sparse, then undetectable as optical sections approached the inner wall of SC. Cell nuclei were closely associated with the TM structural extracellular matrix.
We have used TPEF and optical sectioning to exploit AF as a useful method to visualize the structure of the human conventional aqueous drainage pathway in situ. Ancillary nuclear staining allowed cell association with the AF structures to be seen. This approach revealed a unique 3-D perspective of the TM that is consistent with known TM structural characteristics.
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