title: Two-photon immunofluorescence characterization of the trabecular meshwork in situ.
abstract: OBJECTIVE
  To develop an in situ model to study biological responses and glaucoma pathology in the human trabecular meshwork (TM). Characteristic TM cell- and glaucoma-associated markers were localized in situ in relation to the tissue's autofluorescent structural extracellular matrix (ECM) by two-photon excitation fluorescence optical sectioning (TPEF).
  
  
  METHODS
  Human donor corneoscleral (CS) tissue containing the intact aqueous drainage tract was incubated with dexamethasone (Dex) or TGF-β1, and immunostained for epifluorescence (EF) microscopy with antibodies to myocilin and alpha smooth muscle (α-SMA). Separate specimens were labeled for Type-IV collagen and fibronectin. Nuclei were stained with Hoechst 33342. Multimodal TPEF was used to visualize EF, intravital dyes, and autofluorescence (AF) in situ. Three-dimensional (3D) localization of fluorescence within the TM was analyzed using reconstruction software.
  
  
  RESULTS
  Autofluorescent beams, perforated sheets, and fibers, consistent with the uveal (UV), CS, and juxtacanalicular (JCT) meshwork, respectively, were captured at different depths of the TM. Type-IV collagen EF distinctly outlined the AF beams in a location consistent with basement membrane. Fibronectin EF showed a diffuse reticular pattern throughout the TM. TGF-β1-induced α-SMA expression, which was distributed perinuclearly in cells among autofluorescent structures. Dex-induced myocilin expression had both cytosolic and extracellular distributions.
  
  
  CONCLUSIONS
  The authors have localized markers that are characteristic of TM cells and relevant to glaucoma pathogenesis in situ using multimodal TPEF without conventional histological embedding and sectioning. Protein expression was inducible in situ and could be analyzed with respect to cells and the ECM within the 3D environment of the human TM.
authors.affiliation: Doheny Eye Institute and Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA.
authors.forename: Jose M
authors.initials: JM
authors.lastname: Gonzalez
authors.forename: Martin
authors.initials: M
authors.lastname: Heur
authors.forename: James C H
authors.initials: JC
authors.lastname: Tan
mesh_terms: D000199:Actins / Q000378:metabolism*; D002452:Cell Count; D024141:Collagen Type IV / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism*; D005136:Eye Proteins / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D021621:Imaging, Three-Dimensional; D017785:Photons; D014129:Trabecular Meshwork / Q000737:chemistry* / Q000166:cytology; D000099179:Myocilin
chemical_list: D000199:Actins; D024141:Collagen Type IV; D003598:Cytoskeletal Proteins; D005136:Eye Proteins; D005353:Fibronectins; D006023:Glycoproteins; D000099179:Myocilin; C541116:ACTA2 protein, human