title: Details of the Collagen and Elastin Architecture in the Human Limbal Conjunctiva, Tenon's Capsule and Sclera Revealed by Two-Photon Excited Fluorescence Microscopy.
abstract: OBJECTIVE
  To investigate the architecture and distribution of collagen and elastin in human limbal conjunctiva, Tenon's capsule, and sclera.
  
  
  METHODS
  The limbal conjunctiva, Tenon's capsule, and sclera of human donor corneal buttons were imaged with an inverted two-photon excited fluorescence microscope. No fixation process was necessary. The laser (Ti:sapphire) was tuned at 850 nm for two-photon excitation. Backscatter signals of second harmonic generation (SHG) and autofluorescence (AF) were collected through a 425/30-nm and a 525/45-nm emission filter, respectively. Multiple, consecutive, and overlapping (z-stack) images were acquired. Collagen signals were collected with SHG, whereas elastin signals were collected with AF.
  
  
  RESULTS
  The size and density of collagen bundles varied widely depending on depth: increasing from conjunctiva to sclera. In superficial image planes, collagen bundles were <10 μm in width, in a loose, disorganized arrangement. In deeper image planes (episclera and superficial sclera), collagen bundles were thicker (near 100 μm in width) and densely packed. Comparatively, elastin fibers were thinner and sparse. The orientation of elastin fibers was independent of collagen fibers in superficial layers; but in deep sclera, elastin fibers wove through collagen interbundle gaps. At the limbus, both collagen and elastin fibers were relatively compact and were distributed perpendicular to the limbal annulus.
  
  
  CONCLUSIONS
  Two-photon excited fluorescence microscopy has enabled us to understand in greater detail the collagen and elastin architecture of the human limbal conjunctiva, Tenon's capsule, and sclera.
authors: Department of Ophthalmology, Dongguk University, Ilsan Hospital, Goyang, Gyunggido, South Korea.
authors: Choul Yong
authors: CY
authors: Park
authors: Department of Ophthalmology and Visual Sciences, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York, United States.
authors: Catherine M
authors: CM
authors: Marando
authors: Department of Ophthalmology and Visual Sciences, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York, United States.
authors: Jason A
authors: JA
authors: Liao
authors: Department of Ophthalmology and Visual Sciences, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York, United States.
authors: Jimmy K
authors: JK
authors: Lee
authors: Department of Ophthalmology, Seonam University, Myongji Hospital, Goyang, Gyunggido, South Korea.
authors: Jiwon
authors: J
authors: Kwon
authors: Department of Ophthalmology and Visual Sciences, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York, United States.
authors: Roy S
authors: RS
authors: Chuck
mesh_terms: D000328:Adult; D000368:Aged; D002102:Cadaver; D003094:Collagen / Q000378:metabolism*; D003228:Conjunctiva / Q000166:cytology* / Q000378:metabolism; D004549:Elastin / Q000378:metabolism*; D005260:Female; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008297:Male; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D008875:Middle Aged; D015203:Reproducibility of Results; D012590:Sclera / Q000166:cytology* / Q000378:metabolism; D058475:Tenon Capsule / Q000166:cytology* / Q000378:metabolism
chemical_list: D003094:Collagen; D004549:Elastin