Details of the collagen and elastin architecture in the human limbal conjunctiva, tenon’s capsule and sclera revealed by two-photon excited fluorescence microscopy

Choul Yong Park, Catherine M. Marando, Jason A. Liao, Jimmy K. Lee, Jiwon Kwon, Roy S. Chuck

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

PURPOSE. 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.

Original languageEnglish (US)
Pages (from-to)5602-5610
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume57
Issue number13
DOIs
StatePublished - Oct 1 2016

Fingerprint

Tenon Capsule
Sclera
Elastin
Conjunctiva
Photons
Fluorescence Microscopy
Collagen
Aluminum Oxide
Lasers
Fluorescence

Keywords

  • Collagen
  • Conjunctiva
  • Elastin
  • Sclera
  • Tenon

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Details of the collagen and elastin architecture in the human limbal conjunctiva, tenon’s capsule and sclera revealed by two-photon excited fluorescence microscopy. / Park, Choul Yong; Marando, Catherine M.; Liao, Jason A.; Lee, Jimmy K.; Kwon, Jiwon; Chuck, Roy S.

In: Investigative Ophthalmology and Visual Science, Vol. 57, No. 13, 01.10.2016, p. 5602-5610.

Research output: Contribution to journalArticle

@article{c242db3487a04f7aaef04828f506e395,
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 = "PURPOSE. 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.",
keywords = "Collagen, Conjunctiva, Elastin, Sclera, Tenon",
author = "Park, {Choul Yong} and Marando, {Catherine M.} and Liao, {Jason A.} and Lee, {Jimmy K.} and Jiwon Kwon and Chuck, {Roy S.}",
year = "2016",
month = "10",
day = "1",
doi = "10.1167/iovs.16-19706",
language = "English (US)",
volume = "57",
pages = "5602--5610",
journal = "Investigative Ophthalmology and Visual Science",
issn = "0146-0404",
publisher = "Association for Research in Vision and Ophthalmology Inc.",
number = "13",

}

TY - JOUR

T1 - Details of the collagen and elastin architecture in the human limbal conjunctiva, tenon’s capsule and sclera revealed by two-photon excited fluorescence microscopy

AU - Park, Choul Yong

AU - Marando, Catherine M.

AU - Liao, Jason A.

AU - Lee, Jimmy K.

AU - Kwon, Jiwon

AU - Chuck, Roy S.

PY - 2016/10/1

Y1 - 2016/10/1

N2 - PURPOSE. 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.

AB - PURPOSE. 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.

KW - Collagen

KW - Conjunctiva

KW - Elastin

KW - Sclera

KW - Tenon

UR - http://www.scopus.com/inward/record.url?scp=84994026401&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84994026401&partnerID=8YFLogxK

U2 - 10.1167/iovs.16-19706

DO - 10.1167/iovs.16-19706

M3 - Article

VL - 57

SP - 5602

EP - 5610

JO - Investigative Ophthalmology and Visual Science

JF - Investigative Ophthalmology and Visual Science

SN - 0146-0404

IS - 13

ER -