Detection of subepithelial fibrosis associated with corneal stromal edema by second harmonic generation imaging microscopy

Naoyuki Morishige, Naoyuki Yamada, Shinichiro Teranishi, Tai ichiro Chikama, Teruo Nishida, Atsushi Takahara

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

PURPOSE. Human corneas with or without stromal edema were examined by second harmonic generation (SHG) imaging microscopy to characterize stromal collagen organization. METHODS. Tissue buttons from 31 corneas with stromal edema and 8 normal corneas were fixed, and 3-mm2 blocks were cut and stained with phalloidin, to visualize the cytoskeleton. The blocks were examined by SHG imaging with a laser confocal microscope and a mode-locked titanium:sapphire femtosecond laser. Samples were scanned to a depth of 150 μm from the surface of Bowman's layer, and SHG forward-and backscatter signals were collected. Phalloidin staining was detected by conventional laser confocal microscopy. The three-dimensional structure of the anterior segment of the cornea was reconstructed from stacked SHG images. RESULTS. Three-dimensional reconstruction of SHG signals showed adherence of interwoven collagen lamellae in the anterior stroma to Bowman's layer in both normal and edematous corneas. Abnormal SHG signals at the level of Bowman's layer were observed in edematous corneas; three-dimensional images revealed that these signals were actually localized above Bowman's layer and were indicative of subepithelial fibrosis. Phalloidin staining showed transdifferentiation of stromal cells into fibroblastic cells in edematous corneas. The incidence of subepithelial fibrosis or of fibroblastic cells increased beginning 12 months after the onset of clinical stromal edema. CONCLUSIONS. SHG imaging of the anterior segment of edematous corneas revealed a normal appearance of interwoven collagen lamellae in the anterior stroma. The development of subepithelial fibrosis beginning 12 months after the onset of edema suggests that stromal edema may be a progressive disease.

Original languageEnglish
Pages (from-to)3145-3150
Number of pages6
JournalInvestigative Ophthalmology and Visual Science
Volume50
Issue number7
DOIs
Publication statusPublished - Dec 1 2009

Fingerprint

Corneal Edema
Cornea
Microscopy
Fibrosis
Edema
Phalloidine
Collagen
Confocal Microscopy
Lasers
Staining and Labeling
Three-Dimensional Imaging
Aluminum Oxide
Stromal Cells
Titanium
Cytoskeleton
Incidence

All Science Journal Classification (ASJC) codes

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Detection of subepithelial fibrosis associated with corneal stromal edema by second harmonic generation imaging microscopy. / Morishige, Naoyuki; Yamada, Naoyuki; Teranishi, Shinichiro; Chikama, Tai ichiro; Nishida, Teruo; Takahara, Atsushi.

In: Investigative Ophthalmology and Visual Science, Vol. 50, No. 7, 01.12.2009, p. 3145-3150.

Research output: Contribution to journalArticle

Morishige, Naoyuki ; Yamada, Naoyuki ; Teranishi, Shinichiro ; Chikama, Tai ichiro ; Nishida, Teruo ; Takahara, Atsushi. / Detection of subepithelial fibrosis associated with corneal stromal edema by second harmonic generation imaging microscopy. In: Investigative Ophthalmology and Visual Science. 2009 ; Vol. 50, No. 7. pp. 3145-3150.
@article{54592352123b4dad92cb228bb9d462d6,
title = "Detection of subepithelial fibrosis associated with corneal stromal edema by second harmonic generation imaging microscopy",
abstract = "PURPOSE. Human corneas with or without stromal edema were examined by second harmonic generation (SHG) imaging microscopy to characterize stromal collagen organization. METHODS. Tissue buttons from 31 corneas with stromal edema and 8 normal corneas were fixed, and 3-mm2 blocks were cut and stained with phalloidin, to visualize the cytoskeleton. The blocks were examined by SHG imaging with a laser confocal microscope and a mode-locked titanium:sapphire femtosecond laser. Samples were scanned to a depth of 150 μm from the surface of Bowman's layer, and SHG forward-and backscatter signals were collected. Phalloidin staining was detected by conventional laser confocal microscopy. The three-dimensional structure of the anterior segment of the cornea was reconstructed from stacked SHG images. RESULTS. Three-dimensional reconstruction of SHG signals showed adherence of interwoven collagen lamellae in the anterior stroma to Bowman's layer in both normal and edematous corneas. Abnormal SHG signals at the level of Bowman's layer were observed in edematous corneas; three-dimensional images revealed that these signals were actually localized above Bowman's layer and were indicative of subepithelial fibrosis. Phalloidin staining showed transdifferentiation of stromal cells into fibroblastic cells in edematous corneas. The incidence of subepithelial fibrosis or of fibroblastic cells increased beginning 12 months after the onset of clinical stromal edema. CONCLUSIONS. SHG imaging of the anterior segment of edematous corneas revealed a normal appearance of interwoven collagen lamellae in the anterior stroma. The development of subepithelial fibrosis beginning 12 months after the onset of edema suggests that stromal edema may be a progressive disease.",
author = "Naoyuki Morishige and Naoyuki Yamada and Shinichiro Teranishi and Chikama, {Tai ichiro} and Teruo Nishida and Atsushi Takahara",
year = "2009",
month = "12",
day = "1",
doi = "10.1167/iovs.08-3309",
language = "English",
volume = "50",
pages = "3145--3150",
journal = "Investigative Ophthalmology and Visual Science",
issn = "0146-0404",
publisher = "Association for Research in Vision and Ophthalmology Inc.",
number = "7",

}

TY - JOUR

T1 - Detection of subepithelial fibrosis associated with corneal stromal edema by second harmonic generation imaging microscopy

AU - Morishige, Naoyuki

AU - Yamada, Naoyuki

AU - Teranishi, Shinichiro

AU - Chikama, Tai ichiro

AU - Nishida, Teruo

AU - Takahara, Atsushi

PY - 2009/12/1

Y1 - 2009/12/1

N2 - PURPOSE. Human corneas with or without stromal edema were examined by second harmonic generation (SHG) imaging microscopy to characterize stromal collagen organization. METHODS. Tissue buttons from 31 corneas with stromal edema and 8 normal corneas were fixed, and 3-mm2 blocks were cut and stained with phalloidin, to visualize the cytoskeleton. The blocks were examined by SHG imaging with a laser confocal microscope and a mode-locked titanium:sapphire femtosecond laser. Samples were scanned to a depth of 150 μm from the surface of Bowman's layer, and SHG forward-and backscatter signals were collected. Phalloidin staining was detected by conventional laser confocal microscopy. The three-dimensional structure of the anterior segment of the cornea was reconstructed from stacked SHG images. RESULTS. Three-dimensional reconstruction of SHG signals showed adherence of interwoven collagen lamellae in the anterior stroma to Bowman's layer in both normal and edematous corneas. Abnormal SHG signals at the level of Bowman's layer were observed in edematous corneas; three-dimensional images revealed that these signals were actually localized above Bowman's layer and were indicative of subepithelial fibrosis. Phalloidin staining showed transdifferentiation of stromal cells into fibroblastic cells in edematous corneas. The incidence of subepithelial fibrosis or of fibroblastic cells increased beginning 12 months after the onset of clinical stromal edema. CONCLUSIONS. SHG imaging of the anterior segment of edematous corneas revealed a normal appearance of interwoven collagen lamellae in the anterior stroma. The development of subepithelial fibrosis beginning 12 months after the onset of edema suggests that stromal edema may be a progressive disease.

AB - PURPOSE. Human corneas with or without stromal edema were examined by second harmonic generation (SHG) imaging microscopy to characterize stromal collagen organization. METHODS. Tissue buttons from 31 corneas with stromal edema and 8 normal corneas were fixed, and 3-mm2 blocks were cut and stained with phalloidin, to visualize the cytoskeleton. The blocks were examined by SHG imaging with a laser confocal microscope and a mode-locked titanium:sapphire femtosecond laser. Samples were scanned to a depth of 150 μm from the surface of Bowman's layer, and SHG forward-and backscatter signals were collected. Phalloidin staining was detected by conventional laser confocal microscopy. The three-dimensional structure of the anterior segment of the cornea was reconstructed from stacked SHG images. RESULTS. Three-dimensional reconstruction of SHG signals showed adherence of interwoven collagen lamellae in the anterior stroma to Bowman's layer in both normal and edematous corneas. Abnormal SHG signals at the level of Bowman's layer were observed in edematous corneas; three-dimensional images revealed that these signals were actually localized above Bowman's layer and were indicative of subepithelial fibrosis. Phalloidin staining showed transdifferentiation of stromal cells into fibroblastic cells in edematous corneas. The incidence of subepithelial fibrosis or of fibroblastic cells increased beginning 12 months after the onset of clinical stromal edema. CONCLUSIONS. SHG imaging of the anterior segment of edematous corneas revealed a normal appearance of interwoven collagen lamellae in the anterior stroma. The development of subepithelial fibrosis beginning 12 months after the onset of edema suggests that stromal edema may be a progressive disease.

UR - http://www.scopus.com/inward/record.url?scp=67649977002&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=67649977002&partnerID=8YFLogxK

U2 - 10.1167/iovs.08-3309

DO - 10.1167/iovs.08-3309

M3 - Article

C2 - 19234355

AN - SCOPUS:67649977002

VL - 50

SP - 3145

EP - 3150

JO - Investigative Ophthalmology and Visual Science

JF - Investigative Ophthalmology and Visual Science

SN - 0146-0404

IS - 7

ER -