April 2010
Volume 51, Issue 13
ARVO Annual Meeting Abstract  |   April 2010
Multimodal Imaging of Corneal Cross-Linking Effects
Author Affiliations & Notes
  • O. Stachs
    Department of Ophthalmology, University of Rostock, Rostock, Germany
  • M. Hovakimyan
    Department of Ophthalmology, University of Rostock, Rostock, Germany
  • R. F. Guthoff
    Department of Ophthalmology, University of Rostock, Rostock, Germany
  • G. Hüttmann
    Institute of Biomedical Optics, University of Lübeck, Lübeck, Germany
  • A. Krüger
    Department Biophotonics/Lasermedicine, The Laser Zentrum Hannover, Hannover, Germany
  • P. Steven
    Ophthalmology, Univ Medical Center Schleswig-Holstein, Luebeck, Germany
  • Footnotes
    Commercial Relationships  O. Stachs, None; M. Hovakimyan, None; R.F. Guthoff, None; G. Hüttmann, None; A. Krüger, None; P. Steven, None.
  • Footnotes
    Support  DFG Gant TR37
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 4995. doi:
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    • Get Citation

      O. Stachs, M. Hovakimyan, R. F. Guthoff, G. Hüttmann, A. Krüger, P. Steven; Multimodal Imaging of Corneal Cross-Linking Effects. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4995.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Cross-links between collagen fibrils following riboflavin and UVA application cannot be depicted by confocal in vivo microscopy. Therefore we combined reflective in vivo confocal laser-scanning microscopy (CLSM) with two photon microscopy (TPM) which allows detection of complementary information at the cellular level. Moreover, the TPM provides information about molecular collagen changes.

Methods: : New Zealand white rabbits were subdivided in groups according to different treatment protocol: (I) corneal cross-linking with UVA light (370 nm) and riboflavin after abrasion (standard protocol), (II) same protocol without radiation of UVA, (III) same protocol without removal of epithelium. One week after treatment in vivo CLSM was performed on anesthetized animals. TPM examination including autofluorescence, second sarmonic generation (SHG) and fluoresence lifetime imaging microscopy (FLIM), was conducted on enucleated eyes followed by histology.

Results: : One week after treatment the anterior stroma revealed a complete lack of cells in the group I, whereas other groups showed normal cell distribution (autofluorescence). Protocol (II) and (III) demonstrated a failure of the cross-linking approach as shown by SHG/autofluorescence pattern and fluorescence lifetime ratios. Also in vivo confocal microscopy and histological assessment showed normal corneal morphology and a cell distribution in group II and III comparable to that of the controls.

Conclusions: : Alterations of the collagen matrix were observed only after cross-linking with epithelial abrasion. The combination of CLSM and TPM with SHG and lifetime imaging enables not only a detection of cellular changes after corneal treatment, but allows can also visualise and quantify the alterations of collagen matrix, thus giving direct information about effectiveness of the treatment. The clinical use of autofluorescence/SHG two-photon microscopy would enable for the first time a detection of a therapeutic collagen cross-linking effect immediately after the procedure.

Keywords: cornea: basic science • microscopy: confocal/tunneling • imaging/image analysis: non-clinical 

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