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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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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.
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.
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.
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.
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