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M. Mueller, M. Han, G. Giese, J. Bille, M. Niemz; Revealing The Structural Disorders Of The Keratoconic Cornea With Second Harmonic Generation Microscopy . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2994.
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© ARVO (1962-2015); The Authors (2016-present)
Second harmonic generation (SHG) imaging is a novel method to non–invasively probe the intrastromal structure of corneal tissue. Since fixation, slicing and labelling are not required, the corneal structure can be studied under the conditions closest to its physiological states. We are interested to reveal the structural disorders of the collagen fibres in keratoconic cornea with high resolution SHG imaging.
The keratoconic cornea was obtained from the university eye hospital, University of Frankfurt, Germany. SHG imaging of the collagen fiber network in the keratoconic cornea was performed with an upright Zeiss multiphoton laser scanning microscope (LSM 510 NLO) equipped with a mode–locked femtosecond Ti:Sapphire laser (Coherent Chameleon). The second harmonic signals were collected in the transmission direction.
Second harmonic generation microscopy enables high resolution, strong contrast and large sensing depth corneal imaging. The unique organizations of the collagen fibres in Bowmans membrane, corneal stroma and Descemets membrane were resolved. The lamellar arrangement of collagen fibre is more prominent in the posterior cornea than in the anterior cornea. In the keratoconic cornea, strongly distorted collagen fibres were observed and they were predominantly distributed in the anterior stroma (depth less than 150 micron).
Based on the intrinsic properties of collagen, second harmonic corneal imaging is well suited to non–invasively investigate the structural disorder of the collagen fibre in keratoconic cornea. As a complementary imaging modality for laser scanning confocal and multiphoton fluorescence microscopy, second harmonic generation microscopy is particularly valuable for mini–invasive pathophysiological studies of keratoconus cornea.
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