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M. Han, G. Giese, J. Bille; Non–Invasive Corneal Imaging by Second Harmonic Microscopy . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1080.
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Second harmonic microscopy is a novel method to non–invasively image the intrastromal structure of corneal tissue. Since fixation, slicing and labelling are not required, the corneal structure can be probed under the conditions closest to its physiological states. We are interested to investigate the micro– and macroscopic structure of collagen fibre in cornea and the influences of temperature, fixation, dehydration rate and ultrafast laser surgery to the corneal intrastromal structures.
Excised porcine corneas were probed by a Zeiss multiphoton laser scanning microscope (LSM 510 NLO). Excited by a commercial mode–locked femtosecond Ti:sapphire laser (Coherent Chameleon), second harmonic signals generated by collagen fibres were collected in the transmission direction. Temperature and hydrated rate of corneal tissue were controlled via a custom tissue chamber allowing real time second harmonic imaging. The laser ablation experiments was conducted by a home–made all–solid–state Nd:glass femtosecond laser designed for next generation mini–invasive intrastromal corneal surgery.
Second harmonic microscopy enables high resolution, strong contrast and large sensing depth corneal imaging. Inside the full thickness of the cornea, the orientations and distribution of collagen fibres are nicely visualized. The shrinking and swelling of cornea in responding to temperature, dehydration rate and ultrafast laser ablations can be quantitatively characterized.
Based on the intrinsic properties of collagen, second harmonic corneal imaging is well suited to non–invasively investigate the intrastromal structures of corneal tissue. As a complementary imaging modality for laser scanning confocal and multiphoton fluorescence microscopy, second harmonic corneal imaging is valuable for physiological and pathological corneal studies.
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