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JiaJun Li, Brian Vohnsen; Exploring Directionality In Second-harmonic Cornea Microscopy. Invest. Ophthalmol. Vis. Sci. 2012;53(14):119.
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Two-photon microscopy has become widespread for imaging of biological samples benefitting from increased penetration depth and less photo-bleaching. The related, second-harmonic (SH) optical microscopy is sensitive to the material structure making it ideal to image collagen fibrils without staining due to the nonlinear contrast mechanisms offered. Interpretation of images (forward and backward generated SH) is complicated by the fact that it is a coherent scattering process. The aim of this study is to examine in further detail the directionality of SH radiation produced by collagen in the cornea and examining its potential utility for improved imaging.
A non-commercial SH microscope consisting of an inverted microscope equipped with an ultrafast fs laser (800 nm wavelength) as source has been used to image SH radiation (forward and backward) in post-mortem porcine corneas. The directionality of the SH signal is imaged with a CMOS camera and the corneas are posteriori imaged with lock-in detection using a pair of galvanometric scanners.
The collected SH radiation consists of a dominant axial component supplemented by a cone of radiation as expected from phase-matching conditions. The two radiation patterns have been used in separate for the cornea collagen imaging and for a range of different numerical aperture objectives, and we find that the visibility of the fibrils can be increased when restricting the collection aperture to the on-axis SH radiation.
We have found a strong directionality in SH radiation produced by porcine corneas. Restricting the signal collection optics to the dominant axial component has been found to increase the contrast of the imaged collagen fibrils and this can be further explored towards in-vivo applications.
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