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Second Harmonic Generation Microscopy Of The Human Cornea and Sclera In Vivo


Purpose : Second Harmonic Generation (SHG) microscopy provides high resolution structural images of both the cornea and the sclera without labelling procedures. This is a powerful technique that, as far as we know, has not been yet applied in living human eyes. Here, we present a novel compact SHG microscope developed to image collagen-based ocular structures in human healthy volunteers.

Methods : A research prototype of SHG multiphoton microscope has been designed and built. It is a compact device using a femtosecond Ti:Sapphire laser (800 nm, 76 MHz) as illumination source and an on-air long-working distance objective that allows non-contact imaging of the cornea and sclera. A piezo step-motor is used for optical sectioning and a scanning unit permits imaging of different areas of interest. Ocular movements during image acquisition were minimized by means of a bite-bar and a head fixation system. Images of 100×100 µmwith a resolution of 100×100 px were acquired at a frame rate of 3.3 Hz.

Results : Light safety limits, as well as functionality and sensitivity of the instrument, were carefully established and tested in a previous calibration procedure using ex-vivo fresh ocular tissues. The maximum average laser power used was 20 mW at the corneal plane, 10 times below the maximum permissible exposure (according to ANSI Z136.1-2000). The instrument was successfully applied to obtain images of non-stained collagen-structures in the cornea and sclera of 2 volunteers. Since the measurements were performed without contact, eye movements were the main limiting factor of the quality of the images. Different techniques to compensate motion artefacts were subsequently used to improve the images. A structural analysis of the collagen distribution was carried out for the recorded SHG images.

Conclusions : We presented the first SHG images of anterior ocular structures recorded non-invasively and without physical contact in human subjects. Future additional improvements of this technique could be of potential use for earlier diagnosis of corneal diseases.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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