June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Nonlinear Susceptibility of Collagen fibers in Ocular Tissue With a New IR Laser Source
Author Affiliations & Notes
  • James Andrew Germann
    Instituto de Optica, Consejo Superior de Investigaciones Cientificas, Madrid, Madrid, Spain
  • Gaia Sardiello
    Fyla, Valencia, Spain
  • Sara Otero Barros
    Fyla, Valencia, Spain
  • Susana Marcos
    Instituto de Optica, Consejo Superior de Investigaciones Cientificas, Madrid, Madrid, Spain
    Center for Visual Science, The Institute of Optics, Flaum Eye Institute, University of Rochester, Rochester, New York, United States
  • Footnotes
    Commercial Relationships   James Germann None; Gaia Sardiello Fyla, Code E (Employment); Sara Otero Barros Fyla, Code E (Employment); Susana Marcos None
  • Footnotes
    Support  Horizon 2020 European Project Imcustomeye (H2020-ICT-2017 Ref. 779960); European Research Council 2018-ADG-SILKEYE-833106; Spanish Government PID2020-115191RB-I00; NIH NIE P30EY 001319; Unrestricted Funds Research to Prevent Blindness, NY
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 2393 – A0196. doi:
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    • Get Citation

      James Andrew Germann, Gaia Sardiello, Sara Otero Barros, Susana Marcos; Nonlinear Susceptibility of Collagen fibers in Ocular Tissue With a New IR Laser Source. Invest. Ophthalmol. Vis. Sci. 2022;63(7):2393 – A0196.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Second Harmonic Generation (SHG) can be used to generate confocal images of the collagen architecture of ocular tissue through the depth of said tissue. Typically, SHG images of collagen are collected with a near infrared (IR) source, but the use of longer IR wavelengths is appealing due to deeper penetration and lower potential for photodamage. However, it is unknown if collagen can efficiently create SHG signal using deeper IR wavelengths. To compare how well collagen can convert deeper IR wavelengths, the magnitude of the nonlinear susceptibility (|X2|) of collagen was measured.

Methods : A sample of porcine cornea (n=1) and sclera (n=1, posterior temporal) was optically cleared with Murray’s Clear (2:1 Benzyl Benzoate, Benzyl Alcohol) and placed in a custom built SHG microscope. The laser source was a super-continuum SCHx (Fyla, 18 fs, 80 MHz, 925-1175 nm, 37 mW at sample). Images (160×160 μm, 400×400 pxls, 25 μs dwell time) of collagen fibers were taken at 3 μm steps through the sample. Photons were collected in the forward scattering direction with a 10 μm bandpass filter. Once measurement of the image stack completed, the 10 μm bandpass filter was replaced with another 10 μm bandpass filter and the scan repeated. A total of 12 image stacks of the same tissue volume were taken (emission range 465-585 nm). The total number of photons was counted for each image by summing up the value of the pixels. The number of SHG photons generated was compared to the number of excitation photons and |X2| was computed.

Results : The values of |X2| (units of (eV×cm5/photons×s)0.5) in the wavelength range of 930-1170 nm ranged from 4.87±0.37×10-16 to 7.90±3.27×10-18 in the cornea and from 3.01±0.23×10-15 to 7.30±0.43×10-17 in the sclera. In the cornea and the sclera, the largest |X2| was located in the 930-950 nm range and the smallest was located in the 1054-1074 nm range.

Conclusions : Collagen was able to generate SHG from 930-1170 nm light. The amount of SHG collected was higher in sclera than in cornea, which in turn indicated a higher |X2| value. Included in the calculation of |X2| was the concentration of collagen, which is higher in sclera than in cornea and may explain differences in calculated |X2|. Values of |X2| measured here are from healthy corneas, but irregularities of |X2| measurements could be used to determine irregularities in collagen formation and be a biomarker of ocular diseases.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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