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.