Detection of melanin in the zebrafish retina using photothermal optical coherence tomography

Abstract

Purpose : Optical coherence tomography (OCT) provides depth-resolved images of the retina, but does not allow for quantitative imaging of melanin. We demonstrate a novel technology, photothermal OCT (PT-OCT), to detect melanin in the zebrafish retina.

Methods : Wild-type zebrafish embryos were injected with CRISPR/Cas9 reagents targeting the tyr locus to cause mosaic gene inactivation and disruption of melanin synthesis. Four adult tyrosinase-mosaic zebrafish were imaged with PT-OCT and OCT while anesthetized (n=5 eyes). PT-OCT signal from non-pigmented and pigmented regions of interest (ROIs, 10 A-scans averaged) was compared using the Mann-Whitney test. Optical absorbers such as melanin were detected with PT-OCT by combining an additional laser (λ=685 nm, power up to 4.72 mW at the sample, amplitude modulated at 500 Hz) to a commercial spectral domain OCT system (λ=860nm, 93 nm bandwidth, 0.470 mW at the sample) via a fiber coupler. The OCT phase signal was recorded over time (700 M-scans per A-scan) to compute the PT-OCT signal.

Results : An example en face OCT image of the retinal pigment epithelium (RPE) can be seen in Fig. 1a), where the dark, vertical strip in the bottom-half is an albino region of the RPE. The horizontal red line indicates the position of the B-scan in Fig.1b), where the OCT (gray) and PT-OCT (green) signals are overlaid. The box indicates the location of Fig 1c) and d) where the OCT and PT-OCT signal are presented side-by-side for a non-pigmented region and a pigmented region. The average OCT and PT-OCT signal over depth for both regions can be seen in Fig.1e). An increased PT-OCT signal at the RPE can be seen in the pigmented ROI as compared to the non-pigmented ROI. This increase is statistically significant when comparing all ROIs over all eyes (P<0.001 Mann-Whitney test).

Conclusions : PT-OCT is a novel tool that can localize melanin within the zebrafish retina.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

Fig 1: PT-OCT of melanin in tyrosinase-mosaic zebrafish. A) En face OCT image of the RPE. B) OCT (gray) and PT-OCT signal (green) showing pigmented and non-pigmented regions. C) Separate OCT and D) PT-OCT signal of the boxed area in B). E) OCT (black) and PT-OCT (green) signal averaged over 20 A-scans for (1) non-pigmented and (2) pigmented ROI. RNFL: Retinal nerve fiber layer. OPL: Outer plexiform layer. PR: Photoreceptors. RPE: Retinal pigment epithelium.