Imaging the Adult Zebrafish Cone Photoreceptor Mosaic Using Optical Coherence Tomography (OCT)

Abstract

Purpose : Zebrafish are excellent model organisms for ocular diseases as they provide many experimental opportunities and economic advantages. Optical coherence tomography has been used to image adult zebrafish anterior segment and retina. However, obtaining quantitative data regarding the photoreceptor mosaic has been challenging, due to uncertainty of the lateral scaling in these OCT images. Here we present a method for quantitative in vivo imaging of the adult zebrafish photoreceptor mosaic.

Methods : Seven transgenic fli1:eGFP zebrafish were anesthetized and their eyes imaged with Bioptigen OCT using a telecentric bore to determine axial length. Volumetric scans of the retina were performed using a mouse retinal bore. The eyes were enucleated, the anterior segment removed and fluorescence microscopy performed to visualize the blood vessels. In 9 eyes with strong correlation between OCT and microscopy, a minimum of 3 branch point measurements were made to determine absolute lateral scaling of the OCT image as a function of axial length. A custom program was used to produce en face images of the photoreceptor mosaic. On the five clearest images, cones were manually counted to determine density and Voronoi geometry.

Results : Lateral scaling varied linearly with axial length, ranging from 1.387 um/pix for a 1.618mm eye to 0.808 um/pix for a 2.358mm eye. Mean cone density was 48,607±24,954 cones/mm² and with 58.07±15.87% of cones displaying six sided Voronoi cells. Our data are consistent with previously published histology showing 37,418 cones/mm² and 48.02% six sided Voronoi cells. In some volumes, it was possible to resolve multiple photoreceptor layers reflecting the tiered organization of the zebrafish photoreceptor mosaic, though axial resolution was not sufficient to do this routinely.

Conclusions : OCT is an accurate method to analyze the photoreceptor mosaic in living zebrafish. Determining the absolute scaling of in vivo imagery is critical to being able to use this technology to perform quantitative studies across animals. Future optimization of the optical could provide improved image quality while automation of the en face image software is needed to make the generation of the photoreceptor images practical.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

 

En face OCT scan of adult zebrafish photoreceptor mosaic