Imaging the adult zebrafish cone mosaic using optical coherence tomography

Abstract

Zebrafish (Danio rerio) provide many advantages as a model organism for studying ocular disease and development, and there is great interest in the ability to non-invasively assess their photoreceptor mosaic. Despite recent applications of scanning light ophthalmoscopy, fundus photography, and gonioscopy to in vivo imaging of the adult zebrafish eye, current techniques either lack accurate scaling information (limiting quantitative analyses) or require euthanizing the fish (precluding longitudinal analyses). Here we describe improved methods for imaging the adult zebrafish retina using spectral domain optical coherence tomography (OCT). Transgenic fli1:eGFP zebrafish were imaged using the Bioptigen Envisu R2200 broadband source OCT with a 12-mm telecentric probe to measure axial length and a mouse retina probe to acquire retinal volume scans subtending 1.2 × 1.2 mm nominally. En face summed volume projections were generated from the volume scans using custom software that allows the user to create contours tailored to specific retinal layer(s) of interest. Following imaging, the eyes were dissected for ex vivo fluorescence microscopy, and measurements of blood vessel branch points were compared to those made from the en face OCT images to determine the OCT lateral scale as a function of axial length. Using this scaling model, we imaged the photoreceptor layer of five wild-type zebrafish and quantified the density and packing geometry of the UV cone submosaic. Our in vivo cone density measurements agreed with measurements from previously published histology values. The method presented here allows accurate, quantitative assessment of cone structure in vivo and will be useful for longitudinal studies of the zebrafish cone mosaics.

The Effect of Retinal Melanin on Optical Coherence Tomography Images

“…Zebrafish were imaged with the hand-held Bioptigen Envisu R2200 OCT (Bioptigen) using a 12-mm telecentric lens for axial length measurements and a mouse retina probe for retinal imaging under light-adapted conditions. Axial length measurements were obtained as described previously 27 and used for lateral scaling as detailed by Huckenpahler et al 2 Retinal volume scans were nominally 1.2 × 1.2 mm (1000 A-scans/B-scan; 500 B-scans). Line scans were nominally 1.2 mm (1000 A-scans/B-scan; 40 repeated B-scans).…”

“…Zebrafish volumetric OCT scans were used to generate en face, summed volume projection images for the RPE layer to highlight the patterns in pigmentation using previously described Java software (Oracle Corporation, Redwood Shores, CA). 2,29 En face images for the optic nerve and four surrounding quadrants were aligned in Photoshop (Adobe Systems, San Jose, CA). Averaged line scans were matched to the corresponding B-scan within the volume to locate areas with and without melanin pigment from the en face image for subsequent analysis.…”

“…1 Its superior axial resolution makes it valuable in studying individual retinal layers, and implementation of summed volume projections can provide detailed, layer-specific en face images. 2–5 Despite its use, controversy remains regarding the anatomic origin of the hyperreflective signals corresponding to the photoreceptors and other outer retinal structures on OCT.…”

Repeatability and Reproducibility of In Vivo Cone Density Measurements in the Adult Zebrafish Retina

“…Fourteen WT zebrafish were imaged three times each, with 48 hours between imaging sessions. En face images of the UV cone mosaic were generated from the OCT volumes as previously described (Huckenpahler et al 2016). The en face images for each fish were manually aligned using Photoshop (Adobe Photosystems, San Jose, CA) and a common region of interest (ROI) was cropped (ROI size varied from 12.18–102*10 −3 mm 2 ).…”

“…Recently, an OCT-based method for deriving in vivo estimates of cone density in the adult zebrafish retina was introduced (Huckenpahler et al 2016). This method could allow researchers to quantify photoreceptor changes in response to disease or drug treatment, though the repeatability of these measurements has not been studied.…”