PurposeWe assessed the effect of melanin on the appearance of hyperreflective outer retinal bands in optical coherence tomography (OCT) images.MethodsA total of 23 normal subjects and 51 patients with albinism were imaged using the Bioptigen high-resolution spectral-domain OCT. In addition, three wild type, three albino (slc45a2b4/b4), and eight tyrosinase mosaic zebrafish were imaged with the hand-held Bioptigen Envisu R2200 OCT. To identify pigmented versus nonpigmented regions in the tyrosinase mosaic zebrafish, en face summed volume projections of the retinal pigment epithelium (RPE) were created from volume scans. Longitudinal reflectivity profiles were generated from B-scans to assess the width and maximum intensity of the RPE band in fish, or the presence of one or two RPE/Bruch's membrane (BrM) bands in humans.ResultsThe foveal RPE/BrM appeared as two bands in 71% of locations in patients with albinism and 45% of locations in normal subjects (P = 0.0003). Pigmented zebrafish retinas had significantly greater RPE reflectance, and pigmented regions of mosaic zebrafish also had significantly broader RPE bands than all other groups.ConclusionsThe hyperreflective outer retinal bands in OCT images are highly variable in appearance. We showed that melanin is a major contributor to the intensity and width of the RPE band on OCT. One should use caution in extrapolating findings from OCT images of one or even a few individuals to define the absolute anatomic correlates of the hyperreflective outer retinal bands in OCT images.Translational RelevanceMelanin affects the appearance of the outer retinal bands in OCT images. Use of animal models may help dissect the anatomic correlates of the complex reflective signals in OCT retinal images.

PurposeTo demonstrate and validate that photothermal optical coherence tomography (PT-OCT) can image melanin in the retinal pigment epithelium (RPE) and can observe light-driven melanosome translocation in the zebrafish retina.MethodsA commercial spectral domain OCT system was modified to perform both OCT and PT-OCT. Four adult tyrosinase-mosaic zebrafish with varying levels of melanin expression across their retinas were imaged, and the PT-OCT signal for pigmented and nonpigmented regions were compared. Wild-type dark-adapted (n = 11 fish) and light-adapted (n = 10 fish) zebrafish were also imaged with OCT and PT-OCT. Longitudinal reflectivity and absorption profiles were generated from B-scans to compare the melanin distribution between the two groups.ResultsA significant increase in PT-OCT signal (P < 0.0001, Student's t-test) was observed in pigmented regions of interest (ROI) compared to nonpigmented ROIs in the tyrosinase-mosaic zebrafish, which confirms the PT-OCT signal is specific to melanin in the eye. A significant increase in PT-OCT signal intensity (P < 0.0001, Student's t-test) was also detected in the light-adapted wild-type zebrafish group compared to the dark-adapted group. Additionally, light-adapted zebrafish display more distinct melanin banding patterns than do dark-adapted zebrafish in PT-OCT B-scans.ConclusionsPT-OCT can detect different levels of melanin absorption and characterize pigment distribution in the zebrafish retina, including intracellular changes due to light-driven melanosome translocation within the RPE.Translational RelevancePT-OCT could quantify changes in pigmentation that occur in retinal diseases. The functional information provided by PT-OCT may also enable a better understanding of the anatomical features within conventional OCT images.

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.

Quantification of the human rod and cone photoreceptor mosaic in adaptive optics scanning light ophthalmoscope (AOSLO) images is useful for the study of various retinal pathologies. Subjective and time-consuming manual grading has remained the gold standard for evaluating these images, with no well validated automatic methods for detecting individual rods having been developed. We present a novel deep learning based automatic method, called the rod and cone CNN (RAC-CNN), for detecting and classifying rods and cones in multimodal AOSLO images. We test our method on images from healthy subjects as well as subjects with achromatopsia over a range of retinal eccentricities. We show that our method is on par with human grading for detecting rods and cones.

PurposeTo examine repeatability and reproducibility of ellipsoid zone (EZ) width measurements in patients with retinitis pigmentosa (RP) using a longitudinal reflectivity profile (LRP) analysis.MethodsWe examined Bioptigen optical coherence tomography (OCT) scans from 48 subjects with RP or Usher syndrome. Nominal scan lengths were 6, 7, or 10 mm, and the lateral scale of each scan was calculated using axial length measurements. LRPs were generated from OCT line scans, and the peak corresponding to EZ was manually identified using ImageJ. The locations at which the EZ peak disappeared were used to calculate EZ width. Each scan was analyzed twice by each of two observers, who were masked to their previous measurements and those of the other observer.ResultsOn average, horizontal width (HW) was significantly greater than vertical width (VW), and there was high interocular symmetry for both HW and VW. We observed excellent intraobserver repeatability with intraclass correlation coefficients (ICCs) ranging from 0.996 to 0.998 for HW and VW measurements. Interobserver reproducibility was also excellent for both HW (ICC = 0.989; 95% confidence interval [CI] = 0.983–0.995) and VW (ICC = 0.991; 95% CI = 0.985–0.996), with no significant bias observed between observers.ConclusionsEZ width can be measured using LRPs with excellent repeatability and reproducibility. Our observation of greater HW than VW is consistent with previous observations in RP, though the reason for this anisotropy remains unclear.Translational RelevanceWe describe repeatability and reproducibility of a method for measuring EZ width in patients with RP or Usher syndrome. This approach could facilitate measurement of retinal band thickness and/or intensity.