Quantifying image quality in AOSLO images of photoreceptors.

The use of "quality" to describe the usefulness of an image is ubiquitous but is often subject to domain specific constraints. Despite its continued use as an imaging modality, adaptive optics scanning light ophthalmoscopy (AOSLO) lacks a dedicated metric for quantifying the quality of an image of photoreceptors. Here, we present an approach to evaluating image quality that extracts an estimate of the signal to noise ratio. We evaluated its performance in 528 images of photoreceptors from two AOSLOs, two modalities, and healthy or diseased retinas. The algorithm was compared to expert graders' ratings of the images and previously published image quality metrics. We found no significant difference in the SNR and grades across all conditions. The SNR and the grades of the images were moderately correlated. Overall, this algorithm provides an objective measure of image quality that closely relates to expert assessments of quality in both confocal and split-detector AOSLO images of photoreceptors.

Longitudinal Assessment of OCT-Based Measures of Foveal Cone Structure in Achromatopsia.

Purpose: Achromatopsia (ACHM) is an autosomal recessive retinal disease associated with reduced or absent cone function. There is debate regarding the extent to which cone structure shows progressive degeneration in patients with ACHM. Here, we used optical coherence tomography (OCT) images to evaluate outer nuclear layer (ONL) thickness and ellipsoid zone (EZ) integrity over time in individuals with ACHM. Methods: Sixty-three individuals with genetically confirmed ACHM with follow-up ranging from about 6 months to 10 years were imaged using either Bioptigen or Cirrus OCT. Foveal cone structure was evaluated by assessing EZ integrity and ONL thickness. Results: A total of 470 OCT images were graded, 243 OD and 227 OS. The baseline distribution of EZ grades was highly symmetrical between eyes (P = 0.99) and there was no significant interocular difference in baseline ONL thickness (P = 0.12). The EZ grade remained unchanged over the follow-up period for 60 of 63 individuals. Foveal ONL thickness showed a clinically significant change in only 1 of the 61 individuals analyzed, although detailed adaptive optics imaging revealed no changes in cone density in this individual. Conclusions: ACHM appears to be a generally stable condition, at least over the follow-up period assessed here. As cones are the cellular targets for emerging gene therapies, stable EZ and ONL thickness demonstrate therapeutic potential for ACHM, although other aspects of the visual system need to be considered when determining the best timing for therapeutic intervention.

Peak Cone Density Predicted from Outer Segment Length Measured on Optical Coherence Tomography.

PURPOSE: To compare peak cone density predicted from outer segment length measured on optical coherence tomography with direct measures of peak cone density from adaptive optics scanning light ophthalmoscopy. METHODS: Data from 42 healthy participants with direct peak cone density measures and optical coherence tomography line scans available were used in this study. Longitudinal reflectivity profiles were analyzed using two methods of identifying the boundaries of the ellipsoid and interdigitation zones to estimate maximum outer segment length: peak-to-peak and the slope method. These maximum outer segment length values were then used to predict peak cone density using a previously described geometrical model. A comparison between predicted and direct peak cone density measures was then performed. RESULTS: The mean bias between observers for estimating maximum outer segment length across methods was less than 2 µm. Cone density predicted from the peak-to-peak method against direct cone density measures showed a mean bias of 6,812 cones/mm2 with 50% of participants displaying a 10% difference or less between predicted and direct cone density values. Cone density derived from the slope method showed a mean bias of -17,929 cones/mm2 relative to direct cone density measures, with only 41% of participants demonstrating less than a 10% difference between direct and predicted cone density values. CONCLUSION: Predicted foveal cone density derived from peak-to-peak outer segment length measurements using commercial optical coherence tomography show modest agreement with direct measures of peak cone density from adaptive optics scanning light ophthalmoscopy. The methods used here are imperfect predictors of cone density, however, further exploration of this relationship could reveal a clinically relevant marker of cone structure.

Extracting spacing-derived estimates of rod density in healthy retinae.

Quantification of the rod photoreceptor mosaic using adaptive optics scanning light ophthalmoscopy (AOSLO) remains challenging. Here we demonstrate a method for deriving estimates of rod density and rod:cone ratio based on measures of rod spacing, cone numerosity, and cone inner segment area. Twenty-two AOSLO images with complete rod visualization were used to validate this spacing-derived method for estimating density. The method was then used to estimate rod metrics in an additional 105 images without complete rod visualization. The spacing-derived rod mosaic metrics were comparable to published data from histology. This method could be leveraged to develop large normative databases of rod mosaic metrics, though limitations persist with intergrader variability in assessing cone area and numerosity.

Comparison of Cone Mosaic Metrics From Images Acquired With the SPECTRALIS High Magnification Module and Adaptive Optics Scanning Light Ophthalmoscopy.

Purpose: To compare cone mosaic metrics derived from adaptive optics scanning light ophthalmoscopy (AOSLO) images with those derived from Heidelberg Engineering SPECTRALIS High Magnification Module (HMM) images. Methods: Participants with contiguous cone mosaics had HMM imaging performed at locations superior and temporal to the fovea. These images were registered and averaged offline and then aligned to split-detection AOSLO images; 200 × 200-µm regions of interest were extracted from both modalities. Cones were semi-automatically identified by two graders to provide estimates of cone density and spacing. Results: Thirty participants with contiguous cone mosaics were imaged (10 males, 20 females; age range, 11-67 years). Image quality varied, and 80% of our participants had analyzable HMM images. The intergrader intraclass correlation coefficients for cone metrics were good for both modalities (0.688-0.757 for HMM; 0.805-0.836 for AOSLO). Cone density estimates from HMM images were lower by 2661 cones/mm2 (24.1%) on average compared to AOSLO-derived estimates. Accordingly, HMM estimates of cone spacing were increased on average compared to AOSLO. Conclusions: The cone mosaic can be visualized in vivo using the SPECTRALIS HMM, although image quality is variable and imaging is not successful in every individual. Metrics extracted from HMM images can differ from those from AOSLO, although excellent agreement is possible in individuals with excellent optical quality and precise co-registration between modalities. Translational Relevance: Emerging non-adaptive optics-based photoreceptor imaging is more clinically accessible than adaptive optics techniques and has potential to expand high-resolution imaging in a clinical environment.

Intergrader agreement of foveal cone topography measured using adaptive optics scanning light ophthalmoscopy.

The foveal cone mosaic can be directly visualized using adaptive optics scanning light ophthalmoscopy (AOSLO). Previous studies in individuals with normal vision report wide variability in the topography of the foveal cone mosaic, especially the value of peak cone density (PCD). While these studies often involve a human grader, there have been no studies examining intergrader reproducibility of foveal cone mosaic metrics. Here we re-analyzed published AOSLO foveal cone images from 44 individuals to assess the relationship between the cone density centroid (CDC) location and the location of PCD. Across 5 graders with variable experience, we found a measurement error of 11.7% in PCD estimates and higher intergrader reproducibility of CDC location compared to PCD location (p < 0.0001). These estimates of measurement error can be used in future studies of the foveal cone mosaic, and our results support use of the CDC location as a more reproducible anchor for cross-modality analyses.

Challenges Associated With Ellipsoid Zone Intensity Measurements Using Optical Coherence Tomography.

Translational Relevance: Qualitative evaluation of the ellipsoid zone band on optical coherence tomography is a valuable clinical tool for assessing photoreceptor structure, though more quantitative metrics are emerging. Awareness of the challenges involved in interpreting quantitative metrics is important for their clinical translation.

Automated image processing pipeline for adaptive optics scanning light ophthalmoscopy.

To mitigate the substantial post-processing burden associated with adaptive optics scanning light ophthalmoscopy (AOSLO), we have developed an open-source, automated AOSLO image processing pipeline with both "live" and "full" modes. The live mode provides feedback during acquisition, while the full mode is intended to automatically integrate the copious disparate modules currently used in generating analyzable montages. The mean (±SD) lag between initiation and montage placement for the live pipeline was 54.6 ± 32.7s. The full pipeline reduced overall human operator time by 54.9 ± 28.4%, with no significant difference in resultant cone density metrics. The reduced overhead decreases both the technical burden and operating cost of AOSLO imaging, increasing overall clinical accessibility.

Retinal alterations in patients with Lafora disease.

PURPOSE: Lafora disease is a genetic neurodegenerative metabolic disorder caused by insoluble polyglucosan aggregate accumulation throughout the central nervous system and body. The retina is an accessible neural tissue, which may offer alternative methods to assess neurological diseases quickly and noninvasively. In this way, noninvasive imaging may provide a means to characterize neurodegenerative disease, which enables earlier identification and diagnosis of disease and the ability to monitor disease progression. In this study, we sought to characterize the retina of individuals with Lafora disease using non-invasive retinal imaging. METHODS: One eye of three individuals with genetically confirmed Lafora disease were imaged with optical coherence tomography (OCT) and adaptive optics scanning light ophthalmoscopy (AOSLO). When possible, OCT volume and line scans were acquired to assess total retinal thickness, ganglion cell-inner plexiform layer thickness, and outer nuclear layer + Henle fiber layer thickness. OCT angiography (OCTA) scans were acquired in one subject at the macula and optic nerve head (ONH). AOSLO was used to characterize the photoreceptor mosaic and examine the retinal nerve fiber layer (RNFL). RESULTS: Two subjects with previous seizure activity demonstrated reduced retinal thickness, while one subject with no apparent symptoms had normal retinal thickness. All other clinical measures, as well as parafoveal cone density, were within normal range. Nummular reflectivity at the level of the RNFL was observed using AOSLO in the macula and near the ONH in all three subjects. CONCLUSIONS: This multimodal retinal imaging approach allowed us to observe a number of retinal structural features in all three individuals. Most notably, AOSLO revealed nummular reflectivity within the inner retina of each subject. This phenotype has not been reported previously and may represent a characteristic change produced by the neurodegenerative process.

Interocular symmetry, intraobserver repeatability, and interobserver reliability of cone density measurements in the 13-lined ground squirrel.

BACKGROUND: The 13-lined ground squirrel (13-LGS) possesses a cone-dominant retina that is highly amenable to non-invasive high-resolution retinal imaging. The ability for longitudinal assessment of a cone-dominant photoreceptor mosaic with an adaptive optics scanning light ophthalmoscope (AOSLO) has positioned the 13-LGS to become an accessible model for vision research. Here, we examine the interocular symmetry, repeatability, and reliability of cone density measurements in the 13-LGS. METHODS: Thirteen 13-LGS (18 eyes) were imaged along the vertical meridian with a custom AOSLO. Regions of interest were selected superior and inferior to the optic nerve head, including the cone-rich visual streak. Non-confocal split-detection was used to capture images of the cone mosaic. Five masked observers each manually identified photoreceptors for 26 images three times and corrected an algorithm's cell identification outputs for all 214 images three times. Intraobserver repeatability and interobserver reliability of cone density were characterized using data collected from all five observers, while interocular symmetry was assessed in five animals using the average values of all observers. The distribution of image quality for all images in this study was assessed with open-sourced software. RESULTS: Manual identification was less repeatable than semi-automated correction for four of the five observers. Excellent repeatability was seen from all observers (ICC = 0.997-0.999), and there was good agreement between repeat cell identification corrections in all five observers (range: 9.43-25.71 cells/degree2). Reliability of cell identification was significantly different in two of the five observers, and worst in images taken from hibernating 13-LGS. Interocular symmetry of cone density was seen in the five 13-LGS assessed. Image quality was variable between blur- and pixel intensity-based metrics. CONCLUSIONS: Interocular symmetry with repeatable cone density measurements suggest that the 13-LGS is well-suited for longitudinal examination of the cone mosaic using split-detection AOSLO. Differences in reliability highlight the importance of observer training and automation of AOSLO cell detection. Cone density measurements from hibernating 13-LGS are not repeatable. Additional studies are warranted to assess other metrics of cone health to detect deviations from normal 13-LGS in future models of cone disorder in this species.

The Henle Fiber Layer in Albinism: Comparison to Normal and Relationship to Outer Nuclear Layer Thickness and Foveal Cone Density.

Purpose: Directional optical coherence tomography (D-OCT) allows the visualization of the Henle fiber layer (HFL) in vivo. Here, we used D-OCT to characterize the HFL and outer nuclear layer (ONL) in albinism and examine the relationship between true foveal ONL and peak cone density. Methods: Horizontal D-OCT B-scans were acquired, registered, and averaged for 12 subjects with oculocutaneous albinism and 26 control subjects. Averaged images were manually segmented to extract HFL and ONL thickness. Adaptive optics scanning light ophthalmoscopy was used to acquire images of the foveal cone mosaic in 10 subjects with albinism, from which peak cone density was assessed. Results: Across the foveal region, the HFL topography was different between subjects with albinism and normal controls. In particular, foveal HFL thickness was thicker in albinism than in normal controls (P < 0.0001), whereas foveal ONL thickness was thinner in albinism than in normal controls (P < 0.0001). The total HFL and ONL thickness was not significantly different between albinism and controls (P = 0.3169). Foveal ONL thickness was positively correlated with peak cone density in subjects with albinism (r = 0.8061, P = 0.0072). Conclusions: Foveal HFL and ONL topography are significantly altered in albinism relative to normal controls. Our data suggest that increased foveal cone packing drives the formation of Henle fibers, more so than the lateral displacement of inner retinal neurons (which is reduced in albinism). The ability to quantify foveal ONL and HFL may help further stratify grading schemes used to assess foveal hypoplasia.