Repeatability of in vivo parafoveal cone density and spacing measurements.
ABSTRACT: To assess the repeatability and measurement error associated with cone density and nearest neighbor distance (NND) estimates in images of the parafoveal cone mosaic obtained with an adaptive optics scanning light ophthalmoscope (AOSLO).Twenty-one participants with no known ocular pathology were recruited. Four retinal locations, approximately 0.65° eccentricity from the center of fixation, were imaged 10 times in randomized order with an AOSLO. Cone coordinates in each image were identified using an automated algorithm (with or without manual correction) from which cone density and NND were calculated. Owing to naturally occurring fixational instability, the 10 images recorded from a given location did not overlap entirely. We thus analyzed each image set both before and after alignment.Automated estimates of cone density on the unaligned image sets showed a coefficient of repeatability of 11,769 cones/mm(2) (17.1%). The primary reason for this variability appears to be fixational instability, as aligning the 10 images to include the exact same retinal area results in an improved repeatability of 4358 cones/mm(2) (6.4%) using completely automated cone identification software. Repeatability improved further by manually identifying cones missed by the automated algorithm, with a coefficient of repeatability of 1967 cones/mm(2) (2.7%). NND showed improved repeatability and was generally insensitive to the undersampling by the automated algorithm.As our data were collected in a young, healthy population, this likely represents a best-case estimate for corresponding measurements in patients with retinal disease. Similar studies need to be carried out on other imaging systems (including those using different imaging modalities, wavefront correction technology, and/or image analysis software), as repeatability would be expected to be highly sensitive to initial image quality and the performance of cone identification algorithms. Separate studies addressing intersession repeatability and interobserver reliability are also needed.
Project description: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.
Project description:To characterize the effects of intraframe distortion due to involuntary eye motion on measures of cone mosaic geometry derived from adaptive optics scanning light ophthalmoscope (AOSLO) images.We acquired AOSLO image sequences from 20 subjects at 1.0, 2.0, and 5.0° temporal from fixation. An expert grader manually selected 10 minimally distorted reference frames from each 150-frame sequence for subsequent registration. Cone mosaic geometry was measured in all registered images (n = 600) using multiple metrics, and the repeatability of these metrics was used to assess the impact of the distortions from each reference frame. In nine additional subjects, we compared AOSLO-derived measurements to those from adaptive optics (AO)-fundus images, which do not contain system-imposed intraframe distortions.We observed substantial variation across subjects in the repeatability of density (1.2%-8.7%), inter-cell distance (0.8%-4.6%), percentage of six-sided Voronoi cells (0.8%-10.6%), and Voronoi cell area regularity (VCAR) (1.2%-13.2%). The average of all metrics extracted from AOSLO images (with the exception of VCAR) was not significantly different than those derived from AO-fundus images, though there was variability between individual images.Our data demonstrate that the intraframe distortion found in AOSLO images can affect the accuracy and repeatability of cone mosaic metrics. It may be possible to use multiple images from the same retinal area to approximate a "distortionless" image, though more work is needed to evaluate the feasibility of this approach.Even in subjects with good fixation, images from AOSLOs contain intraframe distortions due to eye motion during scanning. The existence of these artifacts emphasizes the need for caution when interpreting results derived from scanning instruments.
Project description:Purpose:We determine the intersession repeatability of cone measurements via flood-illuminated adaptive optics (AO) imaging in patients with retinitis pigmentosa (RP), to better differentiate variation due to imaging inaccuracies versus pathology-driven change. Methods:A total of 25 4° × 4° AO images were acquired three times on the same day in 10 subjects with RP, registered in i2K Retina, and cones were identified using a custom-built MATLAB algorithm. Nine equally spaced regions of interest were selected for each imaging set. A subset of subjectively "poor" and "good" quality images was selected by three independent graders, analyzed using cone density, cone location similarity (CLS) and cone spacing, and compared to age-matched normals. Results:The coefficient of variation (CoV), repeatability, and percent repeatability of automated cone density were slightly higher in patients with RP compared to age-matched normals, but showed no statistically significant difference. The standard deviation of CLS and cone spacing of nearest-neighbor distance demonstrated a statistically significant difference between good- and poor-quality images. Conclusions:Repeatability of automated cone density measurements in patients with RP is comparable to normals. Misidentification of cones due to image quality variability is a major limitation of automated cone counting algorithms in patients with RP. Our study suggests that CLS and cone spacing metrics could be used to help define image quality and, thus, increase confidence in automated cone counts in patients with RP. Translational Relevance:The novel AO image quality assessment metrics described in our study could help to improve patient image interpretation, prognosis, and longitudinal care.
Project description:Eye motion is a major impediment to the efficient acquisition of high resolution retinal images with the adaptive optics (AO) scanning light ophthalmoscope (AOSLO). Here we demonstrate a solution to this problem by implementing both optical stabilization and digital image registration in an AOSLO. We replaced the slow scanning mirror with a two-axis tip/tilt mirror for the dual functions of slow scanning and optical stabilization. Closed-loop optical stabilization reduced the amplitude of eye-movement related-image motion by a factor of 10-15. The residual RMS error after optical stabilization alone was on the order of the size of foveal cones: ~1.66-2.56 μm or ~0.34-0.53 arcmin with typical fixational eye motion for normal observers. The full implementation, with real-time digital image registration, corrected the residual eye motion after optical stabilization with an accuracy of ~0.20-0.25 μm or ~0.04-0.05 arcmin RMS, which to our knowledge is more accurate than any method previously reported.
Project description:The purpose of this study was to examine cone photoreceptor structure in retinitis pigmentosa (RP) and Usher syndrome using confocal and nonconfocal split-detector adaptive optics scanning light ophthalmoscopy (AOSLO).Nineteen subjects (11 RP, 8 Usher syndrome) underwent ophthalmic and genetic testing, spectral-domain optical coherence tomography (SD-OCT), and AOSLO imaging. Split-detector images obtained in 11 subjects (7 RP, 4 Usher syndrome) were used to assess remnant cone structure in areas of altered cone reflectivity on confocal AOSLO.Despite normal interdigitation zone and ellipsoid zone appearance on OCT, foveal and parafoveal cone densities derived from confocal AOSLO images were significantly lower in Usher syndrome compared with RP. This was due in large part to an increased prevalence of non-waveguiding cones in the Usher syndrome retina. Although significantly correlated to best-corrected visual acuity and foveal sensitivity, cone density can decrease by nearly 38% before visual acuity becomes abnormal. Aberrantly waveguiding cones were noted within the transition zone of all eyes and corresponded to intact inner segment structures. These remnant cones decreased in density and increased in diameter across the transition zone and disappeared with external limiting membrane collapse.Foveal cone density can be decreased in RP and Usher syndrome before visible changes on OCT or a decline in visual function. Thus, AOSLO imaging may allow more sensitive monitoring of disease than current methods. However, confocal AOSLO is limited by dependence on cone waveguiding, whereas split-detector AOSLO offers unambiguous and quantifiable visualization of remnant cone inner segment structure. Confocal and split-detector thus offer complementary insights into retinal pathology.
Project description:To correlate visual function with high-resolution images of retinal structure using adaptive optics scanning laser ophthalmoscopy (AOSLO) in 4 patients with acute zonal occult outer retinopathy (AZOOR).Observational case series.Four women, aged 18 to 51, with acute focal loss of visual field or visual acuity, photopsia, and minimal funduscopic changes were studied with best-corrected visual acuity (BCVA), Goldmann kinetic and automated perimetry and fundus-guided microperimetry, full-field and multifocal electroretinography (ffERG and mfERG), spectral-domain optical coherence tomography (SD-OCT), and AOSLO imaging. Cone spacing was measured in 4 eyes and compared with 27 age-similar normal eyes. Additional functional testing in 1 patient suggested that cones were absent but rods remained. Serum from all patients was analyzed for anti-retinal antibody activity.In all patients vision loss was initially progressive, then stable. Symptoms were unilateral in 2 and bilateral but asymmetric in 2 patients. In each patient, loss of retinal function correlated with structural changes in the outer retina. AOSLO showed focal cone loss in most patients, although in 1 patient with central vision loss such change was absent. In another patient, structural and functional analyses suggested that cones had degenerated but rods remained. Anti-retinal antibody activity against a ?45 kd antigen was detected in 1 of the patients; the other 3 patients showed no evidence of abnormal anti-retinal antibodies.Focal abnormalities of retinal structure correlated with vision loss in patients with AZOOR. High-resolution imaging can localize and demonstrate the extent of outer retinal abnormality in AZOOR patients.
Project description:Purpose:Cone photoreceptor cells can be noninvasively imaged in the living human eye by using nonconfocal adaptive optics scanning ophthalmoscopy split detection. Existing metrics, such as cone density and spacing, are based on simplifying cone photoreceptors to single points. The purposes of this study were to introduce a computer-aided approach for segmentation of cone photoreceptors, to apply this technique to create a normal database of cone diameters, and to demonstrate its use in the context of existing metrics. Methods:Cone photoreceptor segmentation is achieved through a circularly constrained active contour model (CCACM). Circular templates and image gradients attract active contours toward cone photoreceptor boundaries. Automated segmentation from in vivo human subject data was compared to ground truth established by manual segmentation. Cone diameters computed from curated data (automated segmentation followed by manual removal of errors) were compared with histology and published data. Results:Overall, there was good agreement between automated and manual segmentations and between diameter measurements (n = 5191 cones) and published histologic data across retinal eccentricities ranging from 1.35 to 6.35 mm (temporal). Interestingly, cone diameter was correlated to both cone density and cone spacing (negatively and positively, respectively; P < 0.01 for both). Application of the proposed automated segmentation to images from a patient with late-onset retinal degeneration revealed the presence of enlarged cones above individual reticular pseudodrusen (average 23.0% increase, P < 0.05). Conclusions:CCACM can accurately segment cone photoreceptors on split detection images across a range of eccentricities. Metrics derived from this automated segmentation of adaptive optics retinal images can provide new insights into retinal diseases.
Project description:BACKGROUND: Retinitis pigmentosa is characterized by the sequential loss of rod and cone photoreceptors. The preservation of cones would prevent blindness due to their essential role in human vision. Rod-derived Cone Viability Factor is a thioredoxin-like protein that is secreted by rods and is involved in cone survival. To validate the activity of Rod-derived Cone Viability Factors (RdCVFs) as therapeutic agents for treating retinitis Pigmentosa, we have developed e-conome, an automated cell counting platform for retinal flat mounts of rodent models of cone degeneration. This automated quantification method allows for faster data analysis thereby accelerating translational research. METHODS: An inverted fluorescent microscope, motorized and coupled to a CCD camera records images of cones labeled with fluorescent peanut agglutinin lectin on flat-mounted retinas. In an average of 300 fields per retina, nine Z-planes at magnification X40 are acquired after two-stage autofocus individually for each field. The projection of the stack of 9 images is subject to a threshold, filtered to exclude aberrant images based on preset variables. The cones are identified by treating the resulting image using 13 variables empirically determined. The cone density is calculated over the 300 fields. RESULTS: The method was validated by comparison to the conventional stereological counting. The decrease in cone density in rd1 mouse was found to be equivalent to the decrease determined by stereological counting. We also studied the spatiotemporal pattern of the degeneration of cones in the rd1 mouse and show that while the reduction in cone density starts in the central part of the retina, cone degeneration progresses at the same speed over the whole retinal surface. We finally show that for mice with an inactivation of the Nucleoredoxin-like genes Nxnl1 or Nxnl2 encoding RdCVFs, the loss of cones is more pronounced in the ventral retina. CONCLUSION: The automated platform ?-conome used here for retinal disease is a tool that can broadly accelerate translational research for neurodegenerative diseases.
Project description:To study cone photoreceptor structure and function in patients with inherited retinal degenerations treated with sustained-release ciliary neurotrophic factor (CNTF).Two patients with retinitis pigmentosa and one with Usher syndrome type 2 who participated in a phase 2 clinical trial received CNTF delivered by an encapsulated cell technology implant in one eye and sham surgery in the contralateral eye. Patients were followed longitudinally over 30 to 35 months. Adaptive optics scanning laser ophthalmoscopy (AOSLO) provided high-resolution images at baseline and at 3, 6, 12, 18, and 24 months. AOSLO measures of cone spacing and density and optical coherence tomography measures of retinal thickness were correlated with visual function, including visual acuity (VA), visual field sensitivity, and full-field electroretinography (ERG).No significant changes in VA, visual field sensitivity, or ERG responses were observed in either eye of the three patients over 24 months. Outer retinal layers were significantly thicker in CNTF-treated eyes than in sham-treated eyes (P < 0.005). Cone spacing increased by 2.9% more per year in sham-treated eyes than in CNTF-treated eyes (P < 0.001, linear mixed model), and cone density decreased by 9.1%, or 223 cones/degree(2) more per year in sham-treated than in CNTF-treated eyes (P = 0.002, linear mixed model).AOSLO images provided a sensitive measure of disease progression and treatment response in patients with inherited retinal degenerations. Larger studies of cone structure using high-resolution imaging techniques are urgently needed to evaluate the effect of CNTF treatment in patients with inherited retinal degenerations.
Project description:To develop an automated reference frame selection (ARFS) algorithm to replace the subjective approach of manually selecting reference frames for processing adaptive optics scanning light ophthalmoscope (AOSLO) videos of cone photoreceptors.Relative distortion was measured within individual frames before conducting image-based motion tracking and sorting of frames into distinct spatial clusters. AOSLO images from nine healthy subjects were processed using ARFS and human-derived reference frames, then aligned to undistorted AO-flood images by nonlinear registration and the registration transformations were compared. The frequency at which humans selected reference frames that were rejected by ARFS was calculated in 35 datasets from healthy subjects, and subjects with achromatopsia, albinism, or retinitis pigmentosa. The level of distortion in this set of human-derived reference frames was assessed.The average transformation vector magnitude required for registration of AOSLO images to AO-flood images was significantly reduced from 3.33 ± 1.61 pixels when using manual reference frame selection to 2.75 ± 1.60 pixels (mean ± SD) when using ARFS (P = 0.0016). Between 5.16% and 39.22% of human-derived frames were rejected by ARFS. Only 2.71% to 7.73% of human-derived frames were ranked in the top 5% of least distorted frames.ARFS outperforms expert observers in selecting minimally distorted reference frames in AOSLO image sequences. The low success rate in human frame choice illustrates the difficulty in subjectively assessing image distortion.Manual reference frame selection represented a significant barrier to a fully automated image-processing pipeline (including montaging, cone identification, and metric extraction). The approach presented here will aid in the clinical translation of AOSLO imaging.