Quantification of capillary blood flow speeds in diabetic retinopathy using variable interscan time analysis (VISTA) OCTA.

PURPOSE: To investigate retinal capillary plexus capillary flow speed and vessel density in diabetic retinopathy (DR) and normal subjects using variable interscan time analysis (VISTA) optical coherence tomography angiography (OCTA). METHODS: High speed swept source OCTA imaging using multiple interscan times was performed over a 5 mm x 5 mm field-of-view with 600 kHz A-scan rate. Second-generation VISTA OCTA was used to measure a surrogate marker for capillary blood flow speed, VISTA flow speed (VFS), in the superficial and intermediate capillary plexuses, (SCP + ICP)VFS, and deep capillary plexus, DCPVFS. Vessel density was measured using OCTA. RESULTS: Fifty-seven eyes with different DR severity and 37 normal eyes were analyzed. VISTA OCTA provided diverse blood flow speed information, including pseudo-color OCTA and mean flow speed in different regions. Both DCPVFS and DCPVFS/(SCP + ICP)VFS were higher in DR compared to normal eyes. Elevated DCPVFS correlated with decreased DCP vessel density in non-proliferative DR. CONCLUSION: VISTA OCTA can measure a quantitative biomarker for blood flow speed alterations in DR and normal eyes as well as the association with vessel density in different capillary plexuses. VISTA OCTA is promising for studies of pathogenesis and early flow alterations which may precede non-perfusion.

Ensembling U-Nets for microaneurysm segmentation in optical coherence tomography angiography in patients with diabetic retinopathy.

Diabetic retinopathy is one of the leading causes of blindness around the world. This makes early diagnosis and treatment important in preventing vision loss in a large number of patients. Microaneurysms are the key hallmark of the early stage of the disease, non-proliferative diabetic retinopathy, and can be detected using OCT angiography quickly and non-invasively. Screening tools for non-proliferative diabetic retinopathy using OCT angiography thus have the potential to lead to improved outcomes in patients. We compared different configurations of ensembled U-nets to automatically segment microaneurysms from OCT angiography fundus projections. For this purpose, we created a new database to train and evaluate the U-nets, created by two expert graders in two stages of grading. We present the first U-net neural networks using ensembling for the detection of microaneurysms from OCT angiography en face images from the superficial and deep capillary plexuses in patients with non-proliferative diabetic retinopathy trained on a database labeled by two experts with repeats.

Topographic Measurement of the Subretinal Pigment Epithelium Space in Normal Aging and Age-Related Macular Degeneration Using High-Resolution OCT.

Purpose: A micrometer scale hyporeflective band within the retinal pigment epithelium basal lamina - Bruch's membrane complex (RPE-BL-BrM) was topographically measured in aging and age-related macular degeneration (AMD). Methods: In a prospective cross-sectional study, 90 normal eyes from 76 subjects (range = 23-90 years) and 53 dry AMD eyes from 47 subjects (range = 62-91 years) were enrolled. Isotropic volume raster scans over 6 mm × 6 mm (500 × 500 A-scans) were acquired using a high-resolution (2.7 µm axial resolution) spectral-domain optical coherence tomography (SD-OCT) prototype instrument. Six consecutive optical coherence tomography (OCT) volumes were computationally motion-corrected and fused to improve feature visibility. A boundary regression neural network was developed to measure hyporeflective band thickness. Topographic dependence was evaluated over a 6-mm-diameter Early Treatment Diabetic Retinopathy Study (ETDRS) grid. Results: The hyporeflective band thickness map (median of 4.3 µm and 7.8 µm in normal and AMD eyes, respectively) is thicker below and radially symmetric around the fovea. In normal eyes, age-associated differences occur within 0.7 to 2.3 mm from the foveal center (P < 0.05). In AMD eyes, the hyporeflective band is hypothesized to be basal laminar deposits (BLamDs) and is thicker within the 3-mm ETDRS circle (P < 0.0002) compared with normal eyes. The inner ring is the most sensitive location to detect age versus AMD-associated changes within the RPE-BL-BrM. AMD eyes with subretinal drusenoid deposits (SDDs) have a significantly thicker hyporeflective band (P < 0.001) than those without SDDs. Conclusions: The hyporeflective band is a quantifiable biomarker which differentiates AMD from aging. Longitudinal studies are warranted. The hyporeflective band may be a useful biomarker for risk stratification and disease progression.

Rapid examination of lung tissues by nonlinear microscopy.

OBJECTIVES: Traditional histopathology is a time-intensive and labor-intensive process involving tissue formalin fixation, paraffin embedding, and microtoming into thin sections for H&E staining. Frozen section analysis is a modality used during surgery to quickly evaluate tissue, but it has limitations, such as the size and number of the specimens that can be analyzed as well as difficulties with fatty and bony tissues. Our objective was to investigate the performance of nonlinear microscopy, a fluorescence microscopy technique, for the rapid examination of resected lung tumors. METHODS: In this proof-of-principle study, nonlinear microscopy imaging of resected lung tissue was performed on a total of 73 tissue specimens collected from 13 patients who underwent lobectomy, segmentectomy, or wedge resection for pulmonary nodules. RESULTS: Two pathologists reviewed the digital nonlinear microscopy images in comparison to the corresponding histopathologic H&E slides from a variety of pulmonary pathologies. CONCLUSIONS: This study demonstrated that nonlinear microscopy readily replicates traditional H&E staining for both lung tumors and nonneoplastic pulmonary structures. Nonlinear microscopy provides many advantages over frozen section analysis and is an optical imaging platform that has the potential to augment rapid pathologic evaluation of resected tissues in the age of digital pathology.

Data retrieval from archival renal biopsies using nonlinear microscopy.

Thorough examination of renal biopsies may improve understanding of renal disease. Imaging of renal biopsies with fluorescence nonlinear microscopy (NLM) and optical clearing enables three-dimensional (3D) visualization of pathology without microtome sectioning. Archival renal paraffin blocks from 12 patients were deparaffinized and stained with Hoechst and Eosin for fluorescent nuclear and cytoplasmic/stromal contrast, then optically cleared using benzyl alcohol benzyl benzoate (BABB). NLM images of entire biopsy fragments (thickness range 88-660 µm) were acquired using NLM with fluorescent signals mapped to an H&E color scale. Cysts, glomeruli, exudative lesions, and Kimmelstiel-Wilson nodules were segmented in 3D and their volumes, diameters, and percent composition could be obtained. The glomerular count on 3D NLM volumes was high indicating that archival blocks could be a vast tissue resource to enable larger-scale retrospective studies. Rapid optical clearing and NLM imaging enables more thorough biopsy examination and is a promising technique for analysis of archival paraffin blocks.

Rapid Examination of Nonprocessed Renal Cell Carcinoma Using Nonlinear Microscopy.

CONTEXT.­: Histology, the traditional method of examining surgical tissue under a microscope, is a time-consuming process involving the fixation of tissue in formalin, dehydration, embedding in paraffin, and cutting into thin sections for hematoxylin-eosin (H&E) staining. Frozen section analysis is a faster alternative used in surgery to quickly evaluate tissue, but it has limitations, such as the size of the specimens that can be analyzed and difficulties with fatty and bony tissues. OBJECTIVE.­: To rapidly examine nonprocessed kidney tumors using nonlinear microscopy (NLM), a fluorescence microscopy technique that can rapidly visualize fresh or fixed, rapidly stained, nonprocessed tissue resembling H&E histology. This technology eliminates the need for fixation, embedding, microtome sectioning, or slide preparation. DESIGN.­: In this study, a total of 190 tissue specimens were collected from 46 patients who underwent partial or radical nephrectomy. RESULTS.­: Two genitourinary pathologists confirmed that diagnostically important features present in the H&E images could also be identified in the NLM images. CONCLUSIONS.­: The results of this study demonstrated that NLM had a high degree of correspondence with H&E staining for the classical variants of renal cell carcinoma. NLM offers several clinical benefits, such as facilitating rapid renal cell carcinoma diagnosis, assessment of targeted kidney biopsies for both tumor and medical kidney diseases, and collection of fresh renal cell carcinoma tissue for molecular studies.

Assessment of Choriocapillaris Flow Prior to Nascent Geographic Atrophy Development Using Optical Coherence Tomography Angiography.

Purpose: To assess the relationship between choriocapillaris (CC) loss and the development of nascent geographic atrophy (nGA) using optical coherence tomography angiography (OCTA) imaging. Methods: In total, 105 from 62 participants with bilateral large drusen, without late age-related macular degeneration (AMD) or nGA at baseline, were included in this prospective, longitudinal, observational study. Participants underwent swept-source OCTA imaging at 6-month intervals. CC flow deficit percentage (FD%) and drusen volume measurements were determined for the visit prior to nGA development or the second-to-last visit if nGA did not develop. Global and local analyses, the latter based on analyses within superpixels (120 × 120-µm regions), were performed to examine the association between CC FD% and future nGA development. Results: A total of 15 (14%) eyes from 12 (19%) participants developed nGA. There was no significant difference in global CC FD% at the visit prior to nGA development between eyes that developed nGA and those that did not (P = 0.399). In contrast, CC FD% was significantly higher in superpixels that subsequently developed nGA compared to those that did not (P < 0.001), and a model utilizing CC FD% was significantly better at predicting foci of future nGA development at the superpixel level than a model using drusen volume alone (P ≤ 0.040). Conclusions: This study showed that significant impairments in CC blood flow could be detected locally prior to the development of nGA. These findings add to our understanding of the pathophysiologic changes that occur with atrophy development in AMD.

Characterization of the external limbus on corneoscleral topography with ultrawide-field optical coherence tomography.

PURPOSE: To present a new method for 3-dimensional external limbal demarcation on corneoscleral topography derived from optical coherence tomography (OCT). Limbal shape is investigated and compared to other landmarks. METHODS: Images from the anterior segment were obtained with a ultrawide-field (20 mm) OCT. An automated algorithm was developed to demarcate the topographic limbus based on the transition from corneal to scleral curvature. The internal limbus was manually identified as the scleral spur on the OCT images. The external topographic limbus was fit with a circle on a plane. Ellipticity and ovality were defined by the lateral limbal deviation from the best-fit circle. Toricity was defined by the axial deviation from the best-fit plane. Repeatability was assessed by the within-subject standard deviation from two repeated measurements. For comparison, the white-to-white (WTW) diameter was obtained from Pentacam HR. RESULTS: 18 eyes from 11 subjects were analyzed. The topographic limbal diameter was 12.16 ± 0.68 mm (mean ± standard deviation) horizontally and 11.18 ± 0.65 mm vertically. The repeatability for the topographic limbal diameter was 0.054 mm. The internal and WTW horizontal limbal diameters were significantly smaller (linear mixed-effects model (GLMM), p <.017). The vertical internal limbal diameter was significantly larger (GLMM p <.05). The topographic limbus had significant ellipticity (0.25 ± 0.13 mm, wider horizontally, repeatability of 0.07 mm) and toricity (0.15 ± 0.08 mm, flatter horizontally, repeatability of 0.10 mm). Low coefficients of determination were found for the topographical limbus with the internal limbus (R2=0.021 and R2=0.039, for horizontal and vertical diameters respectively) and with the WTW (R2=0.146 for the horizontal diameter). CONCLUSION: The proposed method to demarcate the 3D external topographical limbus is repeatable. The topographic limbal shape and size cannot be accurately derived from WTW nor internal limbus measures. This new technology may improve the process of scleral lens fitting.

Extended and adjustable field-of-view of variable interscan time analysis by ammonite-scanning swept-source optical coherence tomography angiography.

A novel scanning protocol, ammonite scan, is proposed for widefield optical coherence tomography angiography (OCTA) and relative retinal blood flow velocity imaging in the human retina using variable interscan time analysis (VISTA). A repeated circle scan using a 400 kHz swept-source was employed to achieve an interscan time of 1.28 ms. The center of the repeated circular scan continuously moved spirally towards the peripheral region, ensuring an extended and adjustable scan range while preserving the short interscan time. Image artifacts due to eye movement were eliminated via extra motion-correction processing using data redundancy. The relative blood flow velocity in superficial and deep plexus layers was calculated from the VISTA image, and their ratio was used to explore the microvascular flow parameter in the healthy human eye.

Optical Coherence Tomography-History, Evolution, and Future Prospects: 2023 Lasker-DeBakey Clinical Medical Research Award.

In this Viewpoint, 2023 Lasker-DeBakey Clinical Medical Research Award winners James G. Fujimoto, David Huang, and Eric A. Swanson discuss their invention­optical coherence tomography, which allows rapid detection of diseases of the retina that impair vision.

SSN2V: unsupervised OCT denoising using speckle split.

Denoising in optical coherence tomography (OCT) is important to compensate the low signal-to-noise ratio originating from laser speckle. In recent years learning algorithms have been established as the most powerful denoising approach. Especially unsupervised denoising is an interesting topic since it is not possible to acquire noise free scans with OCT. However, speckle in in-vivo OCT images contains not only noise but also information about blood flow. Existing OCT denoising algorithms treat all speckle equally and do not distinguish between the noise component and the flow information component of speckle. Consequently they either tend to either remove all speckle or denoise insufficiently. Unsupervised denoising methods tend to remove all speckle but create results that have a blurry impression which is not desired in a clinical application. To this end we propose the concept, that an OCT denoising method should, besides reducing uninformative noise, additionally preserve the flow-related speckle information. In this work, we present a fully unsupervised algorithm for single-frame OCT denoising (SSN2V) that fulfills these goals by incorporating known operators into our network. This additional constraint greatly improves the denoising capability compared to a network without. Quantitative and qualitative results show that the proposed method can effectively reduce the speckle noise in OCT B-scans of the human retina while maintaining a sharp impression outperforming the compared methods.

Retinal blood flow speed quantification at the capillary level using temporal autocorrelation fitting OCTA [Invited].

Optical coherence tomography angiography (OCTA) can visualize vasculature structures, but provides limited information about blood flow speed. Here, we present a second generation variable interscan time analysis (VISTA) OCTA, which evaluates a quantitative surrogate marker for blood flow speed in vasculature. At the capillary level, spatially compiled OCTA and a simple temporal autocorrelation model, ρ(τ) = exp(-ατ), were used to evaluate a temporal autocorrelation decay constant, α, as the blood flow speed marker. A 600 kHz A-scan rate swept-source OCT prototype instrument provides short interscan time OCTA and fine A-scan spacing acquisition, while maintaining multi mm2 field of views for human retinal imaging. We demonstrate the cardiac pulsatility and assess repeatability of α measured with VISTA. We show different α for different retinal capillary plexuses in healthy eyes and present representative VISTA OCTA in eyes with diabetic retinopathy.

Ultrahigh Resolution OCT Markers of Normal Aging and Early Age-related Macular Degeneration.

Purpose: Ultrahigh resolution spectral domain-OCT (UHR SD-OCT) enables in vivo visualization of micrometric structural markers which differentially associate with normal aging versus age-related macular degeneration (AMD). This study explores the hypothesis that UHR SD-OCT can detect and quantify sub-retinal pigment epithelium (RPE) deposits in early AMD, separating AMD pathology from normal aging. Design: Prospective cross-sectional study. Participants: A total of 53 nonexudative (dry) AMD eyes from 39 patients, and 63 normal eyes from 39 subjects. Methods: Clinical UHR SD-OCT scans were performed using a high-density protocol. Exemplary high-resolution histology and transmission electron microscopy images were obtained from archive donor eyes. Three trained readers evaluated and labeled outer retina morphological features, including the appearance of a hyporeflective split within the RPE-RPE basal lamina (RPE-BL)-Bruch's membrane (BrM) complex on UHR brightness (B)-scans. A semi-automatic segmentation algorithm measured the thickness of the RPE-BL-BrM split/hyporeflective band. Main Outcome Measures: Qualitative description of outer retinal morphological changes on UHR SD-OCT B-scans; the proportion of the RPE-BL-BrM complex with visible split (%) and the thickness of the resulting hyporeflective band (µm). Results: In young normal eyes, UHR SD-OCT consistently revealed an RPE-BL-BrM split/hyporeflective band. Its visibility and thickness were less in eyes of advanced age. However, the split/hyporeflective band was again visible in early AMD eyes. Both qualitative reading and quantitative thickness measurements showed significantly elevated visibility and thickness of the RPE-BL-BrM split/hyporeflective in early AMD eyes compared to age-matched controls. Conclusions: Our imaging results strongly support the hypothesis that appearance of the RPE-BL-BrM split/hyporeflective band in older subjects is dominated by the BL deposit, an indicator of early AMD well known from histology. Ultrahigh resolution SD-OCT can be used to investigate physiological aging as well as early AMD pathology in clinical imaging studies. Developing quantifiable markers associated with disease pathogenesis and progression can facilitate drug discovery, as well as reduce clinical trial times. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Onset and Progression of Persistent Choroidal Hypertransmission Defects in Intermediate Age-Related Macular Degeneration: A Novel Clinical Trial Endpoint.

PURPOSE: The appearance and growth of persistent choroidal hypertransmission defects (hyperTDs) detected on en face swept-source optical coherence tomography (SS-OCT) images from eyes with intermediate age-related macular degeneration (iAMD) were studied to determine if they could serve as novel clinical trial endpoints. DESIGN: Post hoc subgroup analysis of a prospective study. METHODS: Subjects with iAMD underwent 6 × 6 mm SS-OCT angiography imaging at their baseline and follow-up visits. The drusen volumes were obtained using a validated SS-OCT algorithm. Two graders independently evaluated all en face structural images for the presence of persistent hyperTDs. The number and area of all hyperTDs along with drusen volume were obtained from all SS-OCT angiography scans. Eyes were censored from further follow-up once exudative AMD developed. RESULTS: A total of 171 eyes from 121 patients with iAMD were included. Sixty-eight eyes developed at least 1 hyperTD. Within 1 year after developing a hyperTD, 25% of eyes developed new hyperTDs for an average of 0.44 additional hyperTDs. Over 2 years, as hyperTDs appeared, enlarged, and merged, the average area growth rate was 0.220 mm/yr using the square-root transformation strategy. A clinical trial design using the onset and enlargement of these hyperTDs for the study of disease progression in eyes with iAMD is proposed. CONCLUSIONS: The appearance and growth of persistent choroidal hyperTDs in eyes with iAMD can be easily detected and measured using en face OCT imaging and can serve as novel clinical trial endpoints for the study of therapies that may slow disease progression from iAMD to late AMD.

Optical Coherence Tomography Angiography Analysis Toolbox: A Repeatable and Reproducible Software Tool for Quantitative Optical Coherence Tomography Angiography Analysis.

BACKGROUND AND OBJECTIVE: The purpose of this article is to demonstrate the optical coherence tomography angiography (OCTA) Analysis Toolkit (OAT), a custom-designed software package, as a repeatable and reproducible tool for computing OCTA metrics across different devices. MATERIALS AND METHODS: Fourteen participants were imaged using three devices. Foveal avascular zone, vessel index, vessel length index, and vessel diameter index were calculated using the OAT. Repeatability and reproducibility were assessed using the coefficient of variation and interclass correlation coefficient (ICC). RESULTS: Analysis of identical images demonstrated perfect levels of repeatability for all metrics (coefficient of variation 0%), which was a consequence of the software being deterministic (ie, producing the same outputs for the same inputs). Foveal avascular zone ICC values were in the excellent-to-good range (ICC > 0.6) for all devices. All values for vessel index (VI), vessel length index, and vessel diameter index fell in the good-to-fair (ICC > 0.4) or excellent-to-good range, except for vessel index analysis in the Cirrus device (ICC = 0.34). CONCLUSIONS: The OAT appears to be a reliable tool that may enable comparison between OCTA data sets acquired on different imaging instruments, thereby facilitating a more consistent approach to OCTA analysis. [Ophthalmic Surg Lasers Imaging Retina 2023;54:114-122.].

Comparing Accuracies of Length-Type Geographic Atrophy Growth Rate Metrics Using Atrophy-Front Growth Modeling.

Purpose: To compare the accuracies of the previously proposed square-root-transformed and perimeter-adjusted metrics for estimating length-type geographic atrophy (GA) growth rates. Design: Cross-sectional and simulation-based study. Participants: Thirty-eight eyes with GA from 27 patients. Methods: We used a previously developed atrophy-front growth model to provide analytical and numerical evaluations of the square-root-transformed and perimeter-adjusted growth rate metrics on simulated and semisimulated GA growth data. Main Outcome Measures: Comparison of the accuracies of the square-root-transformed and perimeter-adjusted metrics on simulated and semisimulated GA growth data. Results: Analytical and numerical evaluations showed that the accuracy of the perimeter-adjusted metric is affected minimally by baseline lesion area, focality, and circularity over a wide range of GA growth rates. Average absolute errors of the perimeter-adjusted metric were approximately 20 times lower than those of the square-root-transformed metrics, per evaluation on a semisimulated dataset with growth rate characteristics matching clinically observed data. Conclusions: Length-type growth rates have an intuitive, biophysical interpretation that is independent of lesion geometry, which supports their use in clinical trials of GA therapeutics. Taken in the context of prior studies, our analyses suggest that length-type GA growth rates should be measured using the perimeter-adjusted metric, rather than square-root-transformed metrics.

High speed, long range, deep penetration swept source OCT for structural and angiographic imaging of the anterior eye.

This study reports the development of prototype swept-source optical coherence tomography (SS-OCT) technology for imaging the anterior eye. Advances in vertical-cavity surface-emitting laser (VCSEL) light sources, signal processing, optics and mechanical designs, enable a unique combination of high speed, long range, and deep penetration that addresses the challenges of anterior eye imaging. We demonstrate SS-OCT with a 325 kHz A-scan rate, 12.2 µm axial resolution (in air), and 15.5 mm depth range (in air) at 1310 nm wavelength. The ultrahigh 325 kHz A-scan rate not only facilitates biometry measurements by minimizing acquisition time and thus reducing motion, but also enables volumetric OCT for comprehensive structural analysis and OCT angiography (OCTA) for visualizing vasculature. The 15.5 mm (~ 11.6 mm in tissue) depth range spans all optical surfaces from the anterior cornea to the posterior lens capsule. The 1310 nm wavelength range enables structural OCT and OCTA deep in the sclera and through the iris. Achieving high speed and long range requires linearizing the VCSEL wavenumber sweep to efficiently utilize analog-to-digital conversion bandwidth. Dual channel recording of the OCT and calibration interferometer fringe signals, as well as sweep to sweep wavenumber compensation, is used to achieve invariant 12.2 µm (~ 9.1 µm in tissue) axial resolution and optimum point spread function throughout the depth range. Dynamic focusing using a tunable liquid lens extends the effective depth of field while preserving the lateral resolution. Improved optical and mechanical design, including parallax "split view" iris cameras and stable, ergonomic patient interface, facilitates accurate instrument positioning, reduces patient motion, and leads to improved imaging data yield and measurement accuracy. We present structural and angiographic OCT images of the anterior eye, demonstrating the unique imaging capabilities using representative scanning protocols which may be relevant to future research and clinical applications.

Local Geographic Atrophy Growth Rates Not Influenced by Close Proximity to Non-Exudative Type 1 Macular Neovascularization.

Purpose: The local growth rates of geographic atrophy (GA) adjacent to non-exudative type 1 macular neovascularization (MNV) were investigated to determine if MNV influenced GA growth. Methods: Eyes with GA and non-exudative type 1 MNV were followed for at least 1 year. Both GA and the MNV were imaged and measured using swept-source optical coherence tomography angiography (SS-OCTA) scans. Pearson correlations were computed between local growth rates of GA, which were estimated using a biophysical GA growth model, and local distances-to-MNV. Corresponding P values for the null hypothesis of no Pearson correlation were computed using a Monte Carlo approach that adjusts for spatial autocorrelations. Results: Nine eyes were included in this study. There were positive correlations (Pearson's r > 0) between distance-to-MNV and local GA growth in eight (89%) of the eyes; however, in all but one eye (11%), correlations were relatively weak and statistically nonsignificant after Bonferroni correction (corrected P > 0.05). Conclusions: SS-OCTA imaging combined with GA growth modeling and spatial statistical analysis enabled quantitative assessment of correlations between local GA growth rates and local distances-to-MNV. Our results are not consistent with non-exudative type 1 MNV having a strong inhibitory effect on local GA growth rates.