AIM:Over the past several decades, optical sectioning technologies have emerged as valuable tools for evaluating tissue histology. Unlike conventional tissue sectioning, these technologies allow for real-time intraoperative assessments and more efficient tissue triage. In the era of digital pathology, the demand for high-quality, high-throughput optical sectioning platforms is increasing, as they eliminate the need for traditional slide preparation and scanning, potentially transforming anatomical pathology workflows. While non-linear microscopy (NLM) has demonstrated promise in histological evaluation across various tissue types, its application in gastrointestinal tissue assessment remains unexplored. METHODS:This study extends the use of NLM to gastrointestinal histology and develops an image atlas to highlight its potential as an automated digital pathology platform. RESULTS:Our results indicate that NLM generates diagnostic-quality images comparable to traditional H&E slides. Moreover, NLM provides valuable three-dimensional (3D) spatial information, improving clinical evaluations of key histological features such as depth of invasion, lymphovascular and perineural invasion, tumour budding and margin assessment. Time-lapse videos further demonstrate NLM's capability to capture 3D histological structures up to a depth of approximately 100 µm. CONCLUSION:Our findings demonstrate that NLM can serve as an optical sectioning platform for gastrointestinal histology, providing both diagnostic-quality imaging and advanced 3D visualisation. The introduction of an NLM-based atlas has the potential to redefine anatomical pathology workflows and advance digital pathology image analysis.

Purpose:To demonstrate high-resolution, motion-corrected, volume-fused optical coherence tomography (OCT) for assessing longitudinal changes in macular dot form subretinal drusenoid deposits (SDDs). Methods:Six consecutive isotropic volume raster scans over 6 × 6 mm (500 × 500 A-scans) were acquired using a high-resolution (2.7 µm axial resolution) spectral domain OCT prototype instrument. OCT volumes were computationally motion-corrected and fused. The distribution and longitudinal changes in dot SDDs were evaluated using en face OCT in a 50-µm-thick slab, from 27 µm above Bruch's membrane. Results:Computational motion correction and volume fusion methods improve visibility of small en face features and compensate for motion artifacts to facilitate longitudinal assessment. In total, 326 SDDs were identified in a representative series of four eyes from four patients with intermediate age-related macular degeneration (AMD) and assessed with a 3- to 12-month follow-up. Of the SDDs, 85.3% remained stable over the follow-up, while 9.8% regressed, 3.4% fused, and 1.5% new SDDs appeared. Conclusions:Computational motion correction and volume fusion combined with high-resolution OCT B-scans and en face slabs facilitate visualization and longitudinal tracking of focal pathologies, such as SDDs. Translational Relevance:The methods presented have the potential to improve OCT analysis of focal features, such as quantification of SDDs and other AMD biomarkers.

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

Abstract Purpose To assess the repeatability of a microperimetry methodology for quantifying visual function changes in the junctional zone of eyes with geographic atrophy (GA) in the clinical trial context. Methods A post hoc analysis of the OAKS phase III trial was conducted, which enrolled patients with GA secondary to age-related macular degeneration. Microperimetry using a standard 10 − 2 fovea centered grid was performed at baseline and follow-up visits. GA regions were traced on fundus autofluorescence (FAF) images. Two graders independently registered baseline microperimetry images with baseline FAF images in a sampling of 30 eyes from the OAKS study. Agreement between the two graders’ assessments of mean sensitivity and the number of scotomatous points within a ± 250 𝜇m GA junctional zone was assessed. Results The intraclass correlation (ICC) and coefficient of repeatability (CoR) for the mean junctional zone sensitivity were 0.987 and 0.214 dB, respectively. The ICC and CoR for the total number of scotomatous points within the junctional zone were 0.991 and 1.42, respectively. Conclusions The repeatability of the methodology and its compatibility with standard MP acquisitions appear to make it well-suited for identifying and analyzing retinal sensitivity within high-risk areas of the retina. Summary We present a microperimetry-based methodology for assessing visual function changes in the junctional zone of geographic atrophy lesions using a standard 10 − 2 fovea centered grid in a clinical trial context. The approach’s repeatability and compatibility with standard microperimetry grids may make it useful for assessing the effects of GA therapeutics.

A greater understanding of the pathophysiology of AMD has yielded potential therapeutic strategies for slowing vision loss. Structural imaging biomarkers and novel functional outcomes, both under intensive study as surrogate endpoints in AMD clinical trials, are new granular tools needed to assess disease progression and follow response to therapy. These advances have created, for the first time, the possibility to intervene early in AMD, and to measure success in a time frame feasible for clinical trials. At the 2024 Ryan Initiative for Macular Research (RIMR) meeting, a consensus was reached for design of a hypothetical, pragmatic, proof of concept, 24-month, early intervention study to determine the efficacy of an intervention in subjects with intermediate AMD (iAMD). Although further research is needed, using natural history cohorts and intervention trials to validate the proposed trial design and endpoints, this paper presents an AMD expert consensus framework for early AMD clinical trials, that we hope will also be useful for therapeutic developers and regulators. Viable clinical trial strategies are needed to develop treatments that slow the progression of AMD prior to the development of vision-threatening late AMD; this consensus document should aid in reaching this goal.

Visible wavelength optical coherence tomography (vis-OCT) enables high-resolution retinal oximetry in the 500-600nm wavelength range, through spectroscopic measurements of total hemoglobin bound to oxygen (HbO2) relative to total hemoglobin. Current research in vis-OCT uses spectral domain OCT (SD-OCT) with broadband supercontinuum sources. Although these systems afford high axial resolution, they have significant limitations in terms of sensitivity, imaging range, axial scan rate, and commercial scalability. Swept source OCT (SS-OCT) systems using near infrared (NIR) micro-electromechanical systems tunable vertical cavity surface emitting lasers (MEMS-VCSELs) have achieved MHz A-scan rates and meter scale imaging ranges. No MEMS-VCSELs or broadband swept wavelength sources, however, have been demonstrated in 500-600nm range. In this paper, we demonstrate a visible swept source using broadband frequency doubling of an amplified 1050nm MEMS-VCSEL in a thin-film periodically poled lithium niobate (PPLN) waveguide. We show a usable tuning range of 518-544nm, SS-OCT axial resolution in air/tissue of 6.95/5.25 um, >10mm image range, and >5mW peak power over the tuning range. Our PPLN waveguide uses a linearly chirped poling period to broaden the doubling bandwidth, creating strong spectral modulation on the frequency doubled light. Since the spectral modulation is repeatable, and since green wavelength fringes remain phase-continuous across spectral dips, the spectral modulation can be numerically compensated. Some artifacts remain, however, highlighting the need to flatten the spectrum. Additionally, we demonstrate the first MEMS-VCSELs covering 1049-1184nm, corresponding to a more optimal doubled wavelength range of 524.5-592nm.

Purpose:To evaluate visibility of a sub-band posterior to the external limiting membrane (ELM) and assess its age-associated variation. Methods:In a retrospective cross-sectional study, normal eyes were imaged using a high-resolution spectral-domain optical coherence tomography (SD-OCT) prototype (2.7-µm axial resolution). Volume fusion of six sequential scans (each 500 × 500 A-scans over 6 mm × 6 mm) was performed in the motion correction and volume reconstruction in OCT (MoReOCT) framework to enhance feature visibility in OCT. The subjects were divided into three groups: young (21-40 years old), middle (41-60 years old), and older (>60 years old). Three expert graders assessed the visibility of the sub-band on B-scans, and its A-scan intensity relative to ELM intensity (peak intensity ratio) was measured. Results:Forty-four eyes of 44 subjects were imaged. The sub-band, tentatively attributed to the photoreceptor myoid, can be visualized under high-resolution OCT. The B-scan gradings showed that sub-band visibility increased with age (visible in 16.7%, 47.2%, and 66.7% of the young, middle, and older age groups, respectively). The gradings were statistically different among age groups at 1 mm and 2 mm nasal and 1 mm and 2 mm temporal (P < 0.04) from the foveal center. Similarly, the mean peak intensity ratios of the sub-band to the ELM were 71.6%, 77.5%, and 85.2% in the young, middle, and older age groups, respectively, and were positively correlated with age at 1 mm temporal (P = 0.012) and 2 mm temporal (P < 0.001). Conclusions:High-resolution OCT, combined with advanced volume fusion, enables visualization of the photoreceptor myoid and investigation of its age-associated variations. Translational Relevance:Investigating the sub-band can advance our understanding of photoreceptors and their association with aging and disease pathogenesis.

Purpose: To quantify the blood flow speed within retinal microaneurysms (MAs) and investigate the relationship between blood flow speed and clinical characteristics in eyes with diabetic retinopathy (DR). Methods: Variable interscan time analysis (VISTA) quantifies blood flow speed in the vasculature by measuring how fast optical coherence tomography (OCT) angiography (OCTA) saturates for different interscan times. Macular OCTA imaging was performed in eyes with DR using a high-speed swept-source OCT prototype instrument operating at a 600-kHz A-scan rate. The presence of MAs was determined using OCT B-scans, and three-dimensional MA masks were generated. VISTA flow speed (VFS) was determined within MAs and the retinal capillary plexus (RCP). Intraluminal reflectivity, axial location within the RCP, and the presence of intraretinal fluid (IRF) around the MAs were evaluated. Results: A total of 123 MAs were detected from 24 eyes of 20 patients with DR. Mean VFS was 1.26 ms(-1) (95% confidence interval, 1.16-1.35). MAs with medium and high intraluminal reflectivity had slower VFS than those with low intraluminal reflectivity (P < 0.01) and often had slower VFS than the RCP (P < 0.01). Sixty-six MAs were located near IRF and had slower VFS than the other 57 MAs without surrounding IRF (1.16 ms(-1) vs. 1.37 ms(-1); P = 0.03). Conclusions: VISTA OCTA can assess blood flow speed of MAs in relation to other structural features in DR. Decreased blood flow speed in MAs is correlated with the presence of IRF around MAs.

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.

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.

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.