Segmentation of beating embryonic heart structures from 4-D OCT images using deep learning.

Optical coherence tomography (OCT) has been used to investigate heart development because of its capability to image both structure and function of beating embryonic hearts. Cardiac structure segmentation is a prerequisite for the quantification of embryonic heart motion and function using OCT. Since manual segmentation is time-consuming and labor-intensive, an automatic method is needed to facilitate high-throughput studies. The purpose of this study is to develop an image-processing pipeline to facilitate the segmentation of beating embryonic heart structures from a 4-D OCT dataset. Sequential OCT images were obtained at multiple planes of a beating quail embryonic heart and reassembled to a 4-D dataset using image-based retrospective gating. Multiple image volumes at different time points were selected as key-volumes, and their cardiac structures including myocardium, cardiac jelly, and lumen, were manually labeled. Registration-based data augmentation was used to synthesize additional labeled image volumes by learning transformations between key-volumes and other unlabeled volumes. The synthesized labeled images were then used to train a fully convolutional network (U-Net) for heart structure segmentation. The proposed deep learning-based pipeline achieved high segmentation accuracy with only two labeled image volumes and reduced the time cost of segmenting one 4-D OCT dataset from a week to two hours. Using this method, one could carry out cohort studies that quantify complex cardiac motion and function in developing hearts.

Phase-Decorrelation Optical Coherence Tomography Measurement of Cold-Induced Nuclear Cataract.

Purpose: The purpose of this work is to determine the sensitivity of phase-decorrelation optical coherence tomography (OCT) to protein aggregation associated with cataracts in the ocular lens, as compared to OCT signal intensity. Methods: Six fresh porcine globes were held at 4°C until cold cataracts developed. As the globes were re-warmed to ambient temperature, reversing the cold cataract, each lens was imaged repeatedly using a conventional OCT system. Throughout each experiment, the internal temperature of the globe was recorded using a needle-mounted thermocouple. OCT scans were acquired, their temporal fluctuations were analyzed, and the rates of decorrelation were spatially mapped. Both decorrelation and intensity were evaluated as a function of recorded temperature. Results: Both signal decorrelation and intensity were found to change with lens temperature, a surrogate of protein aggregation. However, the relationship between signal intensity and temperature was not consistent across different samples. In contrast, the relationship between decorrelation and temperature was found to be consistent across samples. Conclusions: In this study, signal decorrelation was shown to be a more repeatable metric for quantification of crystallin protein aggregation in the ocular lens than OCT intensity-based metrics. Thus, OCT signal decorrelation measurements could enable more detailed and sensitive study of methods to prevent cataract formation. Translational Relevance: This dynamic light scattering-based approach to early cataract assessment can be implemented on existing clinical OCT systems without hardware additions, so it could quickly become part of a clinical study workflow or an indication for use for a pharmaceutical cataract intervention.

Biomechanics of Ophthalmic Crosslinking.

Crosslinking involves the formation of bonds between polymer chains, such as proteins. In biological tissues, these bonds tend to stiffen the tissue, making it more resistant to mechanical degradation and deformation. In ophthalmology, the crosslinking phenomenon is being increasingly harnessed and explored as a treatment strategy for treating corneal ectasias, keratitis, degenerative myopia, and glaucoma. This review surveys the multitude of exogenous crosslinking strategies reported in the literature, both "light" (involving light energy) and "dark" (involving non-photic chemical processes), and explores their mechanisms, cytotoxicity, and stage of translational development. The spectrum of ophthalmic applications described in the literature is then discussed, with particular attention to proposed therapeutic mechanisms in the cornea and sclera. The mechanical effects of crosslinking are then discussed in the context of their proposed site and scale of action. Biomechanical characterization of the crosslinking effect is needed to more thoroughly address knowledge gaps in this area, and a review of reported methods for biomechanical characterization is presented with an attempt to assess the sensitivity of each method to crosslinking-mediated changes using data from the experimental and clinical literature. Biomechanical measurement methods differ in spatial resolution, mechanical sensitivity, suitability for detecting crosslinking subtypes, and translational readiness and are central to the effort to understand the mechanistic link between crosslinking methods and clinical outcomes of candidate therapies. Data on differences in the biomechanical effect of different crosslinking protocols and their correspondence to clinical outcomes are reviewed, and strategies for leveraging measurement advances predicting clinical outcomes of crosslinking procedures are discussed. Advancing the understanding of ophthalmic crosslinking, its biomechanical underpinnings, and its applications supports the development of next-generation crosslinking procedures that optimize therapeutic effect while reducing complications.

Genetically Encoded Calcium Indicators for In Situ Functional Studies of Corneal Nerves.

Purpose: Millions of people suffer from diseases that involve corneal nerve dysfunction, caused by various conditions, including dry eye syndrome, neurotrophic keratopathy, diabetes, herpes simplex, glaucoma, and Alzheimer's disease. The morphology of corneal nerves has been studied extensively. However, corneal nerve function has only been studied in a limited fashion owing to a lack of tools. Here, we present a new system for studying corneal nerve function. Methods: Optical imaging was performed on the cornea of excised murine globes taken from a model animal expressing a genetically encoded calcium indicator, GCaMP6f, to record calcium transients. A custom perfusion and imaging chamber for ex vivo murine globes was designed to maintain and stabilize the cornea, while allowing the introduction of chemical stimulation during imaging. Results: Imaging of calcium signals in the ex vivo murine cornea was demonstrated. Strong calcium signals with minimal photobleaching were observed in experiments lasting up to 10 minutes. Concentrated potassium and lidocaine solutions both modulated corneal nerve activity. Similar responses were observed in the same neurons across multiple chemical stimulations, suggesting the feasibility of using chemical stimulations to test the response of the corneal nerves. Conclusions: Our studies suggest that this tool will be of great use for studying functional changes to corneal nerves in response to disease and ocular procedures. This process will enable preclinical testing of new ocular procedures to minimize damage to corneal innervation and therapies for diminished neural function.

Semi-automated shear stress measurements in developing embryonic hearts.

Blood-induced shear stress influences gene expression. Abnormal shear stress patterns on the endocardium of the early-stage heart tube can lead to congenital heart defects. To have a better understanding of these mechanisms, it is essential to include shear stress measurements in longitudinal cohort studies of cardiac development. Previously reported approaches are computationally expensive and nonpractical when assessing many animals. Here, we introduce a new approach to estimate shear stress that does not rely on recording 4D image sets and extensive post processing. Our method uses two adjacent optical coherence tomography frames (B-scans) where lumen geometry and flow direction are determined from the structural data and the velocity is measured from the Doppler OCT signal. We validated our shear stress estimate by flow phantom experiments and applied it to live quail embryo hearts where observed shear stress patterns were similar to previous studies.

A Review of Structural and Biomechanical Changes in the Cornea in Aging, Disease, and Photochemical Crosslinking.

The study of corneal biomechanics is motivated by the tight relationship between biomechanical properties and visual function within the ocular system. For instance, variation in collagen fibril alignment and non-enzymatic crosslinks rank high among structural factors which give rise to the cornea's particular shape and ability to properly focus light. Gradation in these and other factors engender biomechanical changes which can be quantified by a wide variety of techniques. This review summarizes what is known about both the changes in corneal structure and associated changes in corneal biomechanical properties in aging, keratoconic, and photochemically crosslinked corneas. In addition, methods for measuring corneal biomechanics are discussed and the topics are related to both clinical studies and biomechanical modeling simulations.

Noninvasive Assessment of Corneal Crosslinking With Phase-Decorrelation Optical Coherence Tomography.

Purpose: There is strong evidence that abnormalities in corneal biomechanical play a causal role in corneal ectasias, such as keratoconus. Additionally, corneal crosslinking (CXL) treatment, which halts progression of keratoconus, directly appeals to corneal biomechanics. However, existing methods of corneal biomechanical assessment have various drawbacks: dependence on IOP, long acquisition times, or limited resolution. Here, we present a method that may avoid these limitations by using optical coherence tomography (OCT) to detect the endogenous random motion within the cornea, which can be associated with stromal crosslinking. Methods: Phase-decorrelation OCT (PhD-OCT), based in the theory of dynamic light scattering, is a method to spatially resolve endogenous random motion by calculating the decorrelation rate, Γ, of the temporally evolving complex-valued OCT signal. PhD-OCT images of ex vivo porcine globes were recorded during CXL and control protocols. In addition, human patients were imaged with PhD-OCT using a clinical OCT system. Results: In both the porcine cornea and the human cornea, crosslinking results in a reduction of Γ (P < 0.0001), indicating more crosslinks. This effect was repeatable in ex vivo porcine corneas (change in average Γ = -41.55 ± 9.64%, n = 5) and not seen after sham treatments (change in average Γ = 2.83 ± 12.56%, n = 5). No dependence of PhD-OCT on IOP was found, and correctable effects were caused by variations in signal-to-noise ratio, hydration, and motion. Conclusions: PhD-OCT may be a useful and readily translatable tool for investigating biomechanical properties of the cornea and for enhancing the diagnosis and treatment of patients.

Complex decorrelation averaging in optical coherence tomography: a way to reduce the effect of multiple scattering and improve image contrast in a dynamic scattering medium.

We demonstrate that complex decorrelation averaging can reduce the effect of multiple scattering and improve optical coherence tomography (OCT) imaging contrast. Complex decorrelation averaging calculates the product of an A-scan and the complex conjugate of a subsequent A-scan. The resultant signal is the product of the amplitudes and the phase difference. All these resulting complex signals at a particular location are then averaged. We take advantage of the fact that complex averaging, in contrast to conventional magnitude averaging, is sensitive to phase decorrelation. Sample motion that increases signal phase variance results in lower signal magnitude after complex averaging. Such motion preferentially results in a faster decorrelation of the multiple scattering signal when compared to the single scattering signal with each scattering event spreading the phase. This indicates that we may reduce multiple scattering by implementing complex decorrelation averaging to preferentially reduce the magnitude of the multiply scattered light signal in OCT images. By adjusting the time between phase-differenced A-scans, one can regulate the amount of measured decorrelation. We have performed experiments on liquid phantoms that give experimental evidence for this hypothesis. A substantial improvement in OCT image contrast using complex decorrelation averaging is demonstrated.