Recent investigations in humans have shown that rod and cone outer segments (ROS and COS, respectively) elongate in response to visual stimuli. Specifically, in phase-based optoretinographic (ORG) the relative phases of backscattered light from photoreceptors' inner segment / outer segments (IS/OS) junction and the COS tips (COST), or ROS tips (ROST), is measured, which allows observation of stimulus-evoked, nanometer-scale changes in the OS length. In this manuscript, we used a cellular resolution AO-OCT system employing an FF-SS-OCT acquisition engine that allowed up to kHz volume acquisition rates, which greatly reduced retinal motion artifacts. ORG responses were recorded in two healthy volunteers, with photopigment bleaching levels in the range of 1-60 %, and modeled using an exponential sum. The proposed harmonic oscillator-based response model allowed us to describe the shape of the cone's ORG responses by amplitudes of deflection and relaxation times. The development of simple quantitative parameters describing the ORG response should benefit future clinical applications and help to track the progress of blinding diseases.
In this presentation we will evaluate the signal processing steps required to obtain stimulus-evoked nm-scale changes in the photoreceptor outer segment (OS) length using full field swept source optical coherence tomography (FF-SS-OCT). Evaluation of stimulus-evoked OS length changes is the basis of photoreceptor optoretinography (ORG), an emerging test of retinal function. To allow cellular resolution, an adaptive optics (AO) enhanced FF-SS-OCT system was constructed. This system allows up to kHz volume acquisition rates, which greatly reduce retinal motion artifacts. Specifically, the effects of wave number calibration, dispersion compensation, and application of short-time Fourier transform (STFT) on image quality metrics, SNR of individual OCT volumes, and extracted phase noise will be presented.
Cellular-resolution imaging of the living human retina requires continuous correction of blur caused by the eye’s dynamic, living optics. Over the past twenty-five years, dozens of labs have employed adaptive optics (AO) to measure and correct this blur in conjunction with retinal imaging modalities such as fundus imaging, scanning light ophthalmoscopy, and optical coherence tomography. While the benefits of AO have become more apparent, the costs of developing AO systems has not fallen substantially. A significant fraction of the cost of an AO system is development of control and analysis software. This software is typically developed by individual investigators, and represents a significant duplication of effort and grant support. Here we present an open-source AO control application and illustrate its performance in conjunction with off-the-shelf optical components.
Assessment of the functional response of photoreceptors plays an important role in assessing and treating vision loss. Optoretinography (ORG) is an emerging non-invasive technique that measures the photoreceptors’ functional response to external light stimuli using optical coherence tomography (OCT) or other phase-sensitive imaging modalities. Recently a novel velocity-based ORG method was demonstrated, illustrating the feasibility of measuring photoreceptor function with clinical-grade OCT systems. Here we test this technique on diseaseaffected retinae of human subjects. The disease-affected retinae exhibited altered responses when compared to a healthy volunteer. The findings indicate promise for this novel tool to find applications in the clinic and clinical research.
While the a-wave of mouse electroretinogram (ERG) occurs within 50 milliseconds after exposure to light, the optoretinogram (ORG) slower than a 20Hz sampling rate could face limitations in observing immediate morphological changes from the retina. In this study, we develop a compact custom-built mouse ORG system based on spectral domain optical coherence tomography (SD-OCT) for 100Hz~1KHz B-scan rates comprised of 100 kHz A-scans. All the optics of the developed ORG system are designed on a 24 x 24 inches optical breadboard to move easily as well as to combine with the ERG system in a dark room. Without using a fundus camera, the OCT system provides en-face images from high-pass filtering and square of the OCT spectral signal for mouse retinal positioning in-vivo before acquiring ORG data. The 490nm LED for light stimulus is generated to make uniform illumination at the mouse retina using the Maxwellian view method. The common path of the OCT scanning light and the visible LED is built with achromatic doublet lens combinations based on optical simulation with Opticstudio® . The developed compact ORG system can not only observe light-evoked responses with 1~10 milliseconds but also be used for the studies of correlations between ORG and ERG in the mouse retina.
The development of functional retinal imaging is of great interest to clinical and experimental ophthalmology because it should improve current clinical practice that relies on a simple evaluation of disease-related changes in retinal morphology. In this presentation, we will review our recent progress in the measurements and interpretation of Optoretinograms (ORG). The ORG comprises light-dependent changes in photoreceptor outer segment length and scattering measured in vivo with submicrometer-resolution by Optical Coherence Tomography (OCT). Our recent data confirm that the mechanisms underlying “slow” ORG response is that extreme phototransduction increases osmotic pressure in the outer segment, driving water entry and swelling to restore osmotic balance. Water movement between different retinal layers was quantified by OCT measurements, and analysis of retinal layer thickness dynamics provided estimates of the water permeability of key barriers between the Choriocapillaris (CC) and photoreceptors, viz., of the CC cell membrane, Bruch’s Membrane (BrM), the RPE layer, and the rod plasma membrane. A model proposed to link the changes in refractive index caused by the osmotically driven water movement between the CC lumen and the outer segment during the ORG predicts the observed BrM scattering changes. Overall, the results and modeling provide new insights into critical water barriers of the posterior eye and an experimental and theoretical toolkit for measuring their permeabilities.
Light-evoked functional retinal imaging is of great interest in clinical and experimental ophthalmology because the alterations in retinal function hold the promise of being potentially more sensitive for disease diagnosis than purely morphology-based assays. Recent progress in Optoretinography (ORG), a technique employing OCT to extract light-evoked functional response of retinal tissue, promises to provide the needed sensitivity to probe early alterations in retinal function. The most promising implementations of the ORG probing of photoreceptor function in clinical settings are using parallel OCT detection schemes such as Line Field and Full Field designs. Herein, we present the instrumentation scheme of a Full-Field-Swept-Source OCT (FF-SS-OCT) system incorporating a high-precision light stimulation channel for facilitating phase-based functional retinal imaging in mice. The assessment of the OCT signal phase errors and their correction using short-time Fourier transform (STFT) is detailed. The performance of the system is investigated using a model eye.
The development of functional retinal imaging is of great interest to clinical and experimental ophthalmology, because it should provide more sensitive tools for ocular diseases diagnostic that would go beyond current gold standard of simple evaluation of the static retinal morphology. In this presentation we will review our recent progress in measurements and interpretation of OCT-based optoretinograms (ORG) i.e., the paradigm of using NIR OCT to measure in vivo bleaching-induced changes in retinal morphology (transient changes in volume of individual neurons, or thickness of retinal layers). Specifically, comparison between different instrumentations used to acquire ORGs and between results acquired using clinical (human) and experimental (animal) systems will be presented. Additionally, intensity-based and phase-based ORG extraction framework will be presented. Finally, we will discuss our findings in the context of current understanding of measured process, being a result osmotically driven water movements between the photoreceptors, and other retinal neurons and its surroundings.
KEYWORDS: Optical coherence tomography, Raster graphics, Speckle pattern, Visualization, Rods, In vivo imaging, Cones, Speckle, Signal analysis, Near infrared
Recent OCT based investigations in humans and in experimental animals have shown that rods and cones outer segments elongate in response to light stimuli. In this manuscript we describe our efforts to implement phase-based “optoretinograms” (ORG) analysis framework to retinal images acquired with standard raster scanning FD-OCT system, that offers much lower phase stability compared to full field or line field OCT acquisition schemes. Our initial results, acquired in anesthetized mice in vivo, showcase successful extraction of phase-based ORG signal and its favorable comparison with intensity-based ORG signal extracted from the same data sets.
Ocular blood flow measurement may have a number of potential applications that explore the relationship between blood flow in the eye and diseases such as: diabetic retinopathy, ocular artery obstruction, hypertensive retinopathy and Alzheimer's disease. Reliable and quantitative method for retinal blood flow estimation is still to be created. Doppler OCT is one of candidates for such a method, but suffers from a number of limitations. Recently we proposed a solution to one of the most prominent artefacts in Doppler OCT, which is the phase wrapping problem. This allows for precise recovery of velocity profile the Doppler OCT technique remains sensitive to temporal dependence of the result on the blood flow velocity changing with the pulse during the OCT measurement. In this report we explore this problem and show that the synchronization of the OCT measurement with heart beats only partially gives control over the acquired blood flows.
We present a modified Fast Phase Unwrapping (FPU) algorithm and its application for calculation of the axial flow velocity and volumetric flow rate in Doppler optical coherence tomography (DOCT). We outline the FPU method and show that it can be implemented in Fourier-domain optical coherence tomography using Fourier transformations (4FT). We present two-dimensional (2D) and three-dimensional (3D) realizations of the algorithm to reconstruct unwrapped phase in numerical simulations, as well as in data collected from phantom. We demonstrate that the phase unwrapping outcomes of the 2D and 3D 4FT FPU algorithms depend on the phase noise in the input data. For low phase noise data both algorithms generate reliable results. With increasing noise, the 2D algorithm starts generating phase unwrapping errors earlier than the 3D version. With the phase noise larger than a limiting value, none of the algorithms provides error-free results. We demonstrate that within their phase noise applicability limits, the phase unwrapping methods enable calculation of volumetric flow rates in the flow phantom even in the presence of phase wraps. We demonstrate that application of phase unwrapping methods enables extension of the measurable flow velocities beyond the phase range limitation of the Doppler OCT data.
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