Chirped flicker optoretinography for in vivo characterization of human photoreceptors' frequency response to light.

Flicker electroretinography (ERG) has served as a valuable noninvasive objective tool for investigating retinal physiological function through the measurement of electrical signals originating from retinal neurons in response to temporally modulated light stimulation. Deficits in the response at certain frequencies can be used as effective biomarkers of cone-pathway dysfunction. In this Letter, we present the progress we made on its optical counterpart-photopic flicker optoretinography (f-ORG). Specifically, we focus on the measurement of the response of light-adapted retinal photoreceptors to a flicker stimulus with chirped frequency modulation. In contrast to measurements performed at discrete frequencies, this technique enables a significantly accelerated characterization of photoreceptor outer segment optical path length modulation amplitudes in the nanometer range as a function of stimulus frequency, enabling the acquisition of the characteristic frequency response in less than 2 sec.

Poster Session: Optoretinography-based frequency characterization of the retinal response to a chirped flickering light.

In this contribution, we present experimental results of in vivo characterization of the photoreceptor's response to a chirped flickering white light stimulating the retina. We acquire the ORG signal with Spatio-Temporal Optical Coherence Tomography (STOC-T) setup, which combines both temporal and coherence gating to overcome limitations present in Full Field Fourier Domain Optical Coherence Tomography. From the acquired volumes, we extract the changes in optical path length (OPL) between the inner and outer photoreceptor junction (ISOS) and the cone outer segment tips (COST). We perform the measurements for frequencies ranging from 5 Hz to 50 Hz. The chirped flickering facilitates significantly shorter data acquisition time. We present results of in vivo measurement from three volunteers. Our results show that we can measure OPL changes between ISOS and COST occurring in response to a chirped flickering stimulation in a reproducible manner and resolve the amplitude of the response in the function of flicker frequency.

In vivo imaging of the human retina using a two-photon excited fluorescence ophthalmoscope.

Noninvasive imaging of endogenous retinal fluorophores, including vitamin A derivatives, is vital to developing new treatments for retinal diseases. Here, we present a protocol for obtaining in vivo two-photon excited fluorescence images of the fundus in the human eye. We describe steps for laser characterization, system alignment, positioning human subjects, and data registration. We detail data processing and demonstrate analysis with example datasets. This technique allays safety concerns by allowing for the acquisition of informative images at low laser exposure. For complete details on the use and execution of this protocol, please refer to Boguslawski et al. (2022).1.

Spatio-temporal optical coherence tomography provides full thickness imaging of the chorioretinal complex.

Despite the rapid development of optical imaging methods, high-resolution in vivo imaging with penetration into deeper tissue layers is still a major challenge. Optical coherence tomography (OCT) has been used successfully for non-invasive human retinal volumetric imaging in vivo, advancing the detection, diagnosis, and monitoring of various retinal diseases. However, there are important limitations of volumetric OCT imaging, especially coherent noise and the limited axial range over which high resolution images can be acquired. The limited range prevents simultaneous measurement of the retina and choroid with adequate lateral resolution. In this article, we address these limitations with a technique that we term spatio-temporal optical coherence tomography (STOC-T), which uses light with controlled spatial and temporal coherence and advanced signal processing methods. STOC-T enabled the acquisition of high-contrast and high-resolution coronal projection images of the retina and choroid at arbitrary depths.

Light-adapted flicker optoretinograms captured with a spatio-temporal optical coherence-tomography (STOC-T) system.

For many years electroretinography (ERG) has been used for obtaining information about the retinal physiological function. More recently, a new technique called optoretinography (ORG) has been developed. In one form of this technique, the physiological response of retinal photoreceptors to visible light, resulting in a nanometric photoreceptor optical path length change, is measured by phase-sensitive optical coherence tomography (OCT). To date, a limited number of studies with phase-based ORG measured the retinal response to a flickering light stimulation. In this work, we use a spatio-temporal optical coherence tomography (STOC-T) system to capture optoretinograms with a flickering stimulus over a 1.7 × 0.85 mm2 area of a light-adapted retina located between the fovea and the optic nerve. We show that we can detect statistically-significant differences in the photoreceptor optical path length (OPL) modulation amplitudes in response to different flicker frequencies and with better signal to noise ratios (SNRs) than for a dark-adapted eye. We also demonstrate the ability to spatially map such response to a patterned stimulus with light stripes flickering at different frequencies, highlighting the prospect of characterizing the spatially-resolved temporal-frequency response of the retina with ORG.

In vivo imaging of the human eye using a 2-photon-excited fluorescence scanning laser ophthalmoscope.

BackgroundNoninvasive assessment of metabolic processes that sustain regeneration of human retinal visual pigments (visual cycle) is essential to improve ophthalmic diagnostics and to accelerate development of new treatments to counter retinal diseases. Fluorescent vitamin A derivatives, which are the chemical intermediates of these processes, are highly sensitive to UV light; thus, safe analyses of these processes in humans are currently beyond the reach of even the most modern ocular imaging modalities.MethodsWe present a compact, 2-photon-excited fluorescence scanning laser ophthalmoscope and spectrally resolved images of the human retina based on 2-photon excitation (TPE) with near-infrared light. A custom Er:fiber laser with integrated pulse selection, along with intelligent postprocessing of data, enables excitation with low laser power and precise measurement of weak signals.ResultsWe demonstrate spectrally resolved TPE fundus images of human subjects. Comparison of TPE data between human and mouse models of retinal diseases revealed similarity with mouse models that rapidly accumulate bisretinoid condensation products. Thus, visual cycle intermediates and toxic byproducts of this metabolic pathway can be measured and quantified by TPE imaging.ConclusionOur work establishes a TPE instrument and measurement method for noninvasive metabolic assessment of the human retina. This approach opens the possibility for monitoring eye diseases in the earliest stages before structural damage to the retina occurs.FundingNIH, Research to Prevent Blindness, Foundation for Polish Science, European Regional Development Fund, Polish National Agency for Academic Exchange, and Polish Ministry of Science and Higher Education.

In vivo imaging of the human cornea with high-speed and high-resolution Fourier-domain full-field optical coherence tomography.

Corneal evaluation in ophthalmology necessitates cellular-resolution and fast imaging techniques that allow for accurate diagnoses. Currently, the fastest volumetric imaging technique is Fourier-domain full-field optical coherence tomography (FD-FF-OCT), which uses a fast camera and a rapidly tunable laser source. Here, we demonstrate high-resolution, high-speed, non-contact corneal volumetric imaging in vivo with FD-FF-OCT that can acquire a single 3D volume with a voxel rate of 7.8 GHz. The spatial coherence of the laser source was suppressed to prevent it from focusing on a spot on the retina, and therefore, exceeding the maximum permissible exposure (MPE). The inherently volumetric nature of FD-FF-OCT data enabled flattening of curved corneal layers. The acquired FD-FF-OCT images revealed corneal cellular structures, such as epithelium, stroma and endothelium, as well as subbasal and mid-stromal nerves.

Source spectrum shaping method for low-coherence interferometry.

Low-coherence interferometry (LCI) suffers mainly from low signal-to-noise (S/N) ratio. By using the source spectrum shaping method (SSSM), the authors can enhance the visibility of the zero-order fringe (V) and S/N ratio in LCI. The presented approach was analyzed numerically for a set of theoretical Gaussian light sources, with different central wavelengths and different spectrum widths. The results have shown a significant improvement of the visibility V. Additionally, a set of commercially available light emitting diodes was analyzed to find the best possible setup. Results of numerical calculations were verified experimentally in an SSSM low coherence Twyman-Green interferometric setup equipped with three light sources.

High-precision topography measurement through accurate in-focus plane detection with hybrid digital holographic microscope and white light interferometer module.

High-precision topography measurement of micro-objects using interferometric and holographic techniques can be realized provided that the in-focus plane of an imaging system is very accurately determined. Therefore, in this paper we propose an accurate technique for in-focus plane determination, which is based on coherent and incoherent light. The proposed method consists of two major steps. First, a calibration of the imaging system with an amplitude object is performed with a common autofocusing method using coherent illumination, which allows for accurate localization of the in-focus plane position. In the second step, the position of the detected in-focus plane with respect to the imaging system is measured with white light interferometry. The obtained distance is used to accurately adjust a sample with the precision required for the measurement. The experimental validation of the proposed method is given for measurement of high-numerical-aperture microlenses with subwavelength accuracy.