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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 2â sec.
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Electrorretinografía , Humanos , Electrorretinografía/métodos , Luz , Estimulación Luminosa , Células Fotorreceptoras de Vertebrados/fisiologíaRESUMEN
The guest editors introduce a feature issue commemorating the 30th anniversary of Optical Coherence Tomography.
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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.
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Luz , Retina , Humanos , Retina/diagnóstico por imagen , Tomografía de Coherencia ÓpticaRESUMEN
This report presents the results of measurements of the two-photon vision threshold for various pulse trains. We employed three pulsed near-infrared lasers and pulse stretchers to obtain variations of the pulse duty cycle parameter over three orders of magnitude. We proposed and extensively described a mathematical model that combines the laser parameters with the visual threshold value. The presented methodology enables one to predict the visual threshold value for a two-photon stimulus for a healthy subject while using a laser source of known parameters. Our findings would be of value to laser engineers and the community interested in nonlinear visual perception.
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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.
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The eye is an ideal organ for imaging by a multi-photon excitation approach, because ocular tissues such as the sclera, cornea, lens and neurosensory retina, are highly transparent to infrared (IR) light. The interface between the retina and the retinal pigment epithelium (RPE) is especially informative, because it reflects the health of the visual (retinoid) cycle and its changes in response to external stress, genetic manipulations, and drug treatments. Vitamin A-derived retinoids, like retinyl esters, are natural fluorophores that respond to multi-photon excitation with near IR light, bypassing the filter-like properties of the cornea, lens, and macular pigments. Also, during natural aging some retinoids form bisretinoids, like diretinoid-pyridiniumethanolamine (A2E), that are highly fluorescent. These bisretinoids appear to be elevated concurrently with aging. Vitamin A-derived retinoids and bisretinoidss are detected by two-photon ophthalmoscopy (2PO), using a new class of light sources with adjustable spatial, temporal, and spectral properties. Furthermore, the two-photon (2P) absorption of IR light by the visual pigments in rod and cone photoreceptors can initiate visual transduction by cis-trans isomerization of retinal, enabling parallel functional studies. Recently we overcame concerns about safety, data interpretation and complexity of the 2P-based instrumentation, the major roadblocks toward advancing this modality to the clinic. These imaging and retina-function assessment advancements have enabled us to conduct the first 2P studies with humans.
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Visión Ocular , Vitamina A , Humanos , Ratones , Animales , Vitamina A/análisis , Retina , Retinoides , Epitelio Pigmentado de la RetinaRESUMEN
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.
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The mechanism underlying the association between elevated red cell distribution width (RDW) and poor prognosis in variety of diseases is unknown although many researchers consider RDW a marker of inflammation. We hypothesized that RDW directly affects intravascular hemodynamics, interactions between circulating cells and vessel wall, inducing local changes predisposing to atherothrombosis. We applied different human and animal models to verify our hypothesis. Carotid plaques harvested from patients with high RDW had increased expression of genes and proteins associated with accelerated atherosclerosis as compared to subjects with low RDW. In microfluidic channels samples of blood from high RDW subjects showed flow pattern facilitating direct interaction with vessel wall. Flow pattern was also dependent on RDW value in mouse carotid arteries analyzed with Magnetic Resonance Imaging. In different mouse models of elevated RDW accelerated development of atherosclerotic lesions in aortas was observed. Therefore, comprehensive biological, fluid physics and optics studies showed that variation of red blood cells size measured by RDW results in increased interactions between vascular wall and circulating morphotic elements which contribute to vascular pathology.
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Aterosclerosis , Índices de Eritrocitos , Animales , Aterosclerosis/patología , Células Sanguíneas , Arterias Carótidas/patología , Eritrocitos/patología , Humanos , Ratones , Pronóstico , Factores de RiesgoRESUMEN
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.
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We report the development of a widely-tunable femtosecond fiber laser system and its application for two-photon vision studies. The source is based on an Er-doped fiber laser with spectral shift up to 2150 nm, followed by a second harmonic generation module to generate a frequency-doubled beam tunable from 872 to 1075 nm. The source delivers sub-230 fs pulses with nearly-constant duration over the entire tuning range, with output powers between 0.68-1.24 mW, which corresponds to a pulse energy of 13.2-24.1 pJ. Such pulse energy is sufficient for employing a system for measurements of two-photon scotopic spectral sensitivity of two-photon vision in humans. The laser parameters allow for very efficient and safe two-photon stimulation of the human visual system, as proved by a good separation between one- and two-photon thresholds for wavelengths below 950 nm, which we have confirmed for 3 healthy subjects.
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We demonstrate in vivo imaging of the ischemic area in the mouse brain after photostroke using a custom prototype Gaussianbeam optical coherence tomography (OCT) setup in which the near infrared imaging beam and the green photoinducing light pass through the same objective lens. The goal of our research was analysis of vascularity of the ischemic area during 2week progress of stroke and correlating the hypo and hyperreflective OCT scattering areas with the location of activated microglia and astroglia. Angiogenesis, which was assessed using angiomaps, showed that the area of vessels in the ischemic center increased until day 7. OCT imaging revealed a heterogeneous scattering signal pattern in the ischemic area. On structural OCT images, we found presence of a core area of ischemia with a hyporeflective OCT signal and a halo of hyperreflective signal around the core. The core signal decreased in size by 70% by day 14. Immunocytochemistry revealed that the hyporeflective area in the ischemic core was associated with microglia/macrophage activation, whereas the hyperreflective signal from the halo came from activated astrocytes.
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Accidente Cerebrovascular , Tomografía de Coherencia Óptica , Animales , Encéfalo/diagnóstico por imagen , Isquemia , Ratones , Accidente Cerebrovascular/diagnóstico por imagen , Tomografía de Coherencia Óptica/métodosRESUMEN
Fourier-domain full-field optical coherence tomography (FD-FF-OCT) is an emerging tool for high-speed eye imaging. However, cross-talk formation in images limits the imaging depth. To this end, we have recently shown that reducing spatial coherence with a fast deformable membrane can suppress the noise but over a limited axial range and with substantial data processing. Here, we demonstrate that a multimode fiber with carefully chosen parameters enables cross-talk-free imaging over a long axial range and without significant artifacts. We also show that it can be used to image the human retina and choroid in vivo with exceptional contrast.
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Retina , Tomografía de Coherencia Óptica , Artefactos , HumanosRESUMEN
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.
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Oftalmoscopios , Imagen Óptica , Retina , Enfermedades de la Retina , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Retina/diagnóstico por imagen , Retina/metabolismo , Enfermedades de la Retina/diagnóstico por imagen , Enfermedades de la Retina/genética , Enfermedades de la Retina/metabolismoRESUMEN
Fourier-domain full-field optical coherence tomography (FD-FF-OCT) has recently emerged as a fast alternative to point-scanning confocal OCT in eye imaging. However, when imaging the cornea with FD-FF-OCT, a spatially coherent laser can focus down on the retina to a spot that exceeds the maximum permissible exposure level. Here we demonstrate that a long multimode fiber with a small core can be used to reduce the spatial coherence of the laser and, thus, enable ultrafast in vivo volumetric imaging of the human cornea without causing risk to the retina.
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Córnea/diagnóstico por imagen , Análisis de Fourier , Tomografía de Coherencia Óptica , Humanos , Procesamiento de Imagen Asistido por ComputadorRESUMEN
Two-photon vision is a phenomenon associated with the perception of short pulses of near-infrared radiation (900-1200 nm) as a visible light. It is caused by the nonlinear process of two-photon absorption by visual pigments. Here we present results showing the influence of pulse duration and repetition rate of short pulsed lasers on the visual threshold. We compared two-photon sensitivity maps of the retina obtained for subjects with normal vision using a cost-effective fiber laser (λc = 1028.4 nm, τp = 12.2 ps, Frep = 19.17 MHz) and a solid-state laser (λc = 1043.3 nm, τp = 0.253 ps, Frep = 62.65 MHz). We have shown that in accordance with the description of two-photon absorption, the average optical power required for two-photon vision for a fiber laser is 4 times greater than that for a solid-state laser. Mean sensitivity measured for the first one is 5.9 ± 2.8 dB lower than for the second but still 17 dB away from the safety limit, confirming that picosecond light sources can be successfully applied in microperimetry. This development would dramatically reduce the cost and complexity of future clinical devices.
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Droplet microfluidics disrupted analytical biology with the introduction of digital polymerase chain reaction and single-cell sequencing. The same technology may also bring important innovation in the analysis of bacteria, including antibiotic susceptibility testing at the single-cell level. Still, despite promising demonstrations, the lack of a high-throughput label-free method of detecting bacteria in nanoliter droplets prohibits analysis of the most interesting strains and widespread use of droplet technologies in analytical microbiology. We use a sensitive and fast measurement of scattered light from nanoliter droplets to demonstrate reliable detection of the proliferation of encapsulated bacteria. We verify the sensitivity of the method by simultaneous readout of fluorescent signals from bacteria expressing fluorescent proteins and demonstrate label-free readout on unlabeled Gram-negative and Gram-positive species. Our approach requires neither genetic modification of the cells nor the addition of chemical markers of metabolism. It is compatible with a wide range of bacterial species of clinical, research, and industrial interest, opening the microfluidic droplet technologies for adaptation in these fields.
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Bacterias Gramnegativas/aislamiento & purificación , Bacterias Grampositivas/aislamiento & purificación , Ensayos Analíticos de Alto Rendimiento , Técnicas Analíticas Microfluídicas , Nanopartículas/química , Análisis de la Célula Individual , Bacterias Gramnegativas/citología , Bacterias Grampositivas/citología , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Corneal biomechanics play a fundamental role in the genesis and progression of corneal pathologies, such as keratoconus; in corneal remodeling after corneal surgery; and in affecting the measurement accuracy of glaucoma biomarkers, such as the intraocular pressure (IOP). Air-puff induced corneal deformation imaging reveals information highlighting normal and pathological corneal response to a non-contact mechanical excitation. However, current commercial systems are limited to monitoring corneal deformation only on one corneal meridian. Here, we present a novel custom-developed swept-source optical coherence tomography (SSOCT) system, coupled with a collinear air-puff excitation, capable of acquiring dynamic corneal deformation on multiple meridians. Backed by numerical simulations of corneal deformations, we propose two different scan patterns, aided by low coil impedance galvanometric scan mirrors that permit an appropriate compromise between temporal and spatial sampling of the corneal deformation profiles. We customized the air-puff module to provide an unobstructed SSOCT field of view and different peak pressures, air-puff durations, and distances to the eye. We acquired multi-meridian corneal deformation profiles (a) in healthy human eyes in vivo, (b) in porcine eyes ex vivo under varying controlled IOP, and (c) in a keratoconus-mimicking porcine eye ex vivo. We detected deformation asymmetries, as predicted by numerical simulations, otherwise missed on a single meridian that will substantially aid in corneal biomechanics diagnostics and pathology screening.
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We present in-vivo imaging of the mouse brain using custom made Gaussian beam optical coherence microscopy (OCM) with 800nm wavelength. We applied new instrumentation to longitudinal imaging of the glioblastoma (GBM) tumor microvasculature in the mouse brain. We have introduced new morphometric biomarkers that enable quantitative analysis of the development of GBM. We confirmed quantitatively an intensive angiogenesis in the tumor area between 3 and 14 days after GBM cells injection confirmed by considerably increased of morphometric parameters. Moreover, the OCM setup revealed heterogeneity and abnormality of newly formed vessels.
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A femtosecond frequency-doubled erbium-doped fiber laser with an adjustable pulse repetition rate is developed and applied in two-photon excited fluorescence microscopy. The all-fiber laser system provides the fundamental pulse at 1560â nm wavelength with 22 fs duration for the second harmonic generation, resulting in 1.35 nJ, 60 fs pulses at 780â nm. The repetition rate is adjusted by a pulse picker unit built-in within the amplifier chain, directly providing transform-limited pulses for any chosen repetition rate between 1 and 12â MHz. We employed the laser source to drive a scanning two-photon excited fluorescence microscope for ex vivo rat skin and other samples' imaging at various pulse repetition rates. Due to compactness, ease of operation, and suitable pulse characteristics, the laser source can be considered as an attractive alternative for Ti:Sapphire laser in biomedical imaging.
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Purpose: To introduce a new approach for keratoconus detection based on corneal microstructure observed in vivo derived from a single Scheimpflug image. Methods: Scheimpflug single-image snapshots from 25 control subjects and 25 keratoconus eyes were analyzed; from each group, five subjects were randomly selected to provide out-of-sample data. Each corneal image was segmented, after which the stromal pixel intensities were statistically modeled with a Weibull distribution. Distribution estimated parameters α and ß, characterizing corneal microstructure, were used in combination with a macrostructure parameter, central corneal thickness (CCT), for the detection of keratoconus. In addition, receiver operating characteristic curves were used to determine the sensitivity and specificity of each parameter for keratoconus detection. Results: The combination of CCT (sensitivity = 88%; specificity = 84%) with microscopic parameters extracted from statistical modeling of light intensity distribution, α (sensitivity = 76%; specificity = 76%) and ß (sensitivity = 96%; specificity = 88%), from a single Scheimpflug image was found to be a successful tool to differentiate between keratoconus and control eyes with no misclassifications (sensitivity = 100%; specificity = 100%) with coefficients of variation up to 2.5%. Conclusions: The combination of microscopic and macroscopic corneal parameters extracted from a static Scheimpflug image is a promising, non-invasive tool to differentiate corneal diseases without the need to perform measurements based on induced deformation of the corneal structure. Translational Relevance: The proposed methodology has the potential to support clinicians in the detection of keratoconus, without compromising patient comfort.