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Human cone outer segment (COS) length changes in response to stimuli bleaching up to 99% of L- and M-cone opsins were measured with high resolution, phase-resolved optical coherence tomography (OCT). Responses comprised a fast phase (â¼5 ms), during which COSs shrink, and two slower phases (1.5 s), during which COSs elongate. The slower components saturated in amplitude (â¼425 nm) and initial rate (â¼3 nm ms-1) and are well described over the 200-fold bleaching range as the sum of two exponentially rising functions with time constants of 80 to 90 ms (component 1) and 1,000 to 1,250 ms (component 2). Measurements with adaptive optics reflection densitometry revealed component 2 to be linearly related to cone pigment bleaching, and the hypothesis is proposed that it arises from cone opsin and disk membrane swelling triggered by isomerization and rate-limited by chromophore hydrolysis and its reduction to membrane-localized all-trans retinol. The light sensitivity and kinetics of component 1 suggested that the underlying mechanism is an osmotic response to an amplified soluble by-product of phototransduction. The hypotheses that component 1 corresponds to G-protein subunits dissociating from the membrane, metabolites of cyclic guanosine monophosphate (cGMP) hydrolysis, or by-products of activated guanylate cyclase are rejected, while the hypothesis that it corresponds to phosphate produced by regulator of G-protein signaling 9 (RGS9)-catalyzed hydrolysis of guanosine triphosphate (GTP) in G protein-phosphodiesterase complexes was found to be consistent with the results. These results provide a basis for the assessment with optoretinography of phototransduction in individual cone photoreceptors in health and during disease progression and therapeutic interventions.
Assuntos
Opsinas dos Cones , GTP Fosfo-Hidrolases , Fosfatos , Proteínas RGS , Células Fotorreceptoras Retinianas Cones , Catálise , Opsinas dos Cones/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Guanosina Monofosfato/metabolismo , Guanosina Trifosfato/metabolismo , Guanilato Ciclase/metabolismo , Humanos , Osmose , Fosfatos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Subunidades Proteicas/metabolismo , Proteínas RGS/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Vitamina A/metabolismoRESUMO
Adaptive optics (AO) based ophthalmic imagers, such as scanning laser ophthalmoscopes (SLO) and optical coherence tomography (OCT), are used to evaluate the structure and function of the retina with high contrast and resolution. Fixational eye movements during a raster-scanned image acquisition lead to intra-frame and intra-volume distortion, resulting in an inaccurate reproduction of the underlying retinal structure. For three-dimensional (3D) AO-OCT, segmentation-based and 3D correlation based registration methods have been applied to correct eye motion and achieve a high signal-to-noise ratio registered volume. This involves first selecting a reference volume, either manually or automatically, and registering the image/volume stream against the reference using correlation methods. However, even within the chosen reference volume, involuntary eye motion persists and affects the accuracy with which the 3D retinal structure is finally rendered. In this article, we introduced reference volume distortion correction for AO-OCT using 3D correlation based registration and demonstrate a significant improvement in registration performance via a few metrics. Conceptually, the general paradigm follows that developed previously for intra-frame distortion correction for 2D raster-scanned images, as in an AOSLO, but extended here across all three spatial dimensions via 3D correlation analyses. We performed a frequency analysis of eye motion traces before and after intra-volume correction and revealed how periodic artifacts in eye motion estimates are effectively reduced upon correction. Further, we quantified how the intra-volume distortions and periodic artifacts in the eye motion traces, in general, decrease with increasing AO-OCT acquisition speed. Overall, 3D correlation based registration with intra-volume correction significantly improved the visualization of retinal structure and estimation of fixational eye movements.
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Imageamento Tridimensional/instrumentação , Óptica e Fotônica , Tomografia de Coerência Óptica/instrumentação , Artefatos , Desenho de Equipamento , Movimentos Oculares/fisiologia , Fixação Ocular/fisiologia , HumanosRESUMO
We propose a versatile 3D phase-imaging microscope platform for real-time imaging of optomicrofluidic devices based on the principle of digital holographic microscopy (DHM). Lab-on-chip microfluidic devices fabricated on transparent polydimethylsiloxane (PDMS) and glass substrates have attained wide popularity in biological sensing applications. However, monitoring, visualization, and characterization of microfluidic devices, microfluidic flows, and the biochemical kinetics happening in these devices is difficult due to the lack of proper techniques for real-time imaging and analysis. The traditional bright-field microscopic techniques fail in imaging applications, as the microfluidic channels and the fluids carrying biological samples are transparent and not visible in bright light. Phase-based microscopy techniques that can image the phase of the microfluidic channel and changes in refractive indices due to the fluids and biological samples present in the channel are ideal for imaging the fluid flow dynamics in a microfluidic channel at high resolutions. This paper demonstrates three-dimensional imaging of a microfluidic device with nanometric depth precisions and high SNR. We demonstrate imaging of microelectrodes of nanometric thickness patterned on glass substrate and the microfluidic channel. Three-dimensional imaging of a transparent PDMS optomicrofluidic channel, fluid flow, and live yeast cell flow in this channel has been demonstrated using DHM. We also quantify the average velocity of fluid flow through the channel. In comparison to any conventional bright-field microscope, the 3D depth information in the images illustrated in this work carry much information about the biological system under observation. The results demonstrated in this paper prove the high potential of DHM in imaging optofluidic devices; detection of pathogens, cells, and bioanalytes on lab-on-chip devices; and in studying microfluidic dynamics in real time based on phase changes.
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Diagnóstico por Imagem/instrumentação , Holografia/instrumentação , Dispositivos Lab-On-A-Chip , Microfluídica/instrumentação , Microscopia/instrumentação , Óptica e Fotônica/instrumentação , Técnicas Biossensoriais , Dimetilpolisiloxanos/química , Eletrodos , Saccharomyces cerevisiae/citologia , Compostos de Estanho/químicaRESUMO
Optoretinography (ORG) has the potential to be an effective biomarker for light-evoked retinal activity owing to its sensitive, objective, and precise localization of retinal function and dysfunction. Many ORG implementations have used adaptive optics (AO) to localize activity on a cellular scale. However, the use of AO restricts field-of-view (FOV) to the isoplanatic angle, necessitating the montaging of multiple regions-of-interest to cover an extended field. In addition, subjects with lens opacities, increased eye movements and decreased mobility pose challenges for effective AO operation. Here, we developed a coarse-scale ORG (CoORG) system without AO, which accommodates FOVs up to 5.5 deg. in a single acquisition. The system is based on a line-scan spectral domain OCT with volume rates of up to 32 Hz (16,000 B-frames per second). For acquiring ORGs, 5.5 deg. wide OCT volumes were recorded after dark adaptation and two different stimulus bleaches. The stimulus-evoked optical phase change was calculated from the reflections encasing the cone outer segments and its variation was assessed vs. eccentricity in 12 healthy subjects. The general behavior of ΔOPL vs. time mimicked published reports. High trial-to-trial repeatability was observed across subjects and with eccentricity. Comparison of ORG between CoORG and AO-OCT based ORG at 1.5°, 2.5°, and 3.5° eccentricity showed an excellent agreement in the same 2 subjects. The amplitude of the ORG response decreased with increasing eccentricity. The variation of ORG characteristics between subjects and versus eccentricity was well explained by the photon density of the stimulus on the retina and the outer segment length. Overall, the high repeatability and rapid acquisition over an extended field enabled the normative characterization of the cone ORG response in healthy eyes, and provides a promising avenue for translating ORG for widespread clinical application.
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Precise control of the temperature rise is a prerequisite for proper photothermal therapy. In retinal laser therapy, the heat deposition is primarily governed by the melanin concentration, which can significantly vary across the retina and from patient to patient. In this work, we present a method for determining the optical and thermal properties of layered materials, directly applicable to the retina, using low-energy laser heating and phase-resolved optical coherence tomography (pOCT). The method is demonstrated on a polymer-based tissue phantom heated with a laser pulse focused onto an absorbing layer buried below the phantom's surface. Using a line-scan spectral-domain pOCT, optical path length changes induced by the thermal expansion were extracted from sequential B-scans. The material properties were then determined by matching the optical path length changes to a thermo-mechanical model developed for fast computation. This method determined the absorption coefficient with a precision of 2.5% and the temperature rise with a precision of about 0.2°C from a single laser exposure, while the peak did not exceed 8°C during 1 ms pulse, which is well within the tissue safety range and significantly more precise than other methods.
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Light propagation in photoreceptor outer segments is affected by photopigment absorption and the phototransduction amplification cascade. Photopigment absorption has been studied using retinal densitometry, while recently, optoretinography (ORG) has provided an avenue to probe changes in outer segment optical path length due to phototransduction. With adaptive optics (AO), both densitometry and ORG have been used for cone spectral classification based on the differential bleaching signatures of the three cone types. Here, we characterize cone classification by ORG, implemented in an AO line-scan optical coherence tomography (OCT), and compare it against densitometry. The cone mosaics of five color normal subjects were classified using ORG showing high probability (â¼0.99), low error (<0.22%), high test-retest reliability (â¼97%), and short imaging durations (< 1 hour). Of these, the cone spectral assignments in two subjects were compared against AO-scanning laser opthalmoscope densitometry. High agreement (mean: 91%) was observed between the two modalities in these two subjects, with measurements conducted 6-7 years apart. Overall, ORG benefits from higher sensitivity and dynamic range to probe cone photopigments compared to densitometry, and thus provides greater fidelity for cone spectral classification.
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Line-scan OCT incorporated with adaptive optics (AO) offers high resolution, speed, and sensitivity for imaging retinal structure and function in vivo. Here, we introduce its implementation with reflective mirror-based afocal telescopes, optimized for imaging light-induced retinal activity (optoretinography) and weak retinal reflections at the cellular scale. A non-planar optical design was followed based on previous recommendations with key differences specific to a line-scan geometry. The three beam paths fundamental to an OCT system -illumination/sample, detection, and reference- were modeled in Zemax optical design software to yield theoretically diffraction-limited performance over a 2.2 deg. field-of-view and 1.5 D vergence range at the eye's pupil. The performance for imaging retinal structure was exemplified by cellular-scale visualization of retinal ganglion cells, macrophages, foveal cones, and rods in human observers. The performance for functional imaging was exemplified by resolving the light-evoked optical changes in foveal cone photoreceptors where the spatial resolution was sufficient for cone spectral classification at an eccentricity 0.3 deg. from the foveal center. This enabled the first in vivo demonstration of reduced S-cone (short-wavelength cone) density in the human foveola, thus far observed only in ex vivo histological preparations. Together, the feasibility for high resolution imaging of retinal structure and function demonstrated here holds significant potential for basic science and translational applications.
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Optoretinography-the non-invasive, optical imaging of light-induced functional activity in the retina-stands to provide a critical biomarker for testing the safety and efficacy of new therapies as well as their rapid translation to the clinic. Optical phase change in response to light, as readily accessible in phase-resolved OCT, offers a path towards all-optical imaging of retinal function. However, typical human eye motion adversely affects phase stability. In addition, recording fast light-induced retinal events necessitates high-speed acquisition. Here, we introduce a high-speed line-scan spectral domain OCT with adaptive optics (AO), aimed at volumetric imaging and phase-resolved acquisition of retinal responses to light. By virtue of parallel acquisition of an entire retinal cross-section (B-scan) in a single high-speed camera frame, depth-resolved tomograms at speeds up to 16 kHz were achieved with high sensitivity and phase stability. To optimize spectral and spatial resolution, an anamorphic detection paradigm was introduced, enabling improved light collection efficiency and signal roll-off compared to traditional methods. The benefits in speed, resolution and sensitivity were exemplified in imaging nanometer-millisecond scale light-induced optical path length changes in cone photoreceptor outer segments. With 660 nm stimuli, individual cone responses readily segregated into three clusters, corresponding to long, middle, and short-wavelength cones. Recording such optoretinograms on spatial scales ranging from individual cones, to 100 µm-wide retinal patches offers a robust and sensitive biomarker for cone function in health and disease.
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Photoreceptors initiate vision by converting photons to electrical activity. The onset of the phototransduction cascade is marked by the isomerization of photopigments upon light capture. We revealed that the onset of phototransduction is accompanied by a rapid (<5 ms), nanometer-scale electromechanical deformation in individual human cone photoreceptors. Characterizing this biophysical phenomenon associated with phototransduction in vivo was enabled by high-speed phase-resolved optical coherence tomography in a line-field configuration that allowed sufficient spatiotemporal resolution to visualize the nanometer/millisecond-scale light-induced shape change in photoreceptors. The deformation was explained as the optical manifestation of electrical activity, caused due to rapid charge displacement following isomerization, resulting in changes of electrical potential and surface tension within the photoreceptor disc membranes. These all-optical recordings of light-induced activity in the human retina constitute an optoretinogram and hold remarkable potential to reveal the biophysical correlates of neural activity in health and disease.
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Transdução de Sinal Luminoso , Células Fotorreceptoras Retinianas Cones , Humanos , Transdução de Sinal Luminoso/fisiologia , Retina/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Tomografia de Coerência Óptica , Visão OcularRESUMO
We demonstrate a single-shot near on-axis digital holographic microscope that uses a constrained optimization approach for retrieval of the complex object function in the hologram plane. The recovered complex object is back-propagated from the hologram plane to image plane using the Fresnel back-propagation algorithm. A numerical aberration compensation algorithm is employed for correcting the aberrations in the object beam. The reference beam angle is calculated automatically using the modulation property of Fourier transform without any additional recording. We demonstrate this approach using a United States Air Force (USAF) resolution target as an object on our digital holographic microscope. We also demonstrate this approach by recovering the quantitative phase images of live yeast cells, red blood cells and dynamics of live dividing yeast cells.