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LED array microscopy is an emerging platform for computational imaging with significant utility for biological imaging. Existing LED array systems often exploit transmission imaging geometries of standard brightfield microscopes that leave the rich backscattered field undetected. This backscattered signal contains high-resolution sample information with superb sensitivity to subtle structural features that make it ideal for biological sensing and detection. Here, we develop an LED array reflectance microscope capturing the sample's backscattered signal. In particular, we demonstrate multimodal brightfield, darkfield, and differential phase contrast imaging on fixed and living biological specimens including Caenorhabditis elegans (C. elegans), zebrafish embryos, and live cell cultures. Video-rate multimodal imaging at 20 Hz records real time features of freely moving C. elegans and the fast beating heart of zebrafish embryos. Our new reflectance mode is a valuable addition to the LED array microscopic toolbox.
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Microscopia/instrumentação , Fenômenos Ópticos , Espalhamento de Radiação , Semicondutores , Sobrevivência Celular , Células HT29 , HumanosRESUMO
Under 980 nm excitation, the temperature dependence of five-photon UV (256 and 276 nm) upconversion luminescence in Yb³âº-Er³âº codoped ß-NaLuF4 nanocrystals was studied from 303 K to 523 K. The 4D(7/2) and 4G(9/2) levels of Er³âº are confirmed to be thermally coupled levels. They are the highest energy states for optical thermometry known so far. By using fluorescence intensity ratio technique, optical temperature sensing characteristics based on the 4D(7/2)/4G(9/2) â 4I(15/2) transitions of Er³âº were reported here for the first time. The obtained sensitivity of this UV-based sensor is higher than that of green-based optical thermometer in low temperature range.
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Beta-NaLuF4:20%Yb,0.5%Tm,x%Gd (x = 10, 20, 30) nanocrystals were synthesized by high temperature thermal decomposition method. X-ray diffraction (XRD) show the as-prepared samples are characteristic of a pure beta-NaLuF4. Transmission electron microscope (TEM) investigations indicate the nanocrystals have particle size of -85 nm with good monodispersity and well-defined crystallographic facets. Particularly, under 980 nm excitation, upconversion (UC) emissions in the UV range of 270-320 nm were observed in these nanocrystals, which further prove that beta-NaLuF4 is an excellent host for building UV compact solid-state lasers or fiber lasers.
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Beta-NaYF4:Yb3+, Tm3+/ZnO core/shell nanoparticles (NPs) were synthesized via a high temperature thermal decomposition method. The as-synthesized core/shell NPs were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), and upconversion luminescence spectra (UCL). Under 980 nm laser excitation, the measured intensity of upconversion luminescence (1I6 --> 3H6, 1I6 --> 3F4, and 1D2 --> 3H6) was different with and without ZnO. During the sample preparations, changing the ratio of the solvent affected the ZnO UV absorption efficiency. The results show that the NIR light can be used as the driving source to excite ZnO, thus extending utility rate to the NIR spectral region and enhancing the light harvest rate.
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A novel near-infrared (NIR)-responsive photocatalyst, ß-NaYF4:Yb(3+),Tm(3+)@ZnO composites, was prepared by a two-step high temperature thermolysis method. In the NIR-responsive photocatalysis, ß-NaYF4:Yb,Tm served as a NIR-to-UV upconverter and provided "UV light" or "necessary energy" to the ZnO catalyst. The energy transfer in the composites and the mixtures of ß-NaYF4:Yb,Tm and ZnO was studied by using steady-state and dynamic fluorescence spectroscopy. The NIR photocatalytic activities were investigated by the decomposition of Rhodamine B. It was found that the energy transfer processes dominated the overall photocatalytic activities, and the generation of hydroxyl radicals was the origin of organic pollutant decomposition under NIR irradiation.
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Self-Biased Magnetic Pendulum Array (SBMPA) is an efficient and portable transmitter in ultralow frequency (ULF). The resonance frequency of SBMPA is affected by the magnetic field of the radially magnetized cylindrical permanent magnets. In order to calculate the resonance frequency, the magnetic field model of a single radially magnetized cylindrical permanent magnet is derived based on the concept of magnetic charge. Then, the influence of the demagnetizing field and external magnetic field on the magnetization of permanent magnets is analyzed, and the magnetic field model of SBMPA is established. The results of the magnetic field model are verified through simulation. The average deviation of magnetic field intensity is determined as 0.021%; thus, the magnetic field model and simulation have consistent results. Finally, the influence of the size and distance of permanent magnets on the resonance frequency is analyzed, which could provide theoretical guidance for the dynamic analysis of SBMPA.
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Accurate segmentation of retinal layer boundaries can facilitate the detection of patients with early ophthalmic disease. Typical segmentation algorithms operate at low resolutions without fully exploiting multi-granularity visual features. Moreover, several related studies do not release their datasets that are key for the research on deep learning-based solutions. We propose a novel end-to-end retinal layer segmentation network based on ConvNeXt, which can retain more feature map details by using a new depth-efficient attention module and multi-scale structures. In addition, we provide a semantic segmentation dataset containing 206 retinal images of healthy human eyes (named NR206 dataset), which is easy to use as it does not require any additional transcoding processing. We experimentally show that our segmentation approach outperforms state-of-the-art approaches on this new dataset, achieving, on average, a Dice score of 91.3% and mIoU of 84.4%. Moreover, our approach achieves state-of-the-art performance on a glaucoma dataset and a diabetic macular edema (DME) dataset, showing that our model is also suitable for other applications. We will make our source code and the NR206 dataset publicly available at (https://github.com/Medical-Image-Analysis/Retinal-layer-segmentation).
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Autoinhibition of kinesin-3 ensures the proper spatiotemporal control of the motor activity for intracellular transport, but the underlying mechanism remains elusive. Here, we determine the full-length structure of kinesin-3 KLP-6 in a compact self-folded state. Unexpectedly, all the internal coiled-coil segments and domains in KLP-6 cooperate to successively lock down the neck and motor domains. The first coiled-coil segment is melted into several short helices that work with the motor domain to restrain the entire neck domain. The second coiled-coil segment associates with its neighboring FHA and MBS domains and integrates with the tail MATH domain to form a supramodule that synergistically wraps around the motor domain to trap the nucleotide and hinder the microtubule binding. This multilevel-lockdown mechanism for autoinhibition could be applicable to other kinesin-3 motors.
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Cinesinas , Microtúbulos , Sequência de Aminoácidos , Microtúbulos/metabolismo , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de ProteínaRESUMO
Purpose: To evaluate the clinical utility of visible light optical coherence tomography (VIS-OCT) and to test whether VIS-OCT reflectivity and spectroscopy of peripapillary retinal nerve fiber layer (pRNFL) are correlated with severity of glaucoma, compared with standard-of-care OCT thickness measurements. Methods: In total 54 eyes (20 normal, 17 suspect/preperimetric glaucoma [GS/PPG], 17 perimetric glaucoma [PG]) were successfully imaged with complete datasets. All the eyes were scanned by a custom-designed dual-channel device that simultaneously acquired VIS-OCT and near-infrared OCT (NIR-OCT) images. A 5 × 5 mm2 scan was taken of the pRNFL. The pRNFL reflectivity was calculated for both channels and the spectroscopic marker was quantified by pVN, defined as the ratio of VIS-OCT to NIR-OCT pRNFL reflectivity. The results were compared with ophthalmic examinations and Zeiss Cirrus OCT. Results: VIS-OCT pRNFL reflectivity significantly, sequentially decreased from normal to GS/PPG to PG, as did NIR-OCT pRNFL reflectivity. The pVN had the same decreasing trend among three groups. Normal and GS/PPG eyes were significantly different in VIS-OCT pRNFL reflectivity (P = 0.002) and pVN (P < 0.001), but were not in NIR-OCT pRNFL reflectivity (P = 0.14), circumpapillary RNFL thickness (P = 0.17), or macular ganglion cell layer and inner plexiform layer thickness (P = 0.07) in a mixed linear regression model. Conclusions: VIS-OCT pRNFL reflectivity and pVN better distinguished GS/PPG from normal eyes than Cirrus OCT thickness measurements. Translational Relevance: VIS-OCT pRNFL reflectivity and pVN could be useful metrics in the early detection of glaucoma upon further longitudinal validation.
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Glaucoma , Hipertensão Ocular , Glaucoma/diagnóstico por imagem , Humanos , Luz , Fibras Nervosas , Células Ganglionares da Retina , Tomografia de Coerência Óptica/métodosRESUMO
The retinal macula is at the center of our visual field, and thus pathological damage in the macula significantly impacts an individual's quality of life. The parafoveal vessels form the inner retina provide oxygen perfusion, and the measurement of parafoveal oxygen saturation (sO2) can evaluate macular metabolism and provide pathophysiological insight. In this paper, for the first time, we present a baseline study of microvascular oxygen saturation (sO2) in perifoveal macular region using visible light optical coherence tomography (VIS-OCT) on normal eyes. The arterial and venous sO2 from all eyes was 92.1 ± 7.1 (vol %) and 48.4 ± 5.0 (vol %) (mean ± SD), respectively. Arteriovenous sO2 difference was 43.8 ± 9.5 (vol %). Marginal correlation was found between venous sO2 and intraocular pressure (IOP) among eyes. No significant correlation was found between sO2 and vessel topological features, including length, diameter, and distance to fovea. This baseline study could serve as a benchmark for the future sO2 investigation of retinal macular pathologies.
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Traditional imaging cytometry uses fluorescence markers to identify specific structures but is limited in throughput by the labeling process. We develop a label-free technique that alleviates the physical staining and provides multiplexed readouts via a deep learning-augmented digital labeling method. We leverage the rich structural information and superior sensitivity in reflectance microscopy and show that digital labeling predicts accurate subcellular features after training on immunofluorescence images. We demonstrate up to three times improvement in the prediction accuracy over the state of the art. Beyond fluorescence prediction, we demonstrate that single cell-level structural phenotypes of cell cycles are correctly reproduced by the digital multiplexed images, including Golgi twins, Golgi haze during mitosis, and DNA synthesis. We further show that the multiplexed readouts enable accurate multiparametric single-cell profiling across a large cell population. Our method can markedly improve the throughput for imaging cytometry toward applications for phenotyping, pathology, and high-content screening.
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Oblique scanning laser ophthalmoscopy (oSLO) is a novel imaging modality to provide volumetric retinal imaging without depth sectioning over a large field of view (FOV). It has been successfully demonstrated in vivo in rodent eyes for volumetric fluorescein angiography (vFA). However, engineering oSLO for human retinal imaging is challenging because of the low numerical aperture (NA) of human ocular optics. To overcome this challenge, we implement optical designs to (a) increase the angle of the intermediate image under Scheimpflug condition, and (b) expand the magnification in the depth dimension with cylindrical lens to enable sufficient sampling density. In addition, we adopt a scanning-and-descaning strategy, resulting in a compact oSLO system. We experimentally show that the current setup can achieve a FOV of ~3 × 6 × 0.8 mm3 , and the transverse and axial resolutions of 7 and 41 µm, respectively. This feasibility study serves an important step for future in vivo human retinal imaging.
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Oftalmoscópios , Óptica e Fotônica , Estudos de Viabilidade , Humanos , Lasers , OftalmoscopiaRESUMO
We report herein the first visible light optical coherence tomography angiography (vis-OCTA) for human retinal imaging. Compared to the existing vis-OCT systems, we devised a spectrometer with a narrower bandwidth to increase the spectral power density for OCTA imaging, while retaining the major spectral contrast in the blood. We achieved a 100 kHz A-line rate, the fastest acquisition speed reported so far for human retinal vis-OCT. We rigorously optimized the imaging protocol such that a single acquisition took < 6 seconds with a field of view (FOV) of 3×7.8 mm2. The angiography enables accurate localization of microvasculature down to the capillary level and thus enables oximetry at vessels < 100 µm in diameter. We demonstrated microvascular hemoglobin oxygen saturation (sO2) at the feeding and draining vessels at the perifoveal region. The longitudinal repeatability was assessed by < 5% coefficient of variation (CV). The unique capabilities of our vis-OCTA system may allow studies on the role of microvascular oxygen in various retinal pathologies.
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Oblique scanning laser ophthalmoscopy (oSLO) is a recently developed technique to provide three-dimensional volumetric fluorescence imaging in retinas over a large field of view, without the need for depth sectioning. In this study, we present volumetric fluorescein angiography (vFA) at 200 B-scans per second in mouse retina in vivo by oSLO. By using a low-cost industrial CMOS camera, imaging speed was improved to 2 volumes per second, â¼10 times more than our previous results. Enabled by the volumetric imaging, we visualized hemodynamics at single capillary level in a depth-dependent manner, and provided methods to quantify capillary hematocrit, absolute capillary blood flow speed, and detection of capillary flow stagnancy and stalling at different vascular layers. The quantitative metrics for capillary hemodynamics enhanced by volumetric imaging can offer valuable insight into vision science and retinal pathologies.
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Biological functions rely on local microvasculature to deliver oxygen and nutrients and carry away metabolic waste. Alterations to local oxygenation levels are manifested in diseases including cancer, diabetes mellitus, etc. The ability to quantify oxygen saturation (sO2) within microvasculature in vivo to assess local tissue oxygenation and metabolic function is highly sought after. Visible light optical coherence tomography (vis-OCT) angiography has shown promise in reaching this goal. However, achieving reliable measurements in small vessels can be challenging due to the reduced contrast and requires data averaging to improve the spectral data quality. Therefore, a method for quality-control of the vis-OCT data from small vessels becomes essential to reject unreliable readings. In this work, we present a quantitative metrics to evaluate the spectral data for a reliable measurement of sO2, including angiography signal to noise ratio (SNR), spectral anomaly detection and discard, and theory-experiment correlation analysis. The thresholds for each quantity can be flexibly adjusted according to different applications and system performance. We used these metrics to measure sO2 of C57BL/6J mouse lower extremity microvasculature and validated it by introducing hyperoxia for expected sO2 changes. After validation, we applied this protocol on C57BL/6J mouse ear microvasculature to conduct in vivo small blood vessel OCT oximetry. This work seeks to standardize the data processing method for in vivo oximetry in small vessels by vis-OCT.
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The retina, as part of the central nervous system, has distinct anatomical and structural properties for its visual function. Light scattering spectroscopy, while widely used for tissue structural characterization and disease diagnosis, has been relatively unexplored in the living retina. Recently, we have developed a fiber-based visible and near-infrared optical coherence tomography system (vnOCT) for in vivo retinal imaging, to uniquely measure a spectroscopic marker (VN ratio) sensitive to nanoscale pathological changes. In the present study, we applied vnOCT in an animal model of glaucoma (dexamethasone-induced ocular hypertension mouse) and tested the capabilities of four optical markers, VN ratio, peripapillary retinal nerve fiber layer (RNFL) thickness, total retinal blood flow, and hemoglobin oxygen saturation ( sO 2 ), for the detection of retinal ganglion cell (RGC) damage in association with ocular hypertension. We found that RNFL-RGC VN ratio and arteriovenous (A-V) sO 2 are capable of detecting early retinal alteration in ocular hypertensive eyes, preceding measurable change of RNFL thickness. This study suggests a potential clinical application of vnOCT in early detection of glaucoma.
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H- and Nb-doped ZnO (HNZO) thin films were fabricated on glass substrates with radio frequency magnetron sputtering. The effect of the flow rate of H2 has been investigated by analyzing the structural, optical, and electrical properties. The incorporation of H during the deposition of Nb-incorporated ZnO films significantly improved their crystallinity, conductivity, and transmittance. The crystallites of the HNZO films were preferentially oriented in the c-axis direction; the films possess high transmittance (approximately 85%) in the visible and near-infrared regions (400 to 1400 nm). The lowest room-temperature resistivity of the HNZO films was measured as 1.28 × 10-3 Ω cm. Such optical and electrical properties along with the remarkable chemical stability of the HNZO films make them a promising candidate for applications in solar cells.
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While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).
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Imagem Multimodal/métodos , Oftalmoscopia/métodos , Retina/diagnóstico por imagem , Tomografia de Coerência Óptica/métodos , Animais , Estudos Transversais , Masculino , RatosRESUMO
Elastic light scattering spectroscopy (ELSS) has been proven a powerful method in measuring tissue structures with exquisite nanoscale sensitivity. However, ELSS contrast in the living human retina has been relatively underexplored, primarily due to the lack of imaging tools with a large spectral bandwidth. Here, we report a simple all fiber-based setup to implement dual-channel visible and near infrared (NIR) optical coherence tomography (vnOCT) for human retinal imaging, bridging over a 300nm spectral gap. Remarkably, the fiber components in our vnOCT system support single-mode propagation for both visible and NIR light, both of which maintain excellent interference efficiencies with fringe visibility of 97% and 90%, respectively. The longitudinal chromatic aberration from the eye is corrected by a custom-designed achromatizing lens. The elegant fiber-based design enables simultaneous imaging for both channels and allows comprehensive ELSS analysis on several important anatomical layers, including nerve fiber layer, outer segment of the photoreceptors and retinal pigment epithelium. This vnOCT platform and method of ELSS analysis open new opportunities in understanding structure-function relationship in the human retina and in exploring new biomarkers for retinal diseases.
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Flow-mediated vasodilation (FMD) is used for assessment of vascular endothelial function in humans as a predictor of cardiovascular events. It has been challenging to carry it on preclinical murine models due to the diminutive size of the femoral artery. Here, we present a new approach to accurately measure the blood velocity and femoral artery diameters of mice by acquiring Doppler optical coherence tomography and optical coherence tomography angiography continuously within 1 single experimental scanning protocol. Using the 3-dimensional imaging and new velocity algorithm, the measurement precision of diameter, blood flow, velocity and wall shear stress are improved to 0.91%, 11.0%, 10.7% and 14.0%, respectively. FMD of healthy mouse femoral artery measured by this method was 11.96% ± 0.98%, which was blunted to 5.69% ± 0.4% by intravenous administration of endothelial nitric oxide synthase inhibitor (L-NG -Nitroarginine methyl ester), in agreement with that reported in the literature.