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Synchrotron radiation can be used as a light source in X-ray microscopy to acquire a high-resolution image of a microscale object for tomography. However, numerous projections must be captured for a high-quality tomographic image to be reconstructed; thus, image acquisition is time consuming. Such dense imaging is not only expensive and time consuming but also results in the target receiving a large dose of radiation. To resolve these problems, sparse acquisition techniques have been proposed; however, the generated images often have many artefacts and are noisy. In this study, a deep-learning-based approach is proposed for the tomographic reconstruction of sparse-view projections that are acquired with a synchrotron light source; this approach proceeds as follows. A convolutional neural network (CNN) is used to first interpolate sparse X-ray projections and then synthesize a sufficiently large set of images to produce a sinogram. After the sinogram is constructed, a second CNN is used for error correction. In experiments, this method successfully produced high-quality tomography images from sparse-view projections for two data sets comprising Drosophila and mouse tomography images. However, the initial results for the smaller mouse data set were poor; therefore, transfer learning was used to apply the Drosophila model to the mouse data set, greatly improving the quality of the reconstructed sinogram. The method could be used to achieve high-quality tomography while reducing the radiation dose to imaging subjects and the imaging time and cost.
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Microscopy by Achromatic X-rays With Emission of Laminar Light (MAXWELL) is a new X-ray/visible technique with attractive characteristics including isotropic resolution in all directions, large-volume imaging and high throughput. An ultrathin, laminar X-ray beam produced by a Wolter type I mirror irradiates the sample stimulating the emission of visible light by scintillating nanoparticles, captured by an optical system. Three-dimensional (3D) images are obtained by scanning the specimen with respect to the laminar beam. We implemented and tested the technique with a high-brightness undulator at SPring-8, demonstrating its validity for a variety of specimens. This work was performed under the Synchrotrons for Neuroscience-an Asia-Pacific Strategic Enterprise (SYNAPSE) collaboration.
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Microscopía , Sincrotrones , Imagenología Tridimensional , Luz , Microscopía/métodos , Tomografía Computarizada por Rayos X/métodos , Rayos XRESUMEN
A temporal focusing multiphoton illumination (TFMI) method is proposed for achieving selective volume illumination (SVI) (i.e., illuminating only the volume of interest) in light-field microscopy (LFM). The proposed method minimizes the background noise of the LFM images and enhances the contrast, and thus improves the imaging quality. Three-dimensional (3D) volumetric imaging is achieved by reconstructing the LFM images using a phase-space deconvolution algorithm. The experimental results obtained using 100-nm fluorescent beads show that the proposed TFMI-LFM system achieves lateral and axial resolutions of 1.2 µm and 1.1 µm, respectively, at the focal plane. Furthermore, the TFMI-LFM system enables 3D images of the single lobe of the drosophila mushroom body with GFP biomarker (OK-107) to be reconstructed in a one-snapshot record.
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The new Brain Imaging Beamline (BIB) of the Taiwan Photon Source (TPS) has been commissioned and opened to users. The BIB and in particular its endstation are designed to take advantage of bright unmonochromatized synchrotron X-rays and target fast 3D imaging, â¼1â ms exposure time plus very high â¼0.3â µm spatial resolution. A critical step in achieving the planned performances was the solution to the X-ray induced damaging problems of the detection system. High-energy photons were identified as their principal cause and were solved by combining tailored filters/attenuators and a high-energy cut-off mirror. This enabled the tomography acquisition throughput to reach >1â mm3â min-1, a critical performance for large-animal brain mapping and a vital mission of the beamline.
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Encéfalo/diagnóstico por imagen , Imagenología Tridimensional , Traumatismos por Radiación/prevención & control , Microtomografía por Rayos X/instrumentación , Animales , Diseño de Equipo , Fotones , Sincrotrones , TaiwánRESUMEN
The high longitudinal and lateral coherence of synchrotron X-rays sources radically transformed radiography. Before them, the image contrast was almost only based on absorption. Coherent synchrotron sources transformed radiography into a multi-faceted tool that can extract information also from "phase" effects. Here, we report a very simple description of the new techniques, presenting them to potential new users without requiring a sophisticated background in advanced physics. We then illustrate the impact of such techniques with a number of examples. Finally, we present the international collaboration SYNAPSE (Synchrotrons for Neuroscience-an Asia-Pacific Strategic Enterprise), which targets the use of phase-contrast radiography to map one full human brain in a few years.
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During this global pandemic, cryo-EM has made a great impact on the structure determination of COVID-19 proteins. However, nearly all high-resolution results are based on data acquired on state-of-the-art microscopes where their availability is restricted to a number of centers across the globe with the studies on infectious viruses being further regulated or forbidden. One potential remedy is to employ multipurpose microscopes. Here, we investigated the capability of 200 kV multipurpose microscopes equipped with a direct electron camera in determining the structures of infectious particles. We used 30 nm particles of the grouper nerve necrosis virus as a test sample and obtained the cryo-EM structure with a resolution as high as â¼2.7 Å from a setting that used electron counting. For comparison, we tested a high-end cryo-EM (Talos Arctica) using a similar virus (Macrobrachium rosenbergii nodavirus) to obtain virtually the same resolution. Those results revealed that the resolution is ultimately limited by the depth of field. Our work updates the density maps of these viruses at the sub-3Å level to allow for building accurate atomic models from de novo to provide structural insights into the assembly of the capsids. Importantly, this study demonstrated that multipurpose TEMs are capable of the high-resolution cryo-EM structure determination of infectious particles and is thus germane to the research on pandemics.
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Microscopía por Crioelectrón , Microscopía Electrónica de Transmisión , SARS-CoV-2/fisiología , Virión/química , COVID-19/patología , COVID-19/virología , Humanos , Imagenología Tridimensional , Modelos Moleculares , SARS-CoV-2/química , SARS-CoV-2/aislamiento & purificaciónRESUMEN
Synchrotron radiation evolved over one-half century into a gigantic worldwide enterprise involving tens of thousands of researchers. Initially, almost all users were physicists. But now they belong to a variety of disciplines: chemistry, materials science, the life sciences, medical research, ecology, cultural heritage and others. This poses a challenge: explaining synchrotron sources without requiring a sophisticated background in theoretical physics. Here this challenge is met with an innovative approach that only involves elementary notions, commonly possessed by scientists of all domains.
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Xenon, an inert anesthetic gas, is increasingly recognized to possess desirable properties including cytoprotective and anti-inflammatory effects. Here we evaluated the effects of xenon on the progression of lupus nephritis (LN) in a mouse model. A two hour exposure of either 70% xenon or 70% nitrogen balanced with oxygen was administered daily for five weeks to female NZB/W F1 mice that had been induced to develop accelerated and severe LN. Xenon treatment improved kidney function and renal histology, and decreased the renal expression of neutrophil chemoattractants, thereby attenuating glomerular neutrophil infiltration. The effects of xenon were mediated primarily by deceasing serum levels of anti-double stranded DNA autoantibody, inhibiting reactive oxygen species production, NF-κB/NLRP3 inflammasome activation, ICAM-1 expression, glomerular deposition of IgG and C3 and apoptosis, in the kidney; and enhancing renal hypoxia inducible factor 1-α expression. Proteomic analysis revealed that the treatment with xenon downregulated renal NLRP3 inflammasome-mediated cellular signaling. Similarly, xenon was effective in improving renal pathology and function in a spontaneous LN model in female NZB/W F1 mice. Thus, xenon may have a therapeutic role in treating LN but further studies are warranted to determine applicability to patients.
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Nefritis Lúpica , Animales , Femenino , Inflamasomas , Riñón , Nefritis Lúpica/tratamiento farmacológico , Ratones , Ratones Endogámicos NZB , FN-kappa B , Proteína con Dominio Pirina 3 de la Familia NLR , Proteómica , XenónRESUMEN
The evolution of flight in feathered dinosaurs and early birds over millions of years required flight feathers whose architecture features hierarchical branches. While barb-based feather forms were investigated, feather shafts and vanes are understudied. Here, we take a multi-disciplinary approach to study their molecular control and bio-architectural organizations. In rachidial ridges, epidermal progenitors generate cortex and medullary keratinocytes, guided by Bmp and transforming growth factor ß (TGF-ß) signaling that convert rachides into adaptable bilayer composite beams. In barb ridges, epidermal progenitors generate cylindrical, plate-, or hooklet-shaped barbule cells that form fluffy branches or pennaceous vanes, mediated by asymmetric cell junction and keratin expression. Transcriptome analyses and functional studies show anterior-posterior Wnt2b signaling within the dermal papilla controls barbule cell fates with spatiotemporal collinearity. Quantitative bio-physical analyses of feathers from birds with different flight characteristics and feathers in Burmese amber reveal how multi-dimensional functionality can be achieved and may inspire future composite material designs. VIDEO ABSTRACT.
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Adaptación Fisiológica , Plumas/anatomía & histología , Plumas/fisiología , Vuelo Animal/fisiología , Animales , Evolución Biológica , Aves/anatomía & histología , Moléculas de Adhesión Celular/metabolismo , Citoesqueleto/metabolismo , Dermis/anatomía & histología , Células Madre/citología , Factores de Tiempo , Transcriptoma/genética , Vía de Señalización Wnt/genéticaRESUMEN
X-ray irradiation has been extensively used in recent years as a fabrication step for nanoparticles and nanoparticle systems. A variant of this technique, continuous-flow X-ray irradiation, has recently been developed, and offers three important advantages: precise control of the irradiation dose, elimination of convection effects in the precursor solution, and suitability for large-scale production. Here, the use of this method to fabricate Au nano-meshes of interest as transparent and flexible electrodes for optoelectronics is reported. The study includes extensive characterization of the synthesis parameters and of the product properties, with rather encouraging results.
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Optical super-resolution microscopy allows nanoscale imaging of protein molecules in intact biological tissues. However, it is still challenging to perform large volume super-resolution imaging for entire animal organs. Here we develop a single-wavelength Bessel lightsheet method, optimized for refractive-index matching with clarified specimens to overcome the aberrations encountered in imaging thick tissues. Using spontaneous blinking fluorophores to label proteins of interest, we resolve the morphology of most, if not all, dopaminergic neurons in the whole adult brain (3.64 × 107 µm3) of Drosophila melanogaster at the nanometer scale with high imaging speed (436 µm3 per second) for localization. Quantitative single-molecule localization reveals the subcellular distribution of a monoamine transporter protein in the axons of a single, identified serotonergic Dorsal Paired Medial (DPM) neuron. Large datasets are obtained from imaging one brain per day to provide a robust statistical analysis of these imaging data.
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Encéfalo/diagnóstico por imagen , Drosophila melanogaster/metabolismo , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Animales , Animales Modificados Genéticamente , Encéfalo/metabolismo , Drosophila melanogaster/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Confocal/instrumentación , Microscopía Fluorescente/instrumentación , Reproducibilidad de los ResultadosRESUMEN
A transmission X-ray microscope has been designed and commissioned at the 18-ID Full-field X-ray Imaging beamline at the National Synchrotron Light Source II. This instrument operates in the 5-11 keV range, and, with the current set of optics, is capable of 30 nm spatial resolution imaging, with a field of view of about 40 µm. For absorption contrast, the minimum exposure time for a single projection image is about 20 ms and an entire 3D tomography data set can be acquired in under 1 min. The system enables tomographic reconstructions with sub-50 nm spatial resolution without the use of markers on the sample or corrections for rotation run-outs.
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Recent advances in super-resolution microscopy allow the localization of single molecules within individual cells but not within multiple whole cells due to weak signals from single molecules and slow acquisition process for point accumulation to reconstruct super-resolution images. Here, we report a fast, large-scale, and three-dimensional super-resolution fluorescence microscope based on single-wavelength Bessel lightsheet to selectively illuminate spontaneous blinking fluorophores tagged to the proteins of interest in space. Critical parameters such as labeling density, excitation power, and exposure time were systematically optimized resulting in a maximum imaging speed of 2.7 × 104 µm3 s-1. Fourier ring correlation analysis revealed a reconstructed image with a lateral resolution of ~75 nm through the accumulation of 250 image volumes on immobilized samples within 15 min. Hence, the designed system could open new insights into the discovery of complex biological structures and live 3D localization imaging.
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Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodos , Células 3T3 , Animales , Células Cultivadas , Colorantes Fluorescentes , Ratones , Neuronas/metabolismo , Neuronas/ultraestructura , Proteínas de Complejo Poro Nuclear/metabolismo , RatasRESUMEN
An error in tomographic reconstruction parameters can result considerable artifacts in the reconstructed image, particularly in micro-computed tomography and nano-computed tomography. This study involved designing an automatic method for efficiently correcting errors resulting from incorrectly determined rotational axes and projection angles. In this method, errors are corrected by minimizing the "total variation" of a reconstructed image, and minimization is accomplished by using the gradient descent method. Compared with two previous methods, the proposed method achieved the best reconstruction results.
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Three-dimensional (3D) histology is the next frontier for modern anatomo-pathology. Characterizing abnormal parameters in a tissue is essential to understand the rationale of pathology development. However, there is no analytical technique, in vivo or histological, that is able to discover such abnormal features and provide a 3D distribution at microscopic resolution. Here, we introduce a unique high-throughput infrared (IR) microscopy method that combines automated image correction and subsequent spectral data analysis for 3D-IR image reconstruction. We performed spectral analysis of a complete organ for a small animal model, a mouse brain with an implanted glioma tumor. The 3D-IR image is reconstructed from 370 consecutive tissue sections and corrected using the X-ray tomogram of the organ for an accurate quantitative analysis of the chemical content. A 3D matrix of 89 × 106 IR spectra is generated, allowing us to separate the tumor mass from healthy brain tissues based on various anatomical, chemical, and metabolic parameters. We demonstrate that quantitative metabolic parameters can be extracted from the IR spectra for the characterization of the brain vs. tumor metabolism (assessing the Warburg effect in tumors). Our method can be further exploited by searching for the whole spectral profile, discriminating tumor vs. healthy tissue in a non-supervised manner, which we call 'spectromics'.
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Determining the filtration function and biochemical status of kidney at the single glomerulus level remains hardly accessible, even from biopsies. Here, we provide evidence that IR spectro-microscopy is a suitable method to account for the filtration capacity of individual glomeruli along with related physio-pathological condition. A â¼4 µm voxel resolution 3D IR image reconstruction is built from consecutive tissue sections, thus, providing a 3D IR spectrum matrix of an individual glomerulus. The filtration capacity of glomeruli was quantitatively determined after BaSO4 perfusion, and additional chemical data could be used to determined oxidative stress effects and fibrosis, thus, combining functional and biochemical information from the same 3D IR spectrum matrix. This analytical approach was applied on mice with unilateral ureteral obstruction (UUO) inducing chronic kidney disease. Compared to the healthy condition, UUO induced a significant drop in glomeruli filtration capacity (-17 ± 8% at day 4 and -48 ± 14% at day 14) and volume (36 ± 10% at day 4 and 67 ± 13% at day 14), along a significant increase of oxidative stress (+61 ± 19% at day 4 and +84 ± 17% at day 14) and a change in the lipid-to-protein ratio (-8.2 ± 3.6% at day 4 and -18.1 ± 5.9% at day 14). Therefore, IR spectro-microscopy might be developed as a new 3D pathology resource for analyzing functional and biochemical parameters of glomeruli.
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Imagenología Tridimensional/métodos , Glomérulos Renales/patología , Insuficiencia Renal Crónica/patología , Espectrofotometría Infrarroja/métodos , Obstrucción Ureteral/patología , Animales , Sulfato de Bario/análisis , Análisis por Conglomerados , Modelos Animales de Enfermedad , Fibrosis , Glomérulos Renales/química , Glomérulos Renales/diagnóstico por imagen , Glomérulos Renales/metabolismo , Masculino , Ratones , Estrés Oxidativo , Insuficiencia Renal Crónica/diagnóstico por imagen , Insuficiencia Renal Crónica/metabolismo , Obstrucción Ureteral/diagnóstico por imagen , Obstrucción Ureteral/metabolismoRESUMEN
The optic lobe is the largest brain area within the central nervous system of cephalopods and it plays important roles in the processing of visual information, the regulation of body patterning, and locomotive behavior. The oval squid Sepioteuthis lessoniana has relatively large optic lobes that are responsible for visual communication via dynamic body patterning. It has been observed that the visual behaviors of oval squids change as the animals mature, yet little is known about how the structure of the optic lobes changes during development. The aim of the present study was to characterize the ontogenetic changes in neural organization of the optic lobes of S. lessoniana from late embryonic stage to adulthood. Magnetic resonance imaging and micro-CT scans were acquired to reconstruct the 3D-structure of the optic lobes and examine the external morphology at different developmental stages. In addition, optic lobe slices with nuclear staining were used to reveal changes in the internal morphology throughout development. As oval squids mature, the proportion of the brain making up the optic lobes increases continuously, and the optic lobes appear to have a prominent dent on the ventrolateral side. Inside the optic lobe, the cortex and the medulla expand steadily from the late embryonic stage to adulthood, but the cell islands in the tangential zone of the optic lobe decrease continuously in parallel. Interestingly, the size of the nuclei of cells within the medulla of the optic lobe increases throughout development. These findings suggest that the optic lobe undergoes continuous external morphological change and internal neural reorganization throughout the oval squid's development. These morphological changes in the optic lobe are likely to be responsible for changes in the visuomotor behavior of oval squids from hatching to adulthood.
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Decapodiformes/anatomía & histología , Decapodiformes/embriología , Embrión no Mamífero/anatomía & histología , Desarrollo Embrionario , Lóbulo Óptico de Animales no Mamíferos/anatomía & histología , Lóbulo Óptico de Animales no Mamíferos/embriología , Animales , Núcleo Celular/metabolismo , Decapodiformes/citología , Embrión no Mamífero/citología , Imagenología Tridimensional , Lóbulo Óptico de Animales no Mamíferos/citologíaRESUMEN
To understand how information flows and is used in the human brain, we must map neural structures at all levels, providing visualizations similar to those of Google Earth for continents, countries, cities, and streets. Unfortunately, the imaging and processing techniques currently used in connectomics projects cannot achieve complete mapping for the brains of large animals within the timespan of a typical research career. However, feasible improvements in x-ray imaging would change this situation. This Q&A discusses synchrotron x-ray tomography, an exciting new approach for in situ mapping of whole-brain wiring diagrams at multiple levels of spatial resolution.
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Conectoma/tendencias , Sincrotrones , Tomografía por Rayos X , Animales , HumanosRESUMEN
High resolution synchrotron microtomography capable of revealing microvessels in three dimensional (3D) establishes distinct imaging markers of mouse kidney disease strongly associated to renal tubulointerstitial (TI) lesions and glomerulopathy. Two complementary mouse models of chronic kidney disease (CKD), unilateral ureteral obstruction (UUO) and focal segmental glomerulosclerosis (FSGS), were used and five candidates of unique 3D imaging markers were identified. Our characterization to differentially reflect the altered microvasculature of renal TI lesions and/or glomerulopathy demonstrated these image features can be used to differentiate the disease status and the possible cause therefore qualified as image markers. These 3D imaging markers were further correlated with the histopathology and renal microvessel-based molecular study using antibodies against vascular endothelial cells (CD31), the connective tissue growth factor or the vascular endothelial growth factor. We also found that these 3D imaging markers individually characterize the development of renal TI lesions or glomerulopathy, quantitative and integrated use of all of them provide more information for differentiating the two renal conditions. Our findings thus establish a practical strategy to characterize the CKD-associated renal injuries by the microangiography-based 3D imaging and highlight the impact of dysfunctional microvasculature as a whole on the pathogenesis of the renal lesions.
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Microvasos/diagnóstico por imagen , Insuficiencia Renal Crónica/diagnóstico por imagen , Insuficiencia Renal Crónica/patología , Tomografía Computarizada por Rayos X/métodos , Algoritmos , Animales , Biomarcadores , Imagenología Tridimensional , Riñón/irrigación sanguínea , Riñón/diagnóstico por imagen , Riñón/patología , Masculino , Ratones Endogámicos BALB C , Neovascularización Patológica/diagnóstico por imagen , SincrotronesRESUMEN
We present a novel ratiometric pH sensor design using water-soluble, dual-emission, Mn(2+)-doped quantum dots (Qdots) decorated with D-penicillamine (DPA-MnQdots). In contrast to more commonly used ratiometric pH sensors that rely on the coupling of two fluorophores, our design uses only a single emitter, which simplifies ratiometric sensing and broadens the applications of the sensor. Our single-emitter DPA-MnQdots exhibit two emission bands, at 510nm (green) and 610nm (red), which are, respectively, attributable to exciton recombination and emission of the Mn(2+) dopants. The emission intensity ratio (I510/I610) of the DPA-MnQdots depends linearly on surrounding pH values within physiological conditions (from pH 4.5 to 8.5). Moreover, the biocompatible DPA-MnQdots were used for long-term monitoring of local pH values in HeLa cells.