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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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Microscopia , Síncrotrons , Imageamento Tridimensional , Luz , Microscopia/métodos , Tomografia Computadorizada por Raios X/métodos , Raios XRESUMO
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 imagem , Imageamento Tridimensional , Lesões por Radiação/prevenção & controle , Microtomografia por Raio-X/instrumentação , Animais , Desenho de Equipamento , Fótons , Síncrotrons , TaiwanRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Mineralized bone forms when collagen-containing osteoid accrues mineral crystals. This is initiated rapidly (primary mineralization), and continues slowly (secondary mineralization) until bone is remodeled. The interconnected osteocyte network within the bone matrix differentiates from bone-forming osteoblasts; although osteoblast differentiation requires EphrinB2, osteocytes retain its expression. Here we report brittle bones in mice with osteocyte-targeted EphrinB2 deletion. This is not caused by low bone mass, but by defective bone material. While osteoid mineralization is initiated at normal rate, mineral accrual is accelerated, indicating that EphrinB2 in osteocytes limits mineral accumulation. No known regulators of mineralization are modified in the brittle cortical bone but a cluster of autophagy-associated genes are dysregulated. EphrinB2-deficient osteocytes displayed more autophagosomes in vivo and in vitro, and EphrinB2-Fc treatment suppresses autophagy in a RhoA-ROCK dependent manner. We conclude that secondary mineralization involves EphrinB2-RhoA-limited autophagy in osteocytes, and disruption leads to a bone fragility independent of bone mass.
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Autofagia/fisiologia , Doenças do Desenvolvimento Ósseo/genética , Calcificação Fisiológica/fisiologia , Efrina-B2/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Autofagossomos/fisiologia , Autofagia/genética , Doenças do Desenvolvimento Ósseo/patologia , Remodelação Óssea/fisiologia , Linhagem Celular , Efrina-B2/genética , Camundongos , Camundongos Endogâmicos C57BL , Osteócitos/metabolismo , Osteócitos/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genética , Proteína rhoA de Ligação ao GTPRESUMO
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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Imageamento Tridimensional/métodos , Glomérulos Renais/patologia , Insuficiência Renal Crônica/patologia , Espectrofotometria Infravermelho/métodos , Obstrução Ureteral/patologia , Animais , Sulfato de Bário/análise , Análise por Conglomerados , Modelos Animais de Doenças , Fibrose , Glomérulos Renais/química , Glomérulos Renais/diagnóstico por imagem , Glomérulos Renais/metabolismo , Masculino , Camundongos , Estresse Oxidativo , Insuficiência Renal Crônica/diagnóstico por imagem , Insuficiência Renal Crônica/metabolismo , Obstrução Ureteral/diagnóstico por imagem , Obstrução Ureteral/metabolismoRESUMO
Mid-infrared (IR), Raman, and X-ray fluorescence (XRF) spectroscopy methods, as well as mass spectrometry (MS), can be used for 3D chemical imaging. These techniques offer an invaluable opportunity to access chemical features of biological samples in a nonsupervised way. The global chemical information they provide enables the exploitation of a large array of chemical species or parameters, so-called 'spectromics'. Extracting chemical data from spectra is critical for the high-quality chemical analysis of biosamples. Furthermore, these are the only currently available techniques that can quantitatively analyze tissue content (e.g., molecular concentrations) and substructures (e.g., cells or blood vessels). The development of chemical-derived biological metadata appears to be a new way to exploit spectral information with machine learning algorithms.
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Encéfalo/diagnóstico por imagem , Encéfalo/ultraestrutura , Imageamento Tridimensional/métodos , Microscopia/métodos , Análise Espectral/métodos , Algoritmos , Animais , Simulação por Computador , Humanos , Espectrometria de Massas/métodos , Camundongos , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Análise Espectral Raman/métodosRESUMO
Asbestos may cause adverse effects, but relationship between mineralogy and texture of fibres versus toxicity is still lacking. Toxicological studies can be interpreted and compared only if quantitative features of fibres are determined. Here, riebeckitic ("crocidolite") amphibole fibres were analysed by XRPD, FTIR, SEM-EDS and EMP-WDS; only crystals with stochiometry Aâ¡BNa2C(Fe2+2.5Mg0.5)CFe3+2TSi8O22W(OH)2 are present in the starting material used for the experiments. Fibres deposited from solutions of 0.1, 1, 10, 25, 50, 75 and 100mg/L were counted by image analysis using SEM images. At 0.1 and 1mg/L the fibres are well separated, whereas between 1 and 10mg/L they start to agglomerate. In-vitro tests performed on fibres deposited at the same mg/L concentrations show that the toxic potential follows a curvilinear increasing trend with a decreasing rate. Since the range of sizes of single fibres and their mineralogy are constant, this decreasing rate can be only attributed to the increasing amount of agglomerated fibres. Hence, single versus agglomerated fibre population is a factor that cannot be neglected in defining the final adverse effects of asbestos. The analytical protocol proposed here is valuable for any aero-dispersed dust, in polluted environments, as well as in the interpretation of experimental studies.
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Currently, only mass-spectrometry (MS) microscopy brings a quantitative analysis of chemical contents of tissue samples in 3D. Here, the reconstruction of a 3D quantitative chemical images of a biological tissue by FTIR spectro-microscopy is reported. An automated curve-fitting method is developed to extract all intense absorption bands constituting IR spectra. This innovation benefits from three critical features: (1) the correction of raw IR spectra to make them quantitatively comparable; (2) the automated and iterative data treatment allowing to transfer the IR-absorption spectrum into a IR-band spectrum; (3) the reconstruction of an 3D IR-band matrix (x, y, z for voxel position and a 4th dimension with all IR-band parameters). Spectromics, which is a new method for exploiting spectral data for tissue metadata reconstruction, is proposed to further translate the related chemical information in 3D, as biochemical and anatomical tissue parameters. An example is given with oxidative stress distribution and the reconstruction of blood vessels in tissues. The requirements of IR microscopy instrumentation to propose 3D digital histology as a clinical routine technology is briefly discussed.
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Imageamento Tridimensional/métodos , Microscopia/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Animais , Encéfalo/irrigação sanguínea , Encéfalo/citologia , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Camundongos , Reconhecimento Automatizado de PadrãoRESUMO
Fasted or weight-category athletes manage their training under strict diet conditions that could impair the stress-recovery balance and result in acute or chronic fatigue. However, to date, no validated biomarker are available to quantify this phenomena. The aim of this study was to assess the validity of a specific index combining plasma albumin and weight change to detect nutrition-related risks of fatigue increase and under-performance in athletes experiencing particular nutritional conditions. An athlete's nutrition risk index (ANRI) equation, based on data from lightweight and heavyweight rowers, was developed using relationship between plasma albumin concentrations combined to weight changes with sport performance and overtraining scores and was tested by odds ratio for failure. The accuracy and sensitivity of this former specific equation was subsequently tested on runners observing the Ramadan-fasting as well as on boxers after a short weight-loss period. Independently of training and performance, lightweight rowers presented lower nutritional parameters than heavyweight (albumin: 37.4 ± 2.7 vs 39.9 ± 1.8 g·L-1, P < 0.05; weight state: 94.5 ± 1.8 vs 99.9 ± 0.9%, P < 0.01). In lightweight, ANRI was related with overtraining score (R2 = 0.21, P < 0.01), risks for failure in competition were enhanced when ANRI increased (OR:2.5, P = 0.03). Relationship of ANRI with overtraining score tended to be also significant in runners (R2 = 0.32, P = 0.06) but not in boxers (P = 0.4). Albumin concentrations combined to weight loss appeared relevant to delineate nutrition-related risks of fatigue and/or competitive failure associated with mid-term diets (about 30 days) as observed in rowers and Ramadan-fasted runners. ANRI may benefit to athletes monitoring by delineating effects of their weight loss program.
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FTIR spectroscopy is used to identify poly-L-lysin fatty-acyl-chain (PLL-FAC) conjugates based on structural differences found between FAC species. Twenty-one PLL-FAC models were used, from C8 to C24, and with up to 5 unsaturation levels (C20:5). Curve fitting of the 3050-2800 cm(-1) spectral interval permitted extraction of IR bands belonging to the stretching vibration modes of methyl, methylene, and alkene groups. Based on molecular structure models in 3D, the number and position of methyl bands could be set according to chain length and unsaturation level. Band positions for ν-(C = C < H), ν(as)(CH3), and ν(as)(CH2) groups did not follow the maximum intensity shift of spectrum curve; it is the underlying band's intensity that is modifying maximum intensity of spectrum curve with respect to chain length and unsaturation level. We thus propose to use FTIR spectroscopy for the production monitoring and the quality control of PLL-FAC conjugates used as nutritional complements, and this should be extended to analysis of fatty acid compounds in general.
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Ácidos Graxos/química , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Ligação de Hidrogênio , Estrutura MolecularRESUMO
BACKGROUND: Nanoparticles can be used for targeted drug delivery, in particular for brain cancer therapy. However, this requires a detailed analysis of nanoparticles from the associated microvasculature to the tumor, not easy because of the required high spatial resolution. The objective of this study is to demonstrate an experimental solution of this problem, based in vivo and post-mortem whole organ imaging plus nanoscale 3-dimensional (3D) X-ray microscopy. RESULTS: The use of gold nanoparticles (AuNPs) as contrast agents paved the way to a detailed high-resolution three dimensional (3D) X-ray and fluorescence imaging analysis of the relation between xenografted glioma cells and the tumor-induced angiogenic microvasculature. The images of the angiogenic microvessels revealed nanoparticle leakage. Complementary tests showed that after endocytotic internalization fluorescent AuNPs allow the visible-light detection of cells. CONCLUSIONS: AuNP-loading of cells could be extended from the case presented here to other imaging techniques. In our study, they enabled us to (1) identify primary glioma cells at inoculation sites in mice brains; (2) follow the subsequent development of gliomas. (3) Detect the full details of the tumor-related microvasculature; (4) Finding leakage of AuNPs from the tumor-related vasculature, in contrast to no leakage from normal vasculature.
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Neoplasias Encefálicas/diagnóstico por imagem , Encéfalo/diagnóstico por imagem , Meios de Contraste/química , Glioma/diagnóstico por imagem , Ouro/química , Nanopartículas Metálicas/química , Animais , Encéfalo/irrigação sanguínea , Encéfalo/patologia , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/patologia , Meios de Contraste/administração & dosagem , Endocitose , Glioma/irrigação sanguínea , Glioma/patologia , Ouro/administração & dosagem , Nanopartículas Metálicas/administração & dosagem , Camundongos , Transplante de Neoplasias , Imagem Óptica/métodos , Tomografia Computadorizada por Raios X/métodosRESUMO
In search of specific label-free biomarkers for differentiation of two oral lesions, namely oral leukoplakia (OLK) and oral squamous-cell carcinoma (OSCC), Fourier-transform infrared (FTIR) spectroscopy was performed on paraffin-embedded tissue sections from 47 human subjects (eight normal (NOM), 16 OLK, and 23 OSCC). Difference between mean spectra (DBMS), Mann-Whitney's U test, and forward feature selection (FFS) techniques were used for optimising spectral-marker selection. Classification of diseases was performed with linear and quadratic support vector machine (SVM) at 10-fold cross-validation, using different combinations of spectral features. It was observed that six features obtained through FFS enabled differentiation of NOM and OSCC tissue (1782, 1713, 1665, 1545, 1409, and 1161 cm(-1)) and were most significant, able to classify OLK and OSCC with 81.3 % sensitivity, 95.7 % specificity, and 89.7 % overall accuracy. The 43 spectral markers extracted through Mann-Whitney's U Test were the least significant when quadratic SVM was used. Considering the high sensitivity and specificity of the FFS technique, extracting only six spectral biomarkers was thus most useful for diagnosis of OLK and OSCC, and to overcome inter and intra-observer variability experienced in diagnostic best-practice histopathological procedure. By considering the biochemical assignment of these six spectral signatures, this work also revealed altered glycogen and keratin content in histological sections which could able to discriminate OLK and OSCC. The method was validated through spectral selection by the DBMS technique. Thus this method has potential for diagnostic cost minimisation for oral lesions by label-free biomarker identification.
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Carcinoma de Células Escamosas/diagnóstico , Leucoplasia Oral/diagnóstico , Neoplasias Bucais/diagnóstico , Boca/patologia , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Biomarcadores Tumorais/análise , Humanos , Sensibilidade e Especificidade , Máquina de Vetores de SuporteRESUMO
Evidence has recently emerged that solid and diffuse tumors produce a specific extracellular matrix (ECM) for division and diffusion, also developing a specific interface with microvasculature. This ECM is mainly composed of collagens and their scaffolding appears to drive tumor growth. Although collagens are not easily analyzable by UV-fluorescence means, FTIR imaging has appeared as a valuable tool to characterize collagen contents in tissues, specially the brain, where ECM is normally devoid of collagen proteins. Here, we used FTIR imaging to characterize collagen content changes in growing glioma tumors. We could determine that C6-derived solid tumors presented high content of triple helix after 8-11 days of growth (typical of collagen fibrils formation; 8/8 tumor samples; 91 % of total variance), and further turned to larger α-helix (days 12-15; 9/10 of tumors; 94 % of variance) and ß-turns (day 18-21; 7/8 tumors; 97 % of variance) contents, which suggest the incorporation of non-fibrillar collagen types in ECM, a sign of more and more organized collagen scaffold along tumor progression. The growth of tumors was also associated to the level of collagen produced (P < 0.05). This study thus confirms that collagen scaffolding is a major event accompanying the angiogenic shift and faster tumor growth in solid glioma phenotypes.
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Neoplasias Encefálicas/diagnóstico , Colágeno/química , Glioma/diagnóstico , Espectroscopia de Infravermelho com Transformada de Fourier , Animais , Neoplasias Encefálicas/química , Neoplasias Encefálicas/genética , Colágeno/genética , Progressão da Doença , Matriz Extracelular/química , Expressão Gênica , Glioma/química , Glioma/genética , Interpretação de Imagem Assistida por Computador , Masculino , Análise de Componente Principal , Estrutura Secundária de Proteína , RatosRESUMO
Amphiboles caused cohorts of deaths in exposed workers, leading to some of the largest class actions in the industry. Once inhaled, these inorganic fibers are thought to be both chemically and morphologically toxic, and their biopersistence in the lungs over decades lead to progressive pathologies, mesothelioma, and asbestosis. However, this exceptionally long chronicity for human pathologies suggests that chemical toxicity is certainly low, suggesting that morphological parameters could be more relevant in the pathology. Here, we developed a 3D Raman/optical imaging methodology in vitro to characterize both morphological and chemical parameters of cell/fiber interactions. We determined that lung cells could vesiculate amphiboles with length below 5 µm or could embed those not exceeding 15 µm in their fibrous extracellular matrix. Lung cells can thus develop defense strategies for handling the biopersistence of inorganic species, which may thus have major impact for biosafety issues related to nanomaterials.
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An original synthesis method based on X-ray irradiation produced gold nanoparticles (AuNPs) with two important properties for biomedical research: intense visible photoluminescence and very high accumulation in cancer cells. The nanoparticles, coated with MUA (11-mercaptoundecanoid acid), are very small (1.4 nm diameter); the above two properties are not present for even slightly larger sizes. The small MUA-AuNPs are non-cytotoxic (except for very high concentrations) and do not interfere with cancer cell proliferation. Multimodality imaging using visible light fluorescence and X-ray microscopy is demonstrated by tracing the nanoparticle-loaded tumor cells.
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Nanopartículas Metálicas/ultraestrutura , Animais , Proliferação de Células/efeitos dos fármacos , Ácidos Graxos , Ouro , Humanos , Luz , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos BALB C , Microscopia/métodos , Microscopia Eletrônica de Transmissão , Neoplasias Experimentais/diagnóstico , Neoplasias Experimentais/patologia , Espectroscopia de Infravermelho com Transformada de Fourier , Compostos de Sulfidrila , Termogravimetria , Distribuição Tecidual , Células Tumorais Cultivadas , Raios XRESUMO
In this contribution we present the design of an original Attenuated Total Reflection (ATR)-based device designed for an IR microscope coupled to a FPA detector and optimized for in-vivo cell imaging. The optical element has been designed to perform real time experiments of cell biochemical processes. The device includes a manually removable Ge-crystal that guarantees an ease manipulation during the cell culture and a large flat surface to support the cell growth and the required change of the culture wells. This layout will allow performing sequential ATR IR imaging with the crystal immersed in the culture wells, minimizing contributions due to water vapors in the optical system. Using existing brilliant synchrotron radiation sources this ATR device may collect images at the surface of the Ge crystal at a sub-cellular spatial resolution with a penetration depth of the evanescent wave inside the sample of ~500 nm within few seconds. A brief summary of the cellular components that should be detected with such optical device is also presented.
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Técnicas Citológicas/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/instrumentação , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Processos de Crescimento Celular , Desenho de Equipamento , Razão Sinal-Ruído , Síncrotrons/instrumentaçãoRESUMO
We show that sufficient concentrations of gold nanoparticles produced by an original synthesis method in EMT-6 and CT-26 cancer cells make it possible to detect the presence, necrosis and proliferation of such cells after inoculation in live mice. We first demonstrated that the nanoparticles do not interfere with the proliferation process. Then, we observed significant differences in the tumor evolution and the angiogenesis process after shallow and deep inoculation. A direct comparison with pathology optical images illustrates the effectiveness of this approach.