RESUMEN
The vagus nerve provides motor, sensory, and autonomic innervation of multiple organs, and electrical vagus nerve stimulation (VNS) provides an adjunctive treatment option for e.g. medication-refractory epilepsy and treatment-resistant depression. The mechanisms of action for VNS are not known, and high-resolution anatomical mapping of the human vagus nerve is needed to better understand its functional organization. Electron microscopy (EM) is required for the detection of both myelinated and unmyelinated axons, but access to well-preserved human vagus nerves for ultrastructural studies is sparse. Intact human vagus nerve samples were procured intra-operatively from deceased organ donors, and tissues were immediately immersion fixed and processed for EM. Ultrastructural studies of cervical and sub-diaphragmatic vagus nerve segments showed excellent preservation of the lamellated wall of myelin sheaths, and the axolemma of myelinated and unmyelinated fibers were intact. Microtubules, neurofilaments, and mitochondria were readily identified in the axoplasm, and the ultrastructural integrity of Schwann cell nuclei, Remak bundles, and basal lamina was also well preserved. Digital segmentation of myelinated and unmyelinated axons allowed for determination of fiber size and myelination. We propose a novel source of human vagus nerve tissues for detailed ultrastructural studies and mapping to support efforts to refine neuromodulation strategies, including VNS.
Asunto(s)
Fibras Nerviosas Mielínicas/ultraestructura , Fibras Nerviosas Amielínicas/ultraestructura , Nervio Vago/ultraestructura , Adulto , Femenino , Humanos , Límite de Detección , Masculino , Microscopía Electrónica/métodos , Microscopía Electrónica/normas , Persona de Mediana Edad , Vaina de Mielina/ultraestructura , Nervio Vago/metabolismoRESUMEN
Segmentation of axons in light and electron micrographs allows for quantitative high-resolution analysis of nervous tissues, but varied axonal dispersion angles result in over-estimates of fiber sizes. To overcome this technical challenge, we developed a novel shape-adjusted ellipse (SAE) determination of axonal size and myelination as an all-inclusive and non-biased tool to correct for oblique nerve fiber presentations. Our new resource was validated by light and electron microscopy against traditional methods of determining nerve fiber size and myelination in rhesus macaques as a model system. We performed detailed segmental mapping and characterized the morphological signatures of autonomic and motor fibers in primate lumbosacral ventral roots (VRs). An en bloc inter-subject variability for the preganglionic parasympathetic fibers within the L7-S2 VRs was determined. The SAE approach allows for morphological ground truth data collection and assignment of individual axons to functional phenotypes with direct implications for fiber mapping and neuromodulation studies.
Asunto(s)
Axones/ultraestructura , Microscopía Electrónica/normas , Fibras Nerviosas Mielínicas/ultraestructura , Raíces Nerviosas Espinales/ultraestructura , Animales , Axones/fisiología , Femenino , Fijadores , Formaldehído , Glutaral , Región Lumbosacra/inervación , Macaca mulatta , Microscopía Electrónica/métodos , Fibras Nerviosas Mielínicas/fisiología , Polímeros , Raíces Nerviosas Espinales/fisiología , Fijación del Tejido/métodosRESUMEN
The analysis of structure factors in 3D cryo-EM Coulomb potential maps and their "enhancement" at the end of the reconstruction process is a well-established practice, normally referred to as sharpening. The aim is to increase contrast and, in this way, to help tracing the atomic model. The most common way to accomplish this enhancement is by means of the so-called B-factor correction, which applies a global filter to boost high frequencies with some dampening considerations related to noise amplification. The results are maps with a better visual aspect and a quasiflat spectrum at medium and high frequencies. This practice is so widespread that most map depositions in the Electron Microscopy Data Base (EMDB) only contain sharpened maps. Here, the use in cryoEM of global B-factor corrections is theoretically and experimentally analyzed. Results clearly illustrate that protein spectra present a falloff. Thus, spectral quasi-flattening may produce protein spectra with distortions when compared with experimental ones, this fact, combined with the practice of reporting only sharpened maps, generates a sub-optimal situation in terms of data preservation, reuse and reproducibility. Now that the field is more advanced, we put forward two suggestions: (1) to use methods which keep more faithfully the original experimental signal properties of macromolecules when "enhancing" the map, and (2) to further stress the need to deposit the original experimental maps without any postprocessing or sharpening, not only the enhanced maps. In the absence of access to these original maps data is lost, preventing their future analysis with new methods.
Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Sustancias Macromoleculares/ultraestructura , Microscopía Electrónica/normas , Conformación Proteica , Microscopía por Crioelectrón , Modelos Moleculares , Programas InformáticosRESUMEN
The high-resolution images provided by the electron microscopy has constituted a limitless source of information in any research field of life and materials science since the early Thirties of the last century. Browsing the scientific literature, electron microscopy was especially popular from the 1970's to 80's, whereas during the 90's, with the advent of innovative molecular techniques, electron microscopy seemed to be downgraded to a subordinate role, as a merely descriptive technique. Ultrastructural histochemistry was crucial to promote the Renaissance of electron microscopy, when it became evident that a precise localization of molecules in the biological environment was necessary to fully understand their functional role. Nowadays, electron microscopy is still irreplaceable for ultrastructural morphology in basic and applied biomedical research, while the application of correlative light and electron microscopy and of refined ultrastructural histochemical techniques gives electron microscopy a central role in functional cell and tissue biology, as a really unique tool for high-resolution molecular biology in situ.
Asunto(s)
Investigación Biomédica/tendencias , Microscopía Electrónica/instrumentación , Microscopía Electrónica/tendencias , Microscopía Electrónica/normasRESUMEN
Recent developments in 3-dimensional electron microcopy (3D-EM) techniques and a concomitant drive to look at complex molecular structures, have led to a rapid increase in the amount of volume data available for biomolecules. This creates a demand for better methods to analyse the data, including improved scores for comparison, classification and integration of data at different resolutions. To this end, we developed and evaluated a set of scoring functions that compare 3D-EM volumes. To test our scores we used a benchmark set of volume alignments derived from the Electron Microscopy Data Bank. We find that the performance of different scores vary with the map-type, resolution and the extent of overlap between volumes. Importantly, adding the overlap information to the local scoring functions can significantly improve their precision and accuracy in a range of resolutions. A combined score involving the local mutual information and overlap (LMI_OV) performs best overall, irrespective of the map category, resolution or the extent of overlap, and we recommend this score for general use. The local mutual information score itself is found to be more discriminatory than cross-correlation coefficient for intermediate-to-low resolution maps or when the map size and density distribution differ significantly. For comparing map surfaces, we implemented two filters to detect the surface points, including one based on the 'extent of surface exposure'. We show that scores that compare surfaces are useful at low resolutions and for maps with evident surface features. All the scores discussed are implemented in TEMPy (http://tempy.ismb.lon.ac.uk/).
Asunto(s)
Imagenología Tridimensional/normas , Sustancias Macromoleculares/química , Microscopía Electrónica/normas , Imagenología Tridimensional/métodos , Microscopía Electrónica/métodos , Modelos MolecularesRESUMEN
Recently, a number of diverse correlative light and electron microscopy (CLEM) protocols have been developed for several model organisms. However, these CLEM methods have largely bypassed plant cell research, with most protocols having little application to plants. Using autophagosome identification as a biological background, we propose and compare two CLEM protocols that can be performed in most plant research laboratories, providing a good compromise that preserves fluorescent signals as well as ultrastructural features. These protocols are based on either the adaptation of a high pressure fixation/GMA acrylic resin embedding method, or on the Tokuyasu approach. Both protocols suitably preserved GFP fluorescence while allowing the observation of cell ultrastructure in plants. Finally, the advantages and disadvantages of these protocols are discussed in the context of multiscale imaging of plant cells.
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Arabidopsis/citología , Microscopía Electrónica/métodos , Autofagosomas , Crioultramicrotomía/métodos , Proteínas Fluorescentes Verdes , Técnicas Histológicas/métodos , Técnicas Histológicas/normas , Microscopía Electrónica/normas , Microscopía Fluorescente/métodos , Raíces de Plantas/citología , Adhesión del Tejido/métodosRESUMEN
Platelet activation has long been known to be accompanied by secretion from at least three types of compartments. These include dense granules, the major source of small molecules; α-granules, the major protein storage organelle; and lysosomes, the site of acid hydrolase storage. Despite ~60 years of research, there are still many unanswered questions about the cell biology of platelet secretion: for example, how are these secretory organelles organized to support cargo release and what are the key routes of cargo release, granule to plasma membrane or granule to canalicular system. Moreover, in recent years, increasing evidence points to the platelet being organized for secretion of the contents from other organelles, namely the dense tubular system (endoplasmic reticulum) and the Golgi apparatus. Conceivably, protein secretion is a widespread property of the platelet and its organelles. In this review, we concentrate on the cell biology of the α-granule and its structure/function relationships. We both review the literature and discuss the wide array of 3-dimensional, high-resolution structural approaches that have emerged in the last few years. These have begun to reveal new and unanticipated outcomes and some of these are discussed. We are hopeful that the next several years will bring rapid advances to this field that will resolve past controversies and be clinically relevant.
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Plaquetas/fisiología , Plaquetas/ultraestructura , Animales , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Humanos , Microscopía Electrónica/métodos , Microscopía Electrónica/normas , Microscopía Fluorescente/métodos , Proteómica/métodos , Vesículas Secretoras/metabolismo , Vesículas Secretoras/ultraestructura , Análisis Espectral/métodos , Relación Estructura-ActividadRESUMEN
Quantitative estimation of myelinated nerve fiber number, together with fiber size parameters, is one of the most important tools for nerve regeneration research. In this study we used a design-based stereological method to evaluate the regenerative process in two experimental paradigms: crush injury and autograft repair. Samples were embedded in resin and morphometric counting and measurements were performed using both light and electron microscopes. Results show a significant difference in myelinated fiber number estimation between light and electron microscopes, especially after autograft repair; light microscope significantly underestimates the number of fibers because of the large number of very small axons that can be detected only in electron microscope. The analysis of the size parameters also shows a higher number of small fibers in electron microscopic analysis, especially in regenerated nerves. This comparative study shows that the integration of data obtained in light microscope with those obtained in electron microscope is necessary in revealing very small myelinated fibers that cannot be detected otherwise. Moreover, the difference in the estimation of total number of myelinated fibers between light and electron microscopes must be considered in data analysis to ensure accurate interpretation of the results.
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Microscopía Electrónica/normas , Microscopía/normas , Fibras Nerviosas Mielínicas/ultraestructura , Regeneración Nerviosa/fisiología , Animales , Femenino , Nervio Mediano/lesiones , Nervio Mediano/fisiología , Nervio Mediano/ultraestructura , Ratas , Ratas WistarRESUMEN
Atomic coordinates in the Worldwide Protein Data Bank (wwPDB) are generally reported to greater precision than the experimental structure determinations have actually achieved. By using information theory and data compression to study the compressibility of protein atomic coordinates, it is possible to quantify the amount of randomness in the coordinate data and thereby to determine the realistic precision of the reported coordinates. On average, the value of each C(α) coordinate in a set of selected protein structures solved at a variety of resolutions is good to about 0.1â Å.
Asunto(s)
Bases de Datos de Proteínas/normas , Interfaz Usuario-Computador , Cristalografía por Rayos X/normas , Diccionarios Químicos como Asunto , Espectroscopía de Resonancia Magnética/normas , Microscopía Electrónica/normas , Valor Predictivo de las Pruebas , Distribución AleatoriaRESUMEN
La cloroquina y sus derivados son fármacos antipalúdicos utilizados en enfermedades del colágeno y afecciones dermatológicas. Describimos un caso de cardiotoxicidad en una mujer de 59 anos diagnosticada de lupus eritematoso discoide y tratada con cloroquina durante 25 anos. La paciente desarrolló trastornos de la conducción y fallo cardiaco, falleciendo finalmente. El corazón mostró hipertrofia biventricular. Describimos el primer caso de auriculopatía inducida por cloroquina. La microscopia óptica y electrónica demostraron vacuolización autofágica de los miocardiocitos auriculares granulares y agranulares, presencia de cuerpos curvilíneos y figuras mieloides, alteraciones mitocondriales y de miofilamentos. La cardiotoxicidad de la cloroquina afectó más intensamente a la aurícula derecha que al resto del corazón. Ello podría explicar, en parte, la aparición de arritmias que ocurre en algunos casos de cardiomiopatía inducida por cloroquina(AU)
The antimalarian agents chloroquine and its derivatives are used in the treatment of connective tissue disease and dermatological disorders. We report a case of carditoxicity in a 59 year-old woman with a 25 year history of chloroquine treatment for a discoid lupus erythematosus. She developed conduction disturbances, cardiac failure and subsequently died. Macroscopically, cardiac biventricular hypertrophy was evident. Light and electronmicroscopic studies of the granular and agranular myocardiocytes of the atria revealed the presence of autophagic vacuoles, curvilinear bodies and myelin-like figures, myofilaments and mitochondrial change. The right atrium was the most damage region of the heart. These lesions may partly explain the appearance of arrhythmias reported in some cases of chloroquine induced myocardiopathy(AU)
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Humanos , Femenino , Persona de Mediana Edad , Cloroquina/efectos adversos , Cloroquina/uso terapéutico , Atrios Cardíacos , Atrios Cardíacos/patología , Enfermedades Cardiovasculares/inducido químicamente , Enfermedades Cardiovasculares/patología , Diagnóstico Diferencial , Paro Cardíaco/inducido químicamente , Paro Cardíaco/complicaciones , Hipertrofia/inducido químicamente , Hipertrofia/complicaciones , Hipertrofia/patología , Cardiomiopatía Hipertrófica/inducido químicamente , Cardiomiopatía Hipertrófica/patología , Microscopía Electrónica/métodos , Microscopía Electrónica/normas , Microscopía ElectrónicaRESUMEN
The biopsy report for nonneoplastic kidney diseases represents a complex integration of clinical data with light, immunofluorescence, and electron microscopic findings. Practice guidelines for the handling and processing of the renal biopsy have previously been created. However, specific guidelines for essential pathologic parameters that should be included in these pathology reports do not exist. The Renal Pathology Society has coordinated an effort through the formation of an ad hoc committee to enumerate the essential elements and pathologic parameters that should be reported for every biopsy specimen. This endeavor aims to establish a minimum reporting standard and to improve communication between pathologists and other physicians. This document represents the collective effort and consensus opinions of this ad hoc committee of the Renal Pathology Society.
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Biopsia/normas , Enfermedades Renales/patología , Riñón/patología , Registros Médicos/normas , Técnica del Anticuerpo Fluorescente/normas , Humanos , Comunicación Interdisciplinaria , Microscopía Electrónica/normas , Microscopía Fluorescente/normas , Valor Predictivo de las PruebasRESUMEN
This Meeting Review describes the proceedings and conclusions from the inaugural meeting of the Electron Microscopy Validation Task Force organized by the Unified Data Resource for 3DEM (http://www.emdatabank.org) and held at Rutgers University in New Brunswick, NJ on September 28 and 29, 2010. At the workshop, a group of scientists involved in collecting electron microscopy data, using the data to determine three-dimensional electron microscopy (3DEM) density maps, and building molecular models into the maps explored how to assess maps, models, and other data that are deposited into the Electron Microscopy Data Bank and Protein Data Bank public data archives. The specific recommendations resulting from the workshop aim to increase the impact of 3DEM in biology and medicine.
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Microscopía Electrónica , Animales , Bases de Datos Factuales/estadística & datos numéricos , Guías como Asunto , Humanos , Almacenamiento y Recuperación de la Información , Sustancias Macromoleculares/química , Microscopía Electrónica/métodos , Microscopía Electrónica/normas , Modelos Moleculares , Conformación Molecular , Anotación de Secuencia MolecularRESUMEN
Introducción. Los neurotequeomas o mixomas de la vaina neural son tumores benignos poco frecuentes, derivados de la vaina de los nervios periféricos, que suelen alojarse en la dermis y en el tejido subcutáneo de la cabeza, el cuello, las extremidades superiores de adultos jóvenes, raramente observados en los miembros inferiores. Objetivo. Se presenta un caso de neurotequeoma celular atípico localizado en el pie de un niño. Caso clínico. Niño de 10 años, que presento nódulo bien circunscrito en dorso de pie derecho de 0,6×0,5cm de diámetro, de 3 años de evolución. Fue resecado realizando el diagnóstico de neurotequeoma celular atípico del pie. Conclusión. Los neurotequeomas son lesiones poco frecuentes, descritas recientemente, de la cual se han descrito algunos casos localizados en el pie. Se han descrito 3 variedades de probable diferente origen, lo que explicaría sus características morfológicas e inmunohistoquímicas particulares. Las características de atipia están en relación con hallazgos morfológicos pero carecen de significado clínico en relación con el comportamiento. Estos tumores pueden confundirse fácilmente con una amplia gama de lesiones tumorales, que pueden excluirse gracias a tinciones de inmunohistoquímica(AU)
Introduction. Neurothekeomas, or nerve sheath myxomas, are rare benign tumours, arising from the sheath of peripheral nerves. They are usually found in young adults, in the dermis and subcutaneous tissue of the head, neck and upper limbs and only rarely occur in the lower limbs. Objective. A case of atypical cellular neurothekeoma of the foot in a child is presented. Case report. A 10 year-old child presented with 3 year history of a well circumscribed nodule on the dorsum of the right foot. A 0.6×0.5 nodule was surgically removed and a diagnosis of atypical cellular neurothekeoma was made. Conclusion. Neurothekeomas are a rare new entity, a few cases of which have been described in the foot. Three distinct varieties have been reported, each of which is probably of a different origin, which would explain their morphological features and immunohistochemical findings. The atypia only refers to morphological findings and is not reflected in their clinical behaviour. These tumours can easily be confused with many other lesions and immunohistochemistry is helpful in the differential diagnosis(AU)
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Humanos , Masculino , Adulto , Mixoma/patología , Neurotecoma/patología , Neoplasias de los Tejidos Blandos/patología , Extremidad Inferior/patología , Microscopía Electrónica/instrumentación , Microscopía Electrónica/tendencias , Inmunohistoquímica/métodos , Inmunohistoquímica/normas , /métodos , Microscopía Electrónica/métodos , Microscopía Electrónica/normas , Microscopía ElectrónicaRESUMEN
This article reviews recent research using a low-energy electron microscope, built by Tromp at IBM, and equipped with an accelerator that permits in situ irradiation with a beam of self-ions. The available ion energies of 20 eV to 5 keV span the range from epitaxial growth by a hyperthermal beam to sputtering at the level of approximately 10 atoms per incident ion. The design criteria and instrument calibration are described. The research described is surface science that requires a vacuum maintained below 10(-10) Torr, with all components contained in the same vacuum. Two general categories of applications are sketched. Experiments that accurately measure important physical quantities include surface mass diffusion over an extended temperature range; determining the critical chemical potential at which island nucleation occurs; observation and explanation of the universal evolution by which adatom and advacancy islands both grow and shrink by beam-driven processes; and the study of sublimation (regarded as negative ion beam intensity). Experiments described here with other goals include beam-assisted synthesis first of large pans and mesas for isolating surface experiments (e.g., nucleation) from the surrounding crystal, and second of Fourier waves on steps, for studies of diffusive relaxation. Operation of exotic structures including Bardeen-Herring sources and Frank growth spirals deformed by crystal anisotropy are also described.
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Microscopía Electrónica/instrumentación , Calibración , Difusión , Diseño de Equipo , Iones/química , Microscopía Electrónica/normas , Nanoestructuras/química , Nanoestructuras/ultraestructura , Dispersión de Radiación , Propiedades de Superficie , Temperatura , VacioRESUMEN
Nuclear morphometry is used to address subtleties of carcinogenesis; it has been proposed for evaluating chemoprevention. An important issue for morphometry concerns control for extraneous sources of variation: fixation, slide cutting and staining. A common strategy has been to standardize the morphometric measures. Morphometric variables--such features as mean nuclear size and staining intensity--are often combined into multivariate indices. In this paper, we consider these variables one by one; any index is to a significant degree dependent on the individual indicators. This paper considers the extent to which statistical adjustment adds to the informational utility of individual indicators. We consider 14 features of 934 prostatic nuclei diagnosed by a single pathologist (Rodolfo Montironi) within a region of either normal tissue or high-grade prostatic intraepithelial neoplasia (HGPIN). HGPIN, a precursor to prostate cancer (PC), has been suggested as a target for PC chemoprevention. We consider a range of adjustment methods: transforming variables into deviations from means or from expected values generated by regression analysis. Our major test of standardization utility is the ability of the variables to deemphasize interindividual differences within diagnostic categories but to distinguish between diagnostic categories.
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Estructuras del Núcleo Celular/ultraestructura , Microscopía Electrónica/normas , Neoplasia Intraepitelial Prostática/ultraestructura , Neoplasias de la Próstata/ultraestructura , Biomarcadores de Tumor/normas , Quimioprevención , Humanos , Masculino , Microscopía Electrónica/métodos , Estándares de ReferenciaRESUMEN
Tobacco mosaic virus (TMV) is widely used as a distance standard in electron microscopy, fiber diffraction, and other imaging techniques. The dimension used as a reference is the pitch of the viral helix, 23 A. This distance, however, has never been measured with any great degree of precision. The helical pitch of TMV has been determined to be 22.92+/-0.03 A by X-ray fiber diffraction methods using highly collimated synchrotron radiation.
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Microscopía Electrónica/normas , Virus del Mosaico del Tabaco/ultraestructura , Virión/ultraestructura , Difracción de Rayos XAsunto(s)
Microscopía Electrónica/normas , Microscopía/normas , Coloración y Etiquetado/métodos , Biomarcadores/análisis , Estructuras Celulares/química , Estructuras Celulares/ultraestructura , Enzimas/análisis , Fluorescencia , Colorantes Fluorescentes/análisis , Oro/química , Oxidación-Reducción , Puntos CuánticosRESUMEN
A simple, fast and efficient noise-reduction protocol for three-dimensional electron tomographic reconstructions of biological material is presented. The approach is based on iterative application of median filtering and shows promise for automatic noise reduction as a pre-processor for automated data analysis tools which aim at segmentation, feature extraction and pattern recognition. The application of this algorithm produces encouraging results for a wide variety of experimental and synthetic electron tomographic reconstructions.