RESUMEN
Large dimension, high-resolution imaging is important for neural circuit visualisation as neurons have both long- and short-range patterns: from axons and dendrites to the numerous synapses at terminal endings. Electron Microscopy (EM) is the favoured approach for synaptic resolution imaging but how such structures can be segmented from high-density images within large volume datasets remains challenging. Fluorescent probes are widely used to localise synapses, identify cell-types and in tracing studies. The equivalent EM approach would benefit visualising such labelled structures from within sub-cellular, cellular, tissue and neuroanatomical contexts. Here we developed genetically-encoded, electron-dense markers using miniSOG. We demonstrate their ability in 1) labelling cellular sub-compartments of genetically-targeted neurons, 2) generating contrast under different EM modalities, and 3) segmenting labelled structures from EM volumes using computer-assisted strategies. We also tested non-destructive X-ray imaging on whole Drosophila brains to evaluate contrast staining. This enabled us to target specific regions for EM volume acquisition.
Asunto(s)
Drosophila/genética , Imagenología Tridimensional/métodos , Microscopía Electrónica/métodos , Neuronas/ultraestructura , Animales , Animales Modificados Genéticamente , Axones/ultraestructura , Dendritas/ultraestructura , Drosophila/ultraestructura , Aumento de la Imagen , Rayos XRESUMEN
The aim of this study was to investigate the capability of X-ray microtomography to obtain information relating to powder characteristics such as wall thickness and solid volume fraction for hollow, polymer-stabilised spray dried dispersion (SDD) particles. SDDs of varying particle properties, with respect to shell wall thickness and degree of particle collapse, were utilised to assess the capability of the approach. The results demonstrate that the approach can provide insight into the morphological characteristics of these hollow particles, and thereby a means to understand/predict the processability and performance characteristics of the bulk material. Quantitative assessments of particle wall thickness, particle/void volume and thereby solid volume fraction were also demonstrated to be achievable. The analysis was also shown to be able to qualitatively assess the impact of the drying rate on the morphological nature of the particle surfaces, thus providing further insight into the final particle shape. The approach demonstrated a practical means to access potentially important particle characteristics for SDD materials which, in addition to the standard bulk powder measurements such as particle size and bulk density, may enable a better understanding of such materials, and their impact on downstream processability and dosage form performance.
Asunto(s)
Polímeros/química , Tecnología Farmacéutica/métodos , Microtomografía por Rayos X/métodos , Tamaño de la Partícula , PolvosRESUMEN
The recently developed three-dimensional electron microscopic (EM) method of serial block-face scanning electron microscopy (SBEM) has rapidly established itself as a powerful imaging approach. Volume EM imaging with this scanning electron microscopy (SEM) method requires intense staining of biological specimens with heavy metals to allow sufficient back-scatter electron signal and also to render specimens sufficiently conductive to control charging artifacts. These more extreme heavy metal staining protocols render specimens light opaque and make it much more difficult to track and identify regions of interest (ROIs) for the SBEM imaging process than for a typical thin section transmission electron microscopy correlative light and electron microscopy study. We present a strategy employing X-ray microscopy (XRM) both for tracking ROIs and for increasing the efficiency of the workflow used for typical projects undertaken with SBEM. XRM was found to reveal an impressive level of detail in tissue heavily stained for SBEM imaging, allowing for the identification of tissue landmarks that can be subsequently used to guide data collection in the SEM. Furthermore, specific labeling of individual cells using diaminobenzidine is detectable in XRM volumes. We demonstrate that tungsten carbide particles or upconverting nanophosphor particles can be used as fiducial markers to further increase the precision and efficiency of SBEM imaging.
Asunto(s)
Encéfalo/ultraestructura , Microscopía Electrónica de Rastreo/métodos , Animales , Imagenología Tridimensional , Ratones , Microscopía Electrónica de Rastreo/instrumentaciónRESUMEN
Modifications to the gene encoding human α-synuclein have been linked to the development of Parkinson's disease. The highly conserved structure of α-synuclein suggests a functional interaction with membranes, and several lines of evidence point to a role in vesicle-related processes within nerve terminals. Using recombinant fusions of human α-synuclein, including new genetic tags developed for correlated light microscopy and electron microscopy (the tetracysteine-biarsenical labeling system or the new fluorescent protein for electron microscopy, MiniSOG), we determined the distribution of α-synuclein when overexpressed in primary neurons at supramolecular and cellular scales in three dimensions (3D). We observed specific association of α-synuclein with a large and otherwise poorly characterized membranous organelle system of the presynaptic terminal, as well as with smaller vesicular structures within these boutons. Furthermore, α-synuclein was localized to multiple elements of the protein degradation pathway, including multivesicular bodies in the axons and lysosomes within neuronal cell bodies. Examination of synapses in brains of transgenic mice overexpressing human α-synuclein revealed alterations of the presynaptic endomembrane systems similar to our findings in cell culture. Three-dimensional electron tomographic analysis of enlarged presynaptic terminals in several brain areas revealed that these terminals were filled with membrane-bounded organelles, including tubulovesicular structures similar to what we observed in vitro. We propose that α-synuclein overexpression is associated with hypertrophy of membrane systems of the presynaptic terminal previously shown to have a role in vesicle recycling. Our data support the conclusion that α-synuclein is involved in processes associated with the sorting, channeling, packaging, and transport of synaptic material destined for degradation.
Asunto(s)
Neuronas/química , Neuronas/metabolismo , Enfermedad de Parkinson/metabolismo , alfa-Sinucleína/análisis , alfa-Sinucleína/biosíntesis , Animales , Células Cultivadas , Células HEK293 , Humanos , Ratones , Ratones Transgénicos , Microscopía Electrónica/métodos , Microscopía de Polarización/métodos , Neuronas/ultraestructura , Enfermedad de Parkinson/patología , Ratas , Ratas Sprague-Dawley , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/ultraestructura , alfa-Sinucleína/genéticaRESUMEN
The hippocampal mossy fiber (MF) terminal is among the largest and most complex synaptic structures in the brain. Our understanding of the development of this morphologically elaborate structure has been limited because of the inability of standard electron microscopy techniques to quickly and accurately reconstruct large volumes of neuropil. Here we use serial block-face electron microscopy (SBEM) to surmount these limitations and investigate the establishment of MF connectivity during mouse postnatal development. Based on volume reconstructions, we find that MF axons initially form bouton-like specializations directly onto dendritic shafts, that dendritic protrusions primarily arise independently of bouton contact sites, and that a dramatic increase in presynaptic and postsynaptic complexity follows the association of MF boutons with CA3 dendritic protrusions. We also identify a transient period of MF bouton filopodial exploration, followed by refinement of sites of synaptic connectivity. These observations enhance our understanding of the development of this highly specialized synapse and illustrate the power of SBEM to resolve details of developing microcircuits at a level not easily attainable with conventional approaches.
Asunto(s)
Microscopía Electrónica/métodos , Fibras Musgosas del Hipocampo/ultraestructura , Fibras Nerviosas/ultraestructura , Sinapsis/ultraestructura , Animales , Animales Recién Nacidos , Axones/ultraestructura , Dendritas/ultraestructura , Procesamiento de Imagen Asistido por Computador , Ratones , Ratones Endogámicos C57BL , Neurópilo/ultraestructura , Terminales Presinápticos/ultraestructura , Seudópodos/ultraestructura , Control de Calidad , Programas InformáticosAsunto(s)
Enfermería de Práctica Avanzada/educación , Educación de Postgrado en Enfermería/organización & administración , Intercambio Educacional Internacional , Enfermeras Clínicas/educación , Aculturación , Barreras de Comunicación , Humanos , Lenguaje , Aprendizaje , Licencia en Enfermería/legislación & jurisprudencia , Enfermeras Clínicas/psicología , Investigación en Educación de Enfermería , Investigación en Evaluación de Enfermería , Investigación Metodológica en Enfermería , Criterios de Admisión Escolar , Aislamiento Social , Estados UnidosRESUMEN
Electron tomography (ET) has been proven an essential technique for imaging the structure of cells beyond the range of the light microscope down to the molecular level. Large-field high-resolution views of biological specimens span more than four orders of magnitude in spatial scale, and, as a consequence, are rather difficult to generate directly. Various techniques have been developed towards generating those views, from increasing the sensor array size to implementing serial sectioning and montaging. Datasets and reconstructions obtained by the latter techniques generate multiple three-dimensional (3D) reconstructions, that need to be combined together to provide all the multiscale information. In this work, we show how to implement montages within TxBR, a tomographic reconstruction software package. This work involves some new application of mathematical concepts related to volume preserving transformations and issues of gauge ambiguity, which are essential problems arising from the nature of the observation in an electron microscope. The purpose of TxBR is to handle those issues as generally as possible in order to correct for most distortions in the 3D reconstructions and allow for a seamless recombination of ET montages.
Asunto(s)
Algoritmos , Tomografía con Microscopio Electrónico/métodos , Imagenología Tridimensional/métodos , Programas Informáticos , Animales , Encéfalo/citología , Drosophila/citología , Drosophila/virología , Virus de Insectos/fisiología , Ratones , Neuronas/ultraestructuraRESUMEN
The striated organelle (SO), a cytoskeletal structure located in the apical region of cochlear and vestibular hair cells, consists of alternating, cross-linked, thick and thin filamentous bundles. In the vestibular periphery, the SO is well developed in both type I and type II hair cells. We studied the 3D structure of the SO with intermediate-voltage electron microscopy and electron microscope tomography. We also used antibodies to α-2 spectrin, one protein component, to trace development of the SO in vestibular hair cells over the first postnatal week. In type I cells, the SO forms an inverted open-ended cone attached to the cell membrane along both its upper and lower circumferences and separated from the cuticular plate by a dense cluster of exceptionally large mitochondria. In addition to contacts with the membrane and adjacent mitochondria, the SO is connected both directly and indirectly, via microtubules, to some stereociliary rootlets. The overall architecture of the apical region in type I hair cells--a striated structure restricting a cluster of large mitochondria between its filaments, the cuticular plate, and plasma membrane--suggests that the SO might serve two functions: to maintain hair-cell shape and to alter transduction by changing the geometry and mechanical properties of hair bundles.
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Citoesqueleto/metabolismo , Células Ciliadas Auditivas/citología , Orgánulos/metabolismo , Actinas/metabolismo , Animales , Membrana Celular/metabolismo , Chinchilla , Microscopía Confocal/métodos , Microscopía Electrónica/métodos , Microscopía Electrónica de Transmisión/métodos , Mitocondrias/metabolismo , Modelos Biológicos , Ratas , Ratas Long-Evans , Estereocilios/metabolismo , Tomografía/métodosRESUMEN
Dominant missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic causes of Parkinson disease (PD) and genome-wide association studies identify LRRK2 sequence variants as risk factors for sporadic PD. Intact kinase function appears critical for the toxicity of LRRK2 PD mutants, yet our understanding of how LRRK2 causes neurodegeneration remains limited. We find that most LRRK2 PD mutants abnormally enhance LRRK2 oligomerization, causing it to form filamentous structures in transfections of cell lines or primary neuronal cultures. Strikingly, ultrastructural analyses, including immuno-electron microscopy and electron microscopic tomography, demonstrate that these filaments consist of LRRK2 recruited onto part of the cellular microtubule network in a well-ordered, periodic fashion. Like LRRK2-related neurodegeneration, microtubule association requires intact kinase function and the WD40 domain, potentially linking microtubule binding and neurodegeneration. Our observations identify a novel effect of LRRK2 PD mutations and highlight a potential role for microtubules in the pathogenesis of LRRK2-related neurodegeneration.
Asunto(s)
Microtúbulos/metabolismo , Mutación/genética , Enfermedad de Parkinson/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Células Cultivadas , Células HEK293 , Células HeLa , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Ratones , Modelos Biológicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Unión Proteica/genética , Multimerización de Proteína , Proteínas Serina-Treonina Quinasas/química , Estructura Terciaria de ProteínaRESUMEN
Recent advances in high-throughput technology facilitate massive data collection and sharing, enabling neuroscientists to explore the brain across a large range of spatial scales. One such form of high-throughput data collection is the construction of large-scale mosaic volumes using light microscopy (Chow et al., 2006; Price et al., 2006). With this technology, researchers can collect and analyze high-resolution digitized volumes of whole brain sections down to 0.2 µm. However, until recently, scientists lacked the tools to easily handle these large high-resolution datasets. Furthermore, artifacts resulting from specimen preparation limited volume reconstruction using this technique to only a single tissue section. In this paper, we carefully describe the steps we used to digitally reconstruct a series of consecutive mouse brain sections labeled with three stains, a stain for blood vessels (DiI), a nuclear stain (TO-PRO-3), and a myelin stain (FluoroMyelin). These stains label important neuroanatomical landmarks that are used for stacking the serial sections during reconstruction. In addition, we show that the use of two software applications, ir-Tweak and Mogrifier, in conjunction with a volume flattening procedure enable scientists to adeptly work with digitized volumes despite tears and thickness variations within tissue sections. These applications make processing large-scale brain mosaics more efficient. When used in combination with new database resources, these brain maps should allow researchers to extend the lifetime of their specimens indefinitely by preserving them in digital form, making them available for future analyses as our knowledge in the field of neuroscience continues to expand.
RESUMEN
Electron microscope tomography [1] has been proven as an essential technique for imaging the structure of cells beyond the range of the light microscope down to the molecular level. However, because of the extreme difference in spatial scales, there is a large gap to be bridged between light and electron microscopy. Various techniques have been developed, including increasing size of the sensor arrays, serial sectioning and montaging. Data sets and reconstructions obtained by the latter techniques generate many 3D reconstructions that need to be glued together to provide information at a larger spatial scale. However, during the course of data acquisition, thin slices may become warped in optical and electron microscope preparations. We review some procedures for de-warping sections and reassembling them into larger reconstructions, and present some data from electron microscopy.
Asunto(s)
Algoritmos , Interpretación de Imagen Asistida por Computador/métodos , Microscopía Electrónica/métodos , Reconocimiento de Normas Patrones Automatizadas/métodos , Técnica de Sustracción , Tomografía Óptica/métodos , Aumento de la Imagen/métodos , Sensibilidad y EspecificidadRESUMEN
The purpose of this prospective descriptive study was to understand 1) the extent to which certified nurse midwives (CNMs) utilize complementary and alternative medicine (CAM), 2) how knowledge regarding CAM is gained, and 3) characteristics of these providers. A survey was designed to determine use of CAM treatment modalities: herbal preparations, pharmacologic/biologic treatments, manual healing/bioelectromagnetic applications, mind-body interventions, and diet and lifestyle. Random sampling was used to select participants (n=500) who were members of the American College of Nurse Midwives. Of those CNMs responding (n=227, 45%), 78% (n=178) reported use of CAM. Herbal preparations were most commonly used (85%), followed by pharmacologic/biologic treatments (82%), mind-body interventions (80%), and manual healing/bioelectromagnetic therapies (47%). Virtually all CAM user respondents utilized at least one diet and lifestyle therapy.
Asunto(s)
Terapias Complementarias/estadística & datos numéricos , Enfermeras Obstetrices/estadística & datos numéricos , Pautas de la Práctica en Enfermería/estadística & datos numéricos , Recolección de Datos , Femenino , Conocimientos, Actitudes y Práctica en Salud , Humanos , Terapia Nutricional/estadística & datos numéricos , Embarazo , Estudios Prospectivos , Sociedades de Enfermería , Estados UnidosRESUMEN
The emergence of electron tomography as a tool for three dimensional structure determination of cells and tissues has brought its own challenges for the preparation of thick sections. High pressure freezing in combination with freeze substitution provides the best method for obtaining the largest volume of well-preserved tissue. However, for deeply embedded, heterogeneous, labile tissues needing careful dissection, such as brain, the damage due to anoxia and excision before cryofixation is significant. We previously demonstrated that chemical fixation prior to high pressure freezing preserves fragile tissues and produces superior tomographic reconstructions compared to equivalent tissue preserved by chemical fixation alone. Here, we provide further characterization of the technique, comparing the ultrastructure of Flock House Virus infected DL1 insect cells that were (1) high pressure frozen without fixation, (2) high pressure frozen following fixation, and (3) conventionally prepared with aldehyde fixatives. Aldehyde fixation prior to freezing produces ultrastructural preservation superior to that obtained through chemical fixation alone that is close to that obtained when cells are fast frozen without fixation. We demonstrate using a variety of nervous system tissues, including neurons that were injected with a fluorescent dye and then photooxidized, that this technique provides excellent preservation compared to chemical fixation alone and can be extended to selectively stained material where cryofixation is impractical.
Asunto(s)
Criopreservación/métodos , Drosophila/ultraestructura , Neuronas/ultraestructura , Fijación del Tejido/métodos , Tomografía/métodos , Animales , Drosophila/virología , Presión , Internalización del VirusRESUMEN
Reactive astrocytes in neurotrauma, stroke, or neurodegeneration are thought to undergo cellular hypertrophy, based on their morphological appearance revealed by immunohistochemical detection of glial fibrillary acidic protein, vimentin, or nestin, all of them forming intermediate filaments, a part of the cytoskeleton. Here, we used a recently established dye-filling method to reveal the full three-dimensional shape of astrocytes assessing the morphology of reactive astrocytes in two neurotrauma models. Both in the denervated hippocampal region and the lesioned cerebral cortex, reactive astrocytes increased the thickness of their main cellular processes but did not extend to occupy a greater volume of tissue than nonreactive astrocytes. Despite this hypertrophy of glial fibrillary acidic protein-containing cellular processes, interdigitation between adjacent hippocampal astrocytes remained minimal. This work helps to redefine the century-old concept of hypertrophy of reactive astrocytes.
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Astrocitos/patología , Lesiones Encefálicas/patología , Animales , Forma de la Célula , Femenino , RatonesRESUMEN
We describe the technique and application of energy filtering, automated most-probable loss (MPL) tomography to intermediate voltage electron microscopy (IVEM). We show that for thick, selectively stained biological specimens, this method produces a dramatic increase in resolution of the projections and the computed volumes versus standard unfiltered transmission electron microscopy (TEM) methods. This improvement in resolution is attributed to the reduction of chromatic aberration, which results from the large percentage of inelastic electron-scattering events for thick specimens. These improvements are particularly evident at the large tilt angles required to improve tomographic resolution in the z-direction. This method effectively increases the usable thickness of selectively stained samples that can be imaged at a given accelerating voltage by dramatically improving resolution versus unfiltered TEM and increasing signal-to-noise versus zero-loss imaging, thereby expanding the utility of the IVEM to deliver information from within specimens up to 3 microm thick.
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Técnicas de Laboratorio Clínico , Microscopía Electrónica de Transmisión/métodos , Tomografía/métodos , Animales , Dendritas/ultraestructura , Electrones , Hipocampo/ultraestructura , Procesamiento de Imagen Asistido por Computador , Ratones , Microscopía Electrónica , Microscopía Electrónica de Transmisión/instrumentación , Modelos Estadísticos , Modelos Teóricos , Dispersión de Radiación , Coloración y EtiquetadoRESUMEN
Growth, loading, and mobilization lead to changes in tendon structure. Recent studies have shown that proteoglycans (PGs) regulate the organization of collagen fibrils, the main structural components of tendons. We hypothesized that moderate exercise alters PG synthesis in the avian gastrocnemius tendon. To test our hypothesis we compared the PG content in gastrocnemius tendons from control 6.5-week-old chickens with that in tendons from 6.5-week-old chickens that underwent exercise. Our results show high levels and a wide variety of glycosaminoglycans (GAGs) in 6.5-week-old tendons. Chondroitin-4-sulfate disaccharide was the major GAG disaccharide in control and exercised 6.5-week-old gastrocnemius tendons. Exercise led to an increase in the size of the tendons, the content of hyaluronic acid, and the level of decorin. High levels of keratan sulfate (KS) were found in the lower halves of gastrocnemius tendons, although the amount of KS decreased with exercise. This corresponded well with lower content of aggrecan in the lower halves of exercised tendons. In conclusion, our data support the hypothesis that exercise alters the content of PGs in chicken tendons.
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Proteínas de la Matriz Extracelular , Esfuerzo Físico/fisiología , Proteoglicanos/metabolismo , Tendones/metabolismo , Agrecanos , Animales , Pollos , Sulfatos de Condroitina/metabolismo , Decorina , Ácido Hialurónico/metabolismo , Sulfato de Queratano/metabolismo , Lectinas Tipo C , Modelos BiológicosRESUMEN
Reaction of a permetallated ethene, (mu4-C=C)Fe2R-mu2Cp*2(CO)10, with diphenylacetylene affords the adduct with a new C2-binding mode, a mu4-dicarbyne complex, (mu4-C-C)Fe2Ru2Cp*2(CO)6(mu-Ph-C[triple bond]C-Ph), containing a dimetallacyclobutariene core.