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1.
Nat Commun ; 11(1): 541, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992713

RESUMO

The fast development of high-resolution electron microscopy (EM) demands a background-noise-free substrate to support the specimens, where atomically thin graphene membranes can serve as an ideal candidate. Yet the preparation of robust and ultraclean graphene EM grids remains challenging. Here we present a polymer- and transfer-free direct-etching method for batch fabrication of robust ultraclean graphene grids through membrane tension modulation. Loading samples on such graphene grids enables the detection of single metal atoms and atomic-resolution imaging of the iron core of ferritin molecules at both room- and cryo-temperature. The same kind of hydrophilic graphene grid allows the formation of ultrathin vitrified ice layer embedded most protein particles at the graphene-water interface, which facilitates cryo-EM 3D reconstruction of archaea 20S proteasomes at a record high resolution of ~2.36 Å. Our results demonstrate the significant improvements in image quality using the graphene grids and expand the scope of EM imaging.


Assuntos
Grafite/química , Microscopia Eletrônica/instrumentação , Microscopia Eletrônica/métodos , Fenômenos Químicos , Microscopia Crioeletrônica/métodos , Elétrons , Desenho de Equipamento , Processamento de Imagem Assistida por Computador , Membranas , Polímeros , Proteínas
2.
Zhongguo Yi Liao Qi Xie Za Zhi ; 43(5): 388-390, 2019 Sep 30.
Artigo em Chinês | MEDLINE | ID: mdl-31625344

RESUMO

OBJECTIVE: Improve the integrity of the digestive electron microscope equipment and reduce the cost of equipment failure maintenance. METHODS: By studying the composition and function of the digestive electron microscope system and analyzing the causes of common faults, a targeted preventive maintenance plan is developed, equipment users are graded, and a training system is established. RESULTS: The user of the device can skillfully analyze the cause of the malfunction and timely deal with the sudden failure of the diagnosis and treatment, thereby reduce the risk of diagnosis and treatment and the investment in hospital maintenance. CONCLUSIONS: Through the analysis and processing of the digestive electron microscope system, point detection leakage, grading training, preventive maintenance can significantly improve the equipment integrity rate, reduce the risk of clinical diagnosis and treatment, effectively reduce the number of equipment failures, and reduce maintenance costs.


Assuntos
Serviço Hospitalar de Engenharia e Manutenção , Microscopia Eletrônica , Falha de Equipamento , Microscopia Eletrônica/instrumentação
3.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 1): 19-32, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30605122

RESUMO

Single-particle analysis by electron microscopy is a well established technique for analyzing the three-dimensional structures of biological macromolecules. Besides its ability to produce high-resolution structures, it also provides insights into the dynamic behavior of the structures by elucidating their conformational variability. Here, the different image-processing methods currently available to study continuous conformational changes are reviewed.


Assuntos
Elétrons , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Imageamento Tridimensional/estatística & dados numéricos , Substâncias Macromoleculares/ultraestrutura , Microscopia Eletrônica/métodos , Proteínas/ultraestrutura , Algoritmos , Humanos , Substâncias Macromoleculares/química , Microscopia Eletrônica/instrumentação , Conformação Molecular , Simulação de Dinâmica Molecular , Análise de Componente Principal , Proteínas/química , Termodinâmica
4.
Methods Mol Biol ; 1880: 199-209, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610698

RESUMO

Live-cell imaging has been widely used to study autophagosome biogenesis and maturation. When combined with correlative electron microscopy, this approach can be extended to reveal ultrastructural details in three dimensions. The resolution of electron microscopy is needed when membrane contact sites and tubular connections between organelles are studied.


Assuntos
Autofagossomos/ultraestrutura , Microscopia Confocal/métodos , Microscopia Eletrônica/métodos , Sobrevivência Celular , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador/métodos , Microscopia Eletrônica/instrumentação , Microtomia/métodos , Proteínas Associadas aos Microtúbulos/análise , Imagem Óptica/métodos , Software
5.
Methods Mol Biol ; 1880: 541-554, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610721

RESUMO

Acute pancreatitis is one of the first pathological processes where autophagy has been described in a human tissue. Autophagy, autodigestion, and cell death are early cellular events in acute pancreatitis. Recent advances in understanding autophagy highlight its importance in pathological conditions. However, methods for monitoring autophagic activity during complex diseases, involving highly differentiated secretory cells, are complicated, and the results are sometimes misinterpreted. Here, we describe methods used to identify autophagic structures and to measure autophagic flux in cultured cell models and animal models of pancreatitis. We also briefly describe the pancreas specific autophagy mouse model that was useful to understand the actual role of autophagy in pancreatitis and to identify a novel selective autophagy pathway named zymophagy. Lastly, we describe the immunomagnetic isolation of autophagosomes and the detection of autophagy in pancreatic tissue samples derived from humans.


Assuntos
Autofagossomos/patologia , Autofagia , Precursores Enzimáticos/metabolismo , Pâncreas/patologia , Pancreatite/patologia , Células Acinares , Animais , Autofagossomos/ultraestrutura , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Linhagem Celular , Ceruletídeo/toxicidade , Modelos Animais de Doenças , Humanos , Lisossomos/metabolismo , Masculino , Camundongos , Microscopia Eletrônica/instrumentação , Microscopia Eletrônica/métodos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Pâncreas/citologia , Pancreatectomia , Pancreatite/induzido quimicamente , Pancreatite/cirurgia , Ratos , Vesículas Secretórias/patologia
6.
Adv Mater ; 31(12): e1806788, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30680803

RESUMO

Owing to their ability to efficiently deliver biological cargo and sense the intracellular milieu, vertical arrays of high aspect ratio nanostructures, known as nanoneedles, are being developed as minimally invasive tools for cell manipulation. However, little is known of the mechanisms of cargo transfer across the cell membrane-nanoneedle interface. In particular, the contributions of membrane piercing, modulation of membrane permeability and endocytosis to cargo transfer remain largely unexplored. Here, combining state-of-the-art electron and scanning ion conductance microscopy with molecular biology techniques, it is shown that porous silicon nanoneedle arrays concurrently stimulate independent endocytic pathways which contribute to enhanced biomolecule delivery into human mesenchymal stem cells. Electron microscopy of the cell membrane at nanoneedle sites shows an intact lipid bilayer, accompanied by an accumulation of clathrin-coated pits and caveolae. Nanoneedles enhance the internalization of biomolecular markers of endocytosis, highlighting the concurrent activation of caveolae- and clathrin-mediated endocytosis, alongside macropinocytosis. These events contribute to the nanoneedle-mediated delivery (nanoinjection) of nucleic acids into human stem cells, which distribute across the cytosol and the endolysosomal system. This data extends the understanding of how nanoneedles modulate biological processes to mediate interaction with the intracellular space, providing indications for the rational design of improved cell-manipulation technologies.


Assuntos
Sistemas de Liberação de Medicamentos/instrumentação , Endocitose/fisiologia , Nanopartículas/química , Agulhas , Silício/química , Cavéolas/metabolismo , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Clatrina/administração & dosagem , Clatrina/metabolismo , Citosol/metabolismo , Endossomos/metabolismo , Humanos , Espaço Intracelular/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Microscopia Eletrônica/instrumentação , Pinocitose/efeitos dos fármacos , Porosidade , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/metabolismo , Propriedades de Superfície
7.
Chromosoma ; 128(1): 7-13, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30175387

RESUMO

The high-order structure of metaphase chromosomes remains still under investigation, especially the 30-nm structure that is still controversial. Advanced 3D imaging has provided useful information for our understanding of this detailed structure. It is evident that new technologies together with improved sample preparations and image analyses should be adequately combined. This mini review highlights 3D imaging used for chromosome analysis so far with future imaging directions also highlighted.


Assuntos
Cromossomos/ultraestrutura , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Imageamento Tridimensional/métodos , Microscopia Eletrônica/métodos , Coloração e Rotulagem/métodos , Animais , Proteínas Cromossômicas não Histona/ultraestrutura , DNA/ultraestrutura , Histonas/ultraestrutura , Hordeum/genética , Hordeum/ultraestrutura , Humanos , Imageamento Tridimensional/instrumentação , Imuno-Histoquímica/métodos , Metáfase , Microscopia de Força Atômica , Microscopia Eletrônica/instrumentação , Manejo de Espécimes/instrumentação , Manejo de Espécimes/métodos
8.
Methods Mol Biol ; 1860: 361-377, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30317518

RESUMO

SNARE-mediated membrane fusion is required for membrane trafficking as well as organelle biogenesis and homeostasis. The membrane fusion reaction involves sequential formation of hemifusion intermediates, whereby lipid monolayers partially mix on route to complete bilayer merger. Studies of the Saccharomyces cerevisiae lysosomal vacuole have revealed many of the fundamental mechanisms that drive the membrane fusion process, as well as features unique to organelle fusion. However, until recently, it has not been amenable to electron microscopy methods that have been invaluable for studying hemifusion in other model systems. Herein, we describe a method to visualize hemifusion intermediates during homotypic vacuole membrane fusion in vitro by transmission electron microscopy (TEM), electron tomography, and cryogenic electron microscopy (cryoEM). This method facilitates acquisition of invaluable ultrastructural data needed to comprehensively understand how fusogenic lipids and proteins contribute to SNARE-mediated membrane fusion-by-hemifusion and the unique features of organelle versus small-vesicle fusion.


Assuntos
Microscopia Eletrônica/métodos , Proteínas SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacúolos/ultraestrutura , Bicamadas Lipídicas/metabolismo , Fusão de Membrana , Microscopia Eletrônica/instrumentação , Ligação Proteica , Proteínas SNARE/ultraestrutura , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Vacúolos/metabolismo
9.
J Hist Biol ; 52(1): 125-160, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-29926225

RESUMO

This paper examines the vital role played by electron microscopy toward the modern definition of viruses, as formulated in the late 1950s. Before the 1930s viruses could neither be visualized by available technologies nor grown in artificial media. As such they were usually identified by their ability to cause diseases in their hosts and defined in such negative terms as "ultramicroscopic" or invisible infectious agents that could not be cultivated outside living cells. The invention of the electron microscope, with magnification and resolution powers several orders of magnitude better than that of optical instruments, opened up possibilities for biological applications. The hitherto invisible viruses lent themselves especially well to investigation with this new instrument. We first offer a historical consideration of the development of the instrument and, more significantly, advances in techniques for preparing and observing specimens that turned the electron microscope into a routine biological tool. We then describe the ways in which the electron microscopic images, or micrographs, functioned as forms of new knowledge about viruses and resulted in a paradigm shift in the very definition of these entities. Micrographs were not mere illustrations since they did the work for the electron microscopists. Drawing extensively on primary publications, we adduce the role of the new instrument in understanding the so-called eclipse phase in virus multiplication and the unexpected spinoffs of data from electron microscopy in naming and classifying viruses. Thus, we show that electron microscopy functioned not only to provide evidence, but also arguments in facilitating a reordering of the world that it brought into the visual realm.


Assuntos
Microscopia Eletrônica/história , Virologia/história , Vírus/ultraestrutura , Artefatos , Bacteriófagos/fisiologia , Bacteriófagos/ultraestrutura , História do Século XX , Microscopia Eletrônica/instrumentação , Fenômenos Fisiológicos Virais , Vírus/classificação
10.
J Biomed Sci ; 25(1): 91, 2018 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-30580764

RESUMO

Extracellular vesicles (EVs) are lipid bilayer-enclosed nanoparticles released by cells. They range from 30 nm to several micrometers in diameter, and ferry biological cargos such as proteins, lipids, RNAs and DNAs for local and distant intercellular communications. EVs have since been found to play a role in development, as well as in diseases including cancers. To elucidate the roles of EVs, researchers have established different methods to visualize and study their spatiotemporal properties. However, since EV are nanometer-sized, imaging them demands a full understanding of each labeling strategy to ensure accurate monitoring. This review covers current and emerging strategies for EV imaging for prospective studies.


Assuntos
Diagnóstico por Imagem/métodos , Vesículas Extracelulares/fisiologia , Microscopia Eletrônica/métodos , Diagnóstico por Imagem/instrumentação , Vesículas Extracelulares/ultraestrutura , Microscopia Eletrônica/instrumentação
11.
Lab Chip ; 18(24): 3840-3848, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30417906

RESUMO

Fluorescence light microscopy (FLM) is commonly used for localizing specific cellular and subcellular targets. Electron microscopy (EM), on the other hand, can reveal ultrastructural details of cellular architectures beyond the limit of optical resolution. Correlative light and electron microscopy (CLEM) that combines the two techniques has proven valuable in various cell biological applications that require both specificity and resolution. Here, we report an efficient and easy-to-use CLEM system, and its applications in studying neuronal synapses. The system utilizes patterned symbols to encode coordinates on micro-fabricated polydimethylsiloxane (PDMS) substrates, on which dissociated primary hippocampal neurons grow and form synaptic connections. After imaging and localizing specifically labeled synapses with FLM, samples are embedded in resin blocks and sectioned for EM analysis. The patterned symbols on PDMS substrates provide coordinate information, allowing efficient co-registration between FLM and EM images with high precision. A custom-developed software package achieves automated EM image collection, FLM/EM alignment, and EM navigation. With this CLEM system, we have obtained high quality electron tomograms of fluorescently labeled synapses along dendrites of hippocampal neurons and analyzed docking statistics of synaptic vesicles (SVs) in different subtypes of excitatory synapses. This technique provides an efficient approach to combine functional studies with ultrastructural analysis of heterogeneous neuronal synapses, as well as other subcellular structures in general.


Assuntos
Técnicas Citológicas/instrumentação , Dimetilpolisiloxanos/química , Processamento de Imagem Assistida por Computador/métodos , Microscopia Eletrônica/instrumentação , Microscopia de Fluorescência/instrumentação , Animais , Células Cultivadas , Técnicas Citológicas/métodos , Desenho de Equipamento , Hipocampo/citologia , Microscopia Eletrônica/métodos , Microscopia de Fluorescência/métodos , Neurônios/citologia , Ratos
12.
Eur J Histochem ; 62(4)2018 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-30418011

RESUMO

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.


Assuntos
Pesquisa Biomédica/tendências , Microscopia Eletrônica/instrumentação , Microscopia Eletrônica/tendências , Microscopia Eletrônica/normas
13.
Nat Methods ; 15(10): 777-780, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30275574

RESUMO

Single-particle reconstruction (SPR) from electron microscopy (EM) images is widely used in structural biology, but it lacks direct information on protein identity. To address this limitation, we developed a computational and analytical framework that reconstructs and coaligns multiple proteins from 2D super-resolution fluorescence images. To demonstrate our method, we generated multicolor 3D reconstructions of several proteins within the human centriole, which revealed their relative locations, dimensions and orientations.


Assuntos
Centríolos/ultraestrutura , Processamento de Imagem Assistida por Computador/métodos , Microscopia Eletrônica/instrumentação , Microscopia Eletrônica/métodos , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Imagem Individual de Molécula/métodos , Algoritmos , Centríolos/metabolismo , Humanos
14.
Curr Opin Microbiol ; 43: 132-138, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29414444

RESUMO

Understanding how microbes utilize their environment is aided by visualizing them in their natural context at high resolution. Correlative imaging enables efficient targeting and identification of labelled viral and bacterial components by light microscopy combined with high resolution imaging by electron microscopy. Advances in genetic and bioorthogonal labelling, improved workflows for targeting and image correlation, and large-scale data collection are increasing the applicability of correlative imaging methods. Furthermore, developments in mass spectroscopy and soft X-ray imaging are expanding the correlative imaging modalities available. Investigating the structure and organization of microbes within their host by combined imaging methods provides important insights into mechanisms of infection and disease which cannot be obtained by other techniques.


Assuntos
Interações entre Hospedeiro e Microrganismos , Microscopia Eletrônica/métodos , Microscopia/métodos , Imagem Individual de Molécula/instrumentação , Bactérias/ultraestrutura , Doenças Transmissíveis/microbiologia , Humanos , Espectrometria de Massas , Microscopia/instrumentação , Microscopia Eletrônica/instrumentação , Imagem Individual de Molécula/métodos , Vírus/ultraestrutura
15.
Methods Cell Biol ; 143: 1-39, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29310772

RESUMO

This chapter gives insight into task-specific methodologies for the evaluation of matrix ultrastructure by light and electron microscopy. It separately considers the isolation and preparation of molecular isolates for negative staining, immunolabeling, rotary shadowing, and single particle analysis. Also considered is the preparation of whole tissues and cultured cells by chemical fixation and cryofixation methodologies. Immunoelectron microscopy for immunoidentification of matrix components may be accomplished en bloc or via section-surface protocols; the advantages and pitfalls in both methodologies are described. Correlative light and electron microscopy, particularly utilizing GFP constructs, demands special consideration in fixation and embedding protocols. TEM imaging methods, including the use of montage software and the acquisition of thick-section tilt series are discussed. The protocols presented in this chapter, with the exception of single particle analysis, are those which are continuously used in our laboratory and represent the latest modifications in our protocols.


Assuntos
Criopreservação/métodos , Matriz Extracelular/ultraestrutura , Microscopia Eletrônica/métodos , Fixação de Tecidos/métodos , Animais , Células Cultivadas/citologia , Células Cultivadas/ultraestrutura , Colágeno/ultraestrutura , Criopreservação/instrumentação , Humanos , Camundongos , Microscopia Eletrônica/instrumentação , Pele/citologia , Pele/ultraestrutura , Coloração e Rotulagem/métodos
16.
Appl Spectrosc ; 71(9): 2051-2075, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28714316

RESUMO

We present a review of the use of selected nanofabricated thin films to deliver a host of capabilities and insights spanning bioanalytical and biophysical chemistry, materials science, and fundamental molecular-level research. We discuss approaches where thin films have been vital, enabling experimental studies using a variety of optical spectroscopies across the visible and infrared spectral range, electron microscopies, and related techniques such as electron energy loss spectroscopy, X-ray photoelectron spectroscopy, and single molecule sensing. We anchor this broad discussion by highlighting two particularly exciting exemplars: a thin-walled nanofluidic sample cell concept that has advanced the discovery horizons of ultrafast spectroscopy and of electron microscopy investigations of in-liquid samples; and a unique class of thin-film-based nanofluidic devices, designed around a nanopore, with expansive prospects for single molecule sensing. Free-standing, low-stress silicon nitride membranes are a canonical structural element for these applications, and we elucidate the fabrication and resulting features-including mechanical stability, optical properties, X-ray and electron scattering properties, and chemical nature-of this material in this format. We also outline design and performance principles and include a discussion of underlying material preparations and properties suitable for understanding the use of alternative thin-film materials such as graphene.


Assuntos
Microscopia Eletrônica/instrumentação , Nanoestruturas , Nanotecnologia/instrumentação , Análise Espectral/instrumentação , Grafite , Técnicas Analíticas Microfluídicas , Compostos de Silício
17.
Methods Mol Biol ; 1471: 187-216, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28349397

RESUMO

Immunolocalization using either fluorescence for light microscopy (LM) or gold particles for electron microscopy (EM) has become a common tool to pinpoint proteins involved in recombination during meiotic prophase. Each method has its advantages and disadvantages. For example, LM immunofluorescence is comparatively easier and higher throughput compared to immunogold EM localization. In addition, immunofluorescence has the advantages that a faint signal can often be enhanced by longer exposure times and colocalization using two (or more) probes with different absorbance and emission spectra is straightforward. However, immunofluorescence is not useful if the object of interest does not label with an antibody probe and is below the resolution of the LM. In comparison, immunogold EM localization is higher resolution than immunofluorescent LM localization, and individual nuclear structures, such as recombination nodules, can be identified by EM regardless of whether they are labeled or not. However, immunogold localization has other disadvantages including comparatively low signal-to-noise ratios, more difficult colocalization using gold particles of different sizes, and the inability to evaluate labeling efficiency before examining the sample using EM (a more expensive and time-consuming technique than LM). Here we describe a method that takes advantage of the good points of both immunofluorescent LM and EM to analyze two classes of late recombination nodules (RNs), only one of which labels with antibodies to MLH1 protein, a marker of crossovers. The method can be used readily with other antibodies to analyze early recombination nodules or other prophase I structures.


Assuntos
Prófase Meiótica I , Microscopia/métodos , Recombinação Genética , Imunofluorescência , Imuno-Histoquímica , Lycopersicon esculentum/citologia , Microscopia/instrumentação , Microscopia Eletrônica/instrumentação , Microscopia Eletrônica/métodos
18.
Rev. iberoam. micol ; 34(1): 36-42, ene.-mar. 2017. ilus
Artigo em Espanhol | IBECS | ID: ibc-160733

RESUMO

Background. Lecanicillium fungicola causes dry bubble disease in Agaricus bisporus mushrooms leading to significant economic losses in commercial production. Aims. To monitor the infection process of L. fungicola in Brazilian strains of A. bisporus. Methods. The interaction between the mycelium of L. fungicola (LF.1) and three strains of A. bisporus (ABI 7, ABI 11/14 and ABI 11/21) was studied. Electron microscopy and X-ray microanalyses of vegetative growth and basidiocarp infection were evaluated. Results. Micrographs show that the vegetative mycelium of the Brazilian strains of A. bisporus is not infected by the parasite. The images show that the pathogen can interlace the hyphae of A. bisporus without causing damage, which contributes to the presence of L. fungicola during the substrate colonization, allowing their presence during primordial formation of A. bisporus. In the basidiocarp, germ tubes form within 16h of infection with L. fungicola and the beginning of penetration takes place within 18h, both without the formation of specialized structures. Conclusions. Scanning electron microscopy enabled the process of colonization and reproduction to be observed within the formation of phialides, conidiophores and verticils of L. fungicola. The formation of calcium oxalate crystals by the pathogen was also visible using the X-ray microanalysis, both at the hyphae in the Petri plate and at basidiocarp infection site (AU)


Antecedentes. Lecanicillium fungicola es el agente causal de la enfermedad de la mole seca en Agaricus bisporus, responsable de importantes pérdidas económicas en la producción comercial de esta seta. Objetivos. Comprobar el proceso de infección de L. fungicola en cepas brasileñas de A. bisporus. Métodos. Se estudió la interacción entre el micelio de L. fungicola (LF.1) y tres cepas de A. bisporus (ABI 7, ABI 11/14 y ABI 11/21). Se evaluaron mediante microscopia electrónica y microanálisis de rayos X el crecimiento vegetativo y la infección de los basidiocarpos. Resultados. Las micrografías muestran que el micelio vegetativo de las cepas brasileñas de A. bisporus no resultó afectado por la infección del parásito. Las imágenes muestran también cómo el agente patógeno puede entrelazar las hifas de A. bisporus sin causar daños, lo que contribuye a la perpetuación de L. fungicola durante la colonización del sustrato y durante la formación de los primordios de A. bisporus. En el basidiocarpo, los tubos germinales se forman después de 16h de la infección con L. fungicola y el comienzo de la penetración tiene lugar tras 18h, sin formación de estructuras especializadas. Conclusiones. La microscopia electrónica permite observar el proceso de colonización y reproducción con la formación de fiálides, conidióforos y verticilos de L. fungicola. La formación de cristales de oxalato de calcio por parte del agente patógeno también fue visible mediante el microanálisis por rayos X, tanto en la infección de las hifas en placa de Petri como en la de los basidiocarpos (AU)


Assuntos
Agaricus campestris/isolamento & purificação , Microanálise por Sonda Eletrônica/instrumentação , Microanálise por Sonda Eletrônica/tendências , Microscopia Eletrônica/instrumentação , Microscopia Eletrônica/métodos , Avaliação de Processos em Cuidados de Saúde/normas , Microanálise por Sonda Eletrônica/métodos , Microanálise por Sonda Eletrônica , Oxalato de Cálcio/uso terapêutico , Fungos/isolamento & purificação
19.
Anal Chem ; 88(22): 11070-11076, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27779407

RESUMO

Superlocalization of immunoplasmonic nanotags on antibody-bound gold-nanoislands (GNIs) along the x and y coordinates was determined using total internal reflection scattering-based super-resolution microscopy (TIRS-SRM) at subdiffraction limit resolution. Individual immunoplasmonic nanotags (20 nm silver nanoparticles) and 100 nm GNIs were selectively acquired in the evanescent field layer by wavelength-dependent plasmonic scattering using two illumination lasers (405 and 635 nm, respectively). α-Fetoprotein (AFP), a liver cancer-related model protein, was immobilized as a target molecule on the GNI arrays. The centroid position of a localized immunoplasmonic nanotag on the GNI was resolved at less than 10 nm of spatial resolution by applying 2D Gaussian fitting to its point spread function. This method showed enhanced sensitive quantification with a limit of detection (LOD) of 7.04 zM (1-2 molecules of AFP/GNI), which was 100-5000000000 times lower than detection limits obtained with previous AFP detection methods. Furthermore, the method was also successfully applied to quantify AFP molecules at the single-molecule level in human serum samples. The wavelength-dependent TIRS-SRM method was demonstrated to be an effective tool for superlocalizing individual protein molecules and interactions in nanoscale regions and was a reliable method for the ultrasensitive quantitative detection of disease-related protein molecules as a nanosensor and for diagnosis at the single-molecule level.


Assuntos
Técnicas Biossensoriais , Nanotecnologia , alfa-Fetoproteínas/análise , Anticorpos/química , Anticorpos/imunologia , Técnicas Biossensoriais/instrumentação , Ouro/química , Humanos , Nanopartículas Metálicas/química , Microscopia Eletrônica/instrumentação , Nanotecnologia/instrumentação , Tamanho da Partícula , Espectrofotometria Ultravioleta/instrumentação , Propriedades de Superfície , alfa-Fetoproteínas/imunologia
20.
Nanomedicine (Lond) ; 11(17): 2359-77, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27488053

RESUMO

Exosomes are cell-secreted nanovesicles present in biological fluids in normal and diseased conditions. Owing to their seminal role in cell-cell communication, emerging evidences suggest that exosomes are fundamental regulators of various diseases. Due to their potential usefulness in disease diagnosis, robust isolation and characterization of exosomes is critical in developing exosome-based assays. In the last few years, different exosome characterization methods, both biophysical and molecular, have been developed to characterize these tiny vesicles. Here, in this review we summarize: first, biophysical techniques based on spectroscopy (e.g., Raman spectroscopy, dynamic light scattering) and other principles, for example, scanning electron microscopy, atomic force microscopy; second, antibody-based molecular techniques including flow cytometry, transmission electron microscopy and third, nanotechnology-dependent exosome characterization methodologies.


Assuntos
Exossomos/química , Exossomos/ultraestrutura , Animais , Fracionamento Químico/instrumentação , Fracionamento Químico/métodos , Difusão Dinâmica da Luz/instrumentação , Difusão Dinâmica da Luz/métodos , Desenho de Equipamento , Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Microscopia de Força Atômica/instrumentação , Microscopia de Força Atômica/métodos , Microscopia Eletrônica/instrumentação , Microscopia Eletrônica/métodos , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Análise Espectral Raman/instrumentação , Análise Espectral Raman/métodos
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