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1.
Nat Commun ; 11(1): 2495, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32427872

RESUMO

Colloidal crystal engineering with nucleic acid-modified nanoparticles is a powerful way for preparing 3D superlattices, which may be useful in many areas, including catalysis, sensing, and photonics. To date, the building blocks studied have been primarily based upon metals, metal oxides, chalcogenide semiconductors, and proteins. Here, we show that metal-organic framework nanoparticles (MOF NPs) densely functionalized with oligonucleotides can be programmed to crystallize into a diverse set of superlattices with well-defined crystal symmetries and compositions. Electron microscopy and small-angle X-ray scattering characterization confirm the formation of single-component MOF superlattices, binary MOF-Au single crystals, and two-dimensional MOF nanorod assemblies. Importantly, DNA-modified porphyrinic MOF nanorods (PCN-222) were assembled into 2D superlattices and found to be catalytically active for the photooxidation of 2-chloroethyl ethyl sulfide (CEES, a chemical warfare simulant of mustard gas). Taken together, these new materials and methods provide access to colloidal crystals that incorporate particles with the well-established designer properties of MOFs and, therefore, increase the scope of possibilities for colloidal crystal engineering with DNA.


Assuntos
Coloides/química , DNA/química , Estruturas Metalorgânicas/química , Nanopartículas/química , Cristalização , DNA/genética , Engenharia/métodos , Microscopia Eletrônica de Transmissão e Varredura/métodos , Nanopartículas/ultraestrutura , Nanotubos/química , Nanotubos/ultraestrutura , Tamanho da Partícula , Espalhamento a Baixo Ângulo , Prata/química , Difração de Raios X
2.
J Mater Chem B ; 7(41): 6399-6411, 2019 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-31642847

RESUMO

Magnesium-yttrium-rare earth element alloys such as WE43 are potential candidates for future bioabsorbable orthopedic implant materials due to their biocompatibility, mechanical properties similar to human bone, and the ability to completely degrade in vivo. Unfortunately, the high corrosion rate of WE43 Mg alloys in physiological environments and subsequent loss of structural integrity limit the wide applications of these materials. In this study, the effect of chemical heterogeneity and microstructure on the corrosion resistance of two alloys with different metallurgical states was investigated: cast (as in traditional preparation) and as-deposited produced by magnetron sputtering. The corrosion behavior was studied by potentiodynamic polarization and electrochemical impedance spectroscopy tests in blood bank buffered saline solution. It was found that the as-deposited alloy showed more than one order of magnitude reduction in corrosion current density compared to the cast alloy, owing to the elimination of micro-galvanic coupling between the Mg matrix and the precipitates. The microstructure and formation mechanism of corrosion products formed on both alloys were discussed based on immersion tests and direct measurements of X-ray photoelectron spectrometry (XPS) and cross-sectional transmission electron microscopy (TEM) analysis.


Assuntos
Ligas/química , Materiais Biocompatíveis/química , Corrosão , Magnésio , Teste de Materiais , Microscopia Eletrônica de Transmissão e Varredura/métodos , Espectroscopia Fotoeletrônica/métodos
3.
Mol Med ; 25(1): 42, 2019 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-31455202

RESUMO

BACKGROUND: HER2 is considered as one of the most important, predictive biomarkers in oncology. The diagnosis of HER2 positive cancer types such as breast- and gastric cancer is usually based on immunohistochemical HER2 staining of tumour tissue. However, the current immunohistochemical methods do not provide localized information about HER2's functional state. In order to generate signals leading to cell growth and proliferation, the receptor spontaneously forms homodimers, a process that can differ between individual cancer cells. MATERIALS AND METHODS: HER2 overexpressing tumour cells were dissociated from formalin-fixed paraffin-embedded (FFPE) patient's biopsy sections, subjected to a heat-induced antigen retrieval procedure, and immobilized on microchips. HER2 was specifically labelled via a two-step protocol involving the incubation with an Affibody-biotin compound followed by the binding of a streptavidin coated quantum dot (QD) nanoparticle. Cells with membrane bound HER2 were identified using fluorescence microscopy, coated with graphene to preserve their hydrated state, and subsequently examined by scanning transmission electron microscopy (STEM) to obtain the locations at the single molecule level. Label position data was statistically analysed via the pair correlation function, yielding information about the presence of HER2 homodimers. RESULTS: Tumour cells from two biopsies, scored HER2 3+, and a HER2 negative control sample were examined. The specific labelling protocol was first tested for a sectioned tissue sample of HER2-overexpressing tumour. Subsequently, a protocol was optimized to study HER2 homodimerization in single cells dissociated from the tissue section. Electron microscopy data showed membrane bound HER2 in average densities of 201-689 proteins/µm2. An automated, statistical analysis of well over 200,000 of measured protein positions revealed the presence of HER2 homodimers in 33 and 55% of the analysed images for patient 1 and 2, respectively. CONCLUSIONS: We introduced an electron microscopy method capable of measuring the positions of individually labelled HER2 proteins in patient tumour cells from which information about the functional status of the receptor was derived. This method could take HER2 testing a step further by examining HER2 homodimerization directly out of tumour tissue and may become important for adjusting a personalized antibody-based drug therapy.


Assuntos
Neoplasias da Mama , Microscopia Eletrônica de Transmissão e Varredura/métodos , Receptor ErbB-2/análise , Receptor ErbB-2/ultraestrutura , Análise de Célula Única/métodos , Biomarcadores Tumorais/análise , Biópsia/métodos , Mama/química , Mama/citologia , Mama/diagnóstico por imagem , Mama/patologia , Neoplasias da Mama/química , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/patologia , Feminino , Grafite , Humanos , Inclusão em Parafina
4.
Methods Cell Biol ; 152: 197-215, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31326021

RESUMO

Electron cryo-tomography using the scanning transmission modality (STEM) enables 3D reconstruction of unstained, vitrified specimens as thick as 1µm or more. Contrast is related to mass/thickness and atomic number, providing quantifiable chemical characterization and mass mapping of intact prokaryotic and eukaryotic cells. Energy dispersive X-ray spectroscopy by STEM provides a simple, on-the-spot chemical identification of the elemental composition in sub-cellular organic bodies or mineral deposits. This chapter provides basic background and practical information for performing cryo-STEM tomography on vitrified biological cells.


Assuntos
Biologia/métodos , Microscopia Crioeletrônica/métodos , Microscopia Eletrônica de Transmissão e Varredura/métodos , Células Eucarióticas/fisiologia , Imageamento Tridimensional/métodos , Células Procarióticas/fisiologia , Espectrometria por Raios X/métodos
5.
Int J Low Extrem Wounds ; 18(3): 323-335, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31140339

RESUMO

Tissue regeneration has become a promising strategy for repairing damaged skin tissues. Among the hydrogels for tissue regeneration applications, topical hydrogels have demonstrated great potential for use as 3D-scaffolds in the burn wound healing process. Currently, no report has been published specifically on icariin-loaded polyvinyl alcohol (PVA)/agar hydrogel on full-thickness burn wounds. In the present study, burn tissue regeneration based on biomimetic hydrogel scaffolds was used for repairing damaged extracellular matrix. Furthermore, a skin burn model was developed in rats, and the icariin-loaded PVA/agar hydrogels were implanted into the damaged portions. The regeneration of the damaged tissues with the help of the icariin-loaded hydrogel group exhibited new translucent skin tissues and repaired extracellular matrix, indicating that the hydrogel can enhance the wound healing process. Moreover, characterization studies such as X-ray diffraction, Fourier-transformed infrared spectroscopy, and differential scanning calorimetry reported the extent of compatibility between icariin and its polymers. Results of the field emission scanning electron microscopy images revealed the extent of the spread of icariin within the polymer-based hydrogel. Furthermore, the wound healing potential, confirmed by histopathological and histochemical findings at the end of 21 days, revealed the visual evidence for the biomimetic property of icariin-loaded PVA/agar hydrogel scaffolds with the extracellular matrix for tissue regeneration.


Assuntos
Queimaduras , Flavonoides/farmacologia , Regeneração/efeitos dos fármacos , Lesões dos Tecidos Moles , Cicatrização , Animais , Queimaduras/patologia , Queimaduras/terapia , Varredura Diferencial de Calorimetria/métodos , Medicamentos de Ervas Chinesas , Excipientes/farmacologia , Microscopia Eletrônica de Transmissão e Varredura/métodos , Modelos Animais , Álcool de Polivinil/farmacologia , Ratos , Lesões dos Tecidos Moles/diagnóstico por imagem , Lesões dos Tecidos Moles/patologia , Lesões dos Tecidos Moles/terapia , Tecidos Suporte , Resultado do Tratamento , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologia
6.
Ultramicroscopy ; 202: 44-50, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30953993

RESUMO

In sample preparation of biological samples for electron microscopy, many types of embedding media are widely used. Unfortunately, none of them is perfectly resistant to beam induced damage. The article is focused on mass loss measuring of pure epoxy resin EMbed 812 that replaced Epon - the most widely used embedding resin for biological electron microscopy, in a form of ultrathin sections with thicknesses ranging from 30 to 100 nm. The STEM imaging was performed in a quantitative way which allowed us to estimate the mass loss directly up to the total dose of 3000 e-/nm2. For data acquisition we used SEM equipped with a commercial STEM detector working at a relatively low acceleration voltage of 30 kV. In this study we estimated the influence of various factors which can affect the endurance of the epoxy resin EMbed 812 ultrathin sections under an electron beam, such as the sample aging, differences between storing the samples in forms of ultrathin sections and whole blocks, ultrathin sections thicknesses, temperature of the sample, probe current, and one or two-sided carbon coating of ultrathin sections. The aim of this work is to investigate beam induced mass loss at electron energies of SEM and find out how to reduce the mass loss.


Assuntos
Resinas Epóxi/química , Microscopia Eletrônica de Transmissão e Varredura/métodos , Elétrons
7.
Micron ; 119: 54-63, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30660856

RESUMO

In the use of solution-based 3D nanoarchitectures for optics, drug delivery, and cancer treatment, the precise nanoparticle architecture morphologies, architecture sizes, interparticle distances, and the assembly stability are all critical to their functionality. 3D nanoparticle architectures in solution are difficult to characterize, as few techniques can provide individualized information on interparticle spacing (defined by linkage molecule), nanoparticle assembly size, morphology, and identification of false aggregation. Bulk characterization techniques, including small angle x-ray scattering, can provide architecture sizes, though they are unable to precisely measure differences within interparticle spacings for individual architectures and can falsely measure assemblies caused by non-linkage grouped nanoparticles. Two solution-based characterization techniques were used to determine which assembly type and linkage length would produce the fastest assembly rate for large DNA-directed gold nanoparticle assemblies. In-situ liquid-cell scanning transmission electron microscopy (LC-STEM), measured interparticle spacings between DNA-functionalized nanoparticles, and fluorescence correlation spectroscopy provided the bulk volume fraction of large and small assemblies for nanoparticle architectures that were assembled using two different types: (1) the hybrid assemblies join two complementary single-stranded DNA linkages, and (2) the bridged assemblies are comprised of single-stranded DNA (bridging component) that is double the length of two different complementary single-stranded DNA-functionalized gold nanoparticles. Assembly times were tested at 24-hrs intervals over 3 days. Statistics derived from the in-situ LC-STEM images provided data for interparticle distance measurements, which identified the fraction of nanoparticles within the images acquired that were at the expected double-stranded DNA-binding distance of the linkages (varied in three distances for each of the two different architectures). In general, longer linkage lengths assembled in the shortest amount of time. The bridged assemblies formed fewer large architectures at 24-hrs but ultimately assembled a greater fraction of nanoparticles, which was due to the longer functionalized DNA lengths for individual nanoparticles. Fluorescence correlation spectroscopy provided a bulk average of the gold nanoparticle assembly sizes over time, which supported the conclusions drawn from the in-situ LC-STEM data. The microscopy provided sub-2 nm precision in the interparticle distances between gold nanoparticles in a solution environment. This coupled microscopy and spectroscopy characterization approach can provide more detailed information than bulk characterization methods.


Assuntos
DNA de Cadeia Simples/metabolismo , Ouro/metabolismo , Nanopartículas Metálicas/ultraestrutura , Microscopia Eletrônica de Transmissão e Varredura/métodos , Espectrometria de Fluorescência/métodos , Cinética , Nanopartículas Metálicas/química , Fatores de Tempo
8.
J Struct Biol ; 206(1): 43-48, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29678776

RESUMO

Intra-neuronal aggregation of α-synuclein into fibrils is the molecular basis for α-synucleinopathies, such as Parkinson's disease. The atomic structure of human α-synuclein (hAS) fibrils was recently determined by Tuttle et al. using solid-state NMR (ssNMR). The previous study found that hAS fibrils are composed of a single protofilament. Here, we have investigated the structure of mouse α-synuclein (mAS) fibrils by STEM and isotope-dilution ssNMR experiments. We found that in contrast to hAS, mAS fibrils consist of two or even three protofilaments which are connected by rather weak interactions in between them. Although the number of protofilaments appears to be different between hAS and mAS, we found that they have a remarkably similar secondary structure and protofilament 3D structure as judged by secondary chemical shifts and intra-molecular distance restraints. We conclude that the two mutant sites between hAS and mAS (positions 53 and 87) in the fibril core region are crucial for determining the quaternary structure of α-synuclein fibrils.


Assuntos
Amiloide/química , Espectroscopia de Ressonância Magnética/métodos , Microscopia Eletrônica de Transmissão e Varredura/métodos , Conformação Molecular , alfa-Sinucleína/química , Amiloide/genética , Amiloide/metabolismo , Animais , Sítios de Ligação/genética , Isótopos de Carbono/química , Isótopos de Carbono/metabolismo , Humanos , Hidrogênio/química , Hidrogênio/metabolismo , Camundongos , Modelos Moleculares , Mutação , Isótopos de Nitrogênio/química , Isótopos de Nitrogênio/metabolismo , Estrutura Secundária de Proteína , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
9.
J Struct Biol ; 206(1): 36-42, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29679649

RESUMO

The C-terminally truncated Y145Stop variant of prion protein (PrP23-144), which is associated with heritable PrP cerebral amyloid angiopathy in humans and also capable of triggering a transmissible prion disease in mice, serves as a useful in vitro model for investigating the molecular and structural basis of amyloid strains and cross-seeding specificities. Here, we determine the protein-solvent interfaces in human PrP23-144 amyloid fibrils generated from recombinant 13C,15N-enriched protein and incubated in aqueous solution containing paramagnetic Cu(II)-EDTA, by measuring residue-specific 15N longitudinal paramagnetic relaxation enhancements using two-dimensional magic-angle spinning solid-state NMR spectroscopy. To further probe the interactions of the amyloid core residues with solvent molecules we perform complementary measurements of amide hydrogen/deuterium exchange detected by solid-state NMR and solution NMR methods. The solvent accessibility data are evaluated in the context of the structural model for human PrP23-144 amyloid.


Assuntos
Amiloide/genética , Proteínas Amiloidogênicas/genética , Códon sem Sentido , Espectroscopia de Ressonância Magnética/métodos , Proteínas Priônicas/genética , Príons/genética , Amiloide/química , Amiloide/metabolismo , Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/metabolismo , Animais , Isótopos de Carbono/química , Isótopos de Carbono/metabolismo , Medição da Troca de Deutério , Humanos , Camundongos , Microscopia Eletrônica de Transmissão e Varredura/métodos , Isótopos de Nitrogênio/química , Isótopos de Nitrogênio/metabolismo , Proteínas Priônicas/química , Proteínas Priônicas/metabolismo , Príons/química , Príons/metabolismo , Soluções/química , Solventes/química
10.
Micron ; 116: 30-35, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30265881

RESUMO

Liquid-cell TEM has enabled an interdisciplinary community of scientists to carry out atomic- / nano-scale studies of solid/liquid interfaces. Nevertheless, the restricted resolution of TEM in liquid media and the necessity to reduce the electron dose to avoid harmful radiolytic effects induced by the beam have limited the use of high resolution imaging to study the atomic structure of nanomaterials in liquid. Here we show that STEM nanodiffraction can be exploited in liquid-cell TEM experiments to overcome these two limitations. We evidence that this technique allows quick analysis of the structure of single gold nanoparticles whatever their zone axis orientation, which substantially increases the percentage of analysable nanostructures with respect to HRTEM investigations. Moreover, STEM nanodiffraction can also be used in very low dose conditions. The electron dose irradiating the analyzed nanostructures during data acquisition can be reduced by almost four orders of magnitude compared to conventional HRTEM analysis. Finally, dynamical analyses in reciprocal space are used to provide new insights into the shape-dependent rotation of nanocrystals in the liquid-cell.


Assuntos
Ouro , Nanopartículas Metálicas/química , Microscopia Eletrônica de Transmissão e Varredura/métodos
11.
Chembiochem ; 20(6): 822-830, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30501011

RESUMO

Staining compounds containing heavy elements (electron dyes) can facilitate the visualization of DNA and related biomolecules by using TEM. However, research into the synthesis and utilization of alternative electron dyes has been limited. Here, we report the synthesis of a novel DNA intercalator molecule, bis-acridine uranyl (BAU). NMR spectroscopy and MS confirmed the validity of the synthetic strategy and gel electrophoresis verified the binding of BAU to DNA. For TEM imaging of DNA, two-dimensional DNA origami nanostructures were used as a robust microscopy test object. By using scanning transmission electron microscopy (STEM) imaging, which is favored over conventional wide-field TEM for improved contrast, and therefore, quantitative image analysis, it is found that the synthesized BAU intercalator can render DNA visible, even at the single-molecule scale. For comparison, other staining compounds with a purported affinity towards DNA, such as dichloroplatinum, cisplatin, osmium tetroxide, and uranyl acetate, have been evaluated. The STEM contrast is discussed in terms of the DNA-dye association constants, number of dye molecules bound per base pair, and the electron-scattering capacity of the metal-containing ligands. These findings pave the way for the future development of electron dyes with specific DNA-binding motifs for high-resolution TEM imaging.


Assuntos
Acridinas/química , Complexos de Coordenação/química , DNA/química , Substâncias Intercalantes/química , Imagem Individual de Molécula/métodos , Acridinas/síntese química , Complexos de Coordenação/síntese química , Substâncias Intercalantes/síntese química , Microscopia Eletrônica de Transmissão e Varredura/métodos , Conformação de Ácido Nucleico , Urânio/química
12.
Methods Mol Biol ; 1894: 247-269, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30547465

RESUMO

In this chapter, we highlight the applications of electron microscopes (EMs) in nanotoxicity assessment. EMs can provide detailed information about the size and morphology of nanomaterials (NMs), their localization in cells and tissues, the nano-bio interactions, as well as the ultrastructural changes induced by NMs exposure. Here, we share with the readers how we prepare the tissue sample, and the different types of EMs used among the nanotoxicologists. It is possible to deploy conventional EMs along or in combination with other analytical techniques, such as electron energy loss spectroscopy (EELS), energy dispersive X-ray spectroscopy (EDS or EDX), and TEM-assisted scanning transmission X-ray microscopy (STXM), toward further elemental and chemical characterization. Appropriate images are inserted to illustrate throughout this chapter.


Assuntos
Técnicas de Preparação Histocitológica/métodos , Microscopia Eletrônica de Transmissão e Varredura/métodos , Nanopartículas/toxicidade , Espectrometria por Raios X/métodos , Espectroscopia de Perda de Energia de Elétrons/métodos , Animais , Linhagem Celular , Técnicas de Preparação Histocitológica/instrumentação , Humanos , Camundongos , Microscopia Eletrônica de Transmissão e Varredura/instrumentação , Espectrometria por Raios X/instrumentação , Espectroscopia de Perda de Energia de Elétrons/instrumentação
13.
Microscopy (Oxf) ; 68(2): 111-121, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30380073

RESUMO

A 3, 3'-diaminobenzidine (DAB)-based method was used to detect the localization of endogenous peroxidase activity in Indian rhinoceros (Rhinoceros unicornis) parotid gland acinar cells. The tissue had previously been resin-embedded in gelatin capsules for routine electron microscopic observations and thus pre-incubation for endogenous peroxidase analysis was not possible. We attempted to demonstrate the relationship between secretory granules (SGs) in resin ultrathin sections of Indian rhinoceros parotid gland acinar cells and endogenous peroxidase activity. A JEM 1400 Plus scanning transmission electron microscope (STEM) was used to conduct energy dispersive X-ray spectroscopy (EDS) analysis of the presence of nitrogen generated by the DAB reaction in bipartite structural SG consisting of a dense body (or core). The mapping patterns of nitrogen were restricted to the dense body. We observed nitrogen localized in the rough endoplasmic reticulum (ER), nuclear envelope (NE) and several components of the Golgi apparatus (G) of rhinoceros parotid gland acinar cells participating in the synthetic pathway of secretory proteins. Moreover, we established a nitrogen-detection method by EDS analysis of rhinoceros parotid gland. The reliability of the method was validated by comparison of the test group (peroxidase detection in ultrathin resin sections) and the control group (ordinary peroxidase detection in semi-thin sections following glutaraldehyde pre-fixation) of rat submandibular gland. The same mapping patterns of nitrogen were detected by DAB reaction in the SG, ER, NE and G in these two groups. Hence, EDS-STEM approaches for endogenous peroxidase post-incubation analysis will prove useful for advanced cytochemical analysis for the identification of any other resin sections.


Assuntos
3,3'-Diaminobenzidina/química , Células Acinares/ultraestrutura , Microscopia Eletrônica de Transmissão e Varredura/métodos , Nitrogênio/análise , Glândula Parótida/diagnóstico por imagem , Peroxidase/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Feminino , Complexo de Golgi/metabolismo , Masculino , Microtomia/métodos , Membrana Nuclear/metabolismo , Glândula Parótida/citologia , Perissodáctilos , Ratos , Ratos Sprague-Dawley , Resinas Sintéticas/química , Fixação de Tecidos/métodos
15.
Ultramicroscopy ; 194: 1-6, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30029082

RESUMO

Soft X-ray spectromicroscopy was applied to study the quantitative distribution of DNA and protein in a mammalian chromosome at the spatial resolution of 100 nm. The quantities of DNA and protein were evaluated using 1s-π* transition in the NEXAFS spectra at the nitrogen K absorption edge. DNA was not uniformly distributed in the chromosome and DNA/protein ratio was less than 0.497. The present analysis revealed the clues to identify other molecules that contribute to the absorption spectrum of the sample. The results suggested that accumulation of the absorption spectra of relevant molecules would support the refinement of the analysis.


Assuntos
Cromossomos de Mamíferos/química , Animais , Células CHO , Linhagem Celular , Cricetulus , DNA/química , Estudos de Avaliação como Assunto , Microscopia Eletrônica de Transmissão e Varredura/métodos , Nitrogênio/química , Proteínas/química , Raios X
16.
Adv Mater ; 30(41): e1706681, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29748979

RESUMO

Electron tomography provides a detailed view into the 3D structure of biological cells and tissues. Physical fixation by vitrification of the aqueous medium provides the most faithful preservation of biological specimens in the native, fully hydrated state. Cryo-microscopy is challenging, however, because of the sensitivity to electron irradiation and due to the weak electron scattering of organic material. Tomography is even more challenging because of the dependence on multiple exposures of the same area. Tomographic imaging is typically performed in wide-field transmission electron microscopy (TEM) mode with phase contrast generated by defocus. Scanning transmission electron microscopy (STEM) is an alternative mode based on detection of scattering from a focused probe beam, without imaging optics following the specimen. While careful configuration of the illumination and detectors is required to generate useful contrast, STEM circumvents the major restrictions of phase contrast TEM to very thin specimens and provides a signal that is more simply interpreted in terms of local composition and density. STEM has gained popularity in recent years for materials science. The extension of STEM to cryomicroscopy and tomography of cells and macromolecules is summarized herein.


Assuntos
Microscopia Crioeletrônica/métodos , Microscopia Eletrônica de Transmissão e Varredura/métodos , Animais , Microscopia Crioeletrônica/instrumentação , Humanos , Microscopia Eletrônica de Transmissão e Varredura/instrumentação
17.
J Struct Biol ; 202(3): 216-228, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29408702

RESUMO

Microscopies based on focused electron probes allow the cell biologist to image the 3D ultrastructure of eukaryotic cells and tissues extending over large volumes, thus providing new insight into the relationship between cellular architecture and function of organelles. Here we compare two such techniques: electron tomography in conjunction with axial bright-field scanning transmission electron microscopy (BF-STEM), and serial block face scanning electron microscopy (SBF-SEM). The advantages and limitations of each technique are illustrated by their application to determining the 3D ultrastructure of human blood platelets, by considering specimen geometry, specimen preparation, beam damage and image processing methods. Many features of the complex membranes composing the platelet organelles can be determined from both approaches, although STEM tomography offers a higher ∼3 nm isotropic pixel size, compared with ∼5 nm for SBF-SEM in the plane of the block face and ∼30 nm in the perpendicular direction. In this regard, we demonstrate that STEM tomography is advantageous for visualizing the platelet canalicular system, which consists of an interconnected network of narrow (∼50-100 nm) membranous cisternae. In contrast, SBF-SEM enables visualization of complete platelets, each of which extends ∼2 µm in minimum dimension, whereas BF-STEM tomography can typically only visualize approximately half of the platelet volume due to a rapid non-linear loss of signal in specimens of thickness greater than ∼1.5 µm. We also show that the limitations of each approach can be ameliorated by combining 3D and 2D measurements using a stereological approach.


Assuntos
Plaquetas/ultraestrutura , Tomografia com Microscopia Eletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Microscopia Eletrônica de Transmissão e Varredura/métodos , Humanos , Imageamento Tridimensional/métodos
18.
J Microsc ; 269(3): 338-345, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29125617

RESUMO

When polymorphonuclear leukocytes (PMNs) phagocytose opsonised zymosan particles (OPZ), free radicals and reactive oxygen species (ROS) are formed in the phagosomes. ROS production is mediated by NADPH oxidase (Nox), which transfers electrons in converting oxygen to superoxide (O2- ). Nox-generated O2- is rapidly converted to other ROS. Free radical-forming secretory vesicles containing the Nox redox center flavocytochrome b558, a membrane protein, and azurophil granules with packaged myeloperoxidase (MPO) have been described. Presuming the probable fusion of these vesicular and granular organelles with phagosomes, the translation process of the enzymes was investigated using energy-filtering and energy-dispersive spectroscopy-scanning transmission electron microscopy. In this work, the primary method for imaging cerium (Ce) ions demonstrated the localisation of H2 O2 generated by phagocytosing PMNs. The MPO activity of the same PMNs was continuously monitored using 0.1% 3,3'-diaminobenzidine-tetrahydrochloride (DAB) and 0.01% H2 O2 . A detailed view of these vesicular and granular structures was created by overlaying each electron micrograph with pseudocolors: blue for Ce and green for nitrogen (N).


Assuntos
Grupo dos Citocromos b/análise , Microscopia Eletrônica de Transmissão e Varredura/métodos , NADPH Oxidases/análise , Neutrófilos/enzimologia , Neutrófilos/fisiologia , Peroxidase/análise , Fagocitose , Análise Espectral/métodos , Cério/análise , Humanos , Processamento de Imagem Assistida por Computador/métodos , Fagossomos/enzimologia
19.
ACS Nano ; 11(12): 12742-12752, 2017 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-29215876

RESUMO

Recent advances in scanning transmission electron and scanning probe microscopies have opened exciting opportunities in probing the materials structural parameters and various functional properties in real space with angstrom-level precision. This progress has been accompanied by an exponential increase in the size and quality of data sets produced by microscopic and spectroscopic experimental techniques. These developments necessitate adequate methods for extracting relevant physical and chemical information from the large data sets, for which a priori information on the structures of various atomic configurations and lattice defects is limited or absent. Here we demonstrate an application of deep neural networks to extract information from atomically resolved images including location of the atomic species and type of defects. We develop a "weakly supervised" approach that uses information on the coordinates of all atomic species in the image, extracted via a deep neural network, to identify a rich variety of defects that are not part of an initial training set. We further apply our approach to interpret complex atomic and defect transformation, including switching between different coordination of silicon dopants in graphene as a function of time, formation of peculiar silicon dimer with mixed 3-fold and 4-fold coordination, and the motion of molecular "rotor". This deep learning-based approach resembles logic of a human operator, but can be scaled leading to significant shift in the way of extracting and analyzing information from raw experimental data.


Assuntos
Aprendizado Profundo , Microscopia Eletrônica de Transmissão e Varredura/métodos , Grafite/química , Humanos , Tamanho da Partícula , Silício/química , Propriedades de Superfície
20.
Proc Natl Acad Sci U S A ; 114(42): 11139-11144, 2017 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-28973937

RESUMO

Metal ions play essential roles in many aspects of biological chemistry. Detecting their presence and location in proteins and cells is important for understanding biological function. Conventional structural methods such as X-ray crystallography and cryo-transmission electron microscopy can identify metal atoms on protein only if the protein structure is solved to atomic resolution. We demonstrate here the detection of isolated atoms of Zn and Fe on ferritin, using cryogenic annular dark-field scanning transmission electron microscopy (cryo-STEM) coupled with single-particle 3D reconstructions. Zn atoms are found in a pattern that matches precisely their location at the ferroxidase sites determined earlier by X-ray crystallography. By contrast, the Fe distribution is smeared along an arc corresponding to the proposed path from the ferroxidase sites to the mineral nucleation sites along the twofold axes. In this case the single-particle reconstruction is interpreted as a probability distribution function based on the average of individual locations. These results establish conditions for detection of isolated metal atoms in the broader context of electron cryo-microscopy and tomography.


Assuntos
Microscopia Crioeletrônica/métodos , Ferro/análise , Microscopia Eletrônica de Transmissão e Varredura/métodos , Zinco/análise , Ferritinas
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