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Combined scanning transmission electron microscopy tilt- and focal series.
Dahmen T, Baudoin JP, Lupini AR, Kübel C, Slusallek P, de Jonge N
(2014) Microsc Microanal 20: 548-60
MeSH Terms: Algorithms, Artifacts, Electron Microscope Tomography, Imaging, Three-Dimensional, Macrophages, Microscopy, Electron, Scanning Transmission, Models, Theoretical, Nanoparticles
Show Abstract · Added May 27, 2014
In this study, a combined tilt- and focal series is proposed as a new recording scheme for high-angle annular dark-field scanning transmission electron microscopy (STEM) tomography. Three-dimensional (3D) data were acquired by mechanically tilting the specimen, and recording a through-focal series at each tilt direction. The sample was a whole-mount macrophage cell with embedded gold nanoparticles. The tilt-focal algebraic reconstruction technique (TF-ART) is introduced as a new algorithm to reconstruct tomograms from such combined tilt- and focal series. The feasibility of TF-ART was demonstrated by 3D reconstruction of the experimental 3D data. The results were compared with a conventional STEM tilt series of a similar sample. The combined tilt- and focal series led to smaller "missing wedge" artifacts, and a higher axial resolution than obtained for the STEM tilt series, thus improving on one of the main issues of tilt series-based electron tomography.
0 Communities
1 Members
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8 MeSH Terms
Whole-cell analysis of low-density lipoprotein uptake by macrophages using STEM tomography.
Baudoin JP, Jerome WG, Kübel C, de Jonge N
(2013) PLoS One 8: e55022
MeSH Terms: Cell Line, Gold, Humans, Lipoproteins, LDL, Macrophages, Metal Nanoparticles, Microscopy, Electron, Scanning Transmission, Protein Transport
Show Abstract · Added March 20, 2014
Nanoparticles of heavy materials such as gold can be used as markers in quantitative electron microscopic studies of protein distributions in cells with nanometer spatial resolution. Studying nanoparticles within the context of cells is also relevant for nanotoxicological research. Here, we report a method to quantify the locations and the number of nanoparticles, and of clusters of nanoparticles inside whole eukaryotic cells in three dimensions using scanning transmission electron microscopy (STEM) tomography. Whole-mount fixed cellular samples were prepared, avoiding sectioning or slicing. The level of membrane staining was kept much lower than is common practice in transmission electron microscopy (TEM), such that the nanoparticles could be detected throughout the entire cellular thickness. Tilt-series were recorded with a limited tilt-range of 80° thereby preventing excessive beam broadening occurring at higher tilt angles. The 3D locations of the nanoparticles were nevertheless determined with high precision using computation. The obtained information differed from that obtained with conventional TEM tomography data since the nanoparticles were highlighted while only faint contrast was obtained on the cellular material. Similar as in fluorescence microscopy, a particular set of labels can be studied. This method was applied to study the fate of sequentially up-taken low-density lipoprotein (LDL) conjugated to gold nanoparticles in macrophages. Analysis of a 3D reconstruction revealed that newly up-taken LDL-gold was delivered to lysosomes containing previously up-taken LDL-gold thereby forming onion-like clusters.
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2 Members
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8 MeSH Terms
A common structure for the potexviruses.
Kendall A, Bian W, Maris A, Azzo C, Groom J, Williams D, Shi J, Stewart PL, Wall JS, Stubbs G
(2013) Virology 436: 173-8
MeSH Terms: Amino Acid Sequence, Binding Sites, Capsid, Capsid Proteins, Cryoelectron Microscopy, Microscopy, Electron, Scanning Transmission, Potexvirus, Protein Structure, Secondary, RNA, Viral, X-Ray Diffraction
Show Abstract · Added February 15, 2016
We have used fiber diffraction, cryo-electron microscopy, and scanning transmission electron microscopy to confirm the symmetry of three potexviruses, potato virus X, papaya mosaic virus, and narcissus mosaic virus, and to determine their low-resolution structures. All three viruses have slightly less than nine subunits per turn of the viral helix. Our data strongly support the view that all potexviruses have approximately the same symmetry. The structures are dominated by a large domain at high radius in the virion, with a smaller domain, which includes the putative RNA-binding site, extending to low radius.
Copyright © 2012 Elsevier Inc. All rights reserved.
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1 Members
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10 MeSH Terms
Fully hydrated yeast cells imaged with electron microscopy.
Peckys DB, Mazur P, Gould KL, de Jonge N
(2011) Biophys J 100: 2522-9
MeSH Terms: Cell Division, Imaging, Three-Dimensional, Microscopy, Electron, Scanning Transmission, Mutation, Organelles, Schizosaccharomyces, Water
Show Abstract · Added March 5, 2014
We demonstrate electron microscopy of fully hydrated eukaryotic cells with nanometer resolution. Living Schizosaccharomyces pombe cells were loaded in a microfluidic chamber and imaged in liquid with scanning transmission electron microscopy (STEM). The native intracellular (ultra)structures of wild-type cells and three different mutants were studied without prior labeling, fixation, or staining. The STEM images revealed various intracellular components that were identified on the basis of their shape, size, location, and mass density. The maximal achieved spatial resolution in this initial study was 32 ± 8 nm, an order of magnitude better than achievable with light microscopy on pristine cells. Light-microscopy images of the same samples were correlated with the corresponding electron-microscopy images. Achieving synergy between the capabilities of light and electron microscopy, we anticipate that liquid STEM will be broadly applied to explore the ultrastructure of live cells.
Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.
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2 Members
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7 MeSH Terms
Three-dimensional locations of gold-labeled proteins in a whole mount eukaryotic cell obtained with 3nm precision using aberration-corrected scanning transmission electron microscopy.
Dukes MJ, Ramachandra R, Baudoin JP, Gray Jerome W, de Jonge N
(2011) J Struct Biol 174: 552-62
MeSH Terms: Animals, COS Cells, Chlorocebus aethiops, Eukaryotic Cells, Gold, Image Processing, Computer-Assisted, Imaging, Three-Dimensional, Microscopy, Electron, Scanning Transmission, Nanoparticles, Proteins
Show Abstract · Added May 27, 2014
Three-dimensional (3D) maps of proteins within the context of whole cells are important for investigating cellular function. However, 3D reconstructions of whole cells are challenging to obtain using conventional transmission electron microscopy (TEM). We describe a methodology to determine the 3D locations of proteins labeled with gold nanoparticles on whole eukaryotic cells. The epidermal growth factor receptors on COS7 cells were labeled with gold nanoparticles, and critical-point dried whole-mount cell samples were prepared. 3D focal series were obtained with aberration-corrected scanning transmission electron microscopy (STEM), without tilting the specimen. The axial resolution was improved with deconvolution. The vertical locations of the nanoparticles in a whole-mount cell were determined with a precision of 3nm. From the analysis of the variation of the axial positions of the labels we concluded that the cellular surface was ruffled. To achieve sufficient stability of the sample under electron beam irradiation during the recording of the focal series, the sample was carbon coated. A quantitative method was developed to analyze the stability of the ultrastructure after electron beam irradiation using TEM. The results of this study demonstrate the feasibility of using aberration-corrected STEM to study the 3D nanoparticle distribution in whole cells.
Copyright © 2011 Elsevier Inc. All rights reserved.
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10 MeSH Terms
Visualizing gold nanoparticle uptake in live cells with liquid scanning transmission electron microscopy.
Peckys DB, de Jonge N
(2011) Nano Lett 11: 1733-8
MeSH Terms: Animals, COS Cells, Cell Membrane, Chlorocebus aethiops, Diffusion, Gold, Materials Testing, Microscopy, Electron, Scanning Transmission, Nanostructures, Solutions
Show Abstract · Added May 27, 2014
The intracellular uptake of 30 nm diameter gold nanoparticles (Au-NPs) was studied at the nanoscale in pristine eukaryotic cells. Live COS-7 cells were maintained in a microfluidic chamber and imaged using scanning transmission electron microscopy. A quantitative image analysis showed that Au-NPs bound to the membranes of vesicles, possibly lysosomes, and occupied 67% of the available surface area. The vesicles accumulated to form a micrometer-sized cluster after 24 h of incubation. Two clusters were analyzed and found to consist of 117 ± 9 and 164 ± 4 NP-filled vesicles.
0 Communities
1 Members
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10 MeSH Terms
Correlative fluorescence microscopy and scanning transmission electron microscopy of quantum-dot-labeled proteins in whole cells in liquid.
Dukes MJ, Peckys DB, de Jonge N
(2010) ACS Nano 4: 4110-6
MeSH Terms: Animals, COS Cells, Chlorocebus aethiops, ErbB Receptors, Eukaryotic Cells, Microfluidic Analytical Techniques, Microscopy, Electron, Scanning Transmission, Microscopy, Fluorescence, Quantum Dots, Staining and Labeling
Show Abstract · Added May 27, 2014
Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7x12 nm were visible in a 5 microm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs.
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10 MeSH Terms
Nanometer-resolution electron microscopy through micrometers-thick water layers.
de Jonge N, Poirier-Demers N, Demers H, Peckys DB, Drouin D
(2010) Ultramicroscopy 110: 1114-9
MeSH Terms: Animals, Electrons, Eukaryotic Cells, Gold, Humans, Microscopy, Electron, Scanning Transmission, Monte Carlo Method, Nanoparticles, Nanotechnology, Water
Show Abstract · Added May 27, 2014
Scanning transmission electron microscopy (STEM) was used to image gold nanoparticles on top of and below saline water layers of several micrometers thickness. The smallest gold nanoparticles studied had diameters of 1.4 nm and were visible for a liquid thickness of up to 3.3 microm. The imaging of gold nanoparticles below several micrometers of liquid was limited by broadening of the electron probe caused by scattering of the electron beam in the liquid. The experimental data corresponded to analytical models of the resolution and of the electron probe broadening as function of the liquid thickness. The results were also compared with Monte Carlo simulations of the STEM imaging on modeled specimens of similar geometry and composition as used for the experiments. Applications of STEM imaging in liquid can be found in cell biology, e.g., to study tagged proteins in whole eukaryotic cells in liquid and in materials science to study the interaction of solid:liquid interfaces at the nanoscale.
Published by Elsevier B.V.
0 Communities
1 Members
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10 MeSH Terms
Three-dimensional scanning transmission electron microscopy of biological specimens.
de Jonge N, Sougrat R, Northan BM, Pennycook SJ
(2010) Microsc Microanal 16: 54-63
MeSH Terms: Animals, Cell Line, Clathrin-Coated Vesicles, Cytoskeleton, Dogs, Golgi Apparatus, Imaging, Three-Dimensional, Mice, Microscopy, Electron, Scanning Transmission
Show Abstract · Added May 27, 2014
A three-dimensional (3D) reconstruction of the cytoskeleton and a clathrin-coated pit in mammalian cells has been achieved from a focal-series of images recorded in an aberration-corrected scanning transmission electron microscope (STEM). The specimen was a metallic replica of the biological structure comprising Pt nanoparticles 2-3 nm in diameter, with a high stability under electron beam radiation. The 3D dataset was processed by an automated deconvolution procedure. The lateral resolution was 1.1 nm, set by pixel size. Particles differing by only 10 nm in vertical position were identified as separate objects with greater than 20% dip in contrast between them. We refer to this value as the axial resolution of the deconvolution or reconstruction, the ability to recognize two objects, which were unresolved in the original dataset. The resolution of the reconstruction is comparable to that achieved by tilt-series transmission electron microscopy. However, the focal-series method does not require mechanical tilting and is therefore much faster. 3D STEM images were also recorded of the Golgi ribbon in conventional thin sections containing 3T3 cells with a comparable axial resolution in the deconvolved dataset.
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9 MeSH Terms
Structure and ultrafast dynamics of white-light-emitting CdSe nanocrystals.
Bowers MJ, McBride JR, Garrett MD, Sammons JA, Dukes AD, Schreuder MA, Watt TL, Lupini AR, Pennycook SJ, Rosenthal SJ
(2009) J Am Chem Soc 131: 5730-1
MeSH Terms: Cadmium, Light, Microscopy, Electron, Scanning Transmission, Nanoparticles, Particle Size, Selenium, Spectrometry, Fluorescence, Time Factors
Show Abstract · Added March 13, 2012
White-light emission from ultrasmall CdSe nanocrystals offers an alternative approach to the realization of solid-state lighting as an appealing technology for consumers. Unfortunately, their extremely small size limits the feasibility of traditional methods for nanocrystal characterization. This paper reports the first images of their structure, which were obtained using aberration-corrected atomic number contrast scanning transmission electron microscopy (Z-STEM). With subangstrom resolution, Z-STEM is one of the few available methods that can be used to directly image the nanocrystal's structure. The initial images suggest that they are crystalline and approximately four lattice planes in diameter. In addition to the structure, for the first time, the exciton dynamics were measured at different wavelengths of the white-light spectrum using ultrafast fluorescence upconversion spectroscopy. The data suggest that a myriad of trap states are responsible for the broad-spectrum emission. It is hoped that the information presented here will provide a foundation for the future development and improvement of white-light-emitting nanocrystals.
0 Communities
2 Members
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8 MeSH Terms