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Showing papers on "Resolution (electron density) published in 2008"


Journal ArticleDOI
08 Feb 2008-Science
TL;DR: 3D stochastic optical reconstruction microscopy (STORM) is demonstrated by using optical astigmatism to determine both axial and lateral positions of individual fluorophores with nanometer accuracy, allowing the 3D morphology of nanoscopic cellular structures to be resolved.
Abstract: Recent advances in far-field fluorescence microscopy have led to substantial improvements in image resolution, achieving a near-molecular resolution of 20 to 30 nanometers in the two lateral dimensions. Three-dimensional (3D) nanoscale-resolution imaging, however, remains a challenge. We demonstrated 3D stochastic optical reconstruction microscopy (STORM) by using optical astigmatism to determine both axial and lateral positions of individual fluorophores with nanometer accuracy. Iterative, stochastic activation of photoswitchable probes enables high-precision 3D localization of each probe, and thus the construction of a 3D image, without scanning the sample. Using this approach, we achieved an image resolution of 20 to 30 nanometers in the lateral dimensions and 50 to 60 nanometers in the axial dimension. This development allowed us to resolve the 3D morphology of nanoscopic cellular structures.

2,589 citations


Journal ArticleDOI
02 Oct 2008-Nature
TL;DR: This work shows how magneto-optical spin detection can be used to determine the location of a spin associated with a single nitrogen-vacancy centre in diamond with nanometre resolution under ambient conditions, and demonstrates the use of a single diamond spin as a scanning probe magnetometer to map nanoscale magnetic field variations.
Abstract: Magnetic resonance imaging and optical microscopy are key technologies in the life sciences. For microbiological studies, especially of the inner workings of single cells, optical microscopy is normally used because it easily achieves resolution close to the optical wavelength. But in conventional microscopy, diffraction limits the resolution to about half the wavelength. Recently, it was shown that this limit can be partly overcome by nonlinear imaging techniques, but there is still a barrier to reaching the molecular scale. In contrast, in magnetic resonance imaging the spatial resolution is not determined by diffraction; rather, it is limited by magnetic field sensitivity, and so can in principle go well below the optical wavelength. The sensitivity of magnetic resonance imaging has recently been improved enough to image single cells, and magnetic resonance force microscopy has succeeded in detecting single electrons and small nuclear spin ensembles. However, this technique currently requires cryogenic temperatures, which limit most potential biological applications. Alternatively, single-electron spin states can be detected optically, even at room temperature in some systems. Here we show how magneto-optical spin detection can be used to determine the location of a spin associated with a single nitrogen-vacancy centre in diamond with nanometre resolution under ambient conditions. By placing these nitrogen-vacancy spins in functionalized diamond nanocrystals, biologically specific magnetofluorescent spin markers can be produced. Significantly, we show that this nanometre-scale resolution can be achieved without any probes located closer than typical cell dimensions. Furthermore, we demonstrate the use of a single diamond spin as a scanning probe magnetometer to map nanoscale magnetic field variations. The potential impact of single-spin imaging at room temperature is far-reaching. It could lead to the capability to probe biologically relevant spins in living cells.

1,814 citations


Journal ArticleDOI
TL;DR: This work describes how spatially structured illumination microscopy can be applied in three dimensions to double the axial as well as the lateral resolution, with true optical sectioning, and has produced the first light microscopy images of the synaptonemal complex in which the lateral elements are clearly resolved.

1,343 citations



Journal ArticleDOI
TL;DR: A light microscope that generates images with translationally invariant 30 × 30 × 75nm resolution over a depth of several micrometers enabling 3D sub-diffraction resolution without compromising speed or sensitivity is reported.
Abstract: Imaging volumes as thick as whole cells at three-dimensional (3D) super-resolution is required to reveal unknown features of cellular organization. We report a light microscope that generates images with translationally invariant 30 x 30 x 75 nm resolution over a depth of several micrometers. This method, named biplane (BP) FPALM, combines a double-plane detection scheme with fluorescence photoactivation localization microscopy (FPALM) enabling 3D sub-diffraction resolution without compromising speed or sensitivity.

824 citations


Journal ArticleDOI
TL;DR: A reconstruction, obtained by using recently developed image processing methods, of the rotavirus inner capsid particle (“double-layer particle” or DLP) at a resolution suitable for interpretation by an atomic model is presented, establishing single-particle reconstruction as a high-resolution technique.
Abstract: Electron cryomicroscopy (cryo-EM) yields images of macromolecular assemblies and their components, from which 3D structures can be determined, by using an image processing method commonly known as "single-particle reconstruction." During the past two decades, this technique has become an important tool for 3D structure determination, but it generally has not been possible to determine atomic models. In principle, individual molecular images contain high-resolution information contaminated by a much higher level of noise. In practice, it has been unclear whether current averaging methods are adequate to extract this information from the background. We present here a reconstruction, obtained by using recently developed image processing methods, of the rotavirus inner capsid particle ("double-layer particle" or DLP) at a resolution suitable for interpretation by an atomic model. The result establishes single-particle reconstruction as a high-resolution technique. We show by direct comparison that the cryo-EM reconstruction of viral protein 6 (VP6) of the rotavirus DLP is similar in clarity to a 3.8-A resolution map obtained from x-ray crystallography. At this resolution, most of the amino acid side chains produce recognizable density. The icosahedral symmetry of the particle was an important factor in achieving this resolution in the cryo-EM analysis, but as the size of recordable datasets increases, single-particle reconstruction also is likely to yield structures at comparable resolution from samples of much lower symmetry. This potential has broad implications for structural cell biology.

361 citations


Journal ArticleDOI
25 Jul 2008-Science
TL;DR: An entirely new generation of instruments enables studies in condensed-matter physics and materials science to be performed at atomic- scale resolution, meeting the growing demand of nanosciences and nanotechnology for the atomic-scale characterization of materials, nanosynthesized products and devices, and the validation of expected functions.
Abstract: Seventy-five years after its invention, transmission electron microscopy has taken a great step forward with the introduction of aberration-corrected electron optics. An entirely new generation of instruments enables studies in condensed-matter physics and materials science to be performed at atomic-scale resolution. These new possibilities are meeting the growing demand of nanosciences and nanotechnology for the atomic-scale characterization of materials, nanosynthesized products and devices, and the validation of expected functions. Equipped with electron-energy filters and electron-energy-loss spectrometers, the new instruments allow studies not only of structure but also of elemental composition and chemical bonding. The energy resolution is about 100 milli-electron volts, and the accuracy of spatial measurements has reached a few picometers. However, understanding the results is generally not straightforward and only possible with extensive quantum-mechanical computer calculations.

290 citations


Journal ArticleDOI
TL;DR: The instrument's new capabilities were exploited to detect a buried Σ3 {112} grain boundary and observe the dynamic arrangements of single atoms and atom pairs with sub-angstrom resolution, an important step toward meeting the challenge of determining the three-dimensional atomic-scale structure of nanomaterials.
Abstract: The ability of electron microscopes to analyze all the atoms in individual nanostructures is limited by lens aberrations. However, recent advances in aberration-correcting electron optics have led to greatly enhanced instrument performance and new techniques of electron microscopy. The development of an ultrastable electron microscope with aberration-correcting optics and a monochromated high-brightness source has significantly improved instrument resolution and contrast. In the present work, we report information transfer beyond 50 pm and show images of single gold atoms with a signal-to-noise ratio as large as 10. The instrument's new capabilities were exploited to detect a buried Sigma3 {112} grain boundary and observe the dynamic arrangements of single atoms and atom pairs with sub-angstrom resolution. These results mark an important step toward meeting the challenge of determining the three-dimensional atomic-scale structure of nanomaterials.

280 citations


Journal ArticleDOI
Tatjana Gericke1, Peter Würtz1, Daniel Reitz1, Tim Langen1, Herwig Ott1 
TL;DR: In this article, the authors used scanning electron microscopy for the detection of single atoms inside a Bose-Einstein condensate with a spatial resolution of better than 150nm.
Abstract: Our knowledge of ultracold quantum gases is strongly influenced by our ability to probe these objects. In situ imaging combined with single-atom sensitivity is an especially appealing scenario, as it can provide direct information on the structure and the correlations of such systems. For a precise characterization a high spatial resolution is mandatory. In particular, the perspective to study quantum gases in optical lattices makes a resolution well below one micrometre highly desirable. Here, we report on a novel microscopy technique, which is based on scanning electron microscopy and allows for the detection of single atoms inside a quantum gas with a spatial resolution of better than 150 nm. We document the great functionality of this technique by precise density measurements of a trapped Bose–Einstein condensate and the first experimental demonstration of single-site addressability in a submicrometre optical lattice. Electron microscopes are regularly used to resolve atoms in solid samples. It turns out that they can also be used to image atoms in a Bose–Einstein condensate—remarkably, without destroying the coherent properties of the condensate.

279 citations


Journal ArticleDOI
TL;DR: Recent findings will be discussed showing that molecular models of proteins can be fitted into depicted organellar ultrastructure of images of frozen hydrated sections, and thus to a better understanding of the function of complex cellular structures.
Abstract: Transmission electron microscopy has provided most of what is known about the ultrastructural organization of tissues, cells, and organelles. Due to tremendous advances in crystallography and magnetic resonance imaging, almost any protein can now be modeled at atomic resolution. To fully understand the workings of biological “nanomachines” it is necessary to obtain images of intact macromolecular assemblies in situ. Although the resolution power of electron microscopes is on the atomic scale, in biological samples artifacts introduced by aldehyde fixation, dehydration and staining, but also section thickness reduces it to some nanometers. Cryofixation by high pressure freezing circumvents many of the artifacts since it allows vitrifying biological samples of about 200 μm in thickness and immobilizes complex macromolecular assemblies in their native state in situ. To exploit the perfect structural preservation of frozen hydrated sections, sophisticated instruments are needed, e.g., high voltage electron microscopes equipped with precise goniometers that work at low temperature and digital cameras of high sensitivity and pixel number. With them, it is possible to generate high resolution tomograms, i.e., 3D views of subcellular structures. This review describes theory and applications of the high pressure cryofixation methodology and compares its results with those of conventional procedures. Moreover, recent findings will be discussed showing that molecular models of proteins can be fitted into depicted organellar ultrastructure of images of frozen hydrated sections. High pressure freezing of tissue is the base which may lead to precise models of macromolecular assemblies in situ, and thus to a better understanding of the function of complex cellular structures.

245 citations


Journal ArticleDOI
TL;DR: In this paper, the authors report their recent progress in the development, optimization, and application of a technique for the 3D high-resolution characterization of crystalline microstructures based on automated serial sectioning using a focused ion beam (FIB) and characterization of the sections by orientation microscopy based on electron backscatter diffraction (EBSD).
Abstract: In the present work, we report our recent progress in the development, optimization, and application of a technique for the three-dimensional (3-D) high-resolution characterization of crystalline microstructures. The technique is based on automated serial sectioning using a focused ion beam (FIB) and characterization of the sections by orientation microscopy based on electron backscatter diffraction (EBSD) in a combined FIB–scanning electron microscope (SEM). On our system, consisting of a Zeiss–Crossbeam FIB-SEM and an EDAX-TSL EBSD system, the technique currently reaches a spatial resolution of 100 · 100 · 100 nm 3 as a standard, but a resolution of 50 · 50 · 50 nm 3 seems to be a realistic optimum. The maximum observable volume is on the order of 50 · 50 · 50 lm 3 . The technique extends all the powerful features of two-dimensional (2-D) EBSD-based orientation microscopy into the third dimension of space. This allows new parameters of the microstructure to be obtained—for example, the full crystallographic characterization of all kinds of interfaces, including the morphology and the crystallographic indices of the interface planes. The technique is illustrated by four examples, including the characterization of pearlite colonies in a carbon steel, of twins in pseudonanocrystalline NiCo thin films, the description of deformation patterns formed under nanoindents in copper single crystals, and the characterization of fatigue cracks in an aluminum alloy. In view of these examples, we discuss the possibilities and limits of the technique. Furthermore, we give an extensive overview of parallel developments of 3-D orientation microscopy (with a focus on the EBSD-based techniques) in other groups.

Journal ArticleDOI
TL;DR: It is shown that a semiempirical scaling factor based on the ratio of the tip contact area diameter to the sample thickness can be used to correct c-AFM current-voltage curves and thus extract mobilities that are in good agreement with values measured in the conventional planar device geometry.
Abstract: We describe local (∼150 nm resolution), quantitative measurements of charge carrier mobility in conjugated polymer films that are commonly used in thin-film transistors and nanostructured solar cel...

Journal ArticleDOI
TL;DR: By using the low voltage HR-SEM technique with extremely high resolution, the influence of the template properties on the structure of the nanocast metal oxides is very clearly shown.
Abstract: We report here a detailed study on the surface topology of well-known ordered mesoporous silica (SBA-15, MCM-41, and KIT-6) and a series of nanocast Co3O4, Co3O4/CoFe2O4 composites by high resolution scanning electron microscopy (HR-SEM). Images of the MCM-41 structure were obtained at a resolution of the pore size, as well as a real space image of the gyroid silica surface of KIT-6 for two different aging temperatures, clearly revealing the differences of the aging procedures. By using the low voltage HR-SEM technique with extremely high resolution, we could very clearly show the influence of the template properties on the structure of the nanocast metal oxides.

Journal ArticleDOI
TL;DR: The cytoplasmic polyhedrosis virus structure revealed a drastic conformational change from a helix to a beta hairpin associated with RNA packaging and replication, coupling of RNA processing and release, and the long sought-after polyhedrin-binding domain.

Journal ArticleDOI
TL;DR: In this article, a gold Fresnel zone plate was used to achieve first-order lateral resolution below 40nm based on the Rayleigh criterion, which was achieved using a phase contrast technique.
Abstract: Substantial improvements in the nanofabrication and characteristics of gold Fresnel zone plates yielded unprecedented resolution levels in hard-x-ray microscopy. Tests performed on a variety of specimens with 8–10keV photons demonstrated a first-order lateral resolution below 40nm based on the Rayleigh criterion. Combined with the use of a phase contrast technique, this makes it possible to view features in the 30nm range; good-quality images can be obtained at video rate, down to 50ms∕frame. The important repercussions on materials science, nanotechnology, and the life sciences are discussed.

Journal ArticleDOI
TL;DR: In this paper, the authors reported an approach based on combining the lateral super resolution provided by photoactivated localization microscopy (PALM) with two-photon temporal focusing that provides optical sectioning.
Abstract: Recent advances in optical microscopy have enabled biological imaging beyond the diffraction limit at nanometer resolution. A general feature of most of the techniques based on photoactivated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM) has been the use of thin biological samples in combination with total internal reflection, thus limiting the imaging depth to a fraction of an optical wavelength. However, to study whole cells or organelles that are typically up to 15 μm deep into the cell, the extension of these methods to a three-dimensional (3D) super resolution technique is required. Here, we report an advance in optical microscopy that enables imaging of protein distributions in cells with a lateral localization precision better than 50 nm at multiple imaging planes deep in biological samples. The approach is based on combining the lateral super resolution provided by PALM with two-photon temporal focusing that provides optical sectioning. We have generated super-resolution images over an axial range of ≈10 μm in both mitochondrially labeled fixed cells, and in the membranes of living S2 Drosophila cells.

Journal ArticleDOI
TL;DR: A versatile soft x-ray diffraction microscope with 70- to 90-nm resolution with high numerical aperture imaging and near-diffraction-limited resolution is reported by using two different tabletop coherent softx-ray sources—a soft x -ray laser and a high-harmonic source.
Abstract: Light microscopy has greatly advanced our understanding of nature. The achievable resolution, however, is limited by optical wavelengths to ≈200 nm. By using imaging and labeling technologies, resolutions beyond the diffraction limit can be achieved for specialized specimens with techniques such as near-field scanning optical microscopy, stimulated emission depletion microscopy, and photoactivated localization microscopy. Here, we report a versatile soft x-ray diffraction microscope with 70- to 90-nm resolution by using two different tabletop coherent soft x-ray sources—a soft x-ray laser and a high-harmonic source. We also use field curvature correction that allows high numerical aperture imaging and near-diffraction-limited resolution of 1.5λ. A tabletop soft x-ray diffraction microscope should find broad applications in biology, nanoscience, and materials science because of its simple optical design, high resolution, large depth of field, 3D imaging capability, scalability to shorter wavelengths, and ultrafast temporal resolution.

Journal ArticleDOI
TL;DR: In this paper, an extension of spectral precision distance microscopy/spectral position determination microscopy (SPDM) was presented, exploiting the novel spectral signature offered by reversible photobleaching of fluorescent proteins.
Abstract: Far-field fluorescence techniques based on the precise determination of object positions have the potential to circumvent the optical resolution limit of direct imaging given by diffraction theory. In order to use localization to obtain structural information far below the diffraction limit, the ‘point-like’ components of the structure have to be detected independently, even if their distance is lower than the conventional optical resolution limit. This goal can be achieved by exploiting various photo-physical properties of the fluorescence labeling (‘spectral signatures’). In first experiments, spectral precision distance microscopy/spectral position determination microscopy (SPDM) was limited to a relatively small number of components to be resolved within the observation volume. Recently, the introduction of photoconvertable molecules has dramatically increased the number of components which can be independently localized. Here, we present an extension of the SPDM concept, exploiting the novel spectral signature offered by reversible photobleaching of fluorescent proteins. In combination with spatially modulated illumination (SMI) microscopy, at the present stage, we have achieved an estimated effective optical resolution of approximately 20 nm in the lateral and 50 nm in the axial direction, or about 1/25th–1/10th of the exciting wavelength.

Journal ArticleDOI
TL;DR: A new type of wide-field fluorescence microscopy is described, which produces 100-nm-scale spatial resolution in all three dimensions, by using structured illumination in a microscope that has two opposing objective lenses.

Journal ArticleDOI
TL;DR: A small gold particle was illuminated with a hard x-ray nanobeam and is reconstructed from its coherent diffraction pattern and a resolution of about 5 nm is achieved in 600 s exposure time.
Abstract: Coherent x-ray diffraction imaging is an x-ray microscopy technique with the potential of reaching spatial resolutions well beyond the diffraction limits of x-ray microscopes based on optics. However, the available coherent dose at modern x-ray sources is limited, setting practical bounds on the spatial resolution of the technique. By focusing the available coherent flux onto the sample, the spatial resolution can be improved for radiation-hard specimens. A small gold particle (size <100 nm) was illuminated with a hard x-ray nanobeam (E=15.25 keV, beam dimensions approximately 100 x 100 nm2) and is reconstructed from its coherent diffraction pattern. A resolution of about 5 nm is achieved in 600 s exposure time.

Journal ArticleDOI
01 Aug 2008-Small
TL;DR: Owing to its sensitivity and noninvasiveness, far-field fluo-rescence microscopy would be almost ideal for biological imaging if the resolution of its established variants were not limited by diffraction.
Abstract: Owing to its sensitivity and noninvasiveness, far-field fluo-rescence microscopy would be almost ideal for biologicalimaging if the resolution of its established variants were notlimitedbydiffractiontoDr l=ð2n sinaÞ,withldenotingthewavelength of light, n the index of refraction, and a theaperture angle of the objective lens.

Journal ArticleDOI
TL;DR: EM-BFACTOR is a program that has been designed to widely facilitate the use of the novel method for objective B-factor determination and contrast restoration introduced by Rosenthal and Henderson, concluding that it helps to unravel the high resolution molecular features concealed in experimental density maps, thereby making them better suited for interpretation.

Journal ArticleDOI
TL;DR: The development of a high-time resolution dynamic transmission electron microscope (DTEM) that captures dynamics in materials with nanosecond time resolution is detailed.

Patent
30 Jun 2008
TL;DR: In this paper, a super-resolution image is produced by dividing a higher resolution image into a set of nonoverlapping rectangular tiles of substantially the same size, and then each pixel in each lower resolution image is mapped to the higher resolution images and it is determined which tiles are mapped to which lower resolution images pixels.
Abstract: Super-resolution images may be produced by dividing a higher resolution image into a set of non-overlapping rectangular tiles of substantially the same size. Then, each pixel in each lower resolution image is mapped to the higher resolution image and it is determined which tiles are mapped to which lower resolution image pixels. A continuous buffer may be allocated for each tile and the relevant lower resolution pixels may be stored, together with optical flow vectors, in that continuous buffer. Then, the determination of gradients may use the information now stored in the buffer to facilitate symmetric multiprocessing using multi-core processors.

Journal ArticleDOI
TL;DR: A fast fitting program, “gmfit”, which employs a Gaussian mixture model (GMM) to represent approximated shapes of the 3D density map and the atomic models and was able to rebuild atomic models of a complex even for maps of 30 Å resolution if sufficient numbers of Gaussian distribution functions were employed for each subunit.

Journal ArticleDOI
TL;DR: Room-temperature aging of zeolite precursor silica sol was followed by SAXS and cryo-TEM, suggesting the formation of predominantly amorphous aggregates before MFI crystallization.
Abstract: Room-temperature aging of zeolite precursor silica sol was followed by SAXS and cryo-TEM. Cryo-TEM imaging of zeolite materials with structural resolution is demonstrated. The results suggest the formation of predominantly amorphous aggregates before MFI crystallization.

Journal ArticleDOI
04 May 2008
TL;DR: By using extreme numerical-aperture solid-immersion microscopy at 1553 nm, this paper demonstrated, under certain circumstances, polarization-sensitive imaging with resolution values approaching 100 nm which substantially surpass the classical scalar diffraction-limit embodied by Sparrowpsilas resolution criterion.
Abstract: By using extreme numerical-aperture solid-immersion microscopy at 1553 nm we demonstrate, under certain circumstances, polarization-sensitive imaging with resolution values approaching 100 nm which substantially surpass the classical scalar diffraction-limit embodied by Sparrowpsilas resolution criterion.

Journal ArticleDOI
TL;DR: Simple analytical expressions are derived for the spatial resolution, contrast and signal-to-noise in X-ray projection images of a generic phase edge that take into account the maximum phase shift generated by the sample and the sharpness of the edge.
Abstract: Simple analytical expressions are derived for the spatial resolution, contrast and signal-to-noise in X-ray projection images of a generic phase edge. The obtained expressions take into account the maximum phase shift generated by the sample and the sharpness of the edge, as well as such parameters of the imaging set-up as the wavelength spectrum and the size of the incoherent source, the source-to-object and object-to-detector distances and the detector resolution. Different asymptotic behavior of the expressions in the cases of large and small Fresnel numbers is demonstrated. The analytical expressions are compared with the results of numerical simulations using Kirchhoff diffraction theory, as well as with experimental X-ray measurements.

Journal ArticleDOI
TL;DR: In this paper, a convergent electron beam is used to scan a cross sectional preparation of a Fe/Au multilayer sample and the differences in the energy-loss spectra induced by the magnetic moments of the Fe atoms can be resolved with a resolution of better than 2 nm.
Abstract: Magnetic circular dichroism (MCD) is a standard technique for the study of magnetic properties of materials in synchrotron beamlines. We present here a scattering geometry in the transmission electron microscope through which MCD can be observed with unprecedented spatial resolution. A convergent electron beam is used to scan a cross sectional preparation of a Fe/Au multilayer sample. Differences in the energy-loss spectra induced by the magnetic moments of the Fe atoms can be resolved with a resolution of better than 2 nm. This is a breakthrough achievement when compared both to the previous energy-loss MCD resolution (200 nm) or the best x-ray MCD experiments (approximately 20 nm).

Book ChapterDOI
01 Jan 2008