Showing papers on "Resolution (electron density) published in 2012"
TL;DR: The method has the sensitivity to measure a 0.1 Å displacement in the oxygen bond length occurring in a time interval of ∼5 fs, which establishes LIED as a promising approach for the imaging of gas-phase molecules with unprecedented spatio-temporal resolution.
Abstract: Molecular structures are imaged with sub-angstrom precision and exposure times of a few femtoseconds. Molecular imaging, or the determination of the positions of atoms in molecules, is an important technique in the physical, chemical and biological sciences. But going beyond mere structure determination, recent technical developments offer the tantalizing prospect of access to ultrafast snapshots of biological molecules and condensed-phase systems undergoing structural changes. One approach uses laser-ionized bursts of coherent electron wave packets to self-interrogate the parent molecular structure. Here, Blaga et al. use this laser-induced electron diffraction (LIED) method to map the structural responses of oxygen and nitrogen molecules to ionization. By measuring a 0.1-angstrom displacement in the oxygen bond length occurring in a time interval of about 5 femtoseconds, the authors establish LIED as a promising approach for imaging of gas-phase molecules with unprecedented spatio-temporal resolution. Establishing the structure of molecules and solids has always had an essential role in physics, chemistry and biology. The methods of choice are X-ray and electron diffraction, which are routinely used to determine atomic positions with sub-angstrom spatial resolution. Although both methods are currently limited to probing dynamics on timescales longer than a picosecond, the recent development of femtosecond sources of X-ray pulses and electron beams suggests that they might soon be capable of taking ultrafast snapshots of biological molecules1,2 and condensed-phase systems3,4,5,6 undergoing structural changes. The past decade has also witnessed the emergence of an alternative imaging approach based on laser-ionized bursts of coherent electron wave packets that self-interrogate the parent molecular structure7,8,9,10,11. Here we show that this phenomenon can indeed be exploited for laser-induced electron diffraction10 (LIED), to image molecular structures with sub-angstrom precision and exposure times of a few femtoseconds. We apply the method to oxygen and nitrogen molecules, which on strong-field ionization at three mid-infrared wavelengths (1.7, 2.0 and 2.3 μm) emit photoelectrons with a momentum distribution from which we extract diffraction patterns. The long wavelength is essential for achieving atomic-scale spatial resolution, and the wavelength variation is equivalent to taking snapshots at different times. We show that the method has the sensitivity to measure a 0.1 A displacement in the oxygen bond length occurring in a time interval of ∼5 fs, which establishes LIED as a promising approach for the imaging of gas-phase molecules with unprecedented spatio-temporal resolution.
498 citations
TL;DR: In this article, the authors demonstrate that reversible photoswitching of a fluorescent protein provides the required nonlinearity at light intensities six orders of magnitude lower than those needed for saturation, and visualize cellular structures by imaging the mammalian nuclear pore and actin cytoskeleton.
Abstract: Using ultralow light intensities that are well suited for investigating biological samples, we demonstrate whole-cell superresolution imaging by nonlinear structured-illumination microscopy. Structured-illumination microscopy can increase the spatial resolution of a wide-field light microscope by a factor of two, with greater resolution extension possible if the emission rate of the sample responds nonlinearly to the illumination intensity. Saturating the fluorophore excited state is one such nonlinear response, and a realization of this idea, saturated structured-illumination microscopy, has achieved approximately 50-nm resolution on dye-filled polystyrene beads. Unfortunately, because saturation requires extremely high light intensities that are likely to accelerate photobleaching and damage even fixed tissue, this implementation is of limited use for studying biological samples. Here, reversible photoswitching of a fluorescent protein provides the required nonlinearity at light intensities six orders of magnitude lower than those needed for saturation. We experimentally demonstrate approximately 40-nm resolution on purified microtubules labeled with the fluorescent photoswitchable protein Dronpa, and we visualize cellular structures by imaging the mammalian nuclear pore and actin cytoskeleton. As a result, nonlinear structured-illumination microscopy is now a biologically compatible superresolution imaging method.
392 citations
TL;DR: In this article, a new form of low-energy transmission Kikuchi diffraction, performed in the SEM Transmission EBSD (t-EBSD) detector and software, has been proposed to capture and analyse the angular intensity variation in large-angle forward scattering of electrons in transmission.
Abstract: Summary
The spatial resolution of electron diffraction within the scanning electron microscope (SEM) has progressed from channelling methods capable of measuring crystallographic characteristics from 10 μm regions to electron backscatter diffraction (EBSD) methods capable of measuring 120 nm particles Here, we report a new form of low-energy transmission Kikuchi diffraction, performed in the SEM Transmission-EBSD (t-EBSD) makes use of an EBSD detector and software to capture and analyse the angular intensity variation in large-angle forward scattering of electrons in transmission, without postspecimen coils We collected t-EBSD patterns from Fe–Co nanoparticles of diameter 10 nm and from 40 nm-thick Ni films with in-plane grain size 15 nm The patterns exhibited contrast similar to that seen in EBSD, but are formed in transmission Monte Carlo scattering simulations showed that in addition to the order of magnitude improvement in spatial resolution from isolated particles, the energy width of the scattered electrons in t-EBSD is nearly two orders of magnitude narrower than that of conventional EBSD This new low-energy transmission diffraction approach builds upon recent progress in achieving unprecedented levels of imaging resolution for materials characterization in the SEM by adding high-spatial-resolution analytical capabilities
355 citations
TL;DR: By illuminating a sample with several uncontrolled random speckles and implementing a blind structured illumination microscopy algorithm, researchers demonstrate that image reconstruction can be achieved without knowing the original illumination pattern, at a resolution two times better than that of conventional wide-field microscopy.
Abstract: By illuminating a sample with several uncontrolled random speckles and implementing a blind structured illumination microscopy algorithm, researchers demonstrate that image reconstruction can be achieved without knowing the original illumination pattern, at a resolution two times better than that of conventional wide-field microscopy.
347 citations
TL;DR: This work demonstrates a form of diffractive imaging that unshackles the image formation process from the constraints of electron optics, improving resolution over that of the lens used by a factor of five and showing for the first time that it is possible to recover the complex exit wave at atomic resolution.
Abstract: Diffractive imaging, in which image-forming optics are replaced by an inverse computation using scattered intensity data, could, in principle, realize wavelength-scale resolution in a transmission electron microscope. However, to date all implementations of this approach have suffered from various experimental restrictions. Here we demonstrate a form of diffractive imaging that unshackles the image formation process from the constraints of electron optics, improving resolution over that of the lens used by a factor of five and showing for the first time that it is possible to recover the complex exit wave (in modulus and phase) at atomic resolution, over an unlimited field of view, using low-energy (30 keV) electrons. Our method, called electron ptychography, has no fundamental experimental boundaries: further development of this proof-of-principle could revolutionize sub-atomic scale transmission imaging.
284 citations
TL;DR: This work shows that it can align frames of movies, recorded with a direct electron detector during beam exposure of rotavirus double-layered particles, thereby greatly reducing image blurring caused by beam-induced motion and sample stage instabilities and increases the efficiency of cryo-EM imaging.
267 citations
TL;DR: An ultra-high vacuum (UHV)-based scanning thermal microscope (SThM) technique that is capable of quantitatively mapping temperature fields with ∼15 mK temperature resolution and ∼10 nm spatial resolution is described.
Abstract: Understanding energy dissipation at the nanoscale requires the ability to probe temperature fields with nanometer resolution. Here, we describe an ultra-high vacuum (UHV)-based scanning thermal microscope (SThM) technique that is capable of quantitatively mapping temperature fields with ∼15 mK temperature resolution and ∼10 nm spatial resolution. In this technique, a custom fabricated atomic force microscope (AFM) cantilever, with a nanoscale Au–Cr thermocouple integrated into the tip of the probe, is used to measure temperature fields of surfaces. Operation in an UHV environment eliminates parasitic heat transport between the tip and the sample enabling quantitative measurement of temperature fields on metal and dielectric surfaces with nanoscale resolution. We demonstrate the capabilities of this technique by directly imaging thermal fields in the vicinity of a 200 nm wide, self-heated, Pt line. Our measurements are in excellent agreement with computational results—unambiguously demonstrating the quanti...
176 citations
TL;DR: The design and performance of a wavelength-dispersive type spectrometer based on the von Hamos geometry equipped with a segmented-type crystal for x-ray diffraction and an energy resolution in the order of 0.25 eV and 1 eV is reported.
Abstract: We report on the design and performance of a wavelength-dispersive type spectrometer based on the von Hamos geometry. The spectrometer is equipped with a segmented-type crystal for x-ray diffraction and provides an energy resolution in the order of 0.25 eV and 1 eV over an energy range of 8000 eV-9600 eV. The use of a segmented crystal results in a simple and straightforward crystal preparation that allows to preserve the spectrometer resolution and spectrometer efficiency. Application of the spectrometer for time-resolved resonant inelastic x-ray scattering and single-shot x-ray emission spectroscopy is demonstrated.
162 citations
TL;DR: In this article, the resolution limits of popular sub-diffraction and sub-wavelength imaging schemes are examined using a unified approach that allows rapid comparison of the relative merits and shortcomings of each technique.
Abstract: The resolution limits of popular sub-diffraction and sub-wavelength imaging schemes are examined using a unified approach that allows rapid comparison of the relative merits and shortcomings of each technique. This is intended to clarify the often confusing and constantly growing array of super-resolution techniques. Specific techniques examined include centroid-based techniques like PALM (photo-activated localization microscopy) and STORM (stochastic optical reconstruction microscopy), structured illumination techniques like SSIM (spatially structured illumination microscopy), STED (stimulated emission depletion), and GSD (ground state depletion), coherent techniques like MRI (magnetic resonance imaging), Rabi gradients, and light shift gradients, as well as quantum-inspired multi-photon techniques. It is found that the ultimate resolution for all these techniques can be described using a simple ratio of an oscillation frequency to an effective decay rate, which can be physically interpreted as the number of oscillations that can be observed before decay (i.e.?the quality factor Q of the imaging transition).
153 citations
TL;DR: The development of subparticle imaging with space, time, and energy resolutions of nanometers, femtoseconds, and millielectron volts is reported, opening the door to various applications in elemental analysis as well as mapping of interfaces and plasmonics.
Abstract: Single-particle imaging of structures has become a powerful methodology in nanoscience and molecular and cell biology. We report the development of subparticle imaging with space, time, and energy resolutions of nanometers, femtoseconds, and millielectron volts, respectively. By using scanning electron probes across optically excited nanoparticles and interfaces, we simultaneously constructed energy-time and space-time maps. Spectrum images were then obtained for the nanoscale dielectric fields, with the energy resolution set by the photon rather than the electron, as demonstrated here with two examples (silver nanoparticles and the metallic copper–vacuum interface). This development thus combines the high spatial resolution of electron microscopy with the high energy resolution of optical techniques and ultrafast temporal response, opening the door to various applications in elemental analysis as well as mapping of interfaces and plasmonics.
149 citations
TL;DR: In infrared supercontinuum radiated from an optical fiber as a promising new light source for infrared microspectroscopy, which allows contact free high resolution hyper spectral infrared microscopy.
Abstract: Combining the molecular specificity of the infrared spectral region with high resolution microscopy has been pursued by researchers for decades. Here we demonstrate infrared supercontinuum radiated from an optical fiber as a promising new light source for infrared microspectroscopy. The supercontinuum light source has a high brightness and spans the infrared region from 1400 nm to 4000 nm. This combination allows contact free high resolution hyper spectral infrared microscopy. The microscope is demonstrated by imaging an oil/water sample with 20 μm resolution.
TL;DR: This work uses a novel AFM mode to probe the localization and interactions of chemical and biological sites on living cells at high speed and high resolution, and demonstrates the ability of the method to quantify and image hydrophobic forces on organic surfaces and on microbial pathogens.
Abstract: Currently, there is a growing need for methods that can quantify and map the molecular interactions of biological samples, both with high-force sensitivity and high spatial resolution. Force-volume imaging is a valuable atomic force microscopy (AFM) modality for probing specific sites on biosurfaces. However, the low speed and poor spatial resolution of this method have severely hampered its widespread use in life science research. We use a novel AFM mode (i.e., peak force tapping with chemically functionalized tips) to probe the localization and interactions of chemical and biological sites on living cells at high speed and high resolution (8 min for 1 μm × 1 μm images at 512 pixels × 512 pixels). First, we demonstrate the ability of the method to quantify and image hydrophobic forces on organic surfaces and on microbial pathogens. Next, we detect single sensor proteins on yeast cells, and we unravel their mechanical properties in relation to cellular function. Owing to its key capabilities (quantitative mapping, resolution of a few nanometers, and true correlation with topography), this novel biochemically sensitive imaging technique is a powerful complement to other advanced AFM modes for quantitative, high-resolution bioimaging.
TL;DR: In this paper, the authors demonstrate x-ray scanning coherent diffraction microscopy (ptychography) with 10nm spatial resolution, clearly exceeding the resolution limits of conventional hard X-ray microscopy, showing that the spatial resolution in a ptychogram is dependent on the shape (structure factor) of a feature and can vary for different features in the object.
Abstract: We demonstrate x-ray scanning coherent diffraction microscopy (ptychography) with 10 nm spatial resolution, clearly exceeding the resolution limits of conventional hard x-ray microscopy. The spatial resolution in a ptychogram is shown to depend on the shape (structure factor) of a feature and can vary for different features in the object. In addition, the resolution and contrast are shown to increase with increasing coherent fluence. For an optimal ptychographic x-ray microscope, this implies a source with highest possible brilliance and an x-ray optic with a large numerical aperture to generate the optimal probe beam.
TL;DR: An electron tomographic method that can be used to determine, from only one viewing direction and with sub-ångström precision, both the position of individual atoms in the plane of observation and their vertical position is presented.
Abstract: A tomography technique based on the idea that Fourier components of scattered electron waves obey a relationship analogous to that expressed in cosmology by Hubble’s law can be used to image at atomic resolution from a single viewing direction. State-of-the-art electron microscopy can readily resolve structures with subatomic resolution, but making three-dimensional images of similar resolution is a tougher challenge. Here, Dirk Van Dyck and Fu-Rong Chen describe an original image-reconstruction method that extracts information on the whereabouts of all atoms, in the plane as well as in the vertical direction, from just one projection. The concept is based on the assumption that each atom acts as a point source, scattering spherical waves that propagate to the detector, where they interfere with spherical waves from other atoms. The observed 'exit wave' contains information on all atoms in the sample, and can be retrieved using appropriate algorithms. The approach is demonstrated experimentally for a two-layered graphene sample. The authors note that their reconstruction technique resembles that used by cosmologists to construct a Hubble plot, hence the name 'Big Bang tomography'. Until now it has not been possible to image at atomic resolution using classical electron tomographic methods1, except when the target is a perfectly crystalline nano-object imaged along a few zone axes2. The main reasons are that mechanical tilting in an electron microscope with sub-angstrom precision over a very large angular range is difficult, that many real-life objects such as dielectric layers in microelectronic devices impose geometrical constraints and that many radiation-sensitive objects such as proteins limit the total electron dose. Hence, there is a need for a new tomographic scheme that is able to deduce three-dimensional information from only one or a few projections. Here we present an electron tomographic method that can be used to determine, from only one viewing direction and with sub-angstrom precision, both the position of individual atoms in the plane of observation and their vertical position. The concept is based on the fact that an experimentally reconstructed exit wave3,4 consists of the superposition of the spherical waves that have been scattered by the individual atoms of the object. Furthermore, the phase of a Fourier component of a spherical wave increases with the distance of propagation at a known ‘phase speed’. If we assume that an atom is a point-like object, the relationship between the phase and the phase speed of each Fourier component is linear, and the distance between the atom and the plane of observation can therefore be determined by linear fitting. This picture has similarities with Big Bang cosmology, in which the Universe expands from a point-like origin such that the distance of any galaxy from the origin is linearly proportional to the speed at which it moves away from the origin (Hubble expansion). The proof of concept of the method has been demonstrated experimentally for graphene with a two-layer structure and it will work optimally for similar layered materials, such as boron nitride and molybdenum disulphide.
TL;DR: The addition of on/off control of molecular emission to maintain concentrations at very low levels in each imaging frame combined with sequential imaging of sparse subsets has enabled the reconstruction of images with resolution far below the optical diffraction limit.
Abstract: In this short review, the general principles are described for obtaining microscopic images with resolution beyond the optical diffraction limit with single molecules. Although it has been known for several decades that single-molecule emitters can blink or turn on and off, in recent work the addition of on/off control of molecular emission to maintain concentrations at very low levels in each imaging frame combined with sequential imaging of sparse subsets has enabled the reconstruction of images with resolution far below the optical diffraction limit. Single-molecule active control microscopy provides a powerful window into information about nanoscale structures that was previously unavailable.
TL;DR: 3D hard X-ray imaging of unstained bacterial cells by a combination of ptychography and tomography is presented and it is shown how the real and reciprocal space approach can be used synergistically on the same sample and with the same setup.
Abstract: Ptychographic coherent X-ray diffractive imaging (PCDI) has been combined with nano-focus X-ray diffraction to study the structure and density distribution of unstained and unsliced bacterial cells, using a hard X-ray beam of 6.2keV photon energy, focused to about 90nm by a Fresnel zone plate lens. While PCDI provides images of the bacteria with quantitative contrast in real space with a resolution well below the beam size at the sample, spatially resolved small angle X-ray scattering using the same Fresnel zone plate (cellular nano-diffraction) provides structural information at highest resolution in reciprocal space up to 2nm−1. We show how the real and reciprocal space approach can be used synergistically on the same sample and with the same setup. In addition, we present 3D hard X-ray imaging of unstained bacterial cells by a combination of ptychography and tomography.
TL;DR: The instrument is presented, allowing a sample to be precisely scanned through a beam while the angle of x-ray incidence can be changed, and achieves a position stability better than 10 nm standard deviation.
Abstract: We present an instrument dedicated to 3D scanning x-ray microscopy, allowing a sample to be precisely scanned through a beam while the angle of x-ray incidence can be changed. The position of the sample is controlled with respect to the beam-defining optics by laser interferometry. The instrument achieves a position stability better than 10 nm standard deviation. The instrument performance is assessed using scanning x-ray diffraction microscopy and we demonstrate a resolution of 18 nm in 2D imaging of a lithographic test pattern while the beam was defined by a pinhole of 3 μm in diameter. In 3D on a test object of copper interconnects of a microprocessor, a resolution of 53 nm is achieved.
TL;DR: The technique extends imaging of unstained and unlabeled macromolecular assemblies in water from the resolution of the light microscope to the nanometerresolution of the electron microscope, suggesting that real-time imaging of protein dynamics is conceptually feasible.
TL;DR: Vaterite, a polymorph of CaCO(3) was first mentioned by H. Vater in 1897, plays key roles in weathering and biomineralization processes, but occurs only in the form of nanosized crystals, unsuitable for structure determination.
Abstract: tion that is fundamental for understanding material properties. Still, a number of compounds have eluded such kinds of analysis because they are nanocrystalline, highly disordered, with strong pseudosymmetries or available only in small amounts in polyphasic or polymorphic systems. These materials are crystallographically intractable with conventional Xray or synchrotron radiation diffraction techniques. Single nanoparticles can be visualized by high-resolution transmission electron microscopy (HR-TEM) up to sub�ngstrom resolution, [2] but obtaining 3D information is still a difficult task, especially for highly beam-sensitive materials and crystal structures with long cell parameters. Electron diffraction (ED) delivers higher resolved data with a significant lower electron dose on the sample, but is biased by a substantial number of missing reflections and the occurrence of dynamic scattering that affects reflection intensities. [3] Therefore, ED is mainly used in combination with Xray powder diffraction and high-resolution electron microscopy. [4]
TL;DR: Small features, so far only visible in transmission electron microscope (TEM) (e.g., the two leaflets of the membrane bi-layer, clathrin coats and cytoskeletal elements), can be resolved directly in the FIB/SEM in the 3D context of whole cells.
Abstract: Focused ion beam/scanning electron microscopy (FIB/SEM) tomography is a novel powerful approach for three-dimensional (3D) imaging of biological samples. Thereby, a sample is repeatedly milled with the focused ion beam (FIB) and each newly produced block face is imaged with the scanning electron microscope (SEM). This process can be repeated ad libitum in arbitrarily small increments allowing 3D analysis of relatively large volumes such as eukaryotic cells. High-pressure freezing and freeze substitution, on the other hand, are the gold standards for electron microscopic preparation of whole cells. In this work, we combined these methods and substantially improved resolution by using the secondary electron signal for image formation. With this imaging mode, contrast is formed in a very small, well-defined area close to the newly produced surface. By using this approach, small features, so far only visible in transmission electron microscope (TEM) (e.g., the two leaflets of the membrane bi-layer, clathrin coats and cytoskeletal elements), can be resolved directly in the FIB/SEM in the 3D context of whole cells.
TL;DR: The application of a recently established extension of the transmission soft X-ray cryo-microscope at the beamline U41-XM of the BESSY II electron storage ring is reported, demonstrating new possibilities to study the role of specific proteins in substructures of adherent cells, especially of the nucleus in toto, accessible to electron microscopy in thinned samples only.
TL;DR: In this article, the authors modify iterative reconstruction methods to improve tolerance to noise in single-shot coherent diffractive imaging at X-ray free-electron laser facilities, which is limited by the low signal-to-noise level of diffraction data at high scattering angles.
Abstract: The resolution of single-shot coherent diffractive imaging at X-ray free-electron laser facilities is limited by the low signal-to-noise level of diffraction data at high scattering angles. The iterative reconstruction methods, which phase a continuous diffraction pattern to produce an image, must be able to extract information from these weak signals to obtain the best quality images. Here we show how to modify iterative reconstruction methods to improve tolerance to noise. The method is demonstrated with the hybrid input-output method on both simulated data and single-shot diffraction patterns taken at the Linac Coherent Light Source.
TL;DR: The results pave the way for the use of such nanoparticles for targeted labeling of surfaces to provide nanoscale mapping of molecular composition, indicated by cathodoluminescence colour, simultaneously acquired with structural electron images in a single instrument.
Abstract: Correlative light and electron microscopy promises to combine molecular specificity with nanoscale imaging resolution. However, there are substantial technical challenges including reliable co-registration of optical and electron images, and rapid optical signal degradation under electron beam irradiation. Here, we introduce a new approach to solve these problems: imaging of stable optical cathodoluminescence emitted in a scanning electron microscope by nanoparticles with controllable surface chemistry. We demonstrate well-correlated cathodoluminescence and secondary electron images using three species of semiconductor nanoparticles that contain defects providing stable, spectrally-distinguishable cathodoluminescence. We also demonstrate reliable surface functionalization of the particles. The results pave the way for the use of such nanoparticles for targeted labeling of surfaces to provide nanoscale mapping of molecular composition, indicated by cathodoluminescence colour, simultaneously acquired with structural electron images in a single instrument.
TL;DR: A full-field transmission X-ray microscope operating continuously from 5 keV to 12 keV with fluorescence mapping capability has been designed and constructed at the Beijing Synchrotron Radiation Facility and the optics design, testing of spatial resolution and fluorescence sensitivity are presented.
Abstract: A full-field transmission X-ray microscope (TXM) operating continuously from 5 keV to 12 keV with fluorescence mapping capability has been designed and constructed at the Beijing Synchrotron Radiation Facility, a first-generation synchrotron radiation facility operating at 2.5 GeV. Spatial resolution better than 30 nm has been demonstrated using a Siemens star pattern in both absorption mode and Zernike phase-contrast mode. A scanning-probe mode fluorescence mapping capability integrated with the TXM has been shown to provide 50 p. p. m. sensitivity for trace elements with a spatial resolution (limited by probing beam spot size) of 20 mm. The optics design, testing of spatial resolution and fluorescence sensitivity are presented here, including performance measurement results.
TL;DR: TomoSTORM, an approach combining single-molecule localization-based super-resolution microscopy with array tomography of structurally intact brain tissue, delineated the course of the membrane and fine-structure of mitochondria in large tissue volumes with a resolution three orders of magnitude better than confocal microscopy.
Abstract: Three-dimensional fluorescence imaging of thick tissue samples with near-molecular resolution remains a fundamental challenge in the life sciences. To tackle this, we developed tomoSTORM, an approach combining single-molecule localization-based super-resolution microscopy with array tomography of structurally intact brain tissue. Consecutive sections organized in a ribbon were serially imaged with a lateral resolution of 28 nm and an axial resolution of 40 nm in tissue volumes of up to 50 µm×50 µm×2.5 µm. Using targeted expression of membrane bound (m)GFP and immunohistochemistry at the calyx of Held, a model synapse for central glutamatergic neurotransmission, we delineated the course of the membrane and fine-structure of mitochondria. This method allows multiplexed super-resolution imaging in large tissue volumes with a resolution three orders of magnitude better than confocal microscopy.
TL;DR: The results are described here with special emphasis on the use of symmetry-based restraints to improve the quality of atomic models derived from density maps of symmetric complexes; on a comparison of the stereochemical quality of Atomic models resulting from different hybrid methods; and on application of MDFF to electron crystallography data.
Abstract: The increasing power and popularity of cryo-electron microscopy (cryo-EM) in structural biology brought about the development of so-called hybrid methods, which permit the interpretation of cryo-EM density maps beyond their nominal resolution in terms of atomic models. The Cryo-EM Modeling Challenge 2010 is the first community effort to bring together developers of hybrid methods as well as cryo-EM experimentalists. Participating in the challenge, the molecular dynamics flexible fitting (MDFF) method was applied to a number of cryo-EM density maps. The results are described here with special emphasis on the use of symmetry-based restraints to improve the quality of atomic models derived from density maps of symmetric complexes; on a comparison of the stereochemical quality of atomic models resulting from different hybrid methods; and on application of MDFF to electron crystallography data.
TL;DR: An adapted sample preparation method based on high-pressure freezing for structure preservation, followed by freeze-substitution for multimodal en-bloc imaging or serial-section imaging is described, which has the potential to extract statistically relevant data of structural details for systems biology.
Abstract: The rationale of correlative light and electron microscopy (CLEM) is to collect data on different information levels--ideally from an identical area on the same sample--with the aim of combining datasets at different levels of resolution to achieve a more holistic view of the hierarchical structural organization of cells and tissues. Modern three-dimensional (3D) imaging techniques in light and electron microscopy opened up new possibilities to expand morphological studies into the third dimension at the nanometer scale and over various volume dimensions. Here, we present two alternative approaches to correlate 3D light microscopy (LM) data with scanning electron microscopy (SEM) volume data. An adapted sample preparation method based on high-pressure freezing for structure preservation, followed by freeze-substitution for multimodal en-bloc imaging or serial-section imaging is described. The advantages and potential applications are exemplarily shown on various biological samples, such as cells, individual organisms, human tissue, as well as plant tissue. The two CLEM approaches presented here are per se not mutually exclusive, but have their distinct advantages. Confocal laser scanning microscopy (CLSM) and focused ion beam-SEM (FIB-SEM) is most suitable for targeted 3D correlation of small volumes, whereas serial-section LM and SEM imaging has its strength in large-area or -volume screening and correlation. The second method can be combined with immunocytochemical methods. Both methods, however, have the potential to extract statistically relevant data of structural details for systems biology.
TL;DR: A superresolution four-wave mixing microscope with spatial resolution approaching 130 nm which represents better than twice the diffraction limit at 800 nm while retaining the ability to acquire materials- and chemical- specific contrast is reported on.
Abstract: We report on the development of a superresolution four-wave mixing microscope with spatial resolution approaching 130 nm which represents better than twice the diffraction limit at 800 nm while retaining the ability to acquire materials- and chemical- specific contrast. The resolution enhancement is achieved by narrowing the microscope’s excitation volume in the focal plane through the combined use of a Toraldo-style pupil phase filter with the multiplicative nature of four-wave mixing.
TL;DR: For the first time, it is demonstrated experimentally that both the spatial resolution and depth contrast can be improved in subsurface microscopy by demodulating the measured near-field signal at higher harmonics of the probe's tapping frequency and by operating at smaller tapping amplitudes.
Abstract: We report an experimental analysis of the capabilities of scattering-type scanning near-field optical microscopy for mapping sub-surface features at varying depths. For the first time, we demonstrate experimentally that both the spatial resolution and depth contrast can be improved in subsurface microscopy by demodulating the measured near-field signal at higher harmonics of the probe’s tapping frequency and by operating at smaller tapping amplitudes. Our findings are qualitatively supported by a simple dipole model.
TL;DR: This work introduces a non-fluorescent analogue to structured illumination microscopy, termed SOIM, where simultaneous oblique illuminations of the sample are used to multiplex high spatial-frequency content into the frequency support of the system.
Abstract: Many biological structures of interest are beyond the diffraction limit of conventional microscopes and their visualization requires application of super-resolution techniques. Such techniques have found remarkable success in surpassing the diffraction limit to achieve sub-diffraction limited resolution; however, they are predominantly limited to fluorescent samples. Here, we introduce a non-fluorescent analogue to structured illumination microscopy, termed structured oblique illumination microscopy (SOIM), where we use simultaneous oblique illuminations of the sample to multiplex high spatial-frequency content into the frequency support of the system. We introduce a theoretical framework describing how to demodulate this multiplexed information to reconstruct an image with a spatial-frequency support exceeding that of the system’s classical diffraction limit. This approach allows enhanced-resolution imaging of non-fluorescent samples. Experimental confirmation of the approach is obtained in a reflection test target with moderate numerical aperture.