Showing papers on "Resolution (electron density) published in 2011"
Journal Article•
TL;DR: The comparison of related genomes has emerged as a powerful lens for genome interpretation as mentioned in this paper, which reveals a small number of new coding exons, candidate stop codon readthrough events and over 10,000 regions of overlapping synonymous constraint within protein-coding exons.
Abstract: The comparison of related genomes has emerged as a powerful lens for genome interpretation. Here we report the sequencing and comparative analysis of 29 eutherian genomes. We confirm that at least 5.5% of the human genome has undergone purifying selection, and locate constrained elements covering ∼4.2% of the genome. We use evolutionary signatures and comparisons with experimental data sets to suggest candidate functions for ∼60% of constrained bases. These elements reveal a small number of new coding exons, candidate stop codon readthrough events and over 10,000 regions of overlapping synonymous constraint within protein-coding exons. We find 220 candidate RNA structural families, and nearly a million elements overlapping potential promoter, enhancer and insulator regions. We report specific amino acid residues that have undergone positive selection, 280,000 non-coding elements exapted from mobile elements and more than 1,000 primate- and human-accelerated elements. Overlap with disease-associated variants indicates that our findings will be relevant for studies of human biology, health and disease.
926 citations
TL;DR: 3D reconstruction of a complex crystalline nanoparticle at atomic resolution is reported, which helps close the gap between the atomic resolution achievable in two-dimensional electron micrographs and the coarser resolution that has hitherto been obtained by conventional electron tomography.
Abstract: Determining the three-dimensional (3D) arrangement of atoms in crystalline nanoparticles is important for nanometre-scale device engineering and also for applications involving nanoparticles, such as optoelectronics or catalysis. A nanoparticle's physical and chemical properties are controlled by its exact 3D morphology, structure and composition. Electron tomography enables the recovery of the shape of a nanoparticle from a series of projection images. Although atomic-resolution electron microscopy has been feasible for nearly four decades, neither electron tomography nor any other experimental technique has yet demonstrated atomic resolution in three dimensions. Here we report the 3D reconstruction of a complex crystalline nanoparticle at atomic resolution. To achieve this, we combined aberration-corrected scanning transmission electron microscopy, statistical parameter estimation theory and discrete tomography. Unlike conventional electron tomography, only two images of the target--a silver nanoparticle embedded in an aluminium matrix--are sufficient for the reconstruction when combined with available knowledge about the particle's crystallographic structure. Additional projections confirm the reliability of the result. The results we present help close the gap between the atomic resolution achievable in two-dimensional electron micrographs and the coarser resolution that has hitherto been obtained by conventional electron tomography.
506 citations
TL;DR: DAOSTORM substantially outperformed the sparse algorithms in simulations at high signal-to-noise ratio typical of STORM data and gave small localization errors similar to the other ‘precise’ algorithm, SA1, together with a sixfold improvement in recall performance.
Abstract: We first investigated the qualitative performance of each algorithm for images of Alexa Fluor 647–immunolabeled microtubules in fixed COS-7 cells. We recorded data at high imaging density using total internal reflection fluorescence microscopy and direct (d)STORM photoswitching conditions5 (100 ms integration time, ~4,000 photons fluorophore–1 frame–1). We plotted localizations on raw images, illustrating the characteristic performance of each algorithm (Fig. 1a). SA1 only localized isolated molecules, which were fitted with small localization error. SA2 localized a larger fraction of the molecules but yielded large localization errors for overlapping molecules. DAOSTORM outperformed both sparse algorithms, identifying almost all molecules with small localization error. We quantified the performance of each algorithm by analyzing simulations of randomly distributed surface-immobilized fluorophores6. We compared observed localizations to simulated positions, calculating the recall5 and localization error at different imaging densities. Recall is the percentage of simulated fluorophores detected. Localization error is the root-mean-square distance between a localization and the simulated position. We also measured the precision5 and redundancy (Supplementary Methods), which did not vary substantially. DAOSTORM substantially outperformed the sparse algorithms in simulations at high signal-to-noise ratio typical of STORM data (bright organic fluorophores, 5,000 photons molecule–1 frame–1; Fig. 1b-c). SA1 showed poor recall at high density, with imaging density at half-maximum recall, ρHM, of 1.2 molecule μm –2. However, SA1 yielded small localization errors even at high imaging density because most overlapping molecules were rejected. SA2 had better recall performance (ρHM = 3.4 molecules μm –2) but gave large localization errors even at low imaging density (>0.1 molecules μm–2). In contrast, DAOSTORM gave small localization errors similar to the other ‘precise’ algorithm, SA1, together with a sixfold improvement in recall performance (ρHM = 7.5 molecules μm –2). For simulations at low signal-to-noise ratio typical of photoactivated localization microscopy data (fluorescent proteins, 200 photons molecule–1 frame–1; DAOSTORM: an algorithm for highdensity super-resolution microscopy
431 citations
400 citations
TL;DR: A novel FTIR system that allows for infrared-spectroscopic nanoimaging of dielectric properties (nano-FTIR) and is envisaged becoming a powerful tool for chemical identification of nanomaterials, as well as for quantitative and contact-free measurement of the local free-carrier concentration and mobility in doped nanostructures.
Abstract: Fourier-transform infrared (FTIR) spectroscopy is a widely used spectroscopic technique, particularly for infrared wavelengths. However, for imaging applications the spatial resolution of FTIR spectrometers is restricted by the diffraction limit. The use of an FTIR spectrometer to pick up the low signal from scanning near-field optical microscopy employing thermal radiation now enables infrared imaging with nanoscale resolution.
272 citations
TL;DR: Electron microscopy has become a major tool for structural biology over the molecular to cellular size range and major developments in instrumentation and methods have advanced the study of single particles in vitrified solution as well as in 3D reconstruction by tomography of irregular objects such as cells or subcellular structures.
Abstract: 1.1. Light and Electron Microscopy and Their Impact in Biology
To fully understand biological processes from the metabolism of a bacterium to the operation of a human brain, it is necessary to know the three-dimensional (3D) spatial arrangement and dynamics of the constituent molecules, how they assemble into complex molecular machines, and how they form functional organelles, cells, and tissues. The methods of X-ray crystallography and NMR spectroscopy can provide detailed information on molecular structure and dynamics. At the cellular level, optical microscopy reveals the spatial distribution and dynamics of molecules tagged with fluorophores. Electron microscopy (EM) overlaps with these approaches, covering a broad range from atomic to cellular structures. The development of cryogenic methods has enabled EM imaging to provide snapshots of biological molecules and cells trapped in a close to native, hydrated state.1,2
Because of the importance of macromolecular assemblies in the machinery of living cells and progress in the EM and image processing methods, EM has become a major tool for structural biology over the molecular to cellular size range. There have been tremendous advances in understanding the 3D spatial organization of macromolecules and their assemblies in cells and tissues, due to developments in both optical and electron microscopy. In light microscopy, super-resolution and single molecule methods have pushed the resolution of fluorescence images to ∼50 nm, using the power of molecular biology to fuse molecules of interest with fluorescent marker proteins.(3) X-ray cryo-tomography is developing as a method for 3D reconstruction of thicker (10 μm) hydrated samples, with resolution reaching the 15 nm resolution range.(4) In EM, major developments in instrumentation and methods have advanced the study of single particles (isolated macromolecular complexes) in vitrified solution as well as in 3D reconstruction by tomography of irregular objects such as cells or subcellular structures.1,5−7 Cryo-sectioning can be used to prepare vitrified sections of cells and tissues that would otherwise be too thick to image by transmission EM (TEM).8,9
In parallel, software improvements have facilitated 3D structure determination from the low contrast, low signal-to-noise ratio (SNR) images of projected densities provided by TEM of biological molecules.10−14 Alignment and classification of images in both 2D and 3D are key methods for improving SNR and detection and sorting of heterogeneity in EM data sets.(14) The resolution of single-particle reconstructions is steadily improving and has gone beyond 4 A for some icosahedral viruses and 5.5 A for asymmetric complexes such as ribosomes, giving a clear view of protein secondary structure elements and, in the best cases, resolving the protein or nucleic acid fold.15,16
182 citations
TL;DR: Flows of micrometer-sized carbon particles or whole blood in a silicone tube and individual red blood cells in mouse ear capillaries were imaged in real time, demonstrating the capability to image highly dynamic processes in vivo at a micrometers-scale resolution.
Abstract: We developed a photoacoustic imaging system that has real-time imaging capability with optical resolution. The imaging system is capable of scanning at 20 Hz over a 9 mm range and up to 40 Hz over a 1 mm scanning range. A focused laser beam provides a lateral resolution of 3.4 μm as measured in an optically nonscattering medium. Flows of micrometer-sized carbon particles or whole blood in a silicone tube and individual red blood cells (RBCs) in mouse ear capillaries were also imaged in real time, demonstrating the capability to image highly dynamic processes in vivo at a micrometer-scale resolution.
171 citations
TL;DR: An approach that corrects for three-dimensional (3D) drift in images of fixed samples without the requirement for fiduciary markers or instrument modifications is discussed and its feasibility in a practical application is shown.
Abstract: The recent development of diffraction-unlimited far-field fluorescence microscopy has overcome the classical resolution limit of ~250 nm of conventional light microscopy by about a factor of ten. The improved resolution, however, reveals not only biological structures at an unprecedented resolution, but is also susceptible to sample drift on a much finer scale than previously relevant. Without correction, sample drift leads to smeared images with decreased resolution, and in the worst case to misinterpretation of the imaged structures. This poses a problem especially for techniques such as Fluorescence Photoactivation Localization Microscopy (FPALM/PALM) or Stochastic Optical Reconstruction Microscopy (STORM), which often require minutes recording time. Here we discuss an approach that corrects for three-dimensional (3D) drift in images of fixed samples without the requirement for fiduciary markers or instrument modifications. Drift is determined by calculating the spatial cross-correlation function between subsets of localized particles imaged at different times. Correction down to ~5 nm precision is achieved despite the fact that different molecules are imaged in each frame. We demonstrate the performance of our drift correction algorithm with different simulated structures and analyze its dependence on particle density and localization precision. By imaging mitochondria with Biplane FPALM we show our algorithm's feasibility in a practical application.
167 citations
IBM1
TL;DR: In this Perspective, the different techniques used for high-resolution molecular imaging, their implementations, advantages and limitations are described, and possible scientific areas of applications are discussed.
Abstract: It has been a long-standing goal to image individual organic molecules with atomic resolution. Although atomic resolution is routinely obtained on many crystalline surfaces by scanning probe microscopy (SPM) methods, clear atomic resolution on molecules has only been achieved in the past two years by means of non-contact atomic force microscopy (NC-AFM) 1,2 and scanning tunnelling hydrogen microscopy (STHM) 3–5 . In these recent works aromatic molecules were imaged, and for the first time the individual atoms within the molecules were resolved. The obtained images directly visualize the molecular structure, for example, showing hexagons at the position of six-membered carbon rings. For both techniques, NC-AFM and STHM, an atomic functionalization of the sensor (that is, the placing of a well-defined atom or molecule at the tip of the scanning probe) was the key for achieving enhanced resolution. In the case of atomic force microscopy (AFM), the tip was modified by deliberately picking up a single carbon monoxide mol ecule, whereas in the case of STHM, the resolution was increased after bringing molecular hydrogen into the gap between the tip and the sample. Both techniques produce high-resolution images that closely resemble the molecular structure. This improved resolution has immediately proved useful for applications to identify the molecular structure of natural compounds 2
165 citations
TL;DR: In this paper, the dependence of short (below 10 min) power fluctuation on PV plant size has been investigated, and an analytic model able to describe the frequency of a given fluctuation for a certain day is proposed.
Abstract: The variable nature of the irradiance can produce significant fluctuations in the power generated by large grid-connected photovoltaic (PV) plants. Experimental 1 s data were collected throughout a year from six PV plants, 18 MWp in total. Then, the dependence of short (below 10 min) power fluctuation on PV plant size has been investigated. The analysis focuses on the study of fluctuation frequency as well as the maximum fluctuation value registered. An analytic model able to describe the frequency of a given fluctuation for a certain day is proposed
TL;DR: Diffraction intensities of lysozyme nanocrystals collected at the Linac Coherent Light Source using 2 keV photons were used for structure determination by molecular replacement and analyzed for radiation damage as a function of pulse length and fluence.
Abstract: X-ray free-electron lasers deliver intense femtosecond pulses that promise to yield high resolution diffraction data of nanocrystals before the destruction of the sample by radiation damage. Diffraction intensities of lysozyme nanocrystals collected at the Linac Coherent Light Source using 2 keV photons were used for structure determination by molecular replacement and analyzed for radiation damage as a function of pulse length and fluence. Signatures of radiation damage are observed for pulses as short as 70 fs. Parametric scaling used in conventional crystallography does not account for the observed effects.
TL;DR: This review summarizes technical developments, both in instrumentation and in computation, that have led to the new structures of icosahedral viruses determined by electron cryo-microscopy, which advance the understanding of virus assembly and cell entry.
TL;DR: The data suggest that under appropriate conditions, TEM imaging with a liquid flow cell is a promising method for understanding the in situ behaviour of nanoscale structures in a prescribed and dynamically changing chemical environment.
Abstract: The imaging of microscopic structures at nanometre-scale spatial resolution in a liquid environment is of interest for a wide range of studies Recently, a liquid flow transmission electron microscopy (TEM) holder equipped with a microfluidic cell has been developed and shown to exhibit flow of nanoparticles through an electron transparent viewing window Here we demonstrate the application of the flow cell system for both scanning and conventional transmission electron microscopy imaging of immobilized nanoparticles with a resolution of a few nanometres in liquid water of micrometre thickness The spatial resolution of conventional TEM bright field imaging is shown to be limited by chromatic aberration due to multiple inelastic scattering in the water, and we demonstrate that the liquid in the cell can be displaced by a gas phase that forms under intense electron irradiation Our data suggest that under appropriate conditions, TEM imaging with a liquid flow cell is a promising method for understanding the in situ behaviour of nanoscale structures in a prescribed and dynamically changing chemical environment
TL;DR: It is demonstrated that spatial information is also encoded in the fluorophore lifetime and that this information can be used to improve the spatial resolution of STED microscopy, and time-gating in the presence of a continuous-wave STED beam produces theoretically unbounded resolution with finite laser power.
Abstract: Stimulated-emission depletion (STED) microscopy improves image resolution by encoding additional spatial information in a second stimulated-decay channel with a spatially-varying strength. Here we demonstrate that spatial information is also encoded in the fluorophore lifetime and that this information can be used to improve the spatial resolution of STED microscopy. By solving a kinetic model for emission in the presence of a time-varying STED pulse, we derive the effective resolution as a function of fluorophore lifetime and pulse duration. We find that the best resolution for a given pulse power is achieved with a pulse of infinitesimally short duration; however, the maximum resolution can be restored for pulses of finite duration by time-gating the fluorescence signal. In parallel, we consider time-gating in the presence of a continuous-wave (CW) STED beam and find that time-gating produces theoretically unbounded resolution with finite laser power. In both cases, the cost of this improved resolution is a reduction in the brightness of the final image. We conclude by discussing situations in which time-gated STED microscopy (T-STED) may provide improved microscope performance beyond an increase in resolution.
TL;DR: This is the first direct comparison of scanning ion conductance microscopy (SICM) with atomic force microscope (AFM) for cell imaging and shows that SICM imaging can provide the true topography of soft samples at a comparable resolution.
Abstract: We present the first direct comparison of scanning ion conductance microscopy (SICM) with atomic force microscopy (AFM) for cell imaging. By imaging the same fibroblast or myoblast cell with both technologies in series, we highlight their advantages and disadvantages with respect to cell imaging. The finite imaging force applied to the sample in AFM imaging results in a coupling of mechanical sample properties into the measured sample topography. For soft samples such as cells this leads to artifacts in the measured topography and to elastic deformation, which we demonstrate by imaging whole fixed cells and cell extensions at high resolution. SICM imaging, on the other hand, has a noncontact character and can provide the true topography of soft samples at a comparable resolution.
TL;DR: In this article, a compact ultrafast electron diffractometer, consisting of a laser-driven rf photocathode that generates 3.0 MeV probe electron pulses, three-stage lens optics, and a custom-built detector for relativistic electrons, was developed.
Abstract: We have developed a compact ultrafast electron diffractometer, consisting of a laser-driven rf photocathode that generates 3.0 MeV probe electron pulses, three-stage lens optics, and a custom-built detector for relativistic electrons. High-quality single-shot transmission electron diffraction has been detected from 180-nm-thick Si single crystals, with an excellent special resolution for diffracted beams; the spot width of 0.02 A−1 is obtained. The pulse width is estimated to be 100 fs in duration. Characteristics of the electron beam and other diffractometer features are discussed.
TL;DR: Atomic force microscopy can visualize almost everything pertinent to structural virology and at resolutions that approach those for electron microscopy (EM).
Abstract: Atomic force microscopy (AFM) can visualize almost everything pertinent to structural virology and at resolutions that approach those for electron microscopy (EM). Membranes have been identified, RNA and DNA have been visualized, and large protein assemblies have been resolved into component substructures. Capsids of icosahedral viruses and the icosahedral capsids of enveloped viruses have been seen at high resolution, in some cases sufficiently high to deduce the arrangement of proteins in the capsomeres as well as the triangulation number (T). Viruses have been recorded budding from infected cells and suffering the consequences of a variety of stresses. Mutant viruses have been examined and phenotypes described. Unusual structural features have appeared, and the unexpectedly great amount of structural nonconformity within populations of particles has been documented. Samples may be imaged in air or in fluids (including culture medium or buffer), in situ on cell surfaces, or after histological procedures. AFM is nonintrusive and nondestructive, and it can be applied to soft biological samples, particularly when the tapping mode is employed. In principle, only a single cell or virion need be imaged to learn of its structure, though normally images of as many as is practical are collected. While lateral resolution, limited by the width of the cantilever tip, is a few nanometers, height resolution is exceptional, at approximately 0.5 nm. AFM produces three-dimensional, topological images that accurately depict the surface features of the virus or cell under study. The images resemble common light photographic images and require little interpretation. The structures of viruses observed by AFM are consistent with models derived by X-ray crystallography and cryo-EM.
21 Oct 2011-Nuclear Instruments & Methods in Physics Research Section A-accelerators Spectrometers Detectors and Associated Equipment
TL;DR: In this article, the authors presented a new high-resolution X-ray imager based on a pnCCD detector and a polycapillary optics, which is operated in split frame mode allowing a high frame rate of 400 Hz with an energy resolution of 152 eV for Mn K α (5.9) at 450 kcps.
Abstract: We present a new high resolution X-ray imager based on a pnCCD detector and a polycapillary optics. The properties of the pnCCD like high quantum efficiency, high energy resolution and radiation hardness are maintained, while color corrected polycapillary lenses are used to direct the fluorescence photons from every spot on a sample to a corresponding pixel on the detector. The camera is sensitive to photons from 3 to 40 keV with still 30% quantum efficiency at 20 keV. The pnCCD is operated in split frame mode allowing a high frame rate of 400 Hz with an energy resolution of 152 eV for Mn K α (5.9 keV) at 450 kcps. In single-photon counting mode (SPC), the time, energy and position of every fluorescence photon is recorded for every frame. A dedicated software enables the visualization of the elements distribution in real time without the need of post-processing the data. A description of the key components including detector, X-ray optics and camera is given. First experiments show the capability of the camera to perform fast full-field X-Ray Fluorescence (FF-XRF) for element analysis. The imaging performance with a magnifying optics (3×) has also been successfully tested.
TL;DR: This work reports on the modeling, fabrication and characterization of zone-doubled Fresnel zone plates for the multi-keV regime (4-12 keV), and demonstrates unprecedented spatial resolution by resolving 15 nm lines and spaces in scanning transmission X-ray microscopy, and focusing diffraction efficiencies of 7.2 keV photon energy.
Abstract: X-ray microscopy based on Fresnel zone plates is a powerful technique for sub-100 nm resolution imaging of biological and inorganic materials. Here, we report on the modeling, fabrication and characterization of zone-doubled Fresnel zone plates for the multi-keV regime (4―12 keV). We demonstrate unprecedented spatial resolution by resolving 15 nm lines and spaces in scanning transmission X-ray microscopy, and focusing diffraction efficiencies of 7.5% at 6.2 keV photon energy. These developments represent a significant step towards 10 nm spatial resolution for hard X-ray energies of up to 12 keV.
TL;DR: The ESPRESSO machine, combination of quick energy-band dispersion measurement and Fermi surface mapping by two-dimensional electron detector for the spin integrated ARPES and the high-efficient spin analysis by the efficient spin detector realizes the comprehensive investigation of spin electronic structure of materials.
Abstract: Highly efficient spin- and angle-resolved photoelectron spectrometer named ESPRESSO (Efficient SPin REsolved SpectroScopy Observation) machine has been developed at the beamline BL-9B in Hiroshima Synchrotron Radiation Center. Combination of high-resolution hemispherical electron analyzer and the high-efficient spin detector based on very low energy electron diffraction by the ferromagnetic target makes the high-energy resolution and angular resolution compatible with spin- and angle-resolved photoemission (SARPES) measurement. 7.5 meV in energy and ±0.18° in angular resolution have been achieved with spin resolution. The ESPRESSO machine, combination of quick energy-band dispersion measurement and Fermi surface mapping by two-dimensional electron detector for the spin integrated ARPES and the high-efficient spin analysis by the efficient spin detector realizes the comprehensive investigation of spin electronic structure of materials.
TL;DR: In this paper, it is demonstrated that scatter dark field imaging is particularly adapted to the study of a material's porosity, and an interferometer, optimized for x-ray energies around 50 keV, enables the investigation of aluminum welding with conventional laboratory xray tubes.
Abstract: X-ray scatter dark field imaging based on the Talbot-Lau interferometer allows for the measurement of ultra–small angle x-ray scattering The latter is related to the variations in the electron density in the sample at the sub- and micron-scale Therefore, information on features of the object below the detector resolution can be revealed In this article, it is demonstrated that scatter dark field imaging is particularly adapted to the study of a material’s porosity An interferometer, optimized for x-ray energies around 50 keV, enables the investigation of aluminum welding with conventional laboratory x-ray tubes The results show an unprecedented contrast between the pool and the aluminum workpiece Our conclusions are confirmed due to micro-tomographic three-dimensional reconstructions of the same object with a microscopic resolution
TL;DR: In this article, a linear phase gradient between the two halves of the objective pupil plane is introduced to split the point spread function into two lateral lobes whose relative position depends on defocus.
Abstract: Localisation microscopy overcomes the diffraction limit by measuring the position of individual molecules to obtain optical images with a lateral resolution better than 30 nm. Single molecule localisation microscopy was originally demonstrated only in two dimensions but has recently been extended to three dimensions. Here we develop a new approach to three-dimensional (3D) localisation microscopy by engineering of the point-spread function (PSF) of a fluorescence microscope. By introducing a linear phase gradient between the two halves of the objective pupil plane the PSF is split into two lateral lobes whose relative position depends on defocus. Calculations suggested that the phase gradient resulting from the very small tolerances in parallelism of conventional slides made from float glass would be sufficient to generate a two-lobed PSF. We demonstrate that insertion of a suitably chosen microscope slide that occupies half the objective aperture combined with a novel fast fitting algorithm for 3D localisation estimation allows nanoscopic imaging with detail resolution well below 100 nm in all three dimensions (standard deviations of 20, 16, and 42 nm in x, y, and z directions, respectively). The utility of the approach is shown by imaging the complex 3D distribution of microtubules in cardiac muscle cells that were stained with conventional near infrared fluorochromes. The straightforward optical setup, minimal hardware requirements and large axial localisation range make this approach suitable for many nanoscopic imaging applications.
TL;DR: The effects of dose and dose-rate were investigated for single-particle cryo-electron microscopy using stroboscopic data collection and a dose- rate effect was observed favoring lower flux densities.
Abstract: Radiation damage is an important resolution limiting factor both in macromolecular X-ray crystallography and cryo-electron microscopy. Systematic studies in macromolecular X-ray crystallography greatly benefited from the use of dose, expressed as energy deposited per mass unit, which is derived from parameters including incident flux, beam energy, beam size, sample composition and sample size. In here, the use of dose is reintroduced for electron microscopy, accounting for the electron energy, incident flux and measured sample thickness and composition. Knowledge of the amount of energy deposited allowed us to compare doses with experimental limits in macromolecular X-ray crystallography, to obtain an upper estimate of radical concentrations that build up in the vitreous sample, and to translate heat-transfer simulations carried out for macromolecular X-ray crystallography to cryo-electron microscopy. Stroboscopic exposure series of 50–250 images were collected for different incident flux densities and integration times from Lumbricus terrestris extracellular hemoglobin. The images within each series were computationally aligned and analyzed with similarity metrics such as Fourier ring correlation, Fourier ring phase residual and figure of merit. Prior to gas bubble formation, the images become linearly brighter with dose, at a rate of approximately 0.1% per 10 MGy. The gradual decomposition of a vitrified hemoglobin sample could be visualized at a series of doses up to 5500 MGy, by which dose the sample was sublimed. Comparison of equal-dose series collected with different incident flux densities showed a dose-rate effect favoring lower flux densities. Heat simulations predict that sample heating will only become an issue for very large dose rates (50 e−A−2 s−1 or higher) combined with poor thermal contact between the grid and cryo-holder. Secondary radiolytic effects are likely to play a role in dose-rate effects. Stroboscopic data collection combined with an improved understanding of the effects of dose and dose rate will aid single-particle cryo-electron microscopists to have better control of the outcome of their experiments.
TL;DR: Progress illustrates the power of cryoEM and ushers it into the arsenal of structural biology, alongside conventional techniques of X-ray crystallography and NMR, as a major tool (and sometimes the preferred one) for the studies of molecular interactions in supramolecular assemblies or machines.
Abstract: Single-particle cryo electron microscopy (cryoEM) is a technique for determining three-dimensional (3D) structures from projection images of molecular complexes preserved in their “native,” noncrystalline state. Recently, atomic or near-atomic resolution structures of several viruses and protein assemblies have been determined by single-particle cryoEM, allowing ab initio atomic model building by following the amino acid side chains or nucleic acid bases identifiable in their cryoEM density maps. In particular, these cryoEM structures have revealed extended arms contributing to molecular interactions that are otherwise not resolved by the conventional structural method of X-ray crystallography at similar resolutions. High-resolution cryoEM requires careful consideration of a number of factors, including proper sample preparation to ensure structural homogeneity, optimal configuration of electron imaging conditions to record high-resolution cryoEM images, accurate determination of image parameters to correct image distortions, efficient refinement and computation to reconstruct a 3D density map, and finally appropriate choice of modeling tools to construct atomic models for functional interpretation. This progress illustrates the power of cryoEM and ushers it into the arsenal of structural biology, alongside conventional techniques of X-ray crystallography and NMR, as a major tool (and sometimes the preferred one) for the studies of molecular interactions in supramolecular assemblies or machines.
TL;DR: The principles of spectrally assigned localization microscopy (SALM) of biological nanostructures are described, focusing on a special SALM approach, spectral precision distance/position determination microscopy(SPDM) with physically modified fluorochromes (SPDMPhymod), based on high‐precision localization of fluorescent molecules.
Abstract: For the improved understanding of biological systems on the nanoscale, it is necessary to enhance the resolution of light microscopy in the visible wavelength range beyond the limits of conventional epifluorescence microscopy (optical resolution of about 200 nm laterally, 600 nm axially). Recently, various far-field methods have been developed allowing a substantial increase of resolution ("superresolution microscopy", or "lightoptical nanoscopy"). This opens an avenue to 'nano-image' intact and even living cells, as well as other biostructures like viruses, down to the molecular detail. Thus, it is possible to combine light optical spatial nanoscale information with ultrastructure analyses and the molecular interaction information provided by molecular cell biology. In this review, we describe the principles of spectrally assigned localization microscopy (SALM) of biological nanostructures, focusing on a special SALM approach, spectral precision distance/position determination microscopy (SPDM) with physically modified fluorochromes (SPDM(Phymod) . Generally, this SPDM method is based on high-precision localization of fluorescent molecules, which can be discriminated using reversibly bleached states of the fluorophores for their optical isolation. A variety of application examples is presented, ranging from superresolution microscopy of membrane and cytoplasmic protein distribution to dual-color SPDM of nuclear proteins. At present, we can achieve an optical resolution of cellular structures down to the 20-nm range, with best values around 5 nm (∼1/100 of the exciting wavelength).
TL;DR: The conversion factor turns out to be simply the sampling rate for the full resolution cases and the introduction of this conversion can compare HSA and Fourier spectral analysis results quantitatively.
Abstract: As the original definition on Hilbert spectrum was given in terms of total energy and amplitude, there is a mismatch between the Hilbert spectrum and the traditional Fourier spectrum, which is defined in terms of energy density. Rigorous definitions of Hilbert energy and amplitude spectra are given in terms of energy and amplitude density in the time-frequency space. Unlike Fourier spectral analysis, where the resolution is fixed once the data length and sampling rate is given, the time-frequency resolution could be arbitrarily assigned in Hilbert spectral analysis (HSA). Furthermore, HSA could also provide zooming ability for detailed examination of the data in a specific frequency range with all the resolution power. These complications have made the conversion between Hilbert and Fourier spectral results difficult and the conversion formula is elusive until now. We have derived a simple relationship between them in this paper. The conversion factor turns out to be simply the sampling rate for the full resolution cases. In case of zooming, there is another additional multiplicative factor. The conversion factors have been tested in various cases including white noise, delta function, and signals from natural phenomena. With the introduction of this conversion, we can compare HSA and Fourier spectral analysis results quantitatively.
TL;DR: In this article, the authors propose an experimental approach for correcting the positioning errors and demonstrate success by two-dimensionally reconstructing both the wavefront of the focused x-ray beam and the complex transmissivity of the weakly scattering objects at the pixel resolution of better than 10 nm in the field of view larger than 5 µm.
Abstract: Ptychographic x-ray diffraction microscopy is a lensless imaging technique with a large field of view and high spatial resolution, which is also useful for characterizing the wavefront of an x-ray probe. The performance of this technique is degraded by positioning errors due to the drift between the sample and illumination optics. We propose an experimental approach for correcting the positioning errors and demonstrate success by two-dimensionally reconstructing both the wavefront of the focused x-ray beam and the complex transmissivity of the weakly scattering objects at the pixel resolution of better than 10 nm in the field of view larger than 5 $\ensuremath{\mu}$m. This method is applicable to not only the observation of organelles inside cells or nano-mesoscale structures buried within bulk materials but also the characterization of probe for single-shot imaging with x-ray free electron lasers.
TL;DR: In this paper, the authors used x-ray diffraction measurements with high energy-resolution and accuracy to study water structure at three different temperatures (7, 25 and 66 C) under normal pressure.
Abstract: We have developed x-ray diffraction measurements with high energy-resolution and accuracy to study water structure at three different temperatures (7, 25 and 66 C) under normal pressure. Using a spherically curved Ge crystal an energy resolution better than 15 eV has been achieved which eliminates influence from Compton scattering. The high quality of the data allows a precise oxygen-oxygen pair correlation function (PCF) to be directly derived from the Fourier transform of the experimental data resolving shell structure out to ~12 {\AA}, i.e. 5 hydration shells. Large-scale molecular dynamics (MD) simulations using the TIP4P/2005 force-field reproduce excellently the experimental shell-structure in the range 4-12 {\AA} although less agreement is seen for the first peak in the PCF. The Local Structure Index [J. Chem. Phys. 104, 7671 (1996)] identifies a tetrahedral minority giving the intermediate-range oscillations in the PCF and a disordered majority providing a more featureless background in this range. The current study supports the proposal that the structure of liquid water, even at high temperatures, can be described in terms of a two-state fluctuation model involving local structures related to the high-density and low-density forms of liquid water postulated in the liquid-liquid phase transition hypothesis.