Showing papers on "Resolution (electron density) published in 2018"

Electron ptychography of 2D materials to deep sub-ångström resolution

Abstract: Aberration-corrected optics have made electron microscopy at atomic resolution a widespread and often essential tool for characterizing nanoscale structures. Image resolution has traditionally been improved by increasing the numerical aperture of the lens (α) and the beam energy, with the state-of-the-art at 300 kiloelectronvolts just entering the deep sub-angstrom (that is, less than 0.5 angstrom) regime. Two-dimensional (2D) materials are imaged at lower beam energies to avoid displacement damage from large momenta transfers, limiting spatial resolution to about 1 angstrom. Here, by combining an electron microscope pixel-array detector with the dynamic range necessary to record the complete distribution of transmitted electrons and full-field ptychography to recover phase information from the full phase space, we increase the spatial resolution well beyond the traditional numerical-aperture-limited resolution. At a beam energy of 80 kiloelectronvolts, our ptychographic reconstruction improves the image contrast of single-atom defects in MoS2 substantially, reaching an information limit close to 5α, which corresponds to an Abbe diffraction-limited resolution of 0.39 angstrom, at the electron dose and imaging conditions for which conventional imaging methods reach only 0.98 angstrom.

X-ray ptychography

Abstract: X-ray ptychographic microscopy combines the advantages of raster scanning X-ray microscopy with the more recently developed techniques of coherent diffraction imaging. It is limited neither by the fabricational challenges associated with X-ray optics nor by the requirements of isolated specimen preparation, and offers in principle wavelength-limited resolution, as well as stable access and solution to the phase problem. In this Review, we discuss the basic principles of X-ray ptychography and summarize the main milestones in the evolution of X-ray ptychographic microscopy and tomography over the past ten years, since its first demonstration with X-rays. We also highlight the potential for applications in the life and materials sciences, and discuss the latest advanced concepts and probable future developments.

High-resolution magnetic resonance spectroscopy using a solid-state spin sensor

Abstract: Quantum systems that consist of solid-state electronic spins can be sensitive detectors of nuclear magnetic resonance (NMR) signals, particularly from very small samples. For example, nitrogen-vacancy centres in diamond have been used to record NMR signals from nanometre-scale samples, with sensitivity sufficient to detect the magnetic field produced by a single protein. However, the best reported spectral resolution for NMR of molecules using nitrogen-vacancy centres is about 100 hertz. This is insufficient to resolve the key spectral identifiers of molecular structure that are critical to NMR applications in chemistry, structural biology and materials research, such as scalar couplings (which require a resolution of less than ten hertz) and small chemical shifts (which require a resolution of around one part per million of the nuclear Larmor frequency). Conventional, inductively detected NMR can provide the necessary high spectral resolution, but its limited sensitivity typically requires millimetre-scale samples, precluding applications that involve smaller samples, such as picolitre-volume chemical analysis or correlated optical and NMR microscopy. Here we demonstrate a measurement technique that uses a solid-state spin sensor (a magnetometer) consisting of an ensemble of nitrogen-vacancy centres in combination with a narrowband synchronized readout protocol to obtain NMR spectral resolution of about one hertz. We use this technique to observe NMR scalar couplings in a micrometre-scale sample volume of approximately ten picolitres. We also use the ensemble of nitrogen-vacancy centres to apply NMR to thermally polarized nuclear spins and resolve chemical-shift spectra from small molecules. Our technique enables analytical NMR spectroscopy at the scale of single cells.

Rapid Structure Determination of Microcrystalline Molecular Compounds Using Electron Diffraction.

Abstract: Chemists of all fields currently publish about 50 000 crystal structures per year, the vast majority of which are X-ray structures. We determined two molecular structures by employing electron rather than X-ray diffraction. For this purpose, an EIGER hybrid pixel detector was fitted to a transmission electron microscope, yielding an electron diffractometer. The structure of a new methylene blue derivative was determined at 0.9 A resolution from a crystal smaller than 1×2 μm2 . Several thousand active pharmaceutical ingredients (APIs) are only available as submicrocrystalline powders. To illustrate the potential of electron crystallography for the pharmaceutical industry, we also determined the structure of an API from its pill. We demonstrate that electron crystallography complements X-ray crystallography and is the technique of choice for all unsolved cases in which submicrometer-sized crystals were the limiting factor.

Phase contrast scanning transmission electron microscopy imaging of light and heavy atoms at the limit of contrast and resolution.

Abstract: Using state of the art scanning transmission electron microscopy (STEM) it is nowadays possible to directly image single atomic columns at sub-A resolution. In standard (high angle) annular dark field STEM ((HA)ADF-STEM), however, light elements are usually invisible when imaged together with heavier elements in one image. Here we demonstrate the capability of the recently introduced Integrated Differential Phase Contrast STEM (iDPC-STEM) technique to image both light and heavy atoms in a thin sample at sub-A resolution. We use the technique to resolve both the Gallium and Nitrogen dumbbells in a GaN crystal in [ $${\bf{10}}\bar{{\bf{1}}}{\bf{1}}$$ ] orientation, which each have a separation of only 63 pm. Reaching this ultimate resolution even for light elements is possible due to the fact that iDPC-STEM is a direct phase imaging technique that allows fine-tuning the microscope while imaging. Apart from this qualitative imaging result, we also demonstrate a quantitative match of ratios of the measured intensities with theoretical predictions based on simulations.

X10 expansion microscopy enables 25-nm resolution on conventional microscopes.

Abstract: Expansion microscopy is a recently introduced imaging technique that achieves super-resolution through physically expanding the specimen by ~4×, after embedding into a swellable gel. The resolution attained is, correspondingly, approximately fourfold better than the diffraction limit, or ~70 nm. This is a major improvement over conventional microscopy, but still lags behind modern STED or STORM setups, whose resolution can reach 20-30 nm. We addressed this issue here by introducing an improved gel recipe that enables an expansion factor of ~10× in each dimension, which corresponds to an expansion of the sample volume by more than 1,000-fold. Our protocol, which we termed X10 microscopy, achieves a resolution of 25-30 nm on conventional epifluorescence microscopes. X10 provides multi-color images similar or even superior to those produced with more challenging methods, such as STED, STORM, and iterative expansion microscopy (iExM). X10 is therefore the cheapest and easiest option for high-quality super-resolution imaging currently available. X10 should be usable in any laboratory, irrespective of the machinery owned or of the technical knowledge.

Expansion Stimulated Emission Depletion Microscopy (ExSTED).

Abstract: Stimulated emission depletion (STED) microscopy is routinely used to resolve the ultrastructure of cells with a ∼10-fold higher resolution compared to diffraction limited imaging. While STED microscopy is based on preparing the excited state of fluorescent probes with light, the recently developed expansion microscopy (ExM) provides subdiffraction resolution by physically enlarging the sample before microscopy. The expansion of the fixed cells by cross-linking and swelling of hydrogels easily enlarges the sample ∼4-fold and hence increases the effective optical resolution by this factor. To overcome the current limits of these complementary approaches, we combined ExM with STED (ExSTED) and demonstrated an increase in resolution of up to 30-fold compared to conventional microscopy (<10 nm lateral and ∼50 nm isotropic). While the increase in resolution is straightforward, we found that high-fidelity labeling via multi-epitopes is required to obtain emitter densities that allow ultrastructural details with ...

Ab initio electron density determination directly from solution scattering data.

Abstract: Using a novel iterative structure factor retrieval algorithm, here I show that electron density can be directly calculated from solution scattering data without modeling The algorithm was validated with experimental data from 12 different biological macromolecules This approach avoids many of the assumptions limiting the resolution and accuracy of modeling algorithms by explicitly calculating electron density This algorithm can be applied to a wide variety of molecular systems

A fully automatic method yielding initial models from high-resolution cryo-electron microscopy maps.

Abstract: We report a fully automated procedure for the optimization and interpretation of reconstructions from cryo-electron microscopy (cryo-EM) data, available in Phenix as phenix.map_to_model. We applied our approach to 476 datasets with resolution of 4.5 A or better, including reconstructions of 47 ribosomes and 32 other protein-RNA complexes. The median fraction of residues in the deposited structures reproduced automatically was 71% for reconstructions determined at resolutions of 3 A or better and 47% for those at resolutions worse than 3 A.

Super-resolution enhancement by quantum image scanning microscopy

Abstract: The principles of quantum optics have yielded a plethora of ideas to surpass the classical limitations of sensitivity and resolution in optical microscopy. While some ideas have been applied in proof-of-principle experiments, imaging a biological sample has remained challenging mainly due to the inherently weak signal measured and the fragility of quantum states of light. In principle, however, these quantum protocols can add new information without sacrificing the classical information and can therefore enhance the capabilities of existing super-resolution techniques. Image scanning microscopy (ISM), a recent addition to the family of super-resolution methods, generates a robust resolution enhancement without sacrificing the signal level. Here we introduce quantum image scanning microscopy (Q-ISM): combining ISM with the measurement of quantum photon correlation allows increasing the resolution of ISM up to two-fold, four times beyond the diffraction limit. We introduce the Q-ISM principle and obtain super-resolved optical images of a biological sample stained with fluorescent quantum dots using photon antibunching, a quantum effect, as a resolution enhancing contrast mechanism.

Multi-slice ptychographic tomography.

Abstract: Ptychography is a form of Coherent Diffractive Imaging, where diffraction patterns are processed by iterative algorithms to recover an image of a specimen. Although mostly applied in two dimensions, ptychography can be extended to produce three dimensional images in two ways: via multi-slice ptychography or ptychographic tomography. Ptychographic tomography relies on 2D ptychography to supply projections to conventional tomographic algorithms, whilst multi-slice ptychography uses the redundancy in ptychographic data to split the reconstruction into a series of axial slices. Whilst multi-slice ptychography can handle multiple-scattering thick specimens and has a much smaller data requirement than ptychographic tomography, its depth resolution is relatively poor. Here we propose an imaging modality that combines the benefits of the two approaches, enabling isotropic 3D resolution imaging of thick specimens with a small number of angular measurements. Optical experiments validate our proposed method.

Pushing the resolution limit by correcting the Ewald sphere effect in single-particle Cryo-EM reconstructions.

Abstract: The Ewald sphere effect is generally neglected when using the Central Projection Theorem for cryo electron microscopy single-particle reconstructions. This can reduce the resolution of a reconstruction. Here we estimate the attainable resolution and report a "block-based" reconstruction method for extending the resolution limit. We find the Ewald sphere effect limits the resolution of large objects, especially large viruses. After processing two real datasets of large viruses, we show that our procedure can extend the resolution for both datasets and can accommodate the flexibility associated with large protein complexes.

meV Resolution in Laser-Assisted Energy-Filtered Transmission Electron Microscopy

Abstract: The electronic, optical, and magnetic properties of quantum solids are determined by their low-energy (<100 meV) many-body excitations. Dynamical characterization and manipulation of such excitations rely on tools that combine nm-spatial, fs-temporal, and meV-spectral resolution. Currently, phonons and collective plasmon resonances can be imaged in nanostructures with atomic (sub-nm) and tens of meV space/energy resolution using state-of-the-art energy-filtered transmission electron microscopy (TEM), but only under static conditions, while fs-resolved measurements are common but lack spatial or energy resolution. Here, we demonstrate a new method of spectrally resolved photon-induced near-field electron microscopy (SRPINEM) that allows us to obtain nm-fs-resolved maps of nanoparticle plasmons with an energy resolution determined by the laser line width (20 meV in this work) and no longer limited by the electron beam and spectrometer energy spreading. This technique can be extended to any optically accessi...

Optical frequency comb Fourier transform spectroscopy with sub-nominal resolution and precision beyond the Voigt profile

Abstract: Broadband precision spectroscopy is indispensable for providing high fidelity molecular parameters for spectroscopic databases. We have recently shown that mechanical Fourier transform spectrometer ...

Evaluating image resolution in stimulated emission depletion microscopy

Abstract: Precise knowledge of the effective spatial resolution in a stimulated emission depletion (STED) microscopy experiment is essential for reliable interpretation of the imaging results. STED microscopy theoretically provides molecular resolution, but practically different factors limit its resolution. Because these factors are related to both the sample and the system, a reliable estimation of the resolution is not straightforward. Here we show a method based on the Fourier ring correlation (FRC), which estimates an absolute resolution value directly from any STED and, more in general, point-scanning microscopy image. The FRC-based resolution metric shows terrific sensitivity to the image signal-to-noise ratio, as well as to all sample and system dependent factors. We validated the method both on commercial and on custom-made microscopes. Since the FRC-based metric can be computed in real time, without any prior information of the system/sample, it can become a fundamental tool for (i) microscopy users to optimize the experimental conditions and (ii) microscopy specialists to optimize the system conditions.

Q&A: Array tomography

Abstract: Array tomography encompasses light and electron microscopy modalities that offer unparalleled opportunities to explore three-dimensional cellular architectures in extremely fine structural and molecular detail. Fluorescence array tomography achieves much higher resolution and molecular multiplexing than most other fluorescence microscopy methods, while electron array tomography can capture three-dimensional ultrastructure much more easily and rapidly than traditional serial-section electron microscopy methods. A correlative fluorescence/electron microscopy mode of array tomography furthermore offers a unique capacity to merge the molecular discrimination strengths of multichannel fluorescence microscopy with the ultrastructural imaging strengths of electron microscopy. This essay samples the first decade of array tomography, highlighting applications in neuroscience.

FIMic: design for ultimate 3D-integral microscopy of in-vivo biological samples.

Abstract: In this work, Fourier integral microscope (FIMic), an ultimate design of 3D-integral microscopy, is presented. By placing a multiplexing microlens array at the aperture stop of the microscope objective of the host microscope, FIMic shows extended depth of field and enhanced lateral resolution in comparison with regular integral microscopy. As FIMic directly produces a set of orthographic views of the 3D-micrometer-sized sample, it is suitable for real-time imaging. Following regular integral-imaging reconstruction algorithms, a 2.75-fold enhanced depth of field and [Formula: see text]-time better spatial resolution in comparison with conventional integral microscopy is reported. Our claims are supported by theoretical analysis and experimental images of a resolution test target, cotton fibers, and in-vivo 3D-imaging of biological specimens.

Near-resonance enhanced label-free stimulated Raman scattering microscopy with spatial resolution near 130 nm.

Abstract: High-resolution optical microscopes that can break 180 nm in spatial resolution set to conventional microscopies are much-needed tools. However, current optical microscopes have to rely on exogenous fluorescent labels to achieve high resolution in biological imaging. Herein, we report near-resonance enhanced label-free stimulated Raman scattering (SRS) microscopy with a lateral resolution near 130 nm, in which the high-resolution image contrast originates directly from a low concentration of endogenous biomolecules, with sensitivity gains of approximately 23 times. Moreover, by using a 0.3-m-long optical fiber, we developed hyperspectral SRS microscopy based on spectral focusing technology. Attributed to enhancements in spatial resolution and sensitivity, we demonstrated high-resolution imaging of three-dimensional structures in single cells and high-resolution mapping of large-scale intact mouse brain tissues in situ. By using enhanced high-resolution hyperspectral SRS, we chemically observed sphingomyelin distributed in the myelin sheath that insulates single axons. Our concept opens the door to biomedical imaging with ~130 nm resolution.

Machining protein microcrystals for structure determination by electron diffraction

Abstract: We demonstrate that ion-beam milling of frozen, hydrated protein crystals to thin lamella preserves the crystal lattice to near-atomic resolution. This provides a vehicle for protein structure determination, bridging the crystal size gap between the nanometer scale of conventional electron diffraction and micron scale of synchrotron microfocus beamlines. The demonstration that atomic information can be retained suggests that milling could provide such detail on sections cut from vitrified cells.

Chiral Discrimination through Bielliptical High-Harmonic Spectroscopy

Abstract: An all-optical technique for distinguishing between chiral isomers of molecules yields much larger effects than current methods, paving the way for subfemtosecond resolution of chiral dynamics

Sparsity-based super-resolution microscopy from correlation information.

Abstract: For more than a century, the wavelength of light was considered to be a fundamental limit on the spatial resolution of optical imaging. Particularly in light microscopy, this limit, known as Abbe's diffraction limit, places a fundamental constraint on the ability to image sub-cellular organelles with high resolution. However, modern microscopy techniques such as STED, PALM, and STORM, manage to recover sub-wavelength information, by relying on fluorescence imaging. Specifically, PALM/STORM acquire large sequences of fluorescence images from molecules attached to the organelles within the imaged specimen, such that in each frame only a small set of fluorophores are active. The position of each fluorophore can be found accurately in each frame, and the image is recovered by superimposing the points from all frames. The resulting grainy image is subsequently smoothed to produce the final super-resolved image with a resolution of tens of nano-meters. However, because PALM/STORM rely on many (>10,000) exposures, they suffer from poor temporal resolution. To address that, super-resolution optical fluctuation imaging (SOFI) was shown to produce sub-diffraction images with increased temporal resolution, by allowing for higher fluorophore density and exploiting the temporal statistics of the emissions. However, the improved temporal resolution of SOFI comes at the expense of its spatial resolution, which is not as high as that of PALM/STORM. Here, we present a new method called SPARCOM: sparsity-based super-resolution correlation microscopy, which combines a shorter integration time than previously reported with spatial resolution comparable to PALM and STORM. SPARCOM relies on sparsity in the correlation domain, exploiting the sparse distribution of fluorescent molecules and the lack of correlation between different emitters. We demonstrate our technique in simulations and in experiments and provide comparisons to state-of-the-art high density methods.

Deep sub-{\AA}ngstrom imaging of 2D materials with a high dynamic range detector

Abstract: Aberration-corrected optics have made electron microscopy at atomic-resolution a widespread and often essential tool for nanocharacterization. Image resolution is dominated by beam energy and the numerical aperture of the lens ({\alpha}), with state-of-the-art reaching ~0.47 {\AA} at 300 keV. Two-dimensional materials are imaged at lower beam energies to avoid knock-on damage, limiting spatial resolution to ~1 {\AA}. Here, by combining a new electron microscope pixel array detector with the dynamic range to record the complete distribution of transmitted electrons and full-field ptychography to recover phase information from the full phase space, we increased the spatial resolution well beyond the traditional lens limitations. At 80 keV beam energy, our ptychographic reconstructions significantly improved image contrast of single-atom defects in MoS2, reaching an information limit close to 5{\alpha}, corresponding to a 0.39 {\AA} Abbe resolution, at the same dose and imaging conditions where conventional imaging modes reach only 0.98 {\AA}.