Showing papers on "Resolution (electron density) published in 2012"

Imaging ultrafast molecular dynamics with laser-induced electron diffraction

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.

Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution.

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.

Transmission EBSD from 10 nm domains in a scanning electron microscope

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

Structured illumination microscopy using unknown speckle patterns

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.

Ptychographic electron microscopy using high-angle dark-field scattering for sub-nanometre resolution imaging

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.

Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution

Abstract: One of the most complex molecular machines of cells is the nuclear pore complex (NPC), which controls all trafficking of molecules in and out of the nucleus. Because of their importance for cellular processes such as gene expression and cytoskeleton organization, the structure of NPCs has been studied extensively during the last few decades, mainly by electron microscopy. We have used super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) to investigate the structure of NPCs in isolated Xenopus laevis oocyte nuclear envelopes, with a lateral resolution of ~15 nm. By generating accumulated super-resolved images of hundreds of NPCs we determined the diameter of the central NPC channel to be 41±7 nm and demonstrate that the integral membrane protein gp210 is distributed in an eightfold radial symmetry. Two-color dSTORM experiments emphasize the highly symmetric NPCs as ideal model structures to control the quality of corrections to chromatic aberration and to test the capability and reliability of super-resolution imaging methods.

Magnetic field imaging with NV ensembles

Abstract: We demonstrate a method of imaging spatially varying magnetic fields using a thin layer of nitrogen-vacancy (NV) centers at the surface of a diamond chip. Fluorescence emitted by the two-dimensional NV ensemble is detected by a CCD array, from which a vector magnetic field pattern is reconstructed. As a demonstration, AC current is passed through wires placed on the diamond chip surface, and the resulting AC magnetic field patterns are imaged using an echo-based technique with sub-micron resolution over a 140 \mu m x 140 \mu m field of view, giving single-pixel sensitivity ~100 nT/\sqrt{Hz}. We discuss ongoing efforts to further improve sensitivity and potential bioimaging applications such as real-time imaging of activity in functional, cultured networks of neurons.

Ultra-high vacuum scanning thermal microscopy for nanometer resolution quantitative thermometry

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...

Wavelet analysis for single molecule localization microscopy

Abstract: Localization of single molecules in microscopy images is a key step in quantitative single particle data analysis. Among them, single molecule based super-resolution optical microscopy techniques require high localization accuracy as well as computation of large data sets in the order of 10(5) single molecule detections to reconstruct a single image. We hereby present an algorithm based on image wavelet segmentation and single particle centroid determination, and compare its performance with the commonly used gaussian fitting of the point spread function. We performed realistic simulations at different signal-to-noise ratios and particle densities and show that the calculation time using the wavelet approach can be more than one order of magnitude faster than that of gaussian fitting without a significant degradation of the localization accuracy, from 1 nm to 4 nm in our range of study. We propose a simulation-based estimate of the resolution of an experimental single molecule acquisition.

A Von Hamos X-Ray Spectrometer Based on a Segmented-Type Diffraction Crystal for Single-Shot X-Ray Emission Spectroscopy and Time-Resolved Resonant Inelastic X-Ray Scattering Studies

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.

The universal scaling laws that determine the achievable resolution in different schemes for super-resolution imaging

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).

Reduced resolution transit delay prescan for quantitative continuous arterial spin labeling perfusion imaging.

Abstract: Arterial spin labeling perfusion MRI can suffer from artifacts and quantification errors when the time delay between labeling and arrival of labeled blood in the tissue is uncertain. This transit delay is particularly uncertain in broad clinical populations, where reduced or collateral flow may occur. Measurement of transit delay by acquisition of the arterial spin labeling signal at many different time delays typically extends the imaging time and degrades the sensitivity of the resulting perfusion images. Acquisition of transit delay maps at the same spatial resolution as perfusion images may not be necessary, however, because transit delay maps tend to contain little high spatial resolution information. Here, we propose the use of a reduced spatial resolution arterial spin labeling prescan for the rapid measurement of transit delay. Approaches to using the derived transit delay information to optimize and quantify higher resolution continuous arterial spin labeling perfusion images are described. Results in normal volunteers demonstrate heterogeneity of transit delay across different brain regions that lead to quantification errors without the transit maps and demonstrate the feasibility of this approach to perfusion and transit delay quantification.

Subparticle Ultrafast Spectrum Imaging in 4D Electron Microscopy

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.

IR microscopy utilizing intense supercontinuum light source.

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.

High-resolution imaging of chemical and biological sites on living cells using peak force tapping atomic force microscopy.

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.

Hard x-ray scanning microscopy with coherent radiation: Beyond the resolution of conventional x-ray microscopes

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.

‘Big Bang’ tomography as a new route to atomic-resolution electron tomography

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.

Microscopy beyond the diffraction limit using actively controlled single molecules

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.

Hard X-ray imaging of bacterial cells: nano-diffraction and ptychographic reconstruction.

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.

An instrument for 3D x-ray nano-imaging.

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.

Ab Initio Structure Determination of Vaterite by Automated Electron Diffraction

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]

High-resolution mapping of human brain metabolites by free induction decay (1)H MRSI at 7 T.

Abstract: This work describes a new approach for high-spatial-resolution (1)H MRSI of the human brain at 7 T. (1)H MRSI at 7 T using conventional approaches, such as point-resolved spectroscopy and stimulated echo acquisition mode with volume head coils, is limited by technical difficulties, including chemical shift displacement errors, B(0)/B(1) inhomogeneities, a high specific absorption rate and decreased T(2) relaxation times. The method presented here is based on free induction decay acquisition with an ultrashort acquisition delay (TE*) of 1.3 ms. This allows full signal detection with negligible T(2) decay or J-modulation. Chemical shift displacement errors were reduced to below 5% per part per million in the in-slice direction and were eliminated in-plane. The B(1) sensitivity was reduced significantly and further corrected using flip angle maps. Specific absorption rate requirements were well below the limit (~20 % = 0.7 W/kg). The suppression of subcutaneous lipid signals was achieved by substantially improving the point-spread function. High-quality metabolic mapping of five important brain metabolites was achieved with high in-plane resolution (64 × 64 matrix with a 3.4 × 3.4 × 12 mm(3) nominal voxel size) in four healthy subjects. The ultrashort TE* increased the signal-to-noise ratio of J-coupled resonances, such as glutamate and myo-inositol, several-fold to enable the mapping of even these metabolites with high resolution. Four measurement repetitions in one healthy volunteer provided proof of the good reproducibility of this method. The high spatial resolution allowed the visualization of several anatomical structures on metabolic maps. Free induction decay MRSI is insensitive to T(2) decay, J-modulation, B(1) inhomogeneities and chemical shift displacement errors, and overcomes specific absorption rate restrictions at ultrahigh magnetic fields. This makes it a promising method for high-resolution (1)H MRSI at 7 T and above.

FIB/SEM tomography with TEM-like resolution for 3D imaging of high-pressure frozen 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.

Noise-robust coherent diffractive imaging with a single diffraction pattern

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.

Correlative light and electron microscopy using cathodoluminescence from nanoparticles with distinguishable colours

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.

A 30 nm-resolution hard X-ray microscope with X-ray fluorescence mapping capability at BSRF

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.

Three-Dimensional, Tomographic Super-Resolution Fluorescence Imaging of Serially Sectioned Thick Samples

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.