Search

Silicon nitride membranes were experimentally obtained as substrates for biological samples, which are examined using a microscope with an operating wavelength of 13.8 nm. The free-hanging films obtained have a size of up to 1.5 × 1.5 mm2, which makes it possible to select an area of interest for investigation on the sample on the order of tens to hundreds of microns. The mechanical strength of the membranes satisfies that the samples do not tear the membranes and withstand transportation. The results obtained are an import-substituting technology for the manufacture of Si3N4 membranes. The resulting membranes have a transparency of more than 40% in the range of the "water transparency window" (2.3–4.4 nm) and EUV (13–15 nm). The developed technology will become the basis for creating cuvettes for living biological samples for soft X-ray microscopy studies. [ABSTRACT FROM AUTHOR]

For soft X-ray microscopy in the approximation of geometrical optics, a theoretical model for the formation of the received image is constructed – a relationship is found between the image recorded on the detector and the 3D distribution of the sample absorption index using a high-aperture mirror objective (NA ¿ 0.3) with a focus depth close to the diffraction limit. The solution of the inverse problem of tomography for determining this 3D distribution from the data of measurements in a high-aperture mirror SXR-microscope is obtained. [ABSTRACT FROM AUTHOR]

A brief description of the concept of a soft X-ray microscope for the Nanoscope station, which is planned to be installed at the SKIF fourth-generation synchrotron, is given. The microscope is designed to study the structure of cells and dynamic processes in them with nanometer spatial resolution. It will use a unique absorption contrast of ~15 between carbon-containing structures and water in the spectral range of the water window, λ = 2.3–4.3 nm, which eliminates the need for contrasting and the use of fluorophores and minimizes the doses of ionizing radiation absorbed in the samples to obtain high-quality 3D images. The scanning and projection schemes of the microscope, their main technical characteristics, including the calculated spectra and parameters of the undulator source are presented, and an estimate of the absorbed doses depending on the resolution is obtained. The main advantage of the proposed concept lies in the use of an objective lens of high-aperture multilayer X-ray mirrors, which makes it possible to clearly visualize the focal section of the sample. Technically simple axial tomography will also be used to reconstruct the three-dimensional structure of frozen or dried samples. In the scanning scheme, due to a low dose of radiation, it will be possible to study living plant cells with a resolution of up to 10 nm, animals with a resolution of up to 80 nm, and cryofixed samples with a resolution of up to 5 nm. In the projection scheme, due to simultaneous observation of the entire focal XY section, the time for obtaining three-dimensional images is significantly reduced, but due to a large dose, it will be oriented mainly on the study of fixed samples. [ABSTRACT FROM AUTHOR]

[Display omitted] • Annealing increases mixing of the layers with formation of TiCr x. • Contribution of TiCr x increases significantly as the period decreases. • Periodicity of all systems are maintained up to 500 °C despite increased mixing. • Short period system with d = 1.41 nm retains an amorphous structure up to 300 °C. The effect of annealing on the intermixing of thin Ti and Cr layers and their crystallinity in multilayer structures was studied using XPS, TEM, XRR and XRD methods. Multilayer structures with varios period values (sum thickness of the Ti and Cr layers) in the range from 1.4 nm to 4.3 nm were considered. The analysis revealed the formation of TiCr x at the interfaces of the layers. The contribution of the formed chromide relative to the base layers increases with decreasing period of the structure. The layers in the structure with thinnest period were found to be X-ray amorphous, while the structure with a thicker period exhibited the appearance of large crystallites of chromium and titanium. Annealing of the multilayer structures induced additional intermixing, leading to an increase in the extension of the chromide regions. Additionally, it resulted in a reduction of internal stresses within the structures. When the multilayer structures were annealed at a temperature of 700 °C, complete degradation occurred, with all layers intermixing with each other and with the Si substrate. [ABSTRACT FROM AUTHOR]

A third‐order aberration analytical analysis method of soft X‐ray optical systems with orthogonal and coplanar arrangement of the main planes of elements is proposed. Firstly, the transfer equations of the aperture ray and the principle ray are derived; then, based on the third‐order aberration theory with the aperture‐ray coordinates on the reference exit wavefront of a plane‐symmetric optical system, the aberration expressions contributed by the wave aberration and defocus of this kind of optical system are studied in detail. Finally, the derived aberration calculation expressions are applied to calculate the aberration of two design examples of such types of optical systems; the images are compared with ray‐tracing results obtained using the Shadow software to validate the aberration expressions. The study shows that the accuracy of the aberration expressions is satisfactory. The analytical analysis method of aberration is helpful in the design and optimization of the soft X‐ray optical systems with orthogonal and coplanar arrangement of the main planes of optical elements. [ABSTRACT FROM AUTHOR]

With the emergence of soft x-ray techniques for imaging cells, there is a pressing need to develop protein localization probes that can be unambiguously identified within the region of x-ray spectrum used for imaging. TiO2 nanocrystal colloids, which have a strong absorption cross-section within the "water-window" region of x-rays, are ideally suited as soft x-ray microscopy probes. To demonstrate their efficacy, TiO2-streptavidin nanoconjugates were prepared and subsequently labeled microtubules polymerized from biotinylated tubulin. The microtubules were imaged using scanning transmission x-ray microscopy (STXM), and the TiO2 nanoparticle tags were specifically identified using x-ray absorption near edge spectroscopy (XANES). These experiments demonstrate that TiO2 nanoparticles are potential probes for protein localization analyses using soft x-ray microscopy.

• Method strong improves the images deconvolution efficiency in soft X-ray microscopy. • The proposed algorithm uses z-tomography data from soft X-ray microscopy. • An equation is proposed for simulating images in high-aperture soft X-Ray microscopes. Multilayer normal-incidence mirrors allow the numerical aperture (NA=0.3–0.5) of a projection lens to be significantly increased in the spectral ranges of the water (λ = 2.3–4.4 nm) and carbon (λ = 4.4–7 nm) windows, in comparison with the Fresnel zone plates. The low depth of focus of high-aperture optics (tens of nm) makes it possible to use z-tomography to reconstruct the structure of samples in soft X-ray microscopy. The presence of strong absorption prevents the direct use of a powerful deconvolution apparatus developed for fluorescence optical microscopy to improve the clarity of the image. In this article, the "intensity restoration algorithm" is proposed that takes into account the absorption effect before standard deconvolution. For an imagine lens with NA = 0.3 and a working wavelength of 3.37 nm, the results of simulating an image of a protein cell and its deconvolutionary processing are presented, before and after applying the proposed method. After its application, the deconvolution efficiency is significantly increased. A "full-period" resolution of 40 nm was obtained for the image of a simulated protein cell. [ABSTRACT FROM AUTHOR]

Cryo-soft X-ray tomography is the unique technology that can image whole intact cells in 3D under normal and pathological conditions without labelling or fixation, at high throughput and spatial resolution. The sample preparation is relatively straightforward; requiring just fast freezing of the specimen before transfer to the microscope for imaging. It is also possible to image chemically fixed samples where necessary. The technique can be correlated with cryo fluorescence microscopy to localize fluorescent proteins to organelles within the whole cell volume. Cryo-correlated light and soft X-ray tomography is particularly useful for the study of gross morphological changes brought about by disease or drugs. For example, viral fluorescent tags can be co-localized to sites of viral replication in the soft X-ray volume. In general this approach is extremely useful in the study of complex 3D organelle structure, nanoparticle uptake or in the detection of rare events in the context of whole cell structure. The main challenge of soft X-ray tomography is that the soft X-ray illumination required for imaging has heretofore only been available at a small number of synchrotron labs worldwide. Recently, a compact device with a footprint small enough to fit in a standard laboratory setting has been deployed ("the SXT-100") and is routinely imaging cryo prepared samples addressing a variety of disease and drug research applications. The SXT-100 facilitates greater access to this powerful technique and greatly increases the scope and throughput of potential research projects. Furthermore, the availability of cryo-soft X-ray tomography in the laboratory will accelerate the development of novel correlative and multimodal workflows by integration with light and electron microscope based approaches. It also allows for co-location of this powerful imaging modality at BSL3 labs or other facilities where safety or intellectual property considerations are paramount. Here we describe the compact SXT-100 microscope along with its novel integrated cryo-fluorescence imaging capability., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Abstract In nanomagnetism, one of the crucial scientific questions is whether magnetic behaviors are deterministic or stochastic on a nanoscale. Apart from the exciting physical issue, this question is also of paramount highest relevance for using magnetic materials in a wealth of technological applications such as magnetic storage and sensor devices. In the past, the research on the stochasticity of a magnetic process has been mainly done by macroscopic measurements, which only offer ensemble-averaged information. To give more accurate answer for the question and to fully understand related underlying physics, the direct observation of statistical behaviors in magnetic structures and magnetic phenomena utilizing advanced characterization techniques is highly required. One of the ideal tools for such study is a full-field soft X-ray microscope since it enables imaging of magnetic structures on the large field of view within a few seconds. Here we review the stochastic behaviors of various magnetic processes including magnetization reversal process in thin films, magnetic domain wall motions in nanowires, and magnetic vortex formations in nanodisks studied by full-field soft X-ray microscopy. The origin triggering the stochastic nature witnessed in each magnetic process and the way to control the intrinsic nature are also discussed. [ABSTRACT FROM AUTHOR]

Abstract The effect of electric current pulses on a sub-100 nm magnetic bubble state in a symmetric Pt/Co multilayer was directly observed using a full-field transmission soft X-ray microscope (MTXM). Field-induced evolution of the magnetic stripe domains into isolated bubbles with their sizes down to 100 nm was imaged under varying external magnetic fields. Electric current pulses were then applied to the created magnetic bubbles, and it was observed that the bubbles could be either created or annihilated by the current pulse depending on the strength of applied magnetic field. The results suggest that the Joule heating plays a critical role in the formation and/or elimination of the bubbles and skyrmions. Finally, the schematic phase diagram for the creation and annihilation of bubbles is presented, suggesting an optimized scheme with the combination of magnetic field and electric current necessary to utilize skyrmions in the practical devices. [ABSTRACT FROM AUTHOR]

Abstract: The study is devoted to the development of a microscope operating at a wavelength of 3.37 nm in the water window. A Schwarzschild lens with a multilayer coating is used as an image-forming objective lens. The microscope has a theoretical resolution of up to 13 nm and a field of view of 7.3-97.5 µm depending on the magnification. A low depth of focus (±37.5 nm) allows one to implement the z-tomography of samples. A schematic diagram of the microscope, the result of aspherizing the mirror collector, and the results of measuring the roughness of the mirror substrates are given. A three-point method for mounting the mirrors and also a magnetic damper operating on Foucault currents are described. The exposure time is estimated. [ABSTRACT FROM AUTHOR]

Radiation damage is an important aspect to be considered when analysing biological samples with X‐ray techniques as it can induce chemical and structural changes in the specimens. This work aims to provide new insights into the soft X‐ray induced radiation damage of the complete sample, including not only the biological tissue itself but also the substrate and embedding medium, and the tissue fixation procedure. Sample preparation and handling involves an unavoidable interaction with the sample matrix and could play an important role in the radiation‐damage mechanism. To understand the influence of sample preparation and handling on radiation damage, the effects of soft X‐ray exposure at different doses on ultralene, paraffin and on paraffin‐embedded rat tissues were studied using Fourier‐transform infrared (FTIR) microspectroscopy and X‐ray microscopy. Tissues were preserved with three different commonly used fixatives: formalin, glutaraldehyde and Karnovsky. FTIR results showed that ultralene and paraffin undergo a dose‐dependent degradation of their vibrational profiles, consistent with radiation‐induced oxidative damage. In addition, formalin fixative has been shown to improve the preservation of the secondary structure of proteins in tissues compared with both glutaraldehyde and Karnovsky fixation. However, conclusive considerations cannot be drawn on the optimal fixation protocol because of the interference introduced by both substrate and embedding medium in the spectral regions specific to tissue lipids, nucleic acids and carbohydrates. Notably, despite the detected alterations affecting the chemical architecture of the sample as a whole, composed of tissue, substrate and embedding medium, the structural morphology of the tissues at the micrometre scale is essentially preserved even at the highest exposure dose. [ABSTRACT FROM AUTHOR]

We developed a soft X-ray microscopic imaging system, CARROT, that combines ptychography and fluorescence X-ray microscopy using a total-reflection Wolter mirror illumination system. The system offers advantages such as achromaticity and long working distances, enabling seamless application to a wide wavelength range. We demonstrated high-resolution imaging of cellular structures and sub-micrometer-scale changes induced by drug administration in mammalian cells. We further showcased the capability to seamlessly switch from ptychography measurements to X-ray fluorescence measurements at different wavelengths to map elemental distributions within cells. Our findings suggest that this system can be used for investigating the correlation between intracellular structures and elemental distribution, as well as for multimodal measurements of visible light fluorescence, Raman scattering, and soft X-rays to provide valuable insights into understanding intracellular dynamics. • New imaging system combines soft X-ray ptychography & fluorescence microscopy. • High-resolution images of drug-treated mammalian cells reveal sub-micrometer changes. • Investigation of the correlation between intracellular structures and elemental distribution. [ABSTRACT FROM AUTHOR]

The microstructure and chemical composition of the discharge products generated in sodium-oxygen batteries are analyzed by transmission X-ray microscopy (TXM) revealing a complex architecture. Unexpectedly, sodium peroxide is detected in the cubic-shaped discharge deposits associated with the often reported sodium superoxide. Analyses on the product distribution show a surface layer rich in decomposition products in contact with an O-deficient region enclosing the bulk of these cubes. In addition, we show that oxygen evolution reaction (OER) occurs by a process that involves oxidation and re-dissolution of the bulk fraction, leading to a contraction of the discharge particles and a composition similar to that found in the intermediate phase of the deposits observed at the end of discharge. All these solid compounds are eventually removed at the end of recharge, but formation of these structures evidence possible irreversible pathways that need to be considered for the cycle life improvement. [ABSTRACT FROM AUTHOR]

In the asexual blood stages of malarial infection, merozoites invade erythrocytes and replicate within a parasitophorous vacuole to form daughter cells that eventually exit (egress) by sequential rupture of the vacuole and erythrocyte membranes. The current model is that PKG, a malarial cGMP-dependent protein kinase, triggers egress, activating malarial proteases and other effectors. Using selective inhibitors of either PKG or cysteine proteases to separately inhibit the sequential steps in membrane perforation, combined with video microscopy, electron tomography, electron energy loss spectroscopy, and soft X-ray tomography of mature intracellular Plasmodium falciparum parasites, we resolve intermediate steps in egress. We show that the parasitophorous vacuole membrane (PVM) is permeabilized 10–30 min before its PKG-triggered breakdown into multilayered vesicles. Just before PVM breakdown, the host red cell undergoes an abrupt, dramatic shape change due to the sudden breakdown of the erythrocyte cytoskeleton, before permeabilization and eventual rupture of the erythrocyte membrane to release the parasites. In contrast to the previous view of PKG-triggered initiation of egress and a gradual dismantling of the host erythrocyte cytoskeleton over the course of schizont development, our findings identify an initial step in egress and show that host cell cytoskeleton breakdown is restricted to a narrow time window within the final stages of egress. [ABSTRACT FROM AUTHOR]

Many signaling molecules involved in G protein-mediated signal transduction, which are present in the lipid rafts and believed to be controlled spatially and temporally, influence the potency and efficacy of neurotransmitter receptors and transporters. This has focus interest on lipid rafts and the notion that these microdomains acts as a kind of signaling platform and thus have an important role in the expression of membrane receptor-mediated signal transduction, cancer, immune responses, neurotransmission, viral infections and various other phenomena due to specific and efficient signaling according to extracellular stimuli. However, the real structure of lipid rafts has not been observed so far due to its small size and a lack of sufficiently sophisticated observation systems. A soft X-ray microscope using a coherent soft X-ray laser in the water window region (2.3–4.4 nm) should prove to be a most powerful tool to observe the dynamic structure of lipid rafts of several tens of nanometers in size in living cells. We have developed for the X-ray microscope a new compact soft X-ray laser using strongly induced plasma high harmonic resonance. We have also developed a time-resolved highly sensitive fluorescence resonance energy transfer (FRET) system and confirmed protein-protein interactions coupled with ligands. The simultaneous use of these new tools for observation of localization of G-protein coupled receptors (GPCRs) in rafts has become an important and optimum tool system to analyze the dynamics of signal transduction through rafts as signaling platform. New technology to visualize rafts is expected to lead to the understanding of those dynamics and innovative development of drug discovery that targets GPCRs localized in lipid rafts.

A method for the accurate evaluation of the organic carbon (OC) content of phytoplankton by soft X-ray microscopy (XM) was developed and applied to the picophytoplanktonSynechococcussp., whose cells are covered by extracellular polysaccharides (EPS). Based on the X-ray absorption coefficients and gray levels of the XM images, the OC content of EPS-covered cells ofSynechococcussp. (0.39–0.47 pg/cell) was found to be 2.0–2.4 times larger than that of EPS-removed cells (0.20 pg/cell). These findings suggest that soft XM could be a useful tool for evaluating the OC content of picophytoplankton and EPS without pretreatment steps. [ABSTRACT FROM PUBLISHER]

Zone-plate-based scanning transmission soft X-ray microspectroscopy (STXM) is a well established technique for high-contrast imaging of sufficiently transparent specimens ( e.g. ultrathin biological tissues, polymer materials, archaeometric specimens or magnetic thin films) with spatial resolutions in the regime of 20 nm and high spectroscopic or chemical sensitivity. However, due to the relatively large depth of focus of zone plates, the resolution of STXM along the optical axis so far stays unambiguously behind for thicker X-ray transparent specimens. This challenge can be addressed by the implementation of a second zone plate in the detection pathway of the beam, resulting in a confocal arrangement. Within this paper a first proof-of-principle study for a confocal STXM (cSTXM) and an elaborate alignment procedure in transmission and fluorescence geometry are presented. Based on first confocal soft X-ray micrographs of well known specimens, the advantage and limitation of cSTXM as well as further development potentials for future applications are discussed. [ABSTRACT FROM AUTHOR]

Imaging is one of the key drivers for new scientific insights - from the observation of distant stars in astronomy to microscopic studies of sub-cellular structures in biology. In the latter case, X-rays are a versatile probe due to their small wavelength and thus high spatial sensitivity. We give an overview of applicable lensless, coherent imaging approaches relying on scattering with a focus on ptychographic microscopy and discuss the experimental requirements for the soft X-ray scattering experiment HORST. Besides the experiment itself, we highlight the importance of sample environments, especially when biological specimens are investigated. Here, the water window is of central importance. In addition to exploitation of the contrast and resolution, resonant ptychography allows to distinguish chemical species at high spatial resolution with both phase and amplitude contrast. [ABSTRACT FROM AUTHOR]

Highlights: [•] Nano-SiO2 (nS) was grafted with two different three-oxy-silanes. [•] The hybrid was inserted on Portland cement pastes and changed the heat development on first hours. [•] Other properties of Portland cement pastes can be modified, according with the type of siloxane. [•] XM-1 in situ images confirm the influence of functionalization of nS surface on C–S–H formation. [Copyright &y& Elsevier]

Many signaling molecules involved in G protein-mediated signal transduction, which are present in the lipid rafts and believed to be controlled spatially and temporally, influence the potency and efficacy of neurotransmitter receptors and transporters. This has focus interest on lipid rafts and the notion that these microdomains acts as a kind of signaling platform and thus have an important role in the expression of membrane receptor-mediated signal transduction, cancer, immune responses, neurotransmission, viral infections and various other phenomena due to specific and efficient signaling according to extracellular stimuli. However, the real structure of lipid rafts has not been observed so far due to its small size and a lack of sufficiently sophisticated observation systems. A soft X-ray microscope using a coherent soft X-ray laser in the water window region (2.3-4.4 nm) should prove to be a most powerful tool to observe the dynamic structure of lipid rafts of several tens of nanometers in size in living cells. We have developed for the X-ray microscope a new compact soft X-ray laser using strongly induced plasma high harmonic resonance. We have also developed a time-resolved highly sensitive fluorescence resonance energy transfer (FRET) system and confirmed protein-protein interactions coupled with ligands. The simultaneous use of these new tools for observation of localization of G-protein coupled receptors (GPCRs) in rafts has become an important and optimum tool system to analyze the dynamics of signal transduction through rafts as signaling platform. New technology to visualize rafts is expected to lead to the understanding of those dynamics and innovative development of drug discovery that targets GPCRs localized in lipid rafts. [ABSTRACT FROM AUTHOR]

Abstract: Cryo transmission X-ray microscopy in the “water window” of photon energies has recently been introduced as a method that exploits the natural contrast of biological samples. We have used cryo tomographic X-ray imaging of the intra-erythrocytic malaria parasite, Plasmodium falciparum, to undertake a survey of the cellular features of this important human pathogen. We examined whole hydrated cells at different stages of growth and defined some of the structures with different X-ray density, including the parasite nucleus, cytoplasm, digestive vacuole and the hemoglobin degradation product, hemozoin. As the parasite develops from an early cup-shaped morphology to a more rounded shape, puncta of hemozoin are formed; these coalesce in the mature trophozoite into a central compartment. In some trophozoite stage parasites we observed invaginations of the parasite surface and, using a selective permeabilization process, showed that these remain connected to the RBC cytoplasm. Some of these invaginations have large openings consistent with phagocytic structures and we observed independent endocytic vesicles in the parasite cytoplasm which appear to play a role in hemoglobin uptake. In schizont stage parasites staggered mitosis was observed and X-ray-dense lipid-rich structures were evident at their apical ends of the developing daughter cells. Treatment of parasites with the antimalarial drug artemisinin appears to affect parasite development and their ability to produce the hemoglobin breakdown product, hemozoin. [ABSTRACT FROM AUTHOR]

Abstract: The high transparency of carbon-containing materials in the spectral region of “carbon window” (λ ∼4.5–5nm) introduces new opportunities for various soft X-ray microscopy applications. The development of efficient multilayer coated X-ray optics operating at the wavelengths of about 4.5nm has stimulated a series of our imaging experiments to study thick biological and synthetic objects. Our experimental set-up consisted of a laser plasma X-ray source generated with the 2nd harmonics of Nd–glass laser, scandium-based thin-film filters, Co/C multilayer mirror and X-ray film UF-4. All soft X-ray images were produced with a single nanosecond exposure and demonstrated appropriate absorption contrast and detector-limited spatial resolution. A special attention was paid to the 3D imaging of thick low-density foam materials to be used in design of laser fusion targets. [ABSTRACT FROM AUTHOR]

Abstract: In recent times, in situ synchrotron-radiation techniques have been used extensively in studies of scientifically and technologically important electrochemical systems. In this review, we showcase the power of electrochemical measurement, either active or passive, in combination with in situ synchrotron-radiation techniques, using research reported on battery systems, corroding metals, ion-selective electrodes and fuel cells. Moreover, we review the specialized electrochemical cells and general design principles utilized by electrochemical synchrotron researchers. [Copyright &y& Elsevier]

Abstract: Novel low-energy X-ray fluorescence (LEXRF) system based on a multiple Si drift detector (SDD) configuration has been developed and implemented in the European TwinMic X-ray microspectroscopy station operating at the Italian synchrotron radiation facility ELETTRA. The setup, hosting up to eight large-area SDDs with specially adapted readout electronics, has demonstrated excellent performance for elemental analysis in the 280–2200eV photon energy range, which covers the K and L edges of light elements, starting from C. The great advantage is the simultaneous acquisition of LEXRF, absorption and phase contrast maps, providing complementary information on elemental composition and morphology of specimen at submicrometer length scales. [Copyright &y& Elsevier]

The combination of high-resolution chemically sensitive soft X-ray microscopy with stereo imaging and processing techniques presented here forms a novel tool for the investigation of aqueous colloidal systems. Information about the spatial distribution within the sample is provided with small calculation effort processing just a pair of stereo micrographs. Thus, the extension towards investigation of dynamical behaviour is possible on the part of the experiment as well as of the processing. The potential of this technique is demonstrated with applications in aqueous soil and clay samples. Within these samples, haematite particles are identified taking advantage of the elemental contrast at the Fe-L edge around E= 707 eV. In combination with stereo microscopy, information about spatial arrangements are revealed and correlated to electrostatic interactions of the different mixtures, addressing to an actual question of soil scientists. The technique allows in-situ sample manipulation, which is demonstrated by a test specimen where particles were added during imaging. [ABSTRACT FROM AUTHOR]

The genetic tractability of the unicellular yeast Schizosaccharomyces pombe has resulted in it becoming an important model organism for the study of many eukaryotic cellular processes, in particular cell division. Over the past few years much progress has been made toward understanding the mechanisms that regulate eukaryotic cell division and the cellular changes that occur—for example, the formation of the cytokinetic contractile ring. However, a full understanding requires both identification of the proteins involved and correlation of this information with images showing the location of molecules in context of the cell architecture. Electron microscopic analyses have revealed exquisite ultrastructural images of cell structure, but this technique typically requires extensive processing—procedures that are labor intensive and time consuming. Imaging techniques that can more rapidly obtain better resolution than light microscopy are needed to advance the use of this model system for precise molecular localization analyses. In this manuscript, we examined S. pombe using soft X-ray tomography, an imaging technique that generates three-dimensional (3-D) images of intact hydrated cells at better than 50 nm isotropic resolution. This technique uses X-rays in the “water window,” where organic material absorbs approximately an order of magnitude more strongly than water, producing high-contrast images of cellular structures. As cells are examined in the absence of any chemical fixatives, stains, or contrast enhancement reagents, the images reflect cellular structures in the near-native state. We conducted preliminary soft X-ray imaging of S. pombe cells before and during cell division that revealed subcellular organelles, the actomyosin ring, and the septum of dividing cells. These images reveal tantalizing details of the cytokinesis process and are the first steps in our goal of generating a portfolio of tomographic images that map the location of labeled molecules into high-resolution 3-D reconstructions of the cell. [ABSTRACT FROM AUTHOR]

We demonstrate compact full-field soft X-ray transmission microscopy with sub 60-nm resolution operating at λ= 2.48 nm. The microscope is based on a 100-Hz regenerative liquid-nitrogen-jet laser-plasma source in combination with a condenser zone plate and a micro-zone plate objective for high-resolution imaging onto a 2048 × 2048 pixel CCD detector. The sample holder is mounted in a helium atmosphere and allows imaging of both dry and wet specimens. The microscope design enables fast sample switching and the sample can be pre-aligned using a visible-light microscope. High-quality images can be acquired with exposure times of less than 5 min. We demonstrate the performance of the microscope using both dry and wet samples. [ABSTRACT FROM AUTHOR]

Colloidal gold is a useful marker for functional-imaging experiments in transmission X-ray microscopy. Due to the low contrast of gold particles with small diameters it is necessary to develop a powerful algorithm to localize the single gold particles. The presented image-analysis algorithm for identifying colloidal gold particles is based on the combination of a threshold with respect to the local absorption and shape discrimination, realized by fitting a Gaussian profile to the identified regions of interest. The shape discrimination provides the possibility of size-selective identification and localization of single colloidal gold particles down to a diameter of 50 nm. The image-analysis algorithm, therefore, has potential for localization studies of several proteins simultaneously and for localization of fiducial markers in X-ray tomography. [ABSTRACT FROM AUTHOR]

Abstract: Scanning transmission X-ray microscopy (STXM) and X-ray photoemission electron microscopy (X-PEEM) are synchrotron based, soft X-ray spectromicroscopy techniques, which provide chemical speciation at better than 50nm spatial resolution based on near edge X-ray absorption spectral (NEXAFS) contrast. The instrumentation and acquisition protocols for these microscopies are described. Methods for converting image sequences to quantitative maps of chemical components are outlined and illustrated with applications to characterization of wet biofilms, optimization of the microstructure of synthetic polymers, and studies of protein interactions with patterned polymer surfaces. [Copyright &y& Elsevier]

The effect of poly(ethylene-co-vinyl acetate) or EVA on the hydration of a pure C3S was investigated by transmission soft X-ray microscopy in highly diluted systems. EVA particles are readily recognizable by their spherical shape and semitransparency to soft X-rays. Polymeric particles adsorb on the surface of hydrating C3S grains, and act as nucleation agents in the development of the composite microstructure. The formation of a polymeric film was observed after a few hours of hydration. Fourier-transform infrared spectroscopy supported the conclusions that a highly soluble phase is formed as a result of EVA-hydrating C3S chemical interactions. [Copyright &y& Elsevier]

Compact narrow-band high-brightness soft X-ray sources based on the Cherenkov effect are very promising. We discuss the theoretical basis for this novel electron-accelerator-based source. We present results of experiments, which confirm the theoretical expectations and demonstrate that intense narrow-band Cherenkov light can be produced at the silicon L-edge (99.7 eV) and, in the water window, at the titanium L-edge (453 eV) and the vanadium L-edge (512 eV). On the basis of theory and experiment, we show that a compact high-brightness Cherenkov source may be realized, which fulfills the requirements for practical soft X-ray microscopy and photoelectron spectroscopy. [ABSTRACT FROM PUBLISHER]

Soft X-ray microscopes can be used to examine whole, hydrated cells up to 10 µm thick and produce images approaching 30 nm resolution. Since cells are imaged in the X-ray transmissive ‘water window’, where organic material absorbs approximately an order of magnitude more strongly than water, chemical contrast enhancement agents are not required to view the distribution of cellular structures. Although living specimens cannot be examined, cells can be rapidly frozen at a precise moment in time and examined in a cryostage, revealing information that most closely approximates that in live cells. In this study, we used a transmission X-ray microscope at photon energies just below the oxygen edge (λ = 2.4 nm) to examine rapidly frozen mouse 3T3 cells and obtained excellent cellular morphology at better than 50 nm lateral resolution. These specimens are extremely stable, enabling multiple exposures with virtually no detectable damage to cell structures. We also show that silver-enhanced, immunogold labelling can be used to localize both cytoplasmic and nuclear proteins in whole, hydrated mammary epithelial cells at better than 50 nm resolution. The future use of X-ray tomography, along with improved zone plate lenses, will enable collection of better resolution (approaching 30 nm), three-dimensional information on the distribution of proteins in cells. [ABSTRACT FROM AUTHOR]