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  • Simultaneous multi‐color imaging using quantum dot structured illumination microscopy
    J. Microsc. (IF 1.813) Pub Date : 2020-01-14
    HUI ZENG; HUAIDONG YANG; GUOXUAN LIU; SICHUN ZHANG; XINRONG ZHANG; YINXIN ZHANG

    Multi‐color structured illumination microscopy (SIM) is a powerful tool used for the investigation of the dynamic interaction between subcellular structures. Nevertheless, most of the multi‐color SIM schemes are currently limited by conventional fluorescent dyes and wavelength dependent optical systems, and can only sequentially record images of different color channels instead of obtaining multi‐color datasets simultaneously. To address these issues, we present a novel multi‐color SIM scheme referred to as quantum dot Structured illumination microscopy (QD‐SIM). QD‐SIM enables simultaneously excitation and collection of multi‐color fluorescent signals. We also propose a theoretical analysis of the image formation in two‐dimensional multi‐color SIM to help combine the optically sectioned and super resolution attributes of SIM. Based on this theory, QD‐SIM enables optically sectioned, super‐resolution, multi‐color simultaneous imaging at a single plane.

    更新日期:2020-01-14
  • Direct measurement of TEM lamella thickness in FIB‐SEM
    J. Microsc. (IF 1.813) Pub Date : 2020-01-13
    A.P. CONLAN; E. TILLOTSON; A. RAKOWSKI; D. COOPER; S.J. HAIGH

    Transmission electron microscope (TEM) specimen preparation by focused ion beam (FIB) milling requires delicate polishing of a thin window of material during the final stages of the procedure. Over or underpolishing is common and requires extra microscope resources to correct. Despite some methods for lamella thickness measurement being available, the majority of users judge the final polishing step subjectively from scanning electron microscope (SEM) images acquired between milling steps. Here we demonstrate successful thickness determination of thin silicon lamellae using calibrated secondary electron detectors in a FIB‐SEM dual‐beam chamber. Unlike previous thickness measurement methods it does not require long acquisition times, the use of in‐chamber scanning transmission electron microscope (STEM) or energy dispersive x‐ray spectroscopy detectors. The calibration aligns a SEM image to an electron energy loss spectroscopy (EELS) map of lamella thickness acquired in a TEM. This calibration reveals the greyscale‐thickness dependence of two secondary electron SEM detectors: the through‐lens detector (TLD) and the in‐chamber electron detector (ICE). It was found that lamella thickness estimation for TLD images is accurate for areas thinner than 0.4 t/λ, whilst ICE images are most accurate for areas thicker than 0.5 t/λ up to 1.1 t/λ. The procedure presented here allows objective lamella thickness determination during the final stages of FIB specimen preparation using conventional imaging modes for common secondary electron detectors.

    更新日期:2020-01-13
  • Combining tensile testing and microscopy to address a diverse range of questions
    J. Microsc. (IF 1.813) Pub Date : 2020-01-13
    SARAH ROBINSON; PAULINE DURAND‐SMET

    Both plants and animals sense and respond to mechanical stresses that arise internally or are externally imposed. In many cases, tissues respond by changing their gene expression or their mechanical properties, which has an impact on how they develop. Many tools have been developed to measure mechanical properties and to investigate responses to mechanical stress. Here we review the state of microscope‐coupled tensile testing at the single‐cell and tissue scale and give a view on future opportunities for extending the technology. Uniaxial tensile testing involves quantifying the deformation of a sample when a force is applied. By varying the amount of force, the speed at which the force is applied or the length of time that it is applied for, many characteristics of the mechanical properties of the sample can be calculated. Tensile testing has been used extensively to measure the mechanical properties of whole tissues or organs. The need for higher resolution data resulted in more researchers using indentation tests to measure mechanical properties instead. Indentation tests provide information at a different scale and are not suitable for answering the same type of questions as tensile testing. Here we discuss that by coupling tensile‐testing machinery with microscopes such as is the case for the Automated Confocal Micro‐Extensometer (ACME) it is possible to obtain tissue‐scale measurements of mechanical properties with cellular resolution. Moreover, to understand and identify the biological processes cells and tissues use to respond to mechanical stress, we need to be able to apply mechanical perturbations to plant samples while recording the induced biological changes with microscopy.

    更新日期:2020-01-13
  • Determining the effect of calcium on cell death rate and perforation formation during leaf development in the novel model system, the lace plant (Aponogeton madagascariensis)
    J. Microsc. (IF 1.813) Pub Date : 2020-01-09
    MEREDITH S. FRASER; ADRIAN N. DAUPHINEE; ARUNIKA H.L.A.N. GUNAWARDENA

    Programmed cell death (PCD) is the destruction of unwanted cells through an intracellularly mediated process. Perforation formation in the lace plant (Aponogeton madagascariensis) provides an excellent model for studying developmentally regulated PCD. Ca2+ fluxes have previously been identified as important signals for PCD in plants and mammals. The fundamental goal of this project was to determine the influence of Ca2+ on the rate of cell death and perforation formation during leaf development in the lace plant. This was investigated using the application of various known calcium modulators including lanthanum III chloride (LaCl3), ruthenium red and calcium ionophore A23187. Detached lace plant leaves at an early stage of development were treated with these modulators in both short‐ and long‐term exposure assays and analysed using live cell imaging. Results from this study indicate that calcium plays a vital role in developmentally regulated PCD in the lace plant as application of the modulators significantly altered the rate of cell death and perforation formation during leaf development. In conclusion, this study exemplifies the suitability of the lace plant for live cell imaging and detached leaf experiments to study cell death and provides insight into the importance of Ca2+ in developmentally regulated PCD in planta.

    更新日期:2020-01-09
  • Electron Compton scattering and the measurement of electron momentum distributions in solids
    J. Microsc. (IF 1.813) Pub Date : 2020-01-06
    A. TALMANTAITE; M.R.C. HUNT; B.G. MENDIS

    Electron Compton scattering is a technique that gives information on the electron momentum density of states and is used to characterize the ground state electronic structure in solids. Extracting the momentum density of states requires us to assume the so‐called ‘impulse approximation’, which is valid for large energy losses. Here, the robustness of the impulse approximation in the low energy transfer regime is tested and confirmed on amorphous carbon films. Compared to traditional Compton measurements, this provides additional benefits of more efficient data collection and a simplified way to probe valence electrons, which govern solid state bonding. However, a potential complication is the increased background from the plasmon signal. To overcome this, a novel plasmon background subtraction routine is proposed for samples that are resistant to beam damage.

    更新日期:2020-01-07
  • Comparison of xenon and gallium sources on the detection and mapping of lithium in Li‐containing materials by using ToF‐SIMS combined FIB‐SEM
    J. Microsc. (IF 1.813) Pub Date : 2020-01-06
    K. B. DERMENCI; H. TESAŘOVÁ; T. ŠAMOŘIL; S. TURAN

    Li can find itself a wide range of applications since it is the lightest metal. However, Li detection by microscopy‐based techniques is problematic because of the highly susceptible nature during electron beam irradiation. ToF‐SIMS is a versatile technique to detect Li but the detection of light materials is also problematic due to the large ion contaminated zone and low sputtering yield. By combining ToF‐SIMS with a recently launched Xe ion source FIB‐SEM, which has small ion contamination and high sputtering yield features, can produce more realistic data at near surface and below the surface region especially for the detection of lightweight materials such as Li. In this study, Li detection and mapping capabilities of ToF‐SIMS attached to the FIB‐SEM with Ga and Xe ion sources were discussed for Al incorporated Li7La3Zr2O12 solid electrolyte sample that contains Li and Al rich regions at triple junctions. In spite of smoother milling from Ga source, Xe performs more precisely in Li mapping. Low ion contaminated zone, high sputtering yield and low straggling obtained from Monte Carlo simulations are the main advantages of Xe ion sources. The Li detection efficiency for Xe is higher than Ga source discriminating the LiAlO2 phase placed at the triple junctions of grains and La2Zr2O7 regions placed at the outer side of LLZO neighbouring the LiAlO2 phase.

    更新日期:2020-01-07
  • Monte Carlo study for correcting the broadened line‐scan profile in scanning electron microscopy
    J. Microsc. (IF 1.813) Pub Date : 2020-01-02
    P. ZHANG

    Line‐scan profile is always broadened due to the probe shape of the primary electron (PE) beam in scanning electron microscopy (SEM), which leads to an inaccurate dimension metrology. Currently, the effective electron beam shape (EEBS) is suggested as the broadening function to overcome this issue for theoretical analysis, rather than the widely used Gaussian profile. However, EEBS is almost impossible to be acquired due to it strongly depends on both the sample topography and the electron beam focusing condition, which makes it is impossible to be applied in practical analysis. Taking the case of gate linewidth measurement, an approach is proposed to find a best‐fit traditional Gaussian profile, which can optimally replace the EEBS in the case of the same sample structure and experimental condition for construction of a database of the parameter in traditional Gaussian profile. This approach is based on the use of the ideal and broadened line‐scan profiles which are both obtained from Monte Carlo (MC) simulation, but respectively by an ideal and a focusing incident electron beam model. The expected value of parameter can be obtained through deconvoluting (here using a maximum‐entropy algorithm) the broadened line‐scan profile then fitting it to the ideal profile. Experimenters can benefit from this database to obtain true line‐scan profiles for accurate gate linewidth measurement. This work should prove useful for samples of other structures and be an extension of the database in the future.

    更新日期:2020-01-02
  • Direct investigation of current transport in cells by conductive atomic force microscopy
    J. Microsc. (IF 1.813) Pub Date : 2019-12-31
    W. ZHAO; L.‐Z. CHEONG; S. XU; W. CUI; S. SONG; C. J. ROURK; C. SHEN

    Currents play critical roles in neurons. Direct observation of current flows in cells at nanometre dimensions and picoampere current resolution is still a daunting task. In this study, we investigated the current flows in hippocampal neurons, PC12 cells and astrocytes in response to voltages applied to the cell membranes using conductive atomic force microscopy (CAFM). The spines in the hippocampal neurons play crucial roles in nerve signal transfer. When the applied voltage was greater than 7.2 V, PC12 cells even show metallic nanowire‐like characteristics. Both the cell body and glial filaments of astrocytes yielded CAFM test results that reflect different electrical conductance. To our best knowledge, the electrical characteristics and current transport through components of cells (especially neurons) in response to an applied external voltage have been revealed for the first time at nanometre dimensions and picoampere current levels. We believe that such studies will pave new ways to study and model the electrical characteristics and physiological behaviours in cells and other biological samples.

    更新日期:2019-12-31
  • Automatic identification of crossovers in cryo‐EM images of murine amyloid protein A fibrils with machine learning
    J. Microsc. (IF 1.813) Pub Date : 2019-12-29
    MATTHIAS WEBER; ALEX BÄUERLE; MATTHIAS SCHMIDT; MATTHIAS NEUMANN; MARCUS FÄNDRICH; TIMO ROPINSKI; VOLKER SCHMIDT

    Detecting crossovers in cryo‐electron microscopy images of protein fibrils is an important step towards determining the morphological composition of a sample. Currently, the crossover locations are picked by hand, which introduces errors and is a time‐consuming procedure. With the rise of deep learning in computer vision tasks, the automation of such problems has become more and more applicable. However, because of insufficient quality of raw data and missing labels, neural networks alone cannot be applied successfully to target the given problem. Thus, we propose an approach combining conventional computer vision techniques and deep learning to automatically detect fibril crossovers in two‐dimensional cryo‐electron microscopy image data and apply it to murine amyloid protein A fibrils, where we first use direct image processing methods to simplify the image data such that a convolutional neural network can be applied to the remaining segmentation problem.

    更新日期:2019-12-30
  • A study on the indexing method of the electron backscatter diffraction pattern assisted by the Kikuchi bandwidth
    J. Microsc. (IF 1.813) Pub Date : 2019-12-29
    F. PENG; Y. ZHANG; J. ZHANG; C. LIN; C. JIANG; H. MIAO; Y. ZENG

    In this study, a new method is established for indexing electron backscatter diffraction (EBSD) patterns assisted by the Kikuchi bandwidth. This method utilises both interplanar angles and interplanar spacings to determine the Miller indices of the Kikuchi bands in EBSD patterns to improve the efficiency and precision of indexing in the EBSD system. Two samples of single‐crystal silicon were investigated to validate the method based on (a) the detection of the edges of the EBSD Kikuchi bands and (b) the calculation of the Kikuchi bandwidths. The relationship between the Kikuchi bandwidth and the interplanar spacing at different positions was established, and the interplanar spacing of the corresponding lattice plane of each Kikuchi band was calculated with the use of the Kikuchi bandwidth information. The relative errors between the theoretical and experimental interplanar spacings are small, with an average relative error of 2.6% and a minimum relative error of 1.04%. The results indicated that the Miller index of each Kikuchi band can be determined accurately with this new method. It is demonstrated that use of this new method improves the efficiency and accuracy of the EBSD system.

    更新日期:2019-12-30
  • Unravelling the ultrastructural details of αT‐catenin‐deficient cell–cell contacts between heart muscle cells by the use of FIB‐SEM
    J. Microsc. (IF 1.813) Pub Date : 2019-12-22
    B. VANSLEMBROUCK; A. KREMER; F. VAN ROY; S. LIPPENS; J. VAN HENGEL

    The intercalated disc is an important structure in cardiomyocytes, as it is essential to maintain correct contraction and proper functioning of the heart. Adhesion and communication between cardiomyocytes are mediated by three main types of intercellular junctions, all residing in the intercalated disc: gap junctions, desmosomes and the areae compositae. Mutations in genes that encode junctional proteins, including αT‐catenin (encoded by CTNNA3), have been linked to arrhythmogenic cardiomyopathy and sudden cardiac death. In mice, the loss of αT‐catenin in cardiomyocytes leads to impaired heart function, fibrosis, changed expression of desmosomal proteins and increased risk for arrhythmias following ischemia‐reperfusion. Currently, it is unclear how the intercalated disc and the intercellular junctions are organised in 3D in the hearts of this αT‐catenin knockout (KO) mouse model. In order to scrutinise this, ventricular cardiac tissue of αT‐catenin KO mice was used for volume electron microscopy (VEM), making use of Focused Ion Beam Scanning Electron Microscopy (FIB‐SEM), allowing a careful 3D reconstruction of the intercalated disc, including gap junctions and desmosomes. Although αT‐catenin KO and control mice display a comparable organisation of the sarcomere and the different intercalated disc regions, the folds of the plicae region of the intercalated disc are longer and more narrow in the KO heart, and the pale region between the sarcomere and the intercalated disc is larger. In addition, αT‐catenin KO intercalated discs appear to have smaller gap junctions and desmosomes in the plicae region, while gap junctions are larger in the interplicae region of the intercalated disc. Although the reason for this remodelling of the ultrastructure after αT‐catenin deletion remains unclear, the excellent resolution of the FIB‐SEM technology allows us to reconstruct details that were not reported before.

    更新日期:2019-12-23
  • Characterisation of the microplasma spraying of biocompatible coating of titanium
    J. Microsc. (IF 1.813) Pub Date : 2019-12-22
    D. L. ALONTSEVA; E. GHASSEMIEH; S. VOINAROVYCH; A. RUSSAKOVA; O. KYSLYTSIA; Y. POLOVETSKYI; A. TOXANBAYEVA

    This paper presents new results of studying the influence of parameters of microplasma spraying (MPS) of Ti wire on the structure and properties of Ti coatings. Based on the design of the experiment and the results of the SEM study, certain spraying modes were chosen to form the desired composition and structure of the Ti coating. The dense sublayer (up to 300 µm thick) provides good adhesion to the substrate, and a porous top layer can accelerate the coated implant ingrowth with the bone. This technology is developed for the manufacture of coated endoprosthesis implants.

    更新日期:2019-12-23
  • Morphology and kinetics of asphalt binder microstructure at gas, liquid and solid interfaces
    J. Microsc. (IF 1.813) Pub Date : 2019-11-12
    A. RAMM; M.C. DOWNER; N. SAKIB; A. BHASIN

    We combined optical and atomic force microscopy to observe morphology and kinetics of microstructures (typically referred to as bees) that formed at free surfaces of unmodified Performance Graded (PG) 64‐22 asphalt binders upon cooling from 150°C to room temperature (RT) at 5°C min–1, and changes in these microstructures when the surface was terminated with a transparent solid (glass) or liquid (glycerol) overlayer. The main findings are: (1) at free binder surfaces, wrinkled microstructures started to form near the crystallization temperature (∼45°C) of saturates such as wax observed by differential scanning calorimetry, then grew to ∼5 µm diameter, ∼25 nm wrinkle amplitude and 10–30% surface area coverage upon cooling to RT, where they persisted indefinitely without observable change in shape or density. (2) Glycerol coverage of the binder surface during cooling reduced wrinkled area and wrinkle amplitude three‐fold compared to free binder surfaces upon initial cooling to RT; continued glycerol coverage at RT eliminated most surface microstructures within ∼4 h. (3) No surface microstructures were observed to form at binder surfaces covered with glass. (4) Submicron bulk microstructures were observed by near‐infrared microscopy beneath the surfaces of all binder samples, with size, shape and density independent of surface coverage. No tendency of such structures to float to the top or sink to the bottom of mm‐thick samples was observed. (5) We attribute the dependence of surface wrinkling on surface coverage to variation in interface tension, based on a thin‐film continuum mechanics model.

    更新日期:2019-12-23
  • A rapid measurement method for structured surface in white light interferometry
    J. Microsc. (IF 1.813) Pub Date : 2019-11-14
    ZILI LEI; XIAOJUN LIU; LI ZHAO; WENJUN YANG; CHENG CHEN; XIAOTING GUO

    White light interferometry (WLI) is an effective and widely‐used technique for structured surface measurement. However, it requires multiframe interferograms with vertical scanning to realise large‐scale measurement, which is time consuming and computationally intensive. This paper proposes a rapid surface measurement method to realise surface recovery with a single interferogram by white light interferometry. First, the feasibility to solve the wrapped phase of a single white‐light interferogram by Hilbert transform is certified. Then, unwrapped phases against zero optical path difference position (OPD) are achieved by a zero optical path difference detection algorithm applied to unwrapping process, which provides efficient surface recovery. To ensure the accuracy of phase solution in the proposed method, the necessary number and width of the interference fringes in the interferogram are analysed and determined based on Hilbert transform and sampling analysis. Finally, measurement results of a standard step sample and a standard reticle template are presented, which prove the accuracy and efficiency of the proposed method.

    更新日期:2019-12-23
  • Steelmaking in India – new evidence from microscopic and archaeometallurgical analysis from middle Ganga plain, Balirajgarh
    J. Microsc. (IF 1.813) Pub Date : 2019-11-27
    V. SINGH; M.R. SINGH

    The present study deliberates on the archaeometallurgical examination of 2300 years old excavated iron nail from India's middle Ganga plain of Balirajgarh. The nail was subjected to multianalytical investigations in order to determine the raw materials used, manufacturing technology and preservation state. The combined analytical techniques optical microscopy, scanning electron microscopy‐energy dispersive X‐ray spectrometry (SEM‐EDX), Vickers hardness and X‐ray diffractometry shed light towards the characterisation and use of the iron artefact. Special attention was paid for qualitative and quantitative analysis of slag inclusions, metal matrix and corrosion products. The presence of heterogeneous microstructure and inclusion of impurities suggests that nail has been produced through the direct process and work hardened. The noncorroded nail is made of hypereutectoid steel and used for building purpose. The study is important to understand the role of technology in the evolution of cultural changes in India that also provides archaeometric data on the method used in the forging work.

    更新日期:2019-12-23
  • Study of SU‐8 photoresist cross‐linking process by atomic force acoustic microscopy
    J. Microsc. (IF 1.813) Pub Date : 2019-12-02
    Y. ZHAO; Y. LIU; Z. WANG; L. WANG; L. LI; F. HOU; Z. SONG; Z. WENG

    In this paper, a method is presented to detect the different phases of epoxy cross‐linking process and the subsurface structures of SU‐8 thin films by atomic force acoustic microscopy (AFAM). The AFAM imaging of SU‐8 thin films was investigated under different exposure and bake conditions. Optimized conditions were obtained for the cross‐linking of SU‐8 thin film at the exposure does of eight laser pulses with the laser fluence 10 mJ cm–2 per pulse and the post exposure bake (PEB) time at 90 s. The subsurface structures of undeveloped SU‐8 thin films were visible in the AFAM images. This method provides an effective and low‐cost way for the determination of different phases of epoxy cross‐linking process in nanostructured compounds, for the non‐destructive testing of subsurface defects, and for the evaluation of the quality of patterned structures.

    更新日期:2019-12-23
  • Expansion microscopy for the analysis of centrioles and cilia
    J. Microsc. (IF 1.813) Pub Date : 2019-11-19
    N. SAHABANDU; D. KONG; V. MAGIDSON; R. NANJUNDAPPA; C. SULLENBERGER; M.R. MAHJOUB; J. LONCAREK

    Centrioles are vital cellular structures that organise centrosomes and cilia. Due to their subresolutional size, centriole ultrastructural features have been traditionally analysed by electron microscopy. Here we present an adaptation of magnified analysis of the proteome expansion microscopy method, to be used for a robust analysis of centriole number, duplication status, length, structural abnormalities and ciliation by conventional optical microscopes. The method allows the analysis of centriole's structural features from large populations of adherent and nonadherent cells and multiciliated cultures. We validate the method using EM and superresolution microscopy and show that it can be used as an affordable and reliable alternative to electron microscopy in the analysis of centrioles and cilia in various cell cultures.

    更新日期:2019-12-23
  • Application of automated electron microscopy imaging and machine learning to characterise and quantify nanoparticle dispersion in aqueous media
    J. Microsc. (IF 1.813) Pub Date : 2019-12-18
    M. ILETT; J. WILLS; P. REES; S. SHARMA; S. MICKLETHWAITE; A. BROWN; R. BRYDSON; N. HONDOW

    For many nanoparticle applications it is important to understand dispersion in liquids. For nanomedicinal and nanotoxicological research this is complicated by the often complex nature of the biological dispersant and ultimately this leads to severe limitations in the analysis of the nanoparticle dispersion by light scattering techniques. Here we present an alternative analysis and associated workflow which utilises electron microscopy. The need to collect large, statistically relevant datasets by imaging vacuum dried, plunge frozen aliquots of suspension was accomplished by developing an automated STEM imaging protocol implemented in an SEM fitted with a transmission detector. Automated analysis of images of agglomerates was achieved by machine learning using two free open‐source software tools: CellProfiler and ilastik. The specific results and overall workflow described enable accurate nanoparticle agglomerate analysis of particles suspended in aqueous media containing other potential confounding components such as salts, vitamins and proteins.

    更新日期:2019-12-19
  • Nanocrystal segmentation in scanning precession electron diffraction data
    J. Microsc. (IF 1.813) Pub Date : 2019-12-09
    T. BERGH, D.N. JOHNSTONE, P. CROUT, S. HØGÅS, P.A. MIDGLEY, R. HOLMESTAD, P.E. VULLUM, A.T.J. VAN HELVOORT

    Scanning precession electron diffraction (SPED) enables the local crystallography of materials to be probed on the nanoscale by recording a two‐dimensional precession electron diffraction (PED) pattern at every probe position as a dynamically rocking electron beam is scanned across the specimen. SPED data from nanocrystalline materials commonly contain some PED patterns in which diffraction is measured from multiple crystals. To analyse such data, it is important to perform nanocrystal segmentation to isolate both the location of each crystal and a corresponding representative diffraction signal. This also reduces data dimensionality significantly. Here, two approaches to nanocrystal segmentation are presented, the first based on virtual dark‐field imaging and the second on non‐negative matrix factorization. Relative merits and limitations are compared in application to SPED data obtained from partly overlapping nanoparticles, and particular challenges are highlighted associated with crystals exciting the same diffraction conditions. It is demonstrated that both strategies can be used for nanocrystal segmentation without prior knowledge of the crystal structures present, but also that segmentation artefacts can arise and must be considered carefully. The analysis workflows associated with this work are provided open‐source.

    更新日期:2019-12-09
  • The importance of being edgy: cell geometric edges as an emerging polar domain in plant cells
    J. Microsc. (IF 1.813) Pub Date : 2019-11-28
    L. ELLIOTT, C. KIRCHHELLE

    Polarity is an essential feature of multicellular organisms and underpins growth and development as well as physiological functions. In polyhedral plant cells, polar domains at different faces have been studied in detail. In recent years, cell edges (where two faces meet) have emerged as discrete spatial domains with distinct biochemical identities. Here, we review and discuss recent advances in our understanding of cell edges as functional polar domains in plant cells and other organisms, highlighting conceptual parallels and open questions regarding edge polarity.

    更新日期:2019-11-29
  • Architecting hierarchical shell porosity of hollow prussian blue‐derived iron oxide for enhanced Li storage
    J. Microsc. (IF 1.813) Pub Date : 2019-10-22
    Z. ZHAO, X. LIU, C. LUAN, X. LIU, D. WANG, T. QIN, L. SUI, W. ZHANG

    Delicate architecture of active material enables improving the performacne of lithium ion batteries. Environmental‐friendly Fe2O3 anode has high theoretical specific capacity (1007 mAh g−1) in lithium ion batteries, but suffers from structural collapsing and poor electronic conductivity. Herein, we design an unique hierarchical iron oxide by regulating the initial precursor prussian blue and targeting hollow‐shell structures with full consideration of temperature controls. Among them, Fe2O3 with a sheet‐crossing structure at 650°C, affords obvious advantages of improved electronic conductivity, short ionic diffusion length, prevented particle agglomeration, and buffer volume change. Thus, we achieve a superior discharge specific capacity of 611 mAh g−1 at 500 mA g−1. Regulating hierarchical structure of prussian blue‐assisted oxides enables effectively enchancing Li storge performance.

    更新日期:2019-11-28
  • Identification of sampling patterns for high‐resolution compressed sensing MRI of porous materials: ‘learning’ from X‐ray microcomputed tomography data
    J. Microsc. (IF 1.813) Pub Date : 2019-11-06
    K. KARLSONS, D.W. DE KORT, A.J. SEDERMAN, M.D. MANTLE, H. DE JONG, M. APPEL, L.F. GLADDEN

    There exists a strong motivation to increase the spatial resolution of magnetic resonance imaging (MRI) acquisitions so that MRI can be used as a microscopy technique in the study of porous materials. This work introduces a method for identifying novel data sampling patterns to achieve undersampling schemes for compressed sensing MRI (CS‐MRI) acquisitions, enabling 3D spatial resolutions of 17.6 µm to be achieved. A data‐driven learning approach is used to derive k‐space undersampling schemes for 3D MRI acquisitions from 3D X‐ray microcomputed tomography (µCT) datasets acquired at a higher spatial resolution than can be acquired using MRI. The performance of the new sampling approach was compared to other, well‐established sampling strategies using simulated MRI data obtained from high‐resolution µCT images of rock core plugs. These simulations were performed for a range of different k‐space sampling fractions (0.125–0.375) using images of Ketton limestone. The method was then extended to consideration of imaging Estaillades limestone and Fontainebleau sandstone. The results show that the new sampling approach performs as well as or better than conventional variable density sampling and without need for time‐consuming parameter optimisation. Further, a bespoke sampling pattern is produced for each rock type. The novel undersampling strategy was employed to acquire 3D magnetic resonance images of a Ketton limestone rock at spatial resolutions of 35 and 17.6 µm. The ability of the k‐space sampling scheme produced using the new approach in enabling reconstruction of the pore space characteristics of the rock was then demonstrated by benchmarking against the pore space statistics obtained from high‐resolution µCT data. The MRI data acquired at 17.6 µm resolution gave excellent agreement with the pore size distribution obtained from the X‐ray microcomputed tomography dataset, while the pore coordination number distribution obtained from the MRI data was slightly skewed to lower coordination numbers. This approach provides a method of producing a k‐space undersampling pattern for MRI acquisition at a spatial resolution for which a fully sampled acquisition at that spatial resolution would be impractically long. The approach can be easily extended to other CS‐MRI techniques, such as spatially resolved flow and relaxation time mapping.

    更新日期:2019-11-28
  • Label‐free superresolution effect of nonlinear reverse saturation absorption thin films through difference method
    J. Microsc. (IF 1.813) Pub Date : 2019-11-06
    C. DING, J. WEI

    Label‐free superresolution effect of nonlinear reverse saturation absorption (NRSA) thin films was investigated through difference method, where a subdiffraction‐limit spot signal was constructed by intensity subtraction of two spots obtained under different laser intensities. By using annealed InSb films as the NRSA thin films, we experimentally obtained the subdiffraction‐limit spot signal, and the optimal reduction ratio was approximately 52.5% of the original spot. Experimental results agreed well with theoretical simulations. The simulation results of a 9‐point array sample showed the capability to achieve nonfluorescence superresolution imaging using the NRSA effect.

    更新日期:2019-11-28
  • Electron microscopy analysis of the thermal phase transition from hydroxyapatita to β‐TCP observed in human teeth
    J. Microsc. (IF 1.813) Pub Date : 2019-11-13
    JOSÉ REYES‐GASGA, NANCY VARGAS BECERRIL

    Samples of enamel and dentin from human molar teeth were heated in air from room temperature (25°C) up to 1200°C and the phase transition from hydroxyapatite (HAP) to tricalcium phosphate (β‐TCP) was recorded. The changes produced in morphology and chemical composition in the tooth during heating were analysed by light microscopy, scanning electron microscopy (SEM), characteristic x‐ray energy dispersion spectroscopy (EDS), x‐ray diffraction (XRD), electron diffraction, transmission electron microscopy (TEM) and high‐resolution electron microscopy (HRTEM). The results indicated a high correlation relationship among Ca content, P content, O content and Na content, and the existence of the Kirkendall effect during the HAP‐ β‐TCP phase transition.

    更新日期:2019-11-28
  • Image scanning difference microscopy
    J. Microsc. (IF 1.813) Pub Date : 2019-11-12
    YUCHEN CHEN, SHAOCONG LIU, CHENGFENG ZHANG, ZHIMIN ZHANG, CUIFANG KUANG, XIANG HAO, XU LIU

    Here, we propose a novel imaging method, which is called image scanning difference microscopy (ISDM), for superresolution imaging. In ISDM, we implement a detector array composed of 19 avalanche photodiodes (APD) rather than single‐point detector in standard confocal microscopy for reconstructing superresolved images with higher signal‐to‐noise ratio (SNR). Combining with our former proposed fluorescence emission difference (FED) method, we have achieved a lateral resolution of 111 nm (∼λ/6) without the damage of image quality, the highest FED resolution to the best of our knowledge. With its simple setup and remarkable performance, we believe that ISDM can become a versatile observation tool in biology and other fundamental studies.

    更新日期:2019-11-28
  • Applications of X‐ray computed tomography for the evaluation of biomaterial‐mediated bone regeneration in critical‐sized defects
    J. Microsc. (IF 1.813) Pub Date : 2019-11-20
    M. PEÑA FERNÁNDEZ, F. WITTE, G. TOZZI

    Bone as such displays an intrinsic regenerative potential following fracture; however, this capacity is limited with large bone defects that cannot heal spontaneously. The management of critical‐sized bone defects remains a major clinical and socioeconomic need with osteoregenerative biomaterials constantly under development aiming at promoting and enhancing bone healing. X‐ray computed tomography (XCT) has become a standard and essential tool for quantifying structure–function relationships in bone and biomaterials, facilitating the development of novel bone tissue engineering strategies. This paper presents recent advancements in XCT analysis of biomaterial‐mediated bone regeneration. As a noninvasive and nondestructive technique, XCT allows for qualitative and quantitative evaluation of three‐dimensional (3D) scaffolds and biomaterial microarchitecture, bone growth into the scaffold as well as the 3D characterisation of biomaterial degradation and bone regeneration in vitro and in vivo. Furthermore, in combination with in situ mechanical testing and digital volume correlation (DVC), XCT demonstrated its potential to better understand the bone–biomaterial interactions and local mechanics of bone regeneration during the healing process in relation to the regeneration achieved in vivo, which will likely provide valuable knowledge for the development and optimisation of novel osteoregenerative biomaterials.

    更新日期:2019-11-20
  • In situ heating TEM observations of evolving nanoscale Al–Mg–Si–Cu precipitates
    J. Microsc. (IF 1.813) Pub Date : 2019-11-18
    JONAS KRISTOFFER SUNDE, SIGURD WENNER, RANDI HOLMESTAD

    This research concerns the precipitation and subsequent dissolution of precipitate phases in an Al–0.86Mg–0.62Si–0.1Cu (at.%) alloy subjected to varying thermal exposure during an in situ heating transmission electron microscope (TEM) experiment. The distribution and crystal structure of precipitates were determined by a scanning diffraction approach at multiple stages, pinpointing the precipitates that underwent phase transformations during heating. Obtained results were compared with TEM studies of the material heated ex situ. This revealed differences in the transformation kinetics of precipitates in an electron transparent lamella (thickness ) to that of macroscopic bulk specimens.

    更新日期:2019-11-18
  • Correlative X‐ray and neutron tomography of root systems using cadmium fiducial markers
    J. Microsc. (IF 1.813) Pub Date : 2019-09-26
    T. CLARK, G. BURCA, R. BOARDMAN, T. BLUMENSATH

    The interactions between plant roots and soil are an area of active research, particularly in terms of water and nutrient uptake. Because noninvasive, in vivo studies are required, tomographic imaging appears an obvious method to use, but no one imaging modality is well suited to capture the complete system. X‐ray imaging gives clear insight to soil structure and composition; however, water is comparatively transparent to X‐rays and biological matter also displays poor contrast with respect to the pores between soil particles. Neutron imaging presents a complementary view where water and biological matter are better distinguished but the soil minerals are not imaged as clearly as they would be with X‐rays.

    更新日期:2019-11-04
  • Hierarchical electrospun tendon‐ligament bioinspired scaffolds induce changes in fibroblasts morphology under static and dynamic conditions
    J. Microsc. (IF 1.813) Pub Date : 2019-08-02
    A. SENSINI, L. CRISTOFOLINI, A. ZUCCHELLI, M.L. FOCARETE, C. GUALANDI, A. DE MORI, A.P. KAO, M. ROLDO, G. BLUNN, G. TOZZI

    The regeneration of injured tendons and ligaments is challenging because the scaffolds needs proper mechanical properties and a biomimetic morphology. In particular, the morphological arrangement of scaffolds is a key point to drive the cells growth to properly regenerate the collagen extracellular matrix. Electrospinning is a promising technique to produce hierarchically structured nanofibrous scaffolds able to guide cells in the regeneration of the injured tissue. Moreover, the dynamic stretching in bioreactors of electrospun scaffolds had demonstrated to speed up cell shape modifications in vitro. The aim of the present study was to combine different imaging techniques such as high‐resolution X‐ray tomography (XCT), scanning electron microscopy (SEM), fluorescence microscopy and histology to investigate if hierarchically structured poly (L‐lactic acid) and collagen electrospun scaffolds can induce morphological modifications in human fibroblasts, while cultured in static and dynamic conditions. After 7 days of parallel cultures, the results assessed that fibroblasts had proliferated on the external nanofibrous sheath of the static scaffolds, elongating themselves circumferentially. The dynamic cultures revealed a preferential axial orientation of fibroblasts growth on the external sheath. The aligned nanofibre bundles inside the hierarchical scaffolds instead, allowed a physiological distribution of the fibroblasts along the nanofibre direction. Inside the dynamic scaffolds, cells appeared thinner compared with the static counterpart. This study had demonstrated that hierarchically structured electrospun scaffolds can induce different fibroblasts morphological modifications during static and dynamic conditions, modifying their shape in the direction of the applied loads.

    更新日期:2019-11-04
  • Imaging techniques for observing laminar geometry in the feather shaft cortex
    J. Microsc. (IF 1.813) Pub Date : 2019-06-24
    CHRISTIAN LAURENT, SHARIF AHMED, RICHARD BOARDMAN, RICHARD COOK, GARETH DYKE, COLIN PALMER, PHILIPP SCHNEIDER, ROELAND DE KAT

    Bird feather shafts are light, stiff and strong, but the fine details of how their structure, mechanics and function relate to one another remains poorly understood. The missing piece in our understanding may be the various fibrous layers that make up the shaft's cortex. Detailed imaging techniques are needed to enable us to capture, analyse and quantify these layers before we can begin to unravel the relationship between their structure, mechanics and function.

    更新日期:2019-11-04
  • Three‐dimensional damage morphologies of thermomechanically deformed sintered nanosilver die attachments for power electronics modules
    J. Microsc. (IF 1.813) Pub Date : 2019-05-20
    P. AGYAKWA, J. DAI, J. LI, B. MOUAWAD, L. YANG, M. CORFIELD, C.M. JOHNSON

    A time‐lapse study of thermomechanical fatigue damage has been undertaken using three‐dimensional X‐ray computer tomography. Morphologies were extracted from tomography data and integrated with data from microscopy modalities at different resolution levels. This enables contextualization of some of the fine‐scale properties which underpin the large‐scale damage observed via tomography. Lateral views of crack development are presented, which show networks analogous to mud‐cracks. Crack fronts which develop in the most porous regions within the sintered attachment layer travel across the boundary into the copper substrate. The propagation characteristics of these cracks within the substrate are analysed. Evidence is provided of heterogeneous densification within the sintered joint under power cycling, and this is shown to play a major role in driving the initiation and propagation of the cracks. Examination of the texture (differing levels of X‐ray absorption) of virtual cross‐sectional images reveals the origins of the nonuniformity of densification. Finally, cracks within the sintered joint are shown to have a negligible impact on the conduction pathway of the joint due to their aspect ratio and orientation with respect to the assembly.

    更新日期:2019-11-04
  • X‐ray microfocus computed tomography: a powerful tool for structural and functional characterisation of 3D printed dosage forms
    J. Microsc. (IF 1.813) Pub Date : 2019-05-17
    C.I. GIOUMOUXOUZIS, O.L. KATSAMENIS, D.G. FATOUROS

    One of the most promising advances in modern pharmaceutical technology is the introduction of three‐dimensional (3D) printing technology for the fabrication of drug products. 3D printed dosage forms have the potential to revolutionise pharmacotherapy as streamlined production of structurally complex formulations with optimal drug releasing properties is now made possible. 3D printed formulations are derived as part of a process where a ‘print‐head’ deposits, or sinters material under computer control to produce a drug carrier. However, this manufacturing route inherently generates objects that deviate from the ideal designed template for reasons specific to the 3D printing method used. This short opinion article discusses the potential of high‐resolution nondestructive 3D (volume) imaging by means of X‐ray microfocus Computed Tomography (μCT) as a Process Analytical Technology for the structural and functional characterisation of 3D printed dosage forms.

    更新日期:2019-11-04
  • The Lost Portrait of Robert Hooke?
    J. Microsc. (IF 1.813) Pub Date : 2019-09-09
    Lawrence R. Griffing

    This letter considers the “Portrait of a Mathematician” attributed to Mary Beale in the 1680s as a likely candidate for a portrait of Robert Hooke made during his lifetime. It closely matches the physical descriptions of Hooke made by his biographers who knew him, Richard Waller (d. 1715) and John Aubrey (1626‐1697). The portrait contains a remarkable diagram, as well as its mechanical analogue, demonstrating the elliptical orbit of a body under constant force similar to an unpublished, unfinished 1685 manuscript by Hooke. It also contains a landscape, which is likely to be Lowther Castle and its associated church. Hooke provided designs for the Lowther castle church renovations completed in 1686. The portraitist, Mary Beale, knew Hooke and was very familiar with the Lowther family, who commissioned thirty portraits from her. The presence of the diagram in the portrait may have been one the reasons it was not purchased and kept by the Royal Society. It might have diminished the legacy of Isaac Newton.

    更新日期:2019-11-04
  • Design of a 3D printed smartphone microscopic system with enhanced imaging ability for biomedical applications
    J. Microsc. (IF 1.813) Pub Date : 2019-09-22
    D. RABHA, A. SARMAH, P. NATH

    Portable, low‐cost smartphone platform microscopic systems have emerged as a potential tool for imaging of various micron and submicron scale particles in recent years (Ozcan; Pirnstill and Coté; Breslauer et al.; Zhu et al.). In most of the reported works, it involves either the use of sophisticated optical set‐ups along with a high‐end computational tool for postprocessing of the captured images, or it requires a high‐end configured smartphone to obtain enhanced imaging of the sample. Present work reports the working of a low‐cost, field‐portable 520× optical microscope using a smartphone. The proposed smartphone microscopic system has been designed by attaching a 3D printed compact optical set‐up to the rear camera of a regular smartphone. By using cloud‐based services, an image processing algorithm has been developed which can be accessed anytime through a mobile broadband network. Using this facility, the quality of the captured images can be further enhanced, thus obviating the need for dedicated computational tools for postprocessing of the images. With the designed microscopic system, an optical resolution ∼2 µm has been obtained. Upon postprocessing, the resolution of the captured images can be improved further. It is envisioned that with properly designed optical set‐up in 3D printer and by developing an image processing application in the cloud, it is possible to obtain a low‐cost, user‐friendly, field‐portable optical microscope on a regular smartphone that performs at par with that of a laboratory‐grade microscope.

    更新日期:2019-11-04
  • A method to simulate the optical image from far‐field scattering numerical data and its application to the total internal reflection microscopy of metallic nanowires
    J. Microsc. (IF 1.813) Pub Date : 2019-09-26
    IGNACIO IGLESIAS, JUAN MUÑOZ, JAIME COLCHERO

    Computational electrodynamics modelling plays an important role in understanding and designing new photonic devices. The results offered by these simulations are usually close‐range field distributions or angular power emission plots. We describe a procedure to compute the optical microscopy image from simulated far‐field scattering data using three‐dimensional discrete Fourier transforms that can be used when the simulation software package do not include proper far‐field to optical imaging projection routines. The method is demonstrated comparing simulated images with real images of nanowires obtained with a total internal reflection microscope.

    更新日期:2019-11-04
  • Volume orientation: a practical solution to analyse the orientation of fibres in composite materials
    J. Microsc. (IF 1.813) Pub Date : 2019-10-03
    F. DE PASCALIS, M. NACUCCHI

    Mechanical properties of fibres reinforced composite materials depend on the type of fibres used, their percentage as well as their arrangement and orientation. As computer technology continues to improve, high‐resolution computed tomography has proven to be an ideal instrument to analyse the structure of this kind of materials. In this context, various approaches have been proposed to detect the fibre orientation distribution and the relative degree of anisotropy of these composite materials. Some of these approaches are based on ‘individual’ measurements that isolate and reconstruct each single fibre and measure its properties. On the other hand, other approaches capture the characteristics of the fibre distribution by means of ‘global’ measurements computed on the entire set of tomographic data. The first methods are more precise but also more complex because they demand a procedure able to segment and separate each single fibre in the polymer, whereas the latter are easier to implement and can be applied even if fibre segmentation and separation is not effective or practicable. In this paper, a global method based on the technique called volume orientation – originally proposed several years ago to study the anisotropy of bone structures – is applied to fibre reinforced composite materials. This new approach does not require data acquired at very high resolution nor very complex procedures for individual segmentation of the fibres, but only binarised data through common thresholding procedures. The effectiveness of the proposed new approach is demonstrated by comparing it to the results obtained from a method based on individual measurements: when resolution and images quality are good enough, the volume orientation method gives results quite similar to the other approach. The analysis of three different case studies demonstrates its flexibility and its validity as an alternative to methods based on the separation of individual fibres, which are not always usable. The samples have been carefully selected in order to range between different attenuation contrast levels and also include a specimen subjected to mechanical testing which can be of great practical interest.

    更新日期:2019-11-04
  • Current outcomes when optimizing ‘standard’ sample preparation for single‐particle cryo‐EM
    J. Microsc. (IF 1.813) Pub Date : 2019-10-07
    B. CARRAGHER, Y. CHENG, A. FROST, R.M. GLAESER, G.C. LANDER, E. NOGALES, H.‐W. WANG

    Although high‐resolution single‐particle cryo‐electron microscopy (cryo‐EM) is now producing a rapid stream of breakthroughs in structural biology, it nevertheless remains the case that the preparation of suitable frozen‐hydrated samples on electron microscopy grids is often quite challenging. Purified samples that are intact and structurally homogeneous – while still in the test tube – may not necessarily survive the standard methods of making extremely thin, aqueous films on grids. As a result, it is often necessary to try a variety of experimental conditions before finally finding an approach that is optimal for the specimen at hand. Here, we summarize some of our collective experiences to date in optimizing sample preparation, in the hope that doing so will be useful to others, especially those new to the field. We also hope that an open discussion of these common challenges will encourage the development of more generally applicable methodology. Our collective experiences span a diverse range of biochemical samples and most of the commonly used variations in how grids are currently prepared. Unfortunately, none of the currently used optimization methods can be said, in advance, to be the one that ultimately will work when a project first begins. Nevertheless, there are some preferred first steps to explore when facing specific problems that can be more generally recommended, based on our experience and that of many others in the cryo‐EM field.

    更新日期:2019-11-04
  • Visualization of ultraviolet absorption distribution beyond the diffraction limit of light by electron‐beam excitation‐assisted optical microscope
    J. Microsc. (IF 1.813) Pub Date : 2019-10-09
    W. INAMI, M. FUKUTA, Y. KAWATA, S. TERAKAWA

    We demonstrated that the high spatial resolution absorption contrast imaging of the crystal of vitamin B9 has absorption at ultraviolet wavelengths. The absorption wavelength matches with the wavelength of the emission of the fluorescent thin film of an electron‐beam excitation‐assisted (EXA) optical microscope. The fine crystal structure was imaged beyond the optical diffraction limit. The image contrast corresponded with the thickness of the crystal. The illumination light is absorbed with the vitamin B9 crystal and the intensity of the transmitted light depends on the thickness of the vitamin B9 crystal. The EXA optical microscope is useful for analysis of growth of a crystal, bioimaging and so on.

    更新日期:2019-11-04
  • Automatic thresholding from the gradients of region boundaries.
    J. Microsc. (IF 1.813) Pub Date : 2016-09-21
    G Landini,D A Randell,S Fouad,A Galton

    We present an approach for automatic threshold segmentation of greyscale images. The procedure is inspired by a reinterpretation of the strategy observed in human operators when adjusting thresholds manually and interactively by means of 'slider' controls. The approach translates into two methods. The first one is suitable for single or multiple global thresholds to be applied globally to images and consists of searching for a threshold value that generates a phase whose boundary coincides with the largest gradients in the original image. The second method is a variation, implemented to operate on the discrete connected components of the thresholded phase (i.e. the binary regions) independently. Consequently, this becomes an adaptive local threshold procedure, which operates relative to regions, rather than to local image subsets as is the case in most local thresholding methods previously published. Adding constraints for specifying certain classes of expected objects in the images can improve the output of the method over the traditional 'segmenting first, then classify' approach.

    更新日期:2019-11-01
  • Estimation of filler macro-dispersion in rubber matrix by radiometric stereo microscopy.
    J. Microsc. (IF 1.813) Pub Date : 2019-02-01
    J Ohser,J Lacayo-Pineda,M Putman,A Rack,D Dobrovolskij

    A new microscopic principle based on radiometric stereo microscopy is presented, which is designed for investigating macro-dispersion of filler in rubber. The image acquisition is combined with a stereological method of estimating the volume-weighted size distribution of the filler particles. Experimental results for carbon black filler in rubber obtained by radiometric stereo microscopy are compared with those from microtomography using synchrotron radiation, and, furthermore, a simulation study is used for evaluation. It turns out that using the new three-dimensional microscopic method, the size distribution of the filler particles can be estimated from fresh cuts of rubber with high accuracy, and thus it is an interesting alternative to well-established dark field microscopy. LAY DESCRIPTION: Macro-dispersion of globular filler particles in a rubber matrix is an important quantity that depends on manufacturing parameters and influences various rubber properties. Therefore, it must be carefully adjusted during the incorporation process and investigated by industrial quality control (ASTM D7723-18). Quality control is usually based on freshly made planar sections so-called fresh cuts through rubber specimen. After stress retention of the rubber one obtains a rough cutting surface in which the filler particles appear as imprints or bumps, called nodges. These nodges can be made visible by classical light microscopy under dark field (DFM) illumination. The systems disperGRADER+ or the disperGRADER Alpha View were specifically designed for rubber inspection. However, it has proved to be very difficult estimating the size distribution of the filler particles from the observed white spots in the DFM image. In any case it is still necessary to compute the size distribution of the filler particles from an estimated size distribution of the section profiles. The latter is numerically unstable, i.e. small errors of the estimated size distribution of the section profiles lead to large errors of the computed filler size distribution. Applying DFM combined with filler dispersion estimation as described in ASTM D7723-18 appears to be a fingerprint method only. For this reason, the new microscope nSPEC 3D was applied for rubber inspection. The principle used for surface imaging is based on radiometric stereo allowing for perfect three-dimensional reconstruction of curved surfaces of fresh cuts. From this reconstruction it is possible to measure the height of particle nodges as well as their volumes. Furthermore, we present a new stereological method for estimating the filler size distribution from samples of the height and the volume of the nodges. Finally, microtomography with synchrotron radiation and computer simulation are applied to evaluate accuracy of the presented method.

    更新日期:2019-11-01
  • Towards robust and versatile single nanoparticle fiducial markers for correlative light and electron microscopy.
    J. Microsc. (IF 1.813) Pub Date : 2019-01-17
    J J H A VAN Hest,A V Agronskaia,J Fokkema,F Montanarella,A Gregorio Puig,C DE Mello Donega,A Meijerink,G A Blab,H C Gerritsen

    Fiducial markers are used in correlated light and electron microscopy (CLEM) to enable accurate overlaying of fluorescence and electron microscopy images. Currently used fiducial markers, e.g. dye-labelled nanoparticles and quantum dots, suffer from irreversible quenching of the luminescence after electron beam exposure. This limits their use in CLEM, since samples have to be studied with light microscopy before the sample can be studied with electron microscopy. Robust fiducial markers, i.e. luminescent labels that can (partially) withstand electron bombardment, are interesting because of the recent development of integrated CLEM microscopes. In addition, nonintegrated CLEM setups may benefit from such fiducial markers. Such markers would allow switching back from EM to LM and are not available yet. Here, we investigate the robustness of various luminescent nanoparticles (NPs) that have good contrast in electron microscopy; 130 nm gold-core rhodamine B-labelled silica particles, 15 nm CdSe/CdS/ZnS core-shell-shell quantum dots (QDs) and 230 nm Y2 O3 :Eu3+ particles. Robustness is studied by measuring the luminescence of (single) NPs after various cycles of electron beam exposure. The gold-core rhodamine B-labelled silica NPs and QDs are quenched after a single exposure to 60 ke- nm-2 with an energy of 120 keV, while Y2 O3 :Eu3+ NPs are robust and still show luminescence after five doses of 60 ke- nm-2 . In addition, the luminescence intensity of Y2 O3 :Eu3+ NPs is investigated as function of electron dose for various electron fluxes. The luminescence intensity initially drops to a constant value well above the single particle detection limit. The intensity loss does not depend on the electron flux, but on the total electron dose. The results indicate that Y2 O3 :Eu3+ NPs are promising as robust fiducial marker in CLEM. LAY DESCRIPTION: Luminescent particles are used as fiducial markers in correlative light and electron microscopy (CLEM) to enable accurate overlaying of fluorescence and electron microscopy images. The currently used fiducial markers, e.g. dyes and quantum dots, loose their luminescence after exposure to the electron beam of the electron microscope. This limits their use in CLEM, since samples have to be studied with light microscopy before the sample can be studied with electron microscopy. Robust fiducial markers, i.e. luminescent labels that can withstand electron exposure, are interesting because of recent developments in integrated CLEM microscopes. Also nonintegrated CLEM setups may benefit from such fiducial markers. Such markers would allow for switching back to fluorescence imaging after the recording of electron microscopy imaging and are not available yet. Here, we investigate the robustness of various luminescent nanoparticles (NPs) that have good contrast in electron microscopy; dye-labelled silica particles, quantum dots and lanthanide-doped inorganic particles. Robustness is studied by measuring the luminescence of (single) NPs after various cycles of electron beam exposure. The dye-labelled silica NPs and QDs are quenched after a single exposure to 60 ke- nm-2 with an energy of 120 keV, while lanthanide-doped inorganic NPs are robust and still show luminescence after five doses of 60 ke- nm-2 . In addition, the luminescence intensity of lanthanide-doped inorganic NPs is investigated as function of electron dose for various electron fluxes. The luminescence intensity initially drops to a constant value well above the single particle detection limit. The intensity loss does not depend on the electron flux, but on the total electron dose. The results indicate that lanthanide-doped NPs are promising as robust fiducial marker in CLEM.

    更新日期:2019-11-01
  • Automated noninvasive epithelial cell counting in phase contrast microscopy images with automated parameter selection.
    J. Microsc. (IF 1.813) Pub Date : 2018-07-13
    R Flight,G Landini,I B Styles,R M Shelton,M R Milward,P R Cooper

    Cell counting is commonly used to determine proliferation rates in cell cultures and for adherent cells it is often a 'destructive' process requiring disruption of the cell monolayer resulting in the inability to follow cell growth longitudinally. This process is time consuming and utilises significant resource. In this study a relatively inexpensive, rapid and widely applicable phase contrast microscopy-based technique has been developed that emulates the contrast changes taking place when bright field microscope images of epithelial cell cultures are defocused. Processing of the resulting images produces an image that can be segmented using a global threshold; the number of cells is then deduced from the number of segmented regions and these cell counts can be used to generate growth curves. The parameters of this method were tuned using the discrete mereotopological relations between ground truth and processed images. Cell count accuracy was improved using linear discriminant analysis to identify spurious noise regions for removal. The proposed cell counting technique was validated by comparing the results with a manual count of cells in images, and subsequently applied to generate growth curves for oral keratinocyte cultures supplemented with a range of concentrations of foetal calf serum. The approach developed has broad applicability and utility for researchers with standard laboratory imaging equipment.

    更新日期:2019-11-01
  • Progress towards a methodology for high throughput 3D reconstruction of soot nanoparticles via electron tomography.
    J. Microsc. (IF 1.813) Pub Date : 2018-01-18
    E Haffner-Staton,A LA Rocca,M W Fay

    The aim of this work is to make progress towards the development of 3D reconstruction as a legitimate alternative to traditional 2D characterization of soot. Time constraints are the greatest opposition to its implementation, as currently reconstruction of a single soot particle takes around 5-6 h to complete. As such, the accuracy and detail gains are currently insufficient to challenge 2D characterization of a representative sample (e.g. 200 particles). This work is a consideration of the optimization of the steps included within the computational reconstruction and manual segmentation of soot particles. Our optimal process reduced the time required by over 70% in comparison to a typical procedure, whilst producing models with no appreciable decrease in quality.

    更新日期:2019-11-01
  • Laser-preparation of geometrically optimised samples for X-ray nano-CT.
    J. Microsc. (IF 1.813) Pub Date : 2017-05-16
    J J Bailey,T M M Heenan,D P Finegan,X Lu,S R Daemi,F Iacoviello,N R Backeberg,O O Taiwo,D J L Brett,A Atkinson,P R Shearing

    A robust and versatile sample preparation technique for the fabrication of cylindrical pillars for imaging by X-ray nano-computed tomography (nano-CT) is presented. The procedure employs simple, cost-effective laser micro-machining coupled with focused-ion beam (FIB) milling, when required, to yield mechanically robust samples at the micrometre length-scale to match the field-of-view (FOV) for nano-CT imaging. A variety of energy and geological materials are exhibited as case studies, demonstrating the procedure can be applied to a variety of materials to provide geometrically optimised samples whose size and shape are tailored to the attenuation coefficients of the constituent phases. The procedure can be implemented for the bespoke preparation of pillars for both lab- and synchrotron-based X-ray nano-CT investigations of a wide range of samples.

    更新日期:2019-11-01
  • Using transmission Kikuchi diffraction to characterise α variants in an α+β titanium alloy.
    J. Microsc. (IF 1.813) Pub Date : 2017-05-05
    V Tong,S Joseph,A K Ackerman,D Dye,T B Britton

    Two phase titanium alloys are important for high-performance engineering components, such as aeroengine discs. The microstructures of these alloys are tailored during thermomechanical processing to precisely control phase fractions, morphology and crystallographic orientations. In bimodal two phase (α + β) Ti-6Al-2Sn-4Zr-2Mo (Ti-6242) alloys there are often three microstructural lengthscales to consider: large (∼10 μm) equiaxed primary α; >200 nm thick plate α with a basketweave morphology; and very fine scaled (<50 nm plate thickness) secondary α that grows between the larger α plates surrounded by retained β. In this work, we utilise high spatial resolution transmission Kikuchi diffraction (TKD, also known as transmission-based electron backscatter diffraction, t-EBSD) and scanning electron microscopy (SEM)-based forward scattering electron imaging to resolve the structures and orientations of basketweave and secondary α in Ti-6242. We analyse the α variants formed within one prior β grain, and test whether existing theories of habit planes of the phase transformation are upheld. Our analysis is important in understanding both the thermomechanical processing strategy of new bimodal two-phase titanium alloys, as well as the ultimate performance of these alloys in complex loading regimes such as dwell fatigue. Our paper champions the significant increase in spatial resolution afforded using transmission techniques, combined with the ease of SEM-based analysis using conventional electron backscatter diffraction (EBSD) systems and forescatter detector (FSD) imaging, to study the nanostructure of real-world engineering alloys.

    更新日期:2019-11-01
  • A novel scale-down cell culture and imaging design for the mechanistic insight of cell colonisation within porous substrate.
    J. Microsc. (IF 1.813) Pub Date : 2017-03-16
    C M Gabbott,Z X Zhou,G X Han,T Sun

    At the core of translational challenges in tissue engineering is the mechanistic understanding of the underpinning biological processes and the complex relationships among components at different levels, which is a challenging task due to the limitations of current tissue culture and assessment methodologies. Therefore, we proposed a novel scale-down strategy to deconstruct complex biomatrices into elementary building blocks, which were resembled by thin modular substrate and then evaluated separately in miniaturised bioreactors using various conventional microscopes. In order to investigate cell colonisation within porous substrate in this proof-of-concept study, TEM specimen supporters (10-30 μm thick) with fine controlled open pores (100∼600 μm) were selected as the modular porous substrate and suspended in 3D printed bioreactor systems. Noninvasive imaging of human dermal fibroblasts cultured on these free-standing substrate using optical microscopes illustrated the complicated dynamic processes used by both individual and coordinated cells to bridge and segment porous structures. Further in situ analysis via SEM and TEM provided high-quality micrographs of cell-cell and cell-scaffold interactions at microscale, depicted cytoskeletal structures in stretched and relaxed areas at nanoscale. Thus this novel scaled-down design was able to improve our mechanistic understanding of tissue formation not only at single- and multiple-cell levels, but also at micro- and nanoscales, which could be difficult to obtain using other methods.

    更新日期:2019-11-01
  • Imaging analysis of the interface between osteoblasts and microrough surfaces of laser-sintered titanium alloy constructs.
    J. Microsc. (IF 1.813) Pub Date : 2017-09-30
    A Cheng,H Chen,Z Schwartz,B D Boyan

    Previous work using focused ion beam (FIB) analysis of osteoblasts on smooth and microrough Ti surfaces showed that the average cell aspect ratio and distance from the surface are greater on the rough surface. In order to better interrogate the relationship between individual cells and their substrate using multiple imaging modalities, we developed a method that tracks the same cell across confocal laser scanning microscopy (CLSM) to correlate surface microroughness with cell morphology and cytoskeleton; scanning electron microscopy (SEM) to provide higher resolution for observation of nanoroughness as well as chemical mapping via energy dispersive X-ray spectroscopy; and transmission electron microscopy (TEM) for high-resolution imaging. FIB was used to prepare thin sections of the cell-material interface for TEM, or for three-dimensional electron tomography. Cells were cultured on laser-sintered Ti-6Al-4V substrates with polished or etched surfaces. Direct cell to surface attachments were observed across surfaces, though bridging across macroscale surface features occurred on rough substrates. Our results show that surface roughness, cell cytoskeleton and gross morphology can be correlated with the cell-material cross-sectional interface at the single cell level across multiple high-resolution imaging modalities. This work provides a platform method for further investigating mechanisms of the cell-material interface.

    更新日期:2019-11-01
  • 3D reconstruction of elastin fibres in coronary adventitia.
    J. Microsc. (IF 1.813) Pub Date : 2016-09-07
    T Luo,H Chen,G S Kassab

    A 3D reconstruction of individual fibres in vascular tissue is necessary to understand the microstructure properties of the vessel wall. The objective of this study is to determine the 3D microstructure of elastin fibres in the adventitia of coronary arteries. Quantification of fibre geometry is challenging due to the complex interwoven structure of the fibres. In particular, accurate linking of gaps remains a significant challenge, and complex features such as long gaps and interwoven fibres have not been adequately addressed by current fibre reconstruction algorithms. We use a novel line Laplacian deformation method, which better deals with fibre shape uncertainty to reconstruct elastin fibres in the coronary adventitia of five swine. A cost function, based on entropy and Euler Spiral, was used in the shortest path search. We find that mean diameter of elastin fibres is 1.67 ± 1.42 μm and fibre orientation is clustered around two major angles of 8.9˚ and 81.8˚. Comparing with CT-FIRE, we find that our method gives more accurate estimation of fibre width. To our knowledge, the measurements obtained using our algorithm represent the first investigation focused on the reconstruction of full elastin fibre length. Our data provide a foundation for a 3D microstructural model of the coronary adventitia to elucidate the structure-function relationship of elastin fibres.

    更新日期:2019-11-01
  • In memoriam - Chris Hawes.
    J. Microsc. (IF 1.813) Pub Date : null
    Ulla Neumann

    更新日期:2019-11-01
  • A step towards intelligent EBSD microscopy: machine-learning prediction of twin activity in MgAZ31.
    J. Microsc. (IF 1.813) Pub Date : 2018-08-09
    Rishabh Sharma,Isaac Chelladurai,Andrew D Orme,Michael P Miles,Christophe Giraud-Carrier,David T Fullwood

    Although microscopy is often treated as a quasi-static exercise for obtaining a snapshot of events and structure, it is clear that a more dynamic approach, involving real-time decision making for guiding the investigation process, may provide deeper insights, more efficiently. On the other hand, many applications of machine learning involve the interpretation of local circumstances from experience gained over many observations; that is, machine learning potentially provides an ideal solution for more efficient microscopy. This paper explores the potential for informing the microscope's observation strategy while characterising critical events. In particular, the identification of regions likely to experience twin activity (twin interaction with grain boundary) in AZ31 magnesium is attempted, from only local information. EBSD-based observations in the neighbourhoods of twin activity are fed into a machine-learning environment to inform the future search for such events, and the accuracy of the resultant decisions is quantified relative to the number of prior observations. The potential for utilising different types of local information, and their resultant value in the prediction process, is also assessed. After applying an attribute selection filter, and various other machine-learning tools, a decision-tree model is able to classify likely neighbourhoods of twin activity with 85% accuracy. The resultant framework provides the first step towards an intelligent microscopy for efficient observation of stochastic events during in situ microscopy campaigns. LAY DESCRIPTION One role of artificial intelligence is to predict future events after learning from many previous observations. In materials science, various phenomena (such as crack nucleation) are difficult to predict because they have been insufficiently observed. Furthermore, observation is difficult, precisely because their location cannot be predicted, leading to a chicken and egg conundrum. This paper applies machine learning to the search for twin nucleation sites in a magnesium alloy, in an attempt to guide the observation of twin nucleation events in a microscope based on previous observations. As more data is obtained, the accuracy of the location prediction will increase. In the current case, the machine-learning tool achieved 85% accuracy for predicting the location of twin interactions with grain boundaries after several thousand observations. The resultant framework provides the first step towards an intelligent microscopy for efficient observation of stochastic events during in situ microscopy campaigns.

    更新日期:2019-11-01
  • Label-free high-speed wide-field imaging of single microtubules using interference reflection microscopy.
    J. Microsc. (IF 1.813) Pub Date : 2018-07-26
    Mohammed Mahamdeh,Steve Simmert,Anna Luchniak,Erik Schäffer,Jonathon Howard

    When studying microtubules in vitro, label free imaging of single microtubules is necessary when the quantity of purified tubulin is too low for efficient fluorescent labelling or there is concern that labelling will disrupt function. Commonly used techniques for observing unlabelled microtubules, such as video enhanced differential interference contrast, dark-field and more recently laser-based interferometric scattering microscopy, suffer from a number of drawbacks. The contrast of differential interference contrast images depends on the orientation of the microtubules, dark-field is highly sensitive to impurities and optical misalignments. In addition, all of these techniques require costly optical components such as Nomarski prisms, dark-field condensers, lasers and laser scanners. Here we show that single microtubules can be imaged at high speed and with high contrast using interference reflection microscopy without the aforementioned drawbacks. Interference reflection microscopy is simple to implement, requiring only the incorporation of a 50/50 mirror instead of a dichroic in a fluorescence microscope, and with appropriate microscope settings has a similar signal-to-noise ratio to differential interference contrast and fluorescence. We demonstrated the utility of interference reflection microscopy by high-speed imaging and tracking of dynamic microtubules at 100 frames per second. In conclusion, the optical quality of interference reflection microscopy falls within the range of other microscope techniques, being inferior to some and superior to others, depending on the metric used and, with minimal microscope modification, can be used to study the dynamics of unlabelled microtubules. LAY DESCRIPTION The cytoskeleton gives a cell its shape and plays a major role in its movement and division. It's also helps organise the content of cells and is the base for intracellular transport. Important components of the cytoskeleton are microtubules, which are hollow cylindrical beams (25 nm in diameter) that assemble from protein building blocks called tubulin. Deficiencies in microtubules are related to many diseases including cancer and Alzheimer. Given their important role, microtubules are heavily investigated in many laboratories. One way to study microtubules is to isolate them from cells and image them using light microscopy. Over the years a number of imaging techniques have been used. These techniques have a number of drawbacks which are addressed by ongoing efforts which this work is a part of. Here, we present a method based on light interference that produce high quality images of microtubules. The technique is cheap and easy to implement making it accessible to a wide base of researchers.

    更新日期:2019-11-01
  • Modelling of focused ion beam induced increases in sample temperature: a case study of heat damage in biological samples.
    J. Microsc. (IF 1.813) Pub Date : 2018-07-19
    A Wolff,N Klingner,W Thompson,Y Zhou,J Lin,Y Y Peng,J A M Ramshaw,Y Xiao

    Ion beam induced heat damage in soft materials and biological samples is not yet well understood in Focused Ion Beam systems (FIBs). The work presented here discusses the physics behind the ion beam - sample interactions and the effects which lead to increases in sample temperature and potential heat damage. A model by which heat damage can be estimated and which allows parameters to be determined that reduce/prevent heat damage was derived from Fourier's law of heat transfer and compared to finite element simulations, numerical modelling results and experiments. The results suggests that ion beam induced heat damage can be prevented/minimised by reducing the ion beam current (local dose rate), decreasing the beam overlap (reduced local ion dose) and by introducing a blur (increased surface cross-section area, reduced local dose) while sputtering, patterning or imaging soft material and nonresin-embedded biological samples using FIBs. LAY DESCRIPTION FIB/SEMs, which combine a scanning electron microscope with a focused ion beam in a single device, have found increasing interest biological research. The device allows to cut samples at precisely selected areas and reveal sub surface information as well as preparing transmission electron microscope samples from bulk materials. Preparing biological samples has proven to be challenging due to the induced heat damage. This work explores the physics behind the sample cutting and proposes a model and a method, based on physical principles which allows the user to estimate the induced heat during the cutting process and to select cutting parameters which avoid heat damage in the sample.

    更新日期:2019-11-01
  • Quantitative 3D comparison of biofilm imaged by X-ray micro-tomography and two-photon laser scanning microscopy.
    J. Microsc. (IF 1.813) Pub Date : 2018-06-22
    A E Larue,P Swider,P Duru,D Daviaud,M Quintard,Y Davit

    Optical imaging techniques for biofilm observation, like laser scanning microscopy, are not applicable when investigating biofilm formation in opaque porous media. X-ray micro-tomography (X-ray CMT) might be an alternative but it finds limitations in similarity of X-ray absorption coefficients for the biofilm and aqueous phases. To overcome this difficulty, barium sulphate was used in Davit et al. (2011) to enable high-resolution 3D imaging of biofilm via X-ray CMT. However, this approach lacks comparison with well-established imaging methods, which are known to capture the fine structures of biofilms, as well as uncertainty quantification. Here, we compare two-photon laser scanning microscopy (TPLSM) images of Pseudomonas Aeruginosa biofilm grown in glass capillaries against X-ray CMT using an improved protocol where barium sulphate is combined with low-gelling temperature agarose to avoid sedimentation. Calibrated phantoms consisting of mono-dispersed fluorescent and X-ray absorbent beads were used to evaluate the uncertainty associated with our protocol along with three different segmentation techniques, namely hysteresis, watershed and region growing, to determine the bias relative to image binarization. Metrics such as volume, 3D surface area and thickness were measured and comparison of both imaging modalities shows that X-ray CMT of biofilm using our protocol yields an accuracy that is comparable and even better in certain respects than TPLSM, even in a nonporous system that is largely favourable to TPLSM.

    更新日期:2019-11-01
  • Mapping birefringence in three dimensions using polarized light field microscopy: the case of the juvenile clamshell.
    J. Microsc. (IF 1.813) Pub Date : 2018-06-22
    M T Tran,R Oldenbourg

    We report methods to generate three-dimensional maps of birefringence, its position and orientation in juvenile shells of the Atlantic hard clamshell (Mercenaria mercenaria). For measuring the retardance and optic axis orientation of curved shell surfaces in three dimensions, we developed enhanced acquisition and processing algorithms and combined results from conventional and light field imaging approaches to reconstruct the three-dimensional shell shape and its anisotropic optical properties. Our work represents the first successful attempt to generate such maps at a spatial resolution of about 2 μm and angular steps of about 9° in terms of the inclination angles of the optic axis. The maps of clamshell birefringence provide structural insights into the early mineralization during juvenile clamshell development.

    更新日期:2019-11-01
  • A new HPF specimen carrier adapter for the use of high-pressure freezing with cryoscanning electron microscope: two applications: stearic acid organization in a hydroxypropyl methylcellulose matrix and mice myocardium.
    J. Microsc. (IF 1.813) Pub Date : 2018-06-15
    B Payre,E Gontier,A Jarray,Y Martinez,J P Laugier,A Delalleau,B M Gaillard,I Anselme,D Goudounèche,I Fourquaux,M Hemati,V Gerbaud,M B Delisle,C Guilbeau-Frugier

    Cryogenic transmission electron microscopy of high-pressure freezing (HPF) samples is a well-established technique for the analysis of liquid containing specimens. This technique enables observation without removing water or other volatile components. The HPF technique is less used in scanning electron microscopy (SEM) due to the lack of a suitable HPF specimen carrier adapter. The traditional SEM cryotransfer system (PP3000T Quorum Laughton, East Sussex, UK; Alto Gatan, Pleasanton, CA, USA) usually uses nitrogen slush. Unfortunately, and unlike HPF, nitrogen slush produces water crystal artefacts. So, we propose a new HPF specimen carrier adapter for sample transfer from HPF system to cryogenic-scanning electronic microscope (Cryo-SEM). The new transfer system is validated using technical two applications, a stearic acid in hydroxypropyl methylcellulose solution and mice myocardium. Preservation of samples is suitable in both cases. Cryo-SEM examination of HPF samples enables a good correlation between acid stearic liquid concentration and acid stearic occupation surface (only for homogeneous solution). For biological samples as myocardium, cytoplasmic structures of cardiomyocyte are easily recognized with adequate preservation of organelle contacts and inner cell organization. We expect this new HPF specimen carrier adapter would enable more SEM-studies using HPF.

    更新日期:2019-11-01
  • An overview of state-of-the-art image restoration in electron microscopy.
    J. Microsc. (IF 1.813) Pub Date : 2018-06-09
    J Roels,J Aelterman,H Q Luong,S Lippens,A Pižurica,Y Saeys,W Philips

    In Life Science research, electron microscopy (EM) is an essential tool for morphological analysis at the subcellular level as it allows for visualization at nanometer resolution. However, electron micrographs contain image degradations such as noise and blur caused by electromagnetic interference, electron counting errors, magnetic lens imperfections, electron diffraction, etc. These imperfections in raw image quality are inevitable and hamper subsequent image analysis and visualization. In an effort to mitigate these artefacts, many electron microscopy image restoration algorithms have been proposed in the last years. Most of these methods rely on generic assumptions on the image or degradations and are therefore outperformed by advanced methods that are based on more accurate models. Ideally, a method will accurately model the specific degradations that fit the physical acquisition settings. In this overview paper, we discuss different electron microscopy image degradation solutions and demonstrate that dedicated artefact regularisation results in higher quality restoration and is applicable through recently developed probabilistic methods.

    更新日期:2019-11-01
  • A multi-emitter fitting algorithm for potential live cell super-resolution imaging over a wide range of molecular densities.
    J. Microsc. (IF 1.813) Pub Date : 2018-05-26
    T Takeshima,T Takahashi,J Yamashita,Y Okada,S Watanabe

    Multi-emitter fitting algorithms have been developed to improve the temporal resolution of single-molecule switching nanoscopy, but the molecular density range they can analyse is narrow and the computation required is intensive, significantly limiting their practical application. Here, we propose a computationally fast method, wedged template matching (WTM), an algorithm that uses a template matching technique to localise molecules at any overlapping molecular density from sparse to ultrahigh density with subdiffraction resolution. WTM achieves the localization of overlapping molecules at densities up to 600 molecules μm-2 with a high detection sensitivity and fast computational speed. WTM also shows localization precision comparable with that of DAOSTORM (an algorithm for high-density super-resolution microscopy), at densities up to 20 molecules μm-2 , and better than DAOSTORM at higher molecular densities. The application of WTM to a high-density biological sample image demonstrated that it resolved protein dynamics from live cell images with subdiffraction resolution and a temporal resolution of several hundred milliseconds or less through a significant reduction in the number of camera images required for a high-density reconstruction. WTM algorithm is a computationally fast, multi-emitter fitting algorithm that can analyse over a wide range of molecular densities. The algorithm is available through the website. https://doi.org/10.17632/bf3z6xpn5j.1.

    更新日期:2019-11-01
  • Automated detection of fluorescent cells in in-resin fluorescence sections for integrated light and electron microscopy.
    J. Microsc. (IF 1.813) Pub Date : 2018-04-27
    J Delpiano,L Pizarro,C J Peddie,M L Jones,L D Griffin,L M Collinson

    Integrated array tomography combines fluorescence and electron imaging of ultrathin sections in one microscope, and enables accurate high-resolution correlation of fluorescent proteins to cell organelles and membranes. Large numbers of serial sections can be imaged sequentially to produce aligned volumes from both imaging modalities, thus producing enormous amounts of data that must be handled and processed using novel techniques. Here, we present a scheme for automated detection of fluorescent cells within thin resin sections, which could then be used to drive automated electron image acquisition from target regions via 'smart tracking'. The aim of this work is to aid in optimization of the data acquisition process through automation, freeing the operator to work on other tasks and speeding up the process, while reducing data rates by only acquiring images from regions of interest. This new method is shown to be robust against noise and able to deal with regions of low fluorescence.

    更新日期:2019-11-01
  • Comparison of hyperspectral classification methods for the analysis of cerium oxide nanoparticles in histological and aqueous samples.
    J. Microsc. (IF 1.813) Pub Date : 2018-04-10
    M P S Idelchik,J Dillon,L Abariute,M A Guttenberg,A Segarceanu,N M Neu-Baker,S A Brenner

    Hyperspectral imaging (HSI) and classification are established methods that are being applied in new ways to the analysis of nanoscale materials in a variety of matrices. Typically, enhanced darkfield microscopy (EDFM)-based HSI data (also known as image datacubes) are collected in the wavelength range of 400-1000 nm for each pixel in a datacube. Utilising different spectral library (SL) creation methods, spectra from pixels in the datacube corresponding to known materials can be collected into reference spectral libraries (RSLs), which can be used to classify materials in datacubes of experimental samples using existing classification algorithms. In this study, EDFM-HSI was used to visualise and analyse industrial cerium oxide (CeO2 ; ceria) nanoparticles (NPs) in rat lung tissues and in aqueous suspension. Rats were exposed to ceria NPs via inhalation, mimicking potential real-world occupational exposures. The lung tissues were histologically prepared: some tissues were stained with hematoxylin and eosin (H&E) and some were left unstained. The goal of this study was to determine how HSI and classification results for ceria NPs were influenced by (1) the use of different RSL creation and classification methods and (2) the application of those methods to samples in different matrices (stained tissue, unstained tissue, or aqueous solution). Three different RSL creation methods - particle filtering (PF), manual selection, and spectral hourglass wizard (SHW) - were utilised to create the RSLs of known materials in unstained and stained tissue, and aqueous suspensions, which were then used to classify the NPs in the different matrices. Two classification algorithms - spectral angle mapper (SAM) and spectral feature fitting (SFF) - were utilised to determine the presence or absence of ceria NPs in each sample. The results from the classification algorithms were compared to determine how each influenced the classification results for samples in different matrices. The results showed that sample matrix and sample preparation significantly influenced the NP classification thresholds in the complex matrices. Moreover, considerable differences were observed in the classification results when utilising each RSL creation and classification method for each type of sample. Results from this study illustrate the importance of appropriately selecting HSI algorithms based on specific material and matrix characteristics in order to obtain optimal classification results. As HSI is increasingly utilised for NP characterisation for clinical, environmental and health and safety applications, this investigation is important for further refining HSI protocols while ensuring appropriate data collection and analysis.

    更新日期:2019-11-01
  • Comparisons of the topographic characteristics and electrical charge distributions among Babesia-infected erythrocytes and extraerythrocytic merozoites using AFM.
    J. Microsc. (IF 1.813) Pub Date : 2018-04-03
    L Scudiero,W De J Mercado-Rojano,A Rudolph,J Wang,J M Laughery,C E Suarez

    Tick-borne Babesia parasites are responsible for costly diseases worldwide. Improved control and prevention tools are urgently needed, but development of such tools is limited by numerous gaps in knowledge of the parasite-host relationships. We hereby used atomic force microscopy (AFM) and frequency-modulated Kelvin probe potential microscopy (FM-KPFM) techniques to compare size, texture, roughness and surface potential of normal and infected Babesia bovis, B. bigemina and B. caballi erythrocytes to better understand the physical properties of these parasites. In addition, AFM and FM-KPFM allowed a detailed view of extraerythrocytic merozoites revealing shape, topography and surface potential of paired and single parasites. B. bovis-infected erythrocytes display distinct surface texture and overall roughness compared to noninfected erythrocytes. Interestingly, B. caballi-infected erythrocytes do not display the surface ridges typical in B. bovis parasites. Observations of extraerythrocytic B. bovis, B. bigemina and B. caballi merozoites using AFM revealed differences in size and shape between these three parasites. Finally, similar to what was previously observed for Plasmodium-infected erythrocytes, FM-KPFM images reveal an unequal electric charge distribution, with higher surface potential above the erythrocyte regions that are likely associated with Babesia parasites than over its remainder regions. In addition, the surface potential of paired extraerythrocytic B. bovis Mo7 merozoites revealed an asymmetric potential distribution. These observations may be important to better understand the unique cytoadhesive properties of B. bovis-infected erythrocytes, and to speculate on the role of differences in the distribution of surface charges in the biology of the parasites.

    更新日期:2019-11-01
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