Showing papers in "Journal of Microscopy in 2009"

Recording and controlling the 4D light field in a microscope using microlens arrays

Abstract: Summary Byinsertingamicrolensarrayattheintermediateimageplane of an optical microscope, one can record four-dimensional light fields of biological specimens in a single snapshot. Unlike a conventional photograph, light fields permit manipulation of viewpoint and focus after the snapshot has been taken, subject to the resolution of the camera and the diffraction limit of the optical system. By inserting a second microlens array and video projector into the microscope’s illumination path, one can control the incident light field falling on the specimen in a similar way. In this paper, we describe a prototype system we have built that implements these ideas, and we demonstrate two applications for it: simulating exotic microscope illumination modalities and correcting for optical aberrations digitally.

A compact STED microscope providing 3D nanoscale resolution.

Abstract: Summary The advent of supercontinuum laser sources has enabled the implementation of compact and tunable stimulated emission depletion fluorescence microscopes for imaging far below the diffraction barrier. Here we report on an enhanced version of this approach displaying an all-physics based resolution down to (19 ± 3) nm in the focal plane. Alternatively, this single objective lens system can be configured for 3D imaging with resolution down to 45 × 45 × 108 nm in a cell. The obtained results can be further improved by mathematical restoration algorithms. The far-field optical nanoscale resolution is attained in a variety of biological samples featuring strong variations in the local density of features.

XuvTools: free, fast and reliable stitching of large 3D datasets.

Abstract: Current biomedical research increasingly requires imaging large and thick 3D structures at high resolution. Prominent examples are the tracking of fine filaments over long distances in brain slices, or the localization of gene expression or cell migration in whole animals like Caenorhabditis elegans or zebrafish. To obtain both high resolution and a large field of view (FOV), a combination of multiple recordings ('tiles') is one of the options. Although hardware solutions exist for fast and reproducible acquisition of multiple 3D tiles, generic software solutions are missing to assemble ('stitch') these tiles quickly and accurately.

Accuracy assessment of elastic strain measurement by EBSD

Abstract: A detailed accuracy analysis of electron backscatter diffraction (EBSD) elastic strain measurement has been carried out using both simulated and experimental patterns. Strains are determined by measuring shifts between two EBSD patterns (one being the reference) over regions of interest (ROI) using an iterative cross-correlation algorithm. An original minimization procedure over 20 regions of interests gives a unique solution for the eight independent components of the deviatoric displacement gradient tensor. It is shown that this method leads to strain measurements on simulated patterns with an accuracy better than 10(-4). The influence of the projection parameters is also investigated. The accuracy assessment is illustrated by two worked examples: (i) four-point bending of a silicon single crystal and (ii) Si(1-x)Ge(x) layers on a Si substrate. Experimental results are compared with finite-element simulations.

Application of environmental scanning electron microscopy to determine biological surface structure

Abstract: The use of environmental scanning electron microscopy in biology is growing as more becomes understood about the advantages and limitations of the technique. These are discussed and we include new evidence about the effect of environmental scanning electron microscopy imaging on the viability of mammalian cells. We show that although specimen preparation for high-vacuum scanning electron microscopy introduces some artefacts, there are also challenges in the use of environmental scanning electron microscopy, particularly at higher resolutions. This suggests the two technologies are best used in combination. We have used human monocyte-derived macrophages as a test sample, imaging their complicated and delicate membrane ruffles and protrusions. We have also explored the possibility of using environmental scanning electron microscopy for dynamic experiments, finding that mammalian cells cannot be imaged and kept alive in the environmental scanning electron microscopy. The dehydration step in which the cell surface is exposed causes irreversible damage, probably via loss of membrane integrity during liquid removal in the specimen chamber. Therefore, mammalian cells should be imaged after fixation where possible to protect against damage as a result of chamber conditions.

Tomography of insulating biological and geological materials using focused ion beam (FIB) sectioning and low-kV BSE imaging.

Abstract: Summary Tomography in a focused ion beam (FIB) scanning electron microscope (SEM) is a powerful method for the characterization of three-dimensional micro- and nanostructures. Although this technique can be routinely applied to conducting materials, FIB–SEM tomography of many insulators, including biological, geological and ceramic samples, is often more difficult because of charging effects that disturb the serial sectioning using the ion beam or the imaging using the electron beam. Here, we show that automatic tomography of biological and geological samples can be achieved by serial sectioning with a focused ion beam and block-face imaging using low-kV backscattered electrons. In addition, a new ion milling geometry is used that reduces the effects of intensity gradients that are inherent in conventional geometry used for FIB–SEM tomography.

Automated tracking of migrating cells in phase-contrast video microscopy sequences using image registration.

Abstract: Analysis of in vitro cell motility is a useful tool for assessing cellular response to a range of factors. However, the majority of cell-tracking systems available are designed primarily for use with fluorescently labelled images. In this paper, five commonly used tracking systems are examined for their performance compared with the use of a novel in-house celltracking system based on the principles of image registration and optical flow. Image registration is a tool commonly used in medical imaging to correct for the effects of patient motion during imaging procedures and works well on low-contrast images, such as those found in bright-field and phase-contrast microscopy. The five cell-tracking systems examined were Retrac, a manual tracking system used as the gold standard; CellTrack, a recently released freely downloadable software system that uses a combination of tracking methods; ImageJ, which is a freely available piece of software with a plug-in for automated tracking (MTrack2) and Imaris and Volocity, both commercially available automated tracking systems. All systemswere used to track migration of human epithelial cells over ten frames of a phase-contrast time-lapse microscopy sequence. This showed that the in-house image-registration system was the most effective of those tested when tracking non-dividing epithelial cells in low-contrast images, with a successful tracking rate of 95%. The performance of the tracking systems was also evaluated by tracking fluorescently labelled epithelial cells imaged with both phase-contrast and confocal microscopy techniques. The results showed that using fluorescence microscopy instead of phase contrast does improve the tracking efficiency for each of the tested systems. For the in-house software, this improvement was relatively small (

A review of high-pressure freezing preparation techniques for correlative light and electron microscopy of the same cells and tissues.

Abstract: In this paper, we review some published studies using correlative light and electron microscopy methods. We further refined our criteria to include only those studies using live cells for light microscope and where high-pressure freezing was the method of specimen preparation for electron microscopy. High-pressure freezing is especially important for some difficult-to-fix samples, and for optimal preservation of ultrastructure in samples larger than a few micrometres. How the light microscope observations are done is completely sample dependent, but the choice of high-pressure freezer depends on the speed required to capture (freeze) the biological event of interest. For events requiring high time resolution (in the 4-5 s range) the Leica EM PACT2 with rapid transfer system works well. For correlative work on structures of interest that are either non-motile or moving slowly (minutes rather than seconds), any make of high-pressure freezer will work. We also report on some efforts to improve the capabilities of the Leica EM PACT2 rapid transfer system.

High-aperture cryogenic light microscopy.

Abstract: We report here the development of instruments and protocols for carrying out high numerical aperture immersion light microscopy on cryogenic specimens. Imaging by this modality greatly increases the lifetimes of fluorescence probes, including those commonly used for protein localization studies, while retaining the ability to image the specimen with high fidelity and spatial resolution. The novel use of a cryogenic immersion fluid also minimizes the refractive index mismatch between the sample and lens, leading to a more efficient coupling of the light from the sample to the image forming system. This enhancement is applicable to both fluorescence and transmitted light microscopy techniques. The design concepts used for the cryogenic microscope can be applied to virtually any existing light-based microscopy technique. This prospect is particularly exciting in the context of 'super-resolution' techniques, where enhanced fluorescence lifetime probes are especially useful. Thus, using this new modality it is now possible to observe dynamic events in a live cell, and then rapidly vitrify the specimen at a specific time point prior to carrying out high-resolution imaging. The techniques described can be used in conjunction with other imaging modalities in correlated studies. We have also developed instrumentation to perform cryo-light imaging together with soft X-ray tomography on the same cryo-fixed specimen as a means of carrying out high content, quantifiable correlated imaging analyses. These methods are equally applicable to correlated light and electron microscopy of frozen biological objects.

Using conventional fluorescent markers for far-field fluorescence localization nanoscopy allows resolution in the 10-nm range

Abstract: We present a novel technique of far-field localization nanoscopy combining spectral precision distance microscopy with widely used fluorochromes like the Green Fluorescent Protein (GFP) derivatives eGFP, EmGFP, Yellow Fluorescent Protein (YFP) and eYFP, synthetic dyes like Alexa 488 and Alexa 568, as well as fluoresceine derivates. Spectral precision distance microscopy allows the surpassing of conventional resolution limits in fluorescence far-field microscopy by precise object localization after the optical isolation of single signals in time. Based on the principles of this technique, our novel nanoscopic method was realized for laser optical precision localization and image reconstruction with highly enhanced optical resolution in intact cells. This allows for spatial assignment of individual fluorescent molecules with nanometre precision. The technique is based on excitation intensity dependent reversible photobleaching of the molecules used combined with fast time sequential imaging under appropriate focusing conditions. A meaningful advantage of the technique is the simple applicability as a universal tool for imaging and investigations to the major part of already available preparations according to standard protocols. Using the above mentioned fluorophores, the positions of single molecules within cellular structures were determined by visible light with an estimated localization precision down to 3 nm; hence distances in the range of 10-30 nm were resolved between individual fluorescent molecules allowing to apply different quantitative structure analysis tools.

Texture heterogeneities in alpha/alpha titanium forging analysed by EBSD-relation to fatigue crack propagation.

Abstract: Summary The microstructure and the local texture of a large IMI 834 forging were characterized using the Electron Back Scattered Diffraction (EBSD) technique. Crystallographic domains called macrozones and formed by a majority of primary αp grains with their axes in nearly the same direction were found. They had a band-like structure, parallel to the axial direction of the forging. The influence of these macrozones on the cold dwell-fatigue properties was studied. Several samples were tested under cold dwell-fatigue conditions. The crack initiation and the short-distance propagation region optically matched a bright region that contained numerous quasi-cleavage facets. The analysis of the EBSD measurements showed that this bright region was enclosed within a sharp textured region with axes at less than 30° from the loading axis. The crystallographic features of the crack nucleation site and the crack propagation path were also analysed.

The weighted Burgers vector: a new quantity for constraining dislocation densities and types using electron backscatter diffraction on 2D sections through crystalline materials

Abstract: The Weighted Burgers Vector (WBV) is defined here as the sum, over all types of dislocations, of [(density of intersections of dislocation lines with a map) x (Burgers vector)]. Here we show that it can be calculated, for any crystal system, solely from orientation gradients in a map view, unlike the full dislocation density tensor, which requires gradients in the third dimension. No assumption is made about gradients in the third dimension and they may be non-zero. The only assumption involved is that elastic strains are small so the lattice distortion is entirely due to dislocations. Orientation gradients can be estimated from gridded orientation measurements obtained by EBSD mapping, so the WBV can be calculated as a vector field on an EBSD map. The magnitude of the WBV gives a lower bound on the magnitude of the dislocation density tensor when that magnitude is defined in a coordinate invariant way. The direction of the WBV can constrain the types of Burgers vectors of geometrically necessary dislocations present in the microstructure, most clearly when it is broken down in terms of lattice vectors. The WBV has three advantages over other measures of local lattice distortion: it is a vector and hence carries more information than a scalar quantity, it has an explicit mathematical link to the individual Burgers vectors of dislocations and, since it is derived via tensor calculus, it is not dependent on the map coordinate system. If a sub-grain wall is included in the WBV calculation, the magnitude of the WBV becomes dependent on the step size but its direction still carries information on the Burgers vectors in the wall. The net Burgers vector content of dislocations intersecting an area of a map can be simply calculated by an integration round the edge of that area, a method which is fast and complements point-by-point WBV calculations.

Confocal mosaicing microscopy in skin excisions: a demonstration of rapid surgical pathology.

Abstract: Precise micro-surgical removal of tumour with minimal damage to the surrounding normal tissue requires a series of excisions, each guided by an examination of frozen histology of the previous. An example is Mohs surgery for the removal of basal cell carcinomas (BCCs) in skin. The preparation of frozen histology is labour-intensive and slow. Confocal microscopy may enable rapid detection of tumours directly in surgical excisions with minimal need for frozen histology. Mosaicing of images enables observation of nuclear and cellular morphology in large areas of surgically excised tissue. In skin, the use of 10-1% acetic acid as a reflectance contrast agent brightens nuclei in 0.5-5 min and enhances nuclear-to-dermis contrast and detectability of BCCs. A tissue fixture was engineered for precisely mounting surgical excisions to enable mosaicing of 36 x 36 images to create a field of view of 12 x 12 mm. This large field of view displays the excision at 2x magnification, similar to that routinely used by Mohs surgeons when examining frozen histology. Comparison of mosaics to histology demonstrates detectability of BCCs. Confocal mosaicing presently requires 9 min, instead of 20-45 min per excision for preparing frozen histology, and thus may provide a means for rapid pathology-at-the-bedside to expedite and guide surgery.

Fast linear feature detection using multiple directional non-maximum suppression.

Abstract: Summary The capacity to detect linear features is central to image analysis, computer vision and pattern recognition and has practical applications in areas such as neurite outgrowth detection, retinal vessel extraction, skin hair removal, plant root analysis and road detection. Linear feature detection often represents the starting point for image segmentation and image interpretation. In this paper, we present a new algorithm for linear feature detection using multiple directional non-maximum suppression with symmetry checking and gap linking. Given its low computational complexity, the algorithm is very fast. We show in several examples that it performs very well in terms of both sensitivity and continuity of detected linear features.

Comparison of different preparation methods of biological samples for FIB milling and SEM investigation.

Abstract: When a new approach in microscopy is introduced, broad interest is attracted only when the sample preparation procedure is elaborated and the results compared with the outcome of the existing methods. In the work presented here we tested different preparation procedures for focused ion beam (FIB) milling and scanning electron microscopy (SEM) of biological samples. The digestive gland epithelium of a terrestrial crustacean was prepared in a parallel for FIB/SEM and transmission electron microscope (TEM). All samples were aldehyde-fixed but followed by different further preparation steps. The results demonstrate that the FIB/SEM samples prepared for conventional scanning electron microscopy (dried) is suited for characterization of those intracellular morphological features, which have membranous/lamellar appearance and structures with composition of different density as the rest of the cell. The FIB/SEM of dried samples did not allow unambiguous recognition of cellular organelles. However, cellular organelles can be recognized by FIB/SEM when samples are embedded in plastic as for TEM and imaged by backscattered electrons. The best results in terms of topographical contrast on FIB milled dried samples were obtained when samples were aldehyde-fixed and conductively stained with the OTOTO method (osmium tetroxide/thiocarbohydrazide/osmium tetroxide/thiocarbohydrazide/osmium tetroxide). In the work presented here we provide evidence that FIB/SEM enables both, detailed recognition of cell ultrastructure, when samples are plastic embedded as for TEM or investigation of sample surface morphology and subcellular composition, when samples are dried as for conventional SEM.

Correlative microscopy: a potent tool for the study of rare or unique cellular and tissue events.

Abstract: Summary Biological studies have relied on two complementary microscope technologies – light (fluorescence) microscopy and electron microscopy. Light microscopy is used to study phenomena at a global scale to look for unique or rare events, and it also provides an opportunity for live imaging, whereas the forte of electron microscopy is the high resolution. Traditionally light and electron microscopy observations are carried out in different populations of cells/tissues and a ‘correlative’ inference is drawn. The advent of true correlative light-electron microscopy has allowed high-resolution imaging by electron microscopy of the same structure observed by light microscopy, and in advanced cases by video microscopy. Thus a rare event captured by low-resolution imaging of a population or transient events captured by live imaging can now also be studied at high resolution by electron microscopy. Here, the potential and difficulties of this approach, along with the most impressive breakthroughs obtained by these methods, are discussed.

Imaging of endodontic biofilms by combined microscopy (FISH/cLSM - SEM).

Abstract: Summary Scanning electron microscopy is a useful imaging approach for the visualization of bacterial biofilms in their natural environments including their medical and dental habitats, because it allows for the exploration of large surfaces with excellent resolution of topographic features. Most biofilms in nature, however, are embedded in a thick layer of extracellular matrix that prevents a clear identification of individual bacteria by scanning electron microscopy. The use of confocal laser scanning microscopy on the other hand in combination with fluorescence in situ hybridization enables thevisualizationofmatrixembeddedbacteriainmulti-layered biofilms. In our study, fluorescence in situ hybridization/ confocal laser scanning microscopy and scanning electron microscopy were applied to visualize bacterial biofilm in endodontic root canals. The resulting fluorescence in situ hybridization/confocal laser scanning microscopy and scanningelectronmicroscopyandpicturesweresubsequently combined into one single image to provide high-resolution information on the location of hidden bacteria. The combined use of scanning electron microscopy and fluorescence in situ hybridization/confocal laser scanning microscopy has the potential to overcome the limits of each single technique.

Self-pressurized rapid freezing (SPRF): a novel cryofixation method for specimen preparation in electron microscopy

Abstract: A method is described for the cryofixation of biological specimens for ultrastructural analysis and immunocytochemical detection studies. The method employs plunge freezing of specimens in a sealed capillary tube into a cryogen such as liquid propane or liquid nitrogen. Using this method a number of single-cell test specimens were well preserved. Also multicellular organisms, such as Caenorhabditis elegans, could be frozen adequately in low ionic strength media or even in water. The preservation of these unprotected specimens is comparable to that achieved with high-pressure freezing in the presence of cryoprotectant. The results are explained by the fact that cooling of water in a confined space below the melting point gives rise to pressure build-up, which may originate from the conversion of a fraction of the water content into low-density hexagonal ice and/or expansion of water during supercooling. Calculations indicate the pressure may be similar in magnitude to that applied in high-pressure freezing. Because the specimens are plunge cooled, suitable cryogens are not limited to liquid nitrogen. It is shown that a range of cryogens and cryogen temperatures can be used successfully. Because the pressure is generated inside the specimen holders as a result of the cooling rather than applied from an external source as in high-pressure freezing, the technique has been referred to as self-pressurized rapid freezing.

High-resolution X-ray tomography of the human inner ear: synchrotron radiation-based study of nerve fibre bundles, membranes and ganglion cells

Abstract: The combination of osmium tetroxide staining and high-resolution tomographic imaging using monochromatic X rays allows visualizing cellular structures of the human inner ear, that is, the organ of Corti, the stria vascularis and further soft tissues of the membranous labyrinth, in three-dimensional space with isotropic micrometre resolution. This approach permits to follow the course of nerve fibre bundles in a major part of the specimen and reveals the detailed three-dimensional arrangement of individual ganglion cells with distinct nuclei by means of X-ray tomography for the first time. The non-destructive neuron cell counting in a selected volume of 125 microm x 800 microm x 600 microm = 0.06 mm(3) gives rise to the estimate that 2000 ganglion cells are present along 1 mm organ of Corti.

In situ observation of intergranular crack nucleation in a grain boundary controlled austenitic stainless steel.

Abstract: Grain boundary engineering has been proposed to increase the lifetime performance of sensitized austenitic stainless steel in aggressive environments. Increased microstructure resistance is typically associated with higher fractions of twin (Sigma3) grain boundaries, but there is uncertainty about the properties and role of other boundaries. To develop predictive models for stress corrosion crack nucleation, more information is required about how grain boundary crystallography and the orientations of the grain boundary plane and its surrounding grains affect crack development. Digital image correlation combined with electron backscatter diffraction has been used to characterize the microstructure and to observe, in situ, the nucleation and propagation of short stress corrosion cracks in thermo-mechanically processed type 304 stainless steel. The crack path and its growth rate have been determined and are found to be influenced by the microstructure.

Ultramicrotomy in the ESEM, a versatile method for materials and life sciences

Abstract: We here present the results of the first materials science analyses obtained with the prototype of a serial block-face sectioning and imaging tool, 3Viewtrade mark of Gatan, Inc (Pleasanton, CA, U.S.A.). It is a specially designed ultramicrotome operating in situ within an environmental scanning electron microscope originally developed for life science research. The microtome removes thin slices from the sample and the environmental scanning electron microscope images each new block surface of the specimen (serial block-face scanning electron microscopy). The Schottky emitter (FEG) of the microscope delivers high spatial resolution and has the advantage of stable performance and high durability. The slice thickness can typically be selected between 50 and 100 nm. It is possible to cut hundreds of slices and simultaneously acquire images with Digital Micrographtrade mark Model 700 (Gatan, Inc.). This article outlines the set-up and describes the automated process. The preparation of specimens for in situ ultramicrotomy is explained and the parameters for good image quality are discussed. In addition, special operative and analytic features of the controlling software are presented. Three different technical materials and one botanical specimen were analyzed delivering first results of this method for materials science and for botany.

A robust method for processing scanning probe microscopy images and determining nanoobject position and dimensions

Abstract: P>Processing of scanning probe microscopy (SPM) images is essential to explore nanoscale phenomena. Image processing and pattern recognition techniques are developed to improve the accuracy and consistency of nanoobject and surface characterization. We present a robust and versatile method to process SPM images and reproducibly estimate nanoobject position and dimensions. This method is using dedicated fits based on the least-square method and the matrix operations. The corresponding algorithms have been implemented in the FabViewer portable application. We illustrate how these algorithms permit not only to correct SPM images but also to precisely determine the position and dimensions of nanocrystals and adatoms on surface. A robustness test is successfully performed using distorted SPM images.

Focused ion beam-scanning electron microscope: exploring large volumes of atherosclerotic tissue.

Abstract: Summary Atherogenesis is a pathological condition in which changes in the ultrastructure and in the localization of proteins occur within the vasculature during all stages of the disease. To gain insight in those changes, high-resolution imaging is necessary. Some of these changes will only be present in a small number of cells, positioned in a ‘sea’ of non-affected cells. To localize this relatively small number of cells, there is a need to first navigate through a large area of the sample and subsequently zoom in onto the area of interest. This approach enables the study of specific cells within their in vivo environment and enables the study of (possible) interactions of these cells with their surrounding cells/environment. The study of a sample in a correlative way using light and electron microscopy is a promising approach to achieve this; however, it is very laborious and additional ultrastructural techniques might be very valuable to find the places of interest. In this report we show that the focused ion beam-scanning electron microscope is a powerful tool to study biological specimens in a correlative way. With this microscope one can scan for the area of interest at low magnification, in this case the atherosclerotic plaque, and subsequently zoom in, for further analysis on an ultrastructural level, rendering valuable and detailed two- and three-dimensional information of, in this case, the endothelial cells and the vessel wall. Moreover, in combination with pre-embedment labelling of surface exposed antigens, the method allows insight into the 3D distribution of these markers.

EBSD for analysing the twinning microstructure in fine-grained TWIP steels and its influence on work hardening

Abstract: A 22 Mn-0.6 C twinning induced plasticity steel with an average grain size of 2.6 μm was deformed in tension at room temperature. The electron backscattered diffraction technique was used to characterize the twinning structure in relation with the local texture evolution. For nanoscale analysis, additional transmission electron microscopy analysis was performed. Nanotwins were activated in the largest grains from the beginning of the deformation. They interacted with a well-developed dislocation structure that induced detectable intragranular orientation variations. With increasing deformation, dense bundles of nanotwins preferentially developed in grains oriented close to the //tensile direction fibre (promoted by the deformation) as well as medium to high angle sub-boundaries. These key features of the twinned microstructure were finally related to the remarkably high strain hardening, which evolved according to different stages.

Photooxidation technology for correlated light and electron microscopy.

Abstract: The combination of the capabilities of light microscopical techniques with the power of resolution of electron microscopy along with technical advances has led to a gradual decline of the gap between classical light and electron microscopy. Among the correlative techniques using the synergistic opportunities, photooxidation methods have been established as valuable tools for visualizing cell structures at both light and electron microscopic level. Fluorescent dyes are used to oxidize the substrate diaminobenzidine, which in its oxidized state forms fine granular precipitates. Stained with osmium, the diaminobenzidine precipitates are well discernible in the electron microscope, thus labelling and defining the cellular structures, which at light microscopy level are recorded by fluorescent probes. The underlying photooxidation reaction is based on the excitation of free oxygen radicals that form upon illumination of fluorochromes; this is a central step in the procedure, which mainly influences the success of the method. This article summarizes basic steps of the technology and progresses, shows efforts and elaborated pathways, and focuses on methodical solutions as to the applicability of different fluorochromes, as well as conditions for fine structural localizations of the reaction products.

A pixel selection rule based on the number of different-phase neighbours for the simulated annealing reconstruction of sandstone microstructure.

Abstract: Summary Sandstone reservoir is one of the main types of oil and gas reservoirs in China It has porous microstructure, which directly affects the transport properties of a sandstone Hence, the study of porous microstructure is important to the exploration and exploitation of oil and gas Three-dimensional microstructure of a sandstone can be reconstructed using the simulated annealing method based on statistical properties of its two-dimensional micrograph The aim of reconstruction is to minimize the discrepancy between the statistical properties of the reconstructed microstructure and those of the two-dimensional image To accelerate the rate of convergence, we proposed a different-phase neighbours (DPNs)-based pixel selection rule to replace the random pixel selection rule of the simulated annealing reconstruction In this rule, pixels with the largest number of DPNs have the largest selection probability The selection probabilities of other pixels are proportional to their DPNs Microstructure reconstructed with the DPNs-based rule is compared with those with the random selection rule and two other biased pixel selection rules The DPNs-based rule is the most effective in enhancing convergence Permeability of the microstructure reconstructed with the DPNs-based rule is estimated by the Kozeny–Carman formula and is in good agreement with the one reconstructed with the random pixel selection rule

Exploring the multi-scale structure of printing paper--a review of modern technology.

Abstract: Summary Printing paper is an important communication and information medium affecting our daily activities in several ways. The structure of paper may affect its optical, mechanical, fluid transport and print properties. In order to achieve a complete understanding of these processes a comprehensive characterization of relevant surface and bulk structures of paper is necessary. The paper grades considered in this study are newsprints, super calendered and coated papers. A detailed description of several image acquisition techniques used to assess the printing paper structures is given, including desktop scanners, profilometry, light microscopy, confocal laser scanning microscopy, scanning electron microscopy, atomic force microscopy, focused-ion-beam, X-ray microtomography and transmission electron microscopy. The presented methods cover a wide range of structure sizes, from the macro- to the sub-nano level. The complementary capabilities of the image acquisition techniques for assessing structural details of paper and prints are discussed.

TIRF microscopy evanescent field calibration using tilted fluorescent microtubules.

Abstract: Summary Totalinternalreflectionfluorescencemicroscopyhasbecomea powerful tool to study the dynamics of sub-cellular structures and single molecules near substrate surfaces. However, the penetration depth of the evanescent field, that is, the distance at which the excitation intensity has exponentially decayed to 1/e, is often left undetermined. This presents a limit on the spatial information about the imaged structures. Here, we present a novel method to quantitatively characterize the illumination in total internal reflection fluorescence microscopy using tilted, fluorescently labelled, microtubules. We find that the evanescent field is well described by a single exponential function, with a penetration depth close to theoretically predicted values. The use of in vitro reconstituted microtubules as nanoscale probes results in a minimal perturbation of the evanescent field; excitation light scattering is eliminated and the refractive index of the sample environment is unchanged. The presented method has the potential to provide a generic tool for in situ calibration of the evanescent field.

Advantages of indium-tin oxide-coated glass slides in correlative scanning electron microscopy applications of uncoated cultured cells.

Abstract: A method of direct visualization by correlative scanning electron microscopy (SEM) and fluorescence light microscopy of cell structures of tissue cultured cells grown on conductive glass slides is described. We show that by growing cells on indium-tin oxide (ITO)-coated glass slides, secondary electron (SE) and backscatter electron (BSE) images of uncoated cells can be obtained in high-vacuum SEM without charging artefacts. Interestingly, we observed that BSE imaging is influenced by both accelerating voltage and ITO coating thickness. By combining SE and BSE imaging with fluorescence light microscopy imaging, we were able to reveal detailed features of actin cytoskeletal and mitochondrial structures in mouse embryonic fibroblasts. We propose that the application of ITO glass as a substrate for cell culture can easily be extended and offers new opportunities for correlative light and electron microscopy studies of adherently growing cells.

Combined EBSD/EDS tomography in a dual‐beam FIB/FEG–SEM

Abstract: Summary An automated method for collecting combined three-dimensional (3D) electron backscatter diffraction (EBSD)/energy dispersive spectroscopy (EDS) data sets on a dual-beam focused ion beam (FIB)/field emission gun scanning electron microscope (FEG–SEM) microscope is described The method uses simple scripting files on the dual beam to move between the EBSD collection and the FIB slicing positions, which are linked to a commercial EBSD data collection programme The EDS data are collected simultaneously with the EBSD patterns analogous to combined two-dimensional (2D) EBSD/EDS The technique has been successfully applied to study both the interdiffusion zone between a coating and a substrate and a complex multi-phase coating on a nickel-based superalloy sample This analysis is shown to enable the complex grain shapes, location of precipitates and phase interconnectivity within these samples to be determined without the ambiguities associated with 2D stereographic analysis