Multiscale iterative voting for differential analysis of stress response for 2D and 3D cell culture models

Focused ion beam-scanning electron microscope (FIB-SEM) tomography is a powerful application in obtaining three-dimensional (3D) information. The FIB creates a cross section and subsequently removes thin slices. The SEM takes images using secondary or backscattered electrons, or maps every slice using X-rays and/or electron backscatter diffraction patterns. The objective of this study is to assess the possibilities of combining FIB-SEM tomography with cathodoluminescence (CL) imaging. The intensity of CL emission is related to variations in defect or impurity concentrations. A potential problem with FIB-SEM CL tomography is that ion milling may change the defect state of the material and the CL emission. In addition the conventional tilted sample geometry used in FIB-SEM tomography is not compatible with conventional CL detectors. Here we examine the influence of the FIB on CL emission in natural diamond and the feasibility of FIB-SEM CL tomography. A systematic investigation establishes that the ion beam influences CL emission of diamond, with a dependency on both the ion beam and electron beam acceleration voltage. CL emission in natural diamond is enhanced particularly at low ion beam and electron beam voltages. This enhancement of the CL emission can be partly explained by an increase in surface defects induced by ion milling. CL emission enhancement could be used to improve the CL image quality. To conduct FIB-SEM CL tomography, a recently developed novel specimen geometry is adopted to enable sequential ion milling and CL imaging on an untilted sample. We show that CL imaging can be manually combined with FIB-SEM tomography with a modified protocol for 3D microstructure reconstruction. In principle, automated FIB-SEM CL tomography should be feasible, provided that dedicated CL detectors are developed that allow subsequent milling and CL imaging without manual intervention, as the current CL detector needs to be manually retracted before a slice can be milled. Due to the required high electron beam acceleration voltage for CL emission, the resolution for FIB-SEM CL tomography is currently limited to several hundreds of nm in XY and up to 650 nm in Z for diamonds. Opaque materials are likely to have an improved Z resolution, as CL emission generated deeper in the material is not able to escape from it.     

Thinning of large mammalian cells for cryo-TEM characterization by cryo-FIB milling

Focused ion beam milling at cryogenic temperatures (cryo-FIB) is a valuable tool that can be used to thin vitreous biological specimens for subsequent imaging and analysis by cryo-transmission electron microscopy (cryo-TEM) in a frozen-hydrated state. This technique offers the potential benefit of eliminating the mechanical artefacts that are typically found with cryo-ultramicrotomy. However, due to the additional complexity in transferring samples in and out of the FIB, contamination and devitrification of the amorphous ice is commonly encountered. To address these problems, we have designed a sample cryo-shuttle that directly and specifically accepts Polara TEM cartridges to simplify the transfer process between FIB and TEM. We optimized several parameters in the cryo-FIB and cryo-TEM processes using the quality of the samples' ice as an indicator and demonstrated high-quality milling with large mammalian cells. By comparing the results from HeLa cells to those from Escherichia coli cells, we discuss some of the artefacts and challenges we have encountered using this technique.     

Far‐reaching geometrical artefacts due to thermal decomposition of polymeric coatings around focused ion beam milled pigment particles

Focused ion beam and scanning electron microscope (FIB‐SEM) instruments are extensively used to characterize nanoscale composition of composite materials, however, their application to analysis of organic corrosion barrier coatings has been limited. The primary concern that arises with use of FIB to mill organic materials is the possibility of severe thermal damage that occurs in close proximity to the ion beam impact. Recent research has shown that such localized artefacts can be mitigated for a number of polymers through cryogenic cooling of the sample as well as low current milling and intelligent ion beam control. Here we report unexpected nonlocalized artefacts that occur during FIB milling of composite organic coatings with pigment particles. Specifically, we show that FIB milling of pigmented polysiloxane coating can lead to formation of multiple microscopic voids within the substrate as far as 5 μm away from the ion beam impact. We use further experimentation and modelling to show that void formation occurs via ion beam heating of the pigment particles that leads to decomposition and vaporization of the surrounding polysiloxane. We also identify FIB milling conditions that mitigate this issue.     

3D ECT reconstruction by an improved Landweber iteration algorithm

Electrical capacitance tomography (ECT) is a relatively mature non-invasive imaging technique that attempts to map dielectric permittivity of materials. Recently, 3D ECT has gained interest because of its potential to generate volumetric images. The study of a fast and accurate image reconstruction algorithm is a challenge task, especially for 3D reconstruction. In this paper, we propose an improved Landweber iteration algorithm. We incorporate an additional acceleration term into the cost function and apply an adaptive threshold operation to the image obtained in each iteration for reducing artefacts. The algorithm proposed is tested by the noise-free and noise-contaminated capacitance data. Sensitivity matrixes and capacitance data of a 3D ECT sensor are obtained by using the finite element (FE) method. Extensive simulations in 3D reconstruction are carried out. The results verify the effectiveness of these improvements. Both the reconstruction time and the artefacts in the reconstructed image are reduced obviously. The experimental results of 3D reconstruction of objects in the shape of letters U and L confirm the effectiveness of the proposed algorithm further.     

Analysis of FIB‐induced damage by electron channelling contrast imaging in the SEM

We have investigated the Ga⁺ ion‐damage effect induced by focused ion beam (FIB) milling in a [001] single crystal of a 316 L stainless steel by the electron channelling contrast imaging (ECCI) technique. The influence of FIB milling on the characteristic electron channelling contrast of surface dislocations was analysed. The ECCI approach provides sound estimation of the damage depth produced by FIB milling. For comparison purposes, we have also studied the same milled surface by a conventional electron backscatter diffraction (EBSD) approach. We observe that the ECCI approach provides further insight into the Ga⁺ ion‐damage phenomenon than the EBSD technique by direct imaging of FIB artefacts in the scanning electron microscope. We envisage that the ECCI technique may be a convenient tool to optimize the FIB milling settings in applications where the surface crystal defect content is relevant.     

Imaging surface and submembranous structures with the atomic force microscope: a study on living cancer cells, fibroblasts and macrophages

Atomic force microscopy (AFM) has been used to image a wide variety of cells. Fixed and dried-coated, wet-fixed or living cells were investigated. The major advantage of AFM over SEM is the avoidance of vacuum and electrons, whereas imaging can be done at environmental pressure and in aqueous conditions. Evidence of the successful application of AFM in biological imaging is provided by comparing results of AFM with SEM and/or TEM. In this study, we investigated surface and submembranous structures of living and glutaraldehyde-fixed colon carcinoma cells, skin fibroblasts and liver macrophages by AFM. Special attention was paid to the correct conditions for the acquisition of images of the surface of these cells, because quality SEM examinations have already been abundantly presented.
AFM imaging of living cells revealed specific structures, such as the cytoskeleton, which were not observed by SEM. Membrane structures, such as ruffles, lamellipodia, microspikes and microvilli, could only clearly be observed after fixing the cells with 0.1% glutaraldehyde. AFM images of living cells were comparable to SEM images of fixed, dried and coated cells, but contained a number of artefacts due to tip–sample interaction. In addition, AFM imaging allowed the visualization of cytoplasmic submembranous structures without the necessity for further preparative steps, allowing us: (i) to follow cytoskeletal changes in fibroblasts under the influence of the microfilament disrupting agent latrunculin A; (ii) to study particle phagocytosis in macrophages. Therefore, in spite of the slow image acquisition of the AFM, the instrument can be used for high-resolution real-time studies of dynamic changes in submembranous structures.     

Silica-rubber microstructure visualised in three dimensions by focused ion beam-scanning electron microscopy

A focused ion beam-scanning electron microscope (FIB-SEM) technique for three-dimensional reconstruction and representation of material microstructures was applied to a silica-filled synthetic rubber for the first time. Backscattered electron imaging allowed differentiation between rubber matrix, silica filler and zinc oxide (used as an activator for the sulphur vulcanisation reaction). Subsequent image processing allowed three-dimensional isosurface model generation of the particulate structure within the rubber composite and separation of zinc oxide from the silica filler. The potential for development and application of this technique using finite element analysis modelling is also highlighted.     

Removing noises caused by motion artefacts in microcirculation maps of human skin in vivo

This paper presents a zero‐padding and cross‐correlation technique‐based correlation mapping optical coherence tomography (ZPCC‐cmOCT) to reconstruct microcirculation maps of human skin in vivo, which can remove the background decorrelation noise caused by motion artefacts. In conventional correlation mapping optical coherence tomography method, the correlation degree of static tissue may be lowered by the motion artefacts due to cardiac and respiratory motion, resulting in background decorrelation noise in microcirculation maps. In zero‐padding and cross‐correlation technique‐based correlation mapping optical coherence tomography method, structural images are first obtained by performing Fourier transform on zero‐padded interference fringes, and then cross‐correlation‐based image registration is utilized to align local areas in two adjacent structural images. Finally, correlation mapping optical coherence tomography method is performed to generate microcirculation maps. Both phantom experiments and in vivo experiments were implemented and the results demonstrate that the proposed method is capable of providing microcirculation maps with the background decorrelation noise removed.     

Automatic macroscopic density artefact removal in a Nissl‐stained microscopic atlas of whole mouse brain

Acquiring a whole mouse brain at the micrometer scale is a complex, continuous and time‐consuming process. Because of defects caused by sample preparation and microscopy, the acquired image data sets suffer from various macroscopic density artefacts that worsen the image quality. We have to develop the available preprocessing methods to improve image quality by removing the artefacts that effect cell segmentation, vascular tracing and visualization. In this study, a set of automatic artefact removal methods is proposed for images obtained by tissue staining and optical microscopy. These methods significantly improve the complicated images that contain various structures, including cells and blood vessels. The whole mouse brain data set with Nissl staining was tested, and the intensity of the processed images was uniformly distributed throughout different brain areas. Furthermore, the processed image data set with its uniform brightness and high quality is now a fundamental atlas for image analysis, including cell segmentation, vascular tracing and visualization.     

Image contrast of dielectric specimens in transmission mode near-field scanning optical microscopy: imaging properties and tip artefacts

Near-field scanning optical microscopy (NSOM) is a scanned probe technique utilizing a subwavelength-sized light source for high-resolution imaging of surfaces. Although NSOM has the potential to exploit and extend the experimental utility of the modern light microscope, the interpretation of image contrast is not straightforward. In near-field microscopy the illumination intensity of the source (probe) is not a constant value, rather it is a function of the probe–sample electronic environment. A number of dielectric specimens have been studied by NSOM to elucidate the contrast role of specimen type, topography and crystallinity; a summary of metallic specimen observations is presented for comparative purposes. Near-field image contrast is found to be a result of lateral changes in optical density and edge scattering for specimens with little sample topography. For surfaces with considerable topography the contributions of topographic (Z) axis contrast to lateral (X,Y) changes in optical density have been characterized. Selected near-field probes have also been shown to exhibit a variety of unusual contrast artefacts. Thorough study of polarization contrast, optical edge (scattering) contrast, as well as molecular orientation in crystalline specimens, can be used to distinguish lateral contrast from topographic components. In a few cases Fourier filtering can be successfully applied to separate the topographic and lateral contrast components.     

Imaging damage evolution in a small particle metal matrix composite

It is difficult to study effectively microstructural damage in metal matrix composites (MMCs) due to artefacts arising from traditional metallographic sample preparation techniques. The sectioning and imaging capabilities of the focused ion beam (FIB) microscope provide an excellent method for studying damage accumulation in MMCs.
The capabilities of the FIB system have been used to carry out a study of damage evolution in a powder-processed/hot-extruded Al2080/SiC_p MMC. Microvoid damage is found to be preserved accurately during FIB sectioning, allowing measurements of the fraction of decohered particles and the void area fraction. These microscopic damage measurements are correlated with the macroscopic damage parameter, D , as determined by density measurements.
Using transmission electron microscopy, the evolution of dislocation structures at the SiC–matrix interfaces has been examined. A previously unreported decohesion mechanism has been observed.     

Restoration of confocal images for quantitative image analysis

Quantitative studies of three-dimensional (3-D) structure of microscopic objects have been made possible through the introduction of microscopic volume imaging techniques, most notably the confocal fluorescence microscope (CFM). Although the CFM is a true volume imager, its specific imaging properties give rise to distortions in the images and hamper subsequent quantitative analysis. Therefore, it is a prerequisite that confocal images are restored prior to analysis. The distortions can be divided into several categories: attenuation of areas in the image due to self-absorption, bleaching effects, geometrical effects and distortions due to diffraction effects. Of these, absorption and diffraction effects are the most important. This paper describes a method aimed at the correction of diffraction-induced distortions. All the steps necessary in restoring confocal images are discussed, including a novel method to measure instrumental properties on a routine basis. To test the restoration procedure an image of a fluorescent planar object was restored. The results show a considerable improvement in the z-resolution and no ringing artefacts. The relevance of the method for image analysis is demonstrated by a comparison of results of applying 3-D texture analysis to restored and unrestored images of a synthetic object. Furthermore, the method can be successfully applied to noisy fluorescence images of biological objects, such as interphase cell nucei.     

Seamless stitching of tile scan microscope images

For diagnostic purposes, optical imaging techniques need to obtain high‐resolution images of extended biological specimens in reasonable time. The field of view of an objective lens, however, is often smaller than the sample size. To image the whole sample, laser scanning microscopes acquire tile scans that are stitched into larger mosaics. The appearance of such image mosaics is affected by visible edge artefacts that arise from various optical aberrations which manifest in grey level jumps across tile boundaries. In this contribution, a technique for stitching tiles into a seamless mosaic is presented. The stitching algorithm operates by equilibrating neighbouring edges and forcing the brightness at corners to a common value. The corrected image mosaics appear to be free from stitching artefacts and are, therefore, suited for further image analysis procedures. The contribution presents a novel method to seamlessly stitch tiles captured by a laser scanning microscope into a large mosaic. The motivation for the work is the failure of currently existing methods for stitching nonlinear, multimodal images captured by our microscopic setups. Our method eliminates the visible edge artefacts that appear between neighbouring tiles by taking into account the overall illumination differences among tiles in such mosaics. The algorithm first corrects the nonuniform brightness that exists within each of the tiles. It then compensates for grey level differences across tile boundaries by equilibrating neighbouring edges and forcing the brightness at the corners to a common value. After these artefacts have been removed further image analysis procedures can be applied on the microscopic images. Even though the solution presented here is tailored for the aforementioned specific case, it could be easily adapted to other contexts where image tiles are assembled into mosaics such as in astronomical or satellite photos.     

Enhancing image contrast of carbon nanotubes on cellular background using helium ion microscope by varying helium ion fluence

Carbon nanotubes (CNTs) have become an important nano entity for biomedical applications. Conventional methods of their imaging, often cannot be applied in biological samples due to an inadequate spatial resolution or poor contrast between the CNTs and the biological sample. Here we report a unique and effective detection method, which uses differences in conductivities of carbon nanotubes and HeLa cells. The technique involves the use of a helium ion microscope to image the sample with the surface charging artefacts created by the He⁺ and neutralised by electron flood gun. This enables us to obtain a few nanometre resolution images of CNTs in HeLa Cells with high contrast, which was achieved by tailoring the He⁺ fluence. Charging artefacts can be efficiently removed for conductive CNTs by a low amount of electrons, the fluence of which is not adequate to discharge the cell surface, resulting in high image contrast. Thus, this technique enables rapid detection of any conducting nano structures on insulating cellular background even in large fields of view and fine spatial resolution. The technique demonstrated has wider applications for researchers seeking enhanced contrast and high‐resolution imaging of any conducting entity in a biological matrix – a commonly encountered issue of importance in drug delivery, tissue engineering and toxicological studies.     

Surface damage induced by FIB milling and imaging of biological samples is controllable

Focused ion beam (FIB) techniques are among the most important tools for the nanostructuring of surfaces. We used the FIB/SEM (scanning electron microscope) for milling and imaging of digestive gland cells. The aim of our study was to document the interactions of FIB with the surface of the biological sample during FIB investigation, to identify the classes of artifacts, and to test procedures that could induce the quality of FIB milled sections by reducing the artifacts. The digestive gland cells were prepared for conventional SEM. During FIB/SEM operation we induced and enhanced artifacts. The results show that FIB operation on biological tissue affected the area of the sample where ion beam was rastering. We describe the FIB-induced surface major artifacts as a melting-like effect, sweating-like effect, morphological deformations, and gallium (Ga(+)) implantation. The FIB induced surface artifacts caused by incident Ga(+) ions were reduced by the application of a protective platinum strip on the surface exposed to the beam and by a suitable selection of operation protocol. We recommend the same sample preparation methods, FIB protocol for milling and imaging to be used also for other biological samples.     

Non‐rigid alignment in electron tomography in materials science

Electron tomography is a key technique that enables the visualization of an object in three dimensions with a resolution of about a nanometre. High‐quality 3D reconstruction is possible thanks to the latest compressed sensing algorithms and/or better alignment and preprocessing of the 2D projections. Rigid alignment of 2D projections is routine in electron tomography. However, it cannot correct misalignments induced by (i) deformations of the sample due to radiation damage or (ii) drifting of the sample during the acquisition of an image in scanning transmission electron microscope mode. In both cases, those misalignments can give rise to artefacts in the reconstruction. We propose a simple‐to‐implement non‐rigid alignment technique to correct those artefacts. This technique is particularly suited for needle‐shaped samples in materials science. It is initiated by a rigid alignment of the projections and it is then followed by several rigid alignments of different parts of the projections. Piecewise linear deformations are applied to each projection to force them to simultaneously satisfy the rigid alignments of the different parts. The efficiency of this technique is demonstrated on three samples, an intermetallic sample with deformation misalignments due to a high electron dose typical to spectroscopic electron tomography, a porous silicon sample with an extremely thin end particularly sensitive to electron beam and another porous silicon sample that was drifting during image acquisitions.     

Low damage lamella preparation of metallic materials by FIB processing with low acceleration voltage and a low incident angle Ar ion milling finish

Metallic materials are known to be very sensitive to Gallium (Ga) focused ion beam (FIB) processing. Crystal defects formed by FIB irradiation degrade the transmission electron microscope image quality, and it is difficult to distinguish original defects from FIB process-induced damage. A solution to this problem is the low acceleration voltage and low incident angle (LVLA) Argon ion milling, which can be incorporated as an extensional countermeasure for FIB damage removal and eventually for preparation of high-quality lamellae. The transmission electron microscope image quality of iron single crystal could be improved by removing crystal defects using the low acceleration voltage and low incident angle Argon ion milling finish. Lamella quality of the processing result was almost similar with that of the conventional electrolytic polishing. As a practical application of the process, low damage lamella of stainless cast steel could be prepared. Effectiveness of the FIB system equipped with the low acceleration voltage and low incident angle Argon ion milling function as a tool to make high-quality metallic material lamellae is illustrated.     

Effects of focused ion beam milling on electron backscatter diffraction patterns in strontium titanate and stabilized zirconia

This study investigates the effect of focused ion beam (FIB) current and accelerating voltage on electron backscatter diffraction pattern quality of yttria-stabilized zirconia (YSZ) and Nb-doped strontium titanate (STN) to optimize data quality and acquisition time for 3D-EBSD experiments by FIB serial sectioning. Band contrast and band slope were used to describe the pattern quality. The FIB probe currents investigated ranged from 100 to 5000 pA and the accelerating voltage was either 30 or 5 kV. The results show that 30 kV FIB milling induced a significant reduction of the pattern quality of STN samples compared to a mechanically polished surface but yielded a high pattern quality on YSZ. The difference between STN and YSZ pattern quality is thought to be caused by difference in the degree of ion damage as their backscatter coefficients and ion penetration depths are virtually identical. Reducing the FIB probe current from 5000 to 100 pA improved the pattern quality by 20% for STN but only showed a marginal improvement for YSZ. On STN, a conductive coating can help to improve the pattern quality and 5 kV polishing can lead to a 100% improvement of the pattern quality relatively to 30 kV FIB milling. For 3D-EBSD experiments of a material such as STN, it is recommended to combine a high kV FIB milling and low kV polishing for each slice in order to optimize the data quality and acquisition time.     

Phase imaging quality improvement by modification of AFM probes’ cantilever

Imaging of the surface of materials by atomic force microscopy under tapping and phase imaging mode, with use of modified probes is addressed. In this study, the circularly shaped holes located in varying distance from the probe base, were cut out by focused ion beam. Such modification was a consequence of the results of the previous experiments (probe tip sharpening and cantilever thinning) where significant improvement of image quality in tapping and phase imaging mode has been revealed. The solution proposed herein gives similar results, but is much simpler from the technological point of view. Shorter exposition time of the tip onto gallium ions during FIB processing allows to reduce material degradation. The aim of this modification was to change harmonic oscillators’ properties in the simplest and fastest way, to obtain stronger signal for higher resonant frequencies, which can be advantageous for improving the quality of imaging in PI mode. Probes shaped in that way were used for AFM investigations with Bruker AFM nanoscope 8. As a testing material, titanium roughness standard sample, supplied by Bruker, was used. The results have shown that the modifications performed within these studies influence the oscillation of the probes, which in some cases may result in deterioration of the imaging quality under tapping mode for one or both self‐resonant frequencies. However, phase imaging results obtained using modified probes are of higher quality. The numerical simulations performed by application of finite element method were used to explain the results obtained experimentally. Phenomenon described within this study allows to apply developed modelling methodology for prediction of effects of various modifications on the probes' tip, and as a result, to predict how proposed modifications will affect AFM imaging quality.     

Electron and ion imaging of gland cells using the FIB/SEM system

The FIB/SEM system was satisfactorily used for scanning ion (SIM) and scanning electron microscopy (SEM) of gland epithelial cells of a terrestrial isopod Porcellio scaber (Isopoda, Crustacea). The interior of cells was exposed by site-specific in situ focused ion beam (FIB) milling. Scanning ion (SI) imaging was an adequate substitution for scanning electron (SE) imaging when charging rendered SE imaging impossible. No significant differences in resolution between the SI and SE images were observed. The contrast on both the SI and SE images is a topographic. The consequences of SI imaging are, among others, introduction of Ga⁺ ions on/into the samples and destruction of the imaged surface. These two characteristics of SI imaging can be used advantageously. Introduction of Ga⁺ ions onto the specimen neutralizes the charge effect in the subsequent SE imaging. In addition, the destructive nature of SI imaging can be used as a tool for the gradual removal of the exposed layer of the imaged surface, uncovering the structures lying beneath. Alternative SEM and SIM in combination with site-specific in situ FIB sample sectioning made it possible to image the submicrometre structures of gland epithelium cells with reproducibility, repeatability and in the same range of magnifications as in transmission electron microscopy (TEM). At the present state of technology, ultrastructural elements imaged by the FIB/SEM system cannot be directly identified by comparison with TEM images.