Three‐dimensional bright‐field scanning transmission electron microscopy elucidate novel nanostructure in microbial biofilms

Bacterial biofilms play key roles in environmental and biomedical processes, and understanding their activities requires comprehension of their nanoarchitectural characteristics. Electron microscopy (EM) is an essential tool for nanostructural analysis, but conventional EM methods are limited in that they either provide topographical information alone, or are suitable for imaging only relatively thin (<300 nm) sample volumes. For biofilm investigations, these are significant restrictions. Understanding structural relations between cells requires imaging of a sample volume sufficiently large to encompass multiple cells and the capture of both external and internal details of cell structure. An emerging EM technique with such capabilities is bright‐field scanning transmission electron microscopy (BF‐STEM) and in the present report BF‐STEM was coupled with tomography to elucidate nanostructure in biofilms formed by the polycyclic aromatic hydrocarbon‐degrading soil bacterium, Delftia acidovorans Cs1‐4. Dual‐axis BF‐STEM enabled high‐resolution 3‐D tomographic recontructions (6–10 nm) visualization of thick (1250 and 1500 nm) sections. The 3‐D data revealed that novel extracellular structures, termed nanopods, were polymorphic and formed complex networks within cell clusters. BF‐STEM tomography enabled visualization of conduits formed by nanopods that could enable intercellular movement of outer membrane vesicles, and thereby enable direct communication between cells. This report is the first to document application of dual‐axis BF‐STEM tomography to obtain high‐resolution 3‐D images of novel nanostructures in bacterial biofilms. Future work with dual‐axis BF‐STEM tomography combined with correlative light electron microscopy may provide deeper insights into physiological functions associated with nanopods as well as other nanostructures.     

Extraction of structural and chemical information from high angle annular dark‐field image by an improved peaks finding method

With the development of spherical aberration (Cs) corrected scanning transmission electron microscopy (STEM), high angle annular dark filed (HAADF) imaging technique has been widely applied in the microstructure characterization of various advanced materials with atomic resolution. However, current qualitative interpretation of the HAADF image is not enough to extract all the useful information. Here a modified peaks finding method was proposed to quantify the HAADF‐STEM image to extract structural and chemical information. Firstly, an automatic segmentation technique including numerical filters and watershed algorithm was used to define the sub‐areas for each atomic column. Then a 2D Gaussian fitting was carried out to determine the atomic column positions precisely, which provides the geometric information at the unit‐cell scale. Furthermore, a self‐adaptive integration based on the column position and the covariance of statistical Gaussian distribution were performed. The integrated intensities show very high sensitivity on the mean atomic number with improved signal‐to‐noise (S/N) ratio. Consequently, the polarization map and strain distributions were rebuilt from a HAADF‐STEM image of the rhombohedral and tetragonal BiFeO₃ interface and a MnO₂ monolayer in LaAlO₃/SrMnO₃/SrTiO₃ heterostructure was discerned from its neighbor TiO₂ layers. Microsc. Res. Tech. 79:820–826, 2016. © 2016 Wiley Periodicals, Inc.     

Tillandsia stricta Sol (Bromeliaceae) leaves as monitors of airborne particulate matter—A comparative SEM methods evaluation: Unveiling an accurate and odd HP‐SEM method

Airborne particulate matter (PM) has been included among the most important air pollutants by governmental environment agencies and academy researchers. The use of terrestrial plants for monitoring PM has been widely accepted, particularly when it is coupled with SEM/EDS. Herein, Tillandsia stricta leaves were used as monitors of PM, focusing on a comparative evaluation of Environmental SEM (ESEM) and High‐Pressure SEM (HPSEM). In addition, specimens air‐dried at formaldehyde atmosphere (AD/FA) were introduced as an SEM procedure. Hydrated specimen observation by ESEM was the best way to get information from T. stricta leaves. If any artifacts were introduced by AD/FA, they were indiscernible from those caused by CPD. Leaf anatomy was always well preserved. PM density was determined on adaxial and abaxial leaf epidermis for each of the SEM proceedings. When compared with ESEM, particle extraction varied from 0 to 20% in air‐dried leaves while 23–78% of particles deposited on leaves surfaces were extracted by CPD procedures. ESEM was obviously the best choice over other methods but morphological artifacts increased in function of operation time while HPSEM operation time was without limit. AD/FA avoided the shrinkage observed in the air‐dried leaves and particle extraction was low when compared with CPD. Structural and particle density results suggest AD/FA as an important methodological approach to air pollution biomonitoring that can be widely used in all electron microscopy labs. Otherwise, previous PM assessments using terrestrial plants as biomonitors and performed by conventional SEM could have underestimated airborne particulate matter concentration.     

Energy dispersive spectroscopy‐scanning transmission electron microscope observations of free radical production in human polymorphonuclear leukocytes phagocytosing non‐opsonized Tannerella forsythia

We investigated the association between human polymorphonuclear leukocytes (PMNs) and non‐opsonized Tannerella forsythia ATCC 43037 displaying a serum‐resistant surface layer (S‐layer). When PMNs were mixed with T. forsythia in suspension, the cells phagocytosed T. forsythia cells. Nitro blue tetrazolium (NBT) reduction, indicative of production, was observed by light microscopy; cerium (Ce) perhydroxide deposition, indicative of H₂O₂ production, was observed by electron microscopy. We examined the relationship between high‐molecular‐weight proteins of the S‐layer and Ce reaction (for T. forsythia phagocytosis) using electron microscopic immunolabeling. Immunogold particles were localized within the PMNs and on cell surfaces, labelling at the same Ce‐reacted sites where the S‐layer was present. We then used energy dispersive spectroscopy (EDS)‐scanning transmission electron microscope (STEM) to perform Ce and nitrogen (N) (for S‐layer immunocytochemistry) elemental analysis on the phagocytosed cells. That is, the elemental mapping and analysis of N by EDS appeared to reflect the presence of the same moieties detected by the 3,3′‐diaminobenzidine‐tetrahydrochloride (DAB) reaction with horseradish peroxidase (HRP)‐conjugated secondary antibodies, instead of immunogold labeling. We focused on the use of EDS‐STEM to visualize the presence of N resulting from the DAB reaction. In a parallel set of experiments, we used EDS‐STEM to perform Ce and gold (Au; from immunogold labeling of the S‐layer) elemental analysis on the same phagocytosing cells.     

Morphology visualization of irregular shape bacteria by electron holography and tomography

In the current work, irregular morphology of Staphylococcus aureus bacteria has been visualized by phase retrieval employing off‐axis electron holography (EH) and 3D reconstruction electron tomography using high‐angle annular dark field scanning transmission electron microscopy (HAADF‐STEM). Bacteria interacting with gold nanoparticles (AuNP) acquired a shrunken or irregular shape due to air dehydration processing. STEM imaging shows the attachment of AuNP on the surface of cells and suggests an irregular 3D morphology of the specimen. The phase reconstruction demonstrates that off‐axis electron holography can reveal with a single hologram the morphology of the specimen and the distribution of the functionalized AuNPs. In addition, EH reduces significantly the acquisition time and the cumulative radiation damage (in three orders of magnitude) over biological samples in comparison with multiple tilted electron expositions intrinsic to electron tomography, as well as the processing time and the reconstruction artifacts that may arise during tomogram reconstruction.     

Revealing local variations in nanoparticle size distributions in supported catalysts: a generic TEM specimen preparation method

The specimen preparation method is crucial for how much information can be gained from transmission electron microscopy (TEM) studies of supported nanoparticle catalysts. The aim of this work is to develop a method that allows for observation of size and location of nanoparticles deposited on a porous oxide support material. A bimetallic Pt‐Pd/Al₂O₃ catalyst in powder form was embedded in acrylic resin and lift‐out specimens were extracted using combined focused ion beam/scanning electron microscopy (FIB/SEM). These specimens allow for a cross‐section view across individual oxide support particles, including the unaltered near surface region of these particles. A site‐dependent size distribution of Pt‐Pd nanoparticles was revealed along the radial direction of the support particles by scanning transmission electron microscopy (STEM) imaging. The developed specimen preparation method enables obtaining information about the spatial distribution of nanoparticles in complex support structures which commonly is a challenge in heterogeneous catalysis.     

Retro‐fitting an older (S)TEM with two Cs aberration correctors for 80 kV and 60 kV operation

For the characterization of light materials using transmission electron microscopy, a low electron acceleration voltage of 80 kV or even 60 kV is attractive due to reduced beam damage to the specimen. The concomitant reduction in resolving power of the microscope can be restored when using spherical aberration (C_s) correctors, which for the most part are only available in the latest and most expensive microscopes. Here, we show that upgrading of existing TEMs is an attractive and cost‐effective alternative. We report on the low‐voltage performance on graphitic material of a JEOL JEM‐2010F built in the early 1990s and retro‐fitted with a conventional imaging C_s corrector and a probe C_s corrector. The performance data show C_s retro‐fitted instruments can compete very favourably against more modern state‐of‐the‐art instruments in both conventional imaging (TEM) and scanning (STEM) modes.     

Quantitative atomic resolution mapping using high-angle annular dark field scanning transmission electron microscopy

A model-based method is proposed to relatively quantify the chemical composition of atomic columns using high angle annular dark field (HAADF) scanning transmission electron microscopy (STEM) images. The method is based on a quantification of the total intensity of the scattered electrons for the individual atomic columns using statistical parameter estimation theory. In order to apply this theory, a model is required describing the image contrast of the HAADF STEM images. Therefore, a simple, effective incoherent model has been assumed which takes the probe intensity profile into account. The scattered intensities can then be estimated by fitting this model to an experimental HAADF STEM image. These estimates are used as a performance measure to distinguish between different atomic column types and to identify the nature of unknown columns with good accuracy and precision using statistical hypothesis testing. The reliability of the method is supported by means of simulated HAADF STEM images as well as a combination of experimental images and electron energy-loss spectra. It is experimentally shown that statistically meaningful information on the composition of individual columns can be obtained even if the difference in averaged atomic number Z is only 3. Using this method, quantitative mapping at atomic resolution using HAADF STEM images only has become possible without the need of simultaneously recorded electron energy loss spectra.     

Structural investigations on green culms and charcoal of Bambusa multiplex

Green culms of Bambusa multiplex and the bamboo charcoal carbonized from the green culms at 700°C have been studied by means of X‐ray diffraction, X‐ray fluorescent element analysis, analytical scanning electron microscopy, and analytical scanning transmission electron microscopy (STEM), aiming at industrial applications as raw materials for functional devices and substances. It is revealed that the green culms and the charcoal contain a significant amount of Si, in particular, ～18 wt % in the skin. The green culms comprise amorphous and crystalline celluloses. The charcoal has a so‐called amorphous structure which is composed of randomly distributed carbon nanotubes and fibers. The growth of Ag‐doped activated charcoal powders that were produced by two different methods using this charcoal powder has also been studied.     

Imaging internal features of whole, unfixed bacteria

Wet scanning-transmission electron microscopy (STEM) is a technique that allows high-resolution transmission imaging of biological samples in a hydrated state, with minimal sample preparation. However, it has barely been used for the study of bacterial cells. In this study, we present an analysis of the advantages and disadvantages of wet STEM compared with standard transmission electron microscopy (TEM). To investigate the potential applications of wet STEM, we studied the growth of polyhydroxyalkanoate and triacylglycerol carbon storage inclusions. These were easily visible inside cells, even in the early stages of accumulation. Although TEM produces higher resolution images, wet STEM is useful when preservation of the sample is important or when studying the relative sizes of different features, since samples do not need to be sectioned. Furthermore, under carefully selected conditions, it may be possible to maintain cell viability, enabling new types of experiments to be carried out. To our knowledge, internal features of bacterial cells have not been imaged previously by this technique.     

Imaging of high-angle annular dark-field scanning transmission electron microscopy and observations of GaN-based violet laser diodes

The first part of this paper is devoted to physics, to explain high‐angle annular dark‐field scanning transmission electron microscopy (HAADF‐STEM) imaging and to interpret why HAADF‐STEM imaging is incoherent, instructing a strict definition of interference and coherence of electron waves. Next, we present our recent investigations of InGaN/GaN multiple quantum wells and AlGaN/GaN strained‐layer superlattice claddings in GaN‐based violet laser diodes, which have been performed by HAADF‐STEM and high‐resolution field‐emission gun scanning electron microscopy.     

Characterization of the μ and P phase precipitates in the CMSX‐4 single crystal superalloy

A combination of scanning electron microscopy (SEM), transmission electron microscopy (TEM) and scanning‐transmission electron microscopy (STEM) using high‐angle annular‐dark‐field (HAADF) imaging, focussed ion beam‐ scanning electron microscopy (FIB‐SEM) tomography, selected area electron diffraction with beam precession (PED), as well as spatially resolved energy‐dispersive X‐ray spectroscopy (EDS) and electron energy loss spectroscopy (EELS), was used to investigate topologically close‐packed (TCP) phases, occurring in the CMSX‐4 superalloy subjected to high temperature annealing and creep deformation. Structural and chemical analyses were performed to identify the TCP phases and provide information concerning the compositional partitioning of elements between them. The results of SEM and FIB‐SEM tomography revealed the presence of merged TCP particles, which were identified by TEM and PED analysis as coprecipitates of the μ and P phases. Inside the TCP particles that were several micrometres in size, platelets of alternating μ and P phases of nanometric width were found. The combination of STEM‐HAADF imaging with spatially resolved EDS and EELS microanalysis allowed determination of the significant partitioning of the constituent elements between the μ and P phases.     

A simple method for exposing plastic embedded structures for scanning electron microscopy

Surfaces of structures which may be identified in sections of plastic embedded tissue can be exposed for examination using scanning electron microscopy by removal of the embedding medium from the sectioned block with sodium methoxide. Correlation of the information provided by sections and scanning electron microscopy is therefore possible. This technique has been used in examining the developing peristome teeth of the moss Funaria hygrometrica.     

Improvements for imaging ceramics sintering in situ in ESEM

Sintering of green samples of alumina produced by ice‐templating was followed in situ in an environmental scanning electron microscope (ESEM) up to temperatures as high as 1375°C. These alumina samples with well‐defined architectures are of great interest in the field of materials science due to their high specific strength (especially in compression), low density and adaptable porosity. For the present study, they also have the advantage to exhibit an important topography, inducing interesting contrast when imaged in an ESEM. Improvements of the imaging conditions in the ESEM were essential to really follow the sintering process involving formation of necks between grains or shift of the centre of grains. This paper describes the improvements made and the results observed on the sintering process of alumina green samples processed by ice‐templating.     

The conversion of a field-emission scanning electron microscope to a high-resolution,high-performance scanning transmission electron microscope,while maintaining original functions

Recently, the reliability of field-emission electron guns has increased. In addition, the cost of computer systems for on-line processing has dropped. Hence, we should now consider the use of scanning transmission electron microscopy (STEM) for routine work, especially, in the field of biology where one may expect to utilize digital image processing techniques. An STEM has been constructed, without disturbing the original functions, by converting a commercial scanning electron microscope equipped with a fieldemission gun. The STEM is generally operated at accelerating voltage 30 kV, focal length 7.5 mm, and beam current 1?2 × 10^?10 A. Several improvements have been incorporated for removing the effects of vibration, contamination, and stray magnetic fields. Also, an adjustable detector aperture was utilized. The modified instrument was connected to an on-line digital image processing system for utilizing the information obtained from STEM images. The advantages of the modified system were studied from various viewpoints.     

A new cryo‐STEM method for imaging food colloids in the scanning electron microscope

A new cryo‐scanning transmission electron microscopy (cryo‐STEM) technique for imaging casein micelles in a field emission scanning electron microscope is presented. Thin films of micellar casein suspensions on lacey carbon grids were prepared using a modified sample holder developed by Gatan UK. Bright and dark field images were obtained at ?135°C showing casein micelles in their frozen hydrated state and in the size range 30–500 nm. Results were compared favorably with published images of casein micelles obtained with conventional cryo‐transmission electron microscopy, suggesting that cryo‐STEM is a useful alternative technique for visualizing food colloids close to their native state. SCANNING 32: 150–154, 2010. © 2010 Wiley Periodicals, Inc.     

Specimen thickness dependence of hydrogen evolution during cryo‐transmission electron microscopy of hydrated soft materials

The evolution of hydrogen from many hydrated cryo‐preserved soft materials under electron irradiation in the transmission electron microscope can be observed at doses of the order of 1000 e nm^?2 and above. Such hydrogen causes artefacts in conventional transmission electron microscope or scanning transmission electron microscopy (STEM) imaging as well as in analyses by electron energy‐loss spectroscopy. Here we show that the evolution of hydrogen depends on specimen thickness. Using wedge‐shaped specimens of frozen‐hydrated Nafion, a perfluorinated ionomer, saturated with the organic solvent DMMP together with both thin and thick sections of frozen‐hydrated porcine skin, we show that there is a thickness below which hydrogen evolution is not detected either by bubble observation in transmission electron microscope image mode or by spectroscopic analysis in STEM electron energy‐loss spectroscopy mode. We suggest that this effect is due to the diffusion of hydrogen, whose diffusivity remains significant even at liquid nitrogen temperature over the length scales and time scales relevant to transmission electron microscopy analysis of thin specimens. In short, we speculate that sufficient hydrogen can diffuse to the specimen surface in thin sections so that concentrations are too low for bubbling or for spectroscopic detection. Significantly, this finding indicates that higher electron doses can be used during the imaging of radiation‐sensitive hydrated soft materials and, consequently, higher spatial resolution can be achieved, if sufficiently thin specimens are used in order to avoid the evolution of hydrogen‐based artefacts.     

Regional differences in the ependyma of the optic tectal ventricle of adult zebrafish with structures referring to brain hydrodynamics

Classical electron microscopic morphological studies provide detailed ultrastructural information, which may lend insights into cellular functions. As a follow‐up to our morphological investigation of the adult zebrafish (Danio rerio) optic tectum, in this study, we have analyzed the ependymal structures lining the surfaces of the tectal ventricle: the torus, tegmental surface of the valvula cerebelli and the periventricular gray zone of the optic tectal cortex. We used toluidine blue stained plastic (semithin) sections for light microscopy and scanning electron microscopy. Our morphological findings of gated entrances and/or egresses indicate that, at least in the adult zebrafish brain, there may be a bidirectional direct flow communication between the ventricular cerebrospinal fluid and the parenchymal interstitial fluid.     

Wet STEM: a new development in environmental SEM for imaging nano-objects included in a liquid phase

Environmental scanning electron microscopy (ESEM) enables wet samples to be observed without potentially damaging sample preparation through the use of partial water vapour pressure in the microscope specimen chamber. However, in the case of latices in colloidal state or microorganisms, samples are not only wet, but made of objects totally submerged in a liquid phase. In this case, under classical ESEM imaging conditions only the top surface of the liquid is imaged, with poor contrast, and possible drifting of objects. The present paper describes experiments using a powerful new Scanning Transmission Electron Microscopy (STEM) imaging system, that allows transmission observations of wet samples in an ESEM. A special device, designed to observe all sorts of objects submerged in a liquid under annular dark-field imaging conditions, is described. Specific features of the device enable to avoid drifting of floating objects which occurs in the case of a large amount of water, thus allowing slow-scan high-definition imaging of particles with a diameter down to few tens of nm. The large potential applications of this new technique are then illustrated, including the imaging of different nano-objects in water. The particular case of grafted latex particles is discussed, showing that it is possible to observe details on their surface when submerged in water. All the examples demonstrate that images acquired in wet STEM mode show particularly good resolution and contrast, without adding enhancing contrast objects, and without staining.     

Quantitative analysis of image contrast in phase contrast STEM for low dose imaging

This work quantitatively evaluates the contrast in phase contrast images of thin vermiculite crystals recorded by TEM and aberration-corrected bright-field STEM. Specimen movement induced by electron irradiation remains a major problem limiting the phase contrast in TEM images of radiation-sensitive specimens. While spot scanning improves the contrast, it does not eliminate the problem. One possibility is to utilise aberration-corrected scanning transmission electron microscopy (STEM) with an Ångstrom-sized probe to illuminate the sample, and thus further reduce irradiation-induced specimen movement. Vermiculite is relatively radiation insensitive in TEM to electron fluences below 100,000 e⁻/Ų and this is likely to be similar for STEM although different damage mechanisms could occur. We compare the performance of a TEM with a thermally assisted field emission electron gun (FEG) and charge coupled device (CCD) image capture to the performance of STEMs with spherical aberration correction, cold field emission electron sources and photomultiplier tube image capture at a range of electron fluences and similar illumination areas. We show that the absolute contrast of the phase contrast images obtained by aberration-corrected STEM is better than that obtained by TEM. Although the STEM contrast is higher, the efficiency of collection of electrons in bright field STEM is still much less than that in bright field TEM (where for thin samples virtually all the electrons contribute to the image), and the SNR of equivalent STEM images is three times lower. This is better than expected, probably due to the absence of a frequency dependent modulation transfer function in the STEM detection system. With optimisation of the STEM bright field collection angles, the efficiency may approach that of bright field TEM, and if reductions in beam-induced specimen movement are found, STEM could surpass the overall performance of TEM.