High‐performance serial block‐face SEM of nonconductive biological samples enabled by focal gas injection‐based charge compensation

A longstanding limitation of imaging with serial block‐face scanning electron microscopy is specimen surface charging. This charging is largely due to the difficulties in making biological specimens and the resins in which they are embedded sufficiently conductive. Local accumulation of charge on the specimen surface can result in poor image quality and distortions. Even minor charging can lead to misalignments between sequential images of the block‐face due to image jitter. Typically, variable‐pressure SEM is used to reduce specimen charging, but this results in a significant reduction to spatial resolution, signal‐to‐noise ratio and overall image quality. Here we show the development and application of a simple system that effectively mitigates specimen charging by using focal gas injection of nitrogen over the sample block‐face during imaging. A standard gas injection valve is paired with a precisely positioned but retractable application nozzle, which is mechanically coupled to the reciprocating action of the serial block‐face ultramicrotome. This system enables the application of nitrogen gas precisely over the block‐face during imaging while allowing the specimen chamber to be maintained under high vacuum to maximise achievable SEM image resolution. The action of the ultramicrotome drives the nozzle retraction, automatically moving it away from the specimen area during the cutting cycle of the knife. The device described was added to a Gatan 3View system with minimal modifications, allowing high‐resolution block‐face imaging of even the most charge prone of epoxy‐embedded biological samples.     

Challenges of microtome‐based serial block‐face scanning electron microscopy in neuroscience

Serial block‐face scanning electron microscopy (SBEM) is becoming increasingly popular for a wide range of applications in many disciplines from biology to material sciences. This review focuses on applications for circuit reconstruction in neuroscience, which is one of the major driving forces advancing SBEM. Neuronal circuit reconstruction poses exceptional challenges to volume EM in terms of resolution, field of view, acquisition time and sample preparation. Mapping the connections between neurons in the brain is crucial for understanding information flow and information processing in the brain. However, information on the connectivity between hundreds or even thousands of neurons densely packed in neuronal microcircuits is still largely missing. Volume EM techniques such as serial section TEM, automated tape‐collecting ultramicrotome, focused ion‐beam scanning electron microscopy and SBEM (microtome serial block‐face scanning electron microscopy) are the techniques that provide sufficient resolution to resolve ultrastructural details such as synapses and provides sufficient field of view for dense reconstruction of neuronal circuits. While volume EM techniques are advancing, they are generating large data sets on the terabyte scale that require new image processing workflows and analysis tools. In this review, we present the recent advances in SBEM for circuit reconstruction in neuroscience and an overview of existing image processing and analysis pipelines.     

An overview of state‐of‐the‐art image restoration in electron microscopy

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

FE‐SEM observation of swelled seaweed using hydrophilic ionic liquid; 1‐butyl‐3‐methylimidazolium tetrafluoroborate

The method to observe the exact morphology of swelled seaweed as an example of biological material by field emission scanning electron microscopy (FE‐SEM) with the aid of hydrophilic ionic liquid (IL); 1‐butyl‐3‐methylimidazolium tetrafluoroborate is reported. Seaweed was first swelled in 3.5% NaCl solution and then treated with the IL and water mixture in 1:7 weight ratios and centrifuged to remove the excess IL solution. Thus treated seaweed maintained its morphology even at high magnification and did not show drying in the FE‐SEM chamber. This observation technique might be useful for various kinds of biological materials to be observed under FE‐SEM. Microsc. Res. Tech., 2013. © 2012 Wiley Periodicals, Inc.     

Reverse Monte Carlo reconstruction algorithm for discrete electron tomography based on HAADF‐STEM atom counting

In this paper, we propose an algorithm to obtain a three‐dimensional reconstruction of a single nanoparticle based on the method of atom counting. The location of atoms in three dimensions has been successfully performed using simulations of high‐angle‐annular‐dark‐field images from only three zone‐axis projections, [110], [310] and [211], for a face‐centred cubic particle. These three orientations are typically accessible by low‐tilt holders often used in high‐performance scanning transmission electron microscopes.     

Aberration‐corrected STEM for atomic‐resolution imaging and analysis

Aberration‐corrected scanning transmission electron microscopes are able to form electron beams smaller than 100 pm, which is about half the size of an average atom. Probing materials with such beams leads to atomic‐resolution images, electron energy loss and energy‐dispersive X‐ray spectra obtained from single atomic columns and even single atoms, and atomic‐resolution elemental maps. We review briefly how such electron beams came about, and show examples of applications. We also summarize recent developments that are propelling aberration‐corrected scanning transmission electron microscopes in new directions, such as complete control of geometric aberration up to fifth order, and ultra‐high‐energy resolution EELS that is allowing vibrational spectroscopy to be carried out in the electron microscope.     

Surface roughness and filler particles characterization of resin‐based composites

The purpose of this study was to evaluate the surface roughness (Ra), and the morphology and composition of filler particles of different composites submitted to toothbrushing and water storage. Disc‐shaped specimens (15 mm × 2 mm) were made from five composites: two conventional (Z100?, and Filtek? Supreme Ultra Universal, 3M), one “quick‐cure” (Estelite ∑ Quick, Tokuyama), one fluoride‐releasing (Beautiful II, Shofu), and one self‐adhering (Vertise Flow, Kerr) composite. Samples were finished/polished using aluminum oxide discs (Sof‐Lex, 3M), and their surfaces were analyzed by profilometry (n = 5) and scanning electron microscopy (SEM; n = 3) at 1 week and after 30,000 toothbrushing cycles and 6‐month water storage. Ra data were analyzed by two‐way analysis of variance and Tukey's test (α = 0.05). Filler particles morphology and composition were analyzed by SEM and X‐ray dispersive energy spectroscopy, respectively. Finishing/polishing resulted in similar Ra for all the composites, while toothbrushing and water storage increased the Ra of all the tested materials, also changing their surface morphology. Beautifil II and Vertise Flow presented the highest Ra after toothbrushing and water storage. Filler particles were mainly composed of silicon, zirconium, aluminum, barium, and ytterbium. Size and morphology of fillers, and composition of the tested composites influenced their Ra when samples were submitted to toothbrushing and water storage.     

Reactions of polypeptide ions with electrons in the gas phase

Reactions of electrons in the energy range below 70 eV with polypeptide cations and anions are reviewed, as well as their applications for the structural analysis of polypeptides. At very low energies (相似文献   

Observation of metal nanoparticles at atomic resolution in Pt‐based cancer chemotherapeutics

The chemotherapeutics cisplatin and oxaliplatin are important tools in the fight against cancer. Both compounds are platinum complexes. Aberration‐corrected scanning transmission electron microscopy using the annular dark‐field imaging mode now routinely provides single‐atom sensitivity with atomic number contrast. Here, this imaging mode is used to directly image the platinum within the two drugs in their dried form on an amorphous carbon support film. The oxaliplatin is found to have wetted the supporting amorphous carbon, forming disordered clusters suggesting that the platinum has remained within the complex. Conversely, the cisplatin sample reveals 1.8‐nm‐diameter metallic platinum clusters. The size and shape of the clusters do not appear to be dependent on drying rate nor formed by beam damage, which may suggest that they were present in the original drug solution.     

Effect of the root canal final rinse protocols on the debris and smear layer removal and on the push‐out strength of an epoxy‐based sealer

The aim of the present study was to evaluate the efficacy of QMiX, SmearClear, and 17% EDTA for the debris and smear layer removal from the root canal and its effects on the push‐out bond strength of an epoxy‐based sealer by scanning electron microscopy (SEM). Forty extracted human canines (n = 10) were assigned to the following final rinse protocols: G1‐distilled water (control), G2–17% EDTA, G3‐SmearClear, and G4‐QMiX. The specimens were submitted to a SEM analysis to evaluate the presence of debris and smear layer, respectively, in the apical or cervical segments. In sequence, forty extracted human maxillary canines with the root canals instrumented were divided into four groups (n = 10) similar to the SEM analysis study. After the filling with AH Plus, the roots were transversally sectioned to obtain dentinal slices. The specimens were submitted to a push‐out bond strength test using an electromechanical testing machine. The statistical analysis for the SEM and push‐out bond strength studies were performed using the Kruskal–Wallis and Dunn tests (α = 5%). There was no difference among the G2, G3, and G4 efficacy in removing the debris and smear layer (P > 0.05). The efficacy of these groups was superior to the control group. The push‐out bond strength values of G2, G3, and G4 were superior to the control group. The ability to remove the debris and smear layer by SmearClear and QMiX was as effective as the 17% EDTA. The final rinse with these solutions promoted similar push‐out bond strength values. Microsc. Res. Tech. 76:533–537, 2013. © 2013 Wiley Periodicals, Inc.     

Performance of signal‐to‐noise ratio estimation for scanning electron microscope using autocorrelation Levinson–Durbin recursion model

A new technique to quantify signal‐to‐noise ratio (SNR) value of the scanning electron microscope (SEM) images is proposed. This technique is known as autocorrelation Levinson–Durbin recursion (ACLDR) model. To test the performance of this technique, the SEM image is corrupted with noise. The autocorrelation function of the original image and the noisy image are formed. The signal spectrum based on the autocorrelation function of image is formed. ACLDR is then used as an SNR estimator to quantify the signal spectrum of noisy image. The SNR values of the original image and the quantified image are calculated. The ACLDR is then compared with the three existing techniques, which are nearest neighbourhood, first‐order linear interpolation and nearest neighbourhood combined with first‐order linear interpolation. It is shown that ACLDR model is able to achieve higher accuracy in SNR estimation.     

Real‐space observation of strong metal‐support interaction: state‐of‐the‐art and what's the next

The real‐space resolving of the encapsulated overlayer in the well‐known model and industry catalysts, ascribed to the advent of dedicated transmission electron microscopy, enables us to probe novel nano/micro architecture chemistry for better application, revisiting our understanding of this key issue in heterogeneous catalysis. In this review, we summarize the latest progress of real‐space observation of SMSI in several well‐known systems mainly covered from the metal catalysts (mostly Pt) supported by the TiO₂, CeO₂ and Fe₃O₄. As a comparison with the model catalyst Pt/Fe₃O₄, the industrial catalyst Cu/ZnO is also listed, followed with the suggested ongoing directions in the field.     

Diffraction effects and inelastic electron transport in angle‐resolved microscopic imaging applications

We analyse the signal formation process for scanning electron microscopic imaging applications on crystalline specimens. In accordance with previous investigations, we find nontrivial effects of incident beam diffraction on the backscattered electron distribution in energy and momentum. Specifically, incident beam diffraction causes angular changes of the backscattered electron distribution which we identify as the dominant mechanism underlying pseudocolour orientation imaging using multiple, angle‐resolving detectors. Consequently, diffraction effects of the incident beam and their impact on the subsequent coherent and incoherent electron transport need to be taken into account for an in‐depth theoretical modelling of the energy‐ and momentum distribution of electrons backscattered from crystalline sample regions. Our findings have implications for the level of theoretical detail that can be necessary for the interpretation of complex imaging modalities such as electron channelling contrast imaging (ECCI) of defects in crystals. If the solid angle of detection is limited to specific regions of the backscattered electron momentum distribution, the image contrast that is observed in ECCI and similar applications can be strongly affected by incident beam diffraction and topographic effects from the sample surface. As an application, we demonstrate characteristic changes in the resulting images if different properties of the backscattered electron distribution are used for the analysis of a GaN thin film sample containing dislocations.     

Backscattered electron image of osmium‐impregnated/macerated tissues as a novel technique for identifying the cis‐face of the Golgi apparatus by high‐resolution scanning electron microscopy

The osmium maceration method with scanning electron microscopy (SEM) enabled to demonstrate directly the three‐dimensional (3D) structure of membranous cell organelles. However, the polarity of the Golgi apparatus (that is, the cis–trans axis) can hardly be determined by SEM alone, because there is no appropriate immunocytochemical method for specific labelling of its cis‐ or trans‐faces. In the present study, we used the osmium impregnation method, which forms deposits of reduced osmium exclusively in the cis‐Golgi elements, for preparation of specimens for SEM. The newly developed procedure combining osmium impregnation with subsequent osmium maceration specifically visualised the cis‐elements of the Golgi apparatus, with osmium deposits that were clearly detected by backscattered electron‐mode SEM. Prolonged osmication by osmium impregnation (2% OsO₄ solution at 40°C for 40 h) and osmium maceration (0.1% OsO₄ solution at 20°C for 24 h) did not significantly impair the 3D ultrastructure of the membranous cell organelles, including the Golgi apparatus. This novel preparation method enabled us to determine the polarity of the Golgi apparatus with enough information about the surrounding 3D ultrastructure by SEM, and will contribute to our understanding of the global organisation of the entire Golgi apparatus in various differentiated cells.