Comparison of three‐dimensional analysis and stereological techniques for quantifying lithium‐ion battery electrode microstructures

Lithium‐ion battery performance is intrinsically linked to electrode microstructure. Quantitative measurement of key structural parameters of lithium‐ion battery electrode microstructures will enable optimization as well as motivate systematic numerical studies for the improvement of battery performance. With the rapid development of 3‐D imaging techniques, quantitative assessment of 3‐D microstructures from 2‐D image sections by stereological methods appears outmoded; however, in spite of the proliferation of tomographic imaging techniques, it remains significantly easier to obtain two‐dimensional (2‐D) data sets. In this study, stereological prediction and three‐dimensional (3‐D) analysis techniques for quantitative assessment of key geometric parameters for characterizing battery electrode microstructures are examined and compared. Lithium‐ion battery electrodes were imaged using synchrotron‐based X‐ray tomographic microscopy. For each electrode sample investigated, stereological analysis was performed on reconstructed 2‐D image sections generated from tomographic imaging, whereas direct 3‐D analysis was performed on reconstructed image volumes. The analysis showed that geometric parameter estimation using 2‐D image sections is bound to be associated with ambiguity and that volume‐based 3‐D characterization of nonconvex, irregular and interconnected particles can be used to more accurately quantify spatially‐dependent parameters, such as tortuosity and pore‐phase connectivity.     

In situ TEM studies of micron‐sized all‐solid‐state fluoride ion batteries: Preparation,prospects, and challenges

Trustworthy preparation and contacting of micron‐sized batteries is an essential task to enable reliable in situ TEM studies during electrochemical biasing. Some of the challenges and solutions for the preparation of all‐solid‐state batteries for in situ TEM electrochemical studies are discussed using an optimized focused ion beam (FIB) approach. In particular redeposition, resistivity, porosity of the electrodes/electrolyte and leakage current are addressed. Overcoming these challenges, an all‐solid‐state fluoride ion battery has been prepared as a model system for in situ TEM electrochemical biasing studies and first results on a Bi/La_0.9Ba_0.1F_2.9 half‐cell are presented. Microsc. Res. Tech. 79:615–624, 2016. © 2016 Wiley Periodicals, Inc.     

Sample preparation and microscopical investigation techniques for metal and ceramics containing graphite and graphene‐like layered particles

Scanning electron microscopy (SEM) techniques are widely used in microstructural investigations of materials since it can provide surface morphology, topography, and chemical information. However, it is important to use correct imaging and sample preparation techniques to reveal the microstructures of materials composed of components with different polishing characteristics such as grey cast iron, graphene platelets (GPLs)‐added SiAlON composite, SiC and B₄C ceramics containing graphite or graphene‐like layered particles. In this study, all microstructural details of gray cast iron were successfully revealed by using argon ion beam milling as an alternative to the standard sample preparation method for cast irons, that is, mechanical polishing followed by chemical etching. The in‐lens secondary electron (I‐L‐SE) image was clearly displayed on the surface details of the graphites that could not be revealed by backscattered electron (BSE) and Everhart–Thornley secondary electron (E‐T SE) images. Mechanical polishing leads to pull‐out of GPLs from SiAlON surface, whereas argon ion beam milling preserved the GPLs and resulted in smooth surface. Grain and grain boundaries of polycrystalline SiC and B₄C were easily revealed by using I‐L SE image in the SEM after only mechanical polishing without any etching process. While the BSE and E‐T SE images did not clearly show the residual graphites in the microstructure, their distribution in the B₄C matrix was fully revealed in the I‐L SE image.     

Scanning (atomic) force microscopy imaging of earthworm haemoglobin calibrated with spherical colloidal gold particles

Scanning (atomic) force microscopy (SFM) permits high-resolution imaging of a biological specimen in physiological solutions. Untreated extracellular haemoglobin molecules of the common North American earthworm, Lumbricus terrestris, were imaged in NH₄Ac solution using calibrated SFM. Individual molecules and their top and side views were clearly identified and were comparable with the images of the same molecule obtained by scanning transmission electron microscopy (STEM). A central depression, the presumed mouth of the hole, was detected. We analysed 75 individual molecules for their lateral dimensions. Compression varied for different molecules, presumably because of the variation of the interaction between the SFM tip and the protein molecule. Two effective heights which correspond to the heights of the points of the haemoglobin molecules first and last touched by the tip, h₁ and h₂, respectively, were measured for each protein and ranged between 1.58 and 16.2 nm for h₁ and 1.23 and 13.6 nm for h₂. The apparent diameter was measured and ranged from 44.9 to 86.6 nm (63.2±10.5 nm, n =75), which is about twice the diameter of the molecule reported by STEM for the top view orientation. The higher the measured effective heights, the worse was the tip convolution effect. In order to determine the tip parameters (semivertical angle, curvature of radius and the cut-off height) and to calibrate images of earthworm haemoglobin molecules, spherical gold particles were scanned as standards. The tip sectional radii at distances of h₁ and h₂ above the tip apex were subtracted from the apparent diameter of the protein. The calibrated lateral dimension was 29.1 ±3.85 nm, which is close to the reported scanning transmission electron microscopy data 30.0 ±0.8 nm. The results presented here demonstrate that the calibration approach of imaging gold particles is practical and relatively accurate. Calibrated SFM imaging can be applied to the study of other biomacromolecules.     

High‐resolution,high‐throughput imaging with a multibeam scanning electron microscope

Electron–electron interactions and detector bandwidth limit the maximal imaging speed of single‐beam scanning electron microscopes. We use multiple electron beams in a single column and detect secondary electrons in parallel to increase the imaging speed by close to two orders of magnitude and demonstrate imaging for a variety of samples ranging from biological brain tissue to semiconductor wafers.     

Evaluation of fracture planes and cell morphology in complementary fractures of cultured cells in the frozen-hydrated state by field-emission secondary electron microscopy: feasibility for ion localization and fluorescence imaging studies

We have employed field-emission secondary electron microscopy (FESEM) for morphological evaluation of freeze-fractured frozen-hydrated renal epithelial LLC-PK₁ cells prepared with our simple cryogenic sandwich-fracture method that does not require any high-vacuum freeze-fracture instrumentation (Chandra et al. (1986) J. Microsc. 144 , 15–37). The cells fractured on the substrate side of the sandwich were matched one-to-one with their corresponding complementary fractured faces on the other side of the sandwich. The FESEM analysis of the frozen-hydrated cells revealed three types of fracture: (i) apical membrane fracture that produces groups of cells together on the substrate fractured at the ectoplasmic face of the plasma membrane; (ii) basal membrane fracture that produces basal plasma membrane-halves on the substrate; and (iii) cross-fracture that passes randomly through the cells. The ectoplasmic face (E-face) and protoplasmic face (P-face) of the membrane were recognized based on the density of intramembranous particles. Feasibility of fractured cells was shown for intracellular ion localization with ion microscopy, and fluorescence imaging with laser scanning confocal microscopy. Ion microscopy imaging of freeze-dried cells fractured at the apical membrane revealed well-preserved intracellular ionic composition of even the most diffusible ions (total concentrations of K⁺, Na⁺ and Ca⁺). Structurally damaged cells revealed lower K⁺ and higher Na⁺ and Ca⁺ contents than in well-preserved cells. Frozen-freeze-dried cells also allowed imaging of fluorescently labelled mitochondria with a laser scanning confocal microscope. Since these cells are prepared without washing away the nutrient medium or using any chemical pretreatment to affect their native chemical and structural makeup, the characterization of fracture faces introduces ideal sample types for chemical and morphological studies with ion and electron microscopes and other techniques such as laser scanning confocal microscopy, atomic force microscopy and near-field scanning optical microscopy.     

Multi-objective optimal design of high frequency probe for scanning ion conductance microscopy

Scanning ion conductance microscopy(SICM) is an emerging non-destructive surface topography characterization apparatus with nanoscale resolution. However, the low regulating frequency of probe in most existing modulated current based SICM systems increases the system noise, and has difficulty in imaging sample surface with steep height changes. In order to enable SICM to have the capability of imaging surfaces with steep height changes, a novel probe that can be used in the modulated current based hopping mode is designed. The design relies on two piezoelectric ceramics with different travels to separate position adjustment and probe frequency regulation in the Z direction. To further improve the resonant frequency of the probe, the material and the key dimensions for each component of the probe are optimized based on the multi-objective optimization method and the finite element analysis. The optimal design has a resonant frequency of above 10 kHz. To validate the rationality of the designed probe, microstructured grating samples are imaged using the homebuilt modulated current based SICM system. The experimental results indicate that the designed high frequency probe can effectively reduce the spike noise by 26% in the average number of spike noise. The proposed design provides a feasible solution for improving the imaging quality of the existing SICM systems which normally use ordinary probes with relatively low regulating frequency.     

Cryo‐scanning x‐ray diffraction microscopy of frozen‐hydrated yeast

We developed cryo‐scanning x‐ray diffraction microscopy, utilizing hard x‐ray ptychography at cryogenic temperature, for the noninvasive, high‐resolution imaging of wet, extended biological samples and report its first frozen‐hydrated imaging. Utilizing phase contrast at hard x‐rays, cryo‐scanning x‐ray diffraction microscopy provides the penetration power suitable for thick samples while retaining sensitivity to minute density changes within unstained samples. It is dose‐efficient and further minimizes radiation damage by keeping the wet samples at cryogenic temperature. We demonstrate these capabilities in two dimensions by imaging unstained frozen‐hydrated budding yeast cells, achieving a spatial resolution of 85 nm with a phase sensitivity of 0.0053 radians. The current work presents the feasibility of cryo‐scanning x‐ray diffraction microscopy for quantitative, high‐resolution imaging of unmodified biological samples extending to tens of micrometres.     

Morphological segmentation of FIB‐SEM data of highly porous media

Nanoporous materials play an important role in modern batteries as well as fuel cells. The materials microstructure needs to be analyzed as it determines the electrochemical properties. However, the microstructure is too fine to be resolved by microcomputed tomography. The method of choice to analyze the microstructure is focused ion beam nanotomography (FIB‐SEM). However, the reconstruction of the porous 3D microstructure from FIB‐SEM image data in general has been an unsolved problem so far. In this paper, we present a new method using morphological operations. First, features are extracted from the data. Subsequently, these features are combined to an initial segmentation, that is then refined by a constrained watershed transformation. We evaluate our method with synthetic data, generated by a simulation of the FIB‐SEM imaging process. We compare the ground truth in the simulated data to the segmentation result. The new method is found to produce a much smaller error than existing techniques.     

High‐temperature oxidation of nickel and chromium studied with an in‐situ environmental scanning electron microscope

Investigations of the morphology of metal oxide scales formed at high temperatures in oxidative environments are usually undertaken after exposure of the samples is completed. In this study, an environmental scanning electron microscope (ESEM) was used as a tool for the in‐situ observation of oxide scale formation. Pure nickel and chromium samples were oxidized at a temperature of 973 K in either pure oxygen or water vapour at a pressure of 667 Pa. The evolution of an oxide scale was followed in‐situ for up to 3 h. The morphology of the developing oxide scales was found to be a function of the metal substrate and the gaseous species. The growth mechanisms of the different metal oxide scales are reviewed and related to the analysed in‐situ images. Emphasis is placed on the relationship between oxidation mechanism and scale morphology. Nickel is seen to oxidise by outward diffusion of nickel probably on oxide grain boundaries when exposed to oxygen. Water vapour changes the scale morphology and a duplex‐type scale arises due to preferential overgrowth. The scale which develops due to chromium oxidation in oxygen is a fine‐grained, thin, and dense layer. In contrast, water vapour leads to whisker growth on chromium and an open, felt‐like structure forms. The applicability of the ESEM to the study of such systems is demonstrated, and its limitations are outlined. The results are encouraging examples of the possibilities which the in‐situ ESEM technique possesses.     

Rapid single‐molecule imaging in cyclic olefin copolymer channels

Rapid preparation of high quality capture surfaces is a major challenge for surface‐based single‐molecule protein binding assays. Here we introduce a simple method to activate microfluidic chambers made from cyclic olefin copolymer for single‐molecule imaging with total internal reflection fluorescence microscopy. We describe a surface coating protocol and demonstrate single‐molecule imaging in off‐the‐shelf microfluidic parts that can be activated for binding assays within a few minutes. As the first example, biotinylated protein directly captured on the neutravidin‐coated surface was detected using fluorescently labeled antibody. We then showed detection of a fusion construct containing green fluorescence protein and verified its single fluorophore behavior by observing stepwise photobleaching events. Finally, a target protein was identified in the crude cell lysate using antibody–sandwich complex formation. In all experiments, controls were completed to ensure that nonspecific binding to the surface was minimal. Based on our results, we conclude that the simple surface preparation described in this paper enables single‐molecule imaging assays without time‐consuming coating procedures. Microsc. Res. Tech. 78:309–316, 2015. © 2015 Wiley Periodicals, Inc.     

Application of sensitive,high‐resolution imaging at a commercial lab‐based X‐ray micro‐CT system using propagation‐based phase retrieval

Several dedicated commercial lab‐based micro‐computed tomography (μCT) systems exist, which provide high‐resolution images of samples, with the capability to also deliver in‐line phase contrast. X‐ray phase contrast is particularly beneficial when visualizing very small features and weakly absorbing samples. The raw measured projections will include both phase and absorption effects. Extending our previous work that addressed the optimization of experimental conditions at the commercial ZEISS Xradia 500 Versa system, single‐distance phase‐contrast imaging is demonstrated on complex biological and material samples. From data captured at this system, we demonstrate extraction of the phase signal or the correction of the mixed image for the phase shift, and show how this procedure increases the contrast and removes artefacts. These high‐quality images, measured without the use of a synchrotron X‐ray source, demonstrate that highly sensitive, micrometre‐resolution imaging of 3D volumes is widely accessible using commercially advanced laboratory devices.     

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.     

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.     

High-resolution water window X-ray imaging of in vivo cells and their products using LiF crystal detectors

High contrast imaging of in vivo Chlorella sorokiniana cells with submicron spatial resolution was obtained with a contact water window X-ray microscopy technique using a point-like, laser-plasma produced, water-window X-ray radiation source, and LiF crystals as detectors. This novel type of X-ray imaging detectors is based on photoluminescence of stable electronic point defects, characterized by high intrinsic resolution. The fluorescence images obtained on LiF crystals exposed in single-shot experiments demonstrate the high sensitivity and dynamic range of this new detector. The powerful performances of LiF crystals allowed us to detect the exudates of Chlorella cells in their living medium and their spatial distribution in situ, without any special sample preparation.     

Integration of a high‐NA light microscope in a scanning electron microscope

We present an integrated light‐electron microscope in which an inverted high‐NA objective lens is positioned inside a scanning electron microscope (SEM). The SEM objective lens and the light objective lens have a common axis and focal plane, allowing high‐resolution optical microscopy and scanning electron microscopy on the same area of a sample simultaneously. Components for light illumination and detection can be mounted outside the vacuum, enabling flexibility in the construction of the light microscope. The light objective lens can be positioned underneath the SEM objective lens during operation for sub‐10 μm alignment of the fields of view of the light and electron microscopes. We demonstrate in situ epifluorescence microscopy in the SEM with a numerical aperture of 1.4 using vacuum‐compatible immersion oil. For a 40‐nm‐diameter fluorescent polymer nanoparticle, an intensity profile with a FWHM of 380 nm is measured whereas the SEM performance is uncompromised. The integrated instrument may offer new possibilities for correlative light and electron microscopy in the life sciences as well as in physics and chemistry.     

Advanced spinning disk‐TIRF microscopy for faster imaging of the cell interior and the plasma membrane

Understanding the cellular processes that occur between the cytosol and the plasma membrane is an important task for biological research. Till now, however, it was not possible to combine fast and high‐resolution imaging of both the isolated plasma membrane and the surrounding intracellular volume. Here, we demonstrate the combination of fast high‐resolution spinning disk (SD) and total internal reflection fluorescence (TIRF) microscopy for specific imaging of the plasma membrane. A customised SD‐TIRF microscope was used with specific design of the light paths that allowed, for the first time, live SD‐TIRF experiments at high acquisition rates. A series of experiments is shown to demonstrate the feasibility and performance of our setup.     

Three‐dimensional structural imaging of starch granules by second‐harmonic generation circular dichroism

Chirality is one of the most fundamental and essential structural properties of biological molecules. Many important biological molecules including amino acids and polysaccharides are intrinsically chiral. Conventionally, chiral species can be distinguished by interaction with circularly polarized light, and circular dichroism is one of the best‐known approaches for chirality detection. As a linear optical process, circular dichroism suffers from very low signal contrast and lack of spatial resolution in the axial direction. It has been demonstrated that by incorporating nonlinear interaction with circularly polarized excitation, second‐harmonic generation circular dichroism can provide much higher signal contrast. However, previous circular dichroism and second‐harmonic generation circular dichroism studies are mostly limited to probe chiralities at surfaces and interfaces. It is known that second‐harmonic generation, as a second‐order nonlinear optical effect, provides excellent optical sectioning capability when combined with a laser‐scanning microscope. In this work, we combine the axial resolving power of second‐harmonic generation and chiral sensitivity of second‐harmonic generation circular dichroism to realize three‐dimensional chiral detection in biological tissues. Within the point spread function of a tight focus, second‐harmonic generation circular dichroism could arise from the macroscopic supramolecular packing as well as the microscopic intramolecular chirality, so our aim is to clarify the origins of second‐harmonic generation circular dichroism response in complicated three‐dimensional biological systems. The sample we use is starch granules whose second‐harmonic generation‐active molecules are amylopectin with both microscopic chirality due to its helical structure and macroscopic chirality due to its crystallized packing. We found that in a starch granule, the second‐harmonic generation for right‐handed circularly polarized excitation is significantly different from second‐harmonic generation for left‐handed one, offering excellent second‐harmonic generation circular dichroism contrast that approaches 100%. In addition, three‐dimensional visualization of second‐harmonic generation circular dichroism distribution with sub‐micrometer spatial resolution is realized. We observed second‐harmonic generation circular dichroism sign change across the starch granules, and the result suggests that in thick biological tissue, second‐harmonic generation circular dichroism arises from macroscopic molecular packing. Our result provides a new method to visualize the organization of three‐dimensional structures of starch granules. The second‐harmonic generation circular dichroism imaging method expands the horizon of nonlinear chiroptical studies from simplified surface/solution environments to complicated biological tissues.