Prelabeling Expansion Single-Molecule Localization Microscopy with Minimal Linkage Error

Expansion microscopy combined with single-molecule localization microscopy (ExSMLM) has a potential for approaching molecular resolution. However, ExSMLM faces multiple challenges such as loss of fluorophores and proteins during polymerization, digestion or denaturation, and an increase in linkage error arising from the distance between the fluorophore and the target molecule. Here, we introduce a trifunctional streptavidin to link the target, fluorophore and gel matrix via a biotinylizable peptide tag. The resultant ExSMLM images of vimentin filaments demonstrated high labeling efficiency and a minimal linkage error of ∼5 nm. Our ExSMLM provides a simple and practical means for fluorescence imaging with molecular resolution.     

Amplified Expansion Stimulated Emission Depletion Microscopy

Expansion microscopy (ExM) enhances spatial resolution by using a swellable polymer that expands the sample volume by a factor of ≈4 in one dimension and a factor of ≈64 in volume. Combining ExM with stimulated emission depletion (STED) microscopy, referred to as ExSTED, increases the resolution to up to 10 nm. However, photobleaching is a critical issue in ExSTED because the sample expansion lowers the fluorophore density whereas high-resolution STED requires high depletion intensity. To overcome these issues, we developed extremely bright expansion nanoscopy by using biotin–avidin signal amplification to increase the labeling density. Our method provides up to sevenfold increases in fluorescence signal intensity in expanded samples, thus enabling the use of STED imaging with maximum depletion intensities of a commercial microscope in the order of GW cm⁻². We demonstrated the method by using biotinylated antibodies and genetic incorporation approaches that allow localization of biotin in a specific molecule or organelle.     

Restructuring butterfat through blending and chemical interesterification. 2. Microstructure and polymorphism

Blending of butterfat with canola oil and subsequent chemical interesterification modified the crystal morphology and X-ray diffraction patterns of butterfat, 90∶10 (w/w), and 80∶20 (w/w) blends of butterfat-canola oil. The morphology of 50∶50 (w/w) was also greatly influenced by interesterification. Polarized light microscopy revealed that addition of canola oil led to gradual aggregation of the crystal structure. Scanning electron microscopy revealed all samples to be mixtures of defined crystalline regions and amorphous areas with greater amorphism as oil content increased. Most samples revealed segregation of solid from liquid. Confocal laser scanning microscopy of butterfat revealed complex aggregated structures that were composed of outwardly radiating filaments from a central nucleus. X-ray diffraction analysis revealed a predominance of β′ and a small proportion of β crystals for all samples examined except interesterified butterfat, which consisted solely of β′ crystals.     

Real-time deflection and friction force imaging by bimorph-based resonance-type high-speed scanning force microscopy in the contact mode

We report herein an alternative high-speed scanning force microscopy method in the contact mode based on a resonance-type piezoelectric bimorph scanner. The experimental setup, the modified optical beam deflection scheme suitable for smaller cantilevers, and a high-speed control program for simultaneous data capture are described in detail. The feature of the method is that the deflection and friction force images of the sample surface can be obtained simultaneously in real time. Images of various samples (e.g., a test grating, a thin gold film, and fluorine-doped tin oxide-coated glass slides) are acquired successfully. The imaging rate is 25 frames per second, and the average scan speed reaches a value of approximately 2.5 cm/s. The method combines the advantages of both observing the dynamic processes of the sample surface and monitoring the frictional properties on the nanometer scale.

PACS

07.79.Lh; 07.79.Sp; 68.37.Ps     

Microscopical study of carbon/carbon composites obtained by chemical vapor infiltration of 0°/0°/90°/90° carbon fiber preforms

The microstructure of carbon/carbon composites obtained by isothermal, isobaric chemical vapor infiltration (CVI) of carbon fiber preforms consisting of aligned fiber bundles separated by fiber fleeces was studied comparatively by polarized light microscopy (PLM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM) combined with selected area electron diffraction (SAED). Deposition rate as well as matrix microstructure do not differ in the aligned fiber bundles and fiber fleeces exhibiting different local surface area/volume ratios. The matrices which are homogeneously textured according to PLM exhibit pronounced spatial texture gradients at the sub-μm-scale if investigated by SAED. The texture gradients appear to be independent on the infiltration time, distance between fibers but evidently depend on the total methane pressure. TEM and SEM observations show a thin high-textured layer between the fiber and the medium-textured transitional layer below the high-textured matrix layer containing columnar grains. This thin layer replicates the surface unevenness of the fiber surface while it is absent at the initial carbon fiber surface before infiltration.     

Synthesis of well-aligned carbon nanotubes with open tips

Large amounts of well-aligned carbon nanotubes (CNTs) with open tips have been produced by pyrolysis of iron(II) phthalocyanine. The aligned CNTs have an average length about 10 μm and diameters ranging from 92 to 229 nm. Some of produced CNTs showed Y-junction structure due to the self-joint growth of two neighboring CNTs. The well-aligned CNTs indicated a bamboo-shaped multiwalled structure and fairly good crystallinity. The aligned CNTs follow tip growth mechanism.     

形态学实验和分析方法在高分子研究中的意义与应用

论述了形态学实验分析方法（X射线衍射、电子显微镜和光学显微镜）的概况及其在高分子研究中的应用，为在高分子材料与工程实践中合理运用该实验方法。掌握正确的图象分析方法提供理论依据。     

Microstructural and Chemical Influences of Silicate Grain-Boundary Phases in Yttria-Stabilized Zirconia

The microstructure and chemistry of grain-boundary phases in silicate-doped Y₂O₃─ZrO₂ ceramics were evaluated by analytical electron microscopy. Two different silicate compositions were used: one an aluminosilicate and the other a borosilicate glass. These grain-boundary phases had a significant impact on the grain morphology, the chemical composition of the grains, and the crystallization of second phases. These results indicate that controlled additions of specific glass phases may provide a means for tailoring the microstructure and physical properties of zirconia ceramics.     

Discrimination of B–C–N nanotubes through energy-filtering electron microscopy

Various multi-walled nanotubes in the B–C–N system are thoroughly investigated using a JEOL-3100FEF high-resolution field emission transmission electron microscope operating at 300 kV and equipped with an in-column built Omega filter. Spatially-resolved B, C and N elemental maps of the nanotubes are constructed. It is realized that a wide variety of tubular arrays composed of B, C and N atoms may exist in the system. Sandwich-like BN-rich and C-rich alternating tubular shells, graphitic C layers inside and outside of pure BN shells induced either by surface contamination, or electron beam irradiation, separation of C-rich and BN-rich tubes and/or BN particles within tubular bunches may take place. One should carefully take these effects into account while analyzing nanotube physical properties, e.g., electrical or optical, rather than simply rely on electron energy loss spectra typically collected from B, C and N containing nanostructures as a whole. Striking dependence of an individual nanotube electrical conductivity on tubular shell chemistry is demonstrated using I–V curve recording in an atomic force microscope.     

Aberrations in confocal spectroscopy of polymeric materials: Erroneous thicknesses and intensities,and loss of resolution

Confocal fluorescence and Raman microscopy are becoming important characterization methods in polymers, especially for blends and films. However, caution must be used in analyzing the data because of the spherical aberration introduced into the illumination and collection of light caused by a mismatch in the indices of refraction of the sample and the design of the microscope objective. In many cases, the measured shape of the region under examination and the measured intensities are rendered invalid because of this aberration. Simultaneously, the axial resolution is degraded because the central light rays and the extreme rays have different focal points. It is shown that the loss of axial resolution can be minimized and the loss in intensity can be either reduced or accounted for. The error in location within the sample and, hence, the shape can be easily corrected. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 81: 1662–1669, 2001     

Electron Microscopy Study of Ordinary Portland Cement and Ordinary Portland Cement–Pulverized Fuel Ash Blended Pastes

Many ordinary portland cement (OPC) OPC–pulverized fuel ash (pfa) blended pastes of various ages have been examined using transmission electron microscopy, energy dispersive X-ray analysis, and scanning electron microscopy. In addition to the phases commonly identified in OPC pastes, such as CH, CSH, AFt, and AFm, we found areas of pooriy crystalline iron-containing material intermixed with CSH and needles of hydrotalcite-like composition. The iron-containing material may be the precursor of hydrogarnet phases identified in older pastes by other authors. In the pfa-blended cements, some of the pfa particles reacted to give a radially fibrillar gel, and others showed areas of well-crystallized hydrogarnet within the clearly defined outer boundary of the particle. Unreactive pfa particles had a rim of fibrillar CSH gel around the particle. The general fine-scale microstructure of the OPC-pfa-blended pastes was similar to that in OPC pastes alone, but the CSH gel material had a lower C:S ratio.     

Polymer-grafted cellulose fibers. II. Polymer localization and induced alterations in fiber morphology

In methyl acrylate- or acrylonitrile-grafted northern softwood Kraft or southern softwood Kraft pulp fibers, polymer grafts are present almost homogeneously throughout the fiber wall, although some surface enhancement is observed. On unhydrolyzed fiber surfaces, acrylonitrile grafts protrude in clumps of tangled polymer tufts whereas methyl acrylate grafts form a more uniform, sometimes knobby surface coating. Grafting followed by hydrolysis causes extensive lengthwise splits in the S1 layer, which pulls away from the S2 layer. In the hydrolyzed solvent-dried fiber, the internal grafted polymer/cellulose domains create a unique filamentous and lamellar periodic substructure in the S2 wall. Coexisting with this substructure are numerous microvoids and occasional large pores. We believe that the enhanced absorbency of these fibers can be attributed to the S1 layer disruption, the presence of osmotically active polymer (sodium polyacrylate) incorporated extensively within the S2 wall, and the presence of a drastically altered, more accessible S2 wall substructure. Analytical electron microscopy is shown to be a useful technique for investigating polymer grafts in cellulose fibers. © 1997 John Wiley & Sons, Inc. J Appl Polym Sci 65:1471–1485, 1997     

Structural Evolution During Formation and Filling of Self-patterned Nanoholes on GaAs (100) Surfaces

Nanohole formation on an AlAs/GaAs superlattice gives insight to both the “drilling” effect of Ga droplets on AlAs as compared to GaAs and the hole-filling process. The shape and depth of the nanoholes formed on GaAs (100) substrates has been studied by the cross-section transmission electron microscopy. The Ga droplets “drill” through the AlAs layer at a much slower rate than through GaAs due to differences in activation energy. Refill of the nanohole results in elongated GaAs mounds along the [01−1] direction. As a result of capillarity-induced diffusion, GaAs favors growth inside the nanoholes, which provides the possibility to fabricate GaAs and AlAs nanostructures.     

Ferroelectric switching and current characteristics dependence on ferroelectric polarization direction of microwave synthesized BiFeO3 nanocubes

Herein, we studied the ferroelectric switching and current characteristics of BiFeO₃ (BFO) nanocubes dispersed on the surface of a Nb-doped SrTiO₃ (Nb:STO) substrate based on the ferroelectric polarization orientation. The microwave synthesis method afforded BFO nanocubes with an average size of ～50 nm, which were dispersed on the Nb:STO substrate surface and the substrate was subsequently subjected to heat treatment at 500 °C for 1 h. The piezoelectric d₃₃ hysteresis loop, ferroelectric domain structure, and ferroelectric polarization switching characteristics of the 50-nm-sized BFO nanocubes were examined using piezoresponse force microscopy. Finally, atomic force microscopy confirmed the dependency of current characteristics on the ferroelectric polarization orientation of the BFO nanocubes, verifying the applicability of BFO nanocubes as storage media for ferroelectric polarization information.     

Comparison of Sizes of Magnesia Crystals Calculated from X-ray Diffraction Line-Broadening and Transmission Electron Microscopy Observations

When MgO is formed by decomposition of MgCO₃ under vacuum, crystal sizes calculated from XRD line breadths are in reasonable agreement with direct TEM observations, 4.4 ± 0.6 nm compared with 3.2 ± 0.8 nm. For aligned MgO crystallites from Mg(OH)₂, the sizes calculated are discordant, 14.0 ± 3.0 nm and 2.6 ± 0.7 nm. The sizes calculated from XRD data are too large because the MgO crystallites form from Mg(OH)₂ in near-perfect alignment.     

Correlative Light and Electron Microscopy Using Frozen Section Obtained Using Cryo-Ultramicrotomy

Immuno-electron microscopy (Immuno-EM) is a powerful tool for identifying molecular targets with ultrastructural details in biological specimens. However, technical barriers, such as the loss of ultrastructural integrity, the decrease in antigenicity, or artifacts in the handling process, hinder the widespread use of the technique by biomedical researchers. We developed a method to overcome such challenges by combining light and electron microscopy with immunolabeling based on Tokuyasu’s method. Using cryo-sectioned biological specimens, target proteins with excellent antigenicity were first immunolabeled for confocal analysis, and then the same tissue sections were further processed for electron microscopy, which provided a well-preserved ultrastructure comparable to that obtained using conventional electron microscopy. Moreover, this method does not require specifically designed correlative light and electron microscopy (CLEM) devices but rather employs conventional confocal and electron microscopes; therefore, it can be easily applied in many biomedical studies.     

Analysis of the microstructural evolution of silicon nitride irradiated with swift Xe ions

The evolution of 220 MeV Xe ion induced radiation damage in polycrystalline Si₃N₄ is studied, within the fluence range 5 × 10¹¹– 2 × 10¹⁴ cm⁻², using transmission electron microscopy techniques. These irradiation conditions allow for the study of track morphology in both crystalline and in radiation-amorphized Si₃N₄. The average track size in the polycrystalline samples is 1.9 ± 0.4 nm and 3.1 ± 0.5 nm in the radiation-amorphized samples. The larger track sizes in the radiation-amorphized material is in agreement with predictions of the inelastic thermal spike model and the role of thermal conductivity in latent track formation.     

High-Resolution Electron Microscopy Observations of Stacking Faults in β-SiC

Structural images of the stacking faults in β-SiC were obtained with a high-resolution electron microscope. Stacking faults initially present in β-SiC powder particles were eliminated as grain growth proceeded at elevated temperatures.     

Microstructural Characterization of Ferroelectric Thin Films in Transverse Section

A technique has been developed for the TEM examination of ferroelectric thin films in transverse section. Some preliminary results are reported for three different thin-film/substrate systems. The microstructures of thin films of lead scandium tantalate deposited onto sapphire and MgO, and lead titanate deposited onto AIN, have been examined, with particular attention being paid to the quality of the thin-film/substrate interfaces and to the changes in the nature of the microstructures of the thin films as a function of distance from their substrates. It is demonstrated that the technique successfully produces adequate electron transparent regions for the characterization of the thin-film/substrate interface of all the samples examined and that it is possible to prepare transverse sections of ferroelectric thin films routinely.