Advantages of stereo imaging of metallic surfaces with low voltage backscattered electrons in a field emission scanning electron microscope

High emission current backscattered electron (HC-BSE) stereo imaging at low accelerating voltages (≤ 5 keV) using a field emission scanning electron microscope was used to display surface structure detail. Samples of titanium with high degrees of surface roughness, for potential medical implant applications, were imaged using the HC-BSE technique at two stage tilts of + 3° and − 3° out of the initial position. A digital stereo image was produced and qualitative height, depth and orientation information on the surface structures was observed. HC-BSE and secondary electron (SE) images were collected over a range of accelerating voltages. The low voltage SE and HC-BSE stereo images exhibited enhanced surface detail and contrast in comparison to high voltage (> 10 keV) BSE or SE stereo images. The low voltage HC-BSE stereo images displayed similar surface detail to the low voltage SE images, although they showed more contrast and directional sensitivity on surface structures. At or below 5 keV, only structures a very short distance into the metallic surface were observed. At higher accelerating voltages a greater appearance of depth could be seen but there was less information on the fine surface detail and its angular orientation. The combined technique of HC-BSE imaging and stereo imaging should be useful for detailed studies on material surfaces and for biological samples with greater contrast and directional sensitivity than can be obtained with current SE or BSE detection modes.     

Back-scatter electron images at 200 kV of subsurface structures in composite materials

Back-scatter electron images of subsurface structures of higher atomic number than the matrix are obtained at accelerating voltages of 50, 100, 150 and 200 kV. Similar back-scatter images of structures on the bottom surface of single crystal foils of copper and mica ranging in thickness from 1 to 4 μm are compared with STEM images. It is concluded that at 200 kV a reasonable resolution of structures down to 1 μm subsurface is possible.     

Nanostructural analysis by atomic force microscopy followed by light microscopy on the same archival slide

Integrated information on ultrastructural surface texture and chemistry increasingly plays a role in the biomedical sciences. Light microscopy provides access to biochemical data by the application of dyes. Ultrastructural representation of the surface structure of tissues, cells, or macromolecules can be obtained by scanning electron microscopy (SEM). However, SEM often requires gold or coal coating of biological samples, which makes a combined examination by light microscopy and SEM difficult. Conventional histochemical staining methods are not easily applicable to biological material subsequent to such treatment. Atomic force microscopy (AFM) gives access to surface textures down to ultrastructural dimensions without previous coating of the sample. A combination of AFM with conventional histochemical staining protocols for light microscopy on a single slide is therefore presented. Unstained cores were examined using AFM (tapping mode) and subsequently stained histochemically. The images obtained by AFM were compared with the results of histochemistry. AFM technology did not interfere with any of the histochemical staining protocols. Ultrastructurally analyzed regions could be identified in light microscopy and histochemical properties of ultrastructurally determined regions could be seen. AFM-generated ultrastructural information with subsequent staining gives way to novel findings in the biomedical sciences. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.     

Focused ion beam scanning electron microscopy in biology

Since the end of the last millennium, the focused ion beam scanning electron microscopy (FIB‐SEM) has progressively found use in biological research. This instrument is a scanning electron microscope (SEM) with an attached gallium ion column and the 2 beams, electrons and ions (FIB) are focused on one coincident point. The main application is the acquisition of three‐dimensional data, FIB‐SEM tomography. With the ion beam, some nanometres of the surface are removed and the remaining block‐face is imaged with the electron beam in a repetitive manner. The instrument can also be used to cut open biological structures to get access to internal structures or to prepare thin lamella for imaging by (cryo‐) transmission electron microscopy. Here, we will present an overview of the development of FIB‐SEM and discuss a few points about sample preparation and imaging.     

SEM electron channelling patterns as a technique for the characterization of ion-implantation damage

Electron channelling patterns (ECPs) formed in back-scattered images in the scanning electron microscope (SEM) have been used occasionally to confirm surface amorphization during ion implantation. In order to place such observations on a more quantitative basis, the study reported here has explored the variation of ECP appearance with both specimen damage levels (and thus subsurface structures) and SEM accelerating voltage (i.e. sampled depth). Polished and annealed (0001) single crystal sapphire discs were implanted to various damage levels up to both subsurface and full surface amorphization. Damage levels were measured independently by Rutherford back-scattering (RBS). Selected-area ECPs were obtained in a Jeol-840 electron microscope operating over the range 5–40 kV in 5-kV steps. Progressive ECP degradation—in terms of high-order line disappearance—was observed with increasing dose, culminating in total pattern loss when full surface amorphization occurred. However, ECP information could still be obtained from the damaged near-surface material even when a subsurface amorphous layer was present, thus demonstrating the shallow retrieval depth of information from the ECP technique. Indeed, because the spatial distribution of damage from ion implantation is both calculable and measurable, these experiments have also allowed us, for the first time, to explore and demonstrate the shallow sample depths from which the majority of ECP contrast originates (< 150 nm in sapphire at an accelerating voltage of 35 kV), even when the beam penetration is considerable by comparison (～ 5 μm). Furthermore, the way in which this sampled depth varies with SEM accelerating voltage is both demonstrated and shown to be a powerful diagnostic technique for studying the distribution of near-surface structural damage.     

扫描电镜的不同含水量植物叶片样品的处理及观察方法研究

为探讨不同含水量植物叶片进行扫描电镜观察的最合适的样品处理方法,及不同电子束加速电压对扫描电镜图像清晰度的影响,对不同含水量叶片进行了比较性研究。并通过理论分析得出：含水量小于75％的植物叶片可不经处理,直接进行观察,大于79％的叶片必须经过样品制备后观察;5—15KV加速电压时获得的图像最清晰。     

Imaging colloidal gold labels in LVSEM

On biological samples, the topographic imaging capabilities of the new generation of scanning electron microscopes (SEM) (those having both field-emission guns and low aberration lenses) rival those of the replica techniques. In addition, they permit the localization of specific molecules on the sample surface using one of several labeling techniques utilizing heavy metal colloids. Normally, colloidal gold can be detected in the SEM both by the secondary electron signal (shape) and by the backscattered electron signal (BSE, Z-contrast). The new instruments seem to produce their best topographic images using low-beam voltage (1–5 kV) where topographic contrast is higher and the required thickness of the metal coating is less (Haggis and Pawley 1988, Ris and Pawley 1988). Although the detection of backscattered electrons is more difficult at low-beam voltage, we are able to show here that the secondary electron (SE) signal produced with a 2–5-kV beam permits the unambiguous detection of gold particles as small as 5 nm on carbon-coated specimens while a 1-kV beam produces a high-quality topographic image of the same sample.     

Effects of interaction volume on X‐ray line‐scans across an ultrasonically consolidated aluminum/copper interface

Diffusion as a bonding mechanism for ultrasonic consolidation of metals is widely debated due to the short weld times and low processing temperatures. To quantify interdiffusion coefficients, X‐ray energy dispersive spectroscopy (XEDS) line‐scans were performed across an Al–Cu interface using the Scanning Electron Microscope (SEM) with accelerating voltages ranging from 6 to 22 KeV in increments of 2 KeV and a step size of 0.05 microns. Higher accelerating voltages resulted in broader concentration profiles, indicating higher apparent interdiffusion coefficients when scanned at the same location of the same sample. This error due to the interaction volume interference was quantified using Monte Carlo simulations. It was found that an accelerating voltage of 22 KeV and diffusion distance less than 5 microns resulted in at least 50% error. Even at a smaller accelerating voltage of 6 KeV, the percent error in calculation of the interdiffusion coefficient for a diffusion distance of 0.5 microns is expected to be 15–20%. An approximate diffusion distance and apparent interdiffusion coefficient for ultrasonically consolidated Al–Cu were 0.503 microns and 0.013 um²/s, respectively. In this study, a methodology is presented that allows one to estimate the error in the calculation of an interdiffusion coefficient from the accelerating voltage used and the diffusion distance measured by the SEM XEDS at that accelerating voltage. SCANNING 35:327‐335, 2013. © 2012 Wiley Periodicals, Inc.     

Traditional coating techniques in scanning electron microscopy compared to uncoated charge compensator technology: Looking at human blood fibrin networks with the ZEISS ULTRA Plus FEG‐SEM

Scanning electron microscopy (SEM) studies surface morphology. Biological material needs to be coated to render the material conductive, and gold coating is traditionally used, although other coating material like carbon and ruthenium vapors may also be used. With modern SEM technology (e.g., ZEISS ULTRA Plus FEG‐SEM), we are able to work at very low kilovolts and also view fine surface structure in much better detail than with previous older technology. Some machines also allow for the study of uncoated material, although this is usually not done with biological material. This study focuses on surface clarity by comparing gold, ruthenium vapor, and carbon coating techniques for biological material. Human fibrin networks are used as example. Uncoated specimens are also viewed with a ZEISS ULTRA Plus FEG‐SEM because of its unique nitrogen charge compensator, and here, the first micrographs for uncoated human fibrin networks versus carbon, gold, and ruthenium coating are shown. We conclude that gold coating for biological material is not preferable with the latest SEM machines, as this method forms gold islands on top of the biological material and therefore produces a false surface morphology. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

拭子喷雾电离-四极杆/静电场轨道阱高分辨质谱法快速筛查化妆品中19种防晒剂

本研究建立了拭子喷雾电离与四极杆/静电场轨道阱高分辨质谱(Q/Orbitrap HRMS)联用法快速筛查化妆品中19种防晒剂.化妆品样品无需前处理,直接用拭子蘸取样品,滴加30μL甲醇-乙腈混合溶剂(4:1,V/V),施加5.0 kV电压,通过拭子喷雾电离方式产生带电液滴.四极杆/静电场轨道阱高分辨质谱采用一级全扫描与...     

Perspective and limitations of cryo-electron microscopy

We investigated the possibility of vitrifying temperature-sensitive lipid phases as well as (small) biological specimens. From a suspension of unilamellar vesicles, prepared from dipalmitoyl-phosphatidylcholine (DPPC), thin aqueous films were formed at various temperatures. With cryo-electron microscopy vesicles were found to be smooth, rippled and faceted or faceted only, depending on the temperature of thin-film formation (318, 312 and 296 K respectively). The morphology and the electron diffraction patterns indicate that membranes can by physically fixed by vitrification in their high-temperature configuration and studied at low temperature by cryo-electron microscopy. This finding suggests that it may also be possible to preserve, in their original state, the more complex membrane systems found in living organisms by initiating rapid-cooling at a physiological temperature. This was explored by vitrification of thin films formed on specimen grids with (human) blood platelets adhering to collagen fibres. Low-temperature observation with an acceleration voltage of 120 kV revealed subcellular details. More details were observed when using higher accelerating voltages (200 and 300 kV) of the electron beam. The results presented in this paper illustrate the great potential of cryo-electron microscopy in the study of membrane dynamics, both in relatively simple model membrane systems and in more complex biological membrane systems.     

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 STEM mass measurement of biological macromolecules in a 300 kV TEM

For almost four decades, the scanning transmission electron microscope (STEM) has made significant contributions to structural biology by providing accurate determinations of the molecular masses of large protein assemblies that have arbitrary shapes and sizes. Nevertheless, STEM mass mapping has been implemented in very few laboratories, most of which have employed cold field‐emission gun (FEG) electron sources operating at acceleration voltages of 100 kV and lower. Here we show that a 300 kV commercial transmission electron microscope (TEM) equipped with a thermally assisted Shottky FEG can also provide accurate STEM mass measurements. Using the recently published database of elastic‐scattering cross sections from the National Institute of Standards and Technology, we show that the measured absolute mass values for tobacco mosaic virus and limpet hemocyanin didecamers agree with the known values to within better than 10%. Applying the established approach, whereby tobacco mosaic virus is added to a specimen as a calibration standard, we find that the measured molecular weight of the hemocyanin assemblies agrees with the known value to within 3%. This accuracy is achievable although only a very small fraction (∼0.002) of the incident probe current of 300 kV electrons is scattered onto the annular dark‐field STEM detector. FEG TEMs operating at intermediate voltages (200–400 kV) are becoming common tools for determining the structure of frozen hydrated protein assemblies. The ability to perform mass determination with the same instrument can provide important complementary information about the numbers of subunits comprising the protein assemblies whose structure is being studied.     

Freeze-fracture of 3T3 cells for high-resolution scanning electron microscopy

Triton-extracted, freeze-fractured 3T3 cells have been examined in the Hitachi S-900 field-emission SEM, after light platinum coating, at low beam voltage to evaluate the performance of the microscope under these conditions. For unstained material fixed in glutaraldehyde alone, high-resolution images can be obtained, at accelerating voltages of 1.5-5kV, after rotary deposition of platinum to an average thickness of 1.5-3nm. Comparisons are made between these results and those of studies by TEM of deep-etch replicas of similar material previously published.     

Tannin-osmium conductive staining of biological specimens for non-coated scanning electron microscopy

Osmication of biological specimens with a mixture of tannic acid, guanidine hydrochloride, arginine hydrochloride and glycine imparted sufficient electron conductivity to permit SEM observations of non-coated samples. No charging was noted at 25 kV acceleration voltage and 1 × 10^?10 A specimen current in rat kidney specimens. The foot-processes of the glomerular podocytes were clearly visible without metal coating. This tannin-osmium method even allowed block staining and enabled continuous observations during and after dissection in the SEM.     

A novel method for the discharge of electrostatic mirror formations in the scanning electron microscope

A method for the removal of electrostatic mirror formations in the scanning electron microscope (SEM) was conceptualised and implemented. This method utilises the controlled rampdown of the primary beam voltage to achieve mirror discharge. It is found that the new technique is effective in enabling imaging from high-beam voltages (20 kV) down to low voltages (1 kV) without the loss of imaging capability due to electrostatic mirrors. The entire discharge cycle could be performed in ≤ 4 min depending on sample and operating conditions. Hence, this technique compares favourably with the time needed to air the specimen chamber for mirror removal, but without the disadvantage of disrupting SEM operation. This method holds great promise in applications where the imaging of insulating samples at both high and low kV ranges are required.     

Voltage-dependent imaging of antimony on the GaAs(110) surface

Using a scanning tunnelling microscope, voltage-dependent imaging of antimony on the GaAs(110) surface has been performed. For negative sample voltages, the images reflect surface dangling bonds. Depending on the magnitude of the voltage, either one or both of the Sb atoms in the surface unit cell are seen. At positive voltage the density of surface-states is observed to be greatly reduced compared to negative voltages. The results are analysed within the context of a simple tight-binding model for a one-dimensional biatomic chain.     

High-power switches based on reversely switched-on dynistors for high-voltage pulse technologies

The results of comparative investigations of commercially produced reversely switched-on dynistors (RSDs) with an operating voltage of 2 kV and 76-mm-diameter structures are presented. The studies were performed in the mode of switching current pulses with an amplitude of 200 kA and a duration of 300 μs. The electric scheme of the power circuit of the generator of high-power high-voltage pulses with a switch on the basis of an assembly of RSDs is considered. RSD switches with an operating current of 250 kA and operating voltages of 12 and 24 kV are described. Some results of using RSD switches in high-voltage pulse technologies are presented.     

Current developments in high voltage electron microscopy

In respect of instrument design two main developments are taking place in high voltage electron microscopy: towards even higher operating voltages (3–5 MV) and towards higher resolving power at moderate voltages (250–600 kV). Applications of existing instruments (650 kV-1.2 MV) are still primarily in metallurgy, especially for radiation damage studies, but their usefulness for biological research is now being actively explored. Microchambers have been developed for observing specimens, both metallurgical and biological, in a controlled gaseous or liquid environment. The prospects for observing living material, except at low magnifications, remain very doubtful on account of ionization effects, but informative work with wet specimens should be possible.     

A small scanning tunnelling microscope with large scan range for biological studies

We have developed a scanning tunnelling microscope specially designed for biological applications presenting some new features: the scanner tube is mounted parallel to the surface of the sample which enables a high resolution optical microscope to be brought close to the sample when working in air or liquids. The maximum scan range is 5×20 μm with a vertical range of 20 μm and the total size of the system does not exceed 10×40 mm. The piezo-sensitivity of the scanner tube versus applied voltage was analysed by interferometry measurements and by using scanning tunnelling microscopes. We found a value for the piezoelectric constant d₁₃ of ?1·71 Å/V at low voltages (under a few volts) going up to ?2 Å/V for higher voltages. Large-scale images of a carbon grid showed a surprisingly good linearity of the scanner tube.