Imaging of immunolabeled membrane receptors in uncoated sem specimens

Epidermal growth factor receptors (EGFR) were labeled with 10 nm immunogold and examined on uncoated specimens of A431 human epidermoid carcinoma cells. A field emission gun and a high-sensitivity YAG ring detector were used to demonstrate the affinity labeling simultaneously in the secondary-electron (SE) and backscattered-electron (BSE) modes with a low accelerating voltage (V_o). At V_o=2kV, the SE and BSE signals were too weak to identify all markers, while at V_o=3–7 kV labeling was observed unambiguously in both the SE and BSE modes with smaller and higher working distances. Increasing the V_o to above 7 kV sometimes provokes instability of the specimens. A V_o of ? 10 kV produces charging artifacts in the SE image, but permits a BSE image of the gold markers providing additional topographic information. In conclusion, immunogold labeling can be used with good results for uncoated specimens.     

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.     

Recent developments and new strategies in scanning electron microscopy*

In addition to improvements in lateral resolution in scanning electron microscopy, recent developments of interest here concern extension of the incident beam energy, E₀, over two decades, from ≈ 20 keV to ≈ 0.1–0.5 keV and the possibility of changing the take-off emission, α, of detected secondary electrons. These two degrees of freedom for image acquisition permit a series of images of the same field of view of a specimen to be obtained, each image of the series differing from the others in some aspect. The origins of these differences are explored in detail and they are tentatively interpreted in terms of the change in the secondary electron emission yield δ vs. E₀, δ = f(E₀), and also of the change in δ vs. α, ∂δ/∂α. Various origins for the chemical contrast and topographic contrast have been identified. Illustrated by correlating a secondary electron image and a backscattered electron image, use of the scatter diagram technique facilitates image comparison. The difference between the lateral resolution and the size of the minimum detectable detail is outlined to avoid possible errors in nanometrology. Some aspects related to charging are also considered and possible causes of contrast reversal are suggested. Finally, the suggested strategy consists of the acquisition of various images of a given specimen by changing one parameter: primary beam energy and take-off angle for conductive specimens; working distance or beam intensity for high-resolution experiments; scanning frequency for insulating specimens.     

Standards for quantification of elements in the otolithic membrane by electron probe X-ray microanalysis: Calibration curves and electron beam sensitivity

An absolute quantitative standardization technique has been developed to measure Ca and K weight fractions (WF) in the otolithic membrane of the saccule and utricle by scanning electron microscopy and electron probe X-ray analysis using the peak-to-background (P/B) ratio method. Microcrystalline salt standards were used to calibrate Ca and K Kα P/B or Y = (P/B) · Z²/A (Z = atomic number; A = atomic weight) against WF at 10, 15, 20 and 25 kV accelerating voltage. The effect of voltage on the calibration, plotting the coefficient of correlation (r) as a function of voltage, was not dependent on the voltage in the range 10–25 kV for Ca standards. K standards were also independent when P/B was corrected for Z²/A. Background counts in the otoconia (B_o) were obtained at 5, 25, 50, 100, 200 and 500 s and used to test the electron beam sensitivity of saccular and utricular otoconia. B_o was not dependent on the spectra acquisition time, with the exception of B_o under Kα K peak in the saccule at 10 kV. Ca and K WF were determined at 10, 15, 20 and 25 kV in the saccule and utricle, showing similar values regardless of the voltage used. This method of calibration offers several advantages, such as stability, homogeneity, known composition of the standards, high reproducibility at different voltages even without Z²/A correction and the similarity between the otoconia and crystal standards. We recommend the application of this method for other elements and biomineral systems.     

Transmission electron microscopy at 20 kV for imaging and spectroscopy

The electron optical performance of a transmission electron microscope (TEM) is characterized for direct spatial imaging and spectroscopy using electrons with energies as low as 20 keV. The highly stable instrument is equipped with an electrostatic monochromator and a C_S-corrector. At 20 kV it shows high image contrast even for single-layer graphene with a lattice transfer of 213 pm (tilted illumination). For 4 nm thick Si, the 200 reflections (271.5 pm) were directly transferred (axial illumination). We show at 20 kV that radiation-sensitive fullerenes (C₆₀) within a carbon nanotube container withstand an about two orders of magnitude higher electron dose than at 80 kV. In spectroscopy mode, the monochromated low-energy electron beam enables the acquisition of EELS spectra up to very high energy losses with exceptionally low background noise. Using Si and Ge, we show that 20 kV TEM allows the determination of dielectric properties and narrow band gaps, which were not accessible by TEM so far. These very first results demonstrate that low kV TEM is an exciting new tool for determination of structural and electronic properties of different types of nano-materials.     

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.     

Dynamical electron diffraction simulation for non‐orthogonal crystal system by a revised real space method

In the transmission electron microscopy, a revised real space (RRS) method has been confirmed to be a more accurate dynamical electron diffraction simulation method for low‐energy electron diffraction than the conventional multislice method (CMS). However, the RRS method can be only used to calculate the dynamical electron diffraction of orthogonal crystal system. In this work, the expression of the RRS method for non‐orthogonal crystal system is derived. By taking Na₂Ti₃O₇ and Si as examples, the correctness of the derived RRS formula for non‐orthogonal crystal system is confirmed by testing the coincidence of numerical results of both sides of Schrödinger equation; moreover, the difference between the RRS method and the CMS for non‐orthogonal crystal system is compared at the accelerating voltage range from 40 to 10 kV. Our results show that the CMS method is almost the same as the RRS method for the accelerating voltage above 40 kV. However, when the accelerating voltage is further lowered to 20 kV or below, the CMS method introduces significant errors, not only for the higher‐order Laue zone diffractions, but also for zero‐order Laue zone. These indicate that the RRS method for non‐orthogonal crystal system is necessary to be used for more accurate dynamical simulation when the accelerating voltage is low. Furthermore, the reason for the increase of differences between those diffraction patterns calculated by the RRS method and the CMS method with the decrease of the accelerating voltage is discussed.     

Primary considerations for image enhancement in high-pressure scanning electron microscopy

The mechanisms of electron beam scattering are examined to evaluate its effect on contrast and resolution in high-pressure scanning electron microscopy (SEM) techniques reported in the literature, such as moist-environment ambient-temperature SEM (MEATSEM) or environmental SEM (ESEM). The elastic and inelastic scattering cross-sections for nitrogen are calculated in the energy range 5–25 keV. The results for nitrogen are verified by measuring the ionization efficiency, and measurements are also made for water vapour. The effect of the scattered beam on the image contrast was assessed and checked experimentally for a step contrast function at 20 kV beam voltage. A considerable degree of beam scattering can be tolerated in high-pressure SEM operation without a significant degradation in resolution. The image formation and detection techniques in high-pressure SEM are considered in detail in the accompanying paper.     

The cutting of ultrathin sections with the thickness less than 20 nm from biological specimens embedded in resin blocks

Low voltage electron microscopes working in transmission mode, like LVEM5 (Delong Instruments, Czech Republic) working at accelerating voltage 5 kV or scanning electron microscope working in transmission mode with accelerating voltage below 1 kV, require ultrathin sections with the thickness below 20 nm. Decreasing of the primary electron energy leads to enhancement of image contrast, which is especially useful in the case of biological samples composed of elements with low atomic numbers. As a result treatments with heavy metals, like post‐fixation with osmium tetroxide or ultrathin section staining, can by omitted. The disadvantage is reduced penetration ability of incident electrons influencing the usable thickness of the specimen resulting in the need of ultrathin sections of under 20 nm thickness. In this study we want to answer basic questions concerning the cutting of extremely ultrathin sections: Is it possible routinely and reproducibly to cut extremely thin sections of biological specimens embedded in commonly used resins with contemporary ultramicrotome techniques and under what conditions? Microsc. Res. Tech. 79:512–517, 2016. © 2016 Wiley Periodicals, Inc.     

Enhanced angular current intensity from Schottky emitters

Even though the Schottky emitter is a high‐brightness source of choice for electron beam systems, its angular current intensity is substantially lower than that of thermionic cathodes, rendering the emitter impractical for applications that require high beam current. In this study, two strategies were attempted to enhance its angular intensity, and their experimental results are reported. The first scheme is to employ a higher extraction field for increasing the brightness. However, the tip shape transformation was found to induce undesirably elevated emission from the facet edges at high fields. The second scheme exploits the fact that the angular intensity is proportional to the square of the electron gun focal length [ Fujita, S. & Shimoyama, H. (2005) Theory of cathode trajectory characterization by canonical mapping transformation. J. Electron Microsc. 54 , 331–343], which can be increased by scaling‐up the emitter tip radius. A high angular current intensity (J_Ω∼ 1.5 mA sr⁻¹) was obtained from a scaled‐up emitter. Preliminary performance tests were conducted on an electron probe‐forming column by substituting the new emitter for the original tungsten filament gun. The beam current up to a few microamperes was achieved with submicron spatial resolution.     

A systematic investigation of the charging effect in scanning electron microscopy for metal nanostructures on insulating substrates

Scanning electron microscopy is perhaps the most important method for investigating and characterizing nanostructures. A well‐known challenge in scanning electron microscopy is the investigation of insulating materials. As insulating materials do not provide a path to ground they accumulate charge, evident as image drift and image distortions. In previous work, we have seen that sample charging in arrays of metal nanoparticles on glass substrates leads to a shrinkage effect, resulting in a measurement error in the nanoparticle dimension of up to 15% at 10 kV and a probe current of 80 ± 10 pA. In order to investigate this effect in detail, we have fabricated metal nanostructures on insulating borosilicate glass using electron beam lithography. Electron beam lithography allows us to tailor the design of our metal nanostructures and the area coverage. The measurements are carried out using two commonly available secondary electron detectors in scanning electron microscopes, namely, an InLens‐ and an Everhart–Thornley detector. We identify and discriminate several contributions to the effect by varying microscope settings, including the size of the aperture, the beam current, the working distance and the acceleration voltage. We image metal nanostructures of various sizes and geometries, investigating the influence of scan‐direction of the electron beam and secondary electron detector used for imaging. The relative measurement error, which we measure as high as 20% for some settings, is found to depend on the acceleration voltage and the type of secondary electron detector used for imaging. In particular, the Everhart–Thornley detectors lower sensitivity to SE₁ electrons increase the magnitude of the shrinkage of up to 10% relative to the InLens measurements. Finally, a method for estimating charge balance in insulating samples is presented.     

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.     

About the role of the various types of secondary electrons (SE1; SE2; SE3) on the performance of LVSEM

The relative weight, δ_Β, of the yield of secondary electrons, SE₂, induced by the backscattered electrons, BSE, with respect to that, δ_P, of secondary electrons, SE₁, induced by the primary electrons, PE, is deduced from simple theoretical considerations. At primary energies E₀ larger than E_M (where the total SE yield δ = δ_P + δ_B is maximum), the dominant role of the backscattering events is established. It is illustrated in SEM by a direct comparison of the contrast between SE images and BSE images obtained at E₀ ～ 5 keV and E₀ ～ 15 keV on a stratified specimen. At energies E₀ less than E_M, the dominant role of SE₁ electrons with respect to SE₂ (and SE₃) is established. It is illustrated by the better practical resolution of diamond images obtained with an in‐lens detection in low voltage SEM E₀ ～ 0.2–1 keV range compared with that obtained with a lateral detector. The present contribution illustrates the improved performance of LVSEM in terms of contrast and of practical resolution as well as the importance of variable voltage methods for subsurface imaging. The common opinion that the practical lateral resolution is given by the incident spot diameter is also reconsidered in LVSEM.     

Rationale for the application of thin,continuous metal films in high magnification electron microscopy

Various metal films of different thicknesses were deposited on to a particle test specimen and their effects on topographic contrast generation and specimen preservation were determined. Tobacco mosaic virus adsorbed on to thin carbon supports or silicon chips was imaged in TEM or high resolution SE-I SEM at a magnification of 350,000×. Tantalum films of 1–2 nm (average mass) thickness produced best contrasts and prevented volume loss of the particles from electron beam damage. Excessively thick films of 5–10 nm thickness blanketed fine structures and caused severe volume losses. Discontinuous 2 nm thick films of gold or platinum decorated the surfaces, caused a loss in topographic contrasts and induced very high volume losses. Thin continuous metal films were necessary to generate high topographic contrast and to prevent volume loss from beam damage by providing sufficient mechanical stability for small topographic features and increased thermal conductivity of the specimen surface.     

Contrast analysis of cryo-images of n-paraffin recorded at 400***kV out to 2·1 Å resolution

N-Paraffin was used as a test specimen for evaluating the relative merits of 400-kV versus 100-kV electron microscopy in recording data for electron crystallographic analysis of beam-sensitive materials. The parameter used for comparison, the relative contrast R, is the ratio of amplitudes from the computed Fourier transform of images and amplitudes from an electron diffraction pattern from the same crystal. R will thus be a measure of the contrast from an experimental image relative to that of a perfect image. Electron diffraction patterns and bright-field images were recorded at 400 kV at a specimen temperature of ?167°C. Using the flood-beam imaging technique the best R-value is 0 08 for all reflections in the resolution zone from 4 to 3 Å. This value is equivalent to that found at 100 kV. In the resolution zone from 3 to 2 Å we have found R — 0 02. Using the spot-scan imaging technique, on the other hand, R was measured to be 0·42 for the reflections between 4- and 3-Å resolution. This amount of relative contrast is 1·7 times that observed at 100 kV. Reflections at 3–2 Å displayed an R-value of 0 05. Besides obtaining higher R-values when applying the spot-scan imaging technique at 400 kV, we observe a higher yield of images with isotropic diffraction and/or higher resolution reflections. Various contrast-attenuating factors, including the modulation transfer function of the photographic film and the cryo-holder, envelope functions for spatial and temporal coherence and lens and high-tension instabilities, the contrast transfer function and lastly the radiation damage effects, have been considered in interpreting the observed image contrast. Overall, use of 400 kV in combination with spot-scan does offer important improvements in contrast levels, which can be very useful in determining the three-dimensional structure from protein crystals.     

Broad beam sources of fast molecules with segmented cold cathodes and emissive grids

Experimental study of fast neutral atom and molecule beam sources with rectangular and circular cross-section of the beam up to 0.8 m² is carried out and the study results are presented. The fast particles are produced as a result of charge exchange collisions between gas molecules and ions accelerated by potential drop between the plasma emitter of the beam source and the secondary plasma inside the processing vacuum chamber. As the emitter is used a glow discharge plasma, whose electrons are confined in an electrostatic trap formed by a cold hollow cathode and an emissive grid, which is negative both to the cathode and to the chamber. In order to prevent from breakdowns between the emitter and the cathode at a current in the cathode circuit up to 10 A as well as between the emitter and the grid at a voltage between them up to 10 kV the cathode and the grid are composed of isolated from each other segments, which are connected to power supplies through resistors. When resistance of the resistorR > U/I ₀, where U is the power supply voltage and I ₀ is the minimal current of stable vacuum arc for a given segment material, then transition from the glow discharge to the steady-state vacuum arc is totally excluded in spite of numerous breakdowns of microsecond duration due to contamination of the source electrodes during its operation with dielectric films and other stimulants of the arc.     

The imaging mechanism of single-walled carbon nanotubes on Si/SiO₂ wafer in scanning electron microscopy

Scanning electron microscopy imaging of both suspended single‐walled carbon nanotubes (SWNTs) and contacted SWNTs with Si/SiO₂ substrate has been studied in this paper. The voltage contrast has been investigated by supplying external electric field around the samples. The results show that the image contrast of SWNTs attributes to both voltage contrast from the area surrounding SWNTs (tens of nanometres in both sides of the SWNTs) and electron beam induced emission from SWNTs themselves under low primary beam energy. Under high primary beam energy, however, EBIE dominates the image contrast due to the fact that the voltage contrast caused by implanted charges of the SiO₂ layer is weakened. Imaging under the primary beam energy lower than 1 keV offers widened diameter of SWNTs, which promises that the SWNTs are observable at very low magnification (lower than 100×). At a larger magnification, however, imaging under the primary beam energy higher than 10 keV can display more realistic images of the SWNTs. In addition, an appropriate external electric field can improve the images.     

A mirror electron microscope for surface analysis

The principle of mirror microscopy has been adapted to provide a relatively low resolution surface microscope (<1000 ×), a large transfer width low energy electron diffractometer and a photoelectron analyser in k_|| space. A focused electron beam of ? 10 kV is decelerated through a Johansson lens, reflected in front of the sample and reaccelerated back through the lens to produce an electron image over a field of view of a few microns. The image can be interpreted as a micrograph of work function variations on the surface if other effects (geometry, magnetic field) are uniform. In the LEED mode, diffracted beams virtually retain their positions on the screen over the whole impact energy range used (0.160 V). Secondary electrons are preferentially focused around the lens-gun electro-optic axis, thus effectively filtering them out from the diffraction pattern. The design has an inherently large coherence length, of up to 10⁴ Å. Photoelectrons can similarly be imaged in k_|| space on the detector plane. The addition of energy filtering at the screen allows the two-dimensional Fermi surface to be imaged.     

Resolution in low voltage scanning electron microscopy

As the energy of an electron beam is reduced, the range falls and the secondary electron yield rises. A low voltage scanning electron microscope can therefore, in principle, examine without damage or charging samples such as insulators, dielectrics or beam sensitive materials. This paper investigates the way in which the choice of beam energy affects the spatial resolution of a secondary electron image. It is shown that for samples which are thin compared to the electron range, the edge resolution and contrast in the image improve with increasing beam energy. In samples that are thicker than the electron range, the resolution can be optimized at either high or low energies, but low energy operation will produce images of higher contrast. At an energy of 2 keV or less beam interaction limited resolutions of the order of 3 nm should be possible.     

A high-voltage power supply of the diagnostic neutral beam injector of the Alcator-Cmod tokamak

A diagnostic neutral beam injector for ensuring the active spectroscopic diagnostics of plasma parameters in the Alcator-Cmod tokamak (Massachusetts Institute of Technology (MIT), Boston, United States) is designed and manufactured at the Institute of Nuclear Physics (Novosibirsk). The energy of fast atoms of the diagnostic injector is determined by the output voltage level of the high-voltage power supply and can vary from 20 to 55 keV. The ion source of the diagnostic neutral beam injector generates proton beams with an equivalent current of up to 7 A. The accelerated protons after the neutralization on a gas target produce streams of neutral particles—fast atoms with an equivalent current of up to 4 A. The diagnostic neutral beam injector is capable of producing 100% energy-modulated fast hydrogen atomic beams, and this is ensured by operation of the high-voltage power supply under the corresponding law. The high-voltage power supply is based on modules consisting of high-frequency transformers and diode rectifiers placed in a sealed tank filled with insulating gas SF₆. The output voltage is smoothly regulated from 20 to 55 kV by IGBT inverters with a pulse-width control energizing the primary windings of the step-up high-frequency transformers. The high-voltage power supply allows the multiple-breakdown operation mode of the load with voltage recovering as the specified time passes after the breakdown. The rated power of the high-voltage power supply is 450 kW. A functional diagram and design of the high-voltage power supply are given.