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.     

Recent advances and applications of high resolution electron microscopy

This review covers several broad areas: firstly, recent developments in HREM instrumentation, and then novel techniques for imaging are discussed, including some of the problems of image interpretation. Applications of HREM techniques to a wide range of materials problems are described and include solid state chemistry, ceramics, semiconductors, metals and natural diamonds. The next generation of high resolution microscopes will operate in the 300–400 kV range, have low C_s objective lenses, and have sufficiently good vacuum to allow the combined use of CBED and EELS facilities with imaging in the sub-2 Å range. Microprocessor control of instrumental parameters such as astigmatism, alignment and defocus are seen as an important way forward in achieving the optimum performance of these instruments.     

Low voltage scanning electron microscopy

The scanning electron microscope (SEM) is usually operated with a beam voltage, V₀, in the range of 10–30 kV, even though many early workers had suggested the use of lower voltages to increase topographic contrast and to reduce specimen charging and beam damage. The chief reason for this contradiction is poor instrumental performance when V₀=1–3 kV, The problems include low source brightness, greater defocusing due to chromatic aberration greater sensitivity to stray fields, and difficulty in collecting the secondary electron signal. Responding to the needs of the semiconductor industry, which uses low V₀ to reduce beam damage, considerable efforts have been made to overcome these problems. The resulting equipment has greatly improved performance at low kV and substantially removes the practical deterrents to operation in this mode. This paper reviews the advantages of low voltage operation, recent progress in instrumentation and describes a prototype instrument designed and built for optimum performance at 1 kV. Other limitations to high resolution topographic imaging such as surface contamination, the de-localized nature of the inelastic scattering event and radiation damage are also discussed.     

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.     

Prerequisites for a Cc/Cs-corrected ultrahigh-resolution TEM

After the introduction of a corrector to compensate for the spherical aberration of a TEM and the acceptance of this new instrumentation for high-resolution CTEM (conventional transmission electron microscope) and STEM (scanning transmission electron microscope) by the electron microscopy community, a demand for even higher resolution far below 1A has emerged. As a consequence several projects around the world have been launched to make these new instruments available and to further push the resolution limits down toward fractions of 1A. For this purpose the so-called TEAM (transmission electron aberration-corrected microscope) has been initiated and is currently under development. With the present paper we give a detailed assessment of the stability required for the base instrument and the electric stability, the manufacturing precision, and feasible semi-automatic alignment procedures for a novel C(c)/C(s)-corrector in order to achieve aberration-free imaging with an information limit of 0.5A at an acceleration voltage of 200 kV according to the goals for the first TEAM instrument. This new aberration corrector, a so-called Achroplanat, in combination with a very stable high-resolution TEM leads to an imaging device with unprecedented resolving power and imaging properties.     

On the benefit of the negative-spherical-aberration imaging technique for quantitative HRTEM

Employing an aberration corrector in a high-resolution transmission electron microscope, the spherical aberration C_S can be tuned to negative values, resulting in a novel imaging technique, which is called the negative C_S imaging (NCSI) technique. The image contrast obtained with the NCSI technique is compared quantitatively with the image contrast formed with the traditional positive C_S imaging (PCSI) technique. For the case of thin objects negative C_S images are superior to positive C_S images concerning the magnitude of the obtained contrast, which is due to constructive rather than destructive superposition of fundamental contrast contributions. As a consequence, the image signal obtained with a negative spherical aberration is significantly more robust against noise caused by amorphous surface layers, resulting in a measurement precision of atomic positions which is by a factor of 2–3 better at an identical noise level. The quantitative comparison of the two alternative C_S-corrected imaging modes shows that the NCSI mode yields significantly more precise results in quantitative high-resolution transmission electron microscopy of thin objects than the traditional PCSI mode.     

Static and dynamic experiments in cryo-electron microscopy: comparative observations using high-vacuum, low-voltage and low-vacuum SEM

We carried out a unique comparative study between three modes of cryo‐scanning electron imaging: high‐vacuum, low‐voltage and low‐vacuum, using ice cream as a model system. Specimens were investigated both with and without a conductive coating (Au/Pd) and at temperatures for which ice either remains fully frozen (< ?110 °C) or undergoes sublimation (?110 to ?90 °C). At high magnification, high‐vacuum imaging of coated specimens gave the best results for ‘static’ specimens (i.e. containing fully frozen ice). Low voltages, such as 1 kV, could be used for imaging uncoated specimens at high vacuum, although slight ‘classical’ charging artefacts remained an issue, and the reduced electron beam penetration tended to decrease the definition between different microstructural features. However, this mode was useful for observing in situ sublimation from uncoated specimens. Low‐vacuum mode, involving small partial pressures of nitrogen gas, was particularly suited to in situ sublimation work: when sublimation was carried out in low vacuum in the absence of an anti‐contaminator plate, sublimation rates were significantly reduced. This is attributed to a small partial pressure of sublimated water vapour remaining near the specimen surface, enhancing thermodynamic stability.     

An optical configuration for fastidious STEM detector calibration and the effect of the objective‐lens pre‐field

In the scanning transmission electron microscope, an accurate knowledge of detector collection angles is paramount in order to quantify signals on an absolute scale. Here we present an optical configuration designed for the accurate measurement of collection angles for both image‐detectors and energy‐loss spectrometers. By deflecting a parallel electron beam, carefully calibrated using a diffraction pattern from a known material, we can directly observe the projection‐distortion in the post‐specimen lenses of probe‐corrected instruments, the 3‐fold caustic when an image‐corrector is fitted, and any misalignment of imaging detectors or spectrometer apertures. We also discuss for the first time, the effect that higher‐order aberrations in the objective‐lens pre‐field has on such an angle‐based detector mapping procedure.     

Direct imaging of local atomic ordering in a Pd-Ni-P bulk metallic glass using Cs-corrected transmission electron microscopy

In amorphous alloys, crystalline atomic clusters as small as 1-2 nm are frequently observed as local lattice fringe images by high-resolution electron microscopy (HREM). These clusters can be understood as local structures of amorphous alloys corresponding to "medium-range-order (MRO)". The MRO structure can be observed only under suitable defocusing conditions of the objective lens in HREM. A clear imaging of the MRO structure is difficult in conventional TEMs, mainly due to the delocalization of the image, caused mainly by the spherical aberration of the objective lens and eventually by the chosen defocus. In the present study, we have examined MRO in a Pd-based bulk metallic glass (Pd(40)Ni(40)P(20)) using a high-resolution TEM (acceleration voltage 200 kV) fitted with a spherical aberration constant corrector (Cs corrector) for aberration correction. We found that when Cs was close to zero and defocus values were near the Gaussian focus, MRO regions with an FCC-Pd structure could be clearly observed with a low image disturbance. Under these conditions, the phase-contrast transfer function was understood to act as an ideal filter function, which distinctly selects specific lattice periods of the FCC-Pd clusters. The obtained atomic images of the glass structure including the FCC-Pd clusters are in good agreement with those expected from image simulation according to our amorphous structure model. In this study, we have demonstrated that the Cs-corrected HREM is a powerful tool to directly image locally ordered structures in metallic glasses.     

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.     

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.     

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.     

In-focus electron microscopy of frozen-hydrated biological samples with a Boersch phase plate

We report the implementation of an electrostatic Einzel lens (Boersch) phase plate in a prototype transmission electron microscope dedicated to aberration-corrected cryo-EM. The combination of phase plate, C_s corrector and Diffraction Magnification Unit (DMU) as a new electron-optical element ensures minimal information loss due to obstruction by the phase plate and enables in-focus phase contrast imaging of large macromolecular assemblies. As no defocussing is necessary and the spherical aberration is corrected, maximal, non-oscillating phase contrast transfer can be achieved up to the information limit of the instrument. A microchip produced by a scalable micro-fabrication process has 10 phase plates, which are positioned in a conjugate, magnified diffraction plane generated by the DMU. Phase plates remained fully functional for weeks or months. The large distance between phase plate and the cryo sample permits the use of an effective anti-contaminator, resulting in ice contamination rates of <0.6 nm/h at the specimen. Maximal in-focus phase contrast was obtained by applying voltages between 80 and 700 mV to the phase plate electrode. The phase plate allows for in-focus imaging of biological objects with a signal-to-noise of 5-10 at a resolution of 2-3 nm, as demonstrated for frozen-hydrated virus particles and purple membrane at liquid-nitrogen temperature.     

Alkyloxy‐s‐triazine derivatives with and without fluorine‐containing substituents as lubricants for steel–steel contact

Three alkyloxy‐s‐triazine derivatives were synthesized and their tribological properties as lubricants for steel–steel contact were evaluated using an Optimol SRV tester at 20°C and 100°C. Their thermal stabilities were also investigated by thermogravimetric analysis. The results show that the three alkyloxy‐s‐triazine lubricants have good thermal stability. Moreover, 2,4,6‐tris(1,1,5‐tri‐H‐octafluoropentyloxy)‐1,3,5‐s‐triazine (FPOT) possesses the best anti‐wear property and good load‐carrying capacity both at 20°C and 100°C. At 20°C the anti‐wear effectiveness of 2,4,6‐tris(n‐pentyloxy)‐1,3,5‐s‐triazine (POT) is the worst, while at 100°C that of the 1,1,5‐tri‐H‐octafluoropentyloxy and/or 1,1,7‐tri‐H‐dodecafluoroheptyloxy tri‐substituted s‐triazine mixture (FMOT) is the worst. In addition, the friction‐reducing properties of the two fluoroalkyloxy‐s‐triazines, FPOT and FMOT, are not as good as those of the non‐fluorine‐containing alkyloxy‐s‐triazine POT. Scanning electron spectroscopy with an energy dispersive analyzer of X‐ray and X‐ray photoelectron spectroscopy analyses of the worn surface indicate that during the rubbing process, tribochemical reactions occur between the lubricants and the metal surface to generate a complex boundary lubrication film comprised of FeF₂, Fe(OH)₂, organofluorine and organonitrogen compounds. Copyright © 2006 John Wiley & Sons, Ltd.     

Multichannel wide‐field microscopic FRET imaging based on simultaneous spectral unmixing of excitation and emission spectra

Simultaneous spectral unmixing of excitation and emission spectra (ExEm unmixing) has inherent ability resolving spectral crosstalks, two key issues of quantitative fluorescence resonance energy transfer (FRET) measurement, of both the excitation and emission spectra between donor and acceptor without additional corrections. We here set up a filter‐based multichannel wide‐field microscope for ExEm unmixing‐based FRET imaging (m‐ExEm‐spFRET) containing a constant system correction factor (f_sc) for a stable system. We performed m‐ExEm‐spFRET with four‐ and two‐wavelength excitation respectively on our system to quantitatively image single living cells expressing FRET tandem constructs, and obtained accurate FRET efficiency (E) and concentration ratio of acceptor to donor (R_C). We also performed m‐ExEm‐spFRET imaging for single living cells coexpressing CFP‐Bax and YFP‐Bax, and found that the E values were about 0 for control cells and about 28% for staurosporin‐treated cells when R_C were larger than 1, indicating that staurosporin induced significant oligomerisation.     

Higher-order aberration corrector for an image-forming system in a transmission electron microscope

We developed a new electron optical system with three dodecapoles to compensate for spherical aberration and six-fold astigmatism, which generally remains in a two-hexapole type corrector. In this study, we applied the corrector for image-forming system in transmission electron microscope. Compensation for higher-order aberration was demonstrated through a diffractogram tableau using a triple three-fold astigmatism field system, which was then compared with a double hexapole field system. Using this electron optical system, six-fold astigmatism was measured to be less than 0.1 mm at an acceleration voltage of 60 kV, showing that the system successfully compensated for six-fold astigmatism.     

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.     

Bright-field TEM imaging of single molecules: Dream or near future?

We examine the suitability of spherical aberration (C(S))-corrected (CS) and uncorrected (UC) transmission electron microscopes (TEM) for conventional bright-field imaging of radiation-sensitive materials. We have chosen an individual molecule suspended in vacuum as a hypothetical example of a well-defined radiation-sensitive sample. We find that for this particular sample, CS instruments provide about 30% improvement over an UC instrument in terms of signal/noise ratio per unit electron dose at 300kV. The lowest imaging doses can be achieved in CS instruments equipped with high-brightness electron source operated at low incident electron energies. Our calculations suggest that it may be possible to image individual, iodine- or bromine-substituted organic molecules in bright-field mode, at doses lower than the accepted values for radiation damage of aromatic molecules.     

Quantitative FRET measurement using emission‐spectral unmixing with independent excitation crosstalk correction

Quantification of fluorescence resonance energy transfer (FRET) needs at least two external samples, an acceptor‐only reference and a linked FRET reference, to calibrate fluorescence signal. Furthermore, all measurements for references and FRET samples must be performed under the same instrumental conditions. Based on a novel notion to predetermine the molar extinction coefficient ratio (R_C) of acceptor‐to‐donor for the correction of acceptor excitation crosstalk, we present here a robust and independent emission‐spectral unmixing FRET methodology, Iem‐spFRET, which can simultaneously measure the E and R_C of FRET sample without any external references, such that Iem‐spFRET circumvents the rigorous restriction of keeping the same imaging conditions for all FRET experiments and thus can be used for the direct measurement of FRET sample. We validate Iem‐spFRET by measuring the absolute E and R_C values of standard constructs with different acceptor‐to‐donor stoichiometry expressed in living cells. Our results demonstrate that Iem‐spFRET is a simple and powerful tool for real‐time monitoring the dynamic intermolecular interaction within single living cells.     

Determining the Surface Electrostatic Potential Ψ s of a Dielectric-Bordering Semiconductor Using the Method of Ψ s "/Ψ s Diagrams

A quantitative approach to determining the integration constant _s0(V _{g 0}) in an expression relating the semiconductor surface potential _s to the voltage V _g applied to the metal–insulator–semiconductor (MIS) structure and its quasi-static capacitance–voltage characteristic C _v(V _g) (normalized to the dielectric capacitance) is described. The method is based on the analysis of experimental functions _s ^"( _s ), where _s ^" = d _s /dV _g, and the same functions calculated for an ideal MIS structure. The obtained function _s (V _g) is a rather exact and complete characteristic of electron properties of the MIS-structure phase boundary (the integrated interface state density, flat-band voltage V _FB, sign and density of the dielectric fixed charge, and variations of these parameters under the action of various factors). Using the example of a particular n-Si MIS structure, it is shown that the method of _s ^"/ _s diagrams ensures a noticeable (up to 0.93 eV) widening of the Si gap sounding region and observation (by the value of the V _FB shift) of very small ( 1 × 10⁷ cm^–2) variations in the charge density at the Si/SiO₂ phase boundary.