Low-voltage Electron Microscopy Research Articles

Urany-Less Low Voltage Transmission Electron Microscopy: A Powerful Tool for Ultrastructural Studying of Cyanobacterial Cells.

Sample preparation protocols for conventional high voltage transmission electron microscopy (TEM) heavily rely on the usage of staining agents containing various heavy metals, most commonly uranyl acetate and lead citrate. However high toxicity, rising legal regulations, and problematic waste disposal of uranyl acetate have increased calls for the reduction or even complete replacement of this staining agent. One of the strategies for uranyless imaging is the employment of low-voltage transmission electron microscopy. To investigate the influence of different imaging and staining strategies on the final image of cyanobacterial cells, samples stained by uranyl acetate with lead citrate, as well as unstained samples, were observed using TEM and accelerating voltages of 200 kV or 25 kV. Moreover, to examine the possibilities of reducing chromatic aberration, which often causes issues when imaging using electrons of lower energies, samples were also imaged using a scanning transmission electron microscopy at 15 kV accelerating voltages. The results of this study demonstrate that low-voltage electron microscopy offers great potential for uranyless electron microscopy.

Assessment Of Left and Right Ventricular Functions In Overweight and Obese Females by Different Echo-Doppler Modalities

Abstract Aim of work Assessment of left and right ventricular functions in overweight and obese females using different echo-Doppler modalities. Methods This study included 32 obese females (group I), 26 overweight females (group II) -both of them have no chronic illnesses- in addition to 25 healthy females (group III) as a control group. All of them had hsCRP level measured. The following parameters were measured using 2D echocardiography: LA, Ao, LA/Ao ratio, LV measures included LV dimensions, 2D LVEF, 2D LV global longitudinal strain (LVGLS), average LV Sm, Em, Am, LV E/Em ratio, LV E, A and LV E/A ratio. RV measures included LV dimensions, RV FAC, RV Tei index, TAPSE, RV Sm, Em, Am, Em/Am ratio, RV E, A, E/A ratio and PASP. Results hsCRP, LA, Ao, LV dimensions, Average LV Am, E/Em, RVD1, RV Tei and PASP were significantly higher in group I compared to group III, but group I had significantly lower LV E/A, LV Sm, Em, LVGLS, RV E/A, Em/Am and RVGLS compared to group III. Group II had significantly higher hsCRP, Ao and RV Tei and significantly lower LV E/A ratio and LV Em compared to group III. On the other hand, group, I had significantly lower average Sm, LVGLS and RVGLS than group II but no significant difference detected between two groups as regard LV and RV diastolic function. Pulse, BMI, BSA, waist circumference, waist to hip ratio, hsCRP were negatively correlated with LVGLS and RVGLS. Cut-off values for BMI showed good sensitivity and specificity to predict impaired LVGLS and RVGLS. Cut-off point 31.9 kg/m2 for impaired LVGLS with sensitivity 80%, specificity 76.81% and p value <0.001, cut-off point >30.73kg/m2 for impaired RVGLS with sensitivity 80%, specificity 69.57% and p value 0.001. Waist circumference was the most sensitive predictor for impaired LVGLS and hsCRP for impaired RVGLS. Conclusion Isolated obesity and overweight are independent predisposing factors for impaired LV and RV systolic and diastolic dysfunctions. TDI and 2D STE are good echo-Doppler modalities to detect subclinical effects on LV and RV functions. Additional Content An author video to accompany this abstract is available on https://academic.oup.com/eurheartjsupp. Please click on the arrow next to ‘More Content’ and then click on ‘Author videos’.

Scanning Transmission Electron Microscopy in a Scanning Electron Microscope for the High-Throughput Imaging of Biological Assemblies

Electron microscopy of soft and biological materials, or "soft electron microscopy", is essential to the characterization of macromolecules. Soft microscopy is governed by enhancing contrast while maintaining low electron doses, and sample preparation and imaging methodologies are driven by the length scale of features of interest. While cryo-electron microscopy offers the highest resolution, larger structures can be characterized efficiently and with high contrast using low-voltage electron microscopy by performing scanning transmission electron microscopy in a scanning electron microscope (STEM-in-SEM). Here, STEM-in-SEM is demonstrated for a four-lobed protein assembly where the arrangement of the proteins in the construct must be examined. STEM image simulations show the theoretical contrast enhancement at SEM-level voltages for unstained structures, and experimental images with multiple STEM modes exhibit the resolution possible for negative-stained proteins. This technique can be extended to complex protein assemblies, larger structures such as cell sections, and hybrid materials, making STEM-in-SEM a valuable high-throughput imaging method.

Imaging atomic motion of light elements in 2D materials with 30 kV electron microscopy.

Scanning transmission electron microscopy (STEM) is the most widespread adopted tool for atomic scale characterization of two-dimensional (2D) materials. However, damage free imaging of 2D materials with electrons has remained problematic even with powerful low-voltage 60 kV-microscopes. An additional challenge is the observation of light elements in combination with heavy elements, particularly when recording fast dynamical phenomena. Here, we demonstrate that 2D WS2 suffers from electron radiation damage during 30 kV-STEM imaging, and we capture beam-induced defect dynamics in real-time by atomic electrostatic potential imaging using integrated differential phase contrast (iDPC)-STEM. The fast imaging of atomic electrostatic potentials with iDPC-STEM reveals the presence and motion of single sulfur atoms near defects and edges in WS2 that are otherwise invisible at the same imaging dose at 30 kV with conventional annular dark-field STEM, and has a vast speed and data processing advantage over electron detector camera based STEM techniques like electron ptychography.

Preparation of water-soluble cellulose derivatives using TEMPO radical-mediated oxidation at extended reaction time

Four types of cellulosic samples, including cotton linters, microcrystalline, never-dried sulfite pulp, and viscose, were tested as potential sources to prepare cellulose-based derivatives, able to be used in homogeneous systems. In this way, by employing water as a solvent, can surpass one of the major drawbacks of native cellulose, its recalcitrance towards common solvents. The chosen derivatization procedure implies the chemical oxidation using 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) radical, sodium hypochlorite, and sodium bromide. Following this treatment, each cellulose sample was processed in such a way as to maximize the content of water-soluble fractions. Therefore, for each cellulose type (except viscose), we could isolate three oxidized samples All of these samples were thoroughly characterized using spectral analyses (FT-IR, 1H NMR, 13C NMR), content of negatively charged groups, and degree of polymerization. The morphology of the oxidized products has been evaluated by means of scanning electron microscopy (SEM), and crystallinity was assessed by using X-ray diffraction (XRD). The thermal analyses for the pure and oxidized cellulose samples were also performed. The limit of the solubility in water for the oxidized fractions was checked by using a low-voltage electron microscopy (LVEM).

Evaluation of Left Atrial Strain Parameter in Coronary Artery Stenosis: A New Tool to Predict LCX Stenosis

Introduction: Speckle tracking echocardiography (STE) based on frame-to-frame tracking of acoustic speckles is a novel angle-independent method, used for evaluating myocardial deformation and recently to assess left atrium (LA) function.Aim: To the best of our knowledge, there is no investigation evaluating the predictor role of LA strain parameters in detection of coronary artery stenosis in ischemic heart disease. In this study, we assessed this parameter for predicting the culprit artery in patients underwent selective coronary angiography (SCA) in Mashhad, Iran. Patients and Methods: This was a case-control study, which was performed in Imam Reza and Quaem Hospitals in Mashhad, Iran between 2017 and 2018 in patients referred for SCA. Subjects with two or three- vessel diseases were excluded .Sixty-five cases were included in the study and divided into four groups according to the vessel involved : Group (1): normal coronary artery subjects (n=36); group (2): patients with left anterior descending (LAD) artery stenosis (n=12); group (3): patients with left circumflex artery (LCX) stenosis (n= 7) and group 4: patients with right coronary artery (RCA) stenosis (n=10). Patients underwent routine TTE and Speckle Tracking Imaging.Results: The mean age of participants was 56.72± 10.69 years old. The male/female ratio was 0.96. Mean absolute value of LA strain was 24.74% in normal subjects, while in groups with stenotic coronary artery disease, less values were defined as 18.63%, 16.03% and 19.05%, respectively (p value=0.05). The amounts of left ventricle end systolic volume (LVESV), LV global longitudinal strain (GLS) and Em of septal annulus were significantly different between normal group and those with LCX stenosis. Among different echocardiographic parameters, LA strain, LVESV and Em of septal annulus were defined as predictors of LCX occlusion with regression analysis. Conclusion: LA strain in subjects with coronary artery stenosis was lower than the strain in normal group and could predict isolated LCX occlusion.

Diagnostic Electron Microscopy of Viruses With Low-voltage Electron Microscopes.

Diagnostic electron microscopy is a useful technique for the identification of viruses associated with human, animal, or plant diseases. The size of virus structures requires a high optical resolution (i.e., about 1 nm), which, for a long time, was only provided by transmission electron microscopes operated at 60 kV and above. During the last decade, low-voltage electron microscopy has been improved and potentially provides an alternative to the use of high-voltage electron microscopy for diagnostic electron microscopy of viruses. Therefore, we have compared the imaging capabilities of three low-voltage electron microscopes, a scanning electron microscope equipped with a scanning transmission detector and two low-voltage transmission electron microscopes, operated at 25 kV, with the imaging capabilities of a high-voltage transmission electron microscope using different viruses in samples prepared by negative staining and ultrathin sectioning. All of the microscopes provided sufficient optical resolution for a recognition of the viruses tested. In ultrathin sections, ultrastructural details of virus genesis could be revealed. Speed of imaging was fast enough to allow rapid screening of diagnostic samples at a reasonable throughput. In summary, the results suggest that low-voltage microscopes are a suitable alternative to high-voltage transmission electron microscopes for diagnostic electron microscopy of viruses.

Visualization of unstained DNA nanostructures with advanced in-focus phase contrast TEM techniques

Over the last few years, tremendous progress has been made in visualizing biologically important macromolecules using transmission electron microscopy (TEM) and understanding their structure-function relation. Yet, despite the importance of DNA in all forms of life, TEM visualization of individual DNA molecules in its native unlabeled form has remained extremely challenging. Here, we present high-contrast images of unstained single-layer DNA nanostructures that were obtained using advanced in-focus phase contrast TEM techniques. These include sub-Ångstrom low voltage electron microscopy (SALVE), the use of a volta-potential phase plate (VPP), and dark-field (DF) microscopy. We discuss the advantages and drawbacks of these techniques for broad applications in structural biology and materials science.

Comparison between a low-voltage benchtop electron microscope and conventional TEM for number size distribution of nearly spherical shape constituent particles of nanomaterial powders and colloids

Nanomaterial powders and colloids are already a large industry and are expected to continue to grow rapidly. In the context of risk assessment associated with nanomaterials, characterization of nanoparticle size and morphology is required. Until now, the best method giving direct access to these parameters has been electron microscopy (EM), in particular, transmission electron microscopy (TEM). Although this method is widely used, several issues are highlighted such as cost, maintenance, sample representativity and damage for sensitive materials. Low-voltage transmission electron microscopes (LVTEMs) could be an alternative approach to solve some of these issues. This paper presents a first comparison between a benchtop LVTEM and a conventional device to determine the number size distribution of the constitutent particles of two polydispersed industrial powders (TiO2 and SiO2) with particle sizes close to 100 nm and two colloids referenced for their particle size (ERM FD 304 and NM 300 K). The samples were prepared with an optimized deposition protocol involving glow discharging and Alcian blue solution pre-treatment on the EM grids. The benchtop LVTEM produced a rather good resolution and the relative differences obtained for the median diameters D50 are generally within ± 15 %. On the basis of these results, benchtop LVTEM could be promoted for identifying nanomaterials within the framework of risk assessment strategy.

Low-voltage coherent electron microscopy based on a highly coherent electron source built from a nanoemitter

It has been a general trend to develop low-voltage electron microscopes due to their high imaging contrast of samples and low radiation damage. Atomic-lattice-resolved transmission electron microscopes with voltages as low as 15–40 kV have been demonstrated. However, achieving an atomic resolution at voltages lower than 10 kV is extremely difficult. An alternative approach is a coherent imaging or phase retrieval imaging, which requires a sufficiently coherent source, an adequately small illumination area on the sample, the detection of high-angle diffraction patterns with a sufficient signal-to-noise ratio, and an appropriate theoretical reconstruction algorithm. This study proposes several transmission-type schemes to achieve coherent imaging of thin materials (less than 5 nm thick) with atomic resolution at voltages lower than 10 kV. Experimental schemes of both lens-less and lens-containing designs and preliminary results based on a highly coherent single-atom electron source are presented. The image plate is designed to be retractable to record the transmission patterns at different positions along the beam propagation direction. In addition, the authors proposed reflection-type coherent electron imaging schemes as novel methods for characterizing surface atomic and electronic structures of materials. The ultimate goal is to achieve high-contrast and high-spatial-resolution imaging of thin materials, such as two-dimensional materials, or molecules, such as organic or biological molecules, under low-dose conditions.

Aberration correction for low voltage optimized transmission electron microscopy

Further development of low voltage electron microscopy leads to an aberration correction of the device in order to improve its spatial resolution. The integration of a corrector to a desktop transmission electron microscope with exclusively low-voltage design seems to be a challenging task. The benefits and potential of the Rose hexapole corrector implemented to such a system are critically considered in this paper. The feasibility of miniaturized corrector suitable for desktop LVEM is especially discussed, including the aspect of corrector contribution to chromatic aberration that appears to be crucial. Optimal corrector parameters and resolution limits of such a system are proposed.•Improved spatial resolution•Spherical aberration correction•Permanent magnet transfer lenses

Low-voltage scanning electron microscopy of multilayer polymer systems

The possibilities of low-voltage scanning electron microscopy for visualization of specific features of the microstructure in internal layers of multilayer polymer films are demonstrated by the example of “chitosan–polyelectrolyte complex–alginic acid” composite. The process of electron beam interaction with a sample at low electron energies is considered. The key parameters of low-voltage electron microscopy, which make it possible to increase the resolution of SEM images of polymer systems, are discussed.

The Boersch effect in a picosecond pulsed electron beam emitted from a semiconductor photocathode

The space charge effect has been clearly observed in the energy distributions of picosecond pulse beams from a spin-polarized electron microscope, and was found to depend upon the quantity of charge per pulse. The non-linear phenomena associated with this effect have also been replicated in beam simulations that take into account of a three-dimensional space charge. The results show that a charge of 500 aC/pulse provides the highest brightness with a 16-ps pulse duration, a 30-keV beam energy, and an emission spot of 1.8 μm. Furthermore, the degeneracy of the wave packet of the pulsed electron beam has been evaluated to be 1.6 × 10−5 with a charge of 100 aC/pulse, which is higher than that for a continuously emitted electron beam despite the low beam energy of 30 keV. The high degeneracy and high brightness contribute to the realization of high temporal and energy resolutions in low-voltage electron microscopy, which will serve to reduce radiolysis damage and enhance scattering contrast.

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.

Left & Right Ventricular Myocardial Performance Index and it’s Relation with TIMI Frame Count in the Coronary Slow Flow Phenomenon

Aim: To investigate the left and right ventricular myocardial performance index in patients with coronary slow flow phenomenon and to determine the relationship between it and thrombolysis in myocardial infarction frame count in SCF patients. Material and methods: 45 patients with slow coronary flow; mean age 51 ± 12 years and 20 subjects with angiographically normal coronary; mean age 52 ± 12 years, were included in the study. All the subjects underwent echocardiography and tissue Doppler imaging to determine left & right ventricular diastolic functions and myocardial performance index (MPI). Results: The TDE parameters obtained from the left and right ventricles showed. LV Sm, and Am, was similar in both the SCF and control groups; however, LV Em, Em/Am, IRT and MPI were significantly different in the SCF group compared to the control. RV wall Sm, and Am were similar in both the SCF and control groups however, RV Em, Em/Am, IRT and MPI were significantly different in the SCF group compared to the control. Patients had significantly higher TFC for each major epicardial coronary artery (P < 0.001). TFC was positively correlated with the left ventricle MPI index (r = 0.61, p < 0.001), the right ventricle MPI index (r = 0.49, p < 0.001), and mitral IVRT (r = 0.61, p < 0.001). There was a strong inverse correlation between mean TFC and left ventricle Ea and Ea/Aa ratio (r = -0.70, p < 0.001; r = -0.45, p = 0.001, respectively). Conclusion: Left and right ventricular diastolic functions deteriorated in the CSF phenomenon and this deterioration was associated with increased TIMI frame count.

Resolution Improvement in Aberration-Corrected Low- Voltage TEM with Monochromator at 60 kV

We have developed a low-voltage electron microscope equipped with a monochromator and Delta-type Cs correctors, which shows atomic resolution at accelerating voltages of 60, 30 and 15 kV. In theory, resolution of TEM images at 60 kV is severely affected by chromatic aberration, which is proven by our calculations of contrast transfer functions and multi-slice image simulation taking chromatic aberration into account with experimental conditions. Experimentally, TEM images of gold nano-particles were observed with non-monochromated and monochromated electron sources at 60 kV. Detectable spatial frequency in the image with the monochromated source was higher than that with non- monochromated source. We have demonstrated that the TEM image resolution at the low- voltage is improved by using a monochromated electron source, which reduce the energy spread of the electron source.

Nanostructures Viewed through Low Voltage Electron Microscopy

Nanostructures Viewed through Low Voltage Electron Microscopy

Energy filtration of secondary and backscattered electrons by the method of the retarding potential in scanning electron and ion microscopy

Data for the study of various contrasts in the methods of scanning electron and ion microscopy with the energy filtration of signals from secondary and reflected electrons are presented. A model of the formation of contrast by secondary electrons in a helium scanning electron microscope is formulated. The possibility of contrast amplification via energy filtration in a helium ion microscope as well as the chance of recording reflected ions by means of secondary electron detection is demonstrated. The recorded data for the material contrast in low-voltage electron microscopy at various acceleration voltages and different modes of energy filtration are given. The possibility of enhancing or reducing material contrast by means of energy filtration is shown.

Electron microscopy of organic–inorganic interfaces: Advantages of low voltage

Low voltage electron microscopy has been applied to many types of materials in the last several decades with great success. The extremely strong interaction of the low voltage electrons with the sample gives high scattering contrast, however it can also result in significant damage of the specimen. Irreparable damage to several types of organic materials results from their large cross section for ionization, or radiolysis, at low voltage. Knock-on damage, which is significant at high voltages for many ceramics, semiconductors, minerals and ordered carbonaceous material such as graphene, is often reduced at low voltages. For organics which damage by radiolysis, measured beam stability increases at higher voltages, but the mass-thickness contrast is also reduced. An increased defocus can be used to generate phase contrast at higher voltages, although it comes at the expense of resolution, as the first zero in the contrast transfer function moves toward larger length scales with increasing defocus. Several examples of low and high voltage (5kV up to 300kV) experimental TEM images of organic-inorganic interfaces are used to demonstrate these phenomena.

Successful application of Low Voltage Electron Microscopy to practical materials problems.

Low-voltage High-Resolution Electron Microscopy (LVHREM) has several advantages, including increased cross-sections for inelastic and elastic scattering, increased contrast per electron, decreased delocalization effects and reduced knock-on damage. Imaging at differing voltages has shown advantages for imaging materials that are knock-on damage sensitive. We show experimentally that different materials systems benefit from low voltage high-resolution microscopy. There are advantages for imaging single layer materials such as graphene at below the knock-on threshold; we present an example of imaging a graphene sheet at 40kV. We have also examined mesoporous silica decorated with Pd nanoparticles and carbon black functionalized with Pd/Pt nanoparticles. In these cases we show that the lower voltage imaging maintains the structure of the surrounding matrix during imaging, whereas aberration correction provides the higher resolution for imaging the nanoparticle lattice. Perhaps surprisingly we show that zeolites damage preferentially by ionization effects (radiolysis). The current literature suggests that below incident energies of 40kV the damage is mainly radiolitic, whereas at incident energies above 200kV the knock-on damage and material sputtering will be the dominant effect. Our experimental observations support this conclusion and the effects we have observed at 40kV are not indicative of knock-on damage. Other nanoscale materials such as thin silicon nanowires also benefit from lower voltage imaging. LVHREM imaging provides an excellent option to avoid beam damage to nanowires; our results suggest that LVHREM is suitable for nanowire-biological composites. Our experimental observations serve as a clear demonstration that even at 40keV accelerating voltage, LVHREM can be used without inducing beam damage to locate dislocations and other crystalline defects, which may have adverse effects on nanowire device performance. Low voltage operation will likely become the new mode of imaging for many electron microscopes, with the instrument being, in essence, tuned to extract all the information possible from each electron that transits the sample.