Low-voltage Transmission Electron Microscopy Research Articles

DIY adapting SEM for low-voltage TEM imaging.

Electron microscopy is essential for examining materials and biological samples at microscopic levels, providing detailed insights. Achieving high-quality imaging is often challenged by the potential damage high-energy beams can cause to sensitive samples. This study compares scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to evaluate image quality, noise levels, and the ability to preserve delicate specimens. We used a modified SEM system with a transmitted electrons conversion accessory, allowing it to operate like a TEM but at lower voltages, thereby reducing sample damage. Our analysis included quantitative assessments of noise levels and texture characteristics such as entropy, contrast, dissimilarity, homogeneity, energy, and correlation. This comprehensive evaluation directly compared traditional TEM and the adapted SEM system across various images. The results showed that TEM provided images with higher clarity and significantly lower noise levels, reinforcing its status as the preferred method for detailed studies. However, the modified SEM system also produced high-quality images at very low acceleration voltages, which is crucial for imaging samples sensitive to high-energy exposure. The texture metrics analysis highlighted the strengths and limitations of each method, with TEM images exhibiting lower entropy and higher homogeneity, indicating smoother and more uniform textures. This study emphasizes the importance of selecting the appropriate electron microscopy method based on research needs, such as sample sensitivity and required detail level. With its conversion accessory, the modified SEM system is a versatile and valuable tool, offering a practical alternative to TEM for various applications. This research enhances our understanding of the capabilities and limitations of SEM and TEM. It paves the way for further innovations in electron microscopy techniques, improving their applicability for studying sensitive materials. RESEARCH HIGHLIGHTS: Our study introduces a modified SEM adapter enabling TEM-like imaging at reduced voltages, effectively minimizing sample damage without compromising image resolution. Through comparative analysis, we found that images from the modified SEM closely match the quality of traditional TEM, showcasing significantly lower noise levels. This advancement underscores the SEM's enhanced capability for detailed structural analysis of sensitive materials, broadening its utility across materials science and biology.

Development of silicon-on-insulator direct electron detector with analog memories in pixels for sub-microsecond imaging.

We have developed a high-speed recordable direct electron detector based on silicon-on-insulator technology. The detector has sixteen analog memories in each pixel to record sixteen images with sub-microsecond temporal resolution. A dedicated data acquisition system has also been developed to display and record the results on a personal computer. The performance of the direct electron detector as an image sensor is evaluated under electron irradiation with an energy of 30 keV in a low-voltage transmission electron microscope equipped with a photocathode electron gun. We demonstrate that the detector can record images at an exposure time of 100 ns and an interval of 900 ns.

Tailored peptide nanomaterials for receptor targeted prostate cancer imaging.

We report the development of a peptide-based optical nanoprobe specifically tailored for prostate cancer imaging. The imaging probe is comprised of cyclic peptide nanotubes, formed via the aqueous co-assembly of four cyclic D,L-alternating octapeptides. The inherent properties of these cyclic building blocks have been carefully selected to enhance their efficacy in imaging applications, through the addition of a cancer targeting peptide and a fluorescent dye. Comprehensive characterization using scanning electron microscopy (FESEM) and low-voltage transmission electron microscopy (LV-TEM) confirms the formation of nanotubes through co-assembly of the cyclic peptides. The resulting nanotubes show an average diameter of 28 nm. Circular dichroism (CD) spectroscopy validates the formation of stable beta-sheet hydrogen bonding structures at both 20 and 37 °C, ensuring their suitability for biomedical applications. Evaluation of PSMA-binding specificity of the resulting peptide nanotubes is assessed using confocal fluorescence microscopy demonstrating receptor-mediated uptake in prostate cancer cells. We anticipate this strategy will provide the basis for the utilization of co-assembled systems for advancing molecular imaging techniques in prostate cancer and other cancers.

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.

Quality assessment of virus-like particle: A new transmission electron microscopy approach.

Transmission electron microscopy (TEM) is a gold standard analytical method for nanoparticle characterization and is playing a valuable role in virus-like particle (VLP) characterization extending to other biological entities such as viral vectors. A dedicated TEM facility is a challenge to both small and medium-sized enterprises (SMEs) and companies operating in low-and-middle income countries (LMICs) due to high start-up and running costs. A low-voltage TEM solution with assisted image acquisition and analysis such as the MiniTEM system, coupled with Vironova Imaging and Analysis Software (VIAS) could provide an affordable and practical alternative. The MiniTEM system has a small footprint and software that enables semi-automated data collection and image analysis workflows using built-in deep learning methods (convolutional neural networks) for automation in analysis, increasing speed of information processing and enabling scaling to larger datasets. In this perspective we outline the potential and challenges in the use of TEM as mainstream analytical tool in manufacturing settings. We highlight the rationale and preliminary findings from our proof-of-concept study aiming to develop a method to assess critical quality attributes (CQAs) of VLPs and facilitate adoption of TEM in manufacturing settings. In our study we explored all the steps, from sample preparation to data collection and analysis using synthetic VLPs as model systems. The applicability of the method in product development was verified at pilot-scale during the technology transfer of dengue VLPs development from a university setting to an LMIC- based vaccine manufacturing company, demonstrating the applicability of this analytical technique to VLP vaccine characterization.

One-Step Growth of Bilayer 2H-1T' MoTe2 van der Waals Heterostructures with Interlayer-Coupled Resonant Phonon Vibration.

2H-1T' MoTe2 van der Waals heterostructures (vdWHs) have promising applications in optoelectronics due to a seamlessly homogeneous semiconductor-metal coupled interface. However, the existing methods to fabricate such vdWHs involved complicated steps that may deteriorate the interfacial coupling and are also lacking precise thickness control capability. Here, a one-step growth method was developed to controllably grow bilayer 2H-1T' MoTe2 vdWHs in the small growth window overlapped for both phases. Atomic-resolution low-voltage transmission electron microscopy shows the distinct moiré patterns in the bilayer vdWHs, revealing the epitaxial nature of the top 2H phase with the lattice parameters regulated by the underneath 1T' phase. Such epitaxially stacked bilayer vdWHs modulate the interlayer coupling by resonating their vibration modes, as unveiled by the angle-resolved polarized Raman spectroscopy and first-principles calculations.

Characterization and Determination of Nanoparticles in Commercial Processed Foods.

A wide variety of foods manufactured by nanotechnology are commercially available on the market and labeled as nanoproducts. However, it is challenging to determine the presence of nanoparticles (NPs) in complex food matrices and processed foods. In this study, top-down-approach-produced (TD)-NP products and nanobubble waters (NBWs) were chosen as representative powdered and liquid nanoproducts, respectively. The characterization and determination of NPs in TD-NP products and NBWs were carried out by measuring constituent particle sizes, hydrodynamic diameters, zeta potentials, and surface chemistry. The results show that most NBWs had different characteristics compared with those of conventional sparkling waters, but nanobubbles were unstable during storage. On the other hand, powdered TD-NP products were found to be highly aggregated, and the constituent particle sizes less than 100 nm were remarkably observed after dispersion compared with counterpart conventional bulk-sized products by scanning electron microscopy at low acceleration voltage and cryogenic transmission electron microscopy. The differences in chemical composition and chemical state between TD-NPs and their counterpart conventional bulk products were also found by X-ray photoelectron spectroscopy. These findings will provide basic information about the presence of NPs in nano-labeled products and be useful to understand and predict the potential toxicity of NPs applied to the food industry.

Evaluation of Ionomer Distribution on Electrocatalysts for Polymer Electrolyte Fuel Cells by Use of a Low Acceleration Voltage Scanning Electron Microscope

The qualitative evaluation of the dispersion of prefluorosulfonic ionomer (PFSI) with different ionomer/carbon mixing ratios (I/C) using electron microscopy was carried out without the use of a stain treatment. Both low acceleration voltage transmission electron microscopy (LAV-TEM) and ultralow acceleration voltage scanning electron microscopy with a retarding method (ULV-SEM) use a characteristically low acceleration voltage, which allows the selective examination of the sensitive ionomer morphology. The high-performance charge-coupled device enables one to obtain high contrast ionomer images without the use of lead or cesium staining, which could otherwise result in morphological changes during these pre-treatments. The electrochemically active surface area of the polymer electrolyte fuel cell using Pt/GCB increased with increasing PFSI content and saturated at an ionomer/carbon weight ratio (I/C) of 1.2, where full coverage of the ionomer was detected by LAV-TEM. The ULV-SEM images showed the obvious occlusion of the primary and secondary pores of the Pt/GCB catalyst layers above I/C = 1.2. The nitrogen gas adsorption measurement, carried out by use of quenched solid-density-functional theory analysis, also supported the occlusion of the primary and secondary pores of the Pt/GCB catalyst layers above I/C = 1.2.

Performance of a silicon-on-insulator direct electron detector in a low-voltage transmission electron microscope.

The performance of a direct electron detector using silicon-on-insulator (SOI) technology in a low-voltage transmission electron microscope (LVTEM) is evaluated. The modulation transfer function and detective quantum efficiency of the detector are measured under backside illumination. The SOI-type detector is demonstrated to have high sensitivity and high efficiency for the direct detection of low-energy electrons. The detector is thus considered suitable for low-dose imaging in an LVTEM.

In-situ formation and evolution of atomic defects in monolayer WSe2 under electron irradiation

Transition metal dichalcogenide (TMD) monolayers such as MoS2, MoSe2, MoTe2, WS2 and WSe2 have attracted significant interest due to their remarkable electronic and optical properties, exhibiting a direct band gap, enabling usability in electronics and optics. Their properties can be altered further by the introduction of lattice defects. In this work, the dynamics of the formation of electron-beam-induced lattice defects in monolayer WSe2 are investigated by in-situ spherical and chromatic aberration-corrected low-voltage transmission electron microscopy. We show and analyze the electron-dose-limited life of a monolayer WSe2 from the formation of isolated Se vacancies over extended defects such as vacancy lines, mirror twin boundaries (MTBs) and inversion domains towards the loss of W atoms leading to the formation of holes and finally the destruction of the monolayer. We identify, moreover, a new type of MTB. Our study extends the basic understanding of defect dynamics in monolayer WSe2, sheds further light on the electron radiation response and suggests new ways for engineering the in-plane architecture of TMDs.

Synchronized Structure and Surface Tension Measurement on Individual Secondary Aerosol Particles by Low-Voltage Transmission Electron Microscopy

A number of physical and chemical models have been built to describe secondary aerosols (SAs) in the atmosphere; however, direct experimental approaches to simultaneously characterizing the chemica...

Improvement of the Performance of Pt Catalysts Supported on Nb-Doped SnO2 Via Well-Controlled Interfaces

Toward the widespread use of polymer electrolyte fuel cells (PEFCs) in electric vehicles, the improvement of the durability and enhancement of the cathode catalyst activity for the oxygen reduction reaction (ORR) are needed. Pt catalysts loaded on graphitized carbon black (Pt/GCB) are promising candidate catalysts with high durability and activity, but the carbon itself has not been able to completely withstand corrosion at high potential. Our group reported that Pt catalysts loaded on Nb-doped SnO2 (Pt/Nb-SnO2), without any carbon additive, showed high electronic conductivity, high ORR activity and high durability (startup/shutdown, load cycling) in membrane electrode assembly (MEA) measurements [1-6]. In the case of a noble metal such as Pt loaded on semiconducting Nb-SnO2, a Schottky barrier would be constructed at the interface between Pt and Nb-SnO2, which tends to decrease the electronic conductivity and electrochemical activity. The electronic conductivity of Pt/Nb-SnO2 with fused-aggregate network structure is enhanced by heat-treatment and high Pt loading (>10 wt%) to equal that of carbon black. XPS spectra for the Pt/Nb-SnO2 catalyst showed that a slight amount of Sn was detected (Fig. 1). The STEM-EDX elemental line analysis indicated that the Sn diffused into the Pt catalyst. We considered that a PtSn interlayer was inserted in the interface between Pt and Nb-SnO2, which would mitigate the effect of the Schottky barrier, induce the electron donation from Pt to Nb-SnO2 and enhance the electronic conductivity. The low cell resistivity of an MEA using our Pt/Nb-SnO2 cathode, as low as that using a Pt/CB cathode, is ascribed to both the enhancement of the electronic conductivity and the construction of electronic conducting pathways by the fused aggregate network structure. Nafion® ionomer film was found to cover uniformly on the hydrophilic surface of the Pt/Nb-SnO2 (Fig. 2), in contrast to the poor coverage of the ionomer on the hydrophobic surface of the Pt/GCB, based on an evaluation with low acceleration voltage transmission electron microscopy. The thin, uniform coverage of the Nafion® ionomer on the Pt/Nb-SnO2 surface helps to construct a membrane electrode assembly (MEA) with low volume ratio of Nafion® to Nb-SnO2 (I/S < 0.20), which increases the apparent mass activity (@ 0.80 V) while maintaining a low Tafel slope by mitigation of the oxygen diffusion overpotential in the Nafion® film with increased porosity in the catalyst layers. This work was partially supported by funds for the “Superlative, Stable, and Scalable Performance Fuel Cell” (SPer-FC) project from the New Energy and Industrial Technology Development Organization (NEDO) of Japan, and JSPS KAKENHI Grant Number 17H03410 from the Ministry of Education, Culture, Sports, Science and Technology. K. Kakinuma, Y. Chino, Y. Senoo, M. Uchida, T. Kamino, H. Uchida, S. Deki, M. Watanabe, Electrochim. Acta, 110, 316 (2013).Y. Senoo, K. Kakinuma, M. Uchida, H. Uchida, S. Deki, M. Watanabe, RSC Adv., 6, 321800 (2014).Y. Chino, K. Taniguchi, Y. Senoo, K. Kakinuma, M. Watanabe, M. Uchida, J. Electrochem. Soc., 162, F736 (2015).Y. Chino, K. Kakinuma, D.A. Tryk, M. Watanabe, M. Uchida, J. Electrochem. Soc., 163, F97 (2016).K. Takahashi, R. Koda, K. Kakinuma, M. Uchida, J. Electrochem. Soc., 164, F235 (2017).K. Kakinuma, R. Kobayashi, A. Iiyama, M. Uchida, J. Electrochem. Soc., 165, J3083 (2018). Figure 1

Reversible superdense ordering of lithium between two graphene sheets.

Many carbon allotropes can act as host materials for reversible lithium uptake1,2, thereby laying the foundations for existing and future electrochemical energy storage. However, insight into how lithium is arranged within these hosts is difficult to obtain from a working system. For example, the use of in situ transmission electron microscopy3-5 to probe light elements (especially lithium)6,7 is severely hampered by their low scattering cross-section for impinging electrons and their susceptibility to knock-on damage8. Here we study the reversible intercalation of lithium into bilayer graphene by in situ low-voltage transmission electron microscopy, using both spherical and chromatic aberration correction9 to enhance contrast and resolution to the required levels. The microscopy is supported by electron energy-loss spectroscopy and density functional theory calculations. On their remote insertion from an electrochemical cell covering one end of the long but narrow bilayer, we observe lithium atoms to assume multi-layered close-packed order between the two carbon sheets. The lithium storage capacity associated with this superdense phase far exceeds that expected from formation of LiC6, which is the densest configuration known under normal conditions for lithium intercalation within bulk graphitic carbon10. Our findings thus point to the possible existence of distinct storage arrangements of ions in two-dimensional layered materials as compared to their bulk parent compounds.

Chromatic- and geometric-aberration-corrected TEM imaging at 80 kV and 20 kV

The interest in light-element nanomaterials stimulated the development of atomic-resolution low-voltage transmission electron microscopy. While geometric-aberration correction made high-resolution imaging at around 80-kV electron acceleration voltage possible, imaging at substantially lower voltages requested the development of chromatic-aberration correction in addition. Recently, the SALVE instrument was introduced with the capability of atomic resolution imaging at electron acceleration voltages from 80 kV down to 20 kV. Here, we show on the example of imaging graphene that at these electron energies, the residual geometric aberrations reintroduce contrast oscillations and delocalization. Also, we show an atomically resolved image of a single vacancy in graphene with a pronounced Jahn-Teller distortion.

Effects of Ionomer Content on Both Performance and Load Cycle Durability for PEFCs Using Pt/Nb-SnO2 Cathode Catalyst Layers

Toward the widespread application of polymer electrolyte fuel cells (PEFCs) in electric vehicles, the enhancement of the cathode catalyst activity for the oxygen reduction reaction (ORR) and the improvement of the durability are needed. Pt catalysts loaded on graphitized carbon black (Pt/GCB) have been confirmed to moderate degradation during startup and shutdown, compared with that of Pt/CB, but these carbons have not been able to completely overcome corrosion at high potential. Electronically conducting oxides, without the use of carbon, such as the Magneli phase titanium oxides and tin dioxide, are promising candidate supports [1,2]. Our group also has reported that Pt catalysts loaded on Nb-doped SnO2 (Pt/Nb-SnO2) and Ta-doped SnO2 (Pt/Ta-SnO2), without any carbon additive, showed high durability during startup/shutdown, while maintaining high ORR activity, by means of rotating disk electrode and membrane electrode assembly (MEA) measurements [3-9]. The high ORR activity of Pt/Nb-SnO2 and Pt/Ta-SnO2 is dependent on a strong interaction between the Pt catalyst and the tin oxide. The low cell resistivity of an MEA using our Pt/Nb-SnO2 cathode, as low as that using a Pt/CB cathode, is ascribed to a unique microstructure involving a fused aggregate network structure, which constructs effective gas diffusion pathways, electronically conducting pathways and high surface area in the catalyst layer (CL). For the further improvement of the cell performance, the optimization of the volume and dispersion of the ionomer in the CL is important. In this research, we have evaluated the cell performance and the load cycle durability of single cells using a Pt/Nb-SnO2 CL with a range of volume ratios of Nafion® ionomer and the support material (I/S). The current-voltage performance and resistivity of the cell using Pt/Nb-SnO2 CL (I/S = 0.24) were equal to those using Pt/GCB CL (I/S = 0.67, optimized I/S ratio, Fig. 1).The apparent mass activity at 0.80 V (MAapp@0.80 V) of the cell using Pt/Nb-SnO2 CL improved with decreasing I/S, and, at I/S = 0.12, approached a value a factor of 2 higher than that using a commercial Pt/GCB CL with an optimized I/S ratio (Fig. 2). The current density at 0.60 V of the cell using Pt/Nb-SnO2 (I/S = 0.12) CL approached the same value of the cell using Pt/GCB CL. The electrochemically active surface area (ECA) of the Pt/Nb-SnO2 CL was twice as large at the initial stage than that of Pt/GCB CL and continued to maintain higher values during a load cycle durability test (0.6 - 1.0 V, 3 s, Fig. 3). We consider that the utilization of Pt and the load-cycle durability of Pt/Nb-SnO2 are higher than those of Pt/GCB. The Nafion® ionomer covered uniformly on the hydrophilic surface of the Pt/Nb-SnO2 (Fig. 4), in contrast to the poor coverage of the ionomer on the hydrophobic surface of the Pt/GCB, based on an evaluation with low acceleration voltage transmission electron microscopy. We suggest that the thin, uniform coverage of the Nafion® ionomer on the Pt/Nb-SnO2 surface, due to the appropriate low level of ionomer, moderates both the oxygen diffusion overpotential in the Nafion® ionomer and the Pt catalyst degradation. This work was partially supported by funds for the “Superlative, Stable, and Scalable Performance Fuel Cell” (SPer-FC) project from the New Energy and Industrial Technology Development Organization (NEDO) of Japan, and JSPS KAKENHI Grant Number 17H03410 from the Ministry of Education, Culture, Sports, Science and Technology. T. Ioroi, Z. Siroma, N. Fujiwara, S. Yamazaki, K. Yasuda, Electrochem. Commun., 7, 183 (2005).A. Masao, S. Noda, F. Takasaki, K. Ito, K. Sasaki, Electrochem. Solid-State Lett., 12, B119 (2009).K. Kakinuma, M. Uchida, T. Kamino, H. Uchida, M. Watanabe, Electrochim. Acta, 56, 2881 (2011).K. Kakinuma, Y. Chino, Y. Senoo, M. Uchida, T. Kamino, H. Uchida, S. Deki, M. Watanabe, Electrochim. Acta, 110, 316 (2013).Y. Senoo, K. Kakinuma, M. Uchida, H. Uchida, S.Deki, M. Watanabe, RSC Adv., 6, 321800 (2014).Y. Senoo, K. Taniguchi, K. Kakinuma, M. Uchida, H. Uchida, S. Deki, M. Watanabe, Electrochem. Commun., 51, 37 (2015).Y. Chino, K. Taniguchi, Y. Senoo, K. Kakinuma, M. Watanabe, M. Uchida, J. Electrochem. Soc., 162, F736 (2015).Y. Chino, K. Kakinuma, D.A. Tryk, M. Watanabe, M. Uchida, J. Electrochem. Soc., 163, F97 (2016).K. Takahashi, R. Koda, K. Kakinuma, M. Uchida, J. Electrochem. Soc., 164, F235 (2017). Figure 1

Direct imaging and determination of the crystal structure of six-layered graphdiyne

Since its discovery, the direct imaging and determination of the crystal structure of few-layer graphdiyne has proven difficult because it is too delicate under irradiation by an electron beam. In this work, the crystal structure of a six-layered graphdiyne nanosheet was directly observed by low-voltage transmission electron microscopy (TEM) using low current density. The combined use of high-resolution TEM (HRTEM) simulation, electron energy-loss spectroscopy, and electron diffraction revealed that the as-synthesized nanosheet was crystalline graphdiyne with a thickness of 2.19 nm (corresponding to a thickness of six layers) and showed ABC stacking. Thus, this work provides direct evidence for the existence and crystal structure of few-layer graphdiyne, which is a new type of two-dimensional carbon material complementary to graphene.

Influence of Ionomer Content on Both Cell Performance and Load Cycle Durability for Polymer Electrolyte Fuel Cells Using Pt/Nb-SnO2 Cathode Catalyst Layers

The steady-state current-voltage performance and load cycle durability of polymer electrolyte fuel cells using Pt catalysts supported on Sn0.96Nb0.04O2-δ (Pt/Nb-SnO2) cathode catalyst layers (CLs), without any carbon additive, were evaluated with various ionomer contents. The apparent mass activity at 0.80 V (MAapp @0.80 V) of the cell using Pt/Nb-SnO2 CL improved with decreasing volume ratio of the Nafion ionomer to the support (I/S). At I/S = 0.12, surprisingly, the MAapp @0.80 V of the cell using Pt/Nb-SnO2 CL approached 2 times higher than that using a commercial Pt catalyst supported on GCB (Pt/GCB) CL with the optimized I/S ratio. The current density at 0.60 V of the cell using Pt/Nb-SnO2 (I/S = 0.12) CL also reached the same value of the cell using Pt/GCB CL. The electrochemically active surface area and MAapp @0.80 V of Pt/Nb-SnO2 CLs during the load cycle durability test maintained higher values than those of Pt/GCB throughout the test, indicating that the Pt/Nb-SnO2 CL had higher durability than the Pt/GCB CL. Evaluation by means of low acceleration voltage transmission electron microscopy proved that the Nafion ionomer covered the hydrophilic surface of the Pt/Nb-SnO2 uniformly, whereas the ionomer did not do so on the hydrophobic surface of the Pt/GCB. The thin, uniform coverage of the Nafion ionomer, which was thus easily obtained on the Pt/Nb-SnO2 surface, was able to lower the overpotential originating from the oxygen diffusion resistance in the Nafion ionomer, thus improving the cell performance while maintaining the high load cycle durability.

Benefits and Limitations of Low-kV Macromolecular Imaging of Frozen-Hydrated Biological Samples

Object contrast is one of the most important parameters of macromolecular imaging. Low-voltage transmission electron microscopy has shown an increased atom contrast for carbon materials, indicating that amplitude contrast contributions increase at a higher rate than phase contrast and inelastic scattering. Here, we studied image contrast using ice-embedded tobacco mosaic virus particles as test samples at 20–80 keV electron energy. The particles showed the expected increase in contrast for lower energies, but at the same time the 2.3-nm-resolution measure decayed more rapidly. We found a pronounced signal loss below 60 keV, and therefore we conclude that increased inelastic scattering counteracts increased amplitude contrast. This model also implies that as long as the amplitude contrast does not increase with resolution, beam damage and multiple scattering will always win over increased contrast at the lowest energies. Therefore, we cannot expect that low-energy imaging of conventionally prepared samples would provide better data than state-of-the-art 200–300 keV imaging.

Single atom spectroscopy: Decreased scattering delocalization at high energy losses, effects of atomic movement and X-ray fluorescence yield

Single atom localization and identification is crucial in understanding effects which depend on the specific local environment of atoms. In advanced nanometer scale materials, the characteristics of individual atoms may play an important role. Here, we describe spectroscopic experiments (electron energy loss spectroscopy, EELS, and Energy Dispersed X-ray spectroscopy, EDX) using a low voltage transmission electron microscope designed towards single atom analysis. For EELS, we discuss the advantages of using lower primary electron energy (30keV and 60keV) and higher energy losses (above 800eV). The effect of atomic movement is considered. Finally, we discuss the possibility of using atomically resolved EELS and EDX data to measure the fluorescence yield for X-ray emission.

Low Voltage Transmission Electron Microscopy in Cell Biology

Low voltage transmission electron microscopy (LVTEM) was employed to examine biological tissues with accelerating voltages as low as 5kV. Tissue preparation was modified to take advantage of the low-voltage techniques. Treatments with heavy metals, such as post-fixation with osmium tetroxide, on block and counterstaining were omitted. Sections (40nm) were thinner than usual and generated highly contrasted images. General appearance of the cells remains similar to that of conventional TEM. New features were however revealed. The matrix of the pancreatic granules displays heterogeneity with partitions that may correspond to the inner-segregation of their secretory proteins. Mitochondria revealed the presence of the ATP synthase granules along their cristea. The nuclear dense chromatin displayed a honeycomb organization while distinct beads, nucleosomes, aligned along thin threads were seen in the dispersed chromatin. Nuclear pore protein complexes revealed their globular nature. The intercalated disks in cardiac muscle displayed their fine structural organization. These features correlate well with data described or predicted by cell and molecular biology. These new aspects are not revealed when thicker and conventionally osmicated tissue sections were examined by LVTEM, indicating that major masking effects are associated with standard TEM techniques. Immunogold was adapted to LVTEM further enhancing its potential in cell biology.