This study demonstrates the accumulation of electron-induced secondary electrons by utilizing a simple geometrical configuration of two branches of a charged insulating biomaterial. The collective motion of these secondary electrons between the branches has been visualized by analyzing the reconstructed amplitude images obtained using in situ electron holography. In order to understand the collective motion of secondary electrons, the trajectories of these electrons around the branches have also been simulated by taking into account the electric field around the charged branches on the basis of Maxwell’s equations.

Transmission electron microscopy (TEM) images of beam sensitive weak-phase objects such as biological cryo samples usually show a very low signal-to-noise ratio. These samples have almost no amplitude contrast and instead structural information is mainly encoded in the phase contrast. To increase the sample contrast in the image, especially for low spatial frequencies, the use of phase plates for close to focus phase contrast enhancement in TEM has long been discussed. Electrostatic phase plates are favorable in particular, as their tunable potential will allow an optimal phase shift adjustment and higher resolution than film phase plates as they avoid additional scattering events in matter. Here we show the first realization of close to focus phase contrast images of actin filament cryo samples acquired using an electrostatic Zach phase plate. Both positive and negative phase contrast is shown, which is obtained by applying appropriate potentials to the phase plate. The dependence of phase contrast improvement on sample orientation with respect to the phase plate is demonstrated and single-sideband artifacts are discussed. Additionally, possibilities to reduce contamination and charging effects of the phase plate are shown.

Zach phase plates (PPs) are promising devices to enhance phase contrast in transmission electron microscopy. The Zach PP shifts the phase of the zero-order beam by a strongly localized inhomogeneous electrostatic potential in the back focal plane of the objective lens. We present substantial improvements of the Zach PP, which overcome previous limitations. The implementation of a microstructured heating device significantly reduces contamination and charging of the PP structure and extends its lifetime. An improved production process allows fabricating PPs with reduced dimensions resulting in lower cut-on frequencies as revealed by simulations of the electrostatic potential. Phase contrast with inversion of PbSe nanoparticles is demonstrated in a standard transmission electron microscope with LaB6 cathode by applying different voltages.

Here we evaluate a new grid substrate developed by ProtoChips Inc. (Raleigh, NC) for cryo-transmission electron microscopy. The new grids are fabricated from doped silicon carbide using processes adapted from the semiconductor industry. A major motivating purpose in the development of these grids was to increase the low-temperature conductivity of the substrate, a characteristic that is thought to affect the appearance of beam-induced movement (BIM) in transmission electron microscope (TEM) images of biological specimens. BIM degrades the quality of data and is especially severe when frozen biological specimens are tilted in the microscope. Our results show that this new substrate does indeed have a significant impact on reducing the appearance and severity of beam-induced movement in TEM images of tilted cryo-preserved samples. Furthermore, while we have not been able to ascertain the exact causes underlying the BIM phenomenon, we have evidence that the rigidity and flatness of these grids may play a major role in its reduction. This improvement in the reliability of imaging at tilt has a significant impact on using data collection methods such as random conical tilt or orthogonal tilt reconstruction with cryo-preserved samples. Reduction in BIM also has the potential for improving the resolution of three-dimensional cryo-reconstructions in general.

Wedge polishing was used to prepare one-dimensional Si n-p junction and Si p-channel metal-oxide-silicon field effect transistor (pMOSFET) samples for precise and quantitative electrostatic potential analysis using off-axis electron holography. To avoid artifacts associated with ion milling, cloth polishing with 0.02-μm colloidal silica suspension was used for final thinning. Uniform thickness and no significant charging were observed by electron holography analysis for samples prepared entirely by this method. The effect of sample thickness was investigated and the minimum thickness for reliable results was found to be ∼160 nm. Below this thickness, measured phase changes were smaller than expected. For the pMOSFET sample, quantitative analysis of two-dimensional electrostatic potential distribution showed that the metallurgical gate length (separation between two extension junctions) was ∼54 nm, whereas the actual gate length was measured to be ∼70 nm by conventional transmission electron microscopy. Thus, source and drain junction encroachment under the gate was 16 nm.

In this article, the secondary electron-emission properties of both vertically and laterally inhomogeneous samples are discussed. To study the effect of surface coverage, the total electron-emission yield of tungsten and niobium samples was measured as a function of primary electron energy and oxide thickness. A method is suggested to avoid charging difficulties during AES measurements of samples that consist of both metal and various insulator parts.

This work demonstrates the possibility of using the Duane–Hunt limit of the bremsstrahlung to determine E2 values of Si3N4 and AlN ceramics. The EDHL versus E0 graph demonstrates that for conductive materials, the experimental curve is parallel to the theoretical (EDHL = E0), but both curves cross in the case of insulators. The intersection points (E2 value), are 3.01 keV for Si3N4 and 2.67 keV for AlN. Imaging of ceramic grain structure at high magnification was performed to demonstrate the validity of the calculated E2 values.

The physical mechanisms involved in electron irradiation of insulating specimens are investigated by combining some simple considerations of solid-state physics (trapping mechanisms of electrons and secondary electron emission) with basic equations of electrostatics. To facilitate the understanding of the involved mechanisms only widely irradiated samples having a uniform distribution of trapping sites are considered. This starting hypothesis allows development of simple models for the trapped charge distributions in ground-coated specimens as investigated in electron probe microanalysis (EPMA) as well as for the bare specimens investigated in scanning electron microscopy (SEM) and environmental SEM (ESEM). Governed by self-regulation processes, the evolution of the electric parameters during the irradiation are also considered for the first time and practical consequences in EPMA, SEM, and ESEM are deduced. In particular, the widespread idea that the noncharging condition of SEM is obtained at a critical energy E2 (where δ + η = 1 with δ and η yields obtained in noncharging experiments) is critically discussed.

A framework is presented for understanding charging processes in low vacuum scanning electron microscopy. We consider the effects of electric fields generated above and below the specimen surface and their effects on various processes taking place in the system. These processes include the formation of an ionic space charge, field-enhanced electron emission, charge trapping and dissipation, and electron–ion recombination. The physical mechanisms behind each of these processes are discussed, as are the microscope operating conditions under which each process is most effective. Readily observable effects on gas gain curves, secondary electron images, and X-ray spectra are discussed.

Kelvin probe microscopy (KPM) is a specialized atomic force microscopy technique in which long-range Coulomb forces between a conductive atomic force probe and a specimen enable the electrical potential at the surface of a specimen to be characterized with high spatial resolution. KPM has been used to characterize nonconductive materials following their exposure to stationary electron beam irradiation in a scanning electron microscope (SEM). Charged beam irradiation of poorly conducting materials results in the trapping of charge at either preexisting or irradiation-induced defects. The reproducible characteristic surface potentials associated with the trapped charge have been mapped using KPM. Potential profiles are calculated and compared with observed potential profiles giving insight into the charging processes and residual trapped charge distributions.

The microanalysis of nonconductive specimen in a scanning electron microscope is limited by charging effects. Using a charge density model for the electric field buildup in a nonconductive specimen irradiated by electrons, a Monte Carlo simulation method has been applied to alumina (Al2O3). The results show a change in the depth distribution for characteristic and bremsstrahlung X-ray, φ(ρz) curves, and ψ(ρz) curves (with absorption) for both elements' Kα lines. The influence of the electric field on the measured X-ray intensity is shown. The dependency of this influence by the three parameters, electron energy, X-ray energy, and charge density, is clarified.

Although the most familiar consequences of specimen charging in transmission electron microscopy can be eliminated by evaporating a thin conducting film (such as a carbon film) onto an insulating specimen or by preparing samples directly on such a conducting film to begin with, a more subtle charging effect still remains. We argue here that specimen charging is in this case likely to produce a dipole sheet rather than a layer of positive charge at the surface of the specimen. A simple model of the factors that control the kinetics of specimen charging, and its neutralization, is discussed as a guide for experiments that attempt to minimize the amount of specimen charging. Believable estimates of the electrostatic forces and the electron optical disturbances that are likely to occur suggest that specimen bending and warping may have the biggest impact on degrading the image quality at high resolution. Electron optical effects are likely to be negligible except in the case of a specimen that is tilted to high angle. A model is proposed to explain how both the mechanical and electron-optical effects of forming a dipole layer would have much greater impact on the image resolution in a direction perpendicular to the tilt axis, a well-known effect in electron microscopy of two-dimensional crystals.

Energy dispersive X-ray spectrometry of uncoated insulators performed at low beam energy (incident energy ≤ 5 keV) and in the variable pressure scanning electron microscope and the environmental scanning electron microscope is subject to spectral artifacts. Charging decelerates the incident beam electrons and reduces the impact energy, lowering the available overvoltage to excite characteristic X-ray peaks. The Duane–Hunt limit of the X-ray bremsstrahlung continuum is commonly used as a diagnostic of charging. Dynamic charging effects can hide the true impact of charging on the X-ray spectrum. Careful examination of the behavior of the X-ray spectrum with time and other variables is needed to avoid spectral artifacts, particularly on relative X-ray intensities.

In the context of electron microscopists' changing attitudes to charging effects, some basic aspects of these phenomenona are surveyed. Methods of mapping internal charge distributions such as doping levels in semiconductors, trap distributions, or internal electric fields in insulators are discussed.

Biological macromolecules embedded in vitreous ice are known to suffer from charging while being imaged in an electron transmission cryomicroscope. We developed an electron beam coater that deposits conductive films onto the surface of frozen-hydrated specimens. The conductive films help to dissipate charge during electron irradiation of poorly conductive ice-embedded biological samples. We observed significant reduction in charging of ice-embedded catalase crystals suspended over holes in a holey carbon film after coating them with a 30-Å-thick layer of an amorphous alloy, Ti88Si12. Images of the crystals after coating showed diffraction spots of up to 3 Å resolution.

Specimens that charge under electron beam irradiation in the scanning electron microscope (SEM) can be stabilized by choosing the beam energy to be such a value that the sum of the secondary and backscatter electron yields is unity, as this establishes a dynamic charge balance. We show here that for pure elements, the energies El and E2, for which charge balance occurs, are related directly to the atomic number of the material. Although generally there is no comparable relation for compounds, we also show that for polymers, the E2 energy is related both to the ratio of the number of valence electrons to molecular weight and to the electro-negativity of the monomer units that form the polymer.