High resolution SEM of bacterial virus T7.

High resolution "low-loss" scanning electron microscopy is a relatively new technique which permits an investigator to examine structures that were formerly visualized exclusively by transmission electron microscopy [1]. This paper presents some images of intact bacterial virus T7, viewed at the ultrastructural level. Due to the high resolution capibility of this technique, and the demanding physical prerequisites for visualization of the specimen, current specimen preparation techniques were modified in order to permit 1--2 nm resolution in surface mode. Using this method of microscopy, it is possible to view clearly this small bacteriophage (the smallest of the T-coliphages), adsorbed to its host bacterium, in a scanning mode at magnification (and resolution) comparable to TEM without resorting to the use of replicas, or reconstruction of a two-dimensional image.     

A high signal-to-noise ratio toroidal electron spectrometer for the SEM

This paper presents a high signal-to-noise ratio electron energy spectrometer attachment for the scanning electron microscope (SEM), designed to measure changes in specimen surface potential from secondary electrons and extract specimen atomic number information from backscattered electrons. Experimental results are presented, which demonstrate that the spectrometer can in principle detect specimen voltage changes well into the sub-mV range, and distinguish close atomic numbers by a signal-to-noise ratio of better than 20. The spectrometer has applications for quantitatively mapping specimen surface voltage and atomic number variations on the nano-scale.     

Imaging of specimens at optimized low and very low energies in scanning electron microscopes.

The modern trend towards low electron energies in scanning electron microscopy (SEM), characterised by lowering the acceleration voltages in low-voltage SEM (LVSEM) or by utilising a retarding-field optical element in low-energy SEM (LESEM), makes the energy range where new contrasts appear accessible. This range is further extended by a scanning low-energy electron microscope (SLEEM) fitted with a cathode lens that achieves nearly constant spatial resolution throughout the energy scale. This enables one to optimise freely the electron beam energy according to the given task. At low energies, there exist classes of image contrast that make particular specimen data visible most effectively or even exclusively within certain energy intervals or at certain energy values. Some contrasts are well understood and can presently be utilised for practical surface examinations, but others have not yet been reliably explained and therefore supplementary experiments are needed.     

Electron backscatter diffraction of grain and subgrain structures — resolution considerations

Characterization of microstructures containing small grains or low-angle grain boundaries by electron backscattered diffraction (EBSD) is limited by the spatial and angular resolution limits of the technique. It was found that the best effective spatial resolution (60 nm) for aluminium alloys in a tungsten-filament scanning electron microscope (SEM) was obtained for an intermediate probe current which provided a compromise between pattern quality and specimen interaction volume. The same specimens and EBSD equipment when used with a field-emission gun SEM showed an improvement in spatial resolution by a factor of 2–3. For characterizing low-angle boundary microstructures, the precision of determining relative orientations is a limiting factor. It was found that the orientation noise was directly related to the probe current and this was interpreted in terms of the effect of probe current on the quality of the diffraction patterns.     

Quantitative measurements of Kikuchi bands in diffraction patterns of backscattered electrons using an electrostatic analyzer

Diffraction patterns of backscattered electrons can provide important crystallographic information with high spatial resolution. Recently, the dynamical theory of electron diffraction was applied to reproduce in great detail backscattering patterns observed in the scanning electron microscope (SEM). However, a fully quantitative comparison of theory and experiment requires angle-resolved measurements of the intensity and the energy of the backscattered electrons, which is difficult to realize in an SEM. This paper determines diffraction patterns of backscattered electrons using an electrostatic analyzer, operating at energies up to 40 keV with sub-eV energy resolution. Measurements are done for different measurement geometries and incoming energies. Generally a good agreement is found between theory and experiment. This spectrometer also allows us to test the influence of the energy loss of the detected electron on the backscattered electron diffraction pattern. It is found that the amplitude of the intensity variation decreases only slowly with increasing energy loss from 0 to 60 eV.     

Beam interactions,contrast and resolution in the SEM

Backscattered and secondary electrons are both used in the SEM for imaging purposes. The backscattered signal is the result of high angle elastic scattering events, while the secondary signal is the result of knock-on inelastic collisions. The characteristic differences between images in the two modes arise from the details of the relevant interactions in the two cases. In order to examine this in a quantitative manner Monte Carlo electron trajectory simulation techniques have been used. Calculations of the ultimate resolution and depth of information of the secondary and backscattered images are presented, together with simulations of the edge brightness effect in high resolution secondary images and an analysis of the microanalytical application of atom number contrast in the backscattered mode.     

Comparison in spatial spreads of secondary electron information between scanning ion and scanning electron microscopy

Monte Carlo simulations have been carried out to compare the spatial spreads of secondary electron (SE) information in scanning ion microscopy (SIM) with scanning electron microscopy (SEM). Under Ga ion impacts, the SEs are excited by three kinds of collision-partners, that is, projectile ion, recoiled target atom, and target electron. The latter two partners dominantly contribute to the total SE yield gamma for the materials of low atomic number Z2. For the materials of high Z2, on the other hand, the projectile ions dominantly contribute to gamma. These Z2 dependencies generally cause the gamma yield to decrease with an increasing Z2, in contrast with the SE yield delta under electron impacts. Most of the SEs are produced in the surface layer of about 5lambda in depth (lambda: the mean free path of SEs), as they are independent of the incident probe. Under 30 keV Ga ion impacts, the spatial spread of SE information is roughly as small as 10 nm, decreasing with an increasing Z2. Under 10 keV electron impacts, the SEI excited by the primary electrons has a small spatial spread of about 5lambda, but the SEII excited by the backscattered electrons has a large one of several 10 to several 100 nanometers, decreasing with an increasing Z2. The main cause of a small spread of SE information at ion impact is the short ranges of the projectile ions returning to the surface to escape as backscattered ions, the recoiled target atoms, and the target electrons in collision cascade. The 30 keV Ga-SIM imaging is better than the 10 keV SEM imaging in spatial resolution for the structure/material measurements. Here, zero-size probes are assumed.     

High-resolution scanning electron microscopy.

The spatial resolution of the scanning electron microscope is limited by at least three factors: the diameter of the electron probe, the size and shape of the beam/specimen interaction volume with the solid for the mode of imaging employed and the Poisson statistics of the detected signal. Any practical consideration of the high-resolution performance of the SEM must therefore also involve a knowledge of the contrast available from the signal producing the image and the radiation sensitivity of the specimen. With state-of-the-art electron optics, resolutions of the order of 1 nm are now possible. The optimum conditions for achieving such performance with the minimum radiation damage to the specimen correspond to beam energies in the range 1-3 keV. Progress beyond this level may be restricted by the delocalization of SE production and ultimate limits to electron-optical performance.     

A methodological basis for SEM autoradiography: biosynthesis and radioligand binding

A method is described for scanning electron microscope (SEM) autoradiography whereby preservation of high resolution cell surface details is retained together with degelatination of the emulsion without gross loss or redistribution of silver grains. This method should provide a convenient medium-sized marker for SEM (using secondary, backscattered electron and X-ray imaging) topographic studies of biosynthesized molecules, and of cell surface receptors and antigens, using indirect or direct labelling procedures with radio-labelled ligands.     

Superposition of chromatic error and beam broadening in transmission electron microscopy of thick carbon and organic specimens.

The chromatic error is calculated using our scattering cross sections obtained from contrast experiments and a distribution function of energy losses from Misell and Burge. The assumed ratio of total inelastic and elastic cross sections was 3.5. Monte-Carlo calculations were performed for the multiple scattering problem of thick carbon specimens using these values for single scattering. As expected, a minimum confusion of the chromatic error disc exists at underfocusing. The half width broadening of an edge is in good agreement with experiments at 100 keV if the experimental method of determining the half width is also taken into consideration theoretically. The lateral displacements of electron paths normal to the direction of the electron incidence, which give rise to poorer resolution at the bottom of a thick specimen in scanning transmission electron microscopy, cannot simply be added to the chromatic error in the normal mode of transmission electron microscopy. Calculations show that there is no difference in edge resolution at 100 keV to be expected, in agreement with experiment. With increasing energy, the influence of beam broadening increases relative to the chromatic error. Considering only the chromatic error (1-2 nm), at 1 MeV and optimum defocus details on the top of a 2 micron specimen will be imaged with nearly twice the value of edge half width as they will at the bottom.     

The measurement of atomic number and composition in an SEM using backscattered detectors

The atomic number dependence of electron backscattering can be used as the basis of a microanalysis technique. The operating procedures and condition for quantitative measurements of specimen atomic number are outlined and an expression relating the accuracy of composition to the atomic number sensitivity has been derived. Some measurements of the spatial resolution of backscattered electron microanalysis are also presented and compared with the resolution of X-ray microanalysis. Although the range of application of this technique is limited, where it can be applied it has the following advantages: (i) higher spatial resolution than X-ray microanalysis for bulk specimens; (ii) very rapid measurement; (iii) can be applied to compounds of low atomic number elements, (e.g. borides, carbides, nitrides, etc.); (iv) specimen preparation is often relatively straightforward.     

New scintillation detector of backscattered electrons for the low voltage SEM

The new scintillation detector of backscattered electrons that is capable of working at primary beam energy as low as 0.5 keV is introduced. Low energy backscattered electrons are accelerated in order to generate a sufficient number of photons. Secondary electrons are deflected back by the energy filter so that the true compositional contrast of the specimen is obtained. The theoretical models of the detector function are described and first demonstration images are presented.     

Enhanced EDX images by fusion of multimodal SEM images using pansharpening techniques

The goal of this paper is to explore the potential interest of image fusion in the context of multimodal scanning electron microscope (SEM) imaging. In particular, we aim at merging the backscattered electron images that usually have a high spatial resolution but do not provide enough discriminative information to physically classify the nature of the sample, with energy‐dispersive X‐ray spectroscopy (EDX) images that have discriminative information but a lower spatial resolution. The produced images are named enhanced EDX. To achieve this goal, we have compared the results obtained with classical pansharpening techniques for image fusion with an original approach tailored for multimodal SEM fusion of information. Quantitative assessment is obtained by means of two SEM images and a simulated dataset produced by a software based on PENELOPE.     

High resolution SEM imaging of gold nanoparticles in cells and tissues

The growing demand of gold nanoparticles in medical applications increases the need for simple and efficient characterization methods of the interaction between the nanoparticles and biological systems. Due to its nanometre resolution, modern scanning electron microscopy (SEM) offers straightforward visualization of metallic nanoparticles down to a few nanometre size, almost without any special preparation step. However, visualization of biological materials in SEM requires complicated preparation procedure, which is typically finished by metal coating needed to decrease charging artefacts and quick radiation damage of biomaterials in the course of SEM imaging. The finest conductive metal coating available is usually composed of a few nanometre size clusters, which are almost identical to the metal nanoparticles employed in medical applications. Therefore, SEM monitoring of metal nanoparticles within cells and tissues is incompatible with the conventional preparation methods. In this work, we show that charging artefacts related to non‐conductive biological specimen can be successfully eliminated by placing the uncoated biological sample on a conductive substrate. By growing the cells on glass pre‐coated with a chromium layer, we were able to observe the uptake of 10 nm gold nanoparticles inside uncoated and unstained macrophages and keratinocytes cells. Imaging in back scattered electrons allowed observation of gold nanoparticles located inside the cells, while imaging in secondary electron gave information on gold nanoparticles located on the surface of the cells. By mounting a skin cross‐section on an improved conductive holder, consisting of a silicon substrate coated with copper, we were able to observe penetration of gold nanoparticles of only 5 nm size through the skin barrier in an uncoated skin tissue. The described method offers a convenient modification in preparation procedure for biological samples to be analyzed in SEM. The method provides high conductivity without application of surface coating and requires less time and a reduced use of toxic chemicals.     

Top-bottom effect in energy-selecting transmission electron microscopy

Energy-selecting TEM can avoid the chromatic error of large specimen thicknesses when selecting an energy window at the most probable energy loss. The resolution is limited by the spatial beam broadening for structures at the top (electron entrance) of the specimen layer. A test experiment with polystyrene spheres of 1.1 μm in diameter shows a blurring of 8 nm when imaging with ΔE = 250 eV and E = 80 keV.     

Characterization of polymer blends and block copolymers by conventional and low voltage SEM

Methods for characterizing block copolymers and polymer blends have been developed. Results from unstained and ruthenium tetroxide-stained samples obtained by scanning electron microscopy at various acceleration voltage and by transmission electron microscopy are presented. The contrast in secondary images between components in stained polymer blends, where one component is preferentially stained, is maximized at higher acceleration voltage (10–25 keV). For measurement of particle size and shape, this is the preferred operating condition. To obtain high-resolution images showing surface topography and fine structure, such as 20 nm domains in block copolymers, low-voltage (<5 keV) imaging is preferred. Observation of the 20 nm domain structure in hydrogenated styrenebutadiene-styrene shows that the spatial resolution now possible by scanning electron microscopy is comparable to that obtained by the traditional method of transmission electron microscopy.     

Theoretical study of the ultimate resolution of SEM

This work aims to clarify the problem of why the ultimate resolution assessed experimentally from the observation of 0.8 nm separation of Au-Pd fine particles is beyond the theoretical resolution limit of scanning electron microscopy (SEM). The correlation between the spatial distribution of secondary electrons on a sample surface and the resolution estimated by edge-to-edge separation in SEM was studied by a Monte Carlo simulation with secondary electron generation included. The result clearly indicates that the edge-to-edge separation can extend beyond the theoretical ultimate resolution, particularly by image processing for contrast expansion and by improving the signal to noise ratio (S/N).     

A model for calculating secondary and backscattered electron yields

Images in the scanning electron microscope (SEM) are formed from both low-energy secondary, and high-energy backscattered, electrons. The quantitative interpretation of SEM images therefore requires a model which can predict the magnitude of both of these signal components for a specimen whose geometry and chemistry is known. It is shown that the combination of a simple electron diffusion model with a Monte Carlo trajectory simulation allows both yields to be calculated, simultaneously, with good accuracy. Data, such as the magnitude and energy of the maximum secondary yield, the secondary variation with tilt, and the contribution of backscattered electrons to the secondary yield coefficient, computed from this model are in excellent agreement with experimental data.     

Objective comparison of scanning ion and scanning electron microscope images

Common and different aspects of scanning electron microscope (SEM) and scanning ion microscope (SIM) images are discussed from a viewpoint of interaction between ion or electron beams and specimens. The SIM images [mostly using 30 keV Ga focused ion beam (FIB)] are sensitive to the sample surface as well as to low-voltage SEM images. Reasons for the SIM images as follows: (1) no backscattered-electron excitation; (2) low yields of backscattered ions; and (3) short ion ranges of 20–40nm, being of the same order of escape depth of secondary electrons (SE) [=(3–5) times the SE mean free path]. Beam charging, channeling, contamination, and surface sputtering are also commented upon.     

High-resolution backscatter electron imaging of colloidal gold in LVSEM

High‐resolution backscatter electron (BSE) imaging of colloidal gold can be accomplished at low voltage using in‐lens or below‐the‐lens FESEMs equipped with either Autrata‐modified yttrium aluminium garnet (YAG) scintillators doped with cerium, or with BSE to secondary electron (SE) conversion plates. The threshold for BSE detection of colloidal gold was 1.8 keV for the YAG detector, and the BSE/SE conversion was sensitive down to 1 keV. Gold particles (6, 12 and 18 nm) have an atomic number of 79 and were clearly distinguished at 500 000× by materials contrast and easily discriminated from cell surfaces coated with platinum with an atomic number of 78. BSE imaging was relatively insensitive to charging, and build up of carbon contamination on the specimen was transparent to the higher energy BSE.