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Serial block-face electron microscopy (SBEM) provides nanoscale 3D ultrastructure of embedded and stained cells and tissues in volumes of up to 107 µm3. In SBEM, electrons with 1–3 keV energies are incident on a specimen block, from which backscattered electron (BSE) images are collected with x, y resolution of 5–10 nm in the block-face plane, and successive layers are removed by an in situ ultramicrotome. Spatial resolution along the z-direction, however, is limited to around 25 nm by the minimum cutting thickness. To improve the z-resolution, we have extracted depth information from BSE images acquired at dual primary beam energies, using Monte Carlo simulations of electron scattering. The relationship between depth of stain and ratio of dual-energy BSE intensities enables us to determine 3D structure with a ×2 improvement in z-resolution. We demonstrate the technique by sub-slice imaging of hepatocyte membranes in liver tissue.

“High resolution SEM imaging” refers to the capability of discerning fine-scale spatial features of a specimen. Such features may be free-standing objects or structures embedded in a matrix. The definition of “fine-scale” depends on the application, which may involve sub-nanometer features in the most extreme cases. The most important factor determining the limit of spatial resolution is the footprint of the incident beam as it enters the specimen. Depending on the level of performance of the electron optics, the limiting beam diameter can be as small as 1 nm or even finer. However, the ultimate resolution performance is likely to be substantially poorer than the beam footprint and will be determined by one or more of several additional factors: (1) delocalization of the imaging signal, which consists of secondary electrons and/or backscattered electrons, due to the physics of the beam electron specimen interactions; (2) constraints imposed on the beam size needed to satisfy the Threshold Equation to establish the visibility for the contrast produced by the features of interest; (3) mechanical stability of the SEM; (4) mechanical stability of the specimen mounting; (5) the vacuum environment and specimen cleanliness necessary to avoid contamination of the specimen; (6) degradation of the specimen due to radiation damage; and (7) stray electromagnetic fields in the SEM environment. Recognizing these factors and minimizing or eliminating their impact is critical to achieving optimum high resolution imaging performance. Because achieving satisfactory high resolution SEM often involves operating at the performance limit of the instrument as well as the technique, the experience may vary from one specimen type to another, with different limiting factors manifesting themselves in different situations. Most importantly, because of the limitations on feature visibility imposed by the Threshold Current/Contrast Equation, for a given choice of operating conditions, there will always be a level of feature contrast below which specimen features will not be visible. Thus, there is always a possible “now you see it, now you don’t” experience lurking when we seek to operate at the limit of the SEM performance envelope.

Electron beams have made possible the development of the versatile, high performance electron microscopes described in the earlier chapters of this book. Techniques for the generation and application of electron beams are now well documented and understood, and a wide variety of images and data can be produced using readily available instruments. While the scanning electron microscope (SEM) is the most widely used tool for high performance imaging and microanalysis, it is not the only option and may not even always be the best instrument to choose to solve a particular problem. In this chapter we will discuss how, by replacing the beam of electrons with a beam of ions, it is possible to produce a high performance microscope which resembles an SEM in many respects and shares some of its capabilities but which also offers additional and important modes of operation.

By selecting the operating parameters of the SEM electron gun, lenses, and apertures, the microscopist controls the characteristics of the focused beam that reaches the specimen surface: energy (typically selected in the range 0.1–30 keV), diameter (0.5 nm to 1 μm or larger), beam current (1 pA to 1 μA), and convergence angle (semi-cone angle 0.001–0.05 rad). In a conventional high vacuum SEM (typically with the column and specimen chamber pressures reduced below 10−3 Pa), the residual atom density is so low that the beam electrons are statistically unlikely to encounter any atoms of the residual gas along the flight path from the electron source to the specimen, a distance of approximately 25 cm.

A conducting or semiconducting specimen must maintain good contact with electrical ground to dissipate the injected beam current. Without such an electrical path, even a highly conducting specimen such as a metal will show charging artifacts, in the extreme case acting as an electron mirror and reflecting the beam off the specimen. A typical strategy is to use an adhesive such as double-sided conducting tape to both grip the specimen to a support, for example, a stub or a planchet, as well as to make the necessary electrical path connection. Note that some adhesives may only be suitable for low magnification (scanned field dimensions greater than 100 × 100 μm, nominally less than 1,000× magnification) and intermediate magnification (scanned field dimensions between 100 μm x 100 μm, nominally less than 1,000X magnification and 10 μm × 10 μm, nominally less than 10,000× magnification) due to dimensional changes which may occur as the adhesive outgases in the SEM leading to image instability such as drift. Good practice is to adequately outgas the mounted specimen in the SEM airlock or a separate vacuum system to minimize contamination in the SEM as well as to minimize further dimensional shrinkage. Note that some adhesive media are also subject to dimensional change due to electron radiation damage during imaging, which can also lead to image drift.

The detection in SEM images of specimen features such as compositional differences, topography (shape, inclination, edges, etc.), and physical differences (crystal orientation, magnetic fields, electrical fields, etc.), depends on satisfying two criteria: (1) establishing the minimum conditions necessary to ensure that the contrast created by the beam–specimen interaction responding to differences in specimen features is statistically significant in the imaging signal (backscattered electrons [BSE], secondary electrons [SE], or a combination) compared to the inevitable random signal fluctuations (noise); and (2) applying appropriate signal processing and digital image processing to render the contrast information that exists in the signal visible to the observer viewing the final image display.

Over the past fifty years the scanning electron microscope (SEM) has established itself as the most versatile and productive tool for imaging and microanalysis in many areas of science and technology, and some seventy-thousand instruments generate millions of micrographs every day. Scanning electron microscopes do, however, have one fundamental limitation in that the only experimental variable available to the operator is the choice of the accelerating voltage. Although the ability to vary beam energy is both necessary and important, it is an unfortunate fact that changing the beam energy also alters many aspects of performance: imaging resolution, relative strength of different signal components, depth of beam penetration, capabilities of the various analytical systems, and the severity of charging and beam-induced damage. This makes it difficult or impossible to optimize the interaction of interest.

Summary Generally, in scanning electron microscopy (SEM) imaging, it is desirable that a high-resolution image be composed mainly of those secondary electrons (SEs) generated by the primary electron beam, denoted SEI. However, in conventional SEM imaging, other, often unwanted, signal components consisting of backscattered electrons (BSEs), and their associated SEs, denoted SEII, are present; these signal components contribute a random background signal that degrades contrast, and therefore signal-to-noise ratio and resolution. Ideally, the highest resolution SEM image would consist only of the SEI component. In SEMs that use conventional pinhole lenses and their associated Everhart–Thornley detectors, the image is composed of several components, including SEI, SEII, and some BSE, depending on the geometry of the detector. Modern snorkel lens systems eliminate the BSEs, but not the SEIIs. We present a microfabricated diaphragm for minimizing the unwanted SEII signal components. We present evidence of improved imaging using a microlithographically generated pattern of Au, about 500 nm thick, that blocks most of the undesired signal components, leaving an image composed mostly of SEIs. We refer to this structure as a “spatial backscatter diaphragm.” SCANNING 35:1-6, 2013. © 2012 Wiley Periodicals, Inc.

The scanning electron microscope (SEM) has become the most widely used of all advanced imaging tools because it offers a unique range of capabilities. It can resolve and image objects with sizes ranging from millimeters to below 1 nm; it offers multiple ways to generate, collect, and display signals; the images produced contain information about the topography, chemical composition, and the magnetic, electrostatic, and crystallographic properties of the sample; and it can generate characteristic x-ray emission from the specimen to provide a quantitative chemical analysis. Unfortunately, one thing that it will be unable to do is maintain its competitive edge in the 21st century. This is because electrons are electromagnetic ‘‘waves’’ and thus the smallest spot, ‘‘d,’’ into which an electron beam can be focused has a diameter of the order of: d 1⁄4 k=a ð1Þ

Because the ability to perform some form of chemical microanalysis has become an essential feature for any microscope, it is necessary to investigate what options are available in the new “ORION” helium ion microscope (HIM). The HIM has the ability to visualize local variations in specimen chemistry in both the ion induced secondary electron and the Rutherford backscattered imaging modes, but this provides only limited and qualitative information. Quantitative, elementally specific, microanalysis could be performed in the HIM using secondary electron spectroscopy, Rutherford backscattered ion spectroscopy, or secondary ion mass spectroscopy, but while each of these options has promise, none of them can presently guarantee either reliable element identification or quantitative analysis across the periodic table.

The scanning helium ion microscope has been used in transmission mode to investigate both the feasibility of this approach and the utility of the signal content and the image information available. Operating at 40 keV the penetration of the ion beam, and the imaging resolution achieved, in MgO crystals was found to be in good agreement with values predicted by Monte Carlo modeling. The bright-field and annular dark-field signals displayed the anticipated contrasts associated with beam absorption and scattering. In addition, the diffraction of the He ion beam within the sample gave rise to crystallographic contrast effects in the form of thickness fringes and dislocation images. Scanning transmission He ion microscopy thus achieves useful sample penetration and provides nanometer scale resolution, high contrast images of crystalline materials and crystal defects even at modest beam energies.

In many applications such as optoelectronic devices, three-dimensional (3D) structures are required. Examples include photonic band gap (PBG) crystals, diffractive optical elements, blazed gratings, MEMS, NEMS, etc. It is known that the performance characteristics of such structures are highly sensitive to their dimensional fidelity. Therefore, it is essential to have a fabrication process by which such 3D structures can be realized with high dimensional accuracy. In this paper, practical methods to control thickness of the remaining resist and etch depth, which may be employed for fabrication of such 3D structures using grayscale electron-beam lithography, are described. Through experiments, explicit control of the remaining resist thickness and etch depth at the resolution of 20nm for the feature sizes of 0.5@mm and 1@mm has been successfully demonstrated. Also, the 1:1 ratio of silicon to resist etching rates was achieved for transferring the remaining resist profile onto the silicon substrate.

The phase-contrast imaging of atomic lattices has now become commonplace for both Transmission Electron Microscopes (TEM) and Scanning Transmission Electron Microscopes (STEMs). Recently, however, bright-field STEM images of multi-wall carbon nanotubes (MWCNTs) recorded from an ultra-high resolution (UHR) in-lens field-emission scanning electron microscope (FE-SEM) operating at 30keV have also demonstrated lattice fringe resolution. One example of such an image containing multiple examples of fringe detail is shown in figure 1. The carbon lattice fringes were analyzed and their origin confirmed by the application of the FFT algorithms in the SMART image analysis program. The resulting power spectrum after thresholding to remove background noise (Figure 2) confirms that phase detail in the image extends down to about 5 Angstroms (0.5nm) and that well defined diffraction spots corresponding to a spacing of 3.4 Angstroms (0.34nm) generated by the (002) basal plane spacing of the graphite lattice are present.

A Monte Carlo simulation of the contrast variation across phase boundaries in backscattered electron images of multiple phase composition systems has been used to develop a model for prediction of the position of the interface based solely on a knowledge of the signal intensity levels on either side of the interface. A wide range of average atomic numbers of the phases on either side of the boundary have been investigated at incident beam energies of 5, 10, 15, and 25 kV and a least-squares minimisation procedure used to optimise the parameters of a generalised model.

A field-emission scanning electron microscope (FESEM) equipped with the standard secondary electron (SE) detector was used to image thin (70-90 nm) and thick (1-3 microns) sections of biological materials that were chemically fixed, dehydrated, and embedded in resin. The preparation procedures, as well as subsequent staining of the sections, were identical to those commonly used to prepare thin sections of biological material for observation with the transmission electron microscope (TEM). The results suggested that the heavy metals, namely, osmium, uranium, and lead, that were used for postfixation and staining of the tissue provided an adequate SE signal that enabled imaging of the cells and organelles present in the sections. The FESEM was also used to image sections of tissues that were selectively stained using cytochemical and immunocytochemical techniques. Furthermore, thick sections could also be imaged in the SE mode. Stereo pairs of thick sections were easily recorded and provided images that approached those normally associated with high-voltage TEM.

Experimental nanotips have shown significant improvement in the resolution performance of a cold field emission scanning electron microscope (SEM). Nanotip electron sources are very sharp electron emitter tips used as a replacement for the conventional tungsten field emission (FE) electron sources. Nanotips offer higher brightness and smaller electron source size. An electron microscope equipped with a nanotip electron gun can provide images with higher spatial resolution and with better signal-to-noise ratio. This could present a considerable advantage over the current SEM electron gun technology if the tips are sufficiently long-lasting and stable for practical use. In this study, an older field-emission critical dimension (CD) SEM was used as an experimental test platform. Substitution of tungsten nanotips for the regular cathodes required modification of the electron gun circuitry and preparation of nanotips that properly fit the electron gun assembly. In addition, this work contains the results of the modeling and theoretical calculation of the electron gun performance for regular and nanotips, the preparation of the SEM including the design and assembly of a measuring system for essential instrument parameters, design and modification of the electron gun control electronics, development of a procedure for tip exchange, and tests of regular emitter, sharp emitter and nanotips. Nanotip fabrication and characterization procedures were also developed. Using a "sharp" tip as an intermediate to the nanotip clearly demonstrated an improvement in the performance of the test SEM. This and the results of the theoretical assessment gave support for the installation of the nanotips as the next step and pointed to potentially even better performance. Images taken with experimental nanotips showed a minimum two-fold improvement in resolution performance than the specification of the test SEM. The stability of the nanotip electron gun was excellent; the tip stayed useful for high-resolution imaging for several hours during many days of tests. The tip lifetime was found to be several months in light use. This paper summarizes the current state of the work and points to future possibilities that will open when electron guns can be designed to take full advantage of the nanotip electron emitters.

Charging is one of the most important problems encountered in scanning electron microscopy and as a result this phenomenon it has received a lot of both theoretical and experimental attention. Despite this, many questions remain about the nature and behavior of charging because of the limitations of the experimental techniques available to study it. For example, although it is now straightforward to determine in situ the surface potential of a sample that is charging during irradiation, it is difficult to measure the lateral extent of the charging, or its persistence once the incident beam is switched off. We describe here a simple technique which provides a rapid way of visualizing the temporal and spatial behavior of charging phenomena.

The relationship between convolution/correlation operation and the data acquisition process of the scanning microscope and spectrometer families is analyzed. It is shown that a coordinate or event sensitive detector, and the intrinsic or extrinsic property of the specimen response, are two important factors in distinguishing the data acquisition mode of such systems. Four types of convolution- and correlation-based modes are extracted to illuminate the physical characteristics of scanning imaging and analysis systems by focusing on the probe, specimen, detector, and their relationships. Criteria for identifying these modes are explored. In addition, the physical meanings of general existing coefficients between the independent variables of convolution and correlation are investigated.

Low voltage scanning electron microscopy (LVSEM) has become the imaging technique of choice for many applications because it offers surface specific information, and both control of charging and a reduction of damage to labile specimens. However fundamental limitations such as the electron wavelength, lens aberrations, and gun brightness, severely limit the performance of the LVSEM, and in addition some capabilities such as microanalysis are difficult or impossible. New technologies, including aberration correction and brighter electron sources are now available, and novel spectroscopic approaches are in development. Together these advances could significantly enhance the performance of the LVSEM. The advent of ultra-high performance ion column imaging systems offers interesting competition to the LVSEM for both imaging and analysis. [DOI: 10.1380/ejssnt.2006.369]

The development of the microcalorimeter energy-dispersive X-ray spectrometer (µ-cal EDS) offers a significant advancement in X-ray microanalysis, especially for electron beam instruments. The benefits are especially pronounced for low voltage (≤5 kV) X-ray microanalysis in the field emission scanning electron microscope (FE-SEM) where the high energy resolution of the µ-cal EDS minimizes the peak overlaps among the myriad of K, L, M and N lines in the 0–5 keV energy range. The availability of L- and M-shell X-ray lines for microanalysis somewhat offsets the absence of X-ray lines traditionally used above 5 keV energy. The benefits and challenges of the µ-cal EDS will be discussed, including P/B ratio for characteristic X-rays, collection angle, count rate capability and the impact of polycapillary X-ray optics on microanalysis.

We have designed a low voltage point source microscope operated with a nanotip field emitter and without any electron optical lenses such that it can be operated in the transmission mode as well as in a reflection mode. An electron gun needed in the reflection mode is the most important difference between the two modes of operation. The magnification of the object wave is achieved by placing the specimen in the divergent electron beam of a nanotip field emitter and observing the object wave using a micro channel plate (MCP) at a great distance from the sample. As no lenses are present a procedure for scaling the magnification has been developed. Since electrons from a point source are highly coherent the out of focus images of the sample are interferograms and contain true three dimensional information of the sample. The transmission mode gives us the following results: (1) Electrons diffracted at an edge of the specimen cause Fresnel fringes in the image plane. (2) An electrically charged holey carbon foil can act in the same way on the electrons as the Young's double slit experiment and results in an interference pattern consisting of parallel fringes. The reflection mode shows us: (1) The image depends on the angle of reflection. (2) An arrangement similar to a field ion microscope resolves single atoms of the specimen. [DOI: 10.1380/ejssnt.2004.81]

We briefly discuss how to set-up our Hitachi HF-2000 transmission electron microscope for medium magnification holography. Then we apply this technique to examine the activation of an as-doped wafer by annealing. We also present voltage profiles of the source-drain region of a CMOS transistor with 75 nm gate architecture taken from an off-the-shelf Intel PIII processor. Special attention is given to the analysis of the reconstructed holograms. [DOI: 10.1380/ejssnt.2004.119]

Serial block face scanning electron microscopy (SBF-SEM) provides nanoscale 3D ultrastructure of entire cells and tissue volumes. In SBF-SEM, an ultramicrotome built into the SEM specimen stage successively removes thin sections from a plastic-embedded, heavy metal-stained specimen. After each cut, the freshly exposed block face is imaged at a low incident electron energy using a backscattered electron detector to provide 3D ultrastructure with a resolution of approximately 5 nm in the plane of the block face and around 25 nm in the perpendicular z-direction, as limited by the slice thickness. We have explored the feasibility of improving the z-resolution in SBF-SEM by recording images at multiple primary beam energies, thus sampling different depths below the block surface.A linear relationship was found between the depth of test structures, generated by Monte Carlo simulations, and the ratio of backscattered image intensities recorded at primary beam energies between 1.4 keV and 6.8 keV. This enabled us to reconstruct the 3D model within a 25-nm surface layer at a z-resolution of around 5 nm. We used a Zeiss Sigma-VP SEM equipped with a Gatan 3View SBF system to acquire 3D data from a specimen consisting of gold spheres embedded in carbon. Experiments were also performed on embedded blocks of stained biological tissues.Although damage of the block under electron irradiation limits the signal to noise ratio, the use of multiple primary beam energies, coupled with a physics-based Monte Carlo model, provides the possibility of obtaining cellular ultrastructure at nearly isotropic 3D spatial resolution.

By applying the basic principles of metrology we discuss how to define the standards that any experimental method to measure resolution has to obey. Our results clearly indicate the need to apply a calibration procedure when designing algorithms to estimate resolution to satisfy accuracy requirements. Similarly, the precision of an algorithm has to be clearly specified. We compare here the performances of a variety of commonly used implementations of published methods, with that of an algorithm based on an approach known to be reliable. Our results confirm that when an algorithm is designed with the clear intent of satisfying metrology requirements, it demonstrates excellent accuracy, precision, and lack of sensitivity to the noise level, as is desirable. As a consequence, the algorithm will have the ability to measure accurately the point spread function convoluted in the image, thus paving the way for quantitative deconvolution techniques.

A simple method is described to determine the effective gas path length when incident electrons scatter in the gas above the specimen. This method is based on the measurement of a characteristic x-ray line emitted from a region close to the incident beam. From various experimental measurements performed on various microscopes, it is shown that the effective gas path length may increase with the chamber pressure and that it is also often dependent of the type of x-ray bullet.

Secondary electron imaging is not possible in the variable pressure scanning electron microscope because the mean free path of the secondaries in the gas is too short to permit them to reach the detector. This paper therefore investigates an alternative strategy for producing an image containing significant amounts of secondary electron contrast. This involves modifying the microscope by the addition of a biased electrode above the sample and then collecting a specimen current signal. This system, originally described by Farley and Shah (1988), is found to produce true secondary electron detail over a wide range of conditions.

Low voltage scanning electron microscopy (LVSEM) is the application of the SEM at beam energies below 5 keV. The fall in electron beam range compared to its magnitude at higher energies leads to significant changes in the beam interaction volume and in the secondary and backscattered electron yields. The topographic and beam penetration contrast effects which dominate images at high energies are replaced by detector collection efficiency contrast effects giving images which are less three dimensional but which contain more detailed information on the surface morphology and, in some circumstances, the surface chemistry of the specimen. In order to observe non-conducting specimens a state of charge balance must be obtained to obviate imaging artifacts. This requires an optimized choice of the incident beam energy, sample tilt, beam current and magnification for each sample. The high stopping power of electrons at low energy can result in enhanced radiation damage. However, because of the small electron range such damage is confined to a thin, near surface, region of the specimen. The combination of a field emission gun and a high performance lens allows the probe size of the instrument to be made almost independent of the chosen beam energy over the range 1–30 keV and probable advance in electron sources and electron optics promise still better levels of performance for the LVSEM.

As noted in the previous section, the present ALIS helium ion source is a descendant of the original work based on FIM technology (for a historical overview see Muller and Tsong 1993) although important research in this area has also been carried out by several other prominent groups (e.g. Orloff and Swanson 1977). In order to be suitable for application, in a high-performance particle beam microscope, the source should ideally not only be bright, but also be as compact as possible to ensure mechanical stability, provide highly stable emission over time periods of several hours, be capable of operating at energies at least in the 10–50 keV range, and be capable of being re-formed and then reused multiple times without a significant change in performance. An overview of history of the helium ion microscope can be found in the literature (Economou 2011), while other technical details can be found in the published patents listed at the end of the bibliography.

A feature of the GFIS ion source is that every aspect of its operation and behavior—from its imaging resolution, the energy range over which it operates, the efficiency of signal production, and the damage it does to the materials that it examines—is ultimately affected by the choice of imaging gas. Ideally, the same source could rapidly be reconfigured to select and generate any one of a number of different ion beams. Because each type of ion has its own strengths and weaknesses, this feature would add substantially to the utility of the ion microscope.

Every microscope requires a high brightness, reliable, stable source of illumination in order to function, and both the quality and the quantity of the illumination provided will determine, and ultimately limit, the performance of the instrument. Each type of microscope will have its own type of illuminating source. For a high-performance scanning electron or ion microscope, the most desirable property of the beam source is that the source must have a high brightness.

For many users, the most important application of an SEM is its ability to identify the chemical composition of a specimen. Energy dispersive spectroscopy (EDS) of the X-rays fluoresced from samples of interest by the incident electron beam provides chemical microanalysis combining unparalleled sensitivity together with high spatial resolution for elements across the entire periodic table. This technique would therefore also be the automatic first choice for microanalysis when using ion beams if it were a viable option.

In this paper we will discuss our recent experimental results, using electron holography and electron interferometry, of studies of ferroelectric domain walls in BaTiO3 and PLZT thin specimens.

The CD-SEM has been the tool of choice for the imaging and metrology of semiconductor devices for the past three decades but now, with critical dimensions at the nanometer scale, electron beam instruments can no longer deliver adequate performance. A scanning microscope using a He+ ion beam offers superior resolution and depth of field, and provides enhanced imaging contrast. Device metrology performed using ion beam imaging produces data which is comparable to or better than that from a conventional CD-SEM although there are significant differences in the experimental conditions required and in the details of image formation. The charging generated by a He+ beam, and the sample damage that it can cause, require care in operation but are not major problems.

An electron holographic method is introduced for the study of ferroelectric domain walls. The method allows not only direct imaging of the domain wall but also the measurement of local spontaneous polarization. Preliminary results of first high-resolution imaging of ferroelectric domain walls by using this technique in BaTiO3 and quantitative measurements of electrostatic field distribution across domain walls are presented.

The electron-optical parameters required to generate an electron hologram are discussed. It is shown that the new generation of commercial field emission transmission electron microscopes (FEG TEM) meet the conditions required. Practical details of the procedures to set up and record a hologram are described. The steps necessary to reconstruct the hologram are also discussed and an outline is provided of a program for the Apple Macintosh™ personal computer which implements these operations. The integrity and accuracy of the reconstruction program has been tested by means of a detailed simulation of high-resolution holograms starting from dynamical multi-slice calculations of the exit wavefront.

Although Scanning Electron Microscopes (SEM) have improved greatly over the last decade the techniques usually employed to measure their performance have not changed significantly in half a century. In particular, describing the imaging performance of an SEM by a single number - its 'resolution' - provides no useful information about its real world imaging capabilities nor about any of the factors that might limit that usefulness of the SEM for tasks such as metrology. The Contrast Transfer Function (CTF) discussed here analyses the way in which the SEM processes signal components of different spatial frequencies. The resultant plot provides information on the noise limited spatial resolution limit, predicts how this will vary with noise level, and provides a powerful general diagnostic capability. This type of measurement, which has become standard practice for transmission electron microscopes, can be performed using the public domain software package IMAGE-J, is rapid, and requires only a specimen offering a broad and flat Fourier spectrum. The capabilities of this approach are demonstrated by a number of examples.

Recent advances in instrumentation have produced the first commercial examples of what can justifiably be called high-resolution scanning electron microscopes. The key components of such instruments are a cold field emission gun, a small-gap immersion probe-forming lens, and a clean dry-pumped vacuum. The performance of these microscopes is characterized by several major features including a spatial resolution, in secondary electron mode on solid specimens, which can exceed 1 nm on a routine basis; an incident probe current density of the order of 10 6 A/cm 2 ; and the ability to maintain these levels of performance over an accelerating voltage range of from 1 to 30 keV. This combination of high resolution, high probe current, low contamination and flexible electron-optical conditions provides many new opportunities for the application of the SEM to materials science, physics, and the life sciences.

SUMMARY Current scanning electron microscopes, equipped with field emission guns and high-performance immersion lenses, can achieve spatial resolutions of the order of 1 nm in both secondary and backscattered imaging modes over a wide range of operating energies. The generation and interpretation of images with nanometre-scale resolution relies on a detailed knowledge, and application, of electron-solid interactions. This paper develops the practical steps required to produce a high-resolution image, and discusses the principles which govern image interpretation. Attention is focused primarily on materials which are low in atomic number and density, such as biological tissue, but the results apply after appropriate scaling of the physical parameters to most other materials.

Until recently high resolution TEM was the only imaging mode capable of probing the atomic lattice structure of crystals composing tooth enamel. Studies designed to determine the polyhedral shape of normal enamel crystals and initiation of carious lesions in enamel crystals were hampered and limited by interpretation of two-dimensional TEM images from thin section and freeze fracture replica specimens lacking depth of field. The newly developed SE-I signal mode for SEM (SE-I/SE-II ratio) can produce images of enamel crystals approaching beam diameter dimensions (0.7–2.0 nm), rivaling the resolution of the TEM technique and generating topographic contrasts for three dimensional imaging at very high magnification (≈1,000,000 X). Ultrathin chromium (Cr) films generate enriched high resolution SE-I contrasts of enamel crystal surfaces and when imaged using an immersion lens field emission SEM operated at high voltage (20–30 KeV) produce unsurpassed topographic contrasts. Since the grain size of Cr is below the resolution of any SEM and is ultrathin (≈1 nm), then SE-I images can provide a more accurate representation of enamel crystal structure than TEM methodologies. Our SE-I SEM observations of normal human enamel crystals reveal fractured spicules which contain angled flat surfaces delineated by a prominent 2 nm wide SE-I edge brightness contrast. Although microscopic observations often show crystals which are hexagonal in cross-section, in both SEM and TEM many other growth habits, including rectangular or irregular crystals (30–40 nm in width) which contain “notches,” are also observed. More detailed morphological studies are therefore required to determine the most likely habit planes and their relevance to the function of the enamel crystals. The granular appearing fine structural contrast imposed onto lattice planes of sectioned enamel in TEM micrographs is also resolved with topographic contrasts in SE-I micrographs. These granules probably represent one or both of the enamel protein classes.

Noise is the single most important limiting factor in scanning electron microscopy. Because of the presence of noise, we are forced to operate the SEM to maximize the available beam current and the beam dose (current × time) at the expense of degraded image resolution, increased charging, and more sample damage. Recent developments in high-performance electron guns, aberration correctors, and lenses are all part of an attempt to attain control of the noise while still achieving ever higher levels of resolution. In this chapter, we will examine noise in the SEM, its origin and properties, its measurement, and how the properties of the detectors used for the collection of secondary emission (SE) electrons and backscatter electrons (BSE) signals affect the noise.

The physical dimensions of nanoscale objects are an important indicator of their functionality. However, measuring feature size from a SEM image is difficult not only because of fundamental considerations, such as the nature of beam interactions and the information transfer properties of the microscope, but because the magnification of the SEM image from which a measurement will be made is completely uncalibrated and additionally is subject to local distortions and variations. Nano-gauges fabricated by electron beam lithography - one or two dimensional structures on the size scale of the objects of interest - provide a local length standards within the image field from which the relative size of features can be accurately determined. In order to provide an absolute measurement of size the dimensions of the nano-gauge structure must themselves be calibrated against some primary standard. Because there are no convenient standards of appropriate scale available we propose that this can be done using a moire fringe technique to bridge the gap between the nanoscale and common length standards such as ruled diffraction gratings.