An annular toroidal backscattered electron energy analyser for use in scanning electron microscopy

A backscattered electron energy spectrometer, based on a toroidal energy analyser and an annular detector, has been devised and adapted for use in a scanning electron microscope. Computer simulations have been carried out for equipotentials and trajectories of electrons in the toroidal deflector, which permit the optimisation of the energy analyser characteristics for special applications. Based upon these results, a device has been built and its efficiency is demonstrated by selected images of a multilayered structure and a series of recorded backscattered electron spectra.     

Topographic and material contrast in low-voltage scanning electron microscopy

Two scintillation backscattered electron (BSE) detectors with a high voltage applied to scintillators were built and tested in a field emission scanning electron microscope (SEM) at low primary beam energies. One detector collects BSE emitted at low take-off angles, the second at high takeoff angles. The low take-off detector gives good topographic tilt contrast, stronger than in the case of the secondary electron (SE) detection and less sensitive to the presence of contamination layers on the surface. The high take-off detector is less sensitive to the topography and can be used for detection of material contrast, but the contrast becomes equivocal at the beam energy of 1 keV or lower.     

Correlative light and backscattered electron microscopy of bone--Part I: Specimen preparation methods

A method for preparing nondecalcified bone and tooth specimens for imaging by both light microscopy (LM) and backscattered electron microscopy in the scanning electron microscope (BSE-SEM) is presented. Bone blocks are embedded in a polymethylmethacrylate (PMMA) mixture and mounted on glass slides using components of a light-cured dental adhesive system. This method of slide preparation allows correlative studies to be carried out between different microscopy modes, using the same histologic section. It also represents a large time savings relative to other mounting methods whose media require long cure times.     

Internal medulla of hair revealed by scanning electron microscopy

The standard scanning electron microscope method for examining surface structure using the secondary electron mode has unexpectedly revealed the internal ladder-type medulla of some animal hairs.     

Fundamental problems of imaging subsurface structures in the backscattered electron mode in scanning electron microscopy

In‐depth imaging of subsurface structures in scanning electron microscopy (SEM) is usually obtained by detecting backscattered electrons (BSE). For a layer‐by‐layer imaging in BSE microtomography, it is preferable to use an energy filtering of BSE. A simple approach is used to estimate the contrast by using backscattering coefficients of bulk materials and the maximum escape depths of the BSE. The contrast obtained by BSE energy filtering is about twice that of the standard BSE method by varying the acceleration voltage. The contrast decreases with increasing information depth. The information depth is about four times smaller than the electron range. The transmission of the spectrometer influences the minimum current of the order of 10^?8 A that is needed to get a contrast of 1%, for example.     

Influence of the angular distribution of backscattered electrons on signals at different take-off angles in low-voltage scanning electron microscopy (LVSEM)

It is well known that the differential Mott cross section for large-angle elastic scattering shows maxima and minima at angles depending on material and electron energy. For electron energies of 10–30 keV, the averaging by frequent elastic scattering processes results in approximate Lambert angular distributions of backscattered electrons (BSE). However, the present Monte Carlo calculations for electron energies E = 1–5 keV and different angles of incidence show strong deviations from a Lambert distribution which increases with decreasing energy. The signals of the BSE detector with five annular segments for different take-off directions show good agreement with the calculations for normal electron incidence.     

Comparison of photon transport efficiency in simple scintillation electron detector configurations for scanning electron microscope

The purpose of this paper is to find some general rules for the design of robust scintillation electron detectors for a scanning electron microscope (SEM) that possesses an efficient light-guiding (LG) system. The paper offers some general instructions on how to avoid the improper design of highly inefficient LG configurations of the detectors. Attention was paid to the relevant optical properties of the scintillator, light guide, and other components used in the LG part of the scintillation detector. Utilizing the optical properties of the detector components, 3D Monte Carlo (MC) simulations of photon transport efficiency in the simple scintillation detector configurations were performed using the computer application called SCIUNI to assess shapes and dimensions of the LG part of the detector. The results of the simulation of both base-guided signal (BGS) configurations for SE detection and edge-guided signal (EGS) configurations for BSE detection are presented. It is demonstrated that the BGS configuration with a matted disc scintillator exit side connected to the cylindrical light guide without optical cement is almost always a sufficiently efficient system with a mean LG efficiency of about 20%. It is simulated that poorly designed EGS strip configurations have an extremely low mean LG efficiency of only 0.01%, which can significantly reduce detector performance. On the other hand, no simple nonoptimized EGS configuration with a light guide widening to a circular or square profile, with a polished cemented scintillator and with an indispensable hole in it has a mean LG efficiency lower than 6.5%.     

Scanning reflection electron microscopy of surface topography by diffusely scattered electrons in the scanning electron microscope

In this paper, the technique of scanning reflection electron microscopy (SREM) by diffusely scattered electrons in the scanning electron microscope is described in detail. A qualitative account of the formation of image contrast in SREM is also described. It is assumed that, for grazing geometry, forward-scattered electrons reflect from regions close to the surface, following a few scattering events within the first few atomic layers, and lose very little energy in the process. The penetration depth of the primary electrons is very limited, resulting in strongly peaked envelopes of forward-scattered electrons. It is also assumed that a surface containing topographic features presents a range of tilt angles, resulting in different reflection coefficients. Tilt contrast results because each facet has a different scattering yield, which is dependent upon local surface inclination. Full details of the instrumentation designed for SREM are described, and to illustrate the technique, results recorded from an epitaxial GaAs on GaAs crystal, Pb₂(Zr,Ta)O₆ thin film on silicon, and SiO₂ amorphous film on silicon are presented.     

Monte Carlo simulation of secondary electron and backscattered electron images in scanning electron microscopy for specimen with complex geometric structure

A new Monte Carlo technique for the simulation of secondary electron (SE) and backscattered electron (BSE) of scanning electron microscopy (SEM) images for an inhomogeneous specimen with a complex geometric structure has been developed. The simulation is based on structure construction modeling with simple geometric structures, as well as on the ray-tracing technique for correction of electron flight-step-length sampling when an electron trajectory crosses the interface of the inhomogeneous structures. This correction is important for the simulation of nanoscale structures of a size comparable with or even less than the electron scattering mean free paths. The physical model for electron transport in solids combines the use of the Mott cross section for electron elastic scattering and a dielectric function approach for electron inelastic scattering, and the cascade SE production is also included.     

A spectroscopic scanning electron microscope design

This paper describes a proposal to improve the design of scanning electron microscopes (SEMs). The design is based upon using an SEM column similar to the conventional one, magnetic sector plates and a mixed field immersion objective lens. The optical axis of the SEM column lies in the horizontal direction and the primary beam is turned through 90 degrees before it reaches the specimen. This arrangement allows for the efficient collection, detection and spectral analysis of the scattered electrons on a hemispherical surface that is located well away from the rest of the SEM column. The proposed SEM design can also be easily extended to incorporate time multiplexed columns and multi-column arrays.     

Surface roughness of preparations for backscattered electron-scanning electron microscopy: the image differences and their Monte Carlo simulation

Patterns and levels of mineralisation in the biological hard tissues have been studied using the backscattered electron (BSE) mode in the scanning electron microscope (SEM). To prevent gross topographic detail overwhelming changes in signal from composition, samples are embedded in polymethylmethacrylate (PMMA) and a flat block surface produced by polishing or micromilling. This study was undertaken to establish the degree of residual topography achieved in these finishing processes. A sample of human rib was embedded in PMMA and prepared, as for examination in the SEM, by polishing on graded abrasives and pre- and, finally, ultramilling. After each preparation step, the block face was imaged using a confocal reflection microscope surface mapping facility. The recorded topographies were used in a Monte Carlo simulation to model the surface interface and thus, for each of the sample preparation techniques, to calculate predicted variations in BSE signal. The latter were compared with experimental data derived under standard operating conditions in the SEM. Micromilling produced block faces with typical peak-trough relief of 80 nm, while hand polishing left occasional scratches 1.5 microns deep with a general undulation of 150-250 nm. Monte Carlo simulations of a rough surface of bone using these data predicted that additional contrast levels of 5% could be expected from micromilled surfaces and > 10% for hand polished samples of bone. Thus, micromilling is the best preparation method for bone, since this tissue develops a collagen orientation-related relief on polishing, which may be largely responsible for the (incorrect) supposition that lamellation in bone is related to changes in net degree of mineralisation.     

A monte carlo study of the position of phase boundaries in backscattered electron images

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.     

Use of backscattered electron detector arrays for forming backscattered electron images in the scanning electron microscope

The backscattered electron (BSE) signal in the scanning electron microscope (SEM) can be used in two different ways. The first is to give a BSE image from an area that is defined by the scanning of the electron beam (EB) over the surface of the specimen. The second is to use an array of small BSE detectors to give an electron backscattering pattern (EBSP) with crystallographic information from a single point. It is also possible to utilize the EBSP detector and computer-control system to give an image from an area on the specimen--for example, to show the orientations of the grains in a polycrystalline sample ("grain orientation imaging"). Some further possibilities based on some other ways for analyzing the output from an EBSP detector array, are described.     

Secondary electron imaging in the variable pressure scanning electron microscope

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.     

Design and characterisation of a single-reflection,solid-state detector with high discrimination against backscattered electrons for cathodoluminescence microscopy

Aportable solid-state detector (SSD) for cathodoluminescence (CL) has been constructed and tested. The detector geometry utilises a parabolic reflector to direct light towards the solid-state detection element. The photo-sensitive area of the solid-state photodiodes is situated at a level in line with or sightly below the top surface of the specimen to minimise the collection of backscattered electrons (BSEs) coming directly from the beam impact point. The components have been integrated into a single unit to enhance portability. In comparison with a commercial CL detection system, the new geometry shows excellent efficiency in rejecting BSE contribution during CL operation. A light collection solid angle close to 1.97π steradian is realised in this geometry, higher than other SSD-CL systems. A method for characterising CL detection system performance has been developed.     

A new method of compositional image formation by a four-element backscattered electron detector in an SEM

A special mixing procedure for signals from a four element backscattered electron (BSE) detector is proposed for compositional image formation when a sample with a rough surface is examined by a scanning electron microscope (SEM). The new method allows appreciable suppression of the influence of the sample surface topography in a compositional mode for take-off angles less than about 30°, relative to the microscope axis. The theoretical approach based on the analysis of BSE angular distribution is compared with the experiment. The mixing procedure uses a dimensionless parameter, which depends mainly on take-off angle. Photographs of the Ge-Zn structure with its rough surface were taken in conventional and proposed compositional modes for take-off angle 11° and electron energy 20 keV and show a considerable suppression of the topographic effect when the new method is used.     

Correlative light and backscattered electron microscopy of bone--part II: automated image analysis

Detailed studies of biological phenomena often involve multiple microscopy and imaging modes and media. For bone biology, various forms of light and electron microscopy are used to study the microscopic structure of bone. Integrating information from the different sources is necessary to understand how different aspects of the bone structure interact. To accomplish this, methods were developed to prepare and image thin sections for correlative light microscopy (LM) and backscattered electron imaging in the scanning electron microscope (BSE-SEM). Images of the same fields of view may then be analyzed for degrees of relationships between specimen features not observed by LM or SEM alone. These methods are applied here to study possible associations between the degree of bone mineralization and pattern of collagen fiber orientation in the mid-shaft of the human femur. The "relational images" obtained allow us to examine the relationship between these two variables, both objectively and quantitatively.     

Light microscopy and scanning electron microscopy: Implications for authentication of misidentified herbal drugs

Plant‐based drugs have reached remarkable acceptability as therapeutic remedy for various diseases due to the adverse effects of contemporary medicines. This increasing popularity of herbal drugs leads to a growing herbal market for the development of plant‐based drugs, nutraceuticals, pharmaceuticals and cosmeceuticals. Herbal drug adulteration is a complex problem which currently has undeniable consequences on health and nutrition. Ambiguities in nomenclature, misidentification and resemblance of colour and texture of the crude herbal drugs are the major causes of adulteration. Three different species commercially marketed under the same trade name Halion are Lepidium apetalum, Asparagus officinalis, and Lepidium didymum. The genuine source of Halion is Lepidium apetalum, which is authenticated by using basic and advanced taxonomic techniques. Morphology, anatomy and palynology of the misidentified sources were done using light and scanning electron microscopic techniques for authentication. This study may help to set microscopic techniques as a tool to achieve quality and standardization of the genuine source of the herbal drug. Phytochemical analysis and biological screening is needed for the further establishment of authenticity and quality of herbal drugs.     

Quantitative DNA measurements in an instrument combining scanning electron microscopy and light microscopy

An instrument for combined scanning electron microscopy (SEM) and light microscopy (LM) to which a photometer unit is attached is described. A special stage in the vacuum chamber of a scanning electron microscope incorporates light microscope optics (objective and condenser) designed for transmission and epi-illumination fluorescence LM. An optical bridge connects these optics to a light microscope, without objective and condenser. The possibility of performing quantitative DNA measurements in this combined microscope (the LM/SEM) was tested using preparations of either chicken erythrocytes, human lymphocytes, or mouse liver cells. The cells were fixed, brought on a cover-glass, quantitatively stained for DNA, dehydrated, and critical point dried (CPD). After mounting the cells were coated with gold. The specimens were brought into the vacuum chamber of the combined microscope and individual cells were studied with SEM and LM. Simultaneously DNA measurements were performed by means of the photometer unit attached to the microscope. It is shown in this study that DNA measurements of cells in the combined microscope give similar results when compared to DNA measurements of embedded cells performed with a conventional fluorescence microscope. Furthermore, it is shown that although the gold layer covering the LM/SEM specimens weakens the fluorescence signal, it does not interfere with the DNA measurements.