Mechanical scanning of the specimen in the scanning electron microscope

A scanning electron microscope (SEM) system equipped with a motor drive specimen stage fully controlled with a personal computer (PC) has been utilized for obtaining ultralow magnification SEM images. This modem motor drive stage works as a mechanical scanning device. To produce ultra-low magnification SEM images, we use a successful combination of the mechanical scanning, electronic scanning, and digital image processing techniques. This new method is extremely labor and time saving for ultra-low magnification and wide-area observation. The option of ultra-low magnification observation (while maintaining the original SEM functions and performance) is important during a scanning electron microscopy session.     

Comparative pollen and foliar micromorphological studies using light microscopy and scanning electron microscopy of some selected species of Lamiaceae from Alpine Zone of Deosai Plateau,Western Himalayas

The study was conducted to highlight a detailed account of morphology of pollen chosen species of Lamiaceae through scanning electron microscopy, and the anatomical characteristics of leaf epidermis of seven species using simple light microscopy. In results, Anisomeles indica and Otostegia aucheri belong to subfamily Lamioideae because it has tricolpate pollen while the rest eight species belong to subfamily Nepetoideae (hexacolpate pollen). The exine sculpturing of pollen of studied species was found to be reticulate. In the family Lamiaceae, four kinds of stomata were found anomocytic, anisocytic, diacytic, and actinocytic, respectively. The cell wall patterns of epidermal cells were irregular or polygonal with straight or undulate walls. It was noted that the variety of the epidermal trichomes seems of taxonomically important for the identification of species of Lamiaceae. Both nonglandular and glandular trichomes were analyzed. The nonglandular trichomes were characterized with long, thin, and pointed apical unicellular cells. The nonglandular trichomes were A‐shaped in Thymus linearis. In Perovskia abrotanoides, stellate glandular trichomes were observed whereas in A. indica and Mentha royleana both glandular and nonglandular trichomes were found. In A. indica, the nonglandular trichomes were sessile and peltate in M. royleana. For the first time in this study, pollen and foliar micromorphological features of selected species of this area are carried out. These taxonomic characters were found to be important in discrimination of species from each other. In future, the detailed study with comprehensive morphology coupled with other important characters is required for delimitation of taxa at various levels.     

The scanning low-energy electron microscope: First attainment of diffraction contrast in the scanning electron microscope

Using small Pb crystals deposited in situ on a partially contaminated Si (100) crystal, we demonstrate that a commercial scanning electron microscope (SEM) can easily be converted into a scanning low-energy electron microscope (SLEEM). Although the contrast mechanism is much more complicated than that in nonscanning LEEM because not only one diffracted monochromatic beam and its close environment are used for imaging, but several diffracted beams and a wide energy spectrum of electrons of different origin (secondary electrons, inelastically andelastically scattered electrons) are used, SLEEM is a valuable addition to the standard SEM because it provides an additional structure- and orientation-sensitive contrast mechanism in crystalline materials, a low sampling depth, and high intensity at low energies.     

Palynological investigation of some selected species of family Fabaceae from Pakistan,using light and scanning electron microscopy techniques

Pollen morphology of 11 species of family Fabaceae that is, Trifolium alexandrinum, Trifolium resupinatum, Arachis hypogaea, Lathyrus aphaca, Medicago lupulina, Vicia sativa, Lathyrus odoratus, Pongamia pinnata, Melilotus indicus, Medicago polymorpha, Medicago sativa from Pakistan has been investigated by light and scanning electron microscopy. Pollen were generally tricolporate, radially symmetrical, isopolar, elliptic in equatorial view and triangular in polar view under LM. Results showed that pollens were per‐prolate (T. alexandrinum), prolate (T. resupinatum, V. sativa, L. odoratus, Melilotus indicus, M. polymorpha, M. sativa) and sub‐prolate (A. hypogaea, L. aphaca, M. lupulina, P. pinnata). The larger polar/equatorial (P/E) ratio was found in T. alexandrinum (2.26 μm) and the smallest was found in M. lupulina (1.21 μm). The exine of T. resupinatum was 3.00 μm in thickness while others posses smaller exine thickness. The larger pore diameter was found in P. pinnata (16.01 μm) while others have smaller. The length of colpi was larger in Arachis hypogaea (32.24) while others posses smaller. Eight types of surface ornamentation (Psilate, faintly rugulate). Perforate and rugulate to verrucate have been observed under SEM. The pollens were europalynous type. Pollen morphology proved to be useful for the specific delimitation and serve as a tool for the identification and classification of taxa at specific and generic levels and can also be used as a key for the taxonomic features. Diversity in exine sculpture is helpful indicative characters for the isolation of closely related species. Hence, it is clear that both qualitative and quantitative characters of pollen can be useful for differentiating between taxa at specific level.     

Macroanatomic,light and scanning electron microscopic studies of the pecten oculi in the stork (Ciconia ciconia)

This study was undertaken to investigate the pecten oculi of stork by using macroscopic, light and electron microscopic techniques. A total of 20 eyes that were obtained from 10 storks were used. The eyes were cleaned and isolated by dissection. After various procedures, four of the pecten oculi were examined by light microscope while the other four with an electron microscope. The remaining 12 eyes were assigned for macroscopic investigation. Pecten oculi of the stork were determined as accordion‐like structures that originated from n. opticus, consisting of 15–17 plica and projecting up to 2/5 of the diameter of the bulbus oculi. Light microscopic examination revealed two types of blood vessels. Afferent–efferent vessels were larger in diamater (40–45 µm), fewer in numbers, and the capillary vessels were smaller in diamater (2–5 µm) and more in numbers. There were granules including amount of melanin pigment at the apical part of the pleats. These granules were fewer and scattered randomly on the basal part of the pleats. As a result, pecten oculi in the stork, which is a migrating bird, were determined to be similar to those of other diurnal birds. Microsc. Res. Tech. 76:963–967, 2013. © 2013 Wiley Periodicals, Inc.     

Comparison of different models for the generation of electron backscattering patterns in the scanning electron microscope

An electron backscattering pattern (EBSP) is formed on a fluorescent (or other) screen from the faster scattered electrons when a single-crystal region of a solid sample is illuminated by a finely focused electron beam (EB). The EBSP is very similar in appearance to the electron channeling pattern (ECP) that is obtained in the scanning electron microscope (SEM) by rocking the beam about a point on the surface of a single crystal. It has been suggested that the mechanisms that give rise to EBSP and ECP are related by reciprocity. If this is indeed the case, then the models that are used to explain them should be the same except for the direction in which the electrons travel through the specimen. The two-event “diffraction model” for EBSP (diffuse scattering followed by diffraction) fails this condition, leading to the conclusion that the “channeling in and channeling out” model for EBSP is more likely to be correct. This has been described rigorously by Reimer (1979, 1985). It is named after the title used by Joy (1994). An attempt is made here to describe this model in a simple way.     

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.     

Integration of a high‐NA light microscope in a scanning electron microscope

We present an integrated light‐electron microscope in which an inverted high‐NA objective lens is positioned inside a scanning electron microscope (SEM). The SEM objective lens and the light objective lens have a common axis and focal plane, allowing high‐resolution optical microscopy and scanning electron microscopy on the same area of a sample simultaneously. Components for light illumination and detection can be mounted outside the vacuum, enabling flexibility in the construction of the light microscope. The light objective lens can be positioned underneath the SEM objective lens during operation for sub‐10 μm alignment of the fields of view of the light and electron microscopes. We demonstrate in situ epifluorescence microscopy in the SEM with a numerical aperture of 1.4 using vacuum‐compatible immersion oil. For a 40‐nm‐diameter fluorescent polymer nanoparticle, an intensity profile with a FWHM of 380 nm is measured whereas the SEM performance is uncompromised. The integrated instrument may offer new possibilities for correlative light and electron microscopy in the life sciences as well as in physics and chemistry.     

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%.     

Reduction of charging effects using vector scanning in the scanning electron microscope.

We describe a vector scanning system to reduce charging effects during scanning electron microscope (SEM) imaging. The vector scan technique exploits the intrinsic charge decay mechanism of the specimen to improve imaging conditions. We compare SEM images obtained by conventional raster scanning versus vector scanning to demonstrate that vector scanning successfully reduces specimen-charging artifacts.     

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.     

A portable scanning electron microscope column design based on the use of permanent magnets

A portable scanning electron microscope (SEM) column design is presented which makes use of permanent magnets. Simulation results predict that such an SEM column is feasible and that it can be compact. The column height is typically less than 12 cm. The column is designed to be modular, so that it can fit onto a wide range of different specimen chamber types, and can also be readily replaced.     

Reproducibility and accuracy of spatial measurements from digitally captured images in the environmental scanning electron microscope

Accurate spatial measurements in a scanning electron microscope (SEM) require calibration of the magnification as a function of working distance and microscope operating conditions. This work presents the results of the calibration of an environmental SEM for the accurate spatial measurement of dimensions and areas in experiments, both for the measurement of strain in steel specimens under applied loads and the measurement of dimensional changes in timber with changes in relative humidity.     

Backscattered electron imaging of cultured cells: application to electron probe X-ray microanalysis using a scanning electron microscope

We report a simple method to study the elemental content in cultured human adherent cells by electron probe X-ray microanalysis with scanning electron microscopy. Cells were adapted to grow on polycarbonate tissue culture cell inserts, washed with distilled water, plunge-frozen with liquid nitrogen and freeze-dried. Unstained, freeze-dried cultured cells were visualized in the secondary and backscattered electron imaging modes of scanning electron microscopy. With backscattered electron imaging it was possible to identify unequivocally major subcellular compartments, i.e. the nucleus, nucleoli and cytoplasm. X-ray microanalysis was used simultaneously to determine the elemental content in cultured cells at the cellular level. In addition, we propose some improvements to optimize backscattered electron and X-ray signal collection. Our findings demonstrate that backscattered electron imaging offers a powerful method to examine whole, freeze-dried cultured cells for scanning electron probe X-ray microanalysis.     

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.     

Application of the scatter diagram technique to the scanning electron microscope: preliminary results from diamond

Using primary beam energies E0 ranging from 0.2 to 15 keV and an in-lens detector, a series of images of the same region of an artificial microstructured diamond sample have been acquired in scanning electron microscopy. Next, the images were analysed by using a scatter diagram technique to underline the topographic contrast change and contrast reversal. The results obtained from 0.5 to 15 keV are discussed with the help of an expression derived from the constant loss model for the secondary electron yield delta of diamond, but including the respective roles of the angle of incidence, i, and of the angle of detection, alpha. More surprising is the quality of images obtained at a beam energy as low as 0.2 keV, and more difficult to explain is the significant contrast change between 0.2 keV and 0.5 keV. For the first time, scatter diagrams are used as a diagnostic tool in scanning electron microscopy, and after some improvements it is hoped that the experimental approach followed here may lead to quantitative estimates of the local tilts of a specimen surface.     

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.