High‐resolution,high‐throughput imaging with a multibeam scanning electron microscope

Electron–electron interactions and detector bandwidth limit the maximal imaging speed of single‐beam scanning electron microscopes. We use multiple electron beams in a single column and detect secondary electrons in parallel to increase the imaging speed by close to two orders of magnitude and demonstrate imaging for a variety of samples ranging from biological brain tissue to semiconductor wafers.     

Signal components in the environmental scanning electron microscope

We demonstrate that the gas-amplified secondary electron signal obtained in the environmental scanning electron microscope has both desired and spurious components. In order to isolate the contributions of backscattered and secondary electrons, two sets of samples were examined. One sample consisted of a pair of materials having similar secondary emission coefficients but different backscatter coefficients, while the other sample had a pair with similar backscatter but different secondary emission coefficients. Our results show how the contribution of the two electron signals varies according to the pressure of the amplifying gas. Backscatter contributions, as well as background due to gas ionization from the primary beam, become significant at higher pressure. Furthermore, we demonstrate that the relative amplification efficiencies of various electron signals are dependent upon the chemistry of the gas.     

Advantages of indium–tin oxide-coated glass slides in correlative scanning electron microscopy applications of uncoated cultured cells

A method of direct visualization by correlative scanning electron microscopy (SEM) and fluorescence light microscopy of cell structures of tissue cultured cells grown on conductive glass slides is described. We show that by growing cells on indium–tin oxide (ITO)-coated glass slides, secondary electron (SE) and backscatter electron (BSE) images of uncoated cells can be obtained in high-vacuum SEM without charging artefacts. Interestingly, we observed that BSE imaging is influenced by both accelerating voltage and ITO coating thickness. By combining SE and BSE imaging with fluorescence light microscopy imaging, we were able to reveal detailed features of actin cytoskeletal and mitochondrial structures in mouse embryonic fibroblasts. We propose that the application of ITO glass as a substrate for cell culture can easily be extended and offers new opportunities for correlative light and electron microscopy studies of adherently growing cells.     

Correlative scanning electron and confocal microscopy imaging of labeled cells coated by indium‐tin‐oxide

Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field‐of‐view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold‐labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium‐tin‐oxide was deposited by ion‐sputtering on gold‐decorated HeLa cells and neurons. Indium‐tin‐oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold‐conjugated markers. Microsc. Res. Tech. 78:433–443, 2015. © 2015 Wiley Periodicals, Inc.     

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.     

Nanotip electron gun for the scanning electron microscope

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.     

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.     

A simple photographic grid system for the correlative examination of cell smears by light and scanning electron microscopy

A simple inexpensive grid system reproduced photographically on black-and-white film provides a support system that allows the same cells to be examined by light and scanning electron microscopy.     

Automated detection of fluorescent cells in in‐resin fluorescence sections for integrated light and electron microscopy

Integrated array tomography combines fluorescence and electron imaging of ultrathin sections in one microscope, and enables accurate high‐resolution correlation of fluorescent proteins to cell organelles and membranes. Large numbers of serial sections can be imaged sequentially to produce aligned volumes from both imaging modalities, thus producing enormous amounts of data that must be handled and processed using novel techniques. Here, we present a scheme for automated detection of fluorescent cells within thin resin sections, which could then be used to drive automated electron image acquisition from target regions via ‘smart tracking’. The aim of this work is to aid in optimization of the data acquisition process through automation, freeing the operator to work on other tasks and speeding up the process, while reducing data rates by only acquiring images from regions of interest. This new method is shown to be robust against noise and able to deal with regions of low fluorescence.     

Assessment of waveguiding properties of gallium oxide nanostructures by angle resolved cathodoluminescence in a scanning electron microscope

Cathodoluminescence (CL) of Ga₂O₃ nanowires and planar microstructures has been studied in a scanning electron microscope, as a function of the orientation angle of the structures relative to the position of the light detection system in the microscope chamber. CL contrast shows a marked dependence on the detection angle due to the waveguiding behaviour of the structures. The angle resolved cathodoluminescence (ARCL) measurements enable to evaluate the optical losses of guided blue-ultraviolet light in nanowires with diameters in the sub-wavelength range, deposited on graphite tape or silicon. In planar, branched feather-like microstructures, ARCL images demonstrate the directional-dependant light guiding behaviour of the nano-branches.     

Palyno‐morphological investigations of subtropical endangered flora of Capparidaceae through light and scanning electron microscopy

The research was performed to investigate pollen morphology of endangered species of Capparidaceae in subtropical regions of Pakistan. The distinguishing characters were investigated by using light microscope and scanning electron microscope. Palynological study is comprised of pollen shape, pollen type, exine sculpturing, polar and equatorial diameter, length and width of colpi, mesocolpium, and exine thickness. In polar view, Cleome viscosa exhibited the highest pollen size 26.4 (32.7–24.5 μm) ±0.776 whereas Capparis spinosa appeared to be the lowest 12.6 (14.5?10.7 μm) ±0.400. In equatorial view, Cleome viscosa had the largest pollen size 17.1 (20.0–15.0 μm) ±0.606 and Capparis spinosa had the smallest pollen size 9.7 (12.50–8.00 μm) ±0.394. The maximum fertility percentage has been observed in Capparis spinosa, that is, 98.96% and minimum in Cleome viscosa, that is, 82.93%. Diagnostic key has been constructed to state the essential diagnostic features by means of which the taxa can be identified. Remarkable variations have been observed in pollen size, shape, and exine sculpturing. All the selected species were tricolporate. Prolate to subprolate pollen were observed. There is a great variation existed in exine sculpturing such as in Capparis decidua and C. sp. nova sculpturing is reticulate, in Capparis himalayensis sculpturing is Scabrate granulate, in Capparis spinosa sculpturing is Psilate, in Cleome viscosa sculpturing is regulate‐reticulate, in Dipterygium glaucum sculpturing is regulate and in Gynandropsis gynandra sculpturing is striate‐regulate. On the basis of overall characteristics of pollen it seems that palynology of this family is helpful at the generic and specific level.     

World wide web-controlled scanning electron microscope

This paper¹ presents a system for remote control of a scanning electron microscope (SEM) over the Internet using the World Wide Web (WWW). The evolution of the SEM to its current incarnation as a PC-SEM is noted, and the World Wide Web is briefly described. The implementation of the authors' system is detailed in terms of configuration and manner of interaction. The potential commercial applications of the research are described. Related work in microscopy and networking fields is considered. A discussion of the advantages of the described system and expected future directions for research and development concludes the paper.     

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.     

Utilization of integrated correlative light and electron microscopy (iCLEM) for imaging sedimentary organic matter

We report here a new microscopic technique for imaging and identifying sedimentary organic matter in geologic materials that combines inverted fluorescence microscopy with scanning electron microscopy and allows for sequential imaging of the same region of interest without transferring the sample between instruments. This integrated correlative light and electron microscopy technique is demonstrated with observations from an immature lacustrine oil shale from the Eocene Green River Mahogany Zone and mid‐oil window paralic shale from the Upper Cretaceous Tuscaloosa Group. This technique has the potential to allow for identification and characterization of organic matter in shale hydrocarbon reservoirs that is not possible using either light or electron microscopy alone, and may be applied to understanding the organic matter type and thermal regime in which organic nanoporosity forms, thereby reducing uncertainty in the estimation of undiscovered hydrocarbon resources.     

Measurements of the surface microroughness with the scanning electron microscope

A three-dimensional reconstruction system for scanning electron microscope consisting of a quadruple scintillator detector, a four-channel frame-grabber and PC-based processing unit was developed. The authors explored a method that takes advantage of the angular distribution of secondary electron emission to obtain quantitative topography contrast and surface profiles. Software processing algorithms were developed to carry out the reconstruction process and compensate for several types of errors, inherent in the method. After pre-calibration, the system allows obtaining surface topography with calculation of microroughness and waviness statistical parameters. The roughness estimation procedure was tested on metal roughness standards for machining and etched silicon surfaces. The main advantages of the system are almost real-time operation and reliable, composition-independent reconstruction for a broad class of materials.     

Palyno‐anatomical studies of monocot taxa and its taxonomic implications using light and scanning electron microscopy

Palyno‐anatomical study of monocots taxa using Light and Scanning Electron Microscopy (SEM) was first time conducted with a view to evaluating their taxonomic significance. Studied plants were collected from different eco‐climatic zones of Pakistan ranges from tropical, sub‐tropical, and moist habitats. The aim of this study is to use palyno‐anatomical features for the correct identification, systematic comparison, and investigation to elucidate the taxonomic significance of these features, which are useful to taxonomists for identifying monocot taxa. A signification variation was observed in quantitative and qualitative characters by using the standard protocol of light microscopy (LM) and SEM. Epidermal cell length varied from maximum in Allium griffthianum (480 ± 35.9) μm at the adaxial surface to minimum in Canna indica (33.6 ± 8.53) μm on abaxial surface. Maximum exine thickness was observed in Canna indica (4.46) μm and minimum in Allium grifthianum (0.8) μm. Variation was observed in shape and exine ornamentation of the pollen, shape of the epidermal cell, number, size, and type of stomata, guard cell shape, and anticlinal wall pattern. Based on these palyno‐anatomical features a taxonomic key was developed, which help in the discrimination of studied taxa. In conclusion, LM and SEM pollen and epidermal morphology is explanatory, significant, and can be of special interest for the plant taxonomist in the correct identification of monocots taxa.     

Imaging deep holes in structures with gaseous secondary electron detection in the environmental scanning electron microscope

The gaseous secondary electron detector (GSED) in the environmental scanning electron microscope (ESEM) permits collection of electron signals from deep inside blind holes in both conducting and insulating materials. The placement of the GSED as the final pressure-limiting aperture of the ESEM creates a situation of apparent illumination along the line of sight of the observer. In principle, any point struck by the primary beam can be imaged. Image quality depends on the depth of the hole. In brass, features at the bottom of a 1.5 mm diameter hole that was 8 mm deep were successfully imaged.     

An automated image alignment system for the scanning electron microscope

We have developed an automated image alignment system for the scanning electron microscope (SEM). This system enables specific locations on a sample to be located and automatically aligned with submicron accuracy. The system comprises a sample stage motorization and control unit together with dedicated imaging electronics and image processing software. The standard SEM sample stage is motorized in the X and Y axes with stepping motors which are fitted with rotary optical encoders. The imaging electronics are interfaced to beam deflection electronics of the SEM and provide the image data for the image processing software. The system initially moves the motorized sample stage to the area of interest and acquires an image. This image is compared with a reference image to determine the required adjustments to the stage position or beam deflection. This procedure is repeated until the area imaged by the SEM matches the reference image. A hierarchical image correlation technique is used to achieve submicron alignment accuracy in a few seconds. The ability to control the SEM beam deflection enables the images to be aligned with an accuracy far exceeding the positioning ability of the SEM stage. The alignment system may be used on a variety of samples without the need for registration or alignment marks since the features in the SEM image are used for alignment. This system has been used for the automatic inspection of devices on semiconductor wafers, and has also enabled the SEM to be used for direct write self-aligned electron beam lithography.     

Specimen‐thickness effects on transmission Kikuchi patterns in the scanning electron microscope

We report the effects of varying specimen thickness on the generation of transmission Kikuchi patterns in the scanning electron microscope. Diffraction patterns sufficient for automated indexing were observed from films spanning nearly three orders of magnitude in thickness in several materials, from 5 nm of hafnium dioxide to 3 μm of aluminum, corresponding to a mass‐thickness range of ～5 to 810 μg cm^–2. The scattering events that are most likely to be detected in transmission are shown to be very near the exit surface of the films. The energies, spatial distribution and trajectories of the electrons that are transmitted through the film and are collected by the detector are predicted using Monte Carlo simulations.     

Contamination in a scanning electron microscope and the influence of specimen cooling

Solutions of the stationary and time-dependent equations of diffusion are presented for contamination when scanning a rectangular area in the scanning electron microscope (SEM). A method is described to record the thickness of the contamination layer by the signal of secondary or backscattered electrons and to measure the influence of electron current density, beam energy, and specimen cooling on the contamination rate.