Compensation of the shadowing error in three-dimensional imaging with a multiple detector scanning electron microscope

A method for three‐dimensional quantitative surface characterization for scanning electron microscopy is presented. The method used a quadruple scintillator detector developed by us. A surface reconstruction algorithm was performed by special software, with new algorithms for error compensation. Among these errors, detector shadowing was of particular importance. This was due to the disturbance in integration continuity when one or more detectors was screened from the flow of electrons. Several methods for the reduction of this error have been proposed and tested by us. The methods were based on software processing of complementary information, such as unshadowed detector signals, shadow depth and modified integration schemes.     

Correlative fluorescence and electron microscopy in tissues: immunocytochemistry

Correlative microscopy is a collection of procedures that rely upon two or more imaging modalities to examine the same specimen. The imaging modalities employed should each provide unique information and the combined correlative data should be more information rich than that obtained by any of the imaging methods alone. Currently the most common form of correlative microscopy combines fluorescence and electron microscopy. While much of the correlative microscopy in the literature is derived from studies of model cell culture systems we have focused, primarily, on correlative microscopy in tissue samples. The use of tissue, particularly human tissue, may add constraints not encountered in cell culture systems. Ultrathin cryosections, typically used for immunoelectron microscopy, have served as the substrate for correlative fluorescence and electron microscopic immunolocalization in our studies. In this work, we have employed the bifunctional reporter FluoroNanogold. This labeling reagent contains both a fluorochrome and a gold-cluster compound and can be imaged by sequential fluorescence and electron microscopy. This approach permits the examination of exactly the same sub-cellular structures in both fluorescence and electron microscopy with a high level of spatial resolution.     

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.     

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.     

Free-standing graphene by scanning transmission electron microscopy

Free-standing graphene sheets have been imaged by scanning transmission electron microscopy (STEM). We show that the discrete numbers of graphene layers enable an accurate calibration of STEM intensity to be performed over an extended thickness and with single atomic layer sensitivity. We have applied this calibration to carbon nanoparticles with complex structures. This leads to the direct and accurate measurement of the electron mean free path. Here, we demonstrate potentials using graphene sheets as a novel mass standard in STEM-based mass spectrometry.     

Construction of 35-millimeter camera adaptors for scanning electron microscopes

Described here is the construction of two 35-mm camera adaptors for attachment to the recording monitors of scanning electron microscopes (SEM). The designs are simple and readily adaptable to almost any SEM. The choice of this camera format for recording SEM images is one of convenience as well as economy and does not sacrifice micrograph quality.     

影响扫描电镜图像质量的因素分析

本文介绍影响扫描电镜图像质量的因素及其对图像质量的影响,分别从加速电压、扫描速度和信噪比、束斑直径、探针电流、消像散校正、工作距离以及反差对比等分析图像质量的变化原因,提出提高图像质量的方法。     

A new method for investigating the undersurface of cell monolayers by scanning electron microscopy

Glow discharge is commonly used for cleaning the inside of coating units and for cleaning hard surfaces before carbon or metal evaporation procedures. In this study it has been used to remove the embedding medium to reveal, for scanning electron microscope (SEM) study, the undersurfaces of Balb/c 3T3 fibroblastic cells that have been cultured on Thermanox discs and embedded in LR White resin. Ten to twenty-minute ionization times were found to reveal the largest area of the undersurface without causing damage to the cells. Chemical etching of the resin was also shown to reveal the undersurface of the cells, but caused some damage, preventing successful re-embedding for transmission electron microscopy, and at higher magnifications revealed less detail. A circular impression within the main outline of the cells was observed in many cells, which is considered to reflect the presence of a nucleus. The undersurfaces of most cells, after applying both methods of etching, displayed a number of very short processes. Subsequent transmission electron microscopy of ultrathin sectioned, re-embedded, areas of the gold sputter-coated blocks revealed the depth of ionization that had occurred and confirmed that the specimens observed in SEM were the undersurfaces of cells. This method can be modified to study the attaching surface of any organism to a substratum.     

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.     

A new rapid method of air-drying for scanning electron microscopy using tetramethylsilane

A new rapid air-drying technique which gives results comparable to critical point-drying is described for scanning electron microscopy, using a trematode parasite, Homalogaster paloniae, as a test specimen.     

Redesign of the scanning electron microscope for parallel energy spectral acquisition

This paper presents a scanning electron microscope (SEM) design that is compatible with parallel electron energy spectrum acquisition. The SEM should in principle be capable of capturing the energy spectrum of all scattered electrons simultaneously, from low energy secondary electrons to elastic backscattered electrons. Preliminary simulation results predict that the beam separator spectrometer will have a relatively high transmission-energy resolution performance, comparable or better than the cylindrical mirror analyzer (CMA), while at the same time being able to capture the entire energy range of scattered electrons.     

Identification of bacteria by studying one section under light microscopy,scanning, and transmission electron microscopy

A method for bacterial identification has been developed by means of studying the same histological sections through several types of microscopy. With this method, one section was processed and analyzed respectively for light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Sections of gingival biopsies were Gram stained and bacteria tentatively identified by LM. Photographs of the sections were taken and presketched transparent acetate sheets (PTAS) were made from the photos. The same section was later prepared for SEM, areas previously thought to contain bacteria were localized by placing the PTAS onto the SEM monitoring screen. The SEM specimens were subsequently processed for TEM, bacteria were located, and micrographs obtained. The results showed that out of ten diseased gingival biopsies observed under the LM, bacteria were found to be present in all the specimens and were identified as both Gram positive and Gram negative. By transferring the section from LM to SEM, the bacteria could be relocated and their morphotype (cocci, rods, etc.) clearly identified in most of the cases. Since cocci may resemble other biological granular structures under SEM, they require further analysis under TEM for additional positive identification. This study demonstrated that the method described here is a useful tool for assessing the presence and identifying bacteria within the gingival tissues.     

Resolution in low voltage scanning electron microscopy

As the energy of an electron beam is reduced, the range falls and the secondary electron yield rises. A low voltage scanning electron microscope can therefore, in principle, examine without damage or charging samples such as insulators, dielectrics or beam sensitive materials. This paper investigates the way in which the choice of beam energy affects the spatial resolution of a secondary electron image. It is shown that for samples which are thin compared to the electron range, the edge resolution and contrast in the image improve with increasing beam energy. In samples that are thicker than the electron range, the resolution can be optimized at either high or low energies, but low energy operation will produce images of higher contrast. At an energy of 2 keV or less beam interaction limited resolutions of the order of 3 nm should be possible.     

A robust focusing and astigmatism correction method for the scanning electron microscope

This paper discusses a new approach to focusing and astigmatism correction based on the fast fourier transforms (FFTs) of scanning electron microscopy (SEM) images. From the FFTs, it is possible to obtain information on the severity of the defocus and astigmatism. This information is then processed by an algorithm to perform real-time focusing and astigmatism correction on the SEM. The algorithm has been tested on defocused and astigmatic images of different samples, including those with highly directional features. Experiments show that the images obtained after running the algorithm can be as good as those that an experienced SEM operator can achieve.     

In situ measurements on individual thin carbon nanotubes using nanomanipulators inside a scanning electron microscope

We demonstrate here a novel method for performing in situ mechanical, electrical and electromechanical measurements on individual thin carbon nanotubes (CNTs) by using nanomanipulators inside a scanning electron microscope. The method includes three key steps: picking up an individual thin CNT from a substrate, connecting the CNT to a second probe or an atomic force microscope cantilever for the measurements and placing the CNT onto a holey carbon film on a transmission electron microscope grid for further structure characterization. With the method, Young’s modulus, the breaking strength and the effects of axial strain on electrical transport properties of individual thin CNTs can be studied. As examples, the mechanical, electrical and electromechanical properties of a double-walled CNT (DWCNT) and a single-walled CNT (SWCNT) were measured. We observed a strain-induced metallic-to-semiconducting transition of the DWCNT and a bandgap increase of the SWCNT. More importantly, the electromechanical properties of the SWCNT were correlated to its chirality determined by electron diffraction. The method enables us to relate mechanical, electrical and electromechanical properties of the measured thin CNTs to their atomic structures.     

Minimizing sample evaporation in the environmental scanning electron microscope

The ElectroScan environmental scanning electron microscope (ESEM) enables wet samples to be observed by eliminating air but allowing water vapour into the sample chamber. However, evaporation from, and condensation on, the sample may occur during the pumpdown sequence used to reach this state, which means that the sample may not be in its natural state when viewed if due care is not taken. In this paper, the pumping system of the ESEM is described mathematically and expressions are derived for the evaporation and condensation. This treatment is then used to calculate the optimum pumpdown sequence. The importance of using the optimized procedure is illustrated by micrographs of fat emulsions.     

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.     

Tomography of asymmetric bulk specimens imaged by scanning electron microscopy

The scanning electron microscope produces nanometer-resolution surface images of biological samples preserved in a life-like state. Extracting three-dimensional information from these two-dimensional images has been the subject of long and ongoing research. We present here a general method and theoretical basis for reconstructing the surfaces of SEM specimens imaged from multiple directions by back-projection. The resulting reconstructions are faithful representations of the original specimen geometry, even when the input images are blurred and have low signal-to-noise ratio.