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.     

Comparative mass measurement of biological macromolecules by scanning transmission electron microscopy

A method is described for measuring the mass/length or mass of molecular assemblies by comparative electron scattering in the STEM. Standard particles whose mass is well established (e.g. TMV or fd bacteriophage) are deposited on the electron microscope grid together with the sample to be measured. Images containing at least one sample and standard and with a clean, contamination-free background are chosen and stored on computer disc and then directly integrated. Use of a comparative technique does not require accurate determination of scattering parameters or instrumental geometry and requires only that the limits of linearity be established. The results of the mass/length measurements on phage pf 1, pili, muscle thick filaments and actin are in good agreement with existing molecular weight data and generally have a standard deviation of about 10%. The results for the total mass measurement of the multisubunit enzymes glutamate dehydrogenase and glutamine synthetase are also close to the literature values for their molecular weights. The results for the spherical, Semliki forest and tomato bushy stunt viruses are lower than expected, possibly reflecting some dissociation during preparation.     

Comparative study of a block copolymer morphology by transmission electron microscopy and scanning force microscopy

Using transmission electron microscopy (TEM) and scanning force microscopy (SFM) together, it was possible to verify important structural features of a nanostructured bulk material such as the kp‐morphology in an ABC triblock copolymer. By applying suitable imaging techniques during the SFM measurements it was possible to determine the morphology without additional manipulation steps in between. In comparison, TEM investigations on this type of material usually require selective staining procedures prior to the measurement. Also electron beam damage is often encountered during TEM measurements especially if components such as poly(methacrylates) are present. In contrast, SFM measurements can be assumed not to significantly change the phase dimensions of the components.     

Automated electron tomography with scanning transmission electron microscopy

We report the successful implementation of a fully automated tomographic data collection system in scanning transmission electron microscopy (STEM) mode. Autotracking is carried out by combining mechanical and electronic corrections for specimen movement. Autofocusing is based on contrast difference of a focus series of a small sample area. The focus gradient that exists in normal images due to specimen tilt is effectively removed by using dynamic focusing. An advantage of STEM tomography with dynamic focusing over TEM tomography is its ability to reconstruct large objects with a potentially higher resolution.     

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.     

Polyethylene glycol (PEG) embedding and subsequent de-embedding as a method for the correlation of light microscopy,scanning electron microscopy,and transmission electron microscopy

The polyethylene glycol (PEG) embedding and subsequent deembedding method was applied to the observation of general tissues in scanning electron microscopy (SEM). Resulting SEM images were of high quality. It was demonstrated that intermicroscopic correlation of images between light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) is easily and reliably done by means of the PEG method. In particular, the exact correlation of immuno-LM with SEM is shown to be of potential value.     

Backscattered electron imaging and scanning transmission electron microscopy imaging of multi-layers

Experimental and theoretical results on image contrast of semiconductor multi-layers in scanning electron microscopy investigation are reported. Two imaging modes have been considered: backscattered electron imaging of bulk specimen and scanning transmission imaging of thinned specimens. The following main results have been reached. The image resolution of the multi-layers is, in both cases, defined by the probe size. The contrast, governed by density and atomic number differences, is affected by the size of the interaction volume in backscattered electron imaging and by the beam broadening in scanning transmission. Operating in the scanning transmission mode, the contrast of bright field images can be easily related to local variation in atomic number and density of the specimen while the dark field image contrast is strongly affected by electron beam energy, detector collection angles and specimen thickness. All these factors are able to produce contrast reversals that are difficult to explain without the support of a suitable simulation code.     

A rapid and simple technique for correlating light microscopy,transmission and scanning electron microscopy of fixed tissues in Epon blocks

A simplified and standardized technique for close correlation between light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM) is described. Perfusion and immersion fixed tissue specimens were embedded in Epon 812 and cut for conventional LM and TEM. The Epon blocks with remaining tissue were thereafter treated with epoxy solvent (ethanol-NaOH solution) for partial epoxy resin removal only (dissolving rate approx 33μm/h). The blocks with partially blotted tissue specimens were then critically point dried and gold coated for SEM. This method, in an easy way, allows repeated observations with LM, TEM and SEM with preserved fine structure and exact correlation. Since the technique is so simple and there is no need for special equipment the method can easily be adopted in all laboratories with basic SEM standards.     

Valence electron energy-loss spectroscopy in monochromated scanning transmission electron microscopy

With the development of monochromators for (scanning) transmission electron microscopes, valence electron energy-loss spectroscopy (VEELS) is developing into a unique technique to study the band structure and optical properties of nanoscale materials. This article discusses practical aspects of spatially resolved VEELS performed in scanning transmission mode and the alignments necessary to achieve the current optimum performance of approximately 0.15 eV energy resolution with an electron probe size of approximately 1 nm. In particular, a collection of basic concepts concerning the acquisition process, the optimization of the energy resolution, the spatial resolution and the data processing are provided. A brief study of planar defects in a Y(1)Ba(2)Cu(3)O(7-)(delta) high-temperature superconductor illustrates these concepts and shows what kind of information can be accessed by VEELS.     

Discrimination of heavy and light elements in a specimen by use of scanning transmission electron microscopy

Heavier elements have a larger scattering cross-section for elastically scattered electrons than lighter ones. Furthermore, the maximum number of scattered electrons is at higher scattering angles for heavier atoms. These differences can be used, in principle, to distinguish heavy and light elements from each other in dark field Scanning Transmission Electron Microscopy (STEM). We have achieved such discrimination in practice by collecting the electrons in a STEM experiment at two different angles. The information about the elemental composition that these two images together contain is visualized by forming linear combinations of the images which are specific for light and heavy elementsrrespectively. The results are demonstrated for a specimen consisting of platinum grains on a holey carbon film and for granulocytes stained with osmium tetroxide.     

The fine structure of fenestrated adrenocortical capillaries revealed by in-lens field-emission scanning electron microscopy and scanning transmission electron microscopy

Cell biologists probing the physiologic movement of macromolecules and solutes across the fenestrated microvascular endothelial cell have used electron microscopy to locate the postulated pore within the fenestrae. Prior to the advent of in-lens field-emission high-resolution scanning electron microscopy (HRSEM) and ultrathin m et al coating technology, quick-freeze, platinum-carbon replica and grazing thin-section transmission electron microscopy (TEM) methods provided two-dimensional or indirect imaging methods. Wedge-shaped octagonal channels composed of fibrils interwoven in a central mesh were depicted as the filtering structures of fenestral diaphragms in images of platinum replicas enhanced by photographic augmentation. However, image accuracy was limited to replication of the cell surface. Subsequent to this, HRSEM technology was developed and provided a high-fidelity, three-dimensional topographic image of the fenestral surface directly from a fixed and dried bulk adrenal specimen coated with a 1 nm chromium film. First described from TEM replicas, the “flower-like” structure comprising the fenestral pores was readily visualized by HRSEM. High-resolution images contained particulate ectodomains on the lumenal surface of the endothelial cell membrane. Particles arranged in a rough octagonal shape formed the fenestral rim. Digital acquisition of analog photographic recordings revealed a filamentous meshwork in the diaphragm, thus confirming and extending observations from replica and grazing section TEM preparations. Endothelial cell pockets, first described in murine renal peritubular capillaries, were observed in rhesus and rabbit adrenocortical capillaries. This report features recent observations of fenestral diaphragms and endothelial pockets fitted with multiple diaphragms utilizing a Schottky field-emission electron microscope. In-lens staging of bulk and thin section specimens allowed tandem imaging in HRSEM and scanning TEM modes at 25 kV.     

Studies of steel fracture by transmission and scanning electron microscopy

Transmission electron micrographs of replicas of the fractured surfaces of steel are compared with scanning electron micrographs of areas of the same surfaces. The advantages of each method are discussed.     

Composition mapping in InGaN by scanning transmission electron microscopy

We suggest a method for chemical mapping that is based on scanning transmission electron microscopy (STEM) imaging with a high-angle annular dark field (HAADF) detector. The analysis method uses a comparison of intensity normalized with respect to the incident electron beam with intensity calculated employing the frozen lattice approximation. This procedure is validated with an In_0.07Ga_0.93N layer with homogeneous In concentration, where the STEM results were compared with energy filtered imaging, strain state analysis and energy dispersive X-ray analysis. Good agreement was obtained, if the frozen lattice simulations took into account static atomic displacements, caused by the different covalent radii of In and Ga atoms. Using a sample with higher In concentration and series of 32 images taken within 42 min scan time, we did not find any indication for formation of In rich regions due to electron beam irradiation, which is reported in literature to occur for the parallel illumination mode. Image simulation of an In_0.15Ga_0.85N layer that was elastically relaxed with empirical Stillinger-Weber potentials did not reveal significant impact of lattice plane bending on STEM images as well as on the evaluated In concentration profiles for specimen thicknesses of 5, 15 and 50 nm. Image simulation of an abrupt interface between GaN and In_0.15Ga_0.85N for specimen thicknesses up to 200 nm showed that artificial blurring of interfaces is significantly smaller than expected from a simple geometrical model that is based on the beam convergence only. As an application of the method, we give evidence for the existence of In rich regions in an InGaN layer which shows signatures of quantum dot emission in microphotoluminescence spectroscopy experiments.     

Characterization of paired helical filaments by scanning transmission electron microscopy

Paired helical filaments (PHFs) are abnormal twisted filaments composed of hyperphosphorylated tau protein. They are found in Alzheimer's disease and other neurodegenerative disorders designated as tauopathies. They are a major component of intracellular inclusions known as neurofibrillary tangles (NFTs). The objective of this review is to summarize various structural studies of PHFs in which using scanning transmission electron microscopy (STEM) has been particularly informative. STEM provides shape and mass per unit length measurements important for studying ultrastructural aspects of filaments. These include quantitative comparisons between dispersed and aggregated populations of PHFs as well as comparative studies of PHFs in Alzheimer's disease and other neurodegenerative disorders. Other approaches are also discussed if relevant or complementary to studies using STEM, e.g., application of a novel staining reagent, Nanovan. Our understanding of the PHF structure and the development of PHFs into NFTs is presented from a historical perspective. Others goals are to describe the biochemical and ultrastructural complexity of authentic PHFs, to assess similarities between authentic and synthetic PHFs, and to discuss recent advances in PHF modeling.     

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.     

Bright-field scanning confocal electron microscopy using a double aberration-corrected transmission electron microscope

Scanning confocal electron microscopy (SCEM) offers a mechanism for three-dimensional imaging of materials, which makes use of the reduced depth of field in an aberration-corrected transmission electron microscope. The simplest configuration of SCEM is the bright-field mode. In this paper we present experimental data and simulations showing the form of bright-field SCEM images. We show that the depth dependence of the three-dimensional image can be explained in terms of two-dimensional images formed in the detector plane. For a crystalline sample, this so-called probe image is shown to be similar to a conventional diffraction pattern. Experimental results and simulations show how the diffracted probes in this image are elongated in thicker crystals and the use of this elongation to estimate sample thickness is explored.     

Investigation of cholesterol,bilirubin, and protein distribution in human gallstones by color cathodoluminescence scanning electron microscopy and transmission electron microscopy

The application of color cathodoluminescent scanning electron microscopy (CCL-SEM) for qualitative luminescence analysis of cholesterol, bilirubin, and protein in human gallstones was demonstrated. Images of these deposits (cholesterol, bilirubin, and protein) were formed in real colors (blue—cholesterol, red, orange—bilirubin, yellow, green—protein) in accordance with the cathodoluminescent spectrum for each control material. The other method described for transmission electron microscopy (TEM) of ultra-thin sections provides more detailed characterization of the ultrastructure of cholesterol-containing regions and their spatial interrelations with bilirubin-containing regions. Using CCL-SEM combined with TEM permits the receipt of more complete information about the chemical composition and ultrastructure of gallstones and may lead to more effective understanding of the pathogenesis of cholesterol cholelithiasis.     

Scanning moiré fringe imaging by scanning transmission electron microscopy

A type of artificial contrast found in annular dark-field imaging is generated by spatial interference between the scanning grating of the electron beam and the specimen atomic lattice. The contrast is analogous to moiré fringes observed in conventional transmission electron microscopy. We propose using this scanning interference for retrieving information about the atomic lattice structure at medium magnifications. Compared with the STEM atomic imaging at high magnifications, this approach might have several advantages including easy observation of lattice discontinuities and reduction of image degradation from carbon contamination and beam damage. Application of the technique to reveal the Burgers vector of misfit dislocations at the interface of epitaxial films is demonstrated and its potential for studying strain fields is discussed.     

Diagnosis of aberrations from crystalline samples in scanning transmission electron microscopy

The Ronchigrams, or shadow images, observed from a thin crystalline sample in a scanning transmission electron microscope characteristically present many sets of fringes, which appear thanks to the coherent interference between the various Bragg-diffracted discs as they overlap in the diffraction plane. A particular region of such patterns can be shown to be independent of the defocus at which they are recorded. The intensity along this so-called achromatic ring depends on the microscope aberrations and can be used to diagnose the wave aberration coefficients, a crucial first step in the operation of an aberration-corrected microscope. A new algorithm is presented that allows the accurate determination of all non-cylindrically symmetric aberrations up to fourth-order from a crystalline sample using this property of the Ronchigram. An experimental procedure for determining the position of and intensity along the achromatic lines, as well as examples of diagnosis from two different crystalline structures, are detailed.     

Experimental quantification of annular dark-field images in scanning transmission electron microscopy

This paper reports on a method to obtain atomic resolution Z-contrast (high-angle annular dark-field) images with intensities normalized to the incident beam. The procedure bypasses the built-in signal processing hardware of the microscope to obtain the large dynamic range necessary for consecutive measurements of the incident beam and the intensities in the Z-contrast image. The method is also used to characterize the response of the annular dark-field detector output, including conditions that avoid saturation and result in a linear relationship between the electron flux reaching the detector and its output. We also characterize the uniformity of the detector response across its entire area and determine its size and shape, which are needed as input for image simulations. We present normalized intensity images of a SrTiO(3) single crystal as a function of thickness. Averaged, normalized atom column intensities and the background intensity are extracted from these images. The results from the approach developed here can be used for direct, quantitative comparisons with image simulations without any need for scaling.