In-lens Field Emission Scanning Electron Research Articles

STEM Imaging of Lattice Fringes and beyond in a UHR In-Lens Field-Emission SEM

Abstract The phase-contrast imaging of atomic lattices has now become commonplace for both Transmission Electron Microscopes (TEM) and Scanning Transmission Electron Microscopes (STEMs). Recently, however, bright-field STEM images of multi-wall carbon nanotubes (MWCNTs) recorded from an ultra-high resolution (UHR) in-lens field-emission scanning electron microscope (FE-SEM) operating at 30keV have also demonstrated lattice fringe resolution. One example of such an image containing multiple examples of fringe detail is shown in figure 1. The carbon lattice fringes were analyzed and their origin confirmed by the application of the FFT algorithms in the SMART image analysis program. The resulting power spectrum after thresholding to remove background noise (Figure 2) confirms that phase detail in the image extends down to about 5 Angstroms (0.5nm) and that well defined diffraction spots corresponding to a spacing of 3.4 Angstroms (0.34nm) generated by the (002) basal plane spacing of the graphite lattice are present.

Localization of soluble major histocompatibility class II-peptide complexes on T cell surface.

Affinity purified major histocompatibility (MHC)-peptide complexes are heterodimeric cell surface glycoproteins and are known to recognize antigen-specific CD4(+) T cell receptors (TCRs). In general, the affinity of MHC-peptide complexes to TCRs are considered very low with a K(D) of 5 x 10(-5) M and, therefore, stabilization of these complexes on T cell surface was not reported earlier. This could be due to (1) incomplete occupancy of MHC molecules with antigenic peptides, (2) variability of the binding constant of peptides to MHC molecules, (3) presence of endogenously bound peptides in MHC preparations, or (4) a combination of these. Using well-characterized HLA-DR2 complex loaded with a high affinity immunodominant epitope analog from human myelin basic protein (MBP), which shows release of gamma-IFN by specific stimulation of transformed human T cell clone (SS8T). The present report demonstrates a method for the localization of bound MHC class II-peptide complexes on T cell surface by backscatter electron imaging using in-lens Field Emission Scanning Electron Microscopy (FESEM). The localization is specific to the complex recognized by the TCR on MHC class II (DR2) and MBP peptide restricted human T cells.

Topographic Imaging of Chromium-Coated Frozen-Hydrated Cell and Macromolecular Complexes by In-Lens Field Emission Scanning Electron Microscopy.

: An in-lens Schottky field emission scanning electron microscope (SEM) combined with a transmission electron microscope (TEM)-type cold-stage and a chromium (Cr) sputter-coating system was developed to rapidly prepare and cryo-image biological specimens to attain accurate nanometer-level structural information. High-resolution topographic images at high primary magnification (>/=200,000x) were digitally recorded with very short dwell times and without beam damage. Plunge freezing in ethane, followed by fracturing, Cr coating, and in-lens cryo-high-resolution scanning electron microscope (HRSEM) imaging directly revealed macromolecular features of yeast cells, platelets, and cell-free elastin analogues. The "vitreous" nature of bulk water in its solid state appeared featureless in cryo-HRSEM images, suggesting that if ice crystals were present they would be </=2-3 nm (the approximate instrument resolution on cryo-specimens). Compared to technically difficult and indirect freeze-fracture TEM replicas, cryo-HRSEM samples are fully hydrated, unfixed, noncryoprotected specimens immersed in featureless ice. The time necessary to cryo-immobilize the specimen and record the image is <3 hr. The hexagonal arrays of intramembrane particles on the protoplasmic face of yeast cells and differences in surface morphology between thrombin-stimulated and quiescent platelets were assessed. A clear interface line between collapsed elastin fibril lacework and vitreous lakes was commonly observed. These experiments demonstrate the feasibility of this technique to rapidly evaluate macromolecular features in cryofixed cells and cell-free systems.

Investigation of the unseeded nucleation of gibbsite, Al(OH) 3, from synthetic bayer liquors

This paper presents the novel application of multi-angle laser light scattering (MALLS) together with transmission electron microscopy (TEM), powder X-ray diffraction (XRD) and In-lens field emission scanning electron microscopy (IFESEM) for studying the unseeded nucleation of gibbsite from synthetic Bayer liquor (sodium aluminate). The effects of supersaturation in the absence or presence of a known inhibitor (sodium gluconate) were examined. In each series of experiments, both homogeneous and heterogeneous primary nucleation mechanisms were observed. For the unpoisoned liquor used in this work the nucleation appeared to be homogeneous for supersaturation ratios greater than 2.51±0.07 and heterogeneous for lower values. From the induction period measurements, the interfacial tension at 60°C was determined to be 45±6 mJ m −2, and used to predict the critical nucleus size of 1.2±0.1 nm. This was then compared to the measured particle sizes from samples examined by TEM (10–40 nm) and IFESEM (∼50 nm). A crystallite size was determined by XRD to be 22±6 nm. The presence of sodium gluconate inhibitor extended the region over which homogeneous nucleation is dominant, but did not affect the measured value of the interfacial tension and hence the critical nucleus size. This is consistent with the poison inhibiting processes on the solid phase and not the solution phase.

Architecture and Polymorphism of Fibrillar Supramolecular Assemblies Produced by in Vitro Aggregation of Human Calcitonin

The human calcitonin (hCT) peptide hormone has a marked tendency to aggregate in aqueous solutions and to form long, thin fibrillar aggregates resulting in viscous and turbid dispersions. In this study, the in vitro aggregation products of hCT were systematically investigated using conventional transmission electron microscopy (CTEM) and in-lens field emission scanning electron microscopy. The mass per length of unstained/air-dried specimens was determined by scanning transmission electron microscopy. Irrespective of the sample preparation method and electron microscopic (EM) imaging mode employed, similar supramolecular assemblies were observed. Based on these EM data, it is proposed that hCT aggregation begins with the formation of approximately 4-nm-thick protofibrils. These protofibrils further interact via lateral association and coiling to form higher-order fibrillar assemblies, i.e., protofibril-ribbons, fibrils, fibril-ribbons, tubes, and multi-stranded cables. The concentrations and history of the aggregated hCT solutions strongly influenced the relative frequency of the various hCT assemblies of hCT revealed distinct helical symmetries at their different levels of aggregation. A hypothetical mechanism assumed for aggregating solutions to form polymorphic fibrillar hCT assemblies is presented in a schematic model, and the supramolecular arrangement of hCT within the various polymorphic fibrillar aggregates is delineated.

High-resolution FEISEM detection of DNA centromeric probes in HeLa metaphase chromosomes.

HeLa metaphase chromosome spreads were hybridized with centromeric biotinylated DNA probes and detected with gold-conjugated anti-biotin antibodies. Chromosomes were observed by an in-lens field emission scanning electron microscope (FEISEM), which permits detection of biological samples without any coating. DNA probes were well localized in the centromeric region of chromosomes and there was clear discrimination between 10 nm fibers that hybridized to DNA probes and those that did not hybridize. This approach shows that in situ hybridization can be directly visualized at the FEISEM level by evaluating only secondary electron emission, which allows physical localization of the hybridized probe with high resolution so that backscatter detection represents only a control. Because chromosomes maintain the 10-nm fiber organization after in situ hybridization procedures, our data suggest that this fiber represents the lowest order of chromatin arrangement that permits transitory DNA denaturation.

Preparation of isolated nuclei from K 562 haemopoietic cell line for high resolution scanning electron microscopy

The aim of the work is to visualise nuclear pore complexes (NPCs) in mammalian cells by high resolution scanning electron microscopy. A detergent-free isolation protocol was employed to obtain clean nuclei from the haemopoietic cell line K 562. Nuclear isolation was performed by mechanical homogenisation under hypotonic conditions followed by purification of the nuclear fraction. The isolated nuclei were attached to silicon chips, fixed, critical point dried, and sputter coated with a thin film (3-4 nm) of tantalum. Analysis of the nuclear surface by scanning electron microscopy (SEM) revealed a strong sensitivity of the outer nuclear membrane (ONM) to disruption during the isolation procedure. A significant reduction of the characteristic pattern of damage to the ONM was achieved by means of an isopicnic centrifugation on an isoosmolar balanced Percoll gradient. Analysis of the population of isolated nuclei by flow cytometry showed no signs of cell cycle specific losses of nuclei during isolation. The SEM investigations of the morphology of the nuclear envelope (NE) and of substructural details of NPCs and polyribosomes were performed using an in-lens field emission scanning electron microscope.

High-Magnification in-lens field-emission SEM of sectioned biological material

Osmium macerated renal specimens are rich in SE-I contrasts at very high magnifications when the ultrathin Chromium (Cr) film/intermediate voltage (15-30 kV) imaging strategy is employed. This technique enables imaging of minute particle domains which contain SE-I edge brightness contrast when staged in an in-lens field emission scanning electron microscope (SEM). The utility of employing methylmethacrylate embedded and sectioned specimens, that were subsequently acetone deembedded and critical point dried on silicon chips, was that they were rendered ideal for ultrahigh resolution SEM studies. The section/SEM method provided an exellent correlate to light microscopy (LM) and transmission electron microscopy (TEM) and may extend topographic image resolution within tissues to the molecular level.Rat kidney was briefly fixed in 0.5% glutaraldehyde/ 0.5% formaldehyde, postfixed in 1.0% OsO4 and osmium macerated (0.1%) prior to the embedding/sectioning/deembedding/drying procedures. Kidney sections ranging from 80 nm to 1.5 μm on silicon chips were thoroughly degassed and coated with a 1 nm fine grain Cr film in a Denton DV-602 sputter system.

Variations in Secondary Electron Emission from MgO Characterized by Secondary Electron Spectroscopy

The recent developments of the in-lens field emission scanning electron microscopes make it possible to image specimen surfaces with subnanometer resolution by collecting high resolution type I secondary electron (SE) signals. SE images with a resolutiori better than 1 nm have also been obtained in scanning transmission electron microscope (STEM) instruments. A problem related to the correct interpretation of these high resolution images is the lack of knowledge on the energies of the collected electrons contributing to the images. On the other hand secondary electron spectroscopy (SES) on clean surfaces has been developed for several years and biased SE imaging of several deposits on metallic and semiconductor crystal surfaces has been demonstrated. The combination of ultra-high resolution SE imaging with SES and Auger electron spectroscopy (AES) can be realized in pur UHV STEM instrument (MIDAS). The description of the performance of the MIDAS is reported elsewhere. We report here some recent results on SE imaging of MgO smoke crystals, in the MIDAS, with energy analysis of the collected secondary electrons.Strong variations of SE signals from different MgO crystals have been observed and the intensity of the collected secondary electrons from some MgO cubes increases under electron beam irradiation. Figure 1 shows a SE image of several MgO cubes, revealing that some of the crystals (indicated by A) have much higher intensity than others (indicated by B) and bright diffuse patches on the dark crystal can be seen (indicated by D). We have used SES to study the variations in SE emission under electron irradiation. The fact that the velocity distribution of the emitted secondary electrons may depend strongly on the surface morphology of the samples studied makes the interpretation of these SE spectra complicated. In order to avoid this problem and other possible complications arising from the detector geometry we acquired SE spectra from a fixed flat area of a single crystal MgO shown in figure 2. The sample was biased at -30 V and the resolution of the SE spectrum is about 1 eV. Figure 3a shows the SE spectra, taken from the exit surface of the crystal. Spectrum 1 was obtained from a poorly emitting MgO cube as that shown in figure 2 (all the spectra were obtained from the circled area). Spectra 2, 3 and 4 were obtained after about 10, 20 and 40 minutes electron irradiation respectively. In order to study spectral shape variations the spectra were normalized so that they all had the same peak intensity. The normalized data were shown in figure 3b.

Ultra-high resolution secondary electron imaging

The recent developments of the in-lens field emission scanning electron microscopes (FESEM) make it possible to image specimen surfaces with subnanometer resolution by collecting type I secondary electrons, with an incident electron probe size of about 0.5 nm in diameter. A resolution of the same order of magnitude has also been obtained in a STEM instrument with secondary electron signals. Both in-lens FESEM and STEM utilize field emission gun as electron beam source (high intensity and small probe size) and high excitation objective lenses to reduce abberations. The examined samples have to be positioned inside the objective pole-pieces. Thus the emitted secondary electrons will experience a strong magnetic field and spiral around the magnetic field lines (cyclotron orbit) before they are collected by the secondary electron detector. The radius and the pitch height of the cyclotron orbit depend on the secondary electron energy and the emission angle with respect to the magnetic field axis.