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.     

Surface structure imaging by electron microscopy

Reconstructed structures at monolayer level on ‘clean and well-defined’ surfaces can be imaged by transmission electron microscopy in fixed beam illumination mode. The specimens are cleaned in-situ in the electron microscope in ultra high vacuum. Transmission electron diffraction pattern intensities can give useful information for determining the surface unit cell size of the structure, and the atom positions (geometric arrangement of atoms in the unit cell) especially those with a large unit cell, since the diffraction intensities are interpreted kinematically. High resolution surface imaging which gives directly the atom positions is tested here for a single monolayer terrace on Ag (111) surface. The result shows the value of HREM for studies of surface crystallography.     

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.     

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.     

Elemental imaging by electron energy loss microscopy

Electron energy loss images obtained by parallel filtration at selected energies are used to derive qualitative and quantitative distributions of elements in specimens as diverse as low alloy steel, cartilage, and ribosomal particles. Partial ionization cross-sections for electron scattering from phosphorus L-shells are calculated.     

The morphology of stigma of Asteraceae observed by scanning electron microscopy

The morphology of stigma has taxonomic values. To further explore the taxonomy of family Asteraceae, the morphological characteristics of stigma of 28 genera, 32 species, and two varieties in the family were observed using scanning electron microscopy. The results indicated that the stigma morphology of these Asteraceae plants could be divided into 10 types, of which eight are reported for the first time. The morphological characteristics of stigma support the close relationship between genera Aster and Erigeron and among genera Sonchus, Taraxacum, and Youngia. Our results enriched the stigma type diversity data and provided a morphological basis for the study of the phylogenetic evolution of Asteraceae.     

Characterization of an oil-degrading Microcoleus consortium by means of confocal scanning microscopy, scanning electron microscopy and transmission electron microscopy

A consortium of microorganisms with the capacity to degrade crude oil has been characterized by means of confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The analysis using CLSM shows that Microcoleus chthonoplastes is the dominant organism in the consortium. This cyanobacterium forms long filaments that group together in bundles inside a mucopolysaccharide sheath. Scanning electron microscopy and transmission electron microscopy have allowed us to demonstrate that this cyanobacterium forms a consortium primarily with three morphotypes of the heterotrophic microorganisms found in the Microcoleus chthonoplastes sheath. The optimal growth of Microcoleus consortium was obtained in presence of light and crude oil, and under anaerobic conditions. When grown in agar plate, only one type of colony (green and filamentous) was observed.     

Crystallographic electron microscopy

We present a family of techniques for the transmission electron microscope that generate surface zone-axis patterns. These patterns display the variation of the diffracted-beam intensity as a function of the angle of the incident electrons. The conditions of the experiments are those of reflection high-energy electron diffraction at near grazing incidence. The techniques are: surface convergent-beam diffraction, a surface analogue of the Tanaka method and a modified double-rocking scheme. Experimental results are presented for diffraction from surfaces of MgO and MoS₂. We anticipate that surface zone-axis patterns (surface ZAPs) will become established as an important tool for surface characterization, especially when used in conjunction with high-resolution surface imaging and surface energy loss spectroscopy; surface ZAPs may be expected to play, in surface analysis, a role analogous to that played by convergent-beam diffraction in normal transmission electron microscopy.     

On the minimum detectable activity of β spectrometers and selective radiometers

A method of β-ray spectrometry is described that consists in measuring the activity of a particular radionuclide in a mixture of a few β-emitting nuclides. The activity of radionuclides simultaneously emitting β and γ rays is determined with a γ spectrometer, and their contribution to the total β spectrum is calculated. Formulas for finding the minimum detectable activity and the error of these measurements are derived. A frequently occurring specific case is considered in which the activity of ⁹⁰Sr radionuclide in equilibrium with ⁹⁰Y is measured against the background of ¹³⁷Cs and ⁴⁰K. The minimum activities that can be measured by the phoswich detector and a detector based on a plastic scintillator are compared. The main sources of errors inherent in this measuring technique are analyzed.     

Semiconductor material assessment by scanning electron microscopy*

Considerable strides have been made in the application of the cathodoluminescent and beam-induced current modes of operation of the scanning electron microscope to the high resolution characterization of semiconductor materials. These techniques can be used to measure parameters such as resistivity, carrier concentration, mobility, composition, diffusion length, minority carrier lifetime, luminescence efficiency, dislocation density and temperature from regions of semiconductors as small as one micron across. The theoretical and experimental factors which limit the range of values over which measurements are possible and the accuracy attainable are discussed in detail. Each technique is illustrated by recent experimental results.     

High resolution imaging by 1 MV electron microscopy

High voltage electron microscopy is well suited to the high resolution imaging of local structural changes in a material. New information at atomic resolution can be obtained on a broad range of material problems. Investigations of the structure of dislocations and planar faults in metallic and covalent crystals is presented.     

Glassy cholesteric structure: thickness variation induced by electron radiation in transmission electron microscopy investigated by atomic force microscopy.

During the observation of glassy cholesteric liquid crystals in transmission electron microscopy (TEM), a new contrast is created or enhanced by electron radiation which has a direct relationship with the periodic microstructure of the specimen. In this paper, we investigate the variations of the sample thickness and mass density as possible causes of this irradiation contrast. By means of observations in atomic force microscopy (AFM) coupled to TEM, we compared the surface corrugations of non-irradiated and irradiated specimens. It is shown that the final contrast is the result of several processes. including fracture during ultramicrotomy and mass loss during irradiation. Mass loss acts as an etching, and hence results in a decrease of the sample thickness. The etching depends on the initial molecular orientation, thus evidencing the latent structure. An electron channelling mechanism is suggested to explain this behaviour.     

Object wave reconstruction by phase-plate transmission electron microscopy

A method is described for the reconstruction of the amplitude and phase of the object exit wave function by phase-plate transmission electron microscopy. The proposed method can be considered as in-line holography and requires three images, taken with different phase shifts between undiffracted and diffracted electrons induced by a suitable phase-shifting device. The proposed method is applicable for arbitrary object exit wave functions and non-linear image formation. Verification of the method is performed for examples of a simulated crystalline object wave function and a wave function acquired with off-axis holography. The impact of noise on the reconstruction of the wave function is investigated.     

Junctional complex revisited by high-resolution scanning electron microscopy

The present study correlates the ultrastructural morphology of junctional complexes as revealed by transmission electron microscopy (TEM) with that observed by high-resolution scanning electron microscopy (HRSEM), thanks to a new modification of the osmium tetroxide maceration technique. The removal of all cytoplasmic organelles by this technique allows the inspection of the inner side of the plasmalemma. With this treatment, a continuous band of tightly packed particles is observed at the most apical portion of lateral membranes. Just below this band, irregular clusters of apparently identical particles are placed all around the cellular contour. The topographical correspondence among these clusters and spot desmosomes seen by TEM identifies them as desmosomes. The continuous band seems to represent the combination of both zonulae, occludens and adherens. Regarding the nature of the particles, we suppose that they probably consist of peripheral membrane proteins clustered at the cytoplasmic surface of intercellular junctions and involved in the linkage between cytoskeleton and plasmalemma.     

Imaging of lubricating oil insolubles by electron microscopy

Many of the insolubles that occur in automotive and industrial lubricating oils are contaminants originating from the machinery being lubricated or the products of combustion. However, some are deliberately introduced by the manufacturer to improve the performance of the lubricant. Many insolubles are too small to be usefully examined in the optical microscope; the greater resolving power of the electron microscope is needed in order to image them. Mineral oil is however one of the most serious contaminants of the electron microscope vacuum and has necessitated the adaptation of existing preparation techniques or the development of new ones in order to image at high magnification the wide variety of insolubles that are present in many modern lubricants. In this report, we describe and illustrate a range of techniques which permits the imaging, spatial distribution and, in some cases, the size distribution of the particles to be obtained.     

Metal oxide systems studied by analytical electron microscopy

Detailed analysis of thin oxide films is providing a better understanding of the mechanisms of oxidation and corrosion. The analytical electron microscope used to study thin layers on iron-chromium alloys has shown that even after oxidation times as short as 10 min at 600°C the ratio of iron to chromium in the surface oxide will be different from that in the underlying alloy and discrete nodules may well have a higher iron content than the protective film in which they are growing. Progressive changes in different morphological features of the oxide films grown at varying times and temperatures can be followed by micro-probe analysis in EMMA 4. Results obtained from the oxidation in air of iron with 10% and iron with 20% added chromium are presented.