Selective contrast in electron microscopy of crystalline cytochrome oxidase

We have used various techniques for preparation of specimens for electron microscopy in order to selectively contrast different regions of vesicle crystals of cytochrome c oxidase dimers. The results are consistent with a dimer composed of two y-shaped monomers [Fuller et al., J. Mol. Biol. 134 (1979) 305] aligned along one pair of arms with the other pair of arms approximately 70 A apart. The four arms of the monomers lie within and perpendicular to the lipid bilayer in which the dimer is embedded, and the arms protrude approximately 25 A from the lipid bilayer on the matrix side of the membrane. The cytoplasmic side domains of the two monomers split away from one another forming a large cleft in the dimer. Monovalent antibodies (Fab fragments) to subunit IV appear to bind to the two monomer arms which are closely apposed across the two-fold axis of the dimer.     

Prospects for lithium imaging using annular bright field scanning transmission electron microscopy: a theoretical study

There is strong interest in lithium imaging, particularly because of its significance in battery materials. However, light atoms only scatter electrons weakly and atomic resolution direct imaging of lithium has proven difficult. This paper explores theoretically the conditions under which lithium columns can be expected to be directly visible using annular bright field scanning transmission electron microscopy. A detailed discussion is given of the controllable parameters and the conditions most favourable for lithium imaging.     

Ultrastructure of activated mouse platelets: a qualitative scanning electron microscopy study

Platelets form an integral part of the coagulation process, and their ultrastructure can provide valuable information regarding diseases associated with hemostasis. During coagulation, platelets aggregate; this aggregation can be achieved in vitro, by adding thrombin to platelet-rich plasma. Previous research showed that human thrombin could be used successfully to activate mouse platelets. When conservative changes are included, the amino acid similarity between human and mouse thrombin is approximately 75%. In this qualitative study, we compare the ultrastructure of mouse platelet aggregates activated by human thrombin as well as two concentrations of mouse thrombin, using the scanning electron microscope. Results show that both human and mouse thrombin activate platelets to form aggregates with typical pseudopodia formation. Magnification up to 250,000x showed membrane morphology with the open canalicular system pores visible in both the mouse- and human-activated platelets. It is therefore concluded that mouse platelets can be successfully aggregated using either mouse or human thrombin.     

The avian erythrocyte: a study of fixation for electron microscopy.

The quality of ultrastructural preservation of the avian erythrocyte achieved using various fixation techniques is evaluated. Different combinations of initial fixatives, buffers and post-fixation procedures were tested as well as variations in fixative osmolarity, pH and temperature. Of the commonly used initial fixatives (glutaraldehyde, acrolein and formaldehyde), 2% glutaraldehyde, alone in a slightly hypertonic buffer containing divalent ions, produced optimum erythrocyte preservation. The osmolarity was balanced using a non-electrolyte such as a sucrose. The addition of 12% hexylene glycol to the buffer solutions also improves erythrocyte preservation, as evidenced by the increased stability of the marginal microtubules, microfilaments and proteinaceous material. The use of Spurr low-viscosity epoxy resin enables the cells to be collected using low gravitational centrifugation.     

Porosity variation in hydroxyapatite and osteoblast morphology: a scanning electron microscopy study

The biocompatibility of hydroxyapatite has been demonstrated by previous studies, with enhancement of osteointegration through the use of porous hydroxyapatite (pHA). Emphasis has been focused on the use of coralline hydroxyapatite or the introduction of macroporosity into synthetic hydroxyapatite. The current study investigates the role of macro‐ and microporosities in synthetic phase‐pure porous hydroxyapatite on the morphological aspects of human osteoblast‐like cells using scanning electron microscopy. Cells were seeded on four different types of porous hydroxyapatite (HA1, HA2, HA3 and HA4) and examined following 1, 2, 14 and 30 days of incubation in vitro. The results indicated that the cells had an affinity to micropores through filopodia extensions, at initial stage of attachment. Cellular proliferation and colonization was evident on all materials with cells forming cellular bridges across the macropores at day 14 with cellular canopy formation covering entire macropores observed by day 30. This study demonstrates that while the introduction of microporosity has no evident effect on cellular morphology at later time points, it seems to play a role in initial cellular anchorage and attachment.     

Determination of the size distribution of lecithin liposomes: a comparative study using freeze fracture, cryoelectron microscopy and dynamic light scattering

The size distribution of liposomes is often determined using freeze fracture, cryoelectron microscopy or dynamic light scattering. However, the resulting size distributions do not directly coincide owing to the different weighting of the techniques. We present several methods which correct for these effects and allow a comparison of liposome size distributions as obtained by freeze fracture, cryoelectron microscopy or dynamic light scattering. These methods are based on theoretical models for the weighting of the size distribution of liposomes, which result from the preparation procedure for freeze fracture electron microscopy and from the measurement by dynamic light scattering. The proposed transformation methods are then experimentally tested with a sample of lecithin liposomes, whose size distribution was determined by dynamic light scattering, freeze fracture and cryoelectron microscopy. Furthermore, the weaknesses of the experimental techniques and hence of the resulting size distributions are discussed.     

Antennal sensilla of two parasitoid wasps: a comparative scanning electron microscopy study

Two closely related parasitoid wasp species, Cotesia glomerata (L.) and Cotesia rubecula (Marshall) (Hymenoptera:Braconidae), are different in their associative learning of plant odors. To provide a solid basis for our research on the mechanisms that underlie this difference, we described the morphology of the antennal sensilla of these two species using scanning electron microscopy complemented with transmission electron microscopy. Female and male antennae of both species have the same six types of sensilla. We classified these sensilla as sensilla trichodea without pores, sensilla trichodea with a tip pore, sensilla trichodea with wall pores, sensilla coeloconica type I, sensilla coeloconica type II, and sensilla placodea. We conclude that the morphology, numbers, and distribution of the sensory receptors are highly similar in these two closely related wasp species. Differences between species and sexes occurred only in sensilla placodea numbers. C. rubecula has more sensilla placodea than C. glomerata and males of both species have a larger number and a higher density of sensilla placodea compared to females of the same species.     

The preparation of leaf surfaces for scanning electron microscopy: a comparative study

Cryopreservation is the superior technique for viewing leaf surfaces in the SEM. Epidermal cells become distorted when freeze dried and disrupt the orientation of epicuticular wax structures. The latter are largely lost during critical point drying. Nevertheless, the appearance of surface structures after subjecting them to each drying method is valuable in interpreting the features observed by cryopreservation.     

Membrane blisters: A fixation artifact a study in fixation for scanning electron microscopy

It is been shown by scanning electron microscopy that fixation in glutaraldehyde followed by fixation in osmium tetroxide results in the presence of membrane blisters on the surface of a variety of cells. Fixation in glutaraldehyde alone or osmium tetroxide alone does not result in such extensive artifacts. The blisters, usually 0.2–0.6 μm in diameter, are seen by transmission electron microscopy to be membrane-bound, virtually empty vesicles. It is concluded that the optimum preservation of the cell surface for scanning electron microscopy is provided by fixation in glutaraldehyde alone.     

Angioarchitecture of squamous cell carcinoma from hamster buccal pouch: a scanning electron microscopy study

Tumoral angiogenesis has been widely studied by histochemical analysis but little has been done regarding morphology of these new vessels. The objective of this study was to perform a qualitative analysis of the angiogenic response to chemical induction with dimethylbenzanthracene (DMBA) and carbamide peroxide of squamous cell carcinoma in pouches of Syrian hamsters after different periods of treatment. Twenty‐four Syrian golden hamsters, divided into three groups of eight animals each, had their right jugal pouches treated with a 5% DMBA solution three times a week and a 10% carbamide peroxide two times a week for 55, 70 and 90 days. The left pouch was considered the control. After tumor induction, five animals in each group had their pouches prepared for analysis under scanning electron microscopy and three animals for analysis under light microscopy. The control pouches showed a vascular system composed by few main vessels running parallel to the longest axis of the pouch with some branches. In the pouches submitted to tumor induction, a well‐differentiated squamous cell carcinoma was present since 55 days induction in all samples. The new vascular system showed the presence of many tortuous vessels and the majority of them were veins and capillaries. Terminal loops were extremely sinuous adopting a glomerular or corkscrew shape. These tumor vessels are different from normal vessels, presenting irregular diameters, outpouchings and constrictions. Angiogenesis of sprouting and intussusceptive kind could be identified in the tumor pouches, and they were more frequent as the tumor developed. SCANNING 31: 188–194, 2009. © 2009 Wiley Periodicals, Inc.     

Image registration in electron microscopy: Application of a robust method

The geometric registration of two electron microscopic images generally is performed by maximizing the cross-correlation coefficient between them. We show that a new similarity measure (the number of sign changes) is useful for performing simultaneously geometric and gray-level registration. This method is robust, which means that it provides a good estimation of the parameters even in the presence of outliers that cannot be described by the registration model.     

Comparison between light and electron microscopy in canine and feline renal pathology: a preliminary study

The aim of this study is to compare the accuracy and clinical use of light and transmission electron microscopy in detecting the early stages of renal pathologies in domestic animals. We examined 30 samples of renal tissue from cats and dogs referred to the Veterinary Hospital of the Department of Animal Pathology for different systemic diseases. The progressions of the kidney pathologies were classified using the scheme system proposed by the International Renal Interest Society. All samples were submitted for conventional histology and ultrastructural examination. Our study shows that electron microscopy is necessary to complete the histological examinations, especially to define early stages of kidney diseases (minimal changes disease, epithelial tubular pathologies, tubular basement membrane and glomerular basement membrane changes). Electron microscopy can be more accurate in defining the level of focal lesion, and permits discrimination between different clinical and pathological alterations such as fibrillary deposits. In conclusion, transmission electron microscopy associated with clinical, histological, histochemical and immunological examinations, is an essential method for diagnosis and prognosis of renal disease.     

Ultrastructural study of the intracellular behavior of four mineral elements in the lactating mammary gland cells: study using conventional transmission electron microscopy

The effects of parenteral injection of aluminum, indium, gadolinium, or terbium in rats have been previously studied in several organs such as the liver, the kidneys, etc., but never in mammary glands. In this work, we have attempted to study the subcellular localization of these elements after their intraperitoneal administration. Their subsequent effects in the lactating mammary gland cells have also been studied. Our results using conventional transmission electron microscopy have shown that the lysosomes of the mammary glandular epithelial cells are the intracellular site of accumulation of the studied elements. Our results have also show intracellular deteriorations such as an expanded ergastoplasm and altered mitochondria after intraperitoneal injection of aluminum and indium.     

Localized etching of silicon in water using a catalytically active platinum-coated atomic force microscopy probe

This paper presents a novel atomic force microscopy (AFM)-based nanofabrication technique for Si in water that is based on highly localized catalytic etching with a Pt-coated AFM probe. It has been shown that nanoscale grooves can be fabricated on the Si surface at room temperature via Pt-assisted catalytic chemical etching in water without the addition of any chemicals. Furthermore, dissolved oxygen (O₂) in water has been found to be a key element for driving the chemical reaction of Si with water in the Si removal process. Experimental results have also suggested that an oscillating cantilever of the Pt-coated AFM probe for the stirring of water is essential in order to overcome the oxygen mass-transfer limitations and enhance the Si removal rate. The elementary chemical reactions taking place during the etching of Si has been estimated on the basis of electrochemical theory. It is proposed that in the first step, dissolved oxygen is reduced and forms hydroxide ions (OH⁻) with water molecules (H₂O) on the surface of the Pt-coated tip. In the second step, Si atoms are oxidized on reaction with OH⁻ ions and water soluble silicates are formed. The catalytic reaction taking place on the surface of a Pt-coated tip can be enhanced by the application of an anodic potential to an additional Pt wire electrode, resulting in a dramatic fifty-fold increase in the Si removal rate.     

Normalizing projection images: a study of image normalizing procedures for single particle three-dimensional electron microscopy

In the process of three-dimensional reconstruction of single particle biological macromolecules several hundreds, or thousands, of projection images are taken from tens or hundreds of independently digitized micrographs. These different micrographs show differences in the background grey level and particle contrast and, therefore, have to be normalized by scaling their pixel values before entering the reconstruction process. In this work several normalization procedures are studied using a statistical comparison framework. We finally show that the use of the different normalization methods affects the reconstruction quality, providing guidance on the choice of normalization procedures.     

Polymer-based materials to be used as the active element in microsensors: a scanning force microscopy study

Polymer-based materials can be incorporated as the active sensing elements in chemiresistor devices. Most of these devices take advantage of the fact that certain polymers will swell when exposed to gaseous analytes. To measure this response, a conducting material such as carbon black is incorporated within the nonconducting polymer matrix. In response to analytes, polymer swelling results in a measurable change in the conductivity of the polymer/carbon composite material. Arrays of these sensors may be used in conjunction with pattern recognition techniques for purposes of analyte recognition and quantification. We have used the technique of scanning force microscopy (SFM) to investigate microstructural changes in carbon-polymer composites formed from the polymers poly (isobutylene) (PIB), poly (vinyl alcohol) (PVA), and poly (ethylene-vinyl acetate) (PEVA) when exposed to the analytes hexane, toluene, water, ethanol, and acetone. Using phase-contrast imaging (PI), changes in the carbon nanoparticle distribution on the surface of the polymer matrix are measured as the polymers are exposed to the analytes in vapor phase. In some but not all cases, the changes were reversible (at the scale of the SFM measurements) upon removal of the analyte vapor. In this paper, we also describe a new type of microsensor based on piezoresistive microcantilever technology. With these new devices, polymeric volume changes accompanying exposure to analyte vapor are measured directly by a piezoresistive microcantilever in direct contact with the polymer. These devices may offer a number of advantages over standard chemiresistor-based sensors.     

Electron microscopy of frozen biological objects: a study using cryosectioning and cryosubstitution

Freezing of bulk biological objects was investigated by X-ray cryodiffraction. Freezing at atmospheric pressure of most microscopic biological samples gives rise to large hexagonal crystals and leads to poor structural preservation of these specimens. High-pressure freezing induces the formation of different ices (hexagonal, cubic and a high-pressure form) consisting of crystals having sizes smaller than those formed at atmospheric pressure. With both freezing methods, a cryoprotectant has to be added to the biological object to avoid the formation of ice crystals. However, special cases can be encountered: some biological objects contain large amounts of natural cryoprotectant or have a low water content. In these cases, vitrification can be achieved, especially using high-pressure freezing. Cryo-sectioning can be performed on vitrified samples, and the sections studied by electron cryomicroscopy. Images and electron diffraction patterns having a resolution better than 2 and 0.2 nm, respectively, can be obtained with such sections. Because samples containing crystalline ices cannot be cryosectioned, their structure has to be studied using cryosubstitution and resin embedding. We show that bacteria, yeast, and ciliate and marine worm elytrum have cellular compartments with an organization that has not been described by classical techniques relying on chemical fixation of the tissues. A high-pressure artefact affecting the Paramecium trichocysts is described. Such artefacts are not general; for example, we show that 70% of high-pressure frozen yeast cells survive successive high-pressure freezing and thawing steps.     

A method of using the incident beam tilt as a eucentric goniometer in transmission electron microscopy

The principal difficulties in constructing and operating a eucentric specimen tilting goniometer in a transmission electron microscope are discussed, together with the goniometric function of the incident beam tilt. The latter function is found easy to operate in a eucentric manner. The imaging beam then will have a non-axial path, which will increase particularly the field chromatic aberration. Earlier, however, a technique for the compensation of the chromatic aberration during displaced aperture dark field image formation has been developed. In combination with this technique, it proved possible to use the ordinary incident beam tilt as a eucentric goniometer. Image sequences were obtained, with accurately varied diffraction conditions. The tilt angles and the direction of the tilt axis can be very accurately determined from the displacements of the diffraction pattern.     

Methodological basis for an autoradiographic demonstration of insulin receptor sites on the surface of whole cells: a study using light- and scanning electron microscopy

An autoradiographic technique is described which allows, on whole cells, the determination of the number of receptor sites which are occupied by ¹²⁵I-insulin according to defined binding conditions. Glutaraldehyde fixation prevents the dissociation of the bound tracer from the receptor, which is the basis for a quantitative correlation between biochemical and autoradiographical data. The homogeneous coating of whole cells with a thin emulsion layer, which was required for these experiments, was achieved by a stripping film technique. An accurate judgment of the coating was possible only with scanning electron microscopic (SEM) techniques. As observed in the SEM, the photographic layer was smooth, uniform and in good contact with the top of the cell. The signal measured in this area was analysed. The majority of the grains also originated from the top side of the cell surface, despite the evidently three-dimensional distribution of the label. As determined in test specimens, 80–85% of the grains originated from 3–4 keV electrons. Due to the short range of these electrons, the grains represent the distribution of the label on the part of the cell surface which is in close contact with the emulsion layer. 15–20% of the grains originated from β-decays with higher energies and add partially to the background. According to these data a determination of the number of receptor sites per unit area of plasma membrane (receptor density) is possible in the surface area of the cell top. Both light microscopy (LM) and SEM were used to analyse autoradiographs. LM analysis is possible in principle but the analytical facilities of SEM are yet superior for the identification of silver grains. The registration of the grains is further facilitated in SEM because of the very large depth of focus.     

Direct imaging of local atomic ordering in a Pd-Ni-P bulk metallic glass using Cs-corrected transmission electron microscopy

In amorphous alloys, crystalline atomic clusters as small as 1-2 nm are frequently observed as local lattice fringe images by high-resolution electron microscopy (HREM). These clusters can be understood as local structures of amorphous alloys corresponding to "medium-range-order (MRO)". The MRO structure can be observed only under suitable defocusing conditions of the objective lens in HREM. A clear imaging of the MRO structure is difficult in conventional TEMs, mainly due to the delocalization of the image, caused mainly by the spherical aberration of the objective lens and eventually by the chosen defocus. In the present study, we have examined MRO in a Pd-based bulk metallic glass (Pd(40)Ni(40)P(20)) using a high-resolution TEM (acceleration voltage 200 kV) fitted with a spherical aberration constant corrector (Cs corrector) for aberration correction. We found that when Cs was close to zero and defocus values were near the Gaussian focus, MRO regions with an FCC-Pd structure could be clearly observed with a low image disturbance. Under these conditions, the phase-contrast transfer function was understood to act as an ideal filter function, which distinctly selects specific lattice periods of the FCC-Pd clusters. The obtained atomic images of the glass structure including the FCC-Pd clusters are in good agreement with those expected from image simulation according to our amorphous structure model. In this study, we have demonstrated that the Cs-corrected HREM is a powerful tool to directly image locally ordered structures in metallic glasses.