Real‐time histological imaging of kidneys stained with food dyes using multiphoton microscopy

We have developed a real‐time imaging technique for diagnosis of kidney diseases which is composed of two steps, staining renal cells safely with food dyes and optical sectioning of living renal tissue to obtain histological images by multiphoton microscopy (MPM). Here, we demonstrated that the MPM imaging with food dyes, including erythrosine and indigo carmine, could be used as fluorescent agents to visualize renal functions and structures such as glomerular bloodstreams, glomerular filtration, and morphology of glomeruli and renal tubules. We also showed that the kidneys of IgA nephropathy model‐mice stained with the food dyes presented histopathological characteristics different from those observed in normal kidneys. The use of the food dyes enhances the quality of tissue images obtained by MPM and offers the potential to contribute to a clinical real‐time diagnosis of kidney diseases. Microsc. Res. Tech. 78:847–858, 2015. © 2015 Wiley Periodicals, Inc.     

Amyloid-beta aggregates formed at polar-nonpolar interfaces differ from amyloid-beta protofibrils produced in aqueous buffers

The deposition of aggregated amyloid-beta (Abeta) peptides in the brain as senile plaques is a pathological hallmark of Alzheimer's disease (AD). Several lines of evidence indicate that fibrillar and, in particular, soluble aggregates of these 40- and 42-residue peptides are important in the etiology of AD. Recent studies also stress that amyloid aggregates are polymorphic and that a single polypeptide can fold into multiple amyloid conformations. Here we review our recent reports that Abeta(1-40) in vitro can form soluble aggregates with predominant beta-structures that differ in stability and morphology. One class of aggregates involved soluble Abeta protofibrils, prepared by vigorous overnight agitation of monomeric Abeta(1-40) in low ionic strength buffers. These aggregates were quite stable and disaggregated to only a limited extent on dilution. A second class of soluble Abeta aggregates was generated at polar-nonpolar interfaces. Aggregation in a two-phase system of buffer over chloroform occurred more rapidly than in buffer alone. In buffered 2% hexafluoroisopropanol (HFIP), microdroplets of HFIP were formed and the half-time for aggregation was less than 10 minutes. Like Abeta protofibrils, these interfacial aggregates showed increased thioflavin T fluorescence and were rich in beta-structure by circular dichroism. However, electron microscopy and atomic force microscopy revealed very different morphologies. The HFIP aggregates formed initial globular clusters that progressed over several days to soluble fibrous aggregates. When diluted out of HFIP these aggregates initially were very unstable and disaggregated completely within 2 minutes. However, their stability increased as they progressed to fibers. It is important to determine whether similar interfacial Abeta aggregates are produced in vivo.     

Correlative microscopy: a potent tool for the study of rare or unique cellular and tissue events

Biological studies have relied on two complementary microscope technologies – light (fluorescence) microscopy and electron microscopy. Light microscopy is used to study phenomena at a global scale to look for unique or rare events, and it also provides an opportunity for live imaging, whereas the forte of electron microscopy is the high resolution. Traditionally light and electron microscopy observations are carried out in different populations of cells/tissues and a 'correlative' inference is drawn. The advent of true correlative light-electron microscopy has allowed high-resolution imaging by electron microscopy of the same structure observed by light microscopy, and in advanced cases by video microscopy. Thus a rare event captured by low-resolution imaging of a population or transient events captured by live imaging can now also be studied at high resolution by electron microscopy. Here, the potential and difficulties of this approach, along with the most impressive breakthroughs obtained by these methods, are discussed.     

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.     

Structure and morphology of the Alzheimer's amyloid fibril

Amyloid fibrils are deposited in a number of diseases, including Alzheimer's disease, Type 2 diabetes, and the transmissible spongiform encephalopathies (TSE). These insoluble deposits are formed from normally soluble proteins that assemble to form fibrous aggregates that accumulate in the tissues. Electron microscopy has been used as a tool to examine the structure and morphology of these aggregates from ex vivo materials, but predominantly from synthetic amyloid fibrils assembled from proteins or peptides in vitro. Electron microscopy has shown that the fibrils are straight, unbranching, and are of a similar diameter (60-100 A) irrespective of the precursor protein. Image processing has enhanced electron micrographs to show that amyloid fibrils appear to be composed of protofilaments wound around one another. In combination with other techniques, including X-ray fiber diffraction and solid state NMR, electron microscopy has revealed that the internal structure of the amyloid fibril is a ladder of beta-sheet structure arranged in a cross-beta conformation.     

Morpho-anatomical characterization of some members of euglenophycota (algae) of north-east Punjab,Pakistan based on light microscopy (LM) and scanning electron microscopy (SEM)

The study was carried out in four districts, that is, Gujranwala, Gujarat, Narowal and Sialkot of Punjab, Pakistan. The sampling was carried out randomly in different seasons from the water bodies especially from wastewater. Twenty-one species belonging to Euglenophycota were identified using light microscopy and scanning electron microscopy from which 04 species belong to genus Phacus, 02 species belonging to Trachelomonas and Euglena based on light microscopy. It was observed that Euglena was the most diverse genus and it is supposed to be the indicator species of the polluted water. It was observed that E. oblonga was found in maximum pH range, that is, 7.0–11.0. Similarly, E. brevicaudatus was found in maximum EC, that is, 169 ± 1.5 ms/cm these outcomes indicated that for internal examination along with LM, SEM was necessary for correct identification of algal sample up to specie level.     

Manufacture and tests of objective zone plates for use in a scanning transmission X-ray microscope

A method of making high resolution zone plates for use as the focusing elements in soft X-ray microscopy is briefly described. Tests carried out on these zone plates indicate a first-order diffraction efficiency of ～0.3% rather than the calculated value of ～0.9%. This indicates that the zones are not positioned as accurately as expected, a conclusion also drawn from tests at optical wavelengths on electron micrographs of the zone plates. Modifications to the manufacturing method to enable zone plates with improved imaging properties to be made are described.     

A multiple technical approach to the study of apoptotic cell micronuclei

Apoptotic micronuclei have been studied, in different cell types, from a morphologic and functional point of view. Conventional electron microscopy, in various staining conditions, selective cytochemistry for DNA, and freeze fracture for the analysis of chromatin fiber organization and size were performed. In situ TdT and Pol I immunofluorescent techniques were carried out to detect double- and single-strand DNA breaking points by confocal laser scanning microscopy. Apoptotic cell ultrathin cryosections were also performed and were analysed by field emission in lens scanning electron microscopy. Double/single strand massively cleaved DNA was detected in micronuclei, with a highly supercoiled, uniformly packed, very dense arrangement.     

Irregular snow crystals: structural features as revealed by low temperature scanning electron microscopy

For nearly 50 years, investigators using light microscopy have vaguely alluded to a unique type of snow crystal that has become known as an irregular snow crystal. However, the limited resolution and depth-of-field of the light microscope has prevented investigators from characterizing these crystals. In this study, a field-emission scanning electron microscope, equipped with a cold stage, was used to document the structural features, physical associations, and atmospheric metamorphosis of irregular snow crystals. The crystals appear as irregular hexagons, measuring 60 to 90 mm across, when viewed from the a-axis. Their length (c-axis) rarely exceeds the diameter. The irregular crystals are occasionally found as secondary particles on other larger forms of snow crystals; however, they most frequently occur in aggregates consisting of more than 100 irregular crystals. In the aggregates, the irregular crystals have their axes oriented parallel to one another and, collectively, tend to form columnar structures. Occasionally, these columnar structures exhibit rounded faces along one side, suggesting atmospheric metamorphoses during formation and descent. In extreme cases of metamorphoses, the aggregates would be difficult to distinguish from graupel. Frost, consisting of irregular crystals, has also been encountered, suggesting that atmospheric conditions that favor their growth can also occur terrestrially.     

The structure of proteoglycan aggregate determined by atomic force microscopy

Proteoglycan aggregate is the major extracellular matrix component in cartilage, comprising about 18% of the dry weight of hyaline cartilage. The proteoglycan aggregate is the major substance in cartilage which resists compression in the joint. The purpose of this study was to utilize the newly developed imaging technique, Atomic force Microscopy (AFM), to visualize the ultrastructure of proteoglycan aggregates. The proteoglycan aggregate molecules were imaged in air using the tapping mode of the AFM. The images illustrated the ultrastructure of the aggregates, especially the individual proteoglycan and the core hyaluronic acid. In addition to the length and width of each molecule, the height of the proteoglycan aggregates and the individual proteoglycans could be directly measured. The images of the ultrastructures of proteoglycan aggregates visualized from the AFM are comparable with those using conventional electron microscopy approaches. Nevertheless, the sample preparation for AFM imaging does not involve fixation, staining, coating, and other routine procedures required for traditional electron microscopy imaging. Thus, this technique could be a simple alternative approach for future analysis of proteoglycan aggregate and its assembly.     

Microtribology friction measurements on microtextured silicon

Abstract

This paper describes the results of microtribology sliding experiments that were carried out on a range of micropatterned silicon surfaces. The objective of this work was to identify critical pattern features that control friction and fluid interaction on surfaces under a broad range of contact conditions and develop models to explain these relationships. These scratch tests were carried out in air, and with deionised water. Tests were carried out under a range of test loads and speeds, from low to high. It was found that in the dry tests, the measured friction was higher on the patterned surfaces than on the non-patterned surface. For the liquid experiments, there was a reduction in friction in some cases, dependent on the pattern and conditions used, indicating that the liquid acted as a lubricant. The greatest reduction in friction was obtained with a herringbone pattern aligned perpendicular to the direction of travel of the probe. However, for higher load conditions, high friction coefficients were measured for many of the patterns. The surfaces of the tested silicon samples were examined with optical microscopy and in high resolution scanning electron microscopy so that the interactions between the probe and the sample could be determined. It was found that the high friction values that had been observed in many experiments were correlated with considerable damage to the structure of the patterned silicon surface.     

Combining FIB milling and conventional Argon ion milling techniques to prepare high‐quality site‐specific TEM samples for quantitative EELS analysis of oxygen in molten iron

This paper reports a procedure to combine the focused ion beam micro‐sampling method with conventional Ar‐milling to prepare high‐quality site‐specific transmission electron microscopy cross‐section samples. The advantage is to enable chemical and structural evaluations of oxygen dissolved in a molten iron sample to be made after quenching and recovery from high‐pressure experiments in a laser‐heated diamond anvil cell. The evaluations were performed by using electron energy‐loss spectroscopy and high‐resolution transmission electron microscopy. The high signal to noise ratios of electron energy‐loss spectroscopy core‐loss spectra from the transmission electron microscopy thin foil, re‐thinned down to 40 nm in thickness by conventional Argon ion milling, provided us with oxygen quantitative analyses of the quenched molten iron phase. In addition, we could obtain lattice‐fringe images using high‐resolution transmission electron microscopy. The electron energy‐loss spectroscopy analysis of oxygen in Fe_0.94O has been carried out with a relative accuracy of 2%, using an analytical procedure proposed for foils thinner than 80 nm. Oxygen K‐edge energy‐loss near‐edge structure also allows us to identify the specific phase that results from quenching and its electronic structure by the technique of fingerprinting of the spectrum with reference spectra in the Fe‐O system.     

Use of high-pressure freeze fixation and freeze fracture electron microscopy to study the influence of the phospholipid molar ratio in the morphology and alignment of bicelles

The high-pressure freeze fixation and freeze fracture electron microscopy techniques were combined with the ³¹P nuclear magnetic resonance to study the morphological transitions of two different dimyristoyl-phosphatidilcholine/dihexanoyl-phosphocholine aggregates by the effect of temperature. Through these techniques, the relationship between magnetic alignment and the morphology of alignable and non-alignable aggregates was evaluated. The micrographs related to the non-alignable dimyristoyl-phosphatidilcholine/dihexanoyl-phosphocholine sample presented rounded objects at a temperature below the dimyristoyl-phosphatidilcholine phase transition (T_m) and, above this temperature an increase of viscosity was followed by the appearance of large elongated aggregates. The micrographs related to the alignable dimyristoyl-phosphatidilcholine/dihexanoyl-phosphocholine sample presented discoidal objects below T_m. Above T_m, when the best alignment was achieved, the images showed large areas of lamellar stacked bilayers and the presence of some multilamellar vesicles. Our results reveal that the composition of the aggregates is a key factor determining the morphological transitions of the bicellar systems. Understanding of the rules governing these transitions is crucial to modulate characteristics of these systems and to adequate them for different applications.     

Characterization of intact subcellular bodies in whole bacteria by cryo-electron tomography and spectroscopic imaging

We illustrate the combined use of cryo‐electron tomography and spectroscopic difference imaging in the study of subcellular structure and subcellular bodies in whole bacteria. We limited our goal and focus to bodies with a distinct elemental composition that was in a sufficiently high concentration to provide the necessary signal‐to‐noise level at the relatively large sample thicknesses of the intact cell. This combination proved very powerful, as demonstrated by the identification of a phosphorus‐rich body in Caulobacter crescentus. We also confirmed the presence of a body rich in carbon, demonstrated that these two types of bodies are readily recognized and distinguished from each other, and provided, for the first time to our knowledge, structural information about them in their intact state. In addition, we also showed the presence of a similar type of phosphorus‐rich body in Deinococcus grandis, a member of a completely unrelated bacteria genus. Cryo‐electron microscopy and tomography allowed the study of the biogenesis and morphology of these bodies at resolutions better than 10 nm, whereas spectroscopic difference imaging provided a direct identification of their chemical composition.     

A simple method for processing individual oocytes and embryos for electron microscopy

A simple method for handling individual specimens that must be processed either for scanning or transmission electron microscopy studies is described. For scanning microscope processing, dehydration is carried out with samples enclosed in small cages made from TAAB capsules in which top and bottom are substituted by plankton nets, and for transmission electron microscopy, samples are pre-embedded in agarose. This procedure significantly reduces mouth pipetting, dissecting microscope observations, is less labour intensive and, most importantly, reduces sample loss.     

Ultrastructural characterization of isolated rat Kupffer cells by transmission X-ray microscopy

The ultrastructure of primary cultured rat Kupffer cells was studied using transmission X-ray microscopy as well as transmission electron microscopy. X-ray microscopical images of intact, hydrated Kupffer cells demonstrated structures such as cell nucleus separated by a nuclear membrane and filaments concentrated in the perinuclear area. Within the cytoplasm, a number of vacuoles were visible; some of these were crescent-shaped vacuoles that were half X-ray lucent, half X-ray dense; others were uniformly dense. The number of crescent-shaped vacuoles was predominant. After phagocytosis of haematite particles, enlarged vacuoles containing the ingested material were visible within the cytoplasm of Kupffer cells while crescent-shaped vacuoles were no longer detectable. Densitometric analysis of the two types of vacuole revealed that the X-ray absorption of the uniform vacuole was approximately half that of the dense part of the crescent-shaped vacuoles. This observation led to speculation on the existence of only one type of vacuole in the cytoplasm of Kupffer cells. The different morphological aspects — crescent-shaped versus uniform vacuoles — might be due to different three-dimensional orientation with respect to the image plane. Using transmission electron microscopy, the morphology of vacuoles differed more widely in diameter, density and shape. Two main types of vacuole were identified: electron-lucent and electron-dense. Based on the observation of only one type of vacuole by transmission X-ray microscopy, the different morphological aspects of vacuoles obtained by transmission electron microscopy could be explained by imaging several different sections of a crescent-shaped vacuole. From the present data it can be concluded that transmission X-ray microscopy is a versatile technique that reveals the ultrastructure of intact, unsectioned biological specimens in their aqueous environment, thereby allowing a more comprehensive interpretation of data obtained by transmission electron microscopy.     

Advantages of the use of intact plant tissues in freeze-fracture electron microscopy

The use of whole, intact plant tissue for freeze-fracture electron microscopy provides important information that cannot be obtained from the use of isolated biological membranes or of artifical (phospholipid) membrane preparations. This is not to imply that these exminations of such preparations are not useful, since it would be difficult to interpret our observations of intact cells and tissues without the analysis of these model systems. Analysis of intact tissue and cells reveals the relative densities of membrane proteins of the different membranes within a cell; the three-dimensional organization of various organelles, especially the endoplasmic reticulum; changes in intramembranous particle (IMP) distribution due to stress or injury; and, in conjunction with the use of filipin, membrane sterol content and relative distribution. It is our intention that this survey of freeze-fracture images of intact plant tissues will illustrate the uniqueness of the information gained from an analysis of whole plant tissues compared to isolated membrane fractions.     

HREM, FIM and tomographic atom probe investigation of Guinier-Preston zones in an Al-1.54at% Cu alloy

An investigation of Guinier-Preston zones in Al-1.54at% Cu alloy annealed for 30h at 100 degrees C was carried out on the same monocrystal with complementary techniques of high-resolution electron microscopy (HREM) and tomographic atom probe-field ion microscopy (TAP-FIM). HREM results show that majority of GP1 zones are monolayers 1-9nm in size. However, some GP2 zones and particles in an intermediate state of growth between GP1 and GP2 stage were also found. From TAP results it follows that GP1 zones with different Cu concentrations ranging from 40% up to 100% Cu coexist. The residual solid solution is very heterogeneous. In the vicinity of GP particles the Cu content in the matrix falls down to zero, the solid solution in other regions contains from 0.7 to 1at% Cu.     

Flat embedding and immunolabelling of SW 1116 colon carcinoma cells in LR white: an improved technique in light and electron microscopy.

Human SW 1116 colon carcinoma cells were grown on matrix-covered coverslips and flat embedded in specially prepared gelatin capsules in the hydrophylic resin LR White. Dehydration and polymerization were carried out so as to maximize preservation of antigenicity. Sections were cut perpendicular to the substratum. To visualize mucin, semithin sections of SW 1116 cells were stained with periodic acid Schiff (PAS) reagent for light microscopy, and ultrathin sections were labelled with a monoclonal mucin antibody (Mab 19-9) and immunogold for electron microscopy. Immunofluorescence was carried out on whole cultured cells using Mab 19-9. The morphological preservation of SW 1116 cells embedded in LR White was comparable to that of Epon-embedded cells. Mucin was localized on the microvillar surface of the apical plasma membrane and occasionally in intercellular spaces between adjacent cells. Mucin was also present in vesicles in the apical and lateral part, and to a lesser extent in the basal part of the cells. We conclude that this new technology significantly improves the morphological preservation of cells and tissues in LR White, while also serving to sustain the antigenicity of cellular antigens.     

船用冷凝器换热管的失效分析

针对船用海水冷凝器铜合金换热管腐蚀失效的问题,对铜合金换热管进行了腐蚀失效分析,进行了换热管失效部位外观形貌分析;对失效的换热管和完好的换热管进行了材料化学成分分析,并与材料的化学成分标准值做了对比;运用扫描电镜对换热管失效部位腐蚀产物进行了射线能谱分析;进行了换热管失效部位腐蚀坑扫描电镜分析。研究结果表明,腐蚀是从换热管内部开始的,并且在换热管腐蚀部位没有检测到砷元素;结合铜合金在海水中的腐蚀失效机理可知,该海水冷凝器换热管失效为换热管材料中砷元素偏析导致的脱锌腐蚀。