Comparison of single‐particle analysis and electron tomography approaches: an overview

Three‐dimensional structure of a wide range of biological specimens can be computed from images collected by transmission electron microscopy. This information integrated with structural data obtained with other techniques (e.g., X‐ray crystallography) helps structural biologists to understand the function of macromolecular complexes and organelles within cells. In this paper, we compare two three‐dimensional transmission electron microscopy techniques that are becoming more and more related (at the image acquisition level as well as the image processing one): electron tomography and single‐particle analysis. The first one is currently used to elucidate the three‐dimensional structure of cellular components or smaller entire cells, whereas the second one has been traditionally applied to structural studies of macromolecules and macromolecular complexes. Also, we discuss possibilities for their integration with other structural biology techniques for an integrative study of living matter from proteins to whole cells.     

Simple technique for high‐throughput marking of distinguishable micro‐areas for microscopy

Today's (nano)‐functional materials, usually exhibiting complex physical properties require local investigation with different microscopy techniques covering different physical aspects such as dipolar and magnetic structure. However, often these must be employed on the very same sample position to be able to truly correlate those different information and corresponding properties. This can be very challenging if not impossible especially when samples lack prominent features for orientation. Here, we present a simple but effective method to mark hundreds of approximately 15×15 μm sample areas at one time by using a commercial transmission electron microscopy grid as shadow mask in combination with thin‐film deposition. Areas can be easily distinguished when using a reference or finder grid structure as shadow mask. We show that the method is suitable to combine many techniques such as light microscopy, scanning probe microscopy and scanning electron microscopy. Furthermore, we find that best results are achieved when depositing aluminium on a flat sample surface using electron‐beam evaporation which ensures good line‐of‐sight deposition. This inexpensive high‐throughput method has several advantageous over other marking techniques such as focused ion‐beam processing especially when batch processing or marking of many areas is required. Nevertheless, the technique could be particularly valuable, when used in junction with, for example focused ion‐beam sectioning to obtain a thin lamellar of a particular pre‐selected area.     

Effects of metals cadmium and chromium alone and in combination on the liver and kidney tissue of male Spraque‐Dawley rats: An ultrastructural and electron‐energy‐loss spectroscopy investigation

Heavy metal pollution has increased in the last decades. Water sources are contaminated and human exposure is often long term exposure to variable amounts of different metals. In this study, male Sprague‐Dawley rats were exposed via oral gavage for 28 days to cadmium (Cd) and chromium (Cr), alone and in combination at concentrations 1000 times the human World Health Organization's acceptable water limits. Rat equivalent dosages were used. Blood markers of liver and kidney function were measured, changes to cellular morphology was determined with transmission electron microscopy and the intracellular metal localisation was determined with the electron energy‐loss spectroscopy and energy filtered transmission electron microscopy analysis. Both Cd and Cr caused changes to the nuclear and mitochondrial membranes and irregular chromatin condensation of hepatocytes. Cr exposure caused dilation of the rough endoplasmic reticulum (rER). The combination caused nuclear and mitochondrial membrane damage as well as irregular chromatin condensation. In the kidney tissue, Cd caused irregular chromatin condensation in the cells of the proximal convoluted tubule (PCT). Cr caused changes to the outer nuclear and mitochondrial membrane and chromatin structure. The combination group caused membrane damage, irregular chromatin condensation and rER changes in the PCT. All the metal groups showed damage to the endothelial cells and pedicles, but not to the mesangial cells. Cd and Cr bio‐accumulation was observed in the nucleus, mitochondria and rER of the liver and kidney and therefore are responsible for the cellular observed damage that can cause functional changes to the tissues and organs.     

Metabolic labeling of Escherichia coli genomic DNA with erythrosine‐11‐dUTP for functional imaging via correlative microscopy

The fluorescent metabolic labeling of microorganisms genome is an advanced imaging technique to observe and study the native shapes, structural changes, functions, and tracking of nucleic acids in single cells or tissues. We have attempted to visualize the newly synthesized DNA within the intact nucleoid of ice‐embedded proliferating cells of Escherichia coli K‐12 (thymidine‐requiring mutant, strain N4316) via correlative light‐electron microscopy. For that purpose, erythrosine‐11‐dUTP was synthesized and used as a modified analog of the exogenous thymidine substrate for metabolic incorporation into the bacterial chromosome. The formed fluorescent genomic DNA during in cellulo polymerase reaction caused a minimal cellular arrest and cytotoxicity of E. coli at certain controlled conditions. The stained cells were visualized in typical red emission color via an epifluorescence microscope. They were further ice‐embedded and examined with a Hilbert differential contrast transmission electron microscopy. At high‐resolution, the ultrastructure of tagged nucleoid appeared with significantly higher electron dense in comparison to the unlabeled one. The enhanced contrast areas in the chromosome were ascribed to the presence of iodine contents from erythrosine dye. The presented labeling approach might be a powerful strategy to reveal the structural and dynamic changes in natural DNA replication including the relationship between newly synthesized in vivo nucleic acid and the physiological state of the cell.     

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.     

Ultra‐thin resin embedding method for scanning electron microscopy of individual cells on high and low aspect ratio 3D nanostructures

The preparation of biological cells for either scanning or transmission electron microscopy requires a complex process of fixation, dehydration and drying. Critical point drying is commonly used for samples investigated with a scanning electron beam, whereas resin‐infiltration is typically used for transmission electron microscopy. Critical point drying may cause cracks at the cellular surface and a sponge‐like morphology of nondistinguishable intracellular compartments. Resin‐infiltrated biological samples result in a solid block of resin, which can be further processed by mechanical sectioning, however that does not allow a top view examination of small cell–cell and cell–surface contacts. Here, we propose a method for removing resin excess on biological samples before effective polymerization. In this way the cells result to be embedded in an ultra‐thin layer of epoxy resin. This novel method highlights in contrast to standard methods the imaging of individual cells not only on nanostructured planar surfaces but also on topologically challenging substrates with high aspect ratio three‐dimensional features by scanning electron microscopy.     

Three‐dimensional bright‐field scanning transmission electron microscopy elucidate novel nanostructure in microbial biofilms

Bacterial biofilms play key roles in environmental and biomedical processes, and understanding their activities requires comprehension of their nanoarchitectural characteristics. Electron microscopy (EM) is an essential tool for nanostructural analysis, but conventional EM methods are limited in that they either provide topographical information alone, or are suitable for imaging only relatively thin (<300 nm) sample volumes. For biofilm investigations, these are significant restrictions. Understanding structural relations between cells requires imaging of a sample volume sufficiently large to encompass multiple cells and the capture of both external and internal details of cell structure. An emerging EM technique with such capabilities is bright‐field scanning transmission electron microscopy (BF‐STEM) and in the present report BF‐STEM was coupled with tomography to elucidate nanostructure in biofilms formed by the polycyclic aromatic hydrocarbon‐degrading soil bacterium, Delftia acidovorans Cs1‐4. Dual‐axis BF‐STEM enabled high‐resolution 3‐D tomographic recontructions (6–10 nm) visualization of thick (1250 and 1500 nm) sections. The 3‐D data revealed that novel extracellular structures, termed nanopods, were polymorphic and formed complex networks within cell clusters. BF‐STEM tomography enabled visualization of conduits formed by nanopods that could enable intercellular movement of outer membrane vesicles, and thereby enable direct communication between cells. This report is the first to document application of dual‐axis BF‐STEM tomography to obtain high‐resolution 3‐D images of novel nanostructures in bacterial biofilms. Future work with dual‐axis BF‐STEM tomography combined with correlative light electron microscopy may provide deeper insights into physiological functions associated with nanopods as well as other nanostructures.     

High-resolution scanning electron microscopy of immunogold-labelled cells by the use of thin plasma coating of osmium

The feasibility of plasma coating of a thin osmium layer for high‐resolution immuno‐scanning electron microscopy of cell surfaces was tested, using Drosophila embryonic motor neurones as a model system. The neuro‐muscular preparations were fixed with formaldehyde and labelled with a neurone‐specific antibody and 10 or 5 nm colloidal gold‐conjugated secondary antibodies. The specimens were post‐fixed with osmium tetroxide and freeze‐dried. Then they were coated with a 1–2 nm thick layer of osmium using a hollow cathode plasma coater. The thin and continuous coating of amorphous osmium gave good signals of gold particles and fine surface structures of neurites in backscattered electron images simultaneously. This method makes it possible to visualize the antigen distribution and the three‐dimensionally complex surface structures of cellular processes with a resolution of several nanometres.     

Molecular ultrastructure of the urothelial surface: Insights from a combination of various microscopic techniques

The urothelium forms the blood–urine barrier, which depends on the complex organization of transmembrane proteins, uroplakins, in the apical plasma membrane of umbrella cells. Uroplakins compose 16 nm intramembrane particles, which are assembled into urothelial plaques. Here we present an integrated survey on the molecular ultrastructure of urothelial plaques in normal umbrella cells with advanced microscopic techniques. We analyzed the ultrastructure and performed measurements of urothelial plaques in the normal mouse urothelium. We used field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), transmission electron microscopy (TEM) on immunolabeled ultrathin sections (immuno‐TEM), and freeze‐fracture replicas (FRIL). We performed immunolabeling of uroplakins for scanning electron microscopy (immuno‐FESEM). All microscopic techniques revealed a variability of urothelial plaque diameters ranging from 332 to 1179 nm. All immunolabeling techniques confirmed the presence of uroplakins in urothelial plaques. FRIL showed the association of uroplakins with 16 nm intramembrane particles and their organization into plaques. Using different microscopic techniques and applied qualitative and quantitative evaluation, new insights into the urothelial apical surface molecular ultrastructure have emerged and may hopefully provide a timely impulse for many ongoing studies. The combination of various microscopic techniques used in this study shows how these techniques complement one another. The described advantages and disadvantages of each technique should be considered for future studies of molecular and structural membrane specializations in other cells and tissues. Microsc. Res. Tech. 77:896–901, 2014. © 2014 Wiley Periodicals, Inc.     

F‐actin and α‐actinin reorganization mediates initial fibroblast interaction with CoCr alloy particles in vitro

Asceptic loosening remains the primary cause for failure of joint implant. The active role of fibroblasts in mediating asceptic loosening is however not well documented. In this study the initial interactions of fibroblasts with metal particles was studied by evaluating changes in the cytoskeletal structure and cytokine level. Murine L929 fibroblasts cultured with cobalt chromium particles were observed by phase contrast and scanning electron microscopy (SEM). Changes in the cytoskeletal rearrangement of F‐actin and α‐actinin focal adhesion plaques were studied by confocal microscopy. Expression of the proinflammatory cytokines IL‐6 and IL‐1α were analyzed by ELISA. The role of actin filaments and microtubules in particle uptake were determined at low temperature and in presence of colchicine and cytochalasin B. Phase contrast and SEM studies reveal that the metal particles adhere to the fibroblasts. The cellular cytoplasm was observed to grow over the particles and is suggestive of particle uptake. Confocal microscopy shows the presence of voids within the F‐actin cytoskeletal framework corresponding to areas occupied by the metal particles, indicating the possible uptake of these particles. Aggregates of α‐actinin into patches at the cell surface were also noted. Adherence and uptake of particles did not occur at low temperature and in presence of cytochalasin B, indicating that it is an active energy‐dependent process involving actin filaments. Changes in the levels of cytokine IL‐6 and IL‐1α were not observed suggesting the role of other cytokine molecules in mediating the inflammatory response to wear debri by fibroblasts. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Immuno-EM using colloidal metal nanoparticles and electron spectroscopic imaging for co-localization at high spatial resolution

Multiple‐labelling immuno‐EM is a powerful tool for localizing and co‐localizing different antigens simultaneously in cells and tissues at high spatial resolution. Commonly used labels for this purpose are differently sized gold spheres. A comparison of results obtained with differently sized markers is often difficult, because the diameters of markers influence labelling efficiency. In the current study, we investigate a method for high‐resolution multiple‐labelling immuno‐EM, using equally sized colloidal markers made of different metals. Energy filtering transmission electron microscopy is used to differentiate particles based on elemental composition. The labels consist of colloidal gold, palladium and platinum‐core gold‐shell particles of approximately 6 nm in diameter, which are conjugated to different primary antibodies. Applicability of the electron spectroscopic imaging, methodology is demonstrated by labelling of actin, α‐actinin and myosin on ultra‐thin cryosections of skeletal muscle tissue.     

Candida albicans biofilms: comparative analysis of room‐temperature and cryofixation for scanning electron microscopy

Biofilms are frequently related to invasive fungal infections and are reported to be more resistant to antifungal drugs than planktonic cells. The structural complexity of the biofilm as well as the presence of a polymeric extracellular matrix (ECM) is thought to be associated with this resistant behavior. Scanning electron microscopy (SEM) after room temperature glutaraldehyde‐based fixation, have been used to study fungal biofilm structure and drug susceptibility but they usually fail to preserve the ECM and, therefore, are not an optimised methodology to understand the complexity of the fungal biofilm. Thus, in this work, we propose a comparative analysis of room‐temperature and cryofixation/freeze substitution of Candida albicans biofilms for SEM observation. Our experiments showed that room‐temperature fixative protocols using glutaraldehyde and osmium tetroxide prior to alcohol dehydration led to a complete extraction of the polymeric ECM of biofilms. ECM from fixative and alcohol solutions were recovered after all processing steps and these structures were characterised by biochemistry assays, transmission electron microscopy and mass spectrometry. Cryofixation techniques followed by freeze‐substitution lead to a great preservation of both ECM structure and C. albicans biofilm cells, allowing the visualisation of a more reliable biofilm structure. These findings reinforce that cryofixation should be the indicated method for SEM sample preparation to study fungal biofilms as it allows the visualisation of the EMC and the exploration of the biofilm structure to its fullest, as its structural/functional role in interaction with host cells, other pathogens and for drug resistance assays.     

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.     

Energy dispersive spectroscopy‐scanning transmission electron microscope observations of free radical production in human polymorphonuclear leukocytes phagocytosing non‐opsonized Tannerella forsythia

We investigated the association between human polymorphonuclear leukocytes (PMNs) and non‐opsonized Tannerella forsythia ATCC 43037 displaying a serum‐resistant surface layer (S‐layer). When PMNs were mixed with T. forsythia in suspension, the cells phagocytosed T. forsythia cells. Nitro blue tetrazolium (NBT) reduction, indicative of production, was observed by light microscopy; cerium (Ce) perhydroxide deposition, indicative of H₂O₂ production, was observed by electron microscopy. We examined the relationship between high‐molecular‐weight proteins of the S‐layer and Ce reaction (for T. forsythia phagocytosis) using electron microscopic immunolabeling. Immunogold particles were localized within the PMNs and on cell surfaces, labelling at the same Ce‐reacted sites where the S‐layer was present. We then used energy dispersive spectroscopy (EDS)‐scanning transmission electron microscope (STEM) to perform Ce and nitrogen (N) (for S‐layer immunocytochemistry) elemental analysis on the phagocytosed cells. That is, the elemental mapping and analysis of N by EDS appeared to reflect the presence of the same moieties detected by the 3,3′‐diaminobenzidine‐tetrahydrochloride (DAB) reaction with horseradish peroxidase (HRP)‐conjugated secondary antibodies, instead of immunogold labeling. We focused on the use of EDS‐STEM to visualize the presence of N resulting from the DAB reaction. In a parallel set of experiments, we used EDS‐STEM to perform Ce and gold (Au; from immunogold labeling of the S‐layer) elemental analysis on the same phagocytosing cells.     

Detection of CD133 (prominin‐1) in a human hepatoblastoma cell line (HuH‐6 clone 5)

We examined CD133 distribution in a human hepatoblastoma cell line (HuH‐6 clone 5). We directly observed the cultured cells on a pressure‐resistant thin film (silicon nitride thin film) in a buffer solution by using the newly developed atmospheric scanning electron microscope (ASEM), which features an open sample dish with a silicon nitride thin film window at its base, through which the scanning electron microscope beam scans samples in solution, from below. The ASEM enabled observation of the ventral cell surface, which could not be observed using standard SEM. However, observation of the dorsal cell surface was difficult with the ASEM. Therefore, we developed a new method to observe the dorsal side of cells by using Aclar® plastic film. In this method, cells are cultured on Aclar plastic film and the dorsal side of cells is in contact with the thin silicon nitride film of the ASEM dish. A preliminary study using the ASEM showed that CD133 was mainly localized in membrane ruffles in the peripheral regions of the cell. Standard transmission electron microscopy and scanning electron microscopy revealed that CD133 was preferentially concentrated in a complex structure comprising filopodia and the leading edge of lamellipodia. We also observed co‐localization of CD133 with F‐actin. An antibody against CD133 decreased cell migration. Methyl‐β‐cyclodextrin treatment decreased cell adhesion as well as lamellipodium and filopodium formation. A decrease in the cholesterol level may perturb CD133 membrane localization. The results suggest that CD133 membrane localization plays a role in tumor cell adhesion and migration. Microsc. Res. Tech. 76:844–852, 2013. © 2013 Wiley Periodicals, Inc.     

Sperm ultrastructure and spermatogenesis in the lizard, <i>Tropidurus itambere</i>

Spermatogenesis, with emphasis on spermiogenesis, is described for the lizard, Tropidurus itambere, using light microscopy, phase contrast and epifluorescence, as well as scanning and transmission electron microscopy. Cellular differentiation involves events of chromatin condensation, nuclear elongation and the formation of structural complexes, such as the acrosomal and axonemal ones. Other new characteristics, exclusive for this species, include various aspects of the subacrosomal granule, the insertion of the proacrosomal vesicle and the development of these structures to participate in the acrosomal complex. Radial projections occur just above the nuclear shoulders, which have been recognized already from the beginning of cellular elongation. The development of the midpiece, the dense bodies, formation of the flagellum and elimination of residual cytoplasm result in the final characterization of the mature spermatozoon. Comparisons between Tropiduridae and other lizard families are made.     

Colloidal particle aggregation in three dimensions

Colloidal systems are of importance not only for everyday products, but also for the development of new advanced materials. In many applications, it is crucial to understand and control colloidal interaction. In this paper, we study colloidal particle aggregation of silica nanoparticles, where the data are given in a three-dimensional micrograph obtained by high-angle annular dark field scanning transmission electron microscopy tomography. We investigate whether dynamic models for particle aggregation, namely the diffusion limited cluster aggregation and the reaction limited cluster aggregation models, can be used to construct structures present in the scanning transmission electron microscopy data. We compare the experimentally obtained silica aggregate to the simulated postaggregated structures obtained by the dynamic models. In addition, we fit static Gibbs point process models, which are commonly used models for point patterns with interactions, to the silica data. We were able to simulate structures similar to the silica structures by using Gibbs point process models. By fitting Gibbs models to the simulated cluster aggregation patterns, we saw that a smaller probability of aggregation would be needed to construct structures similar to the observed silica particle structure.     

Distribution and three‐dimensional appearance of the interstitial cells of Cajal in the rat stomach and duodenum

The relationship between the interstitial cells of Cajal (ICC) and enteric nerves or smooth muscles cells is not fully defined. Presently, distribution and appearance of ICC in the rat stomach and duodenum was studied by immunohistochemistry, electron microscopy, and three‐dimensional reconstruction. c‐kit expressing ICC were regularly observed in the Auerbach's myenteric plexus (AP) of the stomach and duodenum. ICC in stomach and duodenum muscle layers was dissimilarly distributed. c‐kit immunoreactive cells were sparsely distributed in the stomach circular muscle layer but were abundant in the duodenum deep muscular plexus (DMP). Electron microscopy revealed that stomach ICC‐AP were irregular ovals with few cytoplasmic processes, and possessed an electron‐dense cytoplasm, numerous mitochondria, intermediate filaments, and caveolae. Duodenum and stomach ICC‐AP were similar in appearance. Ultrastructure observations and three‐dimensional reconstructions revealed ICC‐AP processes wrapping the nerve fibers and projecting into the space between smooth muscle cells. While ICC‐AP was occasionally close to enteric nerves or smooth muscle cells, no connections were observed. ICC‐DMP in duodenum was elongated and adopted the same cell axis orientation as the circular muscle cells. Unlike ICC‐AP, ICC‐DMP formed gap junctions with smooth muscle cells and had close contact with nerves. These results indicate that ICC‐AP is regularly distributed in stomach and duodenum, while ICC‐DMP is exclusively located in the duodenum. ICC‐DMP, which possess gap junctions and closely contacts nerves, may participate in neuromuscular transmission. Microsc. Res. Tech. 2009. © 2009 Wiley‐Liss, Inc.