Correlative light and electron microscopy reveals discrepancy between gold and fluorescence labelling.

Electron microscopy (EM) is traditionally employed as a follow-up to fluorescence microscopy (FM) to resolve the cellular ultrastructures wherein fluorescently labelled biomolecules reside. In order to translate the information derived from FM studies to EM analysis, biomolecules of interest must be identified in a manner compatible with EM. Although fluorescent signals can serve this purpose when FM is combined with EM in correlative light and electron microscopy (CLEM), the traditional immunogold labelling remains commonly used in this context. In order to investigate how much these two strategies relate, we have directly compared the subcellular localization of on-section fluorescence labelling with on-section immunogold labelling. In addition to antibody labelling of LAMP-1, bioorthogonal click labelling was used to localize soluble cysteine cathepsins or membrane-associated sialylated glycans. We reveal and characterize the existence of inherent discrepancies between the fluorescence signal and the distribution of gold particles in particular in the case of membrane-associated antigens.

Foil-hole and data image quality assessment in 3DEM: Towards high-throughput image acquisition in the electron microscope.

Automatic or semiautomatic data collection approaches on a transmission electron microscope (TEM) for Single Particle Analysis, capable of acquiring large datasets composed of only high quality images, are of great importance to obtain 3D density maps with the highest resolution possible. Typically, this task is performed by an experienced microscopist, who manually decides to keep or discard images according to subjective criteria. Therefore, this methodology is slow, intensive in human work and subjective. In this work, we propose a method to automatically or semiautomatically perform this image selection task. The approach is based on some simple, fast and effective image quality descriptors, which can be computed during acquisition, to characterize foil-hole and data images. The proposed approach has been used to evaluate the quality of different datasets consisting of foil-hole and data images obtained with a FEI Titan Krios electron microscope. The results show that the proposed method is very effective evaluating the quality of foil-hole and data images, as well as predicting the quality of the data images from the foil-hole images.

Conical Fourier shell correlation applied to electron tomograms.

The resolution of electron tomograms is anisotropic due to geometrical constraints during data collection, such as the limited tilt range and single axis tilt series acquisition. Acquisition of dual axis tilt series can decrease these effects. However, in cryo-electron tomography, to limit the electron radiation damage that occurs during imaging, the total dose should not increase and must be fractionated over the two tilt series. Here we set out to determine whether it is beneficial fractionate electron dose for recording dual axis cryo electron tilt series or whether it is better to perform single axis acquisition. To assess the quality of tomographic reconstructions in different directions here we introduce conical Fourier shell correlation (cFSCe/o). Employing cFSCe/o, we compared the resolution isotropy of single-axis and dual-axis (cryo-)electron tomograms using even/odd split data sets. We show that the resolution of dual-axis simulated and cryo-electron tomograms in the plane orthogonal to the electron beam becomes more isotropic compared to single-axis tomograms and high resolution peaks along the tilt axis disappear. cFSCe/o also allowed us to compare different methods for the alignment of dual-axis tomograms. We show that different tomographic reconstruction programs produce different anisotropic resolution in dual axis tomograms. We anticipate that cFSCe/o can also be useful for comparisons of acquisition and reconstruction parameters, and different hardware implementations.

Towards the imaging of Weibel-Palade body biogenesis by serial block face-scanning electron microscopy.

Electron microscopy is used in biological research to study the ultrastructure at high resolution to obtain information on specific cellular processes. Serial block face-scanning electron microscopy is a relatively novel electron microscopy imaging technique that allows three-dimensional characterization of the ultrastructure in both tissues and cells by measuring volumes of thousands of cubic micrometres yet at nanometre-scale resolution. In the scanning electron microscope, repeatedly an image is acquired followed by the removal of a thin layer resin embedded biological material by either a microtome or a focused ion beam. In this way, each recorded image contains novel structural information which can be used for three-dimensional analysis. Here, we explore focused ion beam facilitated serial block face-scanning electron microscopy to study the endothelial cell-specific storage organelles, the Weibel-Palade bodies, during their biogenesis at the Golgi apparatus. Weibel-Palade bodies predominantly contain the coagulation protein Von Willebrand factor which is secreted by the cell upon vascular damage. Using focused ion beam facilitated serial block face-scanning electron microscopy we show that the technique has the sensitivity to clearly reveal subcellular details like mitochondrial cristae and small vesicles with a diameter of about 50 nm. Also, we reveal numerous associations between Weibel-Palade bodies and Golgi stacks which became conceivable in large-scale three-dimensional data. We demonstrate that serial block face-scanning electron microscopy is a promising tool that offers an alternative for electron tomography to study subcellular organelle interactions in the context of a complete cell.

Electron diffraction study of lipids in non-lesional stratum corneum of atopic eczema patients.

Skin barrier impairment is thought to be an important factor in the pathogenesis of atopic eczema (AE). The skin barrier is located in the stratum corneum (SC), consisting of corneocytes embedded in lipids. Ceramides, cholesterol and free fatty acids are the major lipid classes and are crucial for the skin barrier function, but their role in relation to AE is indistinct. Filaggrin is an epidermal barrier protein and common mutations in the filaggrin gene strongly predispose for AE. However, there is no strong evidence that filaggrin mutations are related to the reduced skin barrier in AE. In this study, electron diffraction is used in order to study the lipid organization of control SC and non-lesional SC of AE patients in vivo. An increased presence of the hexagonal lipid organization was observed in non-lesional SC of AE patients, indicating a less dense lipid organization. These changes correlate with a reduced skin barrier function as measured with transepidermal water loss but do not correlate with the presence of filaggrin mutations. These results are indicative for the importance of the lipid organization for a proper skin barrier function.

Mammalian orthoreovirus T3D infects U-118 MG cell spheroids independent of junction adhesion molecule-A.

In the canonical pathway, infection of cells by the wild-type mammalian orthoreovirus Type 3 Dearing (T3D) is dependent on the interaction of the viral spike protein σ1 with the high-affinity cellular receptor junction adhesion molecule-A (JAM-A). We previously demonstrated that the human glioblastoma cell line U-118 MG does not express JAM-A and resists reovirus T3D infection in standard cell culture conditions (SCCC). Heterologous JAM-A expression sensitises U-118 MG cells to reovirus T3D. Here we studied reovirus infection in U-118 MG cells grown in spheroid cultures with the premise that cells in such cultures resemble cells in tumours more than those grown under standard adherent cell culture conditions on a plastic surface. Although the U-118 MG cells in spheroids do not express JAM-A, they are susceptible to reovirus T3D infection. We show that this can be attributed to factors secreted by cells in the spheroids. The concentration of active extracellular proteases cathepsin B and L in the medium of spheroid cultures was increased 19- and 24-fold, respectively, as compared with SCCC. These enzymes can convert the reovirus particles into a form that can infect the U-118 MG cells independent of JAM-A. Taken together, these data demonstrate that infection of tumour cells by wild-type reovirus T3D is not strictly dependent on the expression of JAM-A on the cell surface.

Ultrastructural characterization of maturing iPSC-derived nephron structures upon transplantation.

Pluripotent stem cell-derived kidney organoids hold great promise as a potential auxiliary transplant tissue for individuals with end-stage renal disease and as a platform for studying kidney diseases and drug discovery. To establish accurate models, it is crucial to thoroughly characterize the morphological features and maturation stages of the cellular components within these organoids. Nephrons, the functional units of the kidney, possess distinct morphological structures that directly correlate with their specific functions. High spatial resolution imaging emerges as a powerful technique for capturing ultrastructural details that may go unnoticed with other methods such as immunofluorescent imaging and scRNA sequencing. In our study, we have applied software capable of seamlessly stitching virtual slides generated from electron microscopy, resulting in high-definition overviews of tissue slides. With this technology, we can comprehensively characterize the development and maturation of kidney organoids when transplanted under the renal capsule of mice. These organoids exhibit advanced ultrastructural developments upon transplantation, including the formation of the filtration barrier in the renal corpuscle, the presence of microvilli in the proximal tubule, and various types of cell sub-segmentation in the connecting tubule similarly to those seen in the adult kidney. Such ultrastructural characterization provides invaluable insights into the structural development and functional morphology of nephron segments within kidney organoids and how to advance them by interventions such as a transplantation. Research Highlights High-resolution imaging is crucial to determine morphological maturation of hiPSC-derived kidney organoids. Upon transplantation, refined ultrastructural development of nephron segments was observed, such as the development of the glomerular filtration barrier.

Localization of fluorescently labeled structures in frozen-hydrated samples using integrated light electron microscopy.

Correlative light and electron microscopy is an increasingly popular technique to study complex biological systems at various levels of resolution. Fluorescence microscopy can be employed to scan large areas to localize regions of interest which are then analyzed by electron microscopy to obtain morphological and structural information from a selected field of view at nm-scale resolution. Previously, an integrated approach to room temperature correlative microscopy was described. Combined use of light and electron microscopy within one instrument greatly simplifies sample handling, avoids cumbersome experimental overheads, simplifies navigation between the two modalities, and improves the success rate of image correlation. Here, an integrated approach for correlative microscopy under cryogenic conditions is presented. Its advantages over the room temperature approach include safeguarding the native hydrated state of the biological specimen, preservation of the fluorescence signal without risk of quenching due to heavy atom stains, and reduced photo bleaching. The potential of cryo integrated light and electron microscopy is demonstrated for the detection of viable bacteria, the study of in vitro polymerized microtubules, the localization of mitochondria in mouse embryonic fibroblasts, and for a search into virus-induced intracellular membrane modifications within mammalian cells.

A toolkit for the characterization of CCD cameras for transmission electron microscopy.

Charge-coupled devices (CCD) are nowadays commonly utilized in transmission electron microscopy (TEM) for applications in life sciences. Direct access to digitized images has revolutionized the use of electron microscopy, sparking developments such as automated collection of tomographic data, focal series, random conical tilt pairs and ultralarge single-particle data sets. Nevertheless, for ultrahigh-resolution work photographic plates are often still preferred. In the ideal case, the quality of the recorded image of a vitrified biological sample would solely be determined by the counting statistics of the limited electron dose the sample can withstand before beam-induced alterations dominate. Unfortunately, the image is degraded by the non-ideal point-spread function of the detector, as a result of a scintillator coupled by fibre optics to a CCD, and the addition of several inherent noise components. Different detector manufacturers provide different types of figures of merit when advertising the quality of their detector. It is hard for most laboratories to verify whether all of the anticipated specifications are met. In this report, a set of algorithms is presented to characterize on-axis slow-scan large-area CCD-based TEM detectors. These tools have been added to a publicly available image-processing toolbox for MATLAB. Three in-house CCD cameras were carefully characterized, yielding, among others, statistics for hot and bad pixels, the modulation transfer function, the conversion factor, the effective gain and the detective quantum efficiency. These statistics will aid data-collection strategy programs and provide prior information for quantitative imaging. The relative performance of the characterized detectors is discussed and a comparison is made with similar detectors that are used in the field of X-ray crystallography.

The supramolecular organization of fibrillin-rich microfibrils.

We propose a new model for the alignment of fibrillin molecules within fibrillin microfibrils. Automated electron tomography was used to generate three-dimensional microfibril reconstructions to 18.6-A resolution, which revealed many new organizational details of untensioned microfibrils, including heart-shaped beads from which two arms emerge, and interbead diameter variation. Antibody epitope mapping of untensioned microfibrils revealed the juxtaposition of epitopes at the COOH terminus and near the proline-rich region, and of two internal epitopes that would be 42-nm apart in unfolded molecules, which infers intramolecular folding. Colloidal gold binds microfibrils in the absence of antibody. Comparison of colloidal gold and antibody binding sites in untensioned microfibrils and those extended in vitro, and immunofluorescence studies of fibrillin deposition in cell layers, indicate conformation changes and intramolecular folding. Mass mapping shows that, in solution, microfibrils with periodicities of <70 and >140 nm are stable, but periodicities of approximately 100 nm are rare. Microfibrils comprise two in-register filaments with a longitudinal symmetry axis, with eight fibrillin molecules in cross section. We present a model of fibrillin alignment that fits all the data and indicates that microfibril extensibility follows conformation-dependent maturation from an initial head-to-tail alignment to a stable approximately one-third staggered arrangement.

Small cargo proteins and large aggregates can traverse the Golgi by a common mechanism without leaving the lumen of cisternae.

Procollagen (PC)-I aggregates transit through the Golgi complex without leaving the lumen of Golgi cisternae. Based on this evidence, we have proposed that PC-I is transported across the Golgi stacks by the cisternal maturation process. However, most secretory cargoes are small, freely diffusing proteins, thus raising the issue whether they move by a transport mechanism different than that used by PC-I. To address this question we have developed procedures to compare the transport of a small protein, the G protein of the vesicular stomatitis virus (VSVG), with that of the much larger PC-I aggregates in the same cell. Transport was followed using a combination of video and EM, providing high resolution in time and space. Our results reveal that PC-I aggregates and VSVG move synchronously through the Golgi at indistinguishable rapid rates. Additionally, not only PC-I aggregates (as confirmed by ultrarapid cryofixation), but also VSVG, can traverse the stack without leaving the cisternal lumen and without entering Golgi vesicles in functionally relevant amounts. Our findings indicate that a common mechanism independent of anterograde dissociative carriers is responsible for the traffic of small and large secretory cargo across the Golgi stack.

A new look at Weibel-Palade body structure in endothelial cells using electron tomography.

Multimers of von Willebrand Factor (vWF), a protein mediating blood clotting in response to vascular injury, are stored as tubular structures by endothelial cells in specific organelles, the Weibel-Palade Bodies (WPBs). To date very little is known about the 3D structure of WPBs in relation to the organization of the tubules. Therefore, we have initiated a thorough electron microscopic study in human umbilical vein endothelial cells (HUVECs) using electron tomography to gain further understanding of the ultrastructure of WPBs. We found that in addition to the well-documented cigar-shape, WPBs adopt irregular forms, which appeared to result from homotypic fusion. In transverse views of WPBs the tubular striations appear evenly spaced, which indicates a high level of organization that is likely to involve an underlying scaffold of structural proteins. Additionally, we found that the tubular striations twisted in an orderly fashion, suggesting that they are stored within the WPBs by a spring-loading mechanism. Altogether these data suggest that WPBs undergo a relatively complex maturation process involving homotypic fusion. Although the mechanism of assembly of vWF multimers into tubules is still unknown, the curled arrangement of the tubules within WPBs suggests a high degree of folding of the protein inside the organelle.

The Origin, Dynamic Morphology, and PI4P-Independent Formation of Encephalomyocarditis Virus Replication Organelles.

Picornaviruses induce dramatic rearrangements of endomembranes in the cells that they infect to produce dedicated platforms for viral replication. These structures, termed replication organelles (ROs), have been well characterized for the Enterovirus genus of the Picornaviridae However, it is unknown whether the diverse RO morphologies associated with enterovirus infection are conserved among other picornaviruses. Here, we use serial electron tomography at different stages of infection to assess the three-dimensional architecture of ROs induced by encephalomyocarditis virus (EMCV), a member of the Cardiovirus genus of the family of picornaviruses that is distantly related. Ultrastructural analyses revealed connections between early single-membrane EMCV ROs and the endoplasmic reticulum (ER), establishing the ER as a likely donor organelle for their formation. These early single-membrane ROs appear to transform into double-membrane vesicles (DMVs) as infection progresses. Both single- and double-membrane structures were found to support viral RNA synthesis, and progeny viruses accumulated in close proximity, suggesting a spatial association between RNA synthesis and virus assembly. Further, we explored the role of phosphatidylinositol 4-phosphate (PI4P), a critical host factor for both enterovirus and cardiovirus replication that has been recently found to expedite enterovirus RO formation rather than being strictly required. By exploiting an EMCV escape mutant, we found that low-PI4P conditions could also be overcome for the formation of cardiovirus ROs. Collectively, our data show that despite differences in the membrane source, there are striking similarities in the biogenesis, morphology, and transformation of cardiovirus and enterovirus ROs, which may well extend to other picornaviruses.IMPORTANCE Like all positive-sense RNA viruses, picornaviruses induce the rearrangement of host cell membranes to form unique structures, or replication organelles (ROs), that support viral RNA synthesis. Here, we investigate the architecture and biogenesis of cardiovirus ROs and compare them with those induced by enteroviruses, members of the well-characterized picornavirus genus Enterovirus The origins and dynamic morphologies of cardiovirus ROs are revealed using electron tomography, which points to the endoplasmic reticulum as the donor organelle usurped to produce single-membrane tubules and vesicles that transform into double-membrane vesicles. We show that PI4P, a critical lipid for cardiovirus and enterovirus replication, is not strictly required for the formation of cardiovirus ROs, as functional ROs with typical morphologies are formed under phosphatidylinositol 4-kinase type III alpha (PI4KA) inhibition in cells infected with an escape mutant. Our data show that the transformation from single-membrane structures to double-membrane vesicles is a conserved feature of cardiovirus and enterovirus infections that likely extends to other picornavirus genera.

Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein.

Like all other positive-strand RNA viruses, enteroviruses generate new organelles (replication organelles [ROs]) with a unique protein and lipid composition on which they multiply their viral genome. Suitable tools for live-cell imaging of enterovirus ROs are currently unavailable, as recombinant enteroviruses that carry genes that encode RO-anchored viral proteins tagged with fluorescent reporters have not been reported thus far. To overcome this limitation, we used a split green fluorescent protein (split-GFP) system, comprising a large fragment [strands 1 to 10; GFP(S1-10)] and a small fragment [strand 11; GFP(S11)] of only 16 residues. The GFP(S11) (GFP with S11 fragment) fragment was inserted into the 3A protein of the enterovirus coxsackievirus B3 (CVB3), while the large fragment was supplied by transient or stable expression in cells. The introduction of GFP(S11) did not affect the known functions of 3A when expressed in isolation. Using correlative light electron microscopy (CLEM), we showed that GFP fluorescence was detected at ROs, whose morphologies are essentially identical to those previously observed for wild-type CVB3, indicating that GFP(S11)-tagged 3A proteins assemble with GFP(S1-10) to form GFP for illumination of bona fide ROs. It is well established that enterovirus infection leads to Golgi disintegration. Through live-cell imaging of infected cells expressing an mCherry-tagged Golgi marker, we monitored RO development and revealed the dynamics of Golgi disassembly in real time. Having demonstrated the suitability of this virus for imaging ROs, we constructed a CVB3 encoding GFP(S1-10) and GFP(S11)-tagged 3A to bypass the need to express GFP(S1-10) prior to infection. These tools will have multiple applications in future studies on the origin, location, and function of enterovirus ROs. IMPORTANCE Enteroviruses induce the formation of membranous structures (replication organelles [ROs]) with a unique protein and lipid composition specialized for genome replication. Electron microscopy has revealed the morphology of enterovirus ROs, and immunofluorescence studies have been conducted to investigate their origin and formation. Yet, immunofluorescence analysis of fixed cells results in a rather static view of RO formation, and the results may be compromised by immunolabeling artifacts. While live-cell imaging of ROs would be preferred, enteroviruses encoding a membrane-anchored viral protein fused to a large fluorescent reporter have thus far not been described. Here, we tackled this constraint by introducing a small tag from a split-GFP system into an RO-resident enterovirus protein. This new tool bridges a methodological gap by circumventing the need for immunolabeling fixed cells and allows the study of the dynamics and formation of enterovirus ROs in living cells.

Three-dimensional image reconstruction of large nuclear RNP (lnRNP) particles by automated electron tomography.

Nuclear RNA transcripts of split genes and their splicing products, as well as the general population of nuclear polyadenylated RNA are packaged in multi-component large nuclear ribonucleoprotein (lnRNP) particles. These lnRNP particles, which sediment at the 200 S region in sucrose gradients, contain all U small nuclear RNPs required for precursor messenger RNA (pre-mRNA) splicing and several protein splicing factors, including U2AF and the SR proteins. Electron microscopy of lnRNP particles revealed a large compact structure of 50 nm in diameter. In this study we employed automated computed tomography from electron micrographs for the three-dimensional (3D) image reconstruction of individual lnRNP particles isolated from mammalian cells nuclei and negatively stained. For each particle, a tilt series of 71 images was collected by direct digital recording of the images on a CCD camera attached to a computer controlled TEM facility. The 3D image was reconstructed according to the back projection principle. For rendering, real time display and comparison of the reconstructed particles, interactive computer graphics was employed. The reconstructed 3D images show a compact structure composed of four major subunits connected to each other. Comparison of the reconstructed lnRNP particles revealed morphological similarity of the individual particles, as well as similarity among the sub-structures. Based on these observations we propose a model for the packaging of nuclear pre-mRNAs in lnRNP particles where each substructure represents a functional unit. This model is compatible with the requirements for alternative splicing in multi-intronic pre-mRNAs, and with the fact that the splicing of multi-intronic pre-mRNAs does not occur in a sequential manner.

The C-terminal domain of the Pseudomonas secretin XcpQ forms oligomeric rings with pore activity.

The Pseudomonas secretin XcpQ forms an oligomeric complex, which is involved in the translocation of proteins across the outer membrane via the type II secretion pathway. Pseudomonas aeruginosa produces only small amounts of this complex, 50 to 100 copies per bacterium, and overexpression is lethal to these cells. However, overexpression of Pseudomonas alcaligenes XcpQ could be achieved in the P. alcaligenes mutant strain 537. Protease protection experiments with P. alcaligenes XcpQ showed that the C-terminal domain of XcpQ, which is conserved in all the different members of the secretin family, is largely resistant to proteinase K. This protease-resistant fragment is embedded in the membrane and remains a stable complex, indicating that this domain is involved in complex formation. Both the intact and the protease-protected XcpQ complex showed a tendency to form two-dimensional crystal-like structures. Electron microscopic analysis of these structures showed that the overall oligomeric rings of the intact and of the protease-resistant complex are highly similar. The central cavity of the intact XcpQ complex contains structured mass. Both the intact and the protease-protected XcpQ complex showed pore-forming activity in planar lipid bilayers, consistent with their role as a translocation channel. However, the single-channel conductances observed were not uniform. Together, these results demonstrate that the C-terminal secretin homology domain of XcpQ is the structural domain that forms the channel through which macromolecules are being transported.

Predicted secondary structure of the 20 S proteasome and model structure of the putative peptide channel.

Secondary structure prediction has made great progress in recent years due to the incorporation of evolutionary information, and may be close to a point where (in combination with biochemical and low-resolution structural data) it can guide the modelling of tertiary structure in cases where no model building is possible by homology. Towards this goal it is important to gather information on the performance of prediction methods in advance of the publication of new structures. In anticipation of the soon-to-be-released structure of the 20 S proteasome from Thermoplasma acidophilum, we have applied several widely used secondary structure prediction methods to proteasome sequences and have attempted to model the putative channel in the outer proteasome rings (alpha-rings) based on the obtained predictions.

Automated microscopy for electron tomography.

Instrumentation and methodology for the automatic collection of tomographic tilt series data for the three-dimensional reconstruction of single particles is described. The system consists of a Philips EM 430 TEM, with a Gatan 673 cooled slow-scan CCD camera and a Philips C400 microscope computer control unit attached. The procedure for data collection includes direct digital recording of the images on the CCD camera and the automatic measurement and correction of (a) image shifts resulting from tilting the specimen, (b) variation of defocus and (c) the eucentric height position of the specimen. Experiments are described illustrating the possibilities and limitations of automatic data collection. Data collection at a magnification of 30k shows that the exposure time of the specimen to the beam is reduced by a factor of 10-100 compared to manual operation of the TEM.

von Willebrand factor remodeling during exocytosis from vascular endothelial cells.

BACKGROUND: In vascular endothelial cells, high molecular weight multimers of von Willebrand factor (VWF) are folded into tubular structures for storage in Weibel-Palade bodies. On stimulation, VWF is secreted and forms strings to induce primary hemostasis. The structural changes composing the transition of stored tubular VWF into secreted unfurled VWF strings are still unresolved even though they are vital for normal hemostasis. The secretory pod is a novel structure that we previously described in endothelial cells. It is formed on stimulation and has been postulated to function as a VWF release site. In this study, we investigated the actual formation of secretory pods and the subsequent remodeling of VWF into strings. METHODS: Human umbilical vein endothelial cells were stimulated and studied using various imaging techniques such as live-cell imaging and correlative light and electron microscopy. RESULTS: We found by using live-cell imaging that secretory pods are formed through the coalescence of multiple Weibel-Palade bodies without involvement of other large structures. Secreted VWF expelled from secretory pods was found to adopt a globular conformation. We visualized that VWF strings derive from those globular masses of VWF. Flow experiments showed that, on secretion, the globular masses of VWF move to the edge of the cell, where they anchor and generate VWF strings. CONCLUSION: On secretion, VWF adopts a globular conformation that remodels into strings after translocation and anchoring at the edge of the cell. This finding reveals new pathophysiological mechanisms that could be affected in patients with von Willebrand disease.

STEM tomography in cell biology.

Transmission electron tomography has been used in biological sciences for quite some time and proven to be a valuable tool. However, to date, the different Scanning Transmission modes are almost not used for electron tomography on resin-embedded biological material. We explored different STEM modes on epon-embedded, osmium-uranyl-lead-stained biological material. Bright Field-TEM and High Angle Annular Dark Field-STEM tomograms from the same areas were recorded and compared. Contrast and signal-to-noise ratios were calculated. Template matching was used to validate results obtained in Bright Field-TEM and High Angle Annular Dark Field-STEM tomograms. It is concluded that High Angle Annular Dark Field-STEM gives a five times better contrast and signal-to-noise ratio than Bright Field-TEM. Template matching showed that 1.3 times more information could be extracted from High Angle Annular Dark Field-STEM tomograms than from Bright Field-TEM tomograms.