Three-Dimensional Transmission Electron Microscopy: A Novel Imaging and Characterization Technique with Nanometer Scale Resolution for Materials Science

Three-dimensional transmission electron microscopy (3D-TEM), effectuated by multiple imaging of a sample combined with image analysis, offers a new approach in materials science to obtain 3D information of complex solid materials. Here we report first-of-its-kind results that have been obtained with zeolite materials. Virtual cross-sections and volume rendering of the 3D reconstruction of a metal/zeolite crystal (Ag/NaY) give unequivocal information on the location of the silver particles (10-40 nm in diameter). Virtual cross-sections of the 3D reconstruction of an acid-leached mordenite show the three-dimensional mesoporous channel system (3-20 nm in diameter) with a clarity and definition not seen before

Three-dimensional Transmission Electron Microscopy observations of mesopores in dealuminated zeolite Y**

Supported by NWO under grant 98037. The research of AJK has been made possible by a fellowship of the Royal Netherlands Academy of Arts and Sciences (KNAW). The authors thank J.E.M.J. Raaymakers for the nitrogen physisorption measurements, A.J.M. Mens for the XPS measurements, J.A.R. van Veen and E.J. Creyghton for physical data and useful discussions and Shell International Chemicals and Zeolyst for the samples

Development and Application of 3-Dimensional Transmission Electron Microscopy (3D-TEM) for the Characterization of Metal-Zeolite Catalyst Systems

With electron tomography (3D-TEM) a 3D-reconstruction is calculated from a series of TEM images taken at a tilt angle range (tilting range) of +70° to -70°. The reconstruction can be visualized with contour surfaces that give information about the surface of the sample as well as with slices through the reconstruction that give detailed information on the interior of the sample. Electron tomography gives much more information than Scanning Electron Microscopy (SEM), since SEM gives only information about the surface of a sample. As a case study, the imaging of silver clusters on zeolite NaY is given. The reconstruction shows silver particles at the external surface as well as a silver particle in a mesopore of the zeolite crystallite. It is concluded that 3D-TEM comprises a breakthrough in the characterization of nano-structured solid catalysts

Correlative microscopy for structural microbiology

Microscopic imaging and technolog

Interprofessional teamwork in primary care: the effect of functional heterogeneity on performance and the role of leadership

This study aimed to unravel the complexity of interprofessional teamwork in primary care teams by testing the relationship between functional heterogeneity and team performance through the mediating role of information elaboration, and the moderating roles of directive leadership and participative leadership. The moderated mediation model was validated using survey data from 1105 professionals and 97 supervisors in 143 Dutch primary care teams. The results confirmed the model and showed a significant negative effect of functional heterogeneity on information elaboration, which in turn had a positive effect on team performance. Both directive and participative leadership moderated the negative effect of functional heterogeneity on information elaboration to the extent that the indirect negative effect of functional heterogeneity on team performance became insignificant under high levels of either directive or participative leadership. The theoretical implications of these findings for the literature on healthcare, team diversity, and leadership, as well as the practical implications for policy makers, educationalists and managers of primary care teams, are discussed

Correlated Cryo Super‐Resolution Light and Cryo‐Electron Microscopy on Mammalian Cells Expressing the Fluorescent Protein rsEGFP2

Super‐resolution light microscopy (SRM) enables imaging of biomolecules within cells with nanometer precision. Cryo‐fixation by vitrification offers optimal structure preservation of biological specimens and permits sequential cryo electron microscopy (cryoEM) on the same sample, but is rarely used for SRM due to various technical challenges and the lack of fluorophores developed for vitrified conditions. Here, a protocol to perform correlated cryoSRM and cryoEM on intact mammalian cells using fluorescent proteins and commercially available equipment is described. After cell culture and sample preparation by plunge‐freezing, cryoSRM is performed using the reversibly photoswitchable fluorescent protein rsEGFP2. Next, a super‐resolved image is reconstructed to guide cryoEM imaging to the feature of interest. Finally, the cryoSRM and cryoEM images are correlated to combine information from both imaging modalities. Using this protocol, a localization precision of 30 nm for cryoSRM is routinely achieved. No impediments to successive cryoEM imaging are detected, and the protocol is compatible with a variety of cryoEM techniques. When the optical set‐up and analysis pipeline is established, the total duration of the protocol for experienced cryoEM users is 3 days, not including cell culture. Microscopic imaging and technolog

The preferences of users of electronic medical records in hospitals: Quantifying the relative importance of barriers and facilitators of an innovation

Background: Currently electronic medical records (EMRs) are implemented in hospitals, because of expected benefits for quality and safety of care. However the implementation processes are not unproblematic and are slower than needed. Many of the barriers and facilitators of the adoption of EMRs are identified, but the relative importance of these factors is still undetermined. This paper quantifies the relative importance of known barriers and facilitators of EMR, experienced by the users (i.e., nurses and physicians in hospitals).Methods: A discrete choice experiment (DCE) was conducted among physicians and nurses. Participants answered ten choice sets containing two scenarios. Each scenario included attributes that were based on previously identified barriers in the literature: data entry hardware, technical support, attitude head of department, performance feedback, flexibility of interface, and decision support. Mixed Multinomial Logit analysis was used to determine the relative importance of the attributes.Results: Data on 148 nurses and 150 physicians showed that high flexibility of the interface was the factor with highest relative importance in their preference to use an EMR. For nurses this attribute was followed by support from the head of department, presence of performance feedback from the EMR and presence of decisions support. While for physicians this ordering was different: presence of decision support was relatively more important than performance feedback and support from the head of department.Conclusion: Considering the prominent wish of all the intended users for a flexible interface, currently used EMRs only partially comply with the needs of the users, indicating the need for closer incorporation of user needs during development stages of EMRs. The differences in priorities amongst nurses and physicians show that different users have different needs during the implementation of innovations. Hospital management may use this information to design implementation trajectories to fit the needs of various user groups

Localization of active endogenous and exogenous beta-glucocerebrosidase by correlative light-electron microscopy in human fibroblasts

beta-Glucocerebrosidase (GBA) is the enzyme that degrades glucosylceramide in lysosomes. Defects in GBA that result in overall loss of enzymatic activity give rise to the lysosomal storage disorder Gaucher disease, which is characterized by the accumulation of glucosylceramide in tissue macrophages. Gaucher disease is currently treated by infusion of mannose receptor-targeted recombinant GBA. The recombinant GBA is thought to reach the lysosomes of macrophages, based on the impressive clinical response that is observed in Gaucher patients (type 1) receiving this enzyme replacement therapy. In this study, we used cyclophellitol-derived activity-based probes (ABPs) with a fluorescent reporter that irreversibly bind to the catalytic pocket of GBA, to visualize the active enzymes in a correlative microscopy approach. The uptake of pre-labeled recombinant enzyme was monitored by fluorescence and electron microscopy in human fibroblasts that stably expressed the mannose receptor. The endogenous active enzyme was simultaneously visualized by in situ labeling with the ABP containing an orthogonal fluorophore. This method revealed the efficient delivery of recombinant GBA to lysosomal target compartments that contained endogenous active enzyme

Localization of active endogenous and exogenous beta-glucocerebrosidase by correlative light-electron microscopy in human fibroblasts

beta-Glucocerebrosidase (GBA) is the enzyme that degrades glucosylceramide in lysosomes. Defects in GBA that result in overall loss of enzymatic activity give rise to the lysosomal storage disorder Gaucher disease, which is characterized by the accumulation of glucosylceramide in tissue macrophages. Gaucher disease is currently treated by infusion of mannose receptor-targeted recombinant GBA. The recombinant GBA is thought to reach the lysosomes of macrophages, based on the impressive clinical response that is observed in Gaucher patients (type 1) receiving this enzyme replacement therapy. In this study, we used cyclophellitol-derived activity-based probes (ABPs) with a fluorescent reporter that irreversibly bind to the catalytic pocket of GBA, to visualize the active enzymes in a correlative microscopy approach. The uptake of pre-labeled recombinant enzyme was monitored by fluorescence and electron microscopy in human fibroblasts that stably expressed the mannose receptor. The endogenous active enzyme was simultaneously visualized by in situ labeling with the ABP containing an orthogonal fluorophore. This method revealed the efficient delivery of recombinant GBA to lysosomal target compartments that contained endogenous active enzyme

Correlative cryo super-resolution light and electron microscopy on mammalian cells using fluorescent proteins

Sample fixation by vitrification is critical for the optimal structural preservation of biomolecules and subsequent high-resolution imaging by cryo-correlative light and electron microscopy (cryoCLEM). There is a large resolution gap between cryo fluorescence microscopy (cryoFLM), ~400-nm, and the sub-nanometre resolution achievable with cryo-electron microscopy (cryoEM), which hinders interpretation of cryoCLEM data. Here, we present a general approach to increase the resolution of cryoFLM using cryo-super-resolution (cryoSR) microscopy that is compatible with successive cryoEM investigation in the same region. We determined imaging parameters to avoid devitrification of the cryosamples without the necessity for cryoprotectants. Next, we examined the applicability of various fluorescent proteins (FPs) for single-molecule localisation cryoSR microscopy and found that all investigated FPs display reversible photoswitchable behaviour, and demonstrated cryoSR on lipid nanotubes labelled with rsEGFP2 and rsFastLime. Finally, we performed SR-cryoCLEM on mammalian cells expressing microtubule-associated protein-2 fused to rsEGFP2 and performed 3D cryo-electron tomography on the localised areas. The method we describe exclusively uses commercially available equipment to achieve a localisation precision of 30-nm. Furthermore, all investigated FPs displayed behaviour compatible with cryoSR microscopy, making this technique broadly available without requiring specialised equipment and will improve the applicability of this emerging technique for cellular and structural biology. Microscopic imaging and technolog