Bioimaging data have significant potential for reuse, but unlocking this potential requires systematic archiving of data and metadata in public databases. We propose draft metadata guidelines to begin addressing the needs of diverse communities within light and electron microscopy. We hope this publication and the proposed Recommended Metadata for Biological Images (REMBI) will stimulate discussions about their implementation and future extension.

Efficient design of multi-component food products containing dry and wet components such as biscuits with a moist fruit filling, is of growing interests for food industry. Technology is needed to prevent or reduce water migration from the moist filling to the dry porous cereal material. This can be done by using moisture barrier systems. Knowledge of the microstructure and its relation to water mobility is necessary to develop stable products. This paper describes a study that uses X-ray microtomography (μCT) for the characterisation and visualisation of the 3-D structure of crackers with different porosity, coated biscuit shells and soup inclusions. μCT was used for imaging the inner cellular structure of the cereal matrix or to analyse the integrity of moisture barriers applied on the cereal product. 3-D image analysis methods were developed to obtain quantitative information about the cellular matrix which can be used as input for simulation models for moisture migration. The developed 3-D image analysis method maps the open cellular structure onto a network (graph) representation in which the nodes correspond to the pores and the vertices to the pore-topore interconnection. The pores (nodes) have properties such as volume, surface area and location whereas the vertices have properties such as direct (open connection) and indirect (separated by a single lamella) area. To check the segmentation and network description a model for pore to pore resistance was used. The obtained results demonstrate the potential of μCT and 3-D image analysis for extracting structural information which can be used in models for the moisture penetration in a cellular bakery product

In the biological sciences, data from fluorescence and electron microscopy is correlated to allow fluorescence biomolecule identification within the cellular ultrastructure and/or ultrastructural analysis following live-cell imaging. High-accuracy (sub-100 nm) image overlay requires the addition of fiducial markers, which makes overlay accuracy dependent on the number of fiducials present in the region of interest. Here, we report an automated method for light-electron image overlay at high accuracy, i.e. below 5 nm. Our method relies on direct visualization of the electron beam position in the fluorescence detection channel using cathodoluminescence pointers. We show that image overlay using cathodoluminescence pointers corrects for image distortions, is independent of user interpretation, and does not require fiducials, allowing image correlation with molecular precision anywhere on a sample.

Highlights d MVBs are complex organelles with intraluminal vesicles bound by the limiting membrane d Intraluminal membranes are in a dynamic equilibrium with the limiting membrane d Retrofusion of internal vesicles is controlled by processes used for viral fusion d Exosomes arise from internal MVB vesicles not participating in retrofusion

The endoplasmic reticulum (ER) is the largest organelle contacting virtually every other organelle for information exchange and control of processes such as transport, fusion, and fission. Here, we studied the role of the other organelles on ER network architecture in the cell periphery. We show that the co‐migration of the ER with other organelles, called ER hitchhiking facilitated by late endosomes and lysosomes is a major mechanism controlling ER network architecture. When hitchhiking occurs, emerging ER structures may fuse with the existing ER tubules to alter the local ER architecture. This couples late endosomal/lysosomal positioning and mobility to ER network architecture. Conditions restricting late endosomal movement—including cell starvation—or the depletion of tether proteins that link the ER to late endosomes reduce ER dynamics and limit the complexity of the peripheral ER network architecture. This indicates that among many factors, the ER is controlled by late endosomal movement resulting in an alteration of the ER network architecture.

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The hallmark autoantibodies in rheumatoid arthritis are characterized by variable domain glycans (VDGs). Their abundant occurrence results from the selective introduction of N-linked glycosylation sites during somatic hypermutation, and their presence is predictive for disease development. However, the functional consequences of VDGs on autoreactive B cells remain elusive. Combining crystallography, glycobiology, and functional B cell assays allowed us to dissect key characteristics of VDGs on human B cell biology. Crystal structures showed that VDGs are positioned in the vicinity of the antigen-binding pocket, and dynamic modeling combined with binding assays elucidated their impact on binding. We found that VDG-expressing B cell receptors stay longer on the B cell surface and that VDGs enhance B cell activation. These results provide a rationale on how the acquisition of VDGs might contribute to the breach of tolerance of autoreactive B cells in a major human autoimmune disease.

Today, the resolution in phase-contrast cryo-electron tomography is for a significant part limited by the contrast transfer function (CTF) of the microscope. The CTF is a function of defocus and thus varies spatially as a result of the tilting of the specimen and the finite specimen thickness. Models that include spatial dependencies have not been adopted in daily practice because of their high computational complexity. Here we present an algorithm which reduces the processing time for computing the 'tilted' CTF by more than a factor 100. Our implementation of the full 3D CTF has a processing time on the order of a Radon transform of a full tilt-series. We derive and validate an expression for the damping envelope function describing the loss of resolution due to specimen thickness. Using simulations we quantify the effects of specimen thickness on the accuracy of various forward models. We study the influence of spatially varying CTF correction and subsequent tomographic reconstruction by simulation and present a new approach for space-variant phase-flipping. We show that our CTF correction strategies are successful in increasing the resolution after tomographic reconstruction.