FAST-EM array tomography: a workflow for multibeam volume electron microscopy.

Elucidating the 3D nanoscale structure of tissues and cells is essential for understanding the complexity of biological processes. Electron microscopy (EM) offers the resolution needed for reliable interpretation, but the limited throughput of electron microscopes has hindered its ability to effectively image large volumes. We report a workflow for volume EM with FAST-EM, a novel multibeam scanning transmission electron microscope that speeds up acquisition by scanning the sample in parallel with 64 electron beams.

Extracellular vesicles derived from stressed beta cells mediate monocyte activation and contribute to islet inflammation.

Objective: Beta cell destruction in type 1 diabetes (T1D) results from the combined effect of inflammation and recurrent autoimmunity. In recent years, the role played by beta cells in the development of T1D has evolved from passive victims of the immune system to active contributors in their own destruction. We and others have demonstrated that perturbations in the islet microenvironment promote endoplasmic reticulum (ER) stress in beta cells, leading to enhanced immunogenicity.

DWORF Extends Life Span in a PLN-R14del Cardiomyopathy Mouse Model by Reducing Abnormal Sarcoplasmic Reticulum Clusters.

Background: The p.Arg14del variant of the (phospholamban) gene causes cardiomyopathy, leading to severe heart failure. Calcium handling defects and perinuclear PLN aggregation have both been suggested as pathological drivers of this disease.

Optical STEM detection for scanning electron microscopy.

Recent advances in electron microscopy techniques have led to a significant scale up in volumetric imaging of biological tissue. The throughput of electron microscopes, however, remains a limiting factor for the volume that can be imaged in high resolution within reasonable time. Faster detection methods will improve throughput.

A Kinase Interacting Protein 1 regulates mitochondrial protein levels in energy metabolism and promotes mitochondrial turnover after exercise.

A Kinase Interacting Protein 1 (AKIP1) is a signalling adaptor that promotes mitochondrial respiration and attenuates mitochondrial oxidative stress in cultured cardiomyocytes. We sought to determine whether AKIP1 influences mitochondrial function and the mitochondrial adaptation in response to exercise in vivo. We assessed mitochondrial respiratory capacity, as well as electron microscopy and mitochondrial targeted-proteomics in hearts from mice with cardiomyocyte-specific overexpression of AKIP1 (AKIP1-TG) and their wild type (WT) littermates.

Segmentation in large-scale cellular electron microscopy with deep learning: A literature survey.

Electron microscopy (EM) enables high-resolution imaging of tissues and cells based on 2D and 3D imaging techniques. Due to the laborious and time-consuming nature of manual segmentation of large-scale EM datasets, automated segmentation approaches are crucial. This review focuses on the progress of deep learning-based segmentation techniques in large-scale cellular EM throughout the last six years, during which significant progress has been made in both semantic and instance segmentation.

Pathological ultrastructural alterations of myelinated axons in normal appearing white matter in progressive multiple sclerosis.

Multiple sclerosis (MS) pathophysiology includes inflammation, demyelination and neurodegeneration, but the exact mechanisms of disease initiation and progression are unknown. A major feature of lesions is lack of myelin, which increases axonal energy demand and requires adaptation in number and size of mitochondria. Outside lesions, subtle and diffuse alterations are observed in normal appearing white matter (NAWM) and normal appearing grey matter (NAGM), including increased oxidative stress, reduced axon density and changes in myelin composition and morphology.

mScarlet3: a brilliant and fast-maturing red fluorescent protein.

We report the evolution of mScarlet3, a cysteine-free monomeric red fluorescent protein with fast and complete maturation, as well as record brightness, quantum yield (75%) and fluorescence lifetime (4.0 ns). The mScarlet3 crystal structure reveals a barrel rigidified at one of its heads by a large hydrophobic patch of internal residues.

A Kinase Interacting Protein 1 (AKIP1) promotes cardiomyocyte elongation and physiological cardiac remodelling.

A Kinase Interacting Protein 1 (AKIP1) is a signalling adaptor that promotes physiological hypertrophy in vitro. The purpose of this study is to determine if AKIP1 promotes physiological cardiomyocyte hypertrophy in vivo. Therefore, adult male mice with cardiomyocyte-specific overexpression of AKIP1 (AKIP1-TG) and wild type (WT) littermates were caged individually for four weeks in the presence or absence of a running wheel.

Presence of immunogenic alternatively spliced insulin gene product in human pancreatic delta cells.

Aims/hypothesis: Transcriptome analyses revealed insulin-gene-derived transcripts in non-beta endocrine islet cells. We studied alternative splicing of human INS mRNA in pancreatic islets.

Methods: Alternative splicing of insulin pre-mRNA was determined by PCR analysis performed on human islet RNA and single-cell RNA-seq analysis.

The chaperone DNAJB6 surveils FG-nucleoporins and is required for interphase nuclear pore complex biogenesis.

Biogenesis of nuclear pore complexes (NPCs) includes the formation of the permeability barrier composed of phenylalanine-glycine-rich nucleoporins (FG-Nups) that regulate the selective passage of biomolecules across the nuclear envelope. The FG-Nups are intrinsically disordered and prone to liquid-liquid phase separation and aggregation when isolated. How FG-Nups are protected from making inappropriate interactions during NPC biogenesis is not fully understood.

Microscopic modulation and analysis of islets of Langerhans in living zebrafish larvae.

Microscopic analysis of molecules and physiology in living cells and systems is a powerful tool in life sciences. While in vivo subcellular microscopic analysis of healthy and diseased human organs remains impossible, zebrafish larvae allow studying pathophysiology of many organs using in vivo microscopy. Here, we review the potential of the larval zebrafish pancreas in the context of islets of Langerhans and Type 1 diabetes.

Integrated Array Tomography for 3D Correlative Light and Electron Microscopy.

Volume electron microscopy (EM) of biological systems has grown exponentially in recent years due to innovative large-scale imaging approaches. As a standalone imaging method, however, large-scale EM typically has two major limitations: slow rates of acquisition and the difficulty to provide targeted biological information. We developed a 3D image acquisition and reconstruction pipeline that overcomes both of these limitations by using a widefield fluorescence microscope integrated inside of a scanning electron microscope.

Electron-Beam Induced Luminescence and Bleaching in Polymer Resins and Embedded Biomaterial.

Electron microscopy is crucial for imaging biological ultrastructure at nanometer resolution. However, electron irradiation also causes specimen damage, reflected in structural and chemical changes that can give rise to alternative signals. Here, luminescence induced by electron-beam irradiation is reported across a range of materials widely used in biological electron microscopy.

Early Upper Aerodigestive Tract Cancer Detection Using Electron Microscopy to Reveal Chromatin Packing Alterations in Buccal Mucosa Cells.

A profound characteristic of field cancerization is alterations in chromatin packing. This study aimed to quantify these alterations using electron microscopy image analysis of buccal mucosa cells of laryngeal, esophageal, and lung cancer patients. Analysis was done on normal-appearing mucosa, believed to be within the cancerization field, and not tumor itself.

Optimization of negative stage bias potential for faster imaging in large-scale electron microscopy.

Large-scale electron microscopy (EM) allows analysis of both tissues and macromolecules in a semi-automated manner, but acquisition rate forms a bottleneck. We reasoned that a negative bias potential may be used to enhance signal collection, allowing shorter dwell times and thus increasing imaging speed. Negative bias potential has previously been used to tune penetration depth in block-face imaging.

Sample preparation for energy dispersive X-ray imaging of biological tissues.

Traditional electron microscopy (EM) can be complemented with analytical EM to increase objective sample information enabling feature identification. Energy dispersive X-ray (EDX) imaging provides semi-quantitative elemental composition of the sample with high spatial resolution (~10nm) in ultrathin sections. However, EDX imaging of biological samples is still challenging as a routine method because many elements are at the detection limit for this technique.

Reversible thrombocytopenia during hibernation originates from storage and release of platelets in liver sinusoids.

Immobility is a risk factor for thrombosis due to low blood flow, which may result in activation of the coagulation system, recruitment of platelets and clot formation. Nevertheless, hibernating animals-who endure lengthy periods of immobility-do not show signs of thrombosis throughout or after hibernation. One of the adaptations of hemostasis in hibernators consists of a rapidly reversible reduction of the number of circulating platelets during torpor, i.

Nanobody-Based Probes for Subcellular Protein Identification and Visualization.

Understanding how building blocks of life contribute to physiology is greatly aided by protein identification and cellular localization. The two main labeling approaches developed over the past decades are labeling with antibodies such as immunoglobulin G (IgGs) or use of genetically encoded tags such as fluorescent proteins. However, IgGs are large proteins (150 kDa), which limits penetration depth and uncertainty of target position caused by up to ∼25 nm distance of the label created by the chosen targeting approach.

Vps13 is required for timely removal of nurse cell corpses.

Programmed cell death and consecutive removal of cellular remnants is essential for development. During late stages of oogenesis, the small somatic follicle cells that surround the large nurse cells promote non-apoptotic nurse cell death, subsequently engulf them, and contribute to the timely removal of nurse cell corpses. Here, we identify a role for Vps13 in the timely removal of nurse cell corpses downstream of developmental programmed cell death.

Correction to: Neodymium as an alternative contrast for uranium in electron microscopy.

After publication of our article, it has come to our attention that the legend.

Large-scale electron microscopy database for human type 1 diabetes.

Autoimmune β-cell destruction leads to type 1 diabetes, but the pathophysiological mechanisms remain unclear. To help address this void, we created an open-access online repository, unprecedented in its size, composed of large-scale electron microscopy images ('nanotomy') of human pancreas tissue obtained from the Network for Pancreatic Organ donors with Diabetes (nPOD; www.nanotomy.

Endocytosis of Extracellular Vesicles and Release of Their Cargo from Endosomes.

Extracellular vesicles (EVs), such as exosomes, can mediate long-distance communication between cells by delivering biomolecular cargo. It is speculated that EVs undergo back-fusion at multivesicular bodies (MVBs) in recipient cells to release their functional cargo. However, direct evidence is lacking.