An Overview of Microcrystal Electron Diffraction (MicroED).

The bedrock of drug discovery and a key tool for understanding cellular function and drug mechanisms of action is the structure determination of chemical compounds, peptides, and proteins. The development of new structure characterization tools, particularly those that fill critical gaps in existing methods, presents important steps forward for structural biology and drug discovery. The emergence of microcrystal electron diffraction (MicroED) expands the application of cryo-electron microscopy to include samples ranging from small molecules and membrane proteins to even large protein complexes using crystals that are one-billionth the size of those required for X-ray crystallography. This review outlines the conception, achievements, and exciting future trajectories for MicroED, an important addition to the existing biophysical toolkit.

Mitocytosis, a migrasome-mediated mitochondrial quality-control process.

Damaged mitochondria need to be cleared to maintain the quality of the mitochondrial pool. Here, we report mitocytosis, a migrasome-mediated mitochondrial quality-control process. We found that, upon exposure to mild mitochondrial stresses, damaged mitochondria are transported into migrasomes and subsequently disposed of from migrating cells. Mechanistically, mitocytosis requires positioning of damaged mitochondria at the cell periphery, which occurs because damaged mitochondria avoid binding to inward motor proteins. Functionally, mitocytosis plays an important role in maintaining mitochondrial quality. Enhanced mitocytosis protects cells from mitochondrial stressor-induced loss of mitochondrial membrane potential (MMP) and mitochondrial respiration; conversely, blocking mitocytosis causes loss of MMP and mitochondrial respiration under normal conditions. Physiologically, we demonstrate that mitocytosis is required for maintaining MMP and viability in neutrophils in vivo. We propose that mitocytosis is an important mitochondrial quality-control process in migrating cells, which couples mitochondrial homeostasis with cell migration.

Pollen Cell Wall Patterns Form from Modulated Phases.

The ornately geometric walls of pollen grains have inspired scientists for decades. We show that the evolved diversity of these patterns is entirely recapitulated by a biophysical model in which an initially uniform polysaccharide layer in the extracellular space, mechanically coupled to the cell membrane, phase separates to a spatially modulated state. Experiments reveal this process occurring in living cells. We observe that in ∼10% of extant species, this phase separation reaches equilibrium during development such that individual pollen grains are identical and perfectly reproducible. About 90% of species undergo an arrest of this process prior to equilibrium such that individual grains are similar but inexact copies. Equilibrium patterns have appeared multiple times during the evolution of seed plants, but selection does not favor these states. This framework for pattern development provides a route to rationalizing the surface textures of other secreted structures, such as cell walls and insect cuticle.

Zero-shot denoising of microscopy images recorded at high-resolution limits.

Conventional and electron microscopy visualize structures in the micrometer to nanometer range, and such visualizations contribute decisively to our understanding of biological processes. Due to different factors in recording processes, microscopy images are subject to noise. Especially at their respective resolution limits, a high degree of noise can negatively effect both image interpretation by experts and further automated processing. However, the deteriorating effects of strong noise can be alleviated to a large extend by image enhancement algorithms. Because of the inherent high noise, a requirement for such algorithms is their applicability directly to noisy images or, in the extreme case, to just a single noisy image without a priori noise level information (referred to as blind zero-shot setting). This work investigates blind zero-shot algorithms for microscopy image denoising. The strategies for denoising applied by the investigated approaches include: filtering methods, recent feed-forward neural networks which were amended to be trainable on noisy images, and recent probabilistic generative models. As datasets we consider transmission electron microscopy images including images of SARS-CoV-2 viruses and fluorescence microscopy images. A natural goal of denoising algorithms is to simultaneously reduce noise while preserving the original image features, e.g., the sharpness of structures. However, in practice, a tradeoff between both aspects often has to be found. Our performance evaluations, therefore, focus not only on noise removal but set noise removal in relation to a metric which is instructive about sharpness. For all considered approaches, we numerically investigate their performance, report their denoising/sharpness tradeoff on different images, and discuss future developments. We observe that, depending on the data, the different algorithms can provide significant advantages or disadvantages in terms of their noise removal vs. sharpness preservation capabilities, which may be very relevant for different virological applications, e.g., virological analysis or image segmentation.

New Avenues for Capturing Mineralization Events at Biomaterial Interfaces with Liquid-Transmission Electron Microscopy.

Liquid-transmission electron microscopy (liquid-TEM) provides exciting potential for capturing mineralization events at biomaterial interfaces, though it is largely unexplored. To address this, we established a unique approach to visualize calcium phosphate (CaP)-titanium (Ti) interfacial mineralization events by combining the nanofabrication of Ti lamellae by focused ion beam with in situ liquid-TEM. Multiphasic CaP particles were observed to nucleate, adhere, and form different assemblies onto and adjacent to Ti lamellae. Here, we discuss new approaches for exploring the interaction between biomaterials and liquids at the nanoscale. Driving this technology is crucial for understanding and controlling biomineralization to improve implant osseointegration and direct new pathways for mineralized tissue disease treatment in the future.

Tooth acellular extrinsic fibre cementum incremental lines in humans are formed by parallel branched Sharpey's fibres and not by its mineral phase.

In humans, the growth pattern of the acellular extrinsic fibre cementum (AEFC) has been useful to estimate the age-at-death. However, the structural organization behind such a pattern remains poorly understood. In this study tooth cementum from seven individuals from a Mexican modern skeletal series were analyzed with the aim of unveiling the AEFC collagenous and mineral structure using multimodal imaging approaches. The organization of collagen fibres was first determined using: light microscopy, transmission electron microscopy (TEM), electron tomography, and plasma FIB scanning electron microscopy (PFIB-SEM) tomography. The mineral properties were then investigated using: synchrotron small-angle X-ray scattering (SAXS) for T-parameter (correlation length between mineral particles); synchrotron X-ray diffraction (XRD) for L-parameter (mineral crystalline domain size estimation), alignment parameter (crystals preferred orientation) and lattice parameters a and c; as well as synchrotron X-ray fluorescence for spatial distribution of calcium, phosphorus and zinc. Results show that Sharpey's fibres branched out fibres that cover and uncover other collagen bundles forming aligned arched structures that are joined by these same fibres but in a parallel fashion. The parallel fibres are not set as a continuum on the same plane and when they are superimposed project the AEFC incremental lines due to the collagen birefringence. The orientation of the apatite crystallites is subject to the arrangement of the collagen fibres, and the obtained parameter values along with the elemental distribution maps, revealed this mineral tissue as relatively homogeneous. Therefore, no intrinsic characteristics of the mineral phase could be associated with the alternating AEFC incremental pattern.

Effect of plate orientation on apparent thickness of mineral plates by transmission electron microscopy.

PURPOSE: Transmission electron microscopy (TEM) is widely used to study the ultrastructure of bone. The mineral of bone occurs as polycrystalline mineral plates about 3 to 6 nm in thickness. A problem in using TEM to make quantitative analyses of bone is that the orientation of the plates with respect to the plane of the section being imaged is expected to affect their apparent thickness. The purpose of this study was to test if this was true, if the apparent thickness of plates changed substantially as a result of tilt of the section. METHODS: We prepared TEM sections of samples of cortical human bone by ion beam milling, orienting one section parallel to the collagen fibril axes and one perpendicular to them. We obtained TEM bright field and HAADF images of these sections, tilting the sections up to ± 20° at 2° intervals and measuring the apparent thickness of individual mineral platelets at each angle of tilt. RESULTS: Thickness appears to double as section is tilted ± 20°. True thickness of plates is determined by tilting the section along an axis parallel to the plate orientation and determining the minimum apparent thickness. However, as plates are tilted away from minimum-thickness orientation, they become less well-resolved, disappearing when tilted more than 20°. We therefore also measured apparent thickness of only the darkest (most electron scattering) plate images in an untilted section and obtained the same average thickness as that obtained by tilting. CONCLUSION: We conclude that tilting of the section is not necessary to obtain an accurate measurement of the thickness of mineral plates.

CLEM, a universal tool for analyzing structural organization in thylakoid membranes.

Chlorophyll (Chl) plays a crucial role in photosynthesis, functioning as a photosensitizer. As an integral component of this process, energy absorbed by this pigment is partly emitted as red fluorescence. This signal can be readily imaged by fluorescence microscopy and provides a visualization of photosynthetic activity. However, due to limited resolution, signals cannot be assigned to specific subcellular/organellar membrane structures. By correlating fluorescence micrographs with transmission electron microscopy, researchers can identify sub-cellular compartments and membranes, enabling the monitoring of Chl distribution within thylakoid membrane substructures in cyanobacteria, algae, and higher plant single cells. Here, we describe a simple and effective protocol for correlative light-electron microscopy (CLEM) based on the autofluorescence of Chl and demonstrate its application to selected photosynthetic model organisms. Our findings illustrate the potential of this technique to identify areas of high Chl concentration and photochemical activity, such as grana regions in vascular plants, by mapping stacked thylakoids.

Single-cell visualization and quantification of trace metals in Chlamydomonas lysosome-related organelles.

The acidocalcisome is an acidic organelle in the cytosol of eukaryotes, defined by its low pH and high calcium and polyphosphate content. It is visualized as an electron-dense object by transmission electron microscopy (TEM) or described with mass spectrometry (MS)-based imaging techniques or multimodal X-ray fluorescence microscopy (XFM) based on its unique elemental composition. Compared with MS-based imaging techniques, XFM offers the additional advantage of absolute quantification of trace metal content, since sectioning of the cell is not required and metabolic states can be preserved rapidly by either vitrification or chemical fixation. We employed XFM in Chlamydomonas reinhardtii to determine single-cell and organelle trace metal quotas within algal cells in situations of trace metal overaccumulation (Fe and Cu). We found up to 70% of the cellular Cu and 80% of Fe sequestered in acidocalcisomes in these conditions and identified two distinct populations of acidocalcisomes, defined by their unique trace elemental makeup. We utilized the vtc1 mutant, defective in polyphosphate synthesis and failing to accumulate Ca, to show that Fe sequestration is not dependent on either. Finally, quantitation of the Fe and Cu contents of individual cells and compartments via XFM, over a range of cellular metal quotas created by nutritional and genetic perturbations, indicated excellent correlation with bulk data from corresponding cell cultures, establishing a framework to distinguish the nutritional status of single cells.

Indirect Correlative Light and Electron Microscopy (iCLEM): A Novel Pipeline for Multiscale Quantification of Structure From Molecules to Organs.

Correlative light and electron microscopy (CLEM) methods are powerful methods that combine molecular organization (from light microscopy) with ultrastructure (from electron microscopy). However, CLEM methods pose high cost/difficulty barriers to entry and have very low experimental throughput. Therefore, we have developed an indirect correlative light and electron microscopy (iCLEM) pipeline to sidestep the rate-limiting steps of CLEM (i.e., preparing and imaging the same samples on multiple microscopes) and correlate multiscale structural data gleaned from separate samples imaged using different modalities by exploiting biological structures identifiable by both light and electron microscopy as intrinsic fiducials. We demonstrate here an application of iCLEM, where we utilized gap junctions and mechanical junctions between muscle cells in the heart as intrinsic fiducials to correlate ultrastructural measurements from transmission electron microscopy (TEM), and focused ion beam scanning electron microscopy (FIB-SEM) with molecular organization from confocal microscopy and single molecule localization microscopy (SMLM). We further demonstrate how iCLEM can be integrated with computational modeling to discover structure-function relationships. Thus, we present iCLEM as a novel approach that complements existing CLEM methods and provides a generalizable framework that can be applied to any set of imaging modalities, provided suitable intrinsic fiducials can be identified.

PEGylated Micro/Nanoparticles Based on Biodegradable Poly(Ester Amides): Preparation and Study of the Core-Shell Structure by Synchrotron Radiation-Based FTIR Microspectroscopy and Electron Microscopy.

Surface modification of drug-loaded particles with polyethylene glycol (PEG) chains is a powerful tool that promotes better transport of therapeutic agents, provides stability, and avoids their detection by the immune system. In this study, we used a new approach to synthesize a biodegradable poly(ester amide) (PEA) and PEGylating surfactant. These were employed to fabricate micro/nanoparticles with a core-shell structure. Nanoparticle (NP)-protein interactions and self-assembling were subsequently studied by synchrotron radiation-based FTIR microspectroscopy (SR-FTIRM) and transmission electron microscopy (TEM) techniques. The core-shell structure was identified using IR absorption bands of characteristic chemical groups. Specifically, the stretching absorption band of the secondary amino group (3300 cm-1) allowed us to identify the poly(ester amide) core, while the band at 1105 cm-1 (C-O-C vibration) was useful to demonstrate the shell structure based on PEG chains. By integration of absorption bands, a 2D intensity map of the particle was built to show a core-shell structure, which was further supported by TEM images.

High-Resolution Cryo-Electron Microscopy Structure Determination of Haemophilus influenzae Tellurite-Resistance Protein A via 200 kV Transmission Electron Microscopy.

Membrane proteins constitute about 20% of the human proteome and play crucial roles in cellular functions. However, a complete understanding of their structure and function is limited by their hydrophobic nature, which poses significant challenges in purification and stabilization. Detergents, essential in the isolation process, risk destabilizing or altering the proteins' native conformations, thus affecting stability and functionality. This study leverages single-particle cryo-electron microscopy to elucidate the structural nuances of membrane proteins, focusing on the SLAC1 bacterial homolog from Haemophilus influenzae (HiTehA) purified with diverse detergents, including n-dodecyl ß-D-maltopyranoside (DDM), glycodiosgenin (GDN), ß-D-octyl-glucoside (OG), and lauryl maltose neopentyl glycol (LMNG). This research not only contributes to the understanding of membrane protein structures but also addresses detergent effects on protein purification. By showcasing that the overall structural integrity of the channel is preserved, our study underscores the intricate interplay between proteins and detergents, offering insightful implications for drug design and membrane biology.

Placental Protein 13 and Syncytiotrophoblast Basement Membrane Ultrastructures in Preeclampsia.

Background and Objectives: Preeclampsia has been linked to an inflammatory response that may be brought on by endothelial cell dysfunction. This paper investigates the pathomechanism of syncytiotrophoblast basement membrane (STBM) damage and Placental Protein 13 (PP13) release, which may have a role in systemic endothelial dysfunction in preeclampsia. Materials and Methods: This comparative cross-sectional study involves 54 preeclampsia patients (27 early-onset preeclampsia and 27 late-onset preeclampsia) and 27 pregnant women with normal blood pressure. An enzyme-linked immunosorbent assay was performed to evaluate maternal blood levels of PP13. Following birth, a portion of the placenta was collected for transmission electron microscope (TEM) and immunohistochemical (IHC) analysis. The data were analyzed using STATA version 15. Results: PP13 expression in the placental syncytiotrophoblast was significantly lower in the early-onset preeclampsia, compared to late-onset preeclampsia and normotensive pregnancy, group (p < 0.001). In contrast, serum PP13 levels were found to be the highest in the early-onset preeclampsia group, although no significant difference were found in mean maternal serum levels of PP13 between the three groups. The decreased PP13 expression in placental syncytiotrophoblast can be attributed to the greater extent of damage in the STBM in early-onset preeclampsia that leads to the release of a larger amount of PP13 into maternal circulation. The hypothesis aligns with the TEM analysis results. Preeclamptic pregnancies showed placental syncytiotrophoblast aponeurosis, whereas normotensive pregnancies did not. Placental lesions and STBM shedding were found to be more pronounced in early-onset preeclampsia compared to late-onset preeclampsia. Conclusions: PP13 and STBM damage may play a role in systemic endothelial dysfunction in preeclampsia.

SPIRE-a software tool for bicontinuous phase recognition: application for plastid cubic membranes.

Bicontinuous membranes in cell organelles epitomize nature's ability to create complex functional nanostructures. Like their synthetic counterparts, these membranes are characterized by continuous membrane sheets draped onto topologically complex saddle-shaped surfaces with a periodic network-like structure. Their structure sizes, (around 50-500 nm), and fluid nature make transmission electron microscopy (TEM) the analysis method of choice to decipher their nanostructural features. Here we present a tool, Surface Projection Image Recognition Environment (SPIRE), to identify bicontinuous structures from TEM sections through interactive identification by comparison to mathematical "nodal surface" models. The prolamellar body (PLB) of plant etioplasts is a bicontinuous membrane structure with a key physiological role in chloroplast biogenesis. However, the determination of its spatial structural features has been held back by the lack of tools enabling the identification and quantitative analysis of symmetric membrane conformations. Using our SPIRE tool, we achieved a robust identification of the bicontinuous diamond surface as the dominant PLB geometry in angiosperm etioplasts in contrast to earlier long-standing assertions in the literature. Our data also provide insights into membrane storage capacities of PLBs with different volume proportions and hint at the limited role of a plastid ribosome localization directly inside the PLB grid for its proper functioning. This represents an important step in understanding their as yet elusive structure-function relationship.

A rapid method to visualize human mitochondrial DNA replication through rotary shadowing and transmission electron microscopy.

We report a rapid experimental procedure based on high-density in vivo psoralen inter-strand DNA cross-linking coupled to spreading of naked purified DNA, positive staining, low-angle rotary shadowing, and transmission electron microscopy (TEM) that allows quick visualization of the dynamic of heavy strand (HS) and light strand (LS) human mitochondrial DNA replication. Replication maps built on linearized mitochondrial genomes and optimized rotary shadowing conditions enable clear visualization of the progression of the mitochondrial DNA synthesis and visualization of replication intermediates carrying long single-strand DNA stretches. One variant of this technique, called denaturing spreading, allowed the inspection of the fine chromatin structure of the mitochondrial genome and was applied to visualize the in vivo three-strand DNA structure of the human mitochondrial D-loop intermediate with unprecedented clarity.

Ultrastructural Examination of the Corneal Interface after Predescemetic Deep Anterior Lamellar Keratoplasty (DALK) - A Case Report with Light and Transmission Electron Microscopy.

PURPOSE: To examine corneal buttons with light and transmission electron microscopy (TEM) to visualize the interface area and highlight the ultrastructural corneal changes after deep anterior lamellar keratoplasty (DALK). METHODS: Two patients underwent excimer laser-assisted penetrating repeat keratoplasty after predescemetic DALK. The corneal buttons were examined by light microscopy and TEM. RESULTS: The light microscopic examination of the corneal buttons revealed fragments of a second Descemet's membrane in the central and midperipheral areas (Case 1). In both cases, visualization of the interface area was not possible by light microscopy. The donor and host stroma were tightly attached without dehiscence. TEM identified the interface area by irregularities in the collagen distribution between the donor and host stroma. The thickness of the remaining recipient corneal stroma measured approximately 30 µm (Case 1) and 100 µm (Case 2), respectively. In the host stroma, TEM revealed the absence or degeneration of keratocytes, accumulation of amorphous material between the collagen lamellae, and vacuolar inclusions dispersed in the stroma, forming a band-like zone anterior to Descemet's membrane. CONCLUSION: The interface area after DALK has been mainly investigated by in vivo confocal microscopy. Light microscopy and TEM findings indicate remodeling processes after DALK that are associated with increased keratocyte degeneration and structural alterations of the extracellular matrix in the host stroma. The choice of surgical method may influence the postoperative morphological and functional outcome since these findings were primarily apparent in the remaining host stroma. Therefore, complete exposure of Descemet's membrane is an important prognostic factor for the postoperative visual outcome.

A Disulfide-Stabilized Aß that Forms Dimers but Does Not Form Fibrils.

Aß dimers are a basic building block of many larger Aß oligomers and are among the most neurotoxic and pathologically relevant species in Alzheimer's disease. Homogeneous Aß dimers are difficult to prepare, characterize, and study because Aß forms heterogeneous mixtures of oligomers that vary in size and can rapidly aggregate into more stable fibrils. This paper introduces AßC18C33 as a disulfide-stabilized analogue of Aß42 that forms stable homogeneous dimers in lipid environments but does not aggregate to form insoluble fibrils. The AßC18C33 peptide is readily expressed in Escherichia coli and purified by reverse-phase HPLC to give ca. 8 mg of pure peptide per liter of bacterial culture. SDS-PAGE establishes that AßC18C33 forms homogeneous dimers in the membrane-like environment of SDS and that conformational stabilization of the peptide with a disulfide bond prevents the formation of heterogeneous mixtures of oligomers. Mass spectrometric (MS) studies in the presence of dodecyl maltoside (DDM) further confirm the formation of stable noncovalent dimers. Circular dichroism (CD) spectroscopy establishes that AßC18C33 adopts a ß-sheet conformation in detergent solutions and supports a model in which the intramolecular disulfide bond induces ß-hairpin folding and dimer formation in lipid environments. Thioflavin T (ThT) fluorescence assays and transmission electron microscopy (TEM) studies indicate that AßC18C33 does not undergo fibril formation in aqueous buffer solutions and demonstrate that the intramolecular disulfide bond prevents fibril formation. The recently published NMR structure of an Aß42 tetramer (PDB: 6RHY) provides a working model for the AßC18C33 dimer, in which two ß-hairpins assemble through hydrogen bonding to form a four-stranded antiparallel ß-sheet. It is anticipated that AßC18C33 will serve as a stable, nonfibrilizing, and noncovalent Aß dimer model for amyloid and Alzheimer's disease research.

Laser phase plate for transmission electron microscopy.

Transmission electron microscopy (TEM) of rapidly frozen biological specimens, or cryo-EM, would benefit from the development of a phase plate for in-focus phase contrast imaging. Several types of phase plates have been investigated, but rapid electrostatic charging of all such devices has hindered these efforts. Here, we demonstrate electron phase manipulation with a high-intensity continuous-wave laser beam, and use it as a phase plate for TEM. We demonstrate the laser phase plate by imaging an amorphous carbon film. The laser phase plate provides a stable and tunable phase shift without electrostatic charging or unwanted electron scattering. These results suggest the possibility for dose-efficient imaging of unstained biological macromolecules and cells.

The cryo-EM method microcrystal electron diffraction (MicroED).

In 2013 we established a cryo-electron microscopy (cryo-EM) technique called microcrystal electron diffraction (MicroED). Since that time, data collection and analysis schemes have been fine-tuned, and structures for more than 40 different proteins, oligopeptides and organic molecules have been determined. Here we review the MicroED technique and place it in context with other structure-determination methods. We showcase example structures solved by MicroED and provide practical advice to prospective users.

Software tools for automated transmission electron microscopy.

The demand for high-throughput data collection in electron microscopy is increasing for applications in structural and cellular biology. Here we present a combination of software tools that enable automated acquisition guided by image analysis for a variety of transmission electron microscopy acquisition schemes. SerialEM controls microscopes and detectors and can trigger automated tasks at multiple positions with high flexibility. Py-EM interfaces with SerialEM to enact specimen-specific image-analysis pipelines that enable feedback microscopy. As example applications, we demonstrate dose reduction in cryo-electron microscopy experiments, fully automated acquisition of every cell in a plastic section and automated targeting on serial sections for 3D volume imaging across multiple grids.