CryoCycle your grids: Plunge vitrifying and reusing clipped grids to advance cryoEM democratization.

CryoEM democratization is hampered by access to costly plunge-freezing supplies. We introduce methods, called CryoCycle, for reliably blotting, vitrifying, and reusing clipped cryoEM grids. We demonstrate that vitreous ice may be produced by plunging clipped grids with purified proteins into liquid ethane and that clipped grids may be reused several times for different protein samples. Furthermore, we demonstrate the vitrification of thin areas of cells prepared on gold-coated, pre-clipped grids.

Dynamic PRC1-CBX8 stabilizes a porous structure of chromatin condensates.

The compaction of chromatin is a prevalent paradigm in gene repression. Chromatin compaction is commonly thought to repress transcription by restricting chromatin accessibility. However, the spatial organisation and dynamics of chromatin compacted by gene-repressing factors are unknown. Using cryo-electron tomography, we solved the threedimensional structure of chromatin condensed by the Polycomb Repressive Complex 1 (PRC1) in a complex with CBX8. PRC1-condensed chromatin is porous and stabilised through multivalent dynamic interactions of PRC1 with chromatin. Mechanistically, positively charged residues on the internally disordered regions (IDRs) of CBX8 mask negative charges on the DNA to stabilize the condensed state of chromatin. Within condensates, PRC1 remains dynamic while maintaining a static chromatin structure. In differentiated mouse embryonic stem cells, CBX8-bound chromatin remains accessible. These findings challenge the idea of rigidly compacted polycomb domains and instead provides a mechanistic framework for dynamic and accessible PRC1-chromatin condensates.

Square beams for optimal tiling in transmission electron microscopy.

Imaging large fields of view at a high magnification requires tiling. Transmission electron microscopes typically have round beam profiles; therefore, tiling across a large area is either imperfect or results in uneven exposures, a problem for dose-sensitive samples. Here, we introduce a square electron beam that can easily be retrofitted in existing microscopes, and demonstrate its application, showing that it can tile nearly perfectly and deliver cryo-electron microscopy imaging with a resolution comparable to conventional set-ups.

Square beams for optimal tiling in TEM.

Imaging large fields of view at a high magnification requires tiling. Transmission electron microscopes typically have round beam profiles; therefore, tiling across a large area is either imperfect or results in uneven exposures, a problem on dose-sensitive samples. Here, we introduce a square electron beam that can be easily retrofitted in existing microscopes and demonstrate its application, showing it can tile nearly perfectly and deliver cryo-EM imaging with a resolution comparable to conventional setups.

Square beams for optimal tiling in TEM.

Imaging large fields-of-view at a high magnification requires tiling. Transmission electron microscopes typically have round beam profiles; therefore, tiling across a large field-of-view is either imperfect or results in uneven exposures, which is a problem on dose-sensitive samples. Here we introduce a square electron beam that can be easily retrofitted in existing microscopes and demonstrate its application showing it can tile nearly perfectly and deliver cryo-EM imaging with resolution comparable to conventional setups.

Assessing the Quality of Oxygen Plasma Focused Ion Beam (O-PFIB) Etching on Polypropylene Surfaces Using Secondary Electron Hyperspectral Imaging.

The development of Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM) systems has provided significant advances in the processing and characterization of polymers. A fundamental understanding of ion-sample interactions is still missing despite FIB-SEM being routinely applied in microstructural analyses of polymers. This study applies Secondary Electron Hyperspectral Imaging to reveal oxygen and xenon plasma FIB interactions on the surface of a polymer (in this instance, polypropylene). Secondary Electron Hyperspectral Imaging (SEHI) is a technique housed within the SEM chamber that exhibits multiscale surface sensitivity with a high spatial resolution and the ability to identify carbon bonding present using low beam energies without requiring an Ultra High Vacuum (UHV). SEHI is made possible through the use of through-the-lens detectors (TLDs) to provide a low-pass SE collection of low primary electron beam energies and currents. SE images acquired over the same region of interest from different energy ranges are plotted to produce an SE spectrum. The data provided in this study provide evidence of SEHI's ability to be a valuable tool in the characterization of polymer surfaces post-PFIB etching, allowing for insights into both tailoring polymer processing FIB parameters and SEHI's ability to be used to monitor serial FIB polymer surfaces in situ.

OpenFIBSEM: A universal API for FIBSEM control.

This paper introduces OpenFIBSEM, a universal API to control Focused Ion Beam Scanning Electron Microscopes (FIBSEM). OpenFIBSEM aims to improve the programmability and automation of electron microscopy workflows in structural biology research. The API is designed to be cross-platform, composable, and extendable: allowing users to use any portion of OpenFIBSEM to develop or integrate with other software tools. The package provides core functionality such as imaging, movement, milling, and manipulator control, as well as system calibration, alignment, and image analysis modules. Further, a library of reusable user interface components integrated with napari is provided, ensuring easy and efficient application development. OpenFIBSEM currently supports ThermoFisher and TESCAN hardware, with support for other manufacturers planned. To demonstrate the improved automation capabilities enabled by OpenFIBSEM, several example applications that are compatible with multiple hardware manufacturers are discussed. We argue that OpenFIBSEM provides the foundation for a cross-platform operating system and development ecosystem for FIBSEM systems. The API and applications are open-source and available on GitHub (https://github.com/DeMarcoLab/fibsem).

Helical ultrastructure of the metalloprotease meprin α in complex with a small molecule inhibitor.

The zinc-dependent metalloprotease meprin α is predominantly expressed in the brush border membrane of proximal tubules in the kidney and enterocytes in the small intestine and colon. In normal tissue homeostasis meprin α performs key roles in inflammation, immunity, and extracellular matrix remodelling. Dysregulated meprin α is associated with acute kidney injury, sepsis, urinary tract infection, metastatic colorectal carcinoma, and inflammatory bowel disease. Accordingly, meprin α is the target of drug discovery programs. In contrast to meprin ß, meprin α is secreted into the extracellular space, whereupon it oligomerises to form giant assemblies and is the largest extracellular protease identified to date (~6 MDa). Here, using cryo-electron microscopy, we determine the high-resolution structure of the zymogen and mature form of meprin α, as well as the structure of the active form in complex with a prototype small molecule inhibitor and human fetuin-B. Our data reveal that meprin α forms a giant, flexible, left-handed helical assembly of roughly 22 nm in diameter. We find that oligomerisation improves proteolytic and thermal stability but does not impact substrate specificity or enzymatic activity. Furthermore, structural comparison with meprin ß reveal unique features of the active site of meprin α, and helical assembly more broadly.

Methods of Preparing Nanoscale Vitreous Ice Needles for High-Resolution Cryogenic Characterization.

New high-resolution imaging methods for biological samples such as atom probe tomography (APT), facilitated by the invention of laser-pulsed atom probes and cryo-transfer procedures, have recently emerged. However, ensuring the vitreous state of the fabricated aqueous needle-shaped APT samples remains a challenge despite it being crucial for characterizing biomolecules such as proteins and cellular architectures in their near-native state. Our work investigated three potential approaches: (1) open microcapillary (OMC) method, (2) high-pressure freezing method (HPF), and (3) graphene encapsulation method. Diffraction patterns of the needle specimens acquired by cryo-TEM have demonstrated the vitreous state of the ice needles, although limited to the tip regions, has been achieved with the three proposed approaches. With the capability to prepare vitreous ice needles from hydrated samples of up to ∼200 µm thickness (HPF), combined use of the three approaches opens new avenues for future near-atomic imaging of biological cells in their near-native state.

Anisotropic epitaxial stabilization of a low-symmetry ferroelectric with enhanced electromechanical response.

Piezoelectrics interconvert mechanical energy and electric charge and are widely used in actuators and sensors. The best performing materials are ferroelectrics at a morphotropic phase boundary, where several phases coexist. Switching between these phases by electric field produces a large electromechanical response. In ferroelectric BiFeO3, strain can create a morphotropic-phase-boundary-like phase mixture and thus generate large electric-field-dependent strains. However, this enhanced response occurs at localized, randomly positioned regions of the film. Here, we use epitaxial strain and orientation engineering in tandem-anisotropic epitaxy-to craft a low-symmetry phase of BiFeO3 that acts as a structural bridge between the rhombohedral-like and tetragonal-like polymorphs. Interferometric displacement sensor measurements reveal that this phase has an enhanced piezoelectric coefficient of ×2.4 compared with typical rhombohedral-like BiFeO3. Band-excitation frequency response measurements and first-principles calculations provide evidence that this phase undergoes a transition to the tetragonal-like polymorph under electric field, generating an enhanced piezoelectric response throughout the film and associated field-induced reversible strains. These results offer a route to engineer thin-film piezoelectrics with improved functionalities, with broader perspectives for other functional oxides.

Three-dimensional imaging on a chip using optofluidics light-sheet fluorescence microscopy.

Volumetric, sub-micron to micron level resolution imaging is necessary to assay phenotypes or characteristics at the sub-cellular/organelle scale. However, three-dimensional fluorescence imaging of cells is typically low throughput or compromises on the achievable resolution in space and time. Here, we capitalise on the flow control capabilities of microfluidics and combine it with microoptics to integrate light-sheet based imaging directly into a microfluidic chip. Our optofluidic system flows suspended cells through a sub-micrometer thick light-sheet formed using micro-optical components that are cast directly in polydimethylsiloxane (PDMS). This design ensures accurate alignment, drift-free operation, and easy integration with conventional microfluidics, while providing sufficient spatial resolution, optical sectioning and volumetric data acquisition. We demonstrate imaging rates of 120 ms per cell at sub-µm resolution, that allow extraction of complex cellular phenotypes, exemplified by imaging of cell clusters, receptor distribution, and the analysis of endosomal size changes.

New Insights Into Sperm Ultrastructure Through Enhanced Scanning Electron Microscopy.

The analysis of spermatozoa morphology is fundamental to understand male fertility and the etiology of infertility. Traditionally scanning electron microscopy (SEM) has been used to define surface topology. Recently, however, it has become a critical tool for three-dimensional analysis of internal cellular ultrastructure. Modern SEM provides nanometer-scale resolution, but the meaningfulness of such information is proportional to the quality of the sample preservation. In this study, we demonstrate that sperm quickly and robustly adhere to gold-coated surfaces. Leveraging this property, we developed three step-by-step protocols fulfilling different needs for sperm imaging: chemically fixed monolayers for SEM examination of the external morphology, and two high-pressure freezing-based protocols for fast SEM examination of full cell internal morphology and focused ion-beam SEM tomography. These analyses allow previously unappreciated insights into mouse sperm ultrastructure, including the identification of novel structures within the fibrous sheath and domain-specific interactions between the plasma membrane and exosome-like structures.

The host exosome pathway underpins biogenesis of the human cytomegalovirus virion.

Human Cytomegalovirus (HCMV) infects over half the world's population, is a leading cause of congenital birth defects, and poses serious risks for immuno-compromised individuals. To expand the molecular knowledge governing virion maturation, we analysed HCMV virions using proteomics, and identified a significant proportion of host exosome constituents. To validate this acquisition, we characterized exosomes released from uninfected cells, and demonstrated that over 99% of the protein cargo was subsequently incorporated into HCMV virions during infection. This suggested a common membrane origin, and utilization of host exosome machinery for virion assembly and egress. Thus, we selected a panel of exosome proteins for knock down, and confirmed that loss of 7/9 caused significantly less HCMV production. Saliently, we report that VAMP3 is essential for viral trafficking and release of infectious progeny, in various HCMV strains and cell types. Therefore, we establish that the host exosome pathway is intrinsic for HCMV maturation, and reveal new host regulators involved in viral trafficking, virion envelopment, and release. Our findings underpin future investigation of host exosome proteins as important modulators of HCMV replication with antiviral potential.

Exosome trapping and enrichment using a sound wave activated nano-sieve (SWANS).

Exosomes, a form of extracellular vesicle, are an important precursor in regenerative medicine. Microfluidic methods exist to capture these sub-micrometer sized objects from small quantities of sample, ideal for multiple diagnostic applications. To address the challenge of extraction from large volumes, we use the visual access offered by microfluidic techniques to probe the physical mechanisms behind a method which is compatible with future upscaling. The sound wave actuated nano-sieve uses resonant modes in a packed bed of microparticles to exert trapping forces on nanoparticles. Here, we examine the role of the microparticle size, demonstrating better performance from 15 µm particles than 7 µm particles. When applied to biological samples, we demonstrate for the first time that a packed bed of these larger particles is capable of capturing exosomes and liposomes, the captured particles being on average 20 to 40 times smaller than the pores within the trapped bed.

Capacitive Sensing for Monitoring of Microfluidic Protocols Using Nanoliter Dispensing and Acoustic Mixing.

The development of protocols for bio/chemical reaction requires alternate dispensing and mixing steps. While most microfluidic systems use the opening of additional parts of the channel to allow the ingress of fixed volumes of fluid, this requires knowledge of the protocol before the design of the chip. Our approach of using a microfluidic valve to regulate the flow into an initially empty cavity allows for on-chip protocol development and refinement. Mixing is provided by way of surface acoustic wave excitation; this high-frequency vibration causes steady-state streaming flows. We show that capacitive sensing can be used to measure fluid levels, even if multiple fluid types are used, such that nanoliter dispensing accuracy is achieved. Also, the capacitive readout can be used to establish mixing quality and to monitor temperature fluctuations. These capabilities allow for protocols to be conducted without optical assessment and thus will allow for multiplexing, such that reactions could be conducted, simultaneously, in multiple chambers across a chip.

Hybrid refractive-diffractive axicons for Bessel-beam multiplexing and resolution improvement.

Optical elements rely on refraction, diffraction, or reflection for light manipulation. Fusing diffractive and refractive functions in a single element provides an extra layer of control over the wave propagation, allowing complex beam shaping through self-aligned, monolithic and miniaturized optics. Using gray-scale lithography with high-current focused Xe ion-beams, we realized hybrid refractive-diffractive micro-axicons that feature diffractive gratings engraved on their conical surfaces. Furthermore, we fabricated these devices in lithium niobate, which is a challenging piezo/optoelectronic material for processing with an as-yet unexploited potential in optical applications. The curvilinear surfaces of fabricated micro-axicons with a 230-µm diameter were engraved with diffraction linear and circular gratings of various depths (<400 nm), and the optical performance of these components was characterized, showing excellent agreement with theoretical expectations. The fusing of diffractive elements with carrier refractive surfaces introduces additional or enhanced device functionalities, such as beam multiplexing and resolution improvement. The potential applications of such monolithic and miniaturized hybrid micro-optical components include beamshaping for fluorescence microscopy.

Automated cryo-lamella preparation for high-throughput in-situ structural biology.

Cryo-transmission electron tomography (cryo-ET) in association with cryo-focused ion beam (cryo-FIB) milling enables structural biology studies to be performed directly within the cellular environment. Cryo-preserved cells are milled and a lamella with a typical thickness of 200-300 nm provides an electron transparent window suitable for cryo-ET imaging. Cryo-FIB milling is an effective method, but it is a tedious and time-consuming process, which typically results in ~10 lamellae per day. Here, we introduce an automated method to reproducibly prepare cryo-lamellae on a grid and reduce the amount of human supervision. We tested the routine on cryo-preserved Saccharomyces cerevisiae, mammalian 293 T cells, and lysozyme protein crystals. Here we demonstrate that our method allows an increased throughput, achieving a rate of 5 lamellae/hour without the need to supervise the FIB milling. We demonstrate that the quality of the lamellae is consistent throughout the preparation and their compatibility with cryo-ET analyses.

Assembly and Imaging set up of PIE-Scope.

Cryo-Electron Tomography (cryo-ET) is a method that enables resolving the structure of macromolecular complexes directly in the cellular environment. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-Focused Ion Beam (FIB) milling and correlative light microscopy to ensure that the event of interest is present in the lamella. Here, we present an integrated cryo-FIB and light microscope setup called the Photon Ion Electron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions containing protein complexes of interest. The PIE-scope can be retrofitted on existing microscopes, although the drawings we provide are meant to work on ThermoFisher DualBeams with small mechanical modifications those can be adapted on other brands.