Cryo-electron Microscopy Of Vitreous Sections Research Articles

Molecular Organization of the Skin Barrier.

Cryo-electron microscopy of vitreous sections allows for investigation directly in situ of the molecular architecture of skin. Recently, this technique has contributed to the elucidation of the molecular organization of the skin's permeability barrier and its stepwise formation process. The aim of this review is to provide an overview of the procedure for cryo-electron microscopy of vitreous sections, its analysis using atomic detail molecular dynamics modelling and electron microscopy simulation, and its application in the investigation of the barrier structure and formation process of the skin.

Multiple roads lead to Rome: unique morphology and chemistry of endospores, exospores, myxospores, cysts and akinetes in bacteria.

The production of specialized resting cells is a remarkable survival strategy developed by many organisms to withstand unfavourable environmental factors such as nutrient depletion or other changes in abiotic and/or biotic conditions. Five bacterial taxa are recognized to form specialized resting cells: Firmicutes, forming endospores; Actinobacteria, forming exospores; Cyanobacteria, forming akinetes; the δ-Proteobacterial order Myxococcales, forming myxospores; and Azotobacteraceae, forming cysts. All these specialized resting cells are characterized by low-to-absent metabolic activity and higher resistance to environmental stress (desiccation, heat, starvation, etc.) when compared to vegetative cells. Given their similarity in function, we tested the potential existence of a universal morpho-chemical marker for identifying these specialized resting cells. After the production of endospores, exospores, akinetes and cysts in model organisms, we performed the first cross-species morphological and chemical comparison of bacterial sporulation. Cryo-electron microscopy of vitreous sections (CEMOVIS) was used to describe near-native morphology of the resting cells in comparison to the morphology of their respective vegetative cells. Resting cells shared a thicker cell envelope as their only common morphological feature. The chemical composition of the different specialized resting cells at the single-cell level was investigated using confocal Raman microspectroscopy. Our results show that the different specialized cells do not share a common chemical signature, but rather each group has a unique signature with a variable conservation of the signature of the vegetative cells. Additionally, we present the validation of Raman signatures associated with calcium dipicolinic acid (CaDPA) and their variation across individual cells to develop specific sorting thresholds for the isolation of endospores. This provides a proof of concept of the feasibility of isolating bacterial spores using a Raman-activated cell-sorting platform. This cross-species comparison and the current knowledge of genetic pathways inducing the formation of the resting cells highlights the complexity of this convergent evolutionary strategy promoting bacterial survival.

The Gram-Positive Bacterial Cell Wall.

The chapter about the Gram-positive bacterial cell wall gives a brief historical background on the discovery of Gram-positive cell walls and their constituents and microscopic methods applied for studying the Gram-positive cell envelope. Followed by the description of the different chemical building blocks of peptidoglycan and the biosynthesis of the peptidoglycan layers and high turnover of peptidoglycan during bacterial growth. Lipoteichoic acids and wall teichoic acids are highlighted as major components of the cell wall. Characterization of capsules and the formation of extracellular vesicles by Gram-positive bacteria close the section on cell envelopes which have a high impact on bacterial pathogenesis. In addition, the specialized complex and unusual cell wall of mycobacteria is introduced thereafter. Next a short back view is given on the development of electron microscopic examinations for studying bacterial cell walls. Different electron microscopic techniques and methods applied to examine bacterial cell envelopes are discussed in the view that most of the illustrated methods should be available in a well-equipped life sciences orientated electron microscopic laboratory. In addition, newly developed and mostly well-established cryo-methods like high-pressure freezing and freeze-substitution (HPF-FS) and cryo-sections of hydrated vitrified bacteria (CEMOVIS, Cryo-electron microscopy of vitreous sections) are described. At last, modern cryo-methods like cryo-electron tomography (CET) and cryo-FIB-SEM milling (focus ion beam-scanning electron microscopy) are introduced which are available only in specialized institutions, but at present represent the best available methods and techniques to study Gram-positive cell walls under close-to-nature conditions in great detail and at high resolution.

Hunting for the Adhesion Molecule, Retinoschisin, in Retina using Cemovis

In studying biologically relevant molecules, the ultimate aim is to visualize them in their native environment. One technique that has the potential to resolve individual molecules in cells and tissues is cryo-electron microscopy of vitreous sections (CEMOVIS) combined with tomography. We are interested in revealing the disposition of the retinal adhesion protein, retinoschisin (RS1), within the retina. Immuno-fluorescence and immuno-electron microscopy showed that RS1 is abundant in the intercellular spaces between the inner segments of photoreceptors, and between bipolar cells. Loss-of-function mutations in RS1 cause X-linked retinoschisis (XLRS), a debilitating disease associated with the formation of retinal cavities or cysts (schisis) and vision loss in young males. We solved the structure to 4.1 Å by cryo-electron microscopy (cryoEM), a 16-mer composed of two octameric rings with a total weight of ∼380 kDa. From this structure it is clear that many mutations in the subunit interfaces would preclude assembly of the rings. However, other mutation sites are exposed at the periphery and it was not evident how they would cause disease. In further cryoEM experiments, we observed that RS1 double rings self-assemble into extensive branched networks, suggesting a mechanism for its adhesive function. To ascertain whether this assembly also occurs in situ, we are examining mouse retinal tissue using CEMOVIS and cryo-electron tomography. We obtained images of 50 nm cryosections that show clear distinction of microtubular protofilaments separated by ∼5 nm. This resolution is clearly sufficient to see the RS1 double ring and whether it assembles into networks in the intercellular space between photoreceptors and bipolar cells.

Inroads through the bacterial cell envelope: seeing is believing.

A singular feature of all prokaryotic cells is the presence of a cell envelope composed of a cytoplasmic membrane and a cell wall. The introduction of bacterial cell fractionation techniques in the 1950s and 1960s along with developments in procedures for electron microscopy opened the window towards an understanding of the chemical composition and architecture of the cell envelope. This review traces the contribution of Terry Beveridge in these endeavours, beginning with his doctoral studies in the 1970s on the structure of paracrystalline surface arrays (S-layers), followed by an exploration of cryogenic methods for preserving bacteria for ultrastructural analyses. His insights are reflected in a current example of the contribution of cryo-electron microscopy to S-layer studies - the structure and assembly of the surface array of Caulobacter crescentus. The review then focuses on Terry's contributions to imaging the ultrastructure of bacterial cell envelopes and to the development of cryo-electron microscopy techniques, including the use of CEMOVIS (Cryo-electron Microscopy of Vitreous Sections) to "see" the ultrastructure of the Gram-positive cell envelope - his last scientific endeavour.

Amorphous Calcium Carbonate Granules Form Within an Intracellular Compartment in Calcifying Cyanobacteria.

The recent discovery of cyanobacteria forming intracellular amorphous calcium carbonate (ACC) has challenged the former paradigm suggesting that cyanobacteria-mediated carbonatogenesis was exclusively extracellular. Yet, the mechanisms of intracellular biomineralization in cyanobacteria and in particular whether this takes place within an intracellular microcompartment, remain poorly understood. Here, we analyzed six cyanobacterial strains forming intracellular ACC by transmission electron microscopy. We tested two different approaches to preserve as well as possible the intracellular ACC inclusions: (i) freeze-substitution followed by epoxy embedding and room-temperature ultramicrotomy and (ii) high-pressure freezing followed by cryo-ultramicrotomy, usually referred to as cryo-electron microscopy of vitreous sections (CEMOVIS). We observed that the first method preserved ACC well in 500-nm-thick sections but not in 70-nm-thick sections. However, cell ultrastructures were difficult to clearly observe in the 500-nm-thick sections. In contrast, CEMOVIS provided a high preservation quality of bacterial ultrastructures, including the intracellular ACC inclusions in 50-nm-thick sections. ACC inclusions displayed different textures, suggesting varying brittleness, possibly resulting from different hydration levels. Moreover, an electron dense envelope of ∼2.5 nm was systematically observed around ACC granules in all studied cyanobacterial strains. This envelope may be composed of a protein shell or a lipid monolayer, but not a lipid bilayer as usually observed in other bacteria forming intracellular minerals. Overall, this study evidenced that ACC inclusions formed and were stabilized within a previously unidentified bacterial microcompartment in some species of cyanobacteria.

Nucleosome conformational variability in solution and in interphase nuclei evidenced by cryo-electron microscopy of vitreous sections.

In Eukaryotes, DNA is wound around the histone octamer forming the basic chromatin unit, the nucleosome. Atomic structures have been obtained from crystallography and single particle cryo-electron microscopy (cryoEM) of identical engineered particles. But native nucleosomes are dynamical entities with diverse DNA sequence and histone content, and little is known about their conformational variability, especially in the cellular context. Using cryoEM and tomography of vitreous sections we analyse native nucleosomes, both in vitro, using purified particles solubilized at physiologically relevant concentrations (25–50%), and in situ, within interphase nuclei. We visualize individual nucleosomes at a level of detail that allows us to measure the distance between the DNA gyres wrapped around. In concentrated solutions, we demonstrate a salt-dependent transition, with a high salt compact conformation resembling the canonical nucleosome and an open low salt one, closer to nuclear nucleosomes. Although further particle characterization and cartography are needed to understand the relationship between this conformational variability and chromatin functional states, this work opens a route to chromatin exploration in situ.

Microstructural observation of casein micelles in milk by cryo-electron microscopy of vitreous sections (CEMOVIS).

Casein micelles are present in bovine milk as colloidal particles with diameters of 20-600 nm, which are complex macromolecular assemblies composed of four distinct types of casein and colloidal calcium phosphate (CCP). Multiple structural models of casein micelles have been proposed based on their biochemical or physical properties and observed using electron microscopy. However, the CCP distribution and crosslinking structure between CCP and casein remain unclear. Therefore, the internal structure of casein micelles in raw milk was observed using cryo-electron microscopy of vitreous sections (CEMOVIS) with high precision at high resolution. The results confirmed that the average casein micelle diameter was about 140 nm, and that the CCP diameter in casein micelles was about 2-3 nm, with an average diameter of 2.3 nm. The distribution of CCP in casein micelles was not uniform, with an average interval between CCPs of about 5.4 nm. Areas containing no black particles (attributed to CCP) were present, with an average size of about 19.1 nm. Considering previous reports, these areas possibly correspond to pores or cavities filled with water. Based on differences in the density of structures in casein micelles, we estimated that some of the casein aggregates were able to connect with CCP in a string.

Visualization of adherent cell monolayers by cryo-electron microscopy: A snapshot of endothelial adherens junctions.

Cryo-electron microscopy (cryo-EM) allows the visualization of the cell architecture in its native state. We developed a robust solution to adapt cryo-electron microscopy of vitreous sections (CEMOVIS) to a monolayer of adherent cells using a functionalized polyacrylamide hydrogel growing substrate. We applied this method to reconstitute an endothelial cell monolayer to visualize the morphology of adherens junctions (AJs) which regulate permeability and integrity of the vascular barrier. The fine morphology and ultrastructure of AJs from cultured primary human coronary artery endothelial cells (HCAECs) were analyzed in their native state by using CEMOVIS. Doxycycline and sphingosine-1-phosphate (S1P) are known as efficient regulators of endothelial permeability. Doxycycline and S1P treatments both led to a drastic morphological switch from very uneven to standardized 14-17 nm wide AJs over several microns indicative of a better membrane tethering. Repetitive structures were occasionally noticed within the AJ cleft reflecting a local improved structural organization of VE-cadherin molecules. The ultrastructural stabilization of AJs observed upon treatment likely indicates a better adhesion and thus provides structural clues on the mechanism by which these treatments improve the endothelial barrier function. This method was also successfully extended to a thick epithelial barrier model. We expect our strategy to extend the reliable application of CEMOVIS to virtually any adherent cultured cell systems.

Dormant Bacillus spores protect their DNA in crystalline nucleoids against environmental stress.

Bacterial spores of the genera Bacillus and Clostridium are extremely resistant against desiccation, heat and radiation and involved in the spread and pathogenicity of health relevant species such as Bacillus anthracis (anthrax) or Clostridium botulinum. While the resistance of spores is very well documented, underlying mechanisms are not fully understood. In this study we show, by cryo-electron microscopy of vitreous sections and particular resin thin section electron microscopy, that dormant Bacillus spores possess highly ordered crystalline core structures, which contain the DNA, but only if small acid soluble proteins (SASPs) are present. We found those core structures in spores of all Bacillus species investigated, including spores of anthrax. Similar core structures were detected in Geobacillus and Clostridium species which suggest that highly ordered, at least partially crystalline core regions represent a general feature of bacterial endospores. The crystalline core structures disintegrate in a period during spore germination, when resistance against most stresses is lost. Our results suggest that the DNA is tightly packed into a crystalline nucleoid by binding SASPs, which stabilizes DNA fibrils and protects them against modification. Thus, the crystalline nucleoid seems to be the structural and functional correlate for the remarkable stability of the DNA in bacterial endospores.

Update of technologies for examining the stratum corneum at the molecular level

Understanding the molecular organization of the stratum corneum is still an outstanding problem, despite being both fundamentally and clinically significant. There is a need to develop methodology that yields molecular-level resolution of the stratum corneum components in their native state, without introducing artefacts. We outline here the recent success of cryo-electron microscopy of vitreous sections (CEMOVIS) combined with electron microscopy simulation to elucidate the molecular organization of the stratum corneum in its near-native state. Furthermore, some emerging technologies for studying the physical properties and dynamic behaviour of native stratum corneum at the molecular level are briefly reviewed. These encompass multiphoton microscopy (MPM), polarization transfer solid-state nuclear magnetic resonance (PTssNMR) and PeakForce tapping-mode atomic force microscopy combined with frequency-modulation Kelvin probe force microscopy (KPFM). CEMOVIS combined with electron microscopy simulation allows for molecular structure determination in situ in native stratum corneum, while MPM allows probing of the stratum corneum local physicochemical properties such as fluorophore diffusion coefficients, water content and pH. PTssNMR allows for evaluation of the molecular mobility of stratum corneum keratin and lipid components, and PeakForce KPFM allows for analysis of the local nanomechanical properties of stratum corneum. These emerging techno-logies may contribute to a molecular-level understanding of stratum corneum structure and function in vivo.

Closer to the native state. Critical evaluation of cryo-techniques for Transmission Electron Microscopy: preparation of biological samples.

Over the years Transmission Electron Microscopy (TEM) has evolved into a powerful technique for the structural analysis of cells and tissues at various levels of resolution. However, optimal sample preservation is required to achieve results consistent with reality. During the last few decades, conventional preparation methods have provided most of the knowledge about the ultrastructure of organelles, cells and tissues. Nevertheless, some artefacts can be introduced at all stagesofstandard electron microscopy preparation technique. Instead, rapid freezing techniques preserve biological specimens as close as possible to the native state. Our review focuses on different cryo-preparation approaches, starting from vitrification methods dependent on sample size. Afterwards, we discuss Cryo-Electron Microscopy Of VItreous Sections (CEMOVIS) and the main difficulties associated with this technique. Cryo-Focused Ion Beam (cryo-FIB) is described as a potential alternative for CEMOVIS. Another post-processing route for vitrified samples is freeze substitution and embedding in resin for structural analysis or immunolocalization analysis. Cryo-sectioning according to Tokuyasu is a technique dedicated to high efficiency immunogold labelling. Finally, we introduce hybrid techniques, which combine advantages of primary techniques originally dedicated to different approaches. Hybrid approaches permit to perform the study of difficult-to-fix samples and antigens or help optimize the sample preparation protocol for the integrated Laser and Electron Microscopy (iLEM) technique.

Structural analysis of the PSD-95 cluster by electron tomography and CEMOVIS: a proposal for the application of the genetically encoded metallothionein tag

Postsynaptic density-95 (PSD-95) accumulates at excitatory postsynapses and plays important roles in the clustering and anchoring of numerous proteins at the PSD. However, a detailed ultrastructural analysis of clusters exclusively consisting of PSD-95 has never been performed. Here, we employed a genetically encoded tag, three tandem repeats of metallothionein (3MT), to study the structure of PSD-95 clusters in cells by electron tomography and cryo-electron microscopy of vitreous sections. We also performed conventional transmission electron microscopy (TEM). Cultured hippocampal neurons expressing a fusion protein of PSD-95 coupled to 3MT (PDS-95-3MT) were incubated with CdCl2 to result in the formation of Cd-bound PSD-95-3MT. Two types of electron-dense deposits composed of Cd-bound PSD-95-3MT were observed in these cells by TEM, as reported previously. Electron tomography revealed the presence of membrane-shaped structures representing PSD-95 clusters at the PSD and an ellipsoidal structure located in the non-synaptic cytoplasm. By TEM, the PSD-95 clusters appeared to be composed of a number of dense cores. In frozen hydrated sections, these dense cores were also found beneath the postsynaptic membrane. Taken together, our findings suggest that dense cores of PSD-95 aggregate to form the larger clusters present in the PSD and the non-synaptic cytoplasm.

Chapter 3 - Metallothioneins for Correlative Light and Electron Microscopy

Structural biologists have been working for decades on new strategies to identify proteins in cells unambiguously. We recently explored the possibilities of using the small metal-binding protein, metallothionein (MT), as a tag to detect proteins in transmission electron microscopy. It had been reported that, when fused with a protein of interest and treated in vitro with gold salts, a single MT tag will build an electron-dense gold cluster ~1 nm in diameter; we provided proof of this principle by demonstrating that MT can be used to detect intracellular proteins in bacteria and eukaryotic cells. The method, which is compatible with a variety of sample processing techniques, allows specific detection of proteins in cells with exceptional sensitivity. We illustrated the applicability of the technique in a series of studies to visualize the intracellular distribution of bacterial and viral proteins. Immunogold labeling was fundamental to confirm the specificity of the MT-gold method. When proteins were double-tagged with green fluorescent protein and MT, direct correlative light and electron microscopy allowed visualization of the same macromolecular complexes with different spatial resolutions. MT-gold tagging might also become a useful tool for mapping proteins into the 3D-density maps produced by (cryo)-electron tomography. New protocols will be needed for double or multiple labeling of proteins, using different versions of MT with fluorophores of different colors. Further research is also necessary to render the MT-gold labeling procedure compatible with immunogold labeling on Tokuyasu cryosections and with cryo-electron microscopy of vitreous sections.

A new tool based on two micromanipulators facilitates the handling of ultrathin cryosection ribbons

A close to native structure of bulk biological specimens can be imaged by cryo-electron microscopy of vitreous sections (CEMOVIS). In some cases structural information can be combined with X-ray data leading to atomic resolution in situ. However, CEMOVIS is not routinely used. The two critical steps consist of producing a frozen section ribbon of a few millimeters in length and transferring the ribbon onto an electron microscopy grid. During these steps, the first sections of the ribbon are wrapped around an eyelash (unwrapping is frequent). When a ribbon is sufficiently attached to the eyelash, the operator must guide the nascent ribbon. Steady hands are required. Shaking or overstretching may break the ribbon. In turn, the ribbon immediately wraps around itself or flies away and thereby becomes unusable. Micromanipulators for eyelashes and grids as well as ionizers to attach section ribbons to grids were proposed. The rate of successful ribbon collection, however, remained low for most operators. Here we present a setup composed of two micromanipulators. One of the micromanipulators guides an electrically conductive fiber to which the ribbon sticks with unprecedented efficiency in comparison to a not conductive eyelash. The second micromanipulator positions the grid beneath the newly formed section ribbon and with the help of an ionizer the ribbon is attached to the grid. Although manipulations are greatly facilitated, sectioning artifacts remain but the likelihood to investigate high quality sections is significantly increased due to the large number of sections that can be produced with the reported tool.

The Stack: A New Bacterial Structure Analyzed in the Antarctic Bacterium Pseudomonas deceptionensis M1T by Transmission Electron Microscopy and Tomography

In recent years, improvements in transmission electron microscopy (TEM) techniques and the use of tomography have provided a more accurate view of the complexity of the ultrastructure of prokaryotic cells. Cryoimmobilization of specimens by rapid cooling followed by freeze substitution (FS) and sectioning, freeze fracture (FF) and observation of replica, or cryoelectron microscopy of vitreous sections (CEMOVIS) now allow visualization of biological samples close to their native state, enabling us to refine our knowledge of already known bacterial structures and to discover new ones.Application of these techniques to the new Antarctic cold-adapted bacterium Pseudomonas deceptionensis M1T has demonstrated the existence of a previously undescribed cytoplasmic structure that does not correspond to known bacterial inclusion bodies or membranous formations. This structure, which we term a “stack”, was mainly visualized in slow growing cultures of P . deceptionensis M1T and can be described as a set of stacked membranous discs usually arranged perpendicularly to the cell membrane, but not continuous with it, and found in variable number in different locations within the cell. Regardless of their position, stacks were mostly observed very close to DNA fibers. Stacks are not exclusive to P . deceptionensis M1T and were also visualized in slow-growing cultures of other bacteria. This new structure deserves further study using cryoelectron tomography to refine its configuration and to establish whether its function could be related to chromosome dynamics.

A novel method for high-pressure freezing of adherent cells for frozen hydrated sectioning and CEMOVIS

With the development of Cryo Electron Microscopy Of Vitreous Sections (CEMOVIS), imaging cells in a close to native state has become a reality. However with the commonly used carriers for high-pressure freezing and cryo-sectioning, adherent grown cells either need to be detached from their substrate. Here a new method is presented for high-pressure freezing adherent growing cells for frozen-hydrated sectioning and CEMOVIS. Cells are cultured on golden grids, containing a carbon coated Formvar film, and frozen on a membrane carrier which provides the grids with the structural support needed to withstand the strain of trimming and cryo-sectioning. This method was successfully tested for the two different types of high-pressure freezers, those using a pressure chamber (HPM010, EMHPF, Wohlwend Compact 01/02, HPM100) and those directly pressurizing the sample (EMPact series).

Mechanical Tomography of Human Corneocytes with a Nanoneedle

Atomic force microscopy (AFM) can image biological samples and characterize their mechanical properties. However, the low aspect ratio of standard AFM probes typically limits these measurements to surface properties. Here, the intracellular mechanical behavior of human corneocytes is determined using "nanoneedle" AFM probes. The method evaluates the forces experienced by a nanoneedle as it is pushed into and then retracted from the cell. Indentation loops yield the stiffness profile and information on the elastic and nonelastic mechanical properties at a specific depth below the surface of the corneocytes. A clear difference between the softer ∼50-nm-thick external layer and the more rigid internal structure of corneocytes is apparent, which is consistent with the current understanding of the structure of these cells. There are also significant variations in the mechanical properties of corneocytes from different volunteers. The small diameter of the nanoneedle allows this "mechanical tomography" to be performed with high spatial resolution, potentially offering an opportunity to detect biomechanical changes in corneocytes because of, e.g., environmental factors, aging, or dermatological pathologies.

Contribution of cryoelectron microscopy of vitreous sections to the understanding of biological membrane structure

Using cryoelectron microscopy of vitreous sections, we investigated in situ the ultrastructure of biological membranes, selected from several cell types for their diverse biological functions. Here we describe how to visualize the two membrane leaflets and tightly apposed membranes, lying as close as 1.1 nm apart, by tuning the imaging conditions. We show how defects in membrane stacks may be clues to resolving their structure. Details of membrane proteins are also resolved, as well as protein lattices with correlations between stacked membranes. Imaging the cell in its native hydrated state can now be done in the nanometer resolution range, which should open unique routes for investigating structure-function relationships.

Self-pressurized rapid freezing (SPRF) as a simple fixation method for cryo-electron microscopy of vitreous sections

Cryo-electron microscopy of vitreous sections (CEMOVIS) is currently considered the method of choice to explore cellular ultrastructure at high resolution as close as possible to their native state. Here, we apply a novel, easy-to-use and low-cost freeze fixation method for CEMOVIS, avoiding the use of high-pressure freezing apparatus. Cells are placed in capillary metal tubes, which are tightly closed and plunged directly into liquid ethane cooled by liquid nitrogen. In some parts of the tube, crystalline ice is formed, building up pressure sufficient for the liquid-glass transition of the remaining specimen. We verified the presence of vitreous ice in these preparations using CEMOVIS and electron diffraction. Furthermore, different tube materials being less poisonous than copper were established to minimize physiological alterations of the specimen. Bacteria, yeast and mammalian cells were tested for molecular resolution. The quality of results is equivalent to samples prepared by conventional high pressure freezing apparatus, thus establishing this novel method as fast, easy-to-use and low-cost freeze fixation alternative for cryo-EM.