Cryo-electron microscopy of vitreous sections

Since the beginning of the 1980s, cryo-electron microscopy of a thin film of vitrified aqueous suspension has made it possible to observe biological particles in their native state, in the absence of the usual artefacts of dehydration and staining. Combined with 3-d reconstruction, it has become an important tool for structural molecular biology. Larger objects such as cells and tissues cannot generally be squeezed in a thin enough film. Cryo-electron microscopy of vitreous sections (CEMOVIS) provides then a solution. It requires vitrification of a sizable piece of biological material and cutting it into ultrathin sections, which are observed in the vitrified state. Each of these operations raises serious difficulties that have now been overcome. In general, the native state seen with CEMOVIS is very different from what has been seen before and it is seen in more detail. CEMOVIS will give its full potential when combined with computerized electron tomography for 3-d reconstruction.     

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.     

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.     

Direct visualization of the outer membrane of mycobacteria and corynebacteria in their native state

The cell envelope of mycobacteria, which include the causative agents of tuberculosis and leprosy, is crucial for their success as pathogens. Despite a continued strong emphasis on identifying the multiple chemical components of this envelope, it has proven difficult to combine its components into a comprehensive structural model, primarily because the available ultrastructural data rely on conventional electron microscopy embedding and sectioning, which are known to induce artifacts. The existence of an outer membrane bilayer has long been postulated but has never been directly observed by electron microscopy of ultrathin sections. Here we have used cryo-electron microscopy of vitreous sections (CEMOVIS) to perform a detailed ultrastructural analysis of three species belonging to the Corynebacterineae suborder, namely, Mycobacterium bovis BCG, Mycobacterium smegmatis, and Corynebacterium glutamicum, in their native state. We provide new information that accurately describes the different layers of the mycobacterial cell envelope and challenges current models of the organization of its components. We show a direct visualization of an outer membrane, analogous to that found in gram-negative bacteria, in the three bacterial species examined. Furthermore, we demonstrate that mycolic acids, the hallmark of mycobacteria and related genera, are essential for the formation of this outer membrane. In addition, a granular layer and a low-density zone typifying the periplasmic space of gram-positive bacteria are apparent in CEMOVIS images of mycobacteria and corynebacteria. Based on our observations, a model of the organization of the lipids in the outer membrane is proposed. The architecture we describe should serve as a reference for future studies to relate the structure of the mycobacterial cell envelope to its function.     

Cutting artefacts and cutting process in vitreous sections for cryo-electron microscopy

Cryo-electron microscopy of vitreous sections (CEMOVIS) has recently been shown to provide images of biological specimens with unprecedented quality and resolution. Cutting the sections remains however the major difficulty. Here, we examine the parameters influencing the quality of the sections and analyse the resulting artefacts. They are in particular: knife marks, compression, crevasses, and chatter. We propose a model taking into account the interplay between viscous flow and fracture. We confirm that crevasses are formed on only one side of the section, and define conditions by which they can be avoided. Chatter is an effect of irregular compression due to friction of the section of the knife edge and conditions to prevent this are also explored. In absence of crevasses and chatter, the bulk of the section is compressed approximately homogeneously. Within this approximation, it is possible to correct for compression by a simple linear transformation for the bulk of the section. A research program is proposed to test and refine our understanding of the sectioning process.     

Vitrification of thick samples for soft X-ray cryo-tomography by high pressure freezing

Soft X-ray cryo-microscopy (cryo-XT) offers an ideal complement to electron cryo-microscopy (cryo-EM). Cryo-XT is applicable to samples more than an order of magnitude thicker than cryo-EM, albeit at a more modest resolution of tens of nanometers. Furthermore, the natural contrast obtained in the “water-window” by differential absorption by organic matter vs water yields detailed images of organelles, membranes, protein complexes, and other cellular components. Cryo-XT is thus ideally suited for tomography of eukaryotic cells. The increase in sample thickness places more stringent demands on sample preparation, however. The standard method for cryo-EM, i.e., plunging to a cryogenic fluid such as liquid ethane, is no longer ideally suited to obtain vitrification of thick samples for cryo-XT. High pressure freezing is an alternative approach, most closely associated with freeze-substitution and embedding, or with electron cryo-microscopy of vitreous sections (CEMOVIS). We show here that high pressure freezing can be adapted to soft X-ray tomography of whole vitrified samples, yielding a highly reliable method that avoids crystallization artifacts and potentially offers improved imaging conditions in samples not amenable to plunge-freezing.     

A new approach to improve the quality of ultrathin cryo-sections; its use for immunogold EM and correlative electron cryo-tomography

Cryo-ultramicrotomy can be used to obtain ultrathin cryo-sections from cryo-fixed or aldehyde-fixed cryo-protected vitreous biologic samples. For immuno-gold EM, cryo-sections are retrieved from the cryo-chamber on a droplet of a pick-up solution (paste-like and almost frozen) to which the sections attach. The sections are then placed on an EM specimen grid at room temperature. This procedure compromises the ultrastructure, resulting in folds, holes, and loss of the original material. In this paper we show the critical influence of humidity, stretching, and relief of compression during thawing of the sections. We show a new lift-up hinge device for semi-automated retrieval of cryo-sections that results in significantly improved section quality. This approach was also applied successfully to vitreous sections from high pressure frozen samples. An important advance is that these vitreous cryo-sections can now successfully be post-fixed and immunolabelled after thawing; this allows cryo-EM comparison with adjacent ribbons of sections still in the frozen hydrated state. These findings call for technical innovations aiming at automated cryo-ultramicrotomy in a fully controlled environment for improved localization of proteins within their 'close to native' cellular context and correlative electron cryo-tomography of consecutive ribbons of sections of one frozen hydrated sample.     

Improving the technique of vitreous cryo-sectioning for cryo-electron tomography: Electrostatic charging for section attachment and implementation of an anti-contamination glove box

Cryo-electron tomography of vitreous cryo-sections is the most suitable method for exploring the 3D organization of biological samples that are too large to be imaged in an intact state. Producing good quality vitreous cryo-sections, however, is challenging. Here, we focused on the major obstacles to success: contamination in and around the microtome, and attachment of the ribbon of sections to an electron microscopic grid support film. The conventional method for attaching sections to the grid has involved mechanical force generated by a crude stamping or pressing device, but this disrupts the integrity of vitreous cryo-sections. Furthermore, attachment is poor, and parts of the ribbon of sections are often far from the support film. This results in specimen instability during image acquisition and subsequent difficulty with aligning projection images.Here, we have implemented a protective glove box surrounding the cryo-ultramicrotome that reduces the humidity around and within the microtome during sectioning. We also introduce a novel way to attach vitreous cryo-sections to an EM grid support film using electrostatic charging. The ribbon of vitreous cryo-sections remains in place during transfer and storage and is devoid of stamping related artefacts. We illustrate these improvements by exploring the structure of putative cellular 80S ribosomes within 50 nm, vitreous cryo-sections of Saccharomyces cerevisiae.     

Electron tomography of vitreous sections from cultured mammalian cells

Cryo-electron tomography of appropriately thin, frozen-hydrated biological specimens has excellent potential for investigating the 3D macromolecular architecture of eukaryotic cells and tissues. Since cardiomyocytes are too thick to be visualised in an intact state, we grew immortalised cell line HL-1 to sub-confluency and harvested the cells by enzymatic detachment prior to hyperbaric freezing, ultramicrotomy, and tomography. We improved the efficiency of tomographic acquisition from vitreous cryosections by implementing two new features: (1) fluorescence microscopy at cryogenic temperatures to search for features of interest without expending any of the tolerable electron dose on secondary (non-imaging) tasks, and (2) the use of colloidal gold as fiducial markers. Vital fluorescent staining and subsequent cryo-fluorescence microscopy of vitreous sections were used to localise mitochondria lying in positions suitable for acquiring tilt series, taking into account section flatness, presence of contamination and proximity to grid bars. To provide a simple and robust means of aligning tomograms, we developed a universally applicable protocol for depositing colloidal gold onto vitreous sections, analogous to the method for applying quantum dots described by Masich et al. [Masich, S., Östberg, T., Norlén, L., Shupliakov, O., Daneholt, B., 2006. A procedure to deposit fiducial markers on vitreous cryo-sections for cellular tomography. J. Struct. Biol. 156, 461–468]. Tomograms of thin sections (nominal thickness 65–85 nm) of cardiac mitochondria revealed the interconnectivity of cristae and junctions with the inner mitochondrial membrane. In some cases, ATP synthases could be identified without ambiguity. These findings confirm the feasibility of investigating the structural biology of mammalian cells in three dimensions and at a resolution of 6–8 nm.     

A procedure to deposit fiducial markers on vitreous cryo-sections for cellular tomography

We describe a novel approach for the accurate alignment of images in electron tomography of vitreous cryo-sections. Quantum dots, suspended in organic solvents at cryo-temperatures, are applied directly onto the sections and are subsequently used as fiducial markers to align the tilt series. Data collection can be performed from different regions of the vitreous sections, even when the sections touch the grid only at a few places. We present high-resolution tomograms of some organelles in cryo-sections of human skin cells using this method. The average error in image alignment was about 1nm and the resolution was estimated to be 5-7nm. Thus, the use of section-attached quantum dots as fiducial markers in electron tomography of vitreous cryo-sections facilitates high-resolution in situ 3D imaging of organelles and macromolecular complexes in their native hydrated state.     

Extraction of extendable beaded structures and their identification as fibrillin-containing extracellular matrix microfibrils

High molecular weight aggregates were extracted from human amnion using buffers containing 6 M guanidine hydrochloride. Rotary shadowed preparations and negatively stained samples examined by electron microscopy showed that each aggregate appeared to be a string of globular structures joined by fine filaments, giving the appearance of beads on a string. The periodicity of the beads was variable. A mouse monoclonal antibody directed against a previously characterized pepsin fragment of fibrillin was used with gold-conjugated secondary antibody and immunoelectron microscopy to show that the aggregates contained fibrillin. Similar structures were found in non-denaturing homogenates of skin, tongue, ligament, ciliary zonule, cartilage, and vitreous humor. When immunogold-labeled beaded structures were prepared for electron microscopy in the same manner as tissue, the beaded structures could no longer be seen. Instead, gold-labeled microfibrils were found which appeared to be the same as the fibrillin-containing matrix microfibrils observed in connective tissues and often associated with elastin. Thus, standard TEM protocols including fixation, dehydration, and embedding alter the ultrastructural appearance of microfibrils as compared with negative stain or rotary shadowing techniques. When skin was stretched and prepared for electron microscopy while still under tension, beaded filaments were seen in the tissue sections, but were not visible in non-stretched controls. In addition, when stretched ligament was immunolabeled with antibody directed against fibrillin while still under tension, the periodicity of antibodies along the microfibrils increased compared with non-stretched controls. We propose that microfibrils contain globular structures connected by fine filaments composed at lease in part of highly ordered, periodically distributed fibrillin molecules, whose periodicity is subject to change dependent on the tensional forces applied to the tissue in which they are contained.     

Transmission electron microscopy of the bacterial nucleoid

Water-containing biological material cannot withstand the vacuum of the transmission electron microscope. The classical solution to this problem has been to dehydrate chemically fixed biological samples and then embed them in resin. During such treatment, the bacterial nucleoid is especially prone to aggregation, which affects its global shape and fine structure. Initial attempts to deal with aggregation by optimizing chemical fixation yielded contradictory results. Two decades ago, the situation improved with the introduction of freeze-substitution. This method is based on dehydration of unfixed cryo-immobilized samples at low temperature, which substantially reduces aggregation. As a result, the global shape of the nucleoid can be fairly well defined. Overall, in actively growing bacteria, the nucleoids are dispersed and "coralline" but become more confined when growth ceases. However, it is usually impossible to determine the molecular arrangement of DNA in the nucleoids of freeze-substituted bacteria because crystallization and the subsequent removal of water during substitution result in unavoidable distortions at the ultrastructural level. Recently, cryo-electron microscopy of vitreous sections has enabled the fully hydrated bacterial nucleoid to be studied close to the native state. Such studies have revealed aspects of bacterial nucleoid organization that are not preserved by freeze-substitution, including locally parallel or twisted bundles of DNA filaments, which are more frequently observed once bacterial growth has stopped, whereas in actively growing bacteria, the DNA is seen to be in a mostly disordered pattern.     

Luminal particles within cellular microtubules

The regulation of microtubule dynamics is attributed to microtubule-associated proteins that bind to the microtubule outer surface, but little is known about cellular components that may associate with the internal side of microtubules. We used cryoelectron tomography to investigate in a quantitative manner the three dimensional structure of microtubules in intact mammalian cells. We show that the lumen of microtubules in this native state is filled with discrete, globular particles with a diameter of 7 nm and spacings between 8 and 20 nm in neuronal cells. Cross-sectional views of microtubules confirm the presence of luminal material in vitreous sections of brain tissue. Most of the luminal particles had connections to the microtubule wall, as revealed in tomograms. A higher accumulation of particles was seen near the retracting plus ends of microtubules. The luminal particles were abundant in neurons, but were also observed in other cells, such as astrocytes and stem cells.     

Modifications of the falciform process in the eye of beloniformes (Teleostei: Atherinomorpha): evolution of a curtain-like septum in the eye

In order to comparatively analyze curtain-like septa in the eyes of visually orientated "close-to-surface-predators" among atherinomorph teleosts, we examined the eyes of 24 atherinomorph species under a binocular microscope with regard to the falciform process and related structures in the vitreous cavity. Additionally, falciform process samples were analyzed by transmission electron microscopy. All the studied representatives of the Cyprinodontiformes and Atheriniformes, and of one of the beloniform suborder, Adrianichthyioidei, possess a "typical" processus falciformis. In the eyes of the representatives of the other beloniform suborder, Belonoidei, however, pigmented structures that originate in the region of the optic disc and protrude into the vitreous cavity were noted. In the Hemiramphidae (halfbeaks) and Exocoetidae (flying fishes) these pigmented structures have a more cone-like shape, whereas in the Belonidae (needlefishes) and Scomberesocidae (sauries) horizontally oriented heavily pigmented curtain-like septa occur that divide the vitreous cavity dorsoventrally. It is suggested that the "typical" processus falciformis represents a plesiomorphic feature within the Atherinomorpha, whereas the pigmented modifications of the falciform process must be seen as a synapomorphic character state of the Belonoidei. The curtain-like septum of the Belonidae and Scomberesocidae might have evolved from the cone-like structures that are found in the Exocoetoidea. The functional significance of the pigmented structures in the eye is as yet not clear, except for the curtain-like septum found in Belonidae. It might play a role in visual orientation near the water surface at Snell's window.     

LEBENDNACHWEIS VON EINZELELEMENTEN DES ENDOPLASMATISCHEN RETIKULUMS

By means of a special selective preparation technique, it is possible to investigate in thin sections, by electron microscopy, areas of a cell that have been observed in the living state, by phase-contrast microscopy, up to the time of fixation. Structures recorded in the living state can thus be compared to structures seen in electron micrographs. In cells of the fungus Polystictus versicolor, aggregates of membrane systems as well as single cisternae with a diameter of approximately 200 to 300 A can be detected with phase optics. It can be shown, by calculation, that these structures, which are far below the limit of resolution of the light optical system, give enough contrast to be discernible by phase optics. Thus a basis is provided for observing the dynamics of membrane systems which perhaps may contribute to the analysis of the functional significance of these cell components.     

CEMOVIS on a pathogen: analysis of Bacillus anthracis spores

Background information. Under conditions of starvation, bacteria of Bacillus ssp. are able to form a highly structured cell type, the dormant spore. When the environment presents more favourable conditions, the spore starts to germinate, which will lead to the release of the vegetative form in the life cycle, the bacillus. For Bacillus anthracis, the aetiological agent of anthrax, germination is normally linked to host uptake and represents an important step in the onset of anthrax disease. Morphological studies analysing the organization of the spore and the changes during germination at the electron microscopy level were only previously performed with techniques relying on fixation with aldehydes and osmium, and subsequent dehydration, which can produce artefacts. Results and conclusions. In the present study, we describe the morphology of dormant spores using CEMOVIS (Cryo‐Electron Microscopy of Vitreous Sections). Biosafety measures do not permit freezing of native spores of B. anthracis without chemical fixation. To study the influence of aldehyde fixation on the ultrastructure of the spore, we chose to analyse spores of the closely related non‐pathogen Bacillus cereus T. For none of the investigated structures could we find a difference in morphology induced by aldehyde fixation compared with the native preparations for CEMOVIS. This result legitimizes work with aldehyde‐fixed spores from B. anthracis. Using CEMOVIS, we describe two new structures present in the spore: a rectangular structure, which connects the BclA filaments with the basal layer of the exosporium, and a repetitive structure, which can be found in the terminal layer of the coat. We studied the morphological changes of the spore during germination. After outgrowth of the bacillus, coat and exosporium stay associated, and the layered organization of the coat, as well as the repetitive structure within it, remain unchanged.     

Synaptinemal complexes in the achiasmatic spermatogenesis of Bolbe nigra Giglio-Tos (Mantoidea)

In chiasmatic meiosis of mosquitoes, ascomycetes and lilies the synaptinemal complex (SC) disassociates from the bivalent before metaphase I. Conversely, in the achiasmatic meiosis of Bolbe nigra, the SC remains associated with the bivalent during first metaphase. Light microscopy reveals mid-bodies between disjoining half-bivalents during early first anaphase in Bolbe. Optically controlled serial sections for electron microscopy show that the mid-bodies seen in light micrographs and synaptinemal complexes seen in electron micrographs are the same structure. Electron micrographs indicate that the SC breaks transversely at a point corresponding to the chromosomal kinetochore during anaphase I as the chromatin and the SC begin to separate. During telophase I, SC remnants are at the poles with the chromosomes or between poles. Presently, the evidence is inadequate to state whether the SC serves alternately or simultaneously as a biological contrivance for conjunction and crossing-over or singly as a device for one of these phenomena.Supported by a University of Melbourne Research Fellowship.     

Fine structure of the Deinococcus radiodurans nucleoid revealed by cryoelectron microscopy of vitreous sections

Transmission electron microscopy revealed that the nucleoid of the extremely radioresistant bacteria Deinococcus radiodurans may adopt an unusual ring shape. This led to the hypothesis that the tight toroidal package of the D. radiodurans genome might contribute to radioresistance by preventing diffusion of ends of double-stranded DNA breaks. The molecular arrangement of DNA in the nucleoid, which must be determined to test this hypothesis, is not discernible by conventional methods of electron microscopy. We have applied cryoelectron microscopy of vitreous sections and found that the DNA arrangement in D. radiodurans differs from toroidal spooling. Diffuse coralline nucleoids of exponentially growing D. radiodurans do not reveal any particular molecular order. Electron-dense granules are generally observed in the centers of nucleoids. In stationary-phase cells, the nucleoid segregates from cytoplasm and DNA filaments show locally parallel arrangements, with increasing aspects of cholesteric liquid crystalline phase upon prolonged starvation. The relevance of the observed nucleoid organization to the radiation resistance of D. radiodurans is discussed.     

The future is cold: cryo-preparation methods for transmission electron microscopy of cells

Our knowledge of the organization of the cell is linked, to a great extent, to light and electron microscopy. Choosing either photons or electrons for imaging has many consequences on the image obtained, as well as on the experiment required in order to generate the image. One apparent effect on the experimental side is in the sample preparation, which can be quite elaborate for electron microscopy. In recent years, rapid freezing, cryo-preparation and cryo-electron microscopy have been more widely used because they introduce fewer artefacts during preparation when compared with chemical fixation and room temperature processing. In addition, cryo-electron microscopy allows the visualization of the hydrated specimens. In the present review, we give an introduction to the rapid freezing of biological samples and describe the preparation steps. We focus on bulk samples that are too big to be directly viewed under the electron microscope. Furthermore, we discuss the advantages and limitations of freeze substitution and cryo-electron microscopy of vitreous sections and compare their application to the study of bacteria and mammalian cells and to tomography.