Characterization of the performance of a 200-kV field emission gun for cryo-electron microscopy of biological molecules

The value of an electron microscope equipped with a field emission gun (FEG) was first revealed in materials science applications. More recently, the FEG has played a crucial role in breaking the 10A barrier in single-particle reconstructions of frozen hydrated biological molecules. The standard high-resolution performance tests for electron microscopes are made close to focus, at several hundreds of A underfocus at a magnification of 500,000x or more. While this is appropriate for materials science specimens, it is not suitable for observing frozen hydrated biological specimens with which the optimum underfocus is of the order of 1 micron or so and the magnification is limited by radiation damage to roughly 30,000 to 60,000x. Thus, in order to access the performance of a cryo-electron microscope for high-resolution 3D electron microscopy of biological molecules, additional tests are necessary. We present here resolution tests of a 200-kV FEG using frozen hydrated virus suspensions. The extent and amplitude of the contrast transfer function are used as a test of the performance. We propose that small spherical viruses close to 300A in diameter, such as the picornaviruses or phages, make good specimens for testing the performance of an electron microscope in cryo-mode.     

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.     

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.     

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology ¹. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes ^{2, 3}. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice ⁴, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast ⁵. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes ⁶, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation.In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users.     

Structure determination of macromolecular assemblies by single-particle analysis of cryo-electron micrographs

A new generation of electron microscopes equipped with field emission gun electron sources and the ability to image molecules in their native environment at liquid nitrogen or helium temperatures has enabled the analysis of macromolecular structures at medium resolution (approximately 10 angstroms) and in different conformational states. The amalgamation of electron microscopy and X-ray crystallographic approaches makes it possible to solve structures in the 100-1000 angstroms size range, advancing our understanding of the function of complex assemblies. Many new structures have been solved during the past two years, including one of the smallest complexes to be determined by single-particle cryo-electron microscopy, the transferrin receptor-transferrin complex. Other notable results include the near atomic level resolution structure of the nicotinic acetylcholine receptor in helical arrays and an icosahedral virus structure with an asymmetric polymerase resolved.     

Toward visualization of nanomachines in their native cellular environment

The cellular nanocosm is made up of numerous types of macromolecular complexes or biological nanomachines. These form functional modules that are organized into complex subcellular networks. Information on the ultra-structure of these nanomachines has mainly been obtained by analyzing isolated structures, using imaging techniques such as X-ray crystallography, NMR, or single particle electron microscopy (EM). Yet there is a strong need to image biological complexes in a native state and within a cellular environment, in order to gain a better understanding of their functions. Emerging methods in EM are now making this goal reachable. Cryo-electron tomography bypasses the need for conventional fixatives, dehydration and stains, so that a close-to-native environment is retained. As this technique is approaching macromolecular resolution, it is possible to create maps of individual macromolecular complexes. X-ray and NMR data can be ‘docked’ or fitted into the lower resolution particle density maps to create a macromolecular atlas of the cell under normal and pathological conditions. The majority of cells, however, are too thick to be imaged in an intact state and therefore methods such as ‘high pressure freezing’ with ‘freeze-substitution followed by room temperature plastic sectioning’ or ‘cryo-sectioning of unperturbed vitreous fully hydrated samples’ have been introduced for electron tomography. Here, we review methodological considerations for visualizing nanomachines in a close-to-physiological, cellular context. EM is in a renaissance, and further innovations and training in this field should be fully supported. Robert Feulgen Lecture 2009 presented at the 51st symposium of the Society for Histochemistry in Stubai, Austria, October 7–10, 2009.     

Imaging vesicular dispersions with cold-stage electron microscopy

A fast-freeze, cold-stage transmission electron microscopy technique which can incorporate in situ freeze-drying of the sample is described. Its use in elucidating structure in unstained and stained, hydrated and freeze-dried, aqueous vesicular dispersions of biological and chemical interest is demonstrated with vesicles of l-α-phosphatidylcholine (bovine phosphatidylcholine) and of the synthetic surfactant sodium 4-(1′-heptylnonyl)benzenesulfonate (SHBS). The contrast features observed in transmission electron microscope images of frozen, hydrated samples are identified and explained with the dynamical theory of electron diffraction. Radiolysis by the electron beam is shown to increase contrast in vesicle images and to change their structure and size. Micrographs illustrate the freeze-drying of a dispersion in the microscope; the process causes vesicles to shrink and collapse.     

Specific, sensitive, high-resolution detection of protein molecules in eukaryotic cells using metal-tagging transmission electron microscopy

More than any other methodology, transmission electron microscopy (TEM) has contributed to our understanding of the architecture and organization of cells. With current detection limits approaching atomic resolution, it will ultimately become possible to ultrastructurally image intracellular macromolecular assemblies in situ. Presently, however, methods to unambiguously identify proteins within the crowded environment of the cell's interior are lagging behind. We describe an approach, metal-tagging TEM (METTEM), that allows detection of intracellular proteins in mammalian cells with high specificity, exceptional sensitivity, and at molecular scale resolution. In live cells treated with gold salts, proteins bearing a small metal-binding tag will form 1-nm gold nanoclusters, readily detectable in electron micrographs. The applicability and strength of METTEM is demonstrated by a study of Rubella virus replicase and capsid proteins, which revealed virus-induced cell structures not seen before.     

Perspectives of Molecular and Cellular Electron Tomography

After a general introduction to three-dimensional electron microscopy and particularly to electron tomography (ET), the perspectives of applying ET to native (frozen–hydrated) cellular structures are discussed. In ET, a set of 2-D images of an object is recorded at different viewing directions and is then used for calculating a 3-D image. ET at a resolution of 2–5 nm would allow the 3-D organization of structural cellular components to be studied and would provide important information about spatial relationships and interactions. The question of whether it is a realistic long-term goal to visualize or—by sophisticated pattern recognition methods— identify macromolecules in cells frozenin totoor in frozen sections of cells is addressed. Because of the radiation sensitivity of biological specimens, a prerequisite of application of ET is the automation of the imaging process. Technical aspects of automated ET as realized in Martinsried and experiences are preresented, and limitations of the technique are identified, both theoretically and experimentally. Possible improvements of instrumentation to overcome at least part of the limitations are discussed in some detail. Those means include increasing the accelerating voltage into the intermediate voltage range (300 to 500 kV), energy filtering, the use of a field emission gun, and a liquid-helium-cooled specimen stage. Two additional sections deal with ET of isolated macromolecules and of macromolecular structuresin situ,and one section is devoted to possible methods for the detection of structures in volume data.     

Difficult macromolecular structures determined using X-ray diffraction techniques

Macromolecular crystallography has been, for the last few decades, the main source of structural information of biological macromolecular systems and it is one of the most powerful techniques for the analysis of enzyme mechanisms and macromolecular interactions at the atomic level. In addition, it is also an extremely powerful tool for drug design. Recent technological and methodological developments in macromolecular X-ray crystallography have allowed solving structures that until recently were considered difficult or even impossible, such as structures at atomic or subatomic resolution or large macromolecular complexes and assemblies at low resolution. These developments have also helped to solve the 3D-structure of macromolecules from twin crystals. Recently, this technique complemented with cryo-electron microscopy and neutron crystallography has provided the structure of large macromolecular machines with great precision allowing understanding of the mechanisms of their function.     

The need for a shared database infrastructure: combining X-ray crystallography and electron microscopy

Advances in structural biology are opening greater opportunities for understanding biological structures from the cellular to the atomic level. Particularly promising are the links that can be established between the information provided by electron microscopy and the atomic structures derived from X-ray crystallography and nuclear magnetic resonance spectroscopy. Combining such different kinds of structural data can result in novel biological information on the interaction of biomolecules in large supramolecular assemblies. As a consequence, the need to develop new databases in the field of structural biology that allow for an integrated access to data from all the experimental techniques is becoming critical. Pilot studies performed in recent years have already established a solid background as far as the basic information that an integrated macromolecular structure database should contain, as well as the basic principles for integration. These efforts started in the context of the BioImage project, and resulted in a first complete database prototype that provided a versatile platform for the linking of atomic models or X-ray diffraction data with electron microscopy information. Analysis of the requirements needed to combine data at different levels of resolution have resulted in sets of specifications that make possible the integration of all these different types in the context of a web environment. The case of a structural study linking electron microscopy and X-ray data, which is already contained within the BioImage data base and in the Protein Data Bank, is used here to illustrate the current approach, while a general discussion highlights the urgent need for integrated databases. Received: 26 January 2000 / Revised version: 15 May 2000 / Accepted: 15 May 2000     

3D Ultrastructural Organization of Whole Chlamydomonas reinhardtii Cells Studied by Nanoscale Soft X-Ray Tomography

The complex architecture of their structural elements and compartments is a hallmark of eukaryotic cells. The creation of high resolution models of whole cells has been limited by the relatively low resolution of conventional light microscopes and the requirement for ultrathin sections in transmission electron microscopy. We used soft x-ray tomography to study the 3D ultrastructural organization of whole cells of the unicellular green alga Chlamydomonas reinhardtii at unprecedented spatial resolution. Intact frozen hydrated cells were imaged using the natural x-ray absorption contrast of the sample without any staining. We applied different fiducial-based and fiducial-less alignment procedures for the 3D reconstructions. The reconstructed 3D volumes of the cells show features down to 30 nm in size. The whole cell tomograms reveal ultrastructural details such as nuclear envelope membranes, thylakoids, basal apparatus, and flagellar microtubule doublets. In addition, the x-ray tomograms provide quantitative data from the cell architecture. Therefore, nanoscale soft x-ray tomography is a new valuable tool for numerous qualitative and quantitative applications in plant cell biology.     

Realizing the potential of electron cryo-microscopy

Structural analysis by electron microscopy of biological macromolecules or macromolecular assemblies embedded in rapidly frozen, vitreous ice has made great advances during the last few years. Electron cryo-microscopy, or cryo-EM, can now be used to analyse the structures of molecules arranged in the form of two-dimensional crystals, helical arrays or as single particles with or without symmetry. Although it has been possible, using crystalline or helical specimens, to reach a resolution adequate to build atomic models (4 A), there is every hope this will soon also be possible with single particles. Small and large single particles present different obstacles to progress.     

The Leeuwenhoek lecture 2006. Microscopy goes cold: frozen viruses reveal their structural secrets

The electron microscope provides a powerful tool for investigating the structure of biological complexes such as viruses. A modern instrument is fully capable of atomic resolution on suitable non-biological specimens, but biological materials are difficult to preserve, owing to their fragility, and to image, owing to their radiation, sensitivity. The act of imaging the specimen severely damages it. Originally, samples were prepared by staining with a heavy metal salt, which provides a stable specimen but limits the amount of details that can be retrieved. Now particulate specimens, such as viruses, are prepared by rapid freezing of unstained material and observed in a frozen state with low doses of electrons. The resulting images require extensive computer processing to extract fully detailed three-dimensional information about the specimen. The whole process is referred to as single-particle electron cryomicroscopy. Using this approach, the structure of the human hepatitis B virus core was solved at the level of the protein fold. By comparing maps of RNA- and DNA-containing cores, it was possible to propose a model for the maturation and control of the envelopment of the virus during assembly. These examples show that cryomicroscopy offers great potential for understanding the structure and function of complex biological assemblies.     

The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications

The emergence of electron tomography as a tool for three dimensional structure determination of cells and tissues has brought its own challenges for the preparation of thick sections. High pressure freezing in combination with freeze substitution provides the best method for obtaining the largest volume of well-preserved tissue. However, for deeply embedded, heterogeneous, labile tissues needing careful dissection, such as brain, the damage due to anoxia and excision before cryofixation is significant. We previously demonstrated that chemical fixation prior to high pressure freezing preserves fragile tissues and produces superior tomographic reconstructions compared to equivalent tissue preserved by chemical fixation alone. Here, we provide further characterization of the technique, comparing the ultrastructure of Flock House Virus infected DL1 insect cells that were (1) high pressure frozen without fixation, (2) high pressure frozen following fixation, and (3) conventionally prepared with aldehyde fixatives. Aldehyde fixation prior to freezing produces ultrastructural preservation superior to that obtained through chemical fixation alone that is close to that obtained when cells are fast frozen without fixation. We demonstrate using a variety of nervous system tissues, including neurons that were injected with a fluorescent dye and then photooxidized, that this technique provides excellent preservation compared to chemical fixation alone and can be extended to selectively stained material where cryofixation is impractical.     

On the preparation of cryosections for immunocytochemistry

The key preparation steps in the Tokuyasu thawed frozen section technique for immunocytochemistry, namely freezing, sectioning, thawing, and drying, were studied. A spherical tissue culture cell was used as a model system. The frozen hydrated section technique indicated that glutaraldehyde-fixed, 2.1 M sucrose-infused pellets of cells were routinely vitrified by immersion in liquid nitrogen but water was crystallized when lower sucrose concentrations (0.6-1 M) were used. Quantitative mass measurements showed that the fixed cells are freely permeable to sucrose. The frozen hydrated sections were severely compressed but cell profiles regained their circular appearance upon thawing. The average section thickness of our frozen-hydrated sections was 110 nm: this was reduced to 30-50 nm upon thawing, washing, and air-drying. This change was accompanied by severe drying artifacts. By using the methyl cellulose drying technique, this collapse upon air-drying could be significantly reduced, but not completely prevented, giving an average thickness of 70 nm.     

High-resolution electron cryomicroscopy of macromolecular assemblies

Electron cryomicroscopy is a high-resolution imaging technique that is particularly appropriate for the structural determination of large macromolecular assemblies, which are difficult to study by X-ray crystallography or NMR spectroscopy. For some biological molecules that form two-dimensional crystals, the application of electron cryomicroscopy and image reconstruction can help elucidate structures at atomic resolution. In instances where crystals cannot be formed, atomic-resolution information can be obtained by combining high-resolution structures of individual components determined by X-ray crystallography or NMR with image-derived reconstructions at moderate resolution. This can provide unique and crucial information on the mechanisms of these complexes. Finally, image reconstructions can be used to augment X-ray studies by providing initial models that facilitate phasing of crystals of large macromolecular machines such as ribosomes and viruses.     

Removing Contamination-Induced Reconstruction Artifacts from Cryo-electron Tomograms

Imaging of fully hydrated, vitrified biological samples by electron tomography yields structural information about cellular protein complexes in situ. Here we present a computational procedure that removes artifacts of three-dimensional reconstruction caused by contamination present in samples during imaging by electron microscopy. Applying the procedure to phantom data and electron tomograms of cellular samples significantly improved the resolution and the interpretability of tomograms. Artifacts caused by surface contamination associated with thinning by focused ion beam, as well as those arising from gold fiducial markers and from common, lower contrast contamination, could be removed. Our procedure is widely applicable and is especially suited for applications that strive to reach a higher resolution and involve the use of recently developed, state-of-the-art instrumentation.     

Structural biology of cellular machines

Multi-component macromolecular machines contribute to all essential biological processes, from cell motility and signal transduction to information storage and processing. Structural analysis of assemblies at atomic resolution is emerging as the field of structural cell biology. Several recent studies, including those focused on the ribosome, the acrosomal bundle and bacterial flagella, have demonstrated the ability of a hybrid approach that combines imaging, crystallography and computational tools to generate testable atomic models of fundamental biological machines. A complete understanding of cellular and systems biology will require the detailed structural understanding of hundreds of biological machines. The realization of this goal demands a concerted effort to develop and apply new strategies for the systematic identification, isolation, structural characterization and mechanistic analysis of multi-component assemblies at all resolution ranges. The establishment of a database describing the structural and dynamic properties of protein assemblies will provide novel opportunities to define the molecular and atomic mechanisms controlling overall cell physiology.