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There was a problem filtering reviews right now. Please try again later. Very well written, complete, very helpfull. It describes EM in different organism and give some tips. Worth the money and the investment. Amazon Giveaway allows you to run promotional giveaways in order to create buzz, reward your audience, and attract new followers and customers. Learn more about Amazon Giveaway. Set up a giveaway. There's a problem loading this menu right now. Learn more about Amazon Prime. Get fast, free shipping with Amazon Prime.
Get to Know Us. English Choose a language for shopping. Amazon Music Stream millions of songs. Amazon Advertising Find, attract, and engage customers. Amazon Drive Cloud storage from Amazon. Alexa Actionable Analytics for the Web. AmazonGlobal Ship Orders Internationally. Amazon Inspire Digital Educational Resources. A simple and quick approach is to use LR White resin, which has a low viscosity to speed up diffusion and which can be quickly polymerized using a chemical accelerator Hobot and Newman, Together with a small sample size, overall processing time can be reduced to 1—2 h Laue et al.
The embedding procedure was originally developed for rapid diagnosis of bacterial endospores using thin-section transmission electron microscopy. A Conventional Epon embedding reveals virus particles arrowheads in the endoplasmic reticulum er and different vesicles budding at the reticulum membrane arrows. B Rapid embedding in LR White using 2.
The structural appearance is sufficient to detect all relevant ultrastructural aspects of the flavivirus genesis that are also visible in sections of a conventional Epon embedding compare with A. The structural appearance is sufficient for most questions compare Fig. In addition, on-section immuno-cytochemistry is possible, but most probably restricted to antigens present at a high concentration Laue et al. The following protocol summarizes essential steps of the embedding method: EM of Viruses 13 1.
All manipulations using LR White should be performed in a fume hood. Please consult material data safety sheet or local safety officer in case of any questions regarding lab safety. Fixed samples strong aldehyde fixation is recommended, e. Sample thickness should be not more than 1 mm 0. For preparing suspensions in an agarose gel at a defined thickness, see Laue et al. Washing out of primary fixative with buffer twice for 1 min each. On-section contrasting may not be necessary with these samples.
Dehydration, infiltration, embedding, and polymerization are done on ice. All solutions are used precooled at ice temperature. Incubation in a mixture of ethanol and LR White 1: Mixing of LR White with the accelerator is done in portions of 1 ml using a magnetic stirrer. Precooled LR White is filled in a cylindrical glass vial containing a stirring bar. Finally, the mixture is filled in precooled reaction tubes 0. The mixing of the accelerator with the resin monomer must be done very quickly otherwise polymerization starts only locally.
Transfer of samples into the reaction tubes containing resin mixtures.
Any airtight vial of a volume equal or smaller than 0. Vials with thinner walls than reaction tubes may be removed easily from the polymerized blocks than from the reaction tubes. Polymerization on ice for 15 min. Polymerization in a small volume and on ice or even better, ice water is necessary because the reaction generates heat in a very short time which must be extracted from the resin otherwise bubbles will appear in the polymerized block around the sample rendering ultrathin sectioning difficult.
Residual liquid LR White may be soaked with a tissue before final polymerization is conducted. Sections may be observed without poststaining or after quick on-section staining according to Roth et al. The protocol can be easily tailored to the needs of the sample e. Laboratory facility according to the biosafety class of the virus of interest. No specific materials are needed if the biosafety laboratory is equipped with standard tools pipette, table desk centrifuges, reaction and centrifuge tubes, waste container.
For mixtures of paraformaldehyde with glutaraldehyde e. High-vacuum carbon evaporator e. Glow discharge device e. Grids with a plastic film e. Double-distilled water or equivalent quality is needed for the preparation of all solutions and washing steps. Alcian blue Sigma-Aldrich, No. Solution must be centrifuged to remove crystals. Cleared solution can be stored in the refrigerator for at least 6 months Laue and Bannert, PTA should be adjusted to pH 8.
The alkaline PTA breaks the biomembranes of enveloped viruses thereby revealing internal structures. Same as in Section IV. In addition, secondary antibodies, coupled to colloidal gold e. Bovine serum albumin BSA; e. A, low protease activity as a 0. Glycine, 50 mM in PBS. EM of Viruses D. Ultrathin Section Electron Microscopy Instrumentation: Table desk centrifuge swing-out rotor facilitates collection of cell pellets in low melting point agarose.
Embedding molds different sizes and designs are available; e. D Materials in addition to materials listed in Section IV. Reagents in addition to materials listed in Section IV. In most cases, relevant results can be achieved by using these techniques. Studies may be extended by using more elaborated methods to gain a further quality of results. One of these methods is on-section immunolabeling which allows the combination of structural data provided by ultrathin sections with molecular topology. Starting from chemically fixed samples the embedding procedure must be changed in comparison to the routine protocol.
A resin with a low tendency to co-polymerize with the sample structure, like for instance the LR or Lowicryl resins, should be used together with dehydration and embedding at low temperature Schwarz and Humbel, A useful variant of ultrathin sectioning is the so-called Tokuyasu technique, where the chemically fixed sample is embedded in sucrose, frozen, and sectioned at low temperature compare e. However, the procedure 16 Michael Laue needs particular equipment and experience. Preembedding immunolabeling is another variant, which may be easily combined with standard embedding procedures, if the antigen is accessible for the antibodies e.
An excellent comprehensive overview on most aspects of immunocytochemistry is given in a book by Griffiths and in a review by Skepper A more realistic representation of virus ultrastructure is definitely achieved by using cryopreparation methods. The first step involves the so-called vitrification of samples by cryofixation, which simply means freezing without the formation of ice crystals. Details of the various cryopreparation methods are given in a book by Steinbrecht and Zierold and in a review by Quintana In virology, basically two strategies are used: The bare-grid method is basically used for studying virus structure in suspension e.
The technique needs highly concentrated and purified virus suspensions and particular equipment, such as a plunge-freezer, a cryo-transfer system, and a cryo-electron microscope. Cryo-microscopy can be carried out directly at the thin periphery of the cells Schwartz et al. Because of these reasons, high-pressure freezing is frequently combined with freeze-substitution to replace ice at low temperature Schwarz et al. Freeze-substitution usually ends up in plastic embedding and ultrathin sectioning. Conventional bright-field transmission-electron microscopy reveals a twodimensional projection of all structures illuminated parallel to the electron beam.
Thus, three-dimensional structure is not accessible by this imaging approach. EM of Viruses 17 the specimen reveals three-dimensional information of the structures. Even two images taken at two different illumination angles usually by tilting the sample may be sufficient to generate stereo images for analyzing structures included in the threedimensional volume Heuser, An almost complete reconstruction of the threedimensional structure is achieved by electron tomography Baumeister et al.
A series of two-dimensional images taken with a small increment of the tilt angle is used as a basis for an in silico backprojection of the images into a three-dimensional volume. Image processing, like segmentation and color-coding of individual structures, allows the generation of models providing information on virus structure. Detailed information on electron tomography is available in a comprehensive book by Frank a.
The paper by Geerts et al. An interesting approach to use electron tomography for diagnostic purposes was recently presented by Mast and Demeestre Another method to study virus structure in three dimensions is the so-called single particle analysis. It can be performed with images acquired by negative-staining electron microscopy or by the bare-grid method using cryo-electron microscopy.
The principle behind the method is to record images from different randomly orientated particles and to combine those views in a three-dimensional reconstruction. The method may achieve high resolution, but needs optimal conditions, like highest purity of the original sample and some knowledge about the particles to be studied.
An overview on single-particle analysis is given by Frank b. A particular focus on studying virus morphology by using single-particle analysis and X-ray diffraction may be found in Baker and Johnson Protocols for the preparation and imaging of protein aggregates including viruses are provided by Grassucci et al. For a long time, transmission electron microscopy was the only method to reveal virus ultrastructure at high resolution. Today, other methods such as scanning transmission electron microscopy, scanning force microscopy, and highresolution light microscopy provide information at a high spatial resolution.
Scanning electron microscopy, especially if field-emission systems are used, can give a rapid overview about the events taking place at the surface of cells e. With a resolution of about 1 nm or better, even small viruses can be visualized at a suitable quality Ng et al. The combination of electron microscopy and light microscopy is helpful in many research fields. In virology, especially the dynamics of virus infection, replication and egress can be studied by high-resolution life-cell imaging e. Life-cell imaging of dynamic events may be correlatively combined with an end-point study of the subcellular structure and molecular arrangement by employing high-resolution electron microscopy Brown et al.
Moreover, I thank the numerous colleagues at the Robert Koch Institute for providing interesting samples of which some are depicted in this chapter. Developments of new lowicryl resins for embedding biological specimens at even lower temperatures. Cryo-electron microscopy of viruses. Principles of virus structure determination. Oxford Press, NY, Oxford. Ultracentrifugation of serum samples allows detection of hepatitis C virus RNA in patients with occult hepatitis C.
Electron tomography of molecules and cells. Diagnostic electron microscopy is still a timely and rewarding method. Electron microscopy of viruses. Oxford University Press, Oxford. Involvement of actin filaments in budding of measles virus: Studies on cytoskeletons of infected cells. Jones and Bartlett Publishers, Boston. Virus trafficing—learning from single-virus tracking. A negative staining method for high resolution electron microscopy of viruses.
Studying intracellular transport using high-pressure freezing and correlative light and electron microscopy. Resin development for electron microscopy and an analysis of embedding at low temperature. Oxford 1 26 , — Does the embedding chemistry interact with tissue? Application of transmission electron microscopy to the clinical study of viral and bacterial infections: Two-hour embedding procedure for intracellular detection of viruses by electron microscopy.
EM of Viruses 19 Geerts, W. Virus enrichment using the airfuge for rapid diagnostic EM in infectious diseases.
Arguments pro disinfection in diagnostic electron microscopy: A response to Madeley and Biel. Visualization of macromolecular complexes using cryo-electron microscopy with FEI tecnai transmission electron microscope. Structure of complex viruses and virus-infected cells by electron cryo tomography.
Read the latest chapters of Methods in Cell Biology at www.farmersmarketmusic.com, Elsevier's leading platform of peer-reviewed scholarly literature. Methods in Cell Biology; Electron Microscopy of Model Systems. Electron Microscopy of Model Systems - 1st Edition - ISBN:
Ultramicrotomy for biological electron microscopy. Negative staining and cryoelectron microscopy: My copy is ISBN 1 7]. Negative staining of thinly spread biological samples. Membrane traffic in anaglyph stereo. Electron microscopy for bacterial cells.
Viruses accumulate spontaneously near droplet surfaces: A method to concentrate viruses for electron microscopy. Learning about truth and biases through experience—section surface corrugation, protein denaturation, and staining. Budding of marburgvirus is associated with filopodia. Low-dose automated tomography of frozenhydrated specimens. Visualization of retrovirus budding with correlated light and electron microscopy. Detection limit of negative staining electron microscopy for the diagnosis of bioterrorism-related microorganisms.
Rapid diagnostic thin section electron microscopy of bacterial endospores. Microwave procedures for electron microscopy and resinembedded sections. Electron microscopy of frozen biological suspensions. Electron tomography of negatively stained complex viruses: Application in their diagnosis. Virus particle counting by electron microscopy. Detection and identification of viruses by electron microscopy.
Bioterrorism and electron microscopy differentiation of poxviruses from herpesviruses: Theory and practice of high pressure freezing. Topographic changes in SARS coronavirus-infected cells during late stages of infection. Cryofixation, cryosubstitution, cryoembedding for ultrastructural, immunocytochemical and microanalytical studies.
Contrasting of lowicryl K4M thin sections. Biocidal activities of glutaraldehyde and related compounds. Microwave-assisted tissue processing for same-day EM-diagnosis of potential bioterrorism and clinical samples. Freeze- substitution in virus research: Influence of fixatives and embedding media on immunolabelling of freeze-substituted cells. Immunocytochemical strategies for electron microscopy: Production of novel ebola virus-like particles from cDNAs: An alternative generation of ebola virus generation by reverse genetics.
Cryosectioning fixed and cryoprotected biological material form immunocytochemistry. Extraction of membrane lipids during fixation, dehydration and embedding of acholeplasma laidlawii-cells for electron microscopy. Zernike phase contrast electron microscopy of ice-embedded influenza A virus. Rapid diagnosis of plant virus diseases by transmission electron microscopy.
Plunge Freezing Thin Films B. Limitations of Cryo-EM E. Contributions of Cryo-EM Acknowledgments References Abstract Some bacteria are amongst the most important model organisms for biology and medicine. Methods that involve dehydration and metal stains are widely practiced and have revealed many ultrastructural features, but they can generate misleading artifacts and have failed to preserve important structures such as the bacterial cytoskeleton.
The invention of cryo-electron microscopy, which allows bacterial cells to be imaged in a frozen-hydrated, near-native state without the need for dehydration and stains, has now led to important new insights. Efforts to identify structures and localize specific proteins in cryo-EM images are summarized.
Introduction Bacteria are among the most widely studied biological model systems for many reasons: They are also easy to culture, easy to manipulate genetically, and have quick life cycles. As a result, model bacteria are the focus of many current genomic, transcriptomic, proteomic, and metabolomic projects. A key piece of missing information is, however, how all the molecules within bacterial cells are arranged and how their arrangement supports function.
Early light microscopy LM revealed the amazing diversity of bacterial cell shapes, interesting developmental processes such as sporulation and binary fission, and the variety of reactions bacteria have with different stains. The invention of the electron microscope EM opened the possibility of imaging cells at much higher resolution, but methods had to be developed first to preserve cells within the high vacuum of the microscope column.
These methods have contributed considerably to our knowledge of bacterial ultrastructure, revealing membrane layers, stalks, flagella, pili, fimbriae, phages, and other similar features. Fluorescent light microscopy fLM then basically changed our view of the bacterial cell, showing that a multitude of proteins and also certain genetic loci are spatially and temporally localized Lewis, ; Margolin, ; Thanbichler et al. This was done either by immunofluorescence using fluorescently labeled antibodies or, more 23 2. New Insights from Cryo-Microscopy recently, by genetically fusing the green-fluorescent protein GFP or its derivatives to target proteins.
CryoEM techniques allow cells to be observed in a fully hydrated state, without the need for chemical fixatives or contrast-enhancing metal stains. This approach cryo-electron tomography, or CET has revealed a wealth of new details, including an extensive and complex bacterial cytoskeleton and the architecture of various large macromolecular complexes Li and Jensen, ; Milne and Subramaniam, ; Tocheva et al. Identifying structures and localizing specific proteins remains a challenge, as does imaging thick cells and further increasing resolution.
In this review, we present a technical overview of the different EM methods that are used to image bacteria and discuss their advantages and disadvantages Fig. Mesosomes are convoluted cytoplasmic membranous structures that were seen by methods such as freeze-fracture A and traditional thin-section EM C. A Freeze-etch image of a Bacillus subtilis cell. Cell content cc ; outer surface of plasma membrane opm ; cross-fractured cell wall cw ; mesosome M composed of numerous vesicles.
Cells were fixed with osmium tetroxide before cryo-fixation, producing mesosomes M. C Conventional EM preparation of S. A membranous mesosome is present in the cytoplasm. D Cryo-section through S. No mesosomes are seen. A adapted from Nanninga B—D adapted from Dubochet et al. To illustrate the advantages and limitations of each technique, three specific example ultrastructures will be considered throughout: Methods involving dehydration and metal stains will be described first, followed by cryo-EM methods.
Glow-discharged EM grids are simply floated on a drop of bacterial culture for a few seconds, partially drained, moved onto a drop of staining solution e. New Insights from Cryo-Microscopy 25 B. In summary, this involves chemical fixation, dehydration into transitional solvents, infiltration with resin, embedding and curing of resin, sectioning, and finally heavy metal staining see also Bozzola and Russell and Fig.
The goal of fixation is to immobilize the sample in as native state as possible. The aldehydes cross-link proteins and, to a lesser degree, lipids, carbohydrates and nucleic acids. The result is that the constituents of the cell are linked into a continuous mesh. Secondary fixation with osmium tetroxide oxidizes unsaturated bonds of fatty acids and stabilizes other cell components. In addition, the reduced osmium molecules add density and contrast. Next, the sample is dehydrated by immersion in an organic solvent such as ethanol, acetone, or both, usually in a series of graded steps.
The advantages of this traditional thin-section EM technique are that it employs well-known and commonly practiced methods and it produces a high-contrast, radiation-resistant sample that can be imaged in standard EMs at room temperature, either in 2-D by simple projection or in 3-D through tomography. It was by thin-section EM that the fundamental differences in the cell wall structures of gram-positive and gram-negative organisms were first visualized Bayer, ; Chapman and Hillier, The major disadvantage is, however, that the chemical cross-linking, dehydration, and staining seriously perturb the fine structure of the cells.
These were assumed to be authentic bacterial features with a variety of functions Greenawalt and Whiteside, More advanced preservation methods have since shown that mesosomes are simply artifacts of chemical fixation and dehydration Dubochet et al. New Insights from Cryo-Microscopy 27 of low electron density are seen which in some regions contain aggregated genomic material arrows in Fig. Variations of the chemical fixation procedures were explored to reduce the aggregation of chromatin.
The socalled Ryter-Kellenberger procedure was based on fixation with osmium tetroxide under special conditions, whereas another method used glutaraldehyde fixation and post-fixation with uranyl acetate. Fibrillar structures were detected using both methods Fig. This led to the hypothesis that the results are biased by the fixation and dehydration protocol and that chromatin aggregation is an artifact Eltsov and Zuber, Cryo-Fixation One of the problems of room-temperature chemical fixation is that the fixative cannot reach every molecule simultaneously and cross-link it in its natural position.
Instead, certain structures become cross-linked while others continue diffusing, which can lead to artifactual meshworks and aggregations. Cryo-fixation methods methods that stop molecular motion through cooling can act more instantaneously. If samples are cooled gradually at ambient pressures, however, water will crystallize and denature dissolved macromolecules.
One way to overcome this is to cool the sample so quickly that the water molecules stop moving before they have time to form the hydrogen bond network of crystalline ice. In fact, it has been shown that vitrified cells are preserved so well that many continue living if later thawed Erk et al. The easiest way to increase cooling speed is to minimize the size of the specimen. This of course makes the method especially favorable to bacterial samples. Indeed, the most straight-forward way to vitrify bacteria is to spread them as a thin layer on an EM grid and Fig.
In contrast to mesosomes, cytoskeletal filaments are seen by cryo-EM but not plastic-embedding methods. This is why it was thought for decades that bacterial cells lacked cytoskeletons. To illustrate, images of E. Immunofluorescence light microscopy shows that in E. Almost no internal ultrastructure is visible by negative staining B. A Immunofluorescence of btubA-btubB expressing E. Adapted from Sontag et al. B Negative Staining with uranyl acetate. C Traditional thin-section EM. E Cryo-EM 2-D image of vitreous section. F XY-slice 10 nm through whole cell cryo-electron tomogram.
While each of the established EM methods contributes some information about the organization of chromatin in bacteria, none have revealed it clearly, highlighting our continued need for additional methodological advances. Only glutaraldehyde-prefixed cells show a nucleoid structure using freeze-fracture D. Freeze-substitution reveals a coralline-shaped ribosome-free area with grains and fibers E. Cryo-EM of frozen section shows a dispersed, coralline-shaped ribosome-free area in growing cells G and a more confined ribosome-free area in stationary phase cells H.
A characteristic fine-granularity of DNA structure is seen in the confined nucleoid of stationary phase cells, but it is difficult to interpret F. A Osmium tetroxide fixation of Bacillus megaterium. B Osmium tetroxide fixation of E. D Freezefracture of glutaraldehyde-fixed Streptococcus feacalis. E Freeze-substitution and low-temperature embedding of E. F Nucleoid fine-structure in cryo-section of stationary phase Deinococcus radiodurans.
Dotted and stripy patterns are spaced by regions with indefinable structure asterisks. G Cryo-section of exponentially growing D. Dispersed corraline ribosome-free area RFA; outlined in one cell of the tetrad. H Cryo-section of stationary phase D. Confined, roundish ribosome-free area RFA; outlined in one cell of the tetrad. A adapted from Eltsov and Zuber ; Giesbrecht and Piekarski D adapted from Edelstein et al. F—H adapted from Eltsov and Dubochet New Insights from Cryo-Microscopy 29 plunge freeze them into a cryogen De Carlo, , as will be described in more detail below.
Bacteria can be embedded into agarose before slamming Shimizu and Miyata, In highpressure freezing HPF , the specimen is first enclosed within a small metal aluminum or brass carrier. Because growth media are relatively dilute compared with cell cytoplasm, they have a tendency to crystallize even when the contents of the cell are vitrified. The cell pellet is therefore either resuspended in medium plus a cryo-protectant an anti-freeze agent such as dextran, bovine serum albumin BSA , 1-hexadecene, or other high molecular weight branched sugars such as Ficoll or Mannitol , pelleted again, and transferred to the specimen carrier; or the pellet is added to a sealed pipette-tip, spun through a media-cryo-protectant solution, and transferred into the metal specimen carrier.
Care is taken to avoid drying by maintaining humidity while the specimen carrier is assembled and frozen in the HPF machine. The freezing protocol i. Artifacts can arise from the pelleting of the cells, mixing them with the cryo-protectant, or the impact of high pressure. Besides the major cryo-fixation methods plunge freezing, HPF, and slam freezing, a variety of other freezing protocols have been tried through the years with mixed success. These procedures will therefore be discussed next. The fracturing process typically happens along natural planes of weakness, often through the hydrophobic part of membranes.
As water molecules leave the surface, macromolecular structures like membranes remain and become exposed. A coat of metal typically 0. Subsequently, 20—25 nm carbon is evaporated by rotary shadowing on top of the sample to provide structural support. The sample is then thawed in distilled water and biological material is removed by sulfuric or chromic acid. In addition to surface details Studer et al.
Disadvantages are the rather low resolution based on the grain size of the metal , the unpredictability of the fracturing process, shrinkage due to freeze-drying, stress during preparation, insufficient vitrification i. Depending on the sample preparation and fixation method, even artifacts such as mesosomes can be introduced Fig. Freeze-Substitution Dehydration is one of the most damaging steps in conventional EM. Contrast-enhancing agents such as uranyl acetate or tannic acid may also be added.
At this temperature the solvent melts but the cellular water remains frozen Giddings, Jr. Over time the cellular water is dissolved and replaced by the fixativecontaining solvent. Compared to room-temperature chemical fixation and embedding, freezesubstitution and low-temperature embedding of cryo-fixed cells has been shown to better preserve a multitude of different bacterial structures including membranes, cell walls, and various ultrastructures such as the gliding apparatus of Mycoplasmas Graham, ; McCarren et al.
The individual leaflets of membrane bilayers are often distinguishable, for instance. Mesosomes were not seen in freeze-substituted bacteria, which raised the first serious doubts about their authenticity. The organic solvents used for substitution can still extract or rearrange lipids, though substantially less than at room temperature Paul and Beveridge, Following freezesubstitution, genomic DNA seems to localize in a ribosome-free, coralline-shaped area with a fine structure comprised of a mixture of grains and fibers Hobot et al.
Immunolabeling One of the advantages of room-temperature EM techniques is that they allow the localization of specific antigens through immunolabeling. Immunolabeling approaches can be classified by whether the antibodies are applied before or after the sample is embedded or if the sample is embedded in plastic at all.
Pre-embedding methods are difficult, if not impossible, to apply to bacterial cells, since most bacteria have rigid cells walls that are impervious to all but the most destructive detergents. After the primary and 32 Martin Pilhofer et al. Post-embedding methods are more effective for bacterial cells. The sample is first processed and thin-sectioned as described above, but then the primary and secondary antibodies are applied to the sections. New Insights from Cryo-Microscopy 33 intracellular epitopes to the antibodies without the need for permeabilization, but their antigenicity can be compromised by fixation and embedment.
Special methacrylateembedding resins e. While the location of FtsZ was seen, exemplifying the power of immunoEM, the filaments FtsZ forms were not, presumably because of fixation, dehydration, and embedment Fig. A third approach, which avoids plastic embedment altogether, is the method of Tokuyasu Tokuyasu, and its variants Ladinsky and Howell, ; Slot et al. Cells are first pelleted, mildly fixed with aldehyde, infiltrated with concentrated sucrose i. While this method has only rarely been applied to bacteria, it has accurately defined the presence and locations of certain bacterial antigens Brusca et al.
In a new variant of this Fig. In conventional EM, immunogold labeling can be used to localize proteins, but structures are often not preserved and can be biased by artifacts A. In cryo-EM, large ultrastructures and protein complexes can be identified by their shapes, since macromolecules are preserved in their near-native states B—C , but in other cases, less-direct methods must be used such as manipulating the abundance or stability of a certain gene product D , electron spectroscopic imaging E , or correlated light and electron microscopy F.
A Immunogold labeling of the cell-division protein FtsZ. Note that the filamentous structure of the Z-ring is not preserved. Adapted from Bi and Lutkenhaus Adapted from Komeili et al. Structural core of pyruvate dehydrogenase shown in blue, ribosome in yellow, RNA polymerase in purple, and GroEL in red. The cell membrane is shown in light blue and the rod, a prominent structure filling the space of the tip region, is depicted in green.
Adapted from Kuhner et al. D Segmented tomograms of a Caulobacter wild-type cell left and an FtsZ-overexpressing cell right. Membranes shown in cyan and yellow, FtsZ in red. Adapted from Li et al. E Spectroscopic difference imaging of a Caulobacter crescentus cell. A phosphorus-rich body is indicated by the red arrow. Adapted from Comolli et al. Tomographic slice through a C. The correlation was reproduced in many cells, even after the position of the array was perturbed with mutations.
Adapted from Briegel et al.
This approach has been shown to give better structural preservation with the same labeling efficiency on eukaryotic cells, and may work well for difficult-to-fix materials such as bacteria Stierhof and Schwarz, ; van Donselaar E. Plunge Freezing Thin Films As mentioned above, because bacterial cells are small, they can be efficiently vitrified simply by spreading them into a thin film across an EM grid and plunging them into a cryogen. A few microliters of a bacterial culture is simply applied directly to the grid, or, when working with adherent cells, EM grids can be incubated within the bacterial culture so that the cells can naturally attach to them Seybert et al.
If the sample is to be used for CET, 5 or 10 nm colloidal gold particles are either mixed with the sample, dried onto the grid prior to freezing, or both to serve as fiducial makers for subsequent image alignment. In order to produce the thin film required for vitrification, the grid is blotted briefly 1—2 s with filter paper.
Most laboratories use liquid ethane as a cryogen. Recent advances have shown that a mixture of propane and ethane freezes samples just as effectively as pure ethane, but the mixture does not solidify and is therefore more experimentally convenient Tivol et al. Importantly, plunging non-cryo-protected samples directly into liquid nitrogen is not usually effective because of its lower thermal conductivity.
Many simple plunge freezing machines have been constructed in individual laboratories, but commercial devices are also available Frederik and Hubert, Automatic plunge-freezers allow careful control of parameters such as temperature, humidity, and the extent of blotting prior to freezing. This dramatically improves the efficiency and reproducibility of the method. Plunge-frozen samples are immediately ready for imaging by cryo-EM or they can be stored in liquid nitrogen essentially indefinitely.
New Insights from Cryo-Microscopy 35 B. Cryo-Electron Tomography While tomography is not strictly a cryo-EM method it is routinely used with plasticembedded samples as well , its application to cryo-fixed samples CET is a powerful combination that is opening new windows into bacterial ultrastructure. Frozen grids are loaded at liquid nitrogen temperature into the cryo-stage of a cryo-EM.
The tilt range is limited by either the increasing sample thickness as the cell is rotated or by parts of the grid or grid-holder blocking the beam. For a movie showing a tilt-series of a vitrified bacterium see Movie 1 at http: Since no goniometer is mechanically perfect, the target cell moves laterally and vertically within the column as the sample is tilted. To correct this, the beam and the image have to be shifted and the focus has to be adjusted before each image is acquired.
Several software packages are now available which perform these corrections and acquire tilt-series images automatically Mastronarde, ; Nickell et al. First, the images must be precisely aligned. Changes in specimen height, for instance, cause image rotation, changes in magnification, and changes in focus. Lateral movement of the sample causes shifts between images, and the tiltincrement varies slightly. The translation, rotation, magnification, tilt-axis, and tiltangle of each image is therefore determined by tracking the gold fiducial markers which were added to the sample before freezing throughout the tilt-series.
Automatic EM data collection packages e. Large numbers of tomograms also make it possible to obtain more reliable reconstructions of regular objects through averaging Liu et al. Laser Manipulation of Cells and Tissues: Volume 82 Michael W. Volume 94 Roger D. Methods in Tau Cell Biology: Volume Stuart Feinstein. Nuclear Mechanics and Genome Regulation: Electron Microscopy of Model Systems: Volume 96 Thomas Mueller-Reichert.
Table of contents Introduction: The physics of rapid cooling and its implications for cryo-immobilization of cells 2. Cryo-preparation methodology for plant cell biology 4. Understanding microtubule organizing centers by comparing mutant and wild-type structures with electron tomography 6.
Electron Microscopy of archaea 8. Using electron microscopy to understand functional mechanisms of chromosome alignment on the mitotic spindle Electron tomography of bacterial chemotaxis receptor assemblies How to "read" a vitreous section Electron microscopy of microtubule-based cytoskeletal machinery Reconstructing the endocytotic machinery Localizing Macromolecules in Cells EM analysis of viral morphogenesis Electron tomography of immuno-labeled cryosections Visualizing macromolecules with fluoronanogold: