Electron Microscopy in Cell Biology

1
The Use of Electron Microscopy in Cell Biology

Gareth Grifths
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1 1.1 1.1.1 2 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 3.10 3.10.1 3.10.2 3.11 3.12
Introduction 4 Why Do We Need EM in Cell Biology? The Strategy 5 Specimen Preparation 7
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4 4.1 4.2 4.3 4.4
Ambient Temperature EM Methods 7 Negative Staining of Particles 7 Positive-negative Staining Approaches 9 Classical Resin Embedding 11 Chemical Fixation 12 Postxation 13 Dehydration and Embedding 13 Methacrylate-based Embedding 14 Plastic Sectioning 16 The Tokuyasu Sucrose Embedding Cryosection Method 17 On-section Immunogold-labeling Procedure 20 Step 2 Antibody Labeling 22 Step 3 Gold 22 Preembedding Labeling Methods 23 Immunohistochemical Approaches using Horseradish Peroxidase and Cytochemistry 25 The Use of Microwave Technology for Specimen Preparation 25 Cryo-EM Approaches 26 Vitrication 26 The Bare-grid Method for Particulate Specimens Vitrication of Larger Material 27 The Cellulose Capillary Tube 27
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5 6 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 7 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8
The Fine-needle Biopsy 28 Hydrated Cryosectioning 29 Freeze-substitution 29 Freeze-fracture and Replica Methods 31 Simple Shadowing Methods 31 The Kleinschmidt and other EM Methods for Nucleic Acids EM Autoradiography 33
EM-Visualization at Ambient Temperatures 33
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EM at Cold Temperatures 37 EM Tomography 38 Low- and High-resolution SEM 40 Critical Point Drying 42 Freeze-drying and Cryo-SEM 44 Coating Techniques for Cryo-SEM 47 Scanning Transmission EM 47 X ray Elemental Microanalysis by EM 47 Energy-ltering Transmission Electron Microscopy (EFTEM) Stereology 50 Relative Area and Volume 51 Relative Prole Length and Surface Area 51 Absolute Volume 52 Surfaces and Volumes in Practice 52 Correlative Light and Electron Microscopy 53 Interpretation in EM 54 Which Technique for Which Question? 54 Final Comment 56 Acknowledgments Bibliography 56 56
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Keywords
Transmission Electron Microscopy (TEM) Electrons passing through the specimen form the image. Transmission of electrons through the specimen depends on the accelerating voltage, routinely 80 to 120 KV for specimens thinner than 300 nm, but for thicker specimens, or for higher resolution voltages up to 1000 K can be used.
EM grid A 3-mm diameter, 0.01-mm (1015 m) thick disc of metal (usually copper) with regular perforations. The specimen is placed on the grid and those parts of the specimen that lie across the perforations are visualized in the EM. Scanning Electron Microscopy (SEM) This approach is used to visualize the specimen surface. An electron probe scans the surface and the secondary electrons are recorded in relation to the moving point of the probe. Specimen Preparation The foundation of all EM. These are highly empirical methods that have been developed for all EM approaches, which allow the specimens to be imaged successfully. For TEM, the specimen must either be particulate, generally thinner than 500 nm, or it must be sectioned to that thickness, or thinner, after embedding in plastic, or after cryoimmobilization. Vitrication The process whereby liquid water is cooled so rapidly that the water molecules solidify without crystallization to form amorphous or vitreous ice. A vitried specimen is considered to be cryoimmobilized. Freeze Substitution Vitried specimens are inltrated at low temperatures with solvents, usually with heavy metal stains such as osmium tetroxide or uranyl acetate. Following this low temperature stabilization, the specimen is inltrated with resin at low temperature followed by ultraviolet light polymerization below 20 C, or the specimen temperature is raised to room temperature prior to room temperature resin embedding. Freeze-Fracture/Replica A vitried specimen is planarly split and coated with a thin layer of metal (such as platinum, tungsten, or tantalum). The biological material is digested away and the replica is placed on an EM grid or viewed in the frozen- hydrated state in a SEM. A useful modication, freeze-etching, involves subliming a surface layer of water molecules to reveal details of some internal structures before shadowing. For particles and macromolecular complexes, metal shadowing can be used to form a replica without the freezing step. Glycerol spraying is a related technique where the sample is sprayed into a glycerol suspension and then rotary-shadowed and viewed by TEM. Immunogold Labeling Visualization of antigens on sections, or on the surface of isolated particles using (primary) antibodies. These are detected generally using an additional reaction containing gold particles that have bound (secondary) antibodies, or protein-A, that recognize the primary antibodies.
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Tomography The procedure for collecting multiple tilt images of a set of particles, or structures in (mostly) thick sections or isolated particles and collating the information computationally to provide a three-dimensional model of the structure. Image Analysis Computing methods used to improve the signal-to-noise ratio of EM images. They are based on averaging the information from two-dimensional data sets and can reveal the three-dimensional structure. Stereology A set of sampling tools and geometrical probabilitybased principles that allow one to estimate relative and absolute three-dimensional structural quantities (such as volume, surface, length and number), and spatial relationships, from sectional images.
Most of the organelles discovered after 1945 were rst seen by electron microscopy (EM) in thin sections of resin-embedded cells. Since that time, the technical advances in EM instrumentation and specimen preparation have been enormous, a consequence of physicists, engineers, and biologists working together. These methods now allow cell biologists to visualize unperturbed cell structures over a range of dimensions, from the atomic resolution in the case of single proteins or macromolecular complexes to the range from 2 nm and up in the case of cellular organelles. This level of resolution is far beyond what can be seen by light microscopy. This review will cover the essentials of a range of methods for scientists investigating cell biology using EM. The major emphasis will be on resolution levels poorer than 1 to 3 nm. In all these methods, the most crucial parameter is specimen preparation and, without doubt, the gold- standard reference technique, which involves vitrication by rapid freezing, in conjunction with cryo-EM.
Introduction
1.1
Why Do We Need EM in Cell Biology?
The main goal of this chapter is to convince the reader, especially cell biologists, that electron microscopy (EM) offers them an incredibly powerful set of approaches for analyzing the ultrastructure of cells and their components. Moreover, these
methods have been developed to cover a vast scale of detail, ranging from the atomic level to the level of whole organisms. While high-resolution cryo-EM is thriving, the use of lower resolution EM has dropped considerably in recent times in cutting-edge cell biology research. There is no doubt that this downward trend is a direct effect of the availability of increasingly sophisticated state-of-theart light microscopy (LM) approaches
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that can, for example, detect individual uorescent molecules under ideal conditions. Recently, even the long-considered lower limit to LM resolution (0.2 m) has been reduced for detecting uorescent molecules to below 0.1 m. Such developments, while clearly very important, may even deceive more cell biologists into thinking that LM approaches can now do everything that was earlier considered in the realm of EM. There is, however, a fundamental aw to this argument. Even if one can see a single uorescently labeled molecule in the cell, there is still the fundamental limitation with LM in relating that signal to the dened structures in the cell. An EM-thin section can reveal enormous detail of dened structures, such as membranes, laments, or ribosomes; when this section is labeled with immunogold, the gold particles can be assigned to dened structures with high precision (within 8 to 20 nm). However, at the LM level, the uorescence signal is mostly seen only as a nondescript blob. Often, one compares the immunolabeled signal from one protein in one color with respect to a compartment reference marker seen in a separate color. However, for such a reference marker to be useful, one needs to have shown rst by EM that it is indeed restricted to the structure of interest. Figure 1(a) shows an example of immunouorescence labeling of the Golgi complex, while Fig. 1(b) reveals how much more information becomes available when the same specimen is visualized at the EM level. Both methods provide important information, but at different, complementary scales. State-of-the-art LM is nevertheless, quite justiably at the center stage of cell biology. In contrast to EM analysis, which always presents snap-shots of processes, video analysis by LM can allow one to see
dynamic events in real time within cells. One of the take-home messages of this review is to point out that for modern cell biology studies one needs both LM, to see an overview of the forest, and EM to see the details of the trees. I will try to demonstrate that state-of-the-art EM methods should be standard technology in all laboratories involved in molecular cell biology research. It should also be pointed out that the expertise needed to execute these methods at the highest level is rapidly dwindling, as fewer specialist groups are available to keep these methods alive. I hope this chapter will also contribute to an awareness that this situation can, and must, be reversed.
The Strategy The spectrum of EM techniques of interest to cell biologists is quite broad. For each method, one can easily write a whole book, and many books are indeed available. Several of these methods are technically quite demanding. Given the space limitation and the need to cover almost all techniques, the approach I have taken is to explain the basic concepts behind each approach so that a total beginner could understand how each technique operates and what it is good for. For the most commonly used methods, photographs, and schematic diagrams are provided to show those details that are important for the method to work. The strategy I have followed is to start with live cells or organisms, or isolated biological material in an aqueous solution and describe the two fundamentally different ways of preparing these specimens for transmission EM-conventional chemical methods, and the cryobased approaches. Some of these conventional methods, as well as one partly cryoapproach, can be used for the detection of antigens using
1.1.1
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Fig. 1 Comparison of LM and EM. (a) Shows an immunouorescence micrograph showing labeling for a trans-Golgi protein sialyl transferase. A Hela cell line stably transfected with a construct containing sialyl transferase tagged with a domain from the vesicular stomatitis virus G-protein (provided by Tommy Nilsson) was xed with 3% formaldehyde followed by a permeabilization with 0.1% Triton X-100. The cell was then labeled with a rabbit antibody against G-protein tag followed by a secondary antibody coupled to rhodamine. The typical ribbon-shaped appearance of the red Golgi complex is evident, next to the nucleus. The latter is revealed using DAPI staining for DNA that was done at the end of immunolabeling reaction. (b) Shows a Tokuyasu cryosection of the same specimen labeled with the same antibody and protein-A gold (arrowhead). Note the signicant amount of labeling on the trans-side of the Golgi. There is also sparse label on membranes of some endocytic structures (E). EE represents the early endosomes, evident by their sparse labeling for 5 nm gold-BSA (arrow) that was internalized for 5 min before xation. Bars = (a) 10 m and (b) 100 nm. Micrographs courtesy of Veronika Neubrand and Anja Habermann.
1 m (a)
EE 100 nm E (b)
immunogold labeling; the essence of these methods will be covered. At the end of these preparation methods, the specimens are placed on an EM grid. The two different ways of imaging these specimens at room temperature or using a cryostage are then described. A recently perfected approach for obtaining three-dimensional models from a series of tilted images by tomography will next be summarized, followed by two sets of methods that visualize (mostly) surface structure, namely freeze-fracture (etching)/replica methods for transmission
electron microscopy (TEM) and low- and high-resolution scanning EM. The ability to use scanning transmission EM (STEM) for mass determination and X-ray microanalysis for the detection of some ions will be briey mentioned. The powerful use of stereology to quantify structural parameters from sections will then be outlined. Following a brief description of methods that can be used to correlate LM with EM data, and a note of caution about interpreting thin section images, I end by offering some guidance on which technique is appropriate for which scientic question.
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Specimen Preparation
Before a specimen can be visualized by transmission EM, it must full a crucial criterion it must be very thin (less than about 0.4/0.5 m) for electrons to pass through it. In all transmission-EM approaches, the specimen is mounted on a 3-mm diameter metal (usually copper) grid (0.01 mm in thickness). This supports the specimen between the grid bars and allows electrons to pass through the specimen that lies across the holes in the grid. The grid is usually covered with a thin plastic lm (such as formvar or collodion) and preferably coated with a thin layer of evaporated carbon before attaching the specimen. This provides stability by reducing surface charge buildup. The evaporator is a prominent machine in almost all EM laboratories. In practice, there are two different possibilities for adsorbing the specimen onto the grid. If the specimen is particulate, that is, a discrete entity that is no larger than, at most 0.5 m in height, it can be directly adsorbed onto the grid. Thin regions of whole cells grown on EM-grids can also be analyzed this way, using a newly developed cryo-EM approach (see Sect. 6.1 on Tomography, below). If, however, the structure is larger or, more commonly, one wants to see structures inside organelles, cells, or tissues, one must embed the specimen, or solidify it by vitrication, and then cut thin (usually 50300 nm) sections. An alternative approach is to freeze-fracture the specimen and analyze the fractured surface, or after etching, the subsurface topography. Both TEM and scanning electron microscopy (SEM) can be used to visualize these specimens. The key to the analysis of any cell structure, at any scale, is the ability to x
the structure, either by chemical crosslinking, or, more preferably, by arresting it physically in its aqueous environment by freezing. Although chemical xatives have been used successfully for EM since the early 1940s (see Fig. 17), the introduction of the concept of cooling into the vitried state has now emerged as the fundamental innovation in EM-specimen preparation. The ability to vitrify a specimen within milliseconds, in conjunction with a physical proof that the specimen is indeed in a vitreous state, now offers the best possible method that provides a fundamental frame of reference for all EM-specimen preparation methods. Native biological material is extremely fragile and sensitive to electron beam damage. For this reason, a series of methods have been developed to optimize specimen preparation, the sine qua non of all EM. While one kind of approach, environmental SEM (ESEM) is starting to provide a means of imaging specimens (such as whole cells) in their aqueous environment, this is still in its infancy. This approach will not be discussed further here.
Ambient Temperature EM Methods

3.1
Negative Staining of Particles
The simplest and most rapid approach in EM is classical negative-staining (Figs. 2, 4). This approach is useful for proteins, macromolecular complexes, viruses, (small) bacteria, and isolated organelle fractions. Particles are adsorbed onto EM-grids having a suitable surface. This surface is crucial for success and in most cases the grids are covered
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Fig. 2
Negative staining in practice. In (a) the grids are seen oating on solution, they can be moved from one drop to the next using a ne forceps. (b) After the nal staining reaction, the grid is lifted from the drop of stain and a lter paper is used to remove the excess stain; (c) and (d) show a simple trick to cleanly remove the dried grid from the forceps. A triangular piece of lter paper piece is inserted in the forceps and is then used to gently push out the grid cleanly onto a suitable support.
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with a layer of evaporated carbon on a suitable plastic lm (e.g. formvar). It is often an advantage to glow discharge grids, a procedure whereby air (in a light vacuum) is ionized by 500 to 1500 V, thereby making the grid surface hydrophilic (methods are also available to make grids hydrophobic; these are useful for some specimens). Grids are oated on as little as 3 to 5 L of an aqueous (usually buffered) solution of the specimen for a few seconds to minutes to adsorb the material. The grid plus absorbed particles are then rinsed rapidly with water and oated on a (15%) solution of a heavy metal stain, such as uranyl acetate, sodium phosphotungstate, or ammonium molybdate. If pure water is not desirable, the salts in the buffer (which will react with the heavy metals) can be removed by many rapid changes (1 s each) of the grids on pure stain. After a few seconds to 1 min on the nal drop, the grid is removed with forceps and the excess stain is partially removed with lter paper (Fig. 2). A relatively thick layer (100300 nm) of stain needs to dry down around the specimen. The staining is referred to as negative because the stain lls the spaces around the specimen that are seen outlined in negative contrast. Examples are shown in Fig. 4(ac). Although technically trivial, there are a number of tricks, and in the hands of specialists, this approach can provide a relatively high level of resolution/information.. This approach (and the positive staining discussed below) is easily compatible with immunogold labeling before the drying step. One limitation is that only antigens exposed on the surface of particles can be labeled. In some cases, the particles can be treated with reagents that can open up the particle. In one such example, the reducing agent dithiothreitol (DTT) was
used to expose antigens that are normally buried within vaccinia virus particles (see Fig. 4b).
3.2
Positive-negative Staining Approaches
The adsorption staining procedure developed by Tokuyasu to protect thawed cryosections from drying artifacts is a powerful alternative method that is easier to control and to image than negative staining, especially for beginners. The method is shown in practice in Fig. 3 and schematically in Fig. 8(g). In this approach, the stain adsorbs to some parts of the specimen (positive staining) and can be seen because of the low background contrast. Some negative-staining effects can also be seen, depending on the specimen and the precise conditions used (see the right virus particle in Fig. 4b). This is expected since heavy metal compounds used for negative staining also positively stain biological structures. This method has several advantages over classical negative staining. (1) The stain adsorbs to selected structures, usually by ionic interactions. Therefore, the differences in intensity of staining in different parts of the structures provide additional information not available by negative stain methods. (2) The fact that the overall concentration of heavy metal stain over the grid is far less than in negative staining means that beam damage, often a serious problem in negative staining, is far easier to deal with. (3) This means that it is also easier to collect large data sets, as for tomography due to the reduction in beam-induced damage. Images such as the one shown in Fig. 4(b) are currently being analyzed by this approach. Membrane organelles collapse far less than with normal negative staining
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Fig. 3
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Methylcellulose embedding in practice shows the basic technique involved in methylcellulose embedding. (a) The grid, having been previously rinsed in distilled water, is placed on a solution of methylcellulose and uranyl acetate on ice. (ac) The grid is then looped out and the excess stain is removed by touching the loop onto a surface of lter paper (d) The grid is allowed to air-dry. Ideally, the interference colors should be gold, purple, or blue at the end of the technique. (e) and (f) show removal of the grid from the loop.
(4) Finally, by varying the concentration of the stain, one has the possibility in the Tokuyasu approach to vary the physical appearance of the particle; for example, locally high concentration of uranyl acetate
over some structures can reveal negatively contrasted thin tubules that are often more difcult to visualize by positivestaining methods. An example is given in Fig. 4(b).
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Examples of negative and positive staining for EM. (a) Shows a classical negative staining using phosphotungstic acid of tobacco mosaic virus (TMV). The typical rod shaped appearance of this virus is seen. Some subunits of the virus are also seen end-on. It is evident that these are spherical in prole. (b) Shows vaccinia virus that has been treated with the reducing agent dithiothreitol (DTT). This treatment is known to break disulde bonds within the virus. The virus was then embedded using the metal cellulose/uranyl acetate mixture as shown in Fig. 3. The increased stabilization of structures and the partly positive staining by this procedure allows more details of this relatively large virus to be seen. The arrowheads indicate two round tubular projections that have been ejected from the core of the virus by the DDT treatments. We believe these may facilitate virus entry into cells during infection. The particle on the right shows a side-view of another particle. In (c), the same virus preparation can be seen after conventional negative staining using uranyl acetate. Less ne detail is evident after this approach. Nevertheless, the areas where stain enters the particle can be delineated by the heavy metal stain. The arrowheads indicate apparent openings between the virus and the core of the particle. Bars = 100 nm. (a) Courtesy of Heinz Schwarz; (b) and (c) from the author.
Fig. 4
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3.3
Classical Resin Embedding
The majority of thin section analysis worldwide is still carried out using room-temperature specimen preparation techniques in which chemical xatives,
especially glutaraldehyde, are applied. These approaches are structurally acceptable in so far as the structures they describe have been observed by a cryobased approach; this statement is reasonably valid for all known organelles. For new structures, their acceptance should
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Fig. 4
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be based on conrmation by a cryobased approach, such as freeze-substitution or using hydrated cryosections. By far the most widely used approach for ultrastructural analysis today, as it has been for the past 50 years, is the epoxy resin method. This is a witchs brew of empirically determined steps for converting fresh tissue, cells, or isolated organelles into a thin section. Although the variety of recipes is greater than in most cookbooks, the general principle is as described below. For analyzing repeatedly the same cells or tissues whose ultrastructure is well known, as in pathology, the conventional plastic-section approach is still the method of choice in most laboratories. Many useful textbooks on EM can be found in many established EM laboratories around the world (usually collecting dust!).
3.4
Chemical Fixation
Glutaraldehyde is the chemical xative of choice for preserving structure for conventional EM. The introduction of this xative was, without doubt a revolutionary innovation for EM in cell biology; a number of organelles, such as microtubules, were rst seen only after they could be preserved with this reagent. It is a powerful cross-linker of amino groups, especially in proteins and amino-lipids. Although the chemistry involved in this process is very complex, and complicated more by the fact that the cross-linking coincides with the process of cell death, in practice it is very simple to apply the xative to any open cell system; that is, cells that are accessible to a solution without having a barrier, such as a cell wall or a cuticle to block access. If cells are growing in culture, the best results are usually achieved by adding the
xative to the growth medium directly, for the rst few minutes, and then switching to buffered xative. Any barrier to the solutions, such as cell walls, must be dealt with on an individual basis. The best advice is to check how others have prepared the cells of interest. If one wishes to x a tissue in situ in an animal, the best procedure is usually perfusion, a nontrivial surgical operation. It is important to respect the old rule of thumb that, for optimal results, the specimen pieces should be less than 1 mm in all dimensions to ensure good penetration of reagents. When immunolabeling, as opposed to purely structural analyses, is the goal, it is often the case that extensive crosslinking with glutaraldehyde will tend to sterically hinder the access of antibodies to the antigens. A weaker cross-linker, formaldehyde (which, in solution becomes methylene glycol) is therefore used for this purpose, either by itself, or in combinations with (lower concentrations of) glutaraldehyde. For example, in standard protocols for the Tokuyasu method (see below), cells are xed for 15 to 60 min in 4% formaldehyde and 0.1 to 0.2% glutaraldehyde followed by an overnight (or longer) xation in 4% formaldehyde in a suitable buffer. Aldehyde cross-linking generally is a process that releases protons. This lowering of the pH within cells is unlikely to be protected by the buffer since all the routinely used buffers are charged molecules that are thought to enter cells very inefciently. The cross-linking reactions also consume oxygen. Although this approach can provide a faithful presentation of ultrastructure, there are many known examples of specimen preparationinduced artifacts. This usually becomes evident when a lessperturbing cryobased method is used.
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A classical example is the bacterial mesosome; earlier considered a distinct organelle, it is now known to be a xation artifact, from the use of hydrated cryosections. Another striking example of aldehyde-induced artifacts in the frog retina revealed by high-pressure freezing and freeze-substitution has already been written about.
3.5
this stain for, for example, 1 h; an alternative so-called en bloc procedure that we often use is to leave specimens overnight in a saturated solution of uranyl acetate in 70% ethanol prior to embedding.
3.6
Dehydration and Embedding
Postxation
For pure structural studies, but generally not for immunolabeling, the aldehyde xed tissue is postxed in (0.51%) osmium tetroxide (OsO4 ). One of the very rst micrographs using this xative for EM is shown in Fig. 16. This heavy metal can cross-link lipids and protein and adds general contrast to many organelles, especially membranes (enhancing the trilaminar appearance). It is also clear that artifacts may be induced, the most striking being the proteolysis of structures such as actin laments. It is generally accepted that, when used at the low temperature for freeze-substitution (see below), the use of OsO4 is generally free of artifacts and is a useful addition to enhance contrast. As pointed out below, OsO4 is generally incompatible with the polymerization of many resins used for immunolabeling (such as lowicryl). Concentrations up to 1% in the freeze-substitution schedule may be used without interfering with UV polymerization of these resins. Osmium can also be successfully used with LR white (see below) (see Fig. 7). Many workers add a second postxation step after the OsO4 using uranyl acetate, which binds to negatively charged phosphate groups in phospholipids, is generally an excellent stabilizer of lipids. Tissues are often left in aqueous solutions of
The epoxy resins (e.g. Epon, Spurrs, Araldite) are highly hydrophobic, while many of the methacrylates have a lower hydrophobicity, but are still immiscible with water. For this reason (as in parafn embedding for histology), the water must be replaced (gradually) with an ascending series of ethanol or acetone (which tends to extract more material than ethanol). At the 100% solvent stage, the resin is gradually mixed with the solvent over a period of a few hours until it can be placed in pure resin. At this stage, the specimen in resin is mounted into an embedding mold (Fig. 5b) or other suitable container (e.g. BEEM capsule) and left overnight at room temperature (at which little polymerization occurs) for complete inltration, before being allowed to polymerize for about 12 h at 60 to 70 C. The specimen is now ready to section. The standard epoxy and araldite resins are quite viscous and it is often a problem to penetrate some specimens, such as many plant and insect tissues. In 1969, Arthur Spurr introduced his concoction, known as Spurr resin as a low viscosity epoxy resin alternative. This has become the standard resin for many specimens. Alternative low viscosityembedding media are now also commercially available. For more details there are many classical textbooks available in EM laboratories. For an example of an image of an epoxy resin section, see Fig. 6.
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3.7
Methacrylate-based Embedding
After the introduction of the complete Tokuyasu method in 1978, this approach was the section method of choice for immunolabeling. For the next two to three years, all alternative embedding protocols were far inferior for this purpose. However, in 1980 appeared the rst in a series of papers from Eduard Kellenbergers laboratory in Basel, in which an excellent alternative approach became available. After years of painstaking work in collaboration with an industrial partner (Lowi), Carlemalm et al. were successful in introducing the rst of the Lowicryl resins, the more hydrophilic K4M, and the more hydrophobic HM20. Its great potential for immunogold labeling was shown already in the rst publication. The basic approach is no different to the epoxy protocol outlined above; it is simply a question of which solutions, in which order, and for how long. However, the resins are polymerized by UV light rather than by heat. Although these resins can be used at room temperature, the goal from the outset was to develop these resins predominantly for use at low temperatures. In the initial publications, an approach
known as progressive lowering of temperature (PLT) was introduced. Here, starting at the dehydration step, the temperature is gradually lowered to 35 C. The importance of these resins is that they can be polymerized at this temperature using UV polymerization (although they can also be polymerized by heat). This polymerization usually takes one to three days and often the blocks need additional curing time at room temperature in direct sunlight (without glass). In 1985, this group introduced new resins (K11M) that could be polymerized down to 60 C and HM23, which will polymerize even at 80 C. These became especially useful in combination with freeze-substitution (see below, and Figs. 13, 15b). The important contributions of Bruno Humbel and Martin M ller toward this approach should also u be mentioned. In England, Brian Causton, a chemist, in conjunction with the London Resin (LR) Company, developed an alternative set of resins LR white and LR gold. These resins are now widely used for EM immunolabeling, especially in laboratories that lack the equipment for cryosectioning. As mentioned, LR white is also compatible with osmium tetroxide treatment and recipes exist for providing beautiful preservation
Fig. 5
Plastic sectioning for EM. In (a), a trimmed Epon block is shown in a Leica ultramicrotome being sectioned on a Diatome diamond knife. The 2.5-mm knife-edge is indicated by arrows. A ribbon of sections is seen. This ribbon is only slightly longer in length than the diameter of an EM grid. Normally, one would tease this ribbon away from the knife-edge using a ne eyelash. The grid is then lowered carefully, lm side down, onto the ribbon and pressed gently. The grid is ideally rotated on the surface of the water so that the water can run off smoothly from the surface of the grid. The grid is then raised and air dried. An alternative method that avoids section wrinkles is to dip the grid into the water beneath the sections and then carefully raise the grid at about a 45 angle to sh the ribbon. Photograph courtesy of Robert Ranner Leica Microsystems GmbH, Vienna. (b) Shows specimens that have been polymerized in an embedding mold. The specimens are black following osmium treatment (arrows). A piece of paper with specimen details can be conveniently coembedded with the specimen. Courtesy of Anja Habermann and Maj Britt Hansen.
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in conjunction with immunogold labeling (Fig. 7). All the methacrylate-based resins are sectioned in the same way as other plasticembedded blocks (Fig. 5a). For the more hydrophilic ones, such as Lowicryl K4M, the water level in the trough of the knife boat (Fig. 5a) needs to be lowered,
compared to Epon sectioning, to prevent the sections being pulled behind the knife. All the resins used for EM are potentially toxic, as are the xatives and appropriate caution should be taken when handling them (eye protection, gloves, hood, etc.). Particular care should be
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Fig. 5
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Fig. 6 Example of an epoxy resin section. This shows a section of a J774 macrophage that had internalized BSA-conjugated gold particles for 1 h followed by a chase in medium free of gold for 1 h. Under this condition, the gold is in late endosomes and lysosomes (Ly). Subsequently, 1-m latex beads (B) were added for 1 h followed by a further 3-h chase. Gold can be seen in late endocytic organelles and in the latex bead phagosomes (arrowhead), after a fusion process. Other organelles, Golgi stacks (G), mitochondria (M), and ER are indicated.
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taken when working with the Lowicryl resins that are well known to cause skin problems.
3.8
Plastic Sectioning
The specimen embedded in plastic must next be trimmed to make a small block that can be pyramidal or rectangular at its tip (usually 0.010.1 mm, see Fig. 5a). This can be done manually with razor blades but is much more precisely done using commercially available block trimmers that have metal or diamond blades. The trimmed block is next mounted in an ultramicrotome equipped with a glass knife or, more conveniently using a diamond knife (Fig. 5a). The combination of modern ultramicrotomes and state-of-theart diamond knives now enables sections
as thin as 20 nm to be easily and reproducibly obtained. As shown in Fig. 5(a), during plastic sectioning, the sections are oated on the surface of (clean) distilled water where they can be manipulated with an eyelash and mounted on a carbon and plastic (e.g. formvar) coated grid. In this simple procedure, the grid surface is brought into contact with the sections, which are adsorbed. The sections are routinely stained by oating the grids on a solution of, rst uranyl acetate (which may not be necessary if it was included before embedding), and second with a solution of lead citrate (for a few minutes only) (see Fig. 2a). After rinsing with water and air-drying, the grids are now ready for imaging. When immunogold labeling is desired, sections of methacrylate-embedded material can be labeled, as described below. In this case, the staining with
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Fig. 7
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Example of embedding in LR White. (a) and (b) show rat parotid acinar cells embedded in LR White. Pieces of parotid tissue from a rat were xed in a mixture of 0.5% glutaraldehyde and 4% formaldehyde under microwave irradiation, followed by postxation with 1% osmium tetroxide containing 1.5% potassium ferrocyanide (reduced osmium) under microwave irradiation and continued for 30 min on ice. Tissue blocks were embedded in LR White resin, and ultrathin sections were obtained. The ultrathin section mounted on a (a) nickel grid was pretreated for etching by a saturated aqueous solution of sodium metaperiodate for 30 s, followed by treatment in 1% bovine serum albumin. Immunolabeling was carried out using anti-GF-1. GF-1 is a monoclonal antibody recognizing 105-kDa glycoprotein in the Golgi apparatus of serous exocrine cells. Colloidal gold particles (arrow) can be recognized to distribute on the trans-cisternae of Golgi apparatus (G). The cells in (b) were additionally reacted for the trans-Golgi enzyme thiamine pyrophosphatase (TPPase) subsequent to the primary xation. This reaction product is seen as an electron-dense precipitate in one trans-Golgi cisterna (b) (arrowhead in (b)). The sections were labeled with an antibody GF-1, revealed by immunogold. The gold particles are seen to be restricted to the trans-side of the Golgi stack, colocalizing with the TPPase reaction product (arrowheads). The ER is indicated, as are the secretion granules (SG), the nucleus (N) and mitochondria (M). Micrographs courtesy of Shohei Yamashina.
1 m
1 m
heavy metals is carried out after the immunolabeling. An advantage of this approach for immunolabeling is that the room-temperature cut sections can be stored dry on grids indenitely and still be immunolabeled when desired. These sections can also be powerful tools for use at the immunouorescence level.
3.9
The Tokuyasu Sucrose Embedding Cryosection Method
An elegant and rapid method for visualizing structure and immunolabeling using cryosections was pioneered by Tokuyasu between the late 1960s and 1978. This is now used extensively worldwide.
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In contrast to the hydrated cryosection method (see below), there are two severe restrictions in obtaining cryosections for immunolabeling, in addition to the obvious need for the cryomicrotome. One is that immunolabeling needs to be carried out above 0 C and the other is that ultrathin cryosections of fresh specimens are destroyed when thawed. For this reason, the specimen must be chemically xed prior to sectioning, usually with a light, xation protocol (see above). So, the role of the sectioning process in this approach is simply to be able to cut thin sections of xed cells and tissue that can subsequently be immunolabeled while fully hydrated at ambient temperature. While this may be a disadvantage for high-resolution preservation, it also offers a number of practical advantages in comparison to the hydrated cryosectioning method. First, because the material is chemically xed, the membranes are permeable to high concentrations of sucrose, which is an excellent cryoprotectant. This means that relatively large pieces of specimen (>1 mm3 ) can by vitried routinely by simple immersion in liquid nitrogen (which is a relatively poor coolant). Moreover, sucrose (up to 2.3 M) allows the specimen to be more easily and uniformly thin sectioned in a cryoultramicrotome at, for example, 100 to 120 C. So, although the method involves a vitrication step, it cannot be considered a bone de cryo-EM method. Nevertheless, it is mostly referred to as the cryoimmuno-EM method. The steps in this method are shown schematically in Fig. 8. The trimmed specimen is cut with a glass knife, or now more commonly the superb new generation of diamond knives designed for cryosectioning. The temperature is usually in the range of 90 to
120 C. At these temperatures, water obviously freezes and a major difference between cryo and plastic sectioning is that cryosections are cut on a dry knife (i.e. no water trough). The colder the specimen, the harder the block; for cutting sections for LM, the blocks are made softer by raising the temperature from 50 to 80 C. Many workers also raise the temperature of the block to 80 C for trimming the block before cutting thin sections at colder temperatures. Plasticity of the block is also an important parameter to vary for optimal sectioning properties, and compounds such as polyvinyl pyrrolidine can be added to the sucrose infusion in order to facilitate the sectioning process. At all temperatures, even the best sections are initially compressed. They are then picked up using a 1- to 2-mm loop of thin wire that contains a drop of 2.3 M sucrose (Fig. 8d). The sucrose drop is rapidly brought into the cryochamber and, upon contacting the sections and brought to room temperature, it decompresses; a spherical cell prole that is signicantly compressed after sectioning can now regain its spherical shape. Often, this method leads to overstretching of the sections, causing structural artifacts. For this reason, the use of a mixture of sucrose and methylcellulose was introduced, which could greatly improve structural preservation (see Figs. 1b, 9, 14). Tokuyasu (personal communication) considers the osmotic pressure to be an additional important factor in section pickup. To reduce the impact of high osmotic pressure, using a mixture of 1.7 sucrose solution with the detergent Tween 80, at a nal concentration of 0.01% to retrieve cryosections embedded in 2 to 2.3 M sucrose was recommended; we have found this approach to be very useful. Section compression can also be minimized by reducing the sectioning angle
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(a)
(b)
(c)
(d)
(e)
Fig. 8
(f)
(g)
Schematic illustration of the Tokuyasu method. These drawings, made by Paul Webster, illustrate the basic essential details of the Tokuyasu cryosectioning and labeling method. All steps are carried out within the chamber of the cryoultramicrotome. Prior to step (a), the specimen will have been xed, infused with sucrose, and placed on a specimen pin. (a) Shows the procedure for trimming the block using a trimming device. Glass knives or special diamond trimmers can also be used for this purpose. In (b), the specimen is sectioned on a glass or, in this case, a diamond knife, and the sections are manipulated using an eyelash probe (c). In (d), the sections are picked up on a loop containing 2.3 M sucrose or a mixture of sucrose and methylcellulose. The loop is then touched onto the surface of a grid and the grid is raised and allowed to be oated (section surface down) on a drop of liquid. (e) Shows the procedure for maintaining a moist chamber around the grids. A petri dish is then layered over the grids, and a piece of moist lter paper is added on one side of the petri dish; (f) and (g) show again the principle of drying the grid after embedding in a methyl cellulose mixture (see also Fig. 3).
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of the diamond knife. A new oscillating diamond knife is being developed that already looks highly promising in reducing this problem even further. Once the sections are thawed on the surface of the drop of sucrose (or the mixture with methylcellulose), the loop is brought to the surface of an EM grid and, upon contact the sections are transferred to the grid surface. From now until the nal drying step, the fragile sections must remain hydrated and not be allowed to dry. The universal procedure for immunogold labeling can be applied (see below). It should be noted that in addition to its use for immunolabeling, this method is also an excellent and rapid (12 h) method for preserving and visualizing structures in thin sections by EM. It is surprising that this method has not been more widely used, for example, in pathology, as a rapid diagnostic tool. Either with or without immunolabeling, the nal step in the preparation is to dry/embed the sections. Since, as mentioned, the sections are easily destroyed by air-drying, Tokuyasu spent a number of years developing what in the end turned out to be a surprisingly simple approach to protect the sections from
collapsing during the drying process. His elegant solution was to oat the grids with sections (that were previously oating on pure water) on a mixture of heavy metal stain (usually uranyl acetate) and a polymer (usually methylcellulose, this is more soluble in the cold) for a few minutes. The grids are then looped out using a 3.5-mm loop, the excess solution is removed by blotting and the grid is air-dried/embedded (Figs. 3, 8). After removing the grids from the loop with a forceps, they can be visualized by EM. This simple procedure revolutionized the use of thawed cryosections for immunolabeling at the EM level.
3.10
On-section Immunogold-labeling Procedure
The localization of specic antigens on isolated particles, or on thin sections can be performed using immunogoldlabeling methods. A theoretical drawback of these methods is the need to label the specimen at physiological temperatures, which limits their use for bone de cryobased approaches. Nevertheless, these methods have been very powerful in a
Q4
Q5 Q6
Example of using the Tokuyasu technique. These images show cryosections of glutaraldehyde-xed CHO cells showing the distribution of Prion protein using an anti-PrP C monoclonal antibody Fab fragment (R1). (a) Gold labeled PrPC (arrowheads) was found to be highly enriched in the caveolae at the plasma membrane (PM) (not in this picture) and caveolae-containing membrane structures in the TGN around the pericentriolar region; arrows-centrioles. (b) Endocytosed 5-nm protein-A gold particles (which bind specically to PrPC; arrowheads) indicate the sites of PrPC molecules that had been taken up via caveolae. These results indicate that typical early endosomes do not intersect signicantly with the endocytic pathway of PrPC-containing caveolae. In addition to the typical small tubulo-vesicular structures, many larger multivesicular-bodies (late endosomal/lysosomal proles, L) were loaded with small gold particles representing endocytosed PrPC, whereas the transferrin receptor (labeled with an antibody and large, 10 nm, gold) localized in nearby and distinct structures. As expected, ER, Golgi complex, mitochondria, and nucleus were not labeled, indicating highly specic labeling in these experiments. G: Golgi complex, n: nucleus. Micrographs courtesy of Peter J. Peters.
Fig. 9
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wide spectrum of molecular cell biological applications. The essence of all immunolabeling is the same for all procedures at the LM and EM level. However, the use of sections (for LM or EM) obviates the need for a permeabilization step (see below). Subsequently, a minimum of three incubation steps is required, along with rinsing steps. The rst step is to block nonspecic sites. All proteins (some more than others) have the capacity to adhere
to (most) surfaces and it is generally thought that this is due mostly to ionic and hydrophobic interactions. In practice, one can usually (but not always) block these sites on the sections (and grid) surface by simply oating the grid on a drop of a suitable protein solution for a few minutes. Many groups use different kinds of sera (e.g. goat serum) for this purpose, in which case one must be sure that this does not interfere with the immunolabeling reaction. We prefer to
100 nm (a)
n I G
100 nm (b)
Fig. 9
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use a mixture of sh skin gelatin (from an arctic sh, via the Sigma company) and bovine serum albumin. Whatever one uses, it is recommended to dilute the antibodies and gold reagent in this solution for the subsequent labeling steps. In practice, the whole labeling procedure is most conveniently carried out on the surface of a layer of paralm (see Fig. 2a).
Step 2 Antibody Labeling The grid oating on the block solution is transferred to the surface of a 5- to 10-L drop of the optimal dilution of antibody in the blocking solution and left for 30 to 60 min in a humid chamber on the paralm surface (see Fig. 2a). The problems of antibody specicity must be seriously considered in evaluating EM (as for LM) immunolabeling. It is important to note that apparent proof of specicity by an immunochemical approach such as Western blotting does not necessarily constitute proof of specicity on the surface of a thin section since the conditions facing the antigen are quite different. It is crucial to realize that the complexity of possible interactions on the surface of a section can give rise to subtle and serious artifacts that may fool the observer. It is also important to realize that the numbers of antigens available at the surface of thin sections can be deceptively low. It is crucial to determine the optimal concentration of antibodies for the best signal-to-noise ratio. Above this level, background labeling can increase signicantly. The simplest take-home message to achieve this is to empirically determine the highest concentration of antibody (which, in some cases, may even mean undiluted antibody!) that does not label structures assumed to be free of antigens. The foundation of this assumption is the
3.10.1
available cell biological knowledge on the system. Depending on the gold reagent that will be subsequently used, one may need to use a secondary antibody step. For example, if one uses protein-A gold (which is the preferred reagent in many groups, including ours), and one starts with a mouse antibody (that mostly do not bind protein-A), then an intermediate step of, for example, rabbit anti-mouse is required before applying the protein-A gold. Before and after such a step, a series of rinses on drops of, for example, phosphate-buffered saline (PBS) is required for 10 to 15 min. Alternatively, one can use a secondary antibody conjugated to gold (see below).
Step 3 Gold After the rinses following the last antibody step, the grids are oated on the empirically determined concentration of protein-A gold or an IgG gold conjugate. These conjugates can be easily made in the laboratory or, they can be obtained from many commercial suppliers. In the absence of prior information, the optimal concentration of the gold reagent is the highest concentration that gives negligible labeling in the absence of a primary antibody on the sections that have been exposed to blocking solutions. Colloidal gold particles consist of pure gold, made by reducing gold chloride (HAuCl.2H2 O). A number of methods for preparing these colloids have been around since the days of Michael Faraday but the method of choice now is that introduced by Slot and Geuze. By a simple procedure involving citrate and tannic acid this method produces uniform, spherical particles of any size between about 3 and 17 nm; up to three different sized particles can be routinely used for triple labeling studies. Many proteins
3.10.2
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can be adsorbed fairly stably (but mostly noncovalently onto the surface of gold particles), without loss of activity; the most useful (in our experience) is protein-A. The noncovalent interactions of proteins and other compounds with gold is complex and highly unpredictable; one must be aware of the possibility that any compound bound to gold may elute from the particle surface with increasing storage time. Procedures for single and double labeling have been described. Following the gold incubation, the sections must be rinsed extensively (1520 min) and all salts must be washed away with (preferably double or triple) glass-distilled water prior to nal contrasting with uranyl and/or lead salts. The basic procedures for plastic and cryosections are described above. It should also be noted that any particulate material that can be adsorbed to grids can be labeled, but on their outer, solvent accessible surfaces. Only if the structures can be opened up in some way can one potentially access internally localized antigens.
3.11
distilled water and observe them by light microscopy. When the cells burst (usually after about 10 sfor cultured mammalian cells), xative is added. In this approach, as in all preembedding methods one accepts structural damage at the outset. One attraction of the preembedding approach is that at the end of the labeling procedure, the cells are embedded in conventional epoxy resins, that is, technology available in every EM lab for cell biology. Since the ultimate goal is to see the sites of antigens in the context of cell ultrastructure, it is essential to chemically prex the material before labeling. However, this raises a practical dilemma; if cross-linking is too effective, the antibodies fail to reach many antigens. If, on the other hand, one cross-links too little, cellular ultrastructure is compromised. Since one needs to sequentially introduce antibodies and secondary gold reagents, preembedding labeling is a highly empirical and unpredictable method. Nevertheless, when it works, this approach can provide significant information (see Fig. 10). The essential steps in a typical preembedding protocol are as follows: 1. The cells/tissue slices are xed lightly, usually with 2 to 4% formaldehyde (often mixed with a low concentration of glutaraldehyde), usually for less than 30 min. 2. For labeling intracellular antigens, the cells are permeabilized, most often with 0.1 to 0.3% of the detergent Triton X100 for a few minutes. If the goal is to label antigens on the outer surface of cells this step can be left out. This step, in conjunction with step 1, is difcult to control and is prone to induce artifacts. 3. The permeabilized cells are treated with a protein blocking solution (see labeling of sections above) and then with the
Preembedding Labeling Methods
Whereas most EM labeling protocols these days is done using thawed cryosections or methacrylate sections, there is an alternative set of methods referred to as preembedding labeling that may be preferred for some applications. The principle here is to start with whole cells or tissue slices (e.g. 2050 m thick cryostat or vibratome or tissue/chopper fresh sections) and then treat the material with a solvent or detergent (or even by freeze-thawing) that removes parts of membranes in order to allow antibodies to diffuse into the cell interiors. One simple method that we often use is to put the live cells into
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5 m
Q7
Preembedding labeling. The replication of chromatin in synchronized HeLa cells has been studied using both light and EM immunodetection. The inset shows individual small uorescence foci corresponding to DNA replication sites during early S-phase (100 min after release of cells from a double thymidine block of DNA replication). Biotin-16-deoxy-UTP was used as a marker of the newly synthesized DNA. This was delivered into cells by means of hypotonic shift procedure and was visualized by a secondary, uorescent antibody against biotin. The corresponding EM image is seen in the main gure. For this, the mouse antibiotin was recognized by a 1-nm gold anti-mouse antibody that was subsequently silver-enhanced. The arrow indicates the sites at the periphery of the nucleus (N) where the silver-enhanced gold accumulate, which reects the DNA replication sites corresponding to the immunouorescence foci seen in the inset. Micrographs courtesy of Ivan Raska.
Fig. 10
optimally determined concentration of antibody (in blocking solution) for periods ranging from 30 min to overnight. The excess unbound antibody must be removed by multiple rinsing steps with buffer (e.g. phosphate-buffered saline, PBS). 4. The cells are incubated with a gold reagent (usually for 30 min to 2 h at room temp) that recognizes the antibody (as for section labeling); also here it is crucial to have the optimally determined concentration of marker. The excess is again rinsed away. 5. The cells can now be xed with a high concentration of glutaraldehyde
(0.52% for 3060 min) in order to protect the structure against the subsequent steps of dehydration and embedding. The remaining procedure is identical to the protocol given above for epoxy resin embedding. Accessibility of reagents to antigens is often a severe problem in preembedding labeling, with the difculties increasing as one looks for antigens deeper in the cell, especially within the nucleus. By this approach, a negative result is almost impossible to evaluate; one can never be certain whether or not the reagents had access to the antigens in a dened structure. At the primary antibody level,
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antibody fragments such as Fab (which have a signicant lower avidity than whole IgG) may improve access. At the gold step, it makes sense to use the smallest sizes of gold. An important innovation here was the commercial availability of 1 to 2 nm gold particles bound with secondary antibodies or protein-A. A number of studies have shown that these penetrate signicantly better than 5-nm gold particles. The 1- to 2-nm gold particles are very difcult to see in a typical section. To overcome this problem, metal amplication procedures using silver or gold have long been available and widely used to enhance the size of the gold particles. It should be noted that osmium tetroxide can strip the silver off the gold particles. At the end of this procedure, thin plastic sections of cells are examined in which gold particles provide an indication of the sites of antigens.
3.12
Immunohistochemical Approaches using Horseradish Peroxidase and Cytochemistry
for a number of reasons, this approach cannot be considered a state-of-the-art method for immunolabeling. HRP has also been widely used as a marker of endocytic organelles, and cells will conveniently take up this compound by endocytosis. More recently, cDNAs encoding for this protein as chimeras with different targeting signals have been used to direct the protein to different biosynthetic compartments, such as the endoplasmic reticulum and the Golgi complex. This approach can be very useful to facilitate structural details of these organelles and is likely to be increasingly important as an additional marker for tomographic studies. It has also been used in an interesting method for visualizing endocytic organelles in whole mount cell preparations. For structural identication of membrane compartments an old approach enzyme cytochemistry is still a useful method, although its use has been declining. It is expected that this approach will be used more often for tomographic studies. An example is provided in Fig. 7.
The Use of Microwave Technology for Specimen Preparation In 1970, Mayers rst introduced the idea of using microwaves to enhance the rate and efciency of xative cross-linking. Since that time, this approach has become widely used, not only for the xation step but also for other steps in specimen preparation. Although the precise effects are still not fully understood, specimen exposure to microwaves results in greatly enhanced rates of penetration of chemicals into tissues as well as chemical reaction rates. In the microwave processor, reactions that take hours to occur by diffusion alone can occur in seconds to a few minutes. It is important to note that
3.12.1
Q10
For preembedding labeling, it is also possible to visualize the bound antibody using a secondary antibody bound to horseradish peroxidase (HRP). This reagent penetrates into cells better than gold particles and has the advantage that the enzymatic properties of HRP can be used to oxidize diaminobenzidine (DAB) into an insoluble polymer that binds O5 O4 very well. This cytochemical electron-dense reaction product can be used as an indicator of the site of the antigen. Because of the ability of the reaction product to diffuse, this method is rarely successful for cytoplasmic or nuclear antigens. In the past, it was widely used to localize antigens within the lumen of membrane organelles. However,
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standard kitchen microwave ovens are not recommended, since they are difcult to control and, even with short pulses, tend to overheat the specimen. Specially designed laboratory-grade microwave processors are now available that offer variable, controllable wattage and specimen temperatures. Temperature control is achieved in part by circulating large volumes of water through the microwave chamber to remove excess heat. In our group, following the lead of Paul Webster, we now routinely carry out all steps of epoxy resin embedding using this technology. Whereas the overall process used to take two to three days, it can now be completed in a few hours. The microwave oven is also likely to be an important tool in the future for immunocytochemistry by speeding up the penetration and binding rates of antibodies for immunolabeling. Its role in improving antigen accessibility to antibodies is also being exploited., For an example of tissue sections following microwave-assisted xation, see Fig. 7. It is likely that this technology will play an increasingly important role in many aspects of EM in the future.
whole era of studies that were initiated by Fernandez-Moren in the 1950s. It is estimated that a freezing rate of 105 to 106 C s1 is required to vitrify water at ambient pressures. No method is known that allows cells (or parts of cells) greater than 10 m in thickness to be vitried under these conditions. Only at high pressures can this be achieved to a depth of a few hundreds of microns (see below). Owing to the high rates of cooling that are used, it has been estimated that the process of vitrication arrests the in vivo state within 100 m s1 of the nal perturbation. For this reason, it has evocatively been described as the solidied in vivo state. There are ve different cryo-EM methods we shall discuss: (1) the bare-grid method for isolated particles; (2) hydrated cryosections; (3) freeze-substitution; (4) freeze-fracture and (5) cryo-SEM approaches.
4.2
The Bare-grid Method for Particulate Specimens
Cryo-EM Approaches
4.1
Vitrication
The pioneering ideas of Dubochet on the concept of vitrication was the beginning of a new era in EM. The fact that one requires such a thin specimen for TEM allowed Dubochet and McDowell to vitrify a thin layer of water suspended over the holes on an EM grid. Electron diffraction conrmed that the solidied water was not crystalline but remained amorphous. This breakthrough was the culmination of a
Viruses, small bacteria, isolated organelles, laments, macromolecular complexes, and the like can easily be vitried. The remarkable consequence of the thinness of these specimens is that an almost trivial procedure allows preparations to be routinely vitried. Following the innovation to use a perforated lm grid support, the method involves suspending the specimen on such a grid mounted in a simple guillotine device. For the best results, the atmosphere around the specimen should be kept humid and specialized devices are now available for this purpose. After a brief blotting on one or both surfaces of the specimen, the grid, attached to a forceps, is rapidly shot into a 2-cm deep chamber of liquid ethane cooled by an
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external container of liquid nitrogen. All the subsequent operations such as transporting, storage, mounting in cryoholders and imaging in the electron microscope, are done at liquid nitrogen temperature to avoid devitrication, which occurs above 135 C. The imaging of cryospecimens requires special care because highly energetic electrons, while passing through a sensitive specimen, can easily destroy the ne structure. The so-called low dose imaging has, therefore, been developed (see below). It should be noted that the cryo-EM approach offers the only possibility in EM for visualizing details within the interior of a structure directly. State-ofthe-art examples of images obtained by the bare-grid approach are shown in Figs. 11 and 18(a). This approach has been used for a series of elegant time-resolved analyses, especially by Nigel Unwin and colleagues. For example, the acetylcholine receptor has been visualized in different functional states. The bare-grid approach can also be combined with immunogold labeling and with positive staining using heavy metal salts. This is probably the EM method whose usage is increasing the most in laboratories worldwide.
4.3
Vitrication of Larger Material
The cryopreservation of material larger than 1 m is a topic that has been extensively investigated. Despite earlier complications, it is now generally accepted that the goal is straightforward; the specimen must be vitried if it is to provide a faithful reection of the structure of the in vivo state. The hydrated cryosection method (see below) offers the only approach that can allow sections of native material to be evaluated. It is also the only
method for unambiguously conrming, via electron diffraction, that the vitreous state has indeed been achieved; although large hexagonal ice crystals can be seen directly without diffraction, the presence of the intermediate cubic ice crystals (up to 300 nm) cannot. These can also damage specimens. There are different approaches for vitrifying a layer of biological material thicker than 1 m; one can consider the more traditional methods such as (1) plunging in ethane or propane; (2) jet freezing; (3) slam-freezing on a cooled silver or copper surface. There is now a consensus that all of these approaches unfortunately, fail in practice to give a layer of vitrication that is more than 5 to 10 m. A relatively old method, high-pressure freezing, rst introduced by Moor, has recently emerged as the technique for vitrifying substantial pieces of biological tissue. A reasonable consensus in the eld accepts that a layer up to 200 m (perhaps thicker) can be vitried by this approach. Highpressure freezers are now available from three commercial companies and the application of this approach is growing slowly but steadily, worldwide in established EM laboratories. It should be emphasized that the technology involved is not trivial and there are many technical tricks that can be learned only from the specialists (this is generally true of all EM methods). Examples are shown in Figs. 1315(b). Below, I would like to point out two very powerful adaptations that, in combination with high-pressure freezing, can be very powerful tools.
4.4
Q11
The Cellulose Capillary Tube
Hohenberg has introduced the use of cellulose microcapillary tubes in order to
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Q12
prepare material for HPF. By capillary action, an aqueous suspension can be drawn into these tubes of 200-m diameter, which can be easily sealed by force. The cellulose wall allows molecules up to 10 KDa to diffuse freely through it. Cultured cells can be grown within the tubes on their preferred media prior to freezing (see Fig. 13(a)). For high-pressure freezing, small fragments of these tubes can be conveniently positioned in the specimen holder of the machine. This approach
can also be useful with other methods, for example, epoxy resin embedding or the Tokuyasu cryosection method.
4.5
The Fine-needle Biopsy
A second innovation by Hohenberg is potentially of enormous interest to pathologists. A specially designed jet micro-needle system has been developed that can cut 200-m thick slices of any living tissue.
(a)
(b) Kinesin decoration

Fig. 11
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These slices are ideal for high-pressure freezing. The use of this system for taking micro-biopsy specimens from human patients is now a routine (and for the patient apparently painless) procedure by this group and their medical collaborators.
4.6
Hydrated Cryosectioning
Vitried thick specimens must be sectioned before analysis by TEM. The simplest method in theory, that is by far the most difcult to apply, is the hydrated cryosection method. The theory is indeed simple, one vitries a piece of tissue and prepares cryosections (at a temperature below the recrystallization temperature of vitreous water (<135 C)). These sections can be transferred onto EM-grids kept under liquid nitrogen until they are directly visualized (in the absence of any chemical) at 160 C, or below. In principle, this method allows direct visualization of native structure in the absence of any chemical. In practice, the technique is quite demanding and is hampered by sectioning
problems (cutting artifacts). Foremost among these is the severe compression (routinely 3050%) that in fact accompanies all sectioning methods (in all other sectioning approaches for EM, the sections have the opportunity to stretch (decompress) on aqueous solutions). It seems, however, that in recent years all the major technical problems have found acceptable solutions and the method is now at the stage where it is ready to full its promises. Moreover, new innovations are on the horizon, such as the vibrating diamond knife already mentioned that can considerably reduce the compression artifact and the use of tomography for providing three-dimensional information from such specimens. For recent examples of this approach, see Fig. 12.
4.7
Freeze-substitution
While the frozen-hydrated cryosection approach is currently something for a specialist, the approach of freeze-substitution offers a much easier-to-apply alternative method for visualizing the (close to) in
Fig. 11
Cryo-EM. (a) Cryoelectron micrograph of undecorated microtubules, embedded in vitrous ice. When assembled in vitro, microtubules may be formed from different numbers of protolaments (which can be clearly seen in Fig. 22 after metal shadowing). A computer-assisted 3-D reconstruction in the inset (color) has been carried out on a 15-protolament microtubule. One of them is marked with an arrow. The number of protolaments gives rise to different moiree patterns, which can be seen on a careful inspection of the different tubes. For example, 13-protolament microtubules exhibit very straight lines along the axis, while 15-protolament microtubules show a characteristic alternating pattern of fuzzy and striated regions along the axis. This pattern allows one to identify different types of microtubules. (b) Cryomicrograph of microtubules decorated with kinesin motor domains; the corresponding model is shown in color. The motor domains appear as little globular blobs along the microtubule (arrows). These motor domains also have an intrinsic stabilization effect that creates long isolated protolaments. The inset shows a helical 3-D reconstruction of a microtubule decorated with dimeric ncd domains. Ncd is a kinesin family member that exhibits retrograde directionality, and a unique microtubule-binding pattern, but otherwise shows strong structural similarities to conventional kinesin. It is important to realize that cryo-EM is the only approach that provides information about structural features within an object. Images courtesy of Andy Hoenger.
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20 nm
(b)
(c)
(a)
Fig. 12
50 nm
(d)
(e)
Hydrated cryosections. In this approach, no chemical treatment whatsoever is used. (a) Shows a hydrated cryosection of stallion sperm chromatin after partial decondensation with 10 mM of the reducing agent DTT. The individual DNA laments, whose diameter is 2.7 nm, can be clearly seen, as can their organization into a hexagonally arranged liquid
crystal; (c) and (d) show different orientations of DNA; a diffractogram is evident in (b) showing the hexagonal spacing of the DNA. (e) Shows a hydrated cryosection through puried nucleosome cores that have self-organized in vitro into precisely aligned columns of particles. Both micrographs courtesy of Jacques Dubochet.
vivo structure. Although in this method the specimen is dehydrated and embedded in plastic, a comparison of the data from hydrated cryosections and freezesubstitution has nevertheless convinced most specialists that the latter approach offers many advantages over the former, except at the highest levels of resolution. Freeze-substitution is an old EM method that went out of favor only to emerge over the past decade as the best routine method for preparing sections of faithfully preserved cellular material, thanks especially to the pioneering work by the groups of M ller and Steinu brecht. It can provide not only relatively high quality structural preservation but also the opportunity to label the sections (in contrast to hydrated cryosections) using immunogold methods. The fact that high-pressure freezing, but no other approach, allows vitrication of up to
hundreds of m of depth in the specimen now makes this approach the method of choice for freeze-substitution. The principle behind freeze-substitution is to inltrate the vitried specimen with solvents at low temperature that, over a period of many hours to days, gradually replace the water with the solvent (often mixed with stabilizing xatives, such as OsO4 (that cross-link lipids as the temperature is raised), or uranyl acetate (that can stabilize the head groups of phospholipids). The approach can also be carried out without any chemical xatives. The idea here is to start the process of switching the specimen from a water-containing (vitreous) medium to a solvent that is compatible with the plastic embedding media at low temperatures (see below) while the structure of the biomolecules is still rigid. This is routinely done at, or colder than 80 C. It cannot be ruled out that some cubic ice crystals may form under these conditions but even
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if they do they are not noticeable at the levels of resolution usually desired. In freeze-substitution, the tissue is either chemically xed at low temperature and left to x as the specimen is slowly warmed up, or left unxed and embedded in specially developed methacrylate resins (such as Lowicryl HM23) at 70 to 90 C, using UV polymerization. When labeling of antigens is not required, the specimen is warmed to room temperature, embedded in Epoxy resin, and then sectioned as in the conventional method. Figures 13 and 15 show images made using this approach. An alternative method that is not often used but may be appropriate for some specimens is to combine freeze-drying with freeze-substitution.
4.8
Freeze-fracture and Replica Methods
as evident in the pioneering studies of John Heuser, Tom Reese, Nobutaka Hirokawa and others. The underlying tissue is removed from the metal replica by harsh conditions such as immersion in chromic acid and the ne replica is attached to an EM grid and visualized by ambient temperature TEM. Thus, although this is a bona de cryobased method, the replica of the frozen surface is nally visualized at room temperature. A number of methods have been developed for combining freeze-fracture with immunolabeling. An especially interesting approach has already been developed; this method uses the detergent SDS to partially digest biological material from the replica while retaining integral membrane proteins whose exposed cytoplasmic domain are accessible for immunolabeling. Figure 16 shows two striking examples using this approach.
4.9
Q13
In the 1960s and early 1970s, freezefracture was a widely used approach, especially for the study of membranes. Although now restricted to a few specialist groups in the world, it is still the only method that can allow the investigator to see the insides of membranes. Small pieces of specimen are rst vitried, using a method such as slam-freezing against a cooled copper block or high-pressure freezing. Subsequently, the specimen is fractured, either with a knife, or by physically separating the frozen specimen that is sandwiched between two metal plates. The freshly opened surface is either evaporated with a metal replica directly, to reveal the surface details of the cut specimen, or the specimen is rst allowed to warm up briey to 90 C in order to etch away a layer of water. In this case, the replica also reveals some subsurface details; this is especially useful for visualizing details of the cytoskeleton,
Simple Shadowing Methods
Since the dawn of the EM era, simple metal shadowing methods have proven useful as a substitute for negative staining that show particle surface structure. Any isolated particles, such as those suitable for the baregrid cryo-EM negative-staining approaches can be usefully examined using this approach. When done well, the appearance of the structure can appear more threedimensional than with negative staining. In the simplest instances robust structures can be air-dried before evaporating metal in a unidirectional, or rotary fashion. Better results are usually obtained after a more gentle preparation method, such as freeze-drying. Two examples are shown in Fig. 23. Another related method is glycerol spraying whereby the sample in glycerol is sprayed, then rotary shadowed and viewed
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by TEM. This approach has been widely used to visualize isolated nucleic acids and attached complexes.
4.10
The Kleinschmidt and other EM Methods for Nucleic Acids
A useful method for visualizing DNA or RNA molecules, and attached complexes
was developed by Kleinschmidt in 1959. The molecules are adsorbed to a thin, positively charged protein monolayer spread on a water surface. Cytochrome-C is the most commonly used spreading agent. The lm is then picked up on a specimen grid, contrasted with stain (e.g. uranyl acetate) or by rotary shadowing with platinum, and observed in the microscope. This method may not have the resolution
Tube wall 1 m (a)
PM
1 m (b)
Fig. 13
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of the cryo-EM method but is more suitable for estimating parameters such as the length of the bers. About 10 years after the introduction of the Kleinschmidt method, the heteroduplex analysis for mapping regions of base sequence homology in nucleic acids was established, particularly in combination with formamide as a denaturating agent. The use of a detergent lm (BAC) to replace cytochrome-C later allowed a better resolution of the nucleic acid strands.
4.11
EM Autoradiography
Until the 1970s, the use of EM autoradiography had a central place in cell biological research. In this approach, a radioactive molecule is introduced into the cells/tissues as a pulse of signal; the classical example was the use of radioactive amino acids or sugars that are incorporated into newly synthesized proteins or glycoproteins. By removing the radioactive precursor, a period of chase allows the tracer to be followed sequentially in the cell. This approach was used to follow the ER to the Golgi pathway for secretory proteins and glycoproteins by the groups of Palade and Leblond in the 1960s. At the end of the experiment, the cells/tissues are chemically xed and embedded in epoxy
resins and sectioned. Subsequently, a thin lm of radioactivity- (and light-) sensitive photographic emulsion is layered on the section surface under a red light source in the dark room. The sections must then be incubated for days, weeks, or even months for enough radioactive encounters to interact with the lm. At the end of the incubation, the lm is developed and visualized under the EM. Although in principle an excellent method to follow dened molecules, a signicant disadvantage is the relatively low resolution, at best 0.5 m. This, plus the long waiting period, has resulted in its almost total disappearance from the cell biological scene during the past 20 years. Cell biological problems that used to be analyzed by autoradiography are now better resolved by use of specic antibodies; kinetic results can then be achieved by use of specic inhibitors, such as cycloheximide, that can help to synchronize the synthesis of proteins, for example, through the biosynthetic pathway.
EM-Visualization at Ambient Temperatures
All EMs are rather sophisticated machines because electrons can only be usefully
Fig. 13
High-pressure freezing and freeze-substitution. Shows freeze-substituted specimens following high-pressure freezing. (a) Shows the nematode Diploscapter coronata having been vitried within a capillary tube (the tube wall is indicated); the freeze-substitution was done using osmium in acetone. I-intestine; G, gonad (b) Shows a section through a high-pressure frozen and freeze-substituted yeast cell (Saccharomyces cerevisiae). The specimen was embedded in Lowicryl HM20 resin. Yeast cells are notoriously difcult to preserve by conventional xation methods due to their robust cell wall. This image shows remarkable preservation. The arrowheads indicate direct continuities between the nuclear envelope and the endoplasmic reticulum. This ER, seen in negative contrast, is continuous with a domain of ER that seems to be very closely attached to the plasma membrane (arrowhead adjacent to the plasma membrane (PM)). The nuclear pores are also evident in negative contrast (arrows). Mitochondria (M) and other organelles are also evident. Both images courtesy of Heinz Schwarz.
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FP
PM
200 nm (a)
Fig. 14
Cryo- and plastic-section immunolabeling of African trypanosomes. These gures show the trypanosome Trypanasoma brucei. (a) and (b) show Tokuyasu cryosections of this organism following labeling for the variable surface antigen (VSG). The VSG is seen to be heavily concentrated at the PM, the agellar pocket (FP, the agellum is indicated by F), throughout the Golgi complex (G), as well as in ER and endocytic organelles (not indicated). The arrow in (a) shows a labeled endocytic vesicle in the process of budding from the agellar pocket membrane. Bars300 nm.
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35
Cryo- and plastic-section immunolabeling of African trypanosomes. These gures show the trypanosome Trypanasoma brucei. (a) shows a trypanosome that was xed in 0.05% glutaraldehyde/2% formaldehyde before being embedded in Lowicryl HM 20 after the progressive lowering of temperature method. The sections were labeled with anti-tubulin and donkey anti-mouse gold (12 nm). The array of microtubules beneath the plasma membrane and in the agellum are strongly labeled (arrow). (b) Shows a trypanosome that was vitried by high-pressure freezing followed by freeze-substitution in 0.5% osmium tetroxide, 0.5% gallic acid in acetone and room-temperature embedding in Epon. The excellent preservation of the microtubules is evident by the clear delineation of the tubulin protolaments in cross-section. Micrographs courtesy .. of Christoph Grunfelder, Peter Overath, and Heinz Schwarz.
FP
200 nm (a)
200 nm (b)
transmitted in a rather high vacuum (better than 103 Pa 104 /105 Torr). This high vacuum in the main column also needs to be maintained when a grid is introduced. For this, the grid is rst introduced into a low volume prechamber (airlock) that is quickly pumped to vacuum. The grid can then be carefully inserted into the center of the objective (electromagnetic) lens. At the top of the microscope column is an electron source (tungsten lament, lanthanum hexaboride crystal) or, the (most coherent and expensive) eld-emission lament. The specimen is
observed at acceleration voltages ranging from 80 to 120 kV (standard) to 200 to 400 kV (new intermediate-voltage microscopes) or up to 1 million volts in the high-voltage EM (of which only a small number are available for biological research worldwide; the others have disappeared due to lack of interest). The recent developments in EM tomography will likely see a reversal of this trend, as methods to investigate thicker sections become more useful (see below). The emitted electrons are attracted toward the anode leading to a coherent
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100 nm
(a)
100 nm (b)
Fig. 16
Freeze-fracture immunolabeling. (a) Shows chick liver, tight junctions labeled for the tight junction protein occludin while (b) indicates rat liver, gap junctions double-labeled for connexin 32 (large gold particles), and connexin 26 (small particle). The tissue slices were quick-frozen by contact with a copper block cooled with liquid helium. The frozen samples were fractured in a Balzers BAF 400 T freeze-etch unit at 110 C, replicated by deposition of platinum/carbon (Pt/C) followed by carbon. After thawing and washing with PBS, the pieces of Pt/C replica were transferred to 2.5% SDS containing-buffer for 12 h at room temperature. Subsequently, the replicas were immunogold labeled, rinsed, xed with glutaraldehyde, rinsed again, and picked up on grids. Micrographs courtesy of Kazushi Fujimoto.
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beam of electrons that, with the help of an electromagnetic condenser lens, can then be converged onto the specimen. Electrondense material (biological material heavy metal stains) scatter the electrons, whereas the electron-lucent parts of the specimen allow the electrons to pass down the column where, after magnication by the objective and projector lenses, they form an image on a phosphorescent screen, lm or CCD camera. There are two different types of interactions between electrons and biological material; elastically scattered electrons interact with the specimen with a minimum loss of energy and therefore there is no change in the wavelength of the electrons. This type of scattering is due to a simple deviation of the electrons in the atoms in the specimen. In contrast, inelastically scattered electrons are those that experience energy loss and change of wavelength during their interactions with the electrons of the encountered atoms. These interactions give rise to the secondary events, such as heating the specimen, mass loss, contrast, X-ray emissions and cathodoluminescence. When electrons interact with the phosphorescent screen, the green light emitted by uorescence provides a signal that the human eye sees and the brain needs to interpret. By removing the phosphorescent screen transiently, the electrons can be directed toward photographic emulsion to record the image on lm. Cooled CCD cameras are now rapidly replacing lm but they are very expensive and still do not have the resolution available on lm. It is likely to be only a matter of time before CCD cameras replace lm completely. The inelastically scattered electrons passing through the specimen can damage the specimen, resulting, for example, in mass loss, even in plastic sections. However, in practice, beam damage is not a serious
problem for plastic or Tokuyasu cryosections, at the usual level of resolution needed. For vitried, native specimens this is a very serious concern, as we will now discuss.
EM at Cold Temperatures
Imaging vitreous specimens, either bulk suspension, or hydrated cryosections, is a lot more difcult than imaging specimens at ambient temperatures. This is really not something that can be taught in a manual. Moreover, one has to deal with quite delicate and expensive equipment. A competent cryo-EM specialist needs to understand a lot about the physics of the processes involved. Native biological sections are extremely sensitive to the electron beam, and under the conditions used for ambient temperature specimens, they visibly boil away within seconds. This necessitates low-dose imaging. In the standard approach, the investigator can only surmise that suitable specimens are present at a site selected at low magnication, that is, immediately photographed at higher magnication using the minimum electron dose required for imaging. All focusing and image adjustments are therefore done at sites away from the selected areas. In recent times, the technology available with modern EMs has improved considerably, and is now much more user-friendly. In most cases, the specimen is unstained and to acquire sufcient contrast to see the details is often a problem. For optimal images, one takes advantage of the increase in phase-contrast that is provided by underfocusing. Nevertheless, the fact remains that more and more specialists are now routinely recording images of very high information
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content with this technology. There is no doubt that at the top level the cryoimaging approach provides the highest resolution available in EM today (see Fig. 11). In suitable specimens, by averaging data from large numbers of micrographs, it can now approach (or reach) the atomic level of resolution (0.31 nm) when twodimensional crystals are available. In many cases where a 1 to 3-nm resolution is obtained, the level of information often sufces to help one to t higher resolution X-ray crystallographic data of the same structure into a comprehensive model of the structure. In this way, the details of the (high-resolution) X-ray structure are tted onto its overall shape, which emerges from the (lower resolution) cryo-EM model. It is important to realize that in order to solve a structure to the desired level of resolution, the availability of negatives or online images is merely the beginning of a long, complex, and often tortuous process. In contrast to ambient temperature specimens in which the images are mostly nal, in cryo-EM (as well as in some high-resolution negative stained specimen analysis), there is an increasing number of possibilities to obtain much more relevant structural information by use of computational image processing algorithms. The goal of these procedures is to improve signal-to-noise ratio by averaging a large number of structural units. The two main kinds of approach to obtain this goal are the following: (1) Electron crystallography (including helical reconstructions) of twodimensional crystals and (2) Single particle analysis, including icosahedral particles, where particles imaged at various orientations are collected, averaged and superimposed into a three-dimensional model. Examples of cryoimages obtained using sections are shown in Fig. 12 and from
whole-mounts of particles in Fig. 11 and 19(a) and of cells in Fig. 19(b).

6.1
EM Tomography
In the past, if one wanted a threedimensional view of a structure from thin sections it was necessary to use serial sections. The need to cut and maintain in perfect order up to a hundred or more thin sections is technically quite demanding and, until recently it was very difcult to subsequently align images from each section on top of each other in perfect register. In the past few years, a revolutionary alternative approach has started to have a major impact on EM at all levels. From either a single particle or a relatively thick section, tomography allows one to obtain a three-dimensional reconstruction of the structure of interest. In practice, a device for tilting the specimen, the goniometer stage, allows the grid to be tilted relative to the beam (under standard conditions + and 70 ; new goniometers are now reaching 80 ). Images of all the projections are collected, usually at intervals of 1 to 2 . By backprojecting these images into a threedimensional volume one can construct the original image in three dimension using now standard computational algorithms. The reader is also referred to a whole volume of the Journal of Structural Biology that was dedicated to the exciting developments in this eld (Vol. 138, pp. 1155, 2002). There are a number of problems that can limit this approach. First, since there are a large number of images, the effects of beam damage have to be considered. In practice, this is not a serious problem for the ambient temperature specimens indicated above: one can take 130 to 140
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images, 1 apart. However, for vitried particles or sections, this problem is still a limitation; in practice one is currently restricted to, at most 100 images before the specimen is irreparably damaged. It should be emphasized that a great deal of effort has been made by a few specialists to make this technology available, with important developments in software, automatic image collection and in specimen preparation. The second problem is the missing cone or wedge due to the practical limitation that one cannot tilt the grid 90 there will always be a part of the information that is missed, resulting in loss of resolution. For quasi-regular particles, one can overcome this problem by averaging a number of reconstructions from different viewpoints. For imaging complex structures in sections of cells, it may be possible to combine tomograms from sections that contain the specimen at different orientations. In addition to being used in highresolution EM for reconstructing single particles such as ribosomes this approach, in combination with high-pressure freezing and freeze-substitution, is now being applied to analyze the three-dimensional organization of different structures, such as whole yeast cells, the Golgi complex, mitochondria (see Fig. 18). Most of these studies have used relatively thick (200 300 nm) plastic sections. It should be noted that for the difcult question of deciding whether or not ne tubules connect certain structures (notably the Golgi complex), this approach can still leave open considerable room for subjectivity. In such cases, the use of heavy metal stains (e.g. the cytochemical reaction product of HRP, or other enzyme reaction products) to ll the lumen of tubules (e.g. by expressing
HRP-containing constructs in cells) can greatly aid the analysis. The ability to visualize whole cells directly by a transmission cryo-EM approach can be considered the ultimate goal in electron microscopy. It is now fascinating to consider that, prior to the development of robust methods for preparing thin resin sections of cells, the famous trio of Porter, Claude, and Fullam had made initial attempts in 1945 to visualize cells grown on EM-grids (after osmium tetroxide vapor or formaldehyde primary xation). Despite the harshness of the preparation (by todays standards), what was observed was striking. In the thinner, peripheral regions of the broblast, whose outlines are clearly delineated, one can clearly see mitochondria, laments and so on (see Fig. 17). In an important development, the Baumeister group have succeeded in bringing together a whole battery of technical developments to enable the experiment to be redone with state of cryo-EM tomography. In this publication, they focused on the amoeba, Dictyostelium, that in accompanying videos could be seen to move rapidly across the grid just before vitrication. Although one has to accept the (still signicant) limitation that only cell projections thinner than 400 to 500 nm could be clearly analyzed, the details shown are amazing, at a resolution of 5 nm. It must be realized that this is the rst time it has been possible to look directly inside a relatively unperturbed cell at this level of resolution. An example is given in Fig. 19(b). With more developments, and more widespread use of this approach, (likely aided by higher accelerating voltages and energy ltering) thicker regions of the cell will become more accessible for analysis. The use of hydrated cryosections
Q14
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Fig. 17
One of the rst micrographs of cells. Shows a famous micrograph taken from the paper by Porter, Claude, and Fullam from the Journal of Experimental Medicine (1945). This micrograph is one of the rst ever taken by electron microscopy. The cells were grown on an EM grid, xed in osmium tetroxide, air-dried, and visualized as a whole mount. Micro spikes (arrow) at the leading edge are evident, as is the nucleus and the worm-shaped mitochondria. A ne reticulum beneath the mitochondria is likely to be the endoplasmic reticulum. This image has already been shown in a wonderful book by Christian de Duve in 1984.
1 m
can be expected to complement these efforts. The ability to reconstruct detailed structures (e.g. within the nucleus) will greatly increase the level of information one can obtain from these preparations. Tomography can also greatly simplify the interpretation of the three-dimensional structure from hydrated cryosections.
6.2
Low- and High-resolution SEM
The ability to visualize surface structures by scanning EM is well known, even to many nonscientists, a result of spectacular images of animals, plants, and microorganisms obtained by this approach. Many specimens, such as insects have robust
exoskeletons that can withstand beam damage and only a thin layer of metal needs to be evaporated before visualization; for a beautiful example see Fig. 20. This low-resolution SEM approach method is widely used. At a higher level of resolution, parts of the organ of Corti of the ear are seen in Fig. 21. Over the past 20 years, the use of higher resolution SEM in molecular cell biology has dwindled only a handful of laboratories currently use this approach. This is indeed surprising, and very disappointing given the proven ability of this approach to see high-resolution details of structures such as nuclear pores, bacteriophages (Fig. 22(a, b)) and actin laments (Fig. 22c).
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60 (a)
+60
(b)
(c)
(d)
Fig. 18
(e)
EM Tomography from plastic thick sections. (a) To obtain a three-dimensional reconstruction of an organelle, a tilted-series of images is recorded. A 250-nm section of multilamellar lysosomes in a mouse dendritic cell is shown after high-pressure freezing, freeze-substitution, and resin embedding. This, relatively thick section was tilted from 60 to 60 along two perpendicular axes to produce a tilt-series of 242 images. This panel shows three of these images, collected at 60, 0, and 60 s tilting. The small black dots in the images are 10-nm gold beads on top of the section, which are used as ducial markers (reference structures) for the three-dimensional reconstruction. (b) After calculating the three-dimensional reconstruction from the tilt-series, the three-dimensional volume can be analyzed by displaying it as thin, so-called tomographic slices. The slices can be made along every possible axis. Three slices through the tomographic volume are shown: top: X-Z, middle: X-Y and right: Y-Z. (c) By manually tracing the membranes of the multilamellar lysosomes in the tomographic volume a three-dimensional model is created. (d) Shows a three-dimensional model top view of one of the tomographic slices, while (e) reveals the three-dimensional model view of the endocytic vesicle. The gure was provided by Jean-Luc Murk and Bruno Humbel. From a study by Murk et al., J. Microsc. in press.
In conjunction with immunogold labeling prior to vitrication, the use of cryo-SEM, especially the remarkable new generation of eld-emission (FE) scanning electron microscopes offers the molecular
cell biologist a complementary approach to tomography. SEM visualizes the surface of the structure directly in three dimension. In conjunction with the pioneering efforts of a few specialist groups (especially those
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of Martin M ller, Paul Walther, Hans Ris u and Terry Allen) in developing new approaches for specimen preparation, this method is now capable of extremely highresolution (13 nm) (Fig. 22). A number of publications have shown the great potential of this approach, also for immunogold labeling, especially when gold particles as small as 1 nm are used, that can be visualized in the backscatter mode.
In some studies, Fab domains have been used instead of whole IgG molecules to increase the precision of the labeling.
6.3
Critical Point Drying
The most common method to prepare living specimens for SEM has been chemical xation followed by critical point
100 nm (a)
Actin
VV-Int
ER
MT
R Core Actin PM 100 nm (b)
Fig. 19
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Fig. 20
43
Classical SEM. Shows a beautiful image of Drosophila melanogaster visualized by conventional SEM. The insect was xed with glutaraldehyde, rinsed, critically point-dried and sputter coated using gold-palladium. Fine details of the surface of the insect can be seen in artistic detail. Micrograph .. courtesy of Jurgen Berger (Max Planck Institute for Developmental Biology, Tuebingen, Germany).
drying (CPD). The basic idea of this approach is to prevent drying artifacts, which occurs when the sample is exposed to the surface tension of the liquidgaseous phase border. This method has also been extensively used for preparing material for TEM replicas and for epoxy resin embedding. For this, the specimen is chemically xed and dehydrated in solvents such as ethanol (essentially as for epoxy resin embedding). Later the solvent is replaced by liquid carbon dioxide in the pressure chamber of the critical point drying device. Then, the carbon dioxide is slowly warmed up. This causes the pressure to rise in the
closed system. When the temperature and pressure pass above the so-called critical point of carbon dioxide (31 C and 74 bar), the pressure is carefully reduced and the sample removed once room pressure is reached. The specimen is thus dried without ever having seen the liquidgas phase border, which can cause major distortions to a sensitive specimen. Recent applications of the CPD method in cell biology are the preparation of cytoskeleton elements after detergent treatment and TEM replica. As pointed out by Ris, it is very important to remove every trace of water from the specimen before the next stage, which
Q8
Cryo-EM and tomography. (a) Shows cryo-EM of vitried vaccinia virus using the bare-grid method. This virus is enormously complex as was already evident in Fig 4. Although a tremendous amount of information is present in these particles, at present the detailed threedimensional organization of the virus remains to be deciphered and the tomographic approach is now in progress. (b) Shows the 2002 equivalent of the experiment already carried out. Hela cells were grown on EM-grids and vaccinia virus was added. The goal of this experiment was to visualize intermediates in the entry of the virus into the cell, a highly complex and not understood process. The grid was vitried and examined by cryo-EM. This image is a section through a tomogram (not a physical section) showing details that are not approachable by single projections. The upper particle is, we believe, an intermediate (from VV-Int) in the entry of the viral core and is likely to be still outside the cell. In contrast, the viral core (c) indicated, which is transcriptionally competent, having lost its outer membranes is denitely within the cytoplasm. Actin laments are seen in the lower part of the micrograph, whereas the laments above the core are microtubules. Ribosomes (R) are also evident, as is the ER. This method is the rst approach available for looking inside physically and chemically unperturbed cells by EM at this level of resolution. Bars = 100 nm. Both micrographs courtesy of Marek Cyrklaff; specimen provided by Jacomine Krijnse Locker.
Fig. 19
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Fig. 21
10 m
SEM of the organ of Corti of the mouse ear. These SEM images show a guinea pig cochlea dissected to remove Hensen cells (sensory cells of the ear and the supporting cells). Hensen cells were dissected to give a clear view of the outer hair cells, the sensory cells of the ear (2) and supporting cells. The tops of the outer hair cells have stereocilia attached ((3) the chevron-shaped projections) and the main body of the outer hair cell can be seen below. (1) indicates the outer phalangeal cells of Deiter. The specimen was xed in glutaraldehyde before dissection and then inltrated with tannic acid and osmium tetroxide. This facilitated examination without metal coating in a eld-emission SEM (a Philips XL-30 FESEM operating at 5 kV). This specimen preparation method was a modication of the method of Jongebloed et al. (1999). Micrograph courtesy of Paul Webster.
Q15
for SEM involves coating the specimen with a thin layer of metal (see Fig. 23).
6.4
Freeze-drying and Cryo-SEM
The SEM is able to image the physical surface only. In most biological samples, however, the structures of interest are inside cells and tissue. To get access to the structures of interest, the specimen has to be opened by cracking or by sectioning, or the overlaying water must be removed. This is only possible with a solidstate sample. The most straightforward way to solidify a biological sample is by vitrication. In the cryo-SEM, bulk samples can be investigated over a very large range of magnication, from the resolution level of a dissection microscope up to macromolecular resolution. After being immobilized, by cryoxation the samples can be investigated in the cryo-SEM in
the fully frozen-hydrated or in the partially freeze-dried (deep-etched) state. Many artifacts of conventional chemical xation and dehydration techniques can, thereby, be prevented. Removal of the vitried water is possible by sublimation in the vacuum in the microscope or in a preparation chamber. This preparation step is also called freeze-etching with a partial sublimation of the water, or freeze-drying in the cryoEM literature. When the water is partially removed, the three-dimensional arrangement of the structures in the cytosol can be investigated. Current methodological research focuses on a better control of the ice-sublimation process. This method also has a high, and still not fully used potential for the analysis of in vitro systems in cell biology. Together with Paul Walther, our group is currently investigating actin-membrane interactions using these cryo-SEM approaches.
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100 nm (a)
100 nm (b) 10 nm (c)

Fig. 22
High-resolution cryoscanning EM. (a) and (b) show the T-even bacteriophage Tull 46 after freeze-drying, metal coating and visualization by cryoeld emission SEM. (a) shows an intact particle, whereas (b) is a particle that became disrupted by spesimen preparation. (a) is additionally labelled with an Fab directed towards the tail protein Y of the phage. Individual Fab molecules can be seen decorating pieces of the isolated tail (small arrows) as well as the helical ribbon of the tail protein. In (b) following disruption, the DNA of the phage is clearly evident (large arrow), as are many other structural details of the inner parts of the virus. (c) shows high-resolution image of an actin lament that had been freeze-dried and coated with tantalum-tungsten. ln(a) and (b) by courtesy of Rene Herman, Heinz Schwarz, and Martin Mueller. (c) Courtesy of Paul Walther.
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100 nm (a)
100 nm (b)
100 nm (c)
Fig. 23
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6.5
Coating Techniques for Cryo-SEM
Samples for high-resolution SEM are usually coated with a thin metal layer in order to enhance electrical conductivity and to localize the signal on the surface of the sample. Charging of the sample can also be reduced by working at low accelerating voltage of the primary beam (e.g. 2 kV). It was, however, pointed out, that the excitation volume of the secondary electrons is increased when they interact with an insulator. Therefore, it is advantageous to work with coated (shadowed), conductive samples, even when working at low voltages. Relatively thin samples can ideally be supported by a uniform layer of an electrically conductive material, for example, by 1 nm of tungsten. Bulk cryofractured samples, however, are insulators and very beamsensitive. For these samples, double layer coating has been very useful. The sample is rst coated with a contrast-forming layer of heavy metal (platinum or tungsten), both to enhance electrical conductivity and for mechanical stabilization. A carbon layer with a thickness of 5 to 10 nm is then applied on top of the platinum
layer. As shown in different studies, this layer drastically reduces the effects of beam damage. For the analysis of these samples, the backscattered electron signal has to be used, which is mainly produced by the metal coat that stays in direct contact with the biological structure of interest.
6.6
Scanning Transmission EM
A specialized variation of transmission EM is scanning transmission electron microscopy (STEM, done mostly in the dark-eld mode) in which the specimen on the grid is scanned in a raster pattern with a nely focused electron beam. This offers precise determination of the absolute mass of the specimen irrespective of shape.
6.7
X ray Elemental Microanalysis by EM
X-ray microanalysis of biological specimens can be conducted at two different levels of resolution. Low-resolution microanalysis is typically performed using a scanning electron microscope equipped
Q9
Metal shadowing for EM. (a) Shows the identical TMV particles that were seen by negative staining in Fig. 3. This unpublished image, over 30 years old, was unidirectionally shadowed with platinum. (b) Unidirectional surface shadowing (tantalum/tungsten, elevation angle 45 ) of a microtubule stabilized with small amounts of tau-protein. This protein is essentially invisible due to its highly lamentous structure. This leaves a surface pattern typical for microtubules, exhibiting the outer protolament surface as a continuous axially oriented rim. The arrow shows a protolament splayed out at the end of the microtubule. (c) Unidirectional surface shadowing of tubulin that coassembled with walls monomeric kinesin molecules in vitro. The presence of these molecules renders the outer surface in a completely different way relative to the undecorated microtubules in (b). Instead of an axial striation, one now observes a perpendicular striation with an axial 8-nm repeat (arrows), which corresponds to one motor domain bound to each , -tubulin dimer. This method allows one to clearly distinguish between inner and outer microtubule surface. The inner surface exhibits a 4-nm repeating pattern according to each tubulin monomer. (a) Courtesy of Herman Frank and Heinz Schwarz; (b) and (c) courtesy of Andy Hoenger.
Fig. 23
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with an X-ray detector. This application is typically used to analyze small particulate inclusions in cells and tissues. The analysis of environmental heavy metal contamination, foreign body ingestion, and occupational exposure are the most typical applications. Examination of cells and tissues in the scanning electron microscope using the backscatter electron imaging mode can also be used to provide low-resolution information, for example, inclusions and particles containing high atomic number atoms can be identied in association with particular cell types. X-ray microanalysis of biological specimens can also utilize a transmission electron microscope coupled with an Xray detector, but the demands of specimen preparation and specimen handling are much more challenging. Typically, this high-resolution technique requires the rapid freezing of small, undamaged
d d
biological specimens. The biological specimen requirement involves selecting small (less than 1 mm) specimens, which must be solidied using ultra rapid freezing techniques leading to vitrication. Subsequently, the small specimens are mounted in a cryoultramicrotome using either mechanical clamping or a low temperature cryoglue. The ultrathin sections with thicknesses of about 100 to 120 nm are prepared and transferred to formvar-coated double-folding (sandwich) grids in the cryochamber of the cryoultramicrotome. The grid is next transferred to a cryotransfer stage at 170 C. The cryotransfer stage is inserted into the transmission electron microscope. The transmission electron microscope must be equipped with a liquid nitrogen cooled anticontamination device to prevent the deposition of water vapor onto the specimen from the column of the electron microscope.
(a)
Point counting k
(b)
Intercept counting
a1
a2
a3
a4
Position of the first plane uniform random in [0,k], estimate of volume = k(a1 + a2 + a3 + a4) (c)
Fig. 24
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For X-ray microanalysis, the specimen must be warmed to about 100 C, which allows the water in the specimen to sublime onto the anti contaminator. This freeze-drying step generally requires 45 to 60 min to achieve a specimen that will withstand the high-beam currents necessary for high-resolution X-ray microanalysis. Using this technique allows the analysis of subcellular regions in individual cells such as mitochondria, lysosomes, cytoplasm regions, and nuclear regions. The critical assumptions in this technique are (1) the freezing process does not translocate diffusible elements from their native location; (2) that the cryosectioning process and freeze-drying of the ultrathin section in the electron microscope also do not translocate diffusible elements from their native sites, and (3) that during analysis under the high
electron beam current the diffusible elements also remained in their native location. In practice, these criteria are rarely fullled for small, mobile ions of cell biological interest, such as calcium or magnesium. In general, these technical difculties have limited the applications of this technique in cell biology. An additional limitation is the lack of sensitivity to small changes in concentrations of sodium, chlorine, potassium, and calcium in the small subregions of cells. Under optimal conditions using freeze-dried thin sections, the limit of detection for X-ray analysis in general is about 100 parts per million. It is very difcult to estimate the water content of the subcellular regions so that the dry weight concentration measurement can be converted to a wet weight concentration that is meaningful to biologists.
Principles of Stereology. (a) Shows the principle of estimating area using point counts. A transparent lattice grid is randomly positioned over the images of a structure, in this case, a hypothetical prole through a cell, the nucleus N is shown. The point where the edges of the two lines of the lattice meet each other is considered to be a unique point (arrows). One simply counts the number of points over the structure of interest. In this particular example, there are 13 points in total over the cell, four of which follow the nucleus. If this result were seen after averaging over a number of micrographs, this means that the nucleus represents 4/13 = 30.7% of the cell volume. The number of points P d2 provides an estimate of the area of each structure on the micrographs. (b) Shows the principle of intercept counting to estimate the boundary length of surface. In this example, a square lattice grid with random orientation and position is placed over a piece of string. The arrows indicate the positions at which the lines of the string transect the lines of the grid: These positions (formally dened by the intersection of the edges of the line and one edge of the string) are referred to as intercepts. The length of the line is then given by /4 I d where I is the number of intercepts and d is the distance between the lines of the lattice grid. This principle, rst introduced by Buffon, is an unbiased estimator of the length of the prole, and can be converted to surface area in three dimensions. In this example, if d = 1 cm and there are 21 intercepts, then the length of the string is (3.142)/4 21(Intercepts) 1 cm = 16.495 cm. (c) Shows the principle of the Cavalieri method. The principle here is to section a three-dimensional object with a series of equally spaced sections such that the position of the rst section is random within the interval between the sections. In this example, four sections are made, a1a4, and the volume is then given by the sum of the areas of the object displayed on the sections multiplied by the distance (k) between the sections (section thickness). This method is justiably considered to be a powerful method for stereology, and has been widely used for many different organs, tissues, cells, and organelles. Diagrams courtesy of John Lucocq.
Fig. 24
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Theoretically, the water content could be estimated by making measurements before and after freeze-drying; however, fully hydrated specimens in the electron microscope are intolerant of the high-beam currents that are necessary for X-ray microanalysis. In summary, the most useful methods involving X-ray microanalysis at present are performed in the scanning electron microscope at low resolution for studying occupational exposure, mineralized tissue, and calcication associated with disease processes.
6.8
Stereology
Energy-ltering Transmission Electron Microscopy (EFTEM)
This approach allows improved imaging of thick sections and frozen-hydrated specimens by removing inelastically scattered electrons. The amount of inelastic scattering increases with specimen thickness (in general quite rapidly) and is dependent on the incident electron energy and on the material the beam is interacting with (in general, the heavier the material, the stronger the interaction). In EFTEM, the electrons are separated according to their energies, making it possible to lter out the blurring effects seen with thick sections, as well as retrieve selected chemical or physical information from the specimen. This additional electron selection results in contrast enhancement for all imaging modes and also provides the possibility of selecting electrons with specic scattering effects for imaging, thereby generating object- or elementspecic contrast. For more details as provided by one of the manufacturers of this technology, see (www.leoem.co.uk/temproducts/principle.htm).
Although many cell biologists have yet to realize it, a remarkable set of methods has been developed over the past 30 years for quantifying the sizes, contents, and spatial relationships of biological structures. These methods are known as Stereology, a rigorous, probability-based discipline for estimating three-dimensional structural quantities (including volume, surface area, length, and number) and spatial relationships from sectional or slice images. The methods are design-based, meaning that they rely on the principle of random sampling (especially systematic random sampling) and offer the twin benets of no (or minimal) bias combined with high precision. The work needed to implement them is, surprisingly, less than generally expected, so these methods tend also to be highly efcient. The use of stereological methods will be outlined, as will recently developed approaches for quantifying immunogold particles that are used to detect specic antigens. The sine qua non of stereology is the application of geometrical probes for estimating two-dimensional and threedimensional quantities. Crucial for this goal is unbiased sampling at all levels of specimen preparation and imaging: each specimen (e.g. an organ), and each part of that specimen (e.g. its different tissue compartments), must have an equal chance of being sampled. For some quantities, it is necessary to randomize the orientation of sectioning as well as section position. A number of books and chapters cover these important aspects in great detail. In order to give the avor of how stereology works, we shall consider one method available for determining the absolute volume of any structure and
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two standard methods for determining relative surface area (or length of sectional membrane image) and relative volume (or prole area of sectioned structures). It will be seen that, once the absolute volume of a reference structure (e.g. the nucleus or the cell) is known, it is possible to calculate the absolute total surface and volume of any compartment in the cell using their estimated relative surfaces and volumes. It is equally easy to obtain higherlevel quantitative information about cells in tissues, organs, or whole organisms.
7.1
from randomly positioned sites throughout the specimen, the relative number of test points on each structure is not only an estimator of their relative prole areas but also an estimator of their relative volume in the specimen (in fact, the points then sample three-dimensional space). This simple and efcient pointcounting approach therefore allows the possibility to characterize a set of cells in terms of their relative contents of nucleus, cytosol, mitochondria, Golgi complex, and any other compartments of interest.
7.2
Relative Area and Volume
Relative Prole Length and Surface Area
In order to estimate the areas of the sectional images (proles) of different structures on micrographs, transparent plastic sheets having a uniform array of test points are randomly superimposed over the image (see Fig. 24a). The positioning of the overlay with respect to the specimen must be randomized. These days, this can also be conveniently done by computer. Since each point corresponds to a precise area, the total sectional area of any structure is then directly proportional to the average number of points falling over it. For a set of micrographs, the test points are summed and averaged; relative areas can be equated with relative volumes. The relative numbers of test points falling on different structures within the specimen then provide unbiased estimates of the relative prole areas of these structures. The relative point totals (and relative areas) may be converted into relative volumes (volume densities, volume fractions, or volume proportions) using the principle rst described by the geologist, Delesse, in 1848. If the micrographic images themselves represent a set obtained
There is a simple but precise method for estimating the length of a linear trace (line on a sectional image), irrespective of its shape or orientation. On the basis of the principle rst observed by Buffon in the nineteenth century, a system of parallel test lines is randomly laid over the proles of interest. The lines must be randomized in terms of both their position and orientation. This principle can be simply illustrated with a piece of string, which is crumpled (so as to randomize its orientation) and then laid at on a system of test lines (Fig. 24(b)). The number of times the string is intersected by the test lines (separated by distance, d) is counted and provides a simple and unbiased estimation of the length of string. If the lines on the overlay run in both horizontal and vertical directions, the length of the string is estimated by the formula /4 I d, where I is the number of intersections and d is the distance between the lines of the grid (Fig. 24b). It is easy to visualize the linear traces of membranes on TEM thin sections as the equivalents of pieces of string.
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By an analogous three-dimensional approach based on randomly oriented encounters between section planes and cell or organelle membranes, the relative number of intersections between test lines and different membrane compartments allows one to estimate their relative surface areas, that is, surface of membrane per volume of reference space. This simple and efcient intersection counting approach therefore allows the possibility to characterize cells in terms of the surface areas of their different membranes.
7.3
human brain. It can produce volume estimates with a precision of 5%, or less, from just 5 to 6 sections chosen in a systematicrandom fashion.
7.4
Surfaces and Volumes in Practice
Absolute Volume
Sometimes it is not possible to estimate volume by, say, Archimedes principle of liquid displacement or by weighing, and it is necessary to resort to sectioning. An elegant method for estimating the volume of any structure by sectioning it was invented by Cavalieri in the seventeenth century. It was somehow forgotten until it was rediscovered and was given more general applicability as a stereological principle. The essence of the method is simple. First, the structure is sectioned completely into a uniform random set of slices (Fig. 24c). One can analyze each section but, as already mentioned, a great strength of stereology is the concept of sampling. One systematically selects a subset of (e.g. 1 in every 5) sections. On each sampled section, the area is estimated by, say, the test pointcounting procedure. The combined area of all the sampled sections is then multiplied by the mean distance between selected sections to obtain the Cavalieri estimate of volume. When proper sampling procedures are followed, this is a remarkably efcient and precise estimator of volume, at scales ranging from a lysosome to a
Using the above principles for estimating relative surfaces and volumes, it is easy to appreciate how one could estimate the absolute surface or volume of any cell structure that is larger than the average section thickness (for technical reasons smaller structures are problematical by these methods). For this, one needs to know one quantity in absolute terms. For most cells, biological questions that refer to quantity would pertain to the average volume of the cell (or its nucleus). The Cavalieri principle can be used to estimate cell or nuclear volume by either LM or EM. A rather simple method is to stain cell nuclei with a DNA dye, such as Hoechst dye or Dapi. By confocal microscopy, one can optimally section through a set of nuclei and, knowing the distance between sections (given by differences in the focusing plane) and the areas of sections (as above), one can estimate the average nuclear volume by the Cavalieri principle. It is also possible to use 0.5 to 1 m (semi-thin) section by EM (most nuclei in cells are 10 m or less in diameter so a relatively small number of sections sufce). At 0.3 m (or less), the procedure can be done by EM. Knowing the absolute volume of the nucleus, one would next take random sections at the EM level. For volume estimation, only the position of the sections relative to the specimen needs to be randomized but, for surface estimation, both the position and orientation of the sections must be randomized. At a fairly
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low magnication, the overlay of test points can be used to tell us the relative volume of the nucleus to the whole cell. For example, if on average 40 points hit the nuclear proles and 160 points hit the cytoplasm, the nucleus occupies 40/200 or 20% of the cell volume. If the nuclear volume is known (from above), the absolute volume of the cell is ve times that of the nucleus. The volume of any subcellular compartment of interest can now be estimated by multiplying its volume ratio in the cell (VV ) by total cell volume. Similarly, any surface of interest can be estimated by relating the number of intersections hitting the (membrane) prole of the compartment to the number of points that fall on the entire cell. This gives us the ratio of surface of that membrane to the volume of the cell (SV ). Since S = SV cell volume, this gives an indirect but precise estimate of the absolute surface of the compartment. There are also ingenious methods (again from Gundersen) for estimating particle numerical density (number in a reference volume) or total particle number. Numerical density is estimated by the dissector method, which relies on analysis using two consecutive sections and an unbiased counting frame. If the reference volume is known, an indirect estimate of number is obtained by multiplying this reference volume by particle numerical density. However, number can sometimes be estimated directly using the fractionator method. These two approaches have revolutionized the ability of investigators to provide unbiased estimates of the numbers of cells in different tissues, most prominently in various parts of the brain. Using the test point and intersection counting principles described above, simple methods are available for quantifying immunogold labeling. A major advantage
of the most recent developments is the ability to compare observed distributions of gold particles with predicted distributions obtained by counting test points (organelle compartments) or test-line intersections (membrane compartments). The idea here is to use the uniform distribution of the points and intercepts as indicators of randomness. Their predicted distributions correspond to the patterns expected for random labeling. Comparing observed and predicted distributions allows one to identify compartments that are preferentially labeled and to obtain estimates of their labeling densities and relative labeling indices. A number of stereology courses are offered regularly in Europe and in the United States and the interested reader is well advised to attend such a course. Stereology is not only a set of methods; it offers a completely rigorous way of thinking about good study design in cell and tissue biology. For quantifying structural parameters or for immunolabeling, the use of stereology should be standard. What is most surprising (given the relative lack of its application for cell biology) is that the stereological approach can be so rapidly and efciently applied.
7.5
Correlative Light and Electron Microscopy
It is not widely appreciated that sections cut for immunolabeling at the EM level can also be very useful tools for labeling at the LM level. This is true for plastic sections such as Lowicryl and for Tokuyasu-thawed cryosections. Since the labeling is restricted to one thin layer on the section surface, this approach can provide better resolution than confocal microscopy. Moreover, multiple uorescence labeling with different antibodies on
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different successive serial sections is possible; since each section (or small number of consecutive sections) is labeled independently there is no restriction on the combinations of antibodies used, for example, three different mouse antibodies can be used, each identied using a different, distinguishable uorochrome. DNA stains such as Dapi are also very useful since they can be used to nd the thin sections by light microscopy. Since the sections are thin, a series of sections covers the same structures and the images from the different antibodies can usually be combined to give pseudo-multiple labeling. Recently, an elegant method has been developed that allows visualization of GFPlabeled structures in a living cell and then allows to identify the same cell (using an HRP-based approach) at the EM level.
7.6
think they see a thin tube (in 3D) when they visualize proles of cisternae (especially when the proles are relatively small). Another signicant challenge facing the beginner in interpreting thin sections is how to average information over many cell proles, no two of which can be considered really identical! Biases are rampant in the eld as one or a few representative micrographs are selected for publication. This is especially a problem in presenting immunolabeling images in the absence of quantitation.
7.7
Which Technique for Which Question?
Interpretation in EM
Seeing and understanding something in three dimension is innitely easier than trying to interpret two-dimensional (section) images. Thin sections of cells are highly deceptive because they may seduce the investigator into thinking that the inside of the cell is open to a three-dimensional interpretation. In principle, it is, but the fact remains that it takes many years of looking down the microscope before one can be considered a professional. The loss of the third dimension is a more serious problem than is often imagined. An example was provided of a well known and highly accomplished electron microscopist, who misinterpreted the early thin section images of the ER-cisternae as a system of parallel bers. This example is useful because a large number of (even experienced) cell biologists automatically
Faced with all of these different possibilities for preparing specimens for EM, the beginner may have difculty deciding where to start. The rst advice is to contact a specialist laboratory. Much will then depend on the scale, whether the starting specimen is isolated vesicles, cells, tissues, or an organism, whose size can scale from a virus or bacterium to a whole organism, such as an insect. Starting at the highest scale, the surface of an organism less than about 1 mm can be visualized directly by SEM. Above this size, the specimen must be physically cut at the rst preparation step. An organism as large as 1 mm can be seen at low resolution by conventional ambient temperature SEMs. For higher resolution, the specimen must be available as sets of particles, whole cells, or sections of cells or tissues. In this case, a cryobased specimen preparation is mostly obligatory. For TEM, particles smaller than 0.3 to 0.5 m in height can be directly viewed by cryo-EM or by on-grid negative (positive) staining. The latter should always be the rst approach for any such specimens
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since it can be done in 5 min with simple technology. Freeze-fracture methods can also be applied for isolated particles, especially to see the inner details of membranes in a clearer fashion rather than in the much more limited crosssection prole evident in thin sections. However, the majority of EM studies involve the use of such sections in order to see the detailed ultrastructure within cells. The key question is which approach do I use for (1) structural questions, and (2) immunolabeling questions in the context of ultrastructure. For purely structural questions for best results, the combination of high-pressure freezing and freeze-substitution is recommended. The hydrated cryosection method may be preferred for some questions when one has access to this technology. If one accepts a potentially lower level of specimen quality one can select one of the two sectioning approaches, plastic embedding or Tokuyasu cryosectioning. Which approach is selected is largely a question of taste; at the top level, the results are often very similar, although the Tokuyasu method has the advantage over conventional embedding in that it avoids solvent dehydration. For immunolabeling the rst question is preembedding versus on-section labeling. Preembedding using immunogold is highly empirical and time-consuming, compared to section labeling. However, when it works, one has the advantage that one has the labeled specimen already embedded in (usually) epoxy resin, which can be cut with standard ultramicrotomes that are widely available. When the labeling is not successful, there is a signicantly larger number of variables to adjust than with on-section labeling. If the antigen is within the lumen of a membrane compartment, such as the ER, it makes little sense to try
conventional immunogold preembedding labeling because of the difculties of the antibodies obtaining access to these sites, unless one decides to try to completely open these structures by some means. HRP-based methods can be used better, but one has to accept the fact that this approach is not quantitative and can even be quantitatively misleading. For the majority of localization questions, on-section labeling is the method of choice. In this approach, each part of every structure has, in theory, an equal choice of being expressed to the antibody on the section surface. In practice, however, the situation is more complex. An important question is whether one should use a plastic-section approach (Lowicryl, LR White etc.) or the Tokuyasu cryosection method. The latter method is faster and recommended for antigens that are less likely to be extracted or articially mislocalized during the section pickup stage; such artifacts are especially a danger with small, soluble molecules and lipids. It is generally an excellent method for membrane proteins and cytoskeletal proteins; since there is no embedding medium per se there is generally more access of antibodies to antigens in the section than is the case for plastic sections, where access is strictly surface-limited. While plastic sections tend to label less than cryosections, they have the advantage that the polymerized resin better xes the antigen against redistribution. In conjunction with freeze-substitution, the low temperature Lowicryl resins offer the ultimate level of specimen preparation that is compatible with immunolabeling. For small diffusible molecules and lipids, this is absolutely the method of choice. When all methods fail, even with antibodies that give a good signal by light microscopy, the two most likely reasons
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are, rst, lack of accessibility of the antibody to the antigen, even on the surface of the section, and second, the concentration of available antigen on the section surface is simply below detection. A sensible approach is to use similar sections for immunouorescence analysis that are used for EM; this approach is likely to be more sensitive.
7.8
Final Comment
Given the breadth of techniques available, it is somewhat surprising that the use of more of these EM methods is not sufciently widespread. Rather than being standard methods in every cell biology institute worldwide, the number of specialist groups has been declining (except for the bare-grid cryo-EM method, which is increasing in use). This problem, and the politics and sociology behind it have recently been discussed in detail. The best possibility to learn these techniques is to visit specialist groups. In addition, practical courses are given in many countries, often sponsored by national EM societies. We teach an annual EMBO-sponsored course in Europe every year. For more details see the websites by Paul Webster and Herb Hagler, which give an excellent overview of many of the techniques mentioned in this chapter. See: (http://www.hei.org/research/scientists/ websterbio.htm).and (http://pathcuric1. swmed.edu/Research/haglerEMLab. htm>http://pathcuric1.swmed.edu/Research/haglerEMLab.htm)
specialists for this chapter. I would rst like to thank my colleagues from the annual EMBO-EM course for their support: Heinz Schwarz, Paul Webster, Herb Hagler, John Lucocq, Marek Cyrklaff, Terry Mayhew, Ivan Raska, Peter Peters, Paul Walther and Kiyoteru Tokuyasu. A number of these friends provided material for gures, acknowledged in the gure legends. Collectively, they also helped me enormously in the writing of the text. I would also like to acknowledge the help of Jacques Dubochet, Bill Earnshaw, Helmut Gngi, Kenny Goldie, Andy a Hoenger, Bruno Humbel, Eduard Kellenberger, Kevin Leonard and Werner Villiger for their input, and, in the case of BH, Jacques Dubochet, Andy Hoenger and Bruno Humbel also for providing beautiful micrographs. Additional excellent images were kindly provided by Kazushi Fujimoto, Shohei Yamashima, Hermann Frank, Christoph Gr nfelder, Jean-Luc u Murk, Peter Overath, Veronika Neubrand, Antonino Schepes, Anja Habermann and by Robert Ranner of Leica Microsystems, Vienna. Many thanks go also to Herb Hagler, Paul Webster, Heinz Schwarz, Anja Haberman, and Mark Kuehnel for their great support in the preparation of the gures. Finally, a special thanks to Heinz Schwarz, to whom this chapter is dedicated, for giving me the pleasure of seeing his amazing collection of micrographs, the work of over 30 years, that cover almost all the techniques outlined above.
Bibliography
Acknowledgments
The need to cover such a broad area of EM obliged me to seek the help of many
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Electron Microscopy in Cell Biology

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The Use of Electron Microscopy in Cell Biology

Introduction 4 Why Do We Need EM in Cell Biology? The Strategy 5 Specimen Preparation 7

The Use of Electron Microscopy in Cell Biology

4.5 4.6 4.7 4.8 4.9 4.10 4.11

The Use of Electron Microscopy in Cell Biology

The Use of Electron Microscopy in Cell Biology

Why Do We Need EM in Cell Biology?

The Use of Electron Microscopy in Cell Biology

The Use of Electron Microscopy in Cell Biology

The Use of Electron Microscopy in Cell Biology

Ambient Temperature EM Methods

Negative Staining of Particles

The Use of Electron Microscopy in Cell Biology

The Use of Electron Microscopy in Cell Biology

Positive-negative Staining Approaches

The Use of Electron Microscopy in Cell Biology

The Use of Electron Microscopy in Cell Biology

Classical Resin Embedding

The Use of Electron Microscopy in Cell Biology

The Use of Electron Microscopy in Cell Biology

Dehydration and Embedding

The Use of Electron Microscopy in Cell Biology

The Use of Electron Microscopy in Cell Biology

The Use of Electron Microscopy in Cell Biology

The Use of Electron Microscopy in Cell Biology

The Tokuyasu Sucrose Embedding Cryosection Method

The Use of Electron Microscopy in Cell Biology

The Use of Electron Microscopy in Cell Biology

The Use of Electron Microscopy in Cell Biology

On-section Immunogold-labeling Procedure

The Use of Electron Microscopy in Cell Biology

The Use of Electron Microscopy in Cell Biology

The Use of Electron Microscopy in Cell Biology

Preembedding Labeling Methods

The Use of Electron Microscopy in Cell Biology

The Use of Electron Microscopy in Cell Biology

Immunohistochemical Approaches using Horseradish Peroxidase and Cytochemistry

The Use of Electron Microscopy in Cell Biology

The Bare-grid Method for Particulate Specimens

The Use of Electron Microscopy in Cell Biology

Vitrication of Larger Material

The Cellulose Capillary Tube

Hohenberg has introduced the use of cellulose microcapillary tubes in order to

The Use of Electron Microscopy in Cell Biology

The Fine-needle Biopsy

(b) Kinesin decoration

The Use of Electron Microscopy in Cell Biology

The Use of Electron Microscopy in Cell Biology

The Use of Electron Microscopy in Cell Biology

Freeze-fracture and Replica Methods

Simple Shadowing Methods

The Use of Electron Microscopy in Cell Biology

The Kleinschmidt and other EM Methods for Nucleic Acids

Tube wall 1 m (a)

The Use of Electron Microscopy in Cell Biology

EM-Visualization at Ambient Temperatures

The Use of Electron Microscopy in Cell Biology

The Use of Electron Microscopy in Cell Biology

The Use of Electron Microscopy in Cell Biology

The Use of Electron Microscopy in Cell Biology

The Use of Electron Microscopy in Cell Biology

whole-mounts of particles in Fig. 11 and 19(a) and of cells in Fig. 19(b).

The Use of Electron Microscopy in Cell Biology