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The cytoskeleton is a complex of detergent-insoluble components of the cytoplasm playing critical roles in cell motility, shape generation, and mechanical properties of a cell. Fibrillar polymers-actin filaments, microtubules, and intermediate filaments- are major constituents of the cytoskeleton, which constantly change their organization during cellular activities. The actin cytoskeleton is especially polymorphic, as actin filaments can form multiple higher order assemblies performing different functions. Structural information about cytoskeleton organization is critical for understanding its functions and mechanisms underlying various forms of cellular activity. Because of the nanometer-scale thickness of cytoskeletal fibers, electron microscopy (EM) is a key tool to determine the structure of the cytoskeleton.
This article describes application of rotary shadowing (or metal replica) EM for visualization of the cytoskeleton. The procedure is applicable to thin cultured cells growing on glass coverslips and consists of detergent extraction of cells to expose their cytoskeleton, chemical fixation to provide stability, ethanol dehydration and critical point drying to preserve three-dimensionality, rotary shadowing with platinum to create contrast, and carbon coating to stabilize replicas. This technique provides easily interpretable three-dimensional images, in which individual cytoskeletal fibers are clearly resolved, and individual proteins can be identified by immunogold labeling. More importantly, replica EM is easily compatible with live cell imaging, so that one can correlate the dynamics of a cell or its components, e.g., expressed fluorescent proteins, with high resolution structural organization of the cytoskeleton in the same cell.
Electron microscopy (EM) has been instrumental in discovering the cytoskeleton in the first place, and also in investigating its structural organization in different cells and conditions. The initial progress in the cytoskeletal studies closely paralleled the development of EM techniques. Thus, the introduction of heavy metal fixation led to the discovery of actin filaments in non-muscle cells (1), while the discovery of microtubules (2) was made possible after the introduction of aldehyde fixation (3).
A great value of EM is its ability to obtain structural information at a high resolution level, which for biological samples is limited by a sample preparation procedure rather than by the power of a transmission electron microscope (TEM). However, vacuum in the TEM column and electron beam irradiation impose strict restrictions on how samples should be prepared, which in turn greatly affect the quality of images and the rate of success. A large number of different EM protocols have been developed over the years to improve the quality of samples and the amount of collected information, and to avoid artifacts. Each technique has its pluses and minuses, making it more suitable for some applications than for others.
The thin sectioning technique was initially a dominant way to visualize the cytoskeleton (4, 5). It involves the embedding of chemically fixed specimens into a resin, followed by thin sectioning to allow for beam penetration. Contrast is generated by positive staining of the sections with heavy metal salts, and the limited ability of stains to bind bioorganic material reduces the resolution of this technique. Thin sections provide a 2D view of the sample at a single plane, and a series of sections is required to retrieve the 3D information. Such reconstruction works well with relatively large and simple objects, but is not efficient in revealing the details of complex and delicately organized cytoskeletal structures, such as, for example, the actin filament networks in lamellipodia of locomoting cells.
Different versions of whole mount EM have been used to investigate the structural organization of the cytoskeleton in its entirety. Thus, the structural arrangement of actin filaments in lamellipodia was first visualized by the negative staining EM of cultured cells (6). In this technique, partially permeabilized cells growing on EM grids are immersed into a heavy metal stain solution, which is blotted off shortly after, and the samples are dried in open air. The dried stain generates a dark amorphous background on which the structures appear as translucent shapes. Negative staining EM provides high resolution and allows one to see the thin regions of a cell all the way through. Weaknesses of the procedure include sample flattening during air drying, relatively low contrast, and low stability of the samples.
In cryo EM technique, the samples are quickly frozen (to prevent ice crystal formation) and viewed while still embedded in amorphous ice, either as whole mounts or after cryosectioning, so that they remain hydrated and the proteins retain their natural conformation (7, 8). To view frozen samples, a TEM should be equipped with a chilled sample holder and electron beam power and the observation time should be minimized to keep the specimen frozen. No contrasting procedures are used in this technique except for the specimen’s own contrast, which is quite low. Therefore, significant image processing is required for the presentation and analysis of images. The major limitation of the technique is the significant difficulty in obtaining successful samples.
In metal replica EM, heavy metals are evaporated onto a 3D sample at an angle, which reveals its surface topography (9). The quality of the samples is greatly enhanced if rotary, and not unilateral, coating is used, as it helps to avoid deep featureless shadows. As metal coating is not cohesive, it is subsequently stabilized by a layer of carbon, which keeps the metal grains together, and it is fairly transparent for the electron beam. The coated sample, or just a metal-carbon replica, is subsequently removed from its original support and placed onto EM grids. The resolution of replica EM is quite high, but it depends on the metal grain size, the thickness of the coating, and the angle of shadowing. Platinum is the most popular metal, as it provides a good compromise between the grain size and ease of evaporation. The replica technique was initially introduced to study freeze-fractured samples (10), but it is applicable for a large range of samples, such as single molecules (11, 12), cells (13–15), and tissues (16, 17). This approach can reveal the 3D structure in great detail, but it is limited by the depth of shadowing penetration.
For our studies of the cytoskeleton organization in cultured cells, we chose platinum replica EM, in which detergent extraction is used to expose the cytoskeleton; chemical fixation helps to preserve the sample structure; ethanol dehydration followed by critical point drying (CPD) preserves the cell’s 3D organization; and rotary shadowing with platinum creates contrast. Over the years, we have found a good combination of individual steps to develop a reliable and relatively simple protocol that consistently produces highly informative images with excellent yield that can be combined with immunochemistry (18–20). However, this approach is not universal, but is limited to relatively thin samples attached to glass surfaces. Also, because of extensive fixation and dehydration, it cannot achieve the molecular level of resolution, but is optimal for analyses of the fine architecture of macromolecular assemblies.
As EM, in general, cannot work with live samples, investigators can only guess the kind of activity the cell was involved in at the moment of fixation, and what it would do next. A partial solution for this problem is provided by correlative light and EM, in which the dynamics of a living cell is followed by time-lapse optical imaging, and the same sample is subsequently analyzed by EM. Our EM protocol made it possible to perform correlative light and EM routinely, as it allowed us to obtain high quality structural information for a cell of interest with high probability (15, 18–20). Several other EM techniques have also been used in a correlative approach, including thin sections of resin-embedded samples (21–24), cryosections (25, 26), and negatively contrasted cells (27), although for correlative cryo EM, light microscopy served only to survey the sample rather than to study its dynamics.
In a basic form, replica EM can be used to study the cytoskeleton architecture in a cell population. In an advanced form, it can be combined with immunogold staining to detect specific proteins in the cytoskeleton (Fig. 1), and with light microscopy to correlate the cytoskeleton organization with cell behavior or with the distribution and dynamics of fluorescent probes (Figs. 1 and and2).2). The basic procedure consisting of extraction, fixation, dehydration, CPD, metal shadowing, and preparation of replicas is described first, and it is followed by the description of the advanced applications.
A major source of artifacts in this technique is the failure to perform a genuine CPD, which may occur if wet samples are transiently exposed to air, or water is not fully exchanged to ethanol or ethanol to CO2, or if the dried samples absorb ambient humidity. In order to get a high quality preparation, it is critical not to allow a liquid-gas interface to touch the samples at any point during the procedure. Practically, it means keeping the cells away from air while they are wet, and away from water, while they are dry. Changes of solutions need to be done quickly, with a layer of liquid always being retained above the cells. After drying, the cells should be kept at low humidity until they are coated with carbon.
Detergent extraction is used to expose the internal cytoskeletal structures, while the carrier buffer is designed to maximally preserve them until fixation. Additional preservation may be achieved using specific and non-specific stabilizers.
Chemical fixation provides cytoskeletal structures with physical resistance against subsequent procedures, especially dehydration and CPD. It is a three-step procedure using different fixatives: glutaraldehyde, tannic acid and uranyl acetate.
Drying of the samples is necessary to expose the surfaces for metal coating in a vacuum. However, plain drying in the open air generates major structural distortions. When the liquid-gas interface passes through the samples, the forces of the surface tension that are enormous at the cellular scale flatten the samples. During CPD, the temperature and pressure of a liquid are raised above its critical point, at which the phase boundary and surface tension do not exist. In this state, the liquid can well be considered as compressed gas. When the pressure is released, the samples remain dry with the 3D organization intact, because they never experienced the surface tension. Carbon dioxide has reasonably low values of critical point pressure and temperature that can be tolerated by biological samples. However, a direct transfer of the samples from water to CO2 is not possible and ethanol, which is freely miscible with both water and CO2, is used as an intermediate. For dehydration and CPD, the coverslips are stacked in the sample holder with pieces of lens tissue as spacers, and processed simultaneously.
Platinum shadowing generates the contrast of the samples. The angle and the thickness of the coating are critical parameters influencing the quality of the image. Lower angles provide higher contrast, but do not penetrate deep into the sample. Thinner coats provide higher resolution, but lower contrast. For cellular studies, we shadow platinum at a ~45° angle with rotation to achieve a ~2 nm thickness of the coat, which is controlled by the thickness monitor. Carbon is applied from the top of the samples with a thickness of 3.5–5 nm. The basic steps of coating are listed below. Use the equipment manual for detailed operation.
The release of the replicas from the coverslips is achieved by floating the coverslips onto the surface of hydrofluoric acid solution, which dissolves glass. After that, the replicas are washed and mounted on EM grids.
Structural information has much greater value if the identity of the structures is known. Immunostaining is a conventional way to identify cellular components. For EM purposes, the antibodies are labeled with electron-dense markers. A popular marker, colloidal gold, has a higher electron density than platinum and thus is appropriate for platinum replica EM. For successful immunogold replica EM, a primary antibody should work after glutaraldehyde fixation, which optimally preserves the structure (see Note 23).
The correlative light and EM combines the advantages of both the microscopic techniques, namely, the high spatial resolution of EM and the high temporal resolution of live imaging. In this procedure, the cell dynamics is recorded by light microscopy, and then the same cell is analyzed by EM. The correlative analysis is extremely important from at least two points of view: to control for potential artifacts and to establish functional connections between the cytoskeletal organization and the cell’s motile behavior or the dynamics of cytoskeletal components (28, 29). Modifications of the basic procedure as required for correlative EM are described below.
The author acknowledges the current support from NIH grant R01 GM 70898.
1Small coverslips allow for better exchange of solutions during dehydration and CPD, and thus for better quality of samples at the end.
2Stock solutions with a concentration of more than 2× change pH significantly after dilution to the working concentration. Free acid PIPES is not soluble in water and forms a milky suspension, but becomes soluble upon neutralization. KOH granules can be used for neutralization initially, until the solution almost clears. However, remember to allow enough time for the granules to dissolve before adding more. Finish the pH adjustment with 1 N KOH. KOH is preferable over NaOH, because K+-containing buffer, more faithfully imitates the cytoplasm composition.
3PEG is a non-specific stabilizer of the cytoskeleton; phalloidin and taxol are specific stabilizers of actin filaments and microtubules, respectively.
4Commercially available etched coverslips are not suitable for replica EM, as the marks are not visible in TEM.
5Rinsing with PBS is optional, but if omitted, the extraction solution at the next step should be added in sufficient quantity to overcome the potentially harmful effects from the remaining medium and serum.
6The choice of the extraction solution depends on a cell type and a goal. For a new experimental system, try different options in the preliminary experiments. Basic extraction solution (Triton X-100 in PEM) gives a better clarity of the cytoskeleton, but it is easier to damage the cells during extraction. If using this protocol, handle the samples extremely gently, and use phalloidin and taxol to better preserve the actin filaments and microtubules, respectively. The addition of PEG to the extraction solution provides for better preservation of the cells, but it also retains many cytoskeleton-associated components, which may partially obscure the filament arrangement. Such an effect is increased with PEG concentration and molecular weight, but PEGs in the range of 20,000–40,000 act similarly. We typically use 2% PEG (35,000). Phalloidin and taxol are not as necessary in this case. For extremely fragile and poorly attached cells, low concentrations of glutaraldehyde can be used as stabilizing supplements for the extraction solution. In this case, the detergent and fixative compete with each other, and the results depend on their ratio. The extraction solution containing 0.5% Triton X-100 and 0.25% glutaraldehyde in PEM buffer worked well in our experiments.
7For PEG-containing extraction solutions, use a longer washing time, at least 1 min in each change. If drugs are used during extraction, add them also to the rinsing buffer in a four fold to five fold lower concentration.
8It is convenient to use a multiwell plate with numbered wells (24-well for 6–8 mm coverslips or 12-well for 9–12 mm coverslips) to transfer the samples. This makes it possible to combine samples from different experiments for EM processing while keeping parallel samples in the original container as a backup.
9Uranyl acetate and tannic acid react with each other and form a precipitate. Extensive washing is important to avoid the formation of debris on the samples.
10Pieces of lens tissue slightly larger, than the holder’s bottom area, will make minor wrinkles which promote a looser packing of the coverslips in the holder and facilitate the liquid exchange.
11The acceptable number of samples for a load depends on the sizes of the holder and the coverslips. For an 18 × 12 mm holder and ~7 × 7 mm coverslips, the maximum load is 12. For larger coverslips, the load should be decreased. Larger holders may accept more samples, especially if the coverslips are staggered.
12It is not necessary to dry the beakers before the next incubation, as the ethanol concentration may not be exact, except for 100% ethanol, when it is better to dry the beakers and scaffolds with tissue. Incubation for 5 min is minimal. For larger coverslips or greater loads, increase the incubation time.
13The process of CPD is most commonly used for scanning EM and production of microelectronics. Consequently, the protocols suggested by the manufacturers or incorporated into automated procedures of CPDs are designed for those applications. Replica TEM, however, is more demanding in terms of sample quality. We adjusted the CPD processing to fully remove all traces of ethanol from the samples before bringing the CO2 to the critical point; this helps to eliminate minor artifacts that appear as a fusion of closely positioned filaments in the cytoskeleton. The CPD operation described here is applicable to manual CPDs, such as Samdri PVD-3D (Tousimis), which we use in the lab, or to semi-automatic CPDs switched to a manual mode of operation, e.g., Sam-dri-795 (Tousimis).
14Lower temperatures are acceptable, but the diffusion of ethanol from the samples will be slower, so that longer washing time is needed. Warming up the chamber till the ambient temperature is allowed if the outlet valve of the CPD is closed, and the CO2 remains pressurized and in liquid form. However, it is important to cool down the chamber back to 15°C before opening the outlet valve for purging out the ethanol–CO2 mixture.
15Letting the liquid level go below the samples will irreversibly damage them. On the other hand, too low a rate of liquid exchange is also a mistake. Adjust the outlet valve to get a steady-state liquid level, about halfway from the top of the holder to the top of the chamber. This will also make the liquid mixing more efficient. Although the shaking step sounds a bit amusing, it does make a difference by helping to remove the ethanol from the samples.
16Fast release of pressure may cause condensation of CO2 back to liquid state and ruin the dried samples.
17Conventional Scotch double-sided tape becomes too sticky in a vacuum, preventing the safe detachment of the samples after coating. To avoid this problem, sandwich the double-sided tape between the glued parts of two Post-It notes, with the sticky sides exposed.
18Humidity in the room should be below 35%; the 35–50% humidity level may be acceptable, but much caution and the speedy mounting of the samples is required; humidity >50% is not acceptable. Try to run a powerful dehumidifier in the latter case.
19If the evaporator is not equipped with a thickness monitor, the thickness of the coating may be adjusted in the preliminary experiments based on the amount of coating material loaded (for platinum) or used (carbon) for evaporation.
20To safely float a coverslip, grab it with the forceps from the top for parallel edges, lift the coverslip, and carefully place it onto the liquid surface, keeping it in a horizontal position. Practice first by placing a coverslip onto a clean solid surface. If the replica does not fall apart along the scratches, use the platinum loop to reach the replica from below, lightly touch it and pull or shake it to detach it from other pieces. Extreme care should be used not to ruin the replicas with these manipulations.
21Water has much greater surface tension than HF, which may cause severe replica breakage, if detergent is not added. Test the detergent concentration before applying it to the samples. An overdose of detergent causes shrinkage and drowning of the replicas. Stock solutions should be changed at least every 2 weeks. Old detergents leave contamination on the samples, looking like semi-transparent films between filaments. Household non-colored detergent, such as Ivory, works fine. Triton X-100 can also be used, but it should be changed more frequently.
22Sometimes, replicas appear to be repelled by the grid, making it difficult to establish the initial contact between a replica piece and a grid. Try to gently guide a piece of replica to the wall of the well to restrict its motility, and then pick it up. However, there is a danger of smashing the replica against the wall with this approach. In severe cases, use glow discharge to treat grids.
23The efficiency of staining may be improved if the cells are fixed with a lower (e.g., 0.2%) glutaraldehyde concentration before staining. For some antibodies that do not work after glutaraldehyde fixation, it may be possible to stain unfixed samples by incubating them with primary antibodies diluted in PEM for 10–15 min, then fixing with glutaraldehyde, and quenching and staining with a secondary antibody.
24Gold size of 10–20 nm is optimal for this technique, as smaller particles are poorly visible, and larger particles are too disruptive for an image.
25The coverslips can be mounted either inside or outside the dish, but inside mounting is more convenient at later stages, when the centerpiece of the coverslip needs to be cut out. For mounting, use a minimal amount of grease, just sufficient to seal the dish; excessive grease causes complications at later stages. Commercially available glass-bottom dishes have coverslips permanently glued to the bottom, which makes it difficult to remove them for EM processing.
26Cutting under water is more difficult than in the air; therefore, use a sharp diamond pencil and avoid glass crumbs.
27To reduce the effect of shaky hands, hold a razor blade with one hand with the sharp blade corner pointing down; stabilize the blade by putting the index finger of the other hand onto the blunt blade corner pointing up; rest the forearms on the table and the other fingers of both hands on the microscope stage and/or dish edges; keep the blade above the sample and find its unfocused image in the microscope; slowly bring down the sharp corner of the blade until it almost comes to focus; bring the blade corner to a region where a cut is to be made; under microscope control, bring it down to the sample and make a scratch.