Replicas, Shadowing, and Negative Staining 271 CHAPTER 8 TECHNIQUES Vacuum Evaporation Vacuum evaporators are used to coat substrates or specimens with carbon and/or metals and to pro­ duce pure carbon substrates for specimen support. The duration of evaporation, temperature of electrodes during evaporation (amperage used), angle of evaporation, vacuum at the time of evaporation, material being evaporated, and the temperature of the stage holding the specimen or support being coated are all factors for the end-product desired. As stated earlier, the shortest duration that delivers a usable coat, the lowest source (electrode) temperature that will cause evaporation, the largest angle between the source and substrate, the highest vacuum, the metal with the smallest grain structure, and a cooled stage will work together to produce the finest grain structure on the coated substrate. However, since other factors must be considered, all of these conditions cannot always be met. When shadowing a substrate with topography (e.g., a freeze-fracture specimen), contrast is developed by the dep­ osition of metal/carbon on one side of a particulate specimen, with a clear area behind the specimen that is not coated. This area is known as the shadow. Taller objects (like protozoan cells) are typically shadowed from one direction with metal at an angle of 45°, followed by a stabilizing carbon coat from sources directly overhead. Alternatively, carbon and metal are deposited simultaneously at a 45" angle. Specimens with little relief (nucleic acids, bacterial pili, viruses) are shadowed at a lower (10°) angle (Fig. 165A). If specimens are supported on a stage that is rotated rapidly during evaporation, the specimen is coated from all sides, producing very little shadow, but giving overall bulk to the sample (Fig. 1658). Carbon is evaporated onto most metal coatings to provide further mechanical stability to the coat. It is also evaporated onto plastic film-coated grids to provide mechanical stability and electrical conductivity to decrease thermal drift when the electron beam interacts with the coated grid. The barest perceptible car­ bon coat is usually adequate for this purpose. Finally, as already described in the section on film-coating grids, thicker carbon coats can be evapo­ rated onto mica, which are then stripped from the mica substrate. The carbon film is then used to coat grids, ultimately serving as a support film for specimens. A variety of metals requiring different evaporative temperatures can be used in shadowing. Those requiring higher evaporative temperatures have a finer grain structure. Using metal alloys or evaporating a metal and carbon at the same time produces finer-grain coatings, because nucleation of single metals takes place more rapidly than mixtures, resulting in larger deposited grains. For freeze-fracturing and similar applications, the objective is to produce an electron-transparent replica of the specimen surface. The specimen is digested with acids or bases so that only the replicated sur­ face of the specimen is examined with the electron microscope. In the past, vacuum evaporators were used to coat SEM samples by attaching the samples to rotating, tilting stages during the coating process in an attempt to coat all the hills and valleys in typical SEM speci­ mens with lots of topographic detail. With the advent of sputter coaters that have nondirectional metal deposition on sample surfaces, the vacuum evaporator has been retired from most SEM specimen preparations. Shadow Casting 1. Applications and Objectives Shadow casting metals onto specimens at high or low angles from a fixed direction is used to develop contrast in a specimen (tall or short, respectively) as a result of topographic differences in specimen details. Shadow casting of specimens on a rotating stage confers bulk to a sample with little topography. It is also possible to calculate the height of specimen structures by measuring shadow length and relating it to the angle of shadow. M. J. Dykstra et al., Biological Electron Microscopy © Michael J. Dykstra 2003 272 Chapter 8 (A) Figure 165. Escherichia coli coated with platinum-palladium. (A) Low-angle. fixed-direction coating. 51.474 x. (B) Low-angle, rotary coating. 65,337X. 2. Materials Needed • Vacuum evaporator • Carbon electrodes • Metal electrodes of choice • Specimen (dried or, in the case of freeze-fracture, frozen) • Grids • Pieces of 3 X 5 in. (7.6 X 12.7 cm) card stock • Carbon electrode sharpener • 400-grit emery paper Replicas, Shadowing, and Negative Staining 273 3. Procedure Carbon Electrode Preparation 1. Polish the ends of both carbon electrodes by first sanding them on a piece of 400-grit emery paper on a counter top and then polishing the sanded surface on a piece of cardboard or card stock until the sanded surface is shiny. 2. Use the electrode sharpener to sharpen the polished end of one electrode so that the final electrode has a reduced-diameter end about 5 mm long (Fig. 160). 3. Install the carbon rods with the sharpened rod in the driven electrode and the other attached to the fixed electrode. The rods should meet midway between the two electrodes, which are spread as far apart as possible. After the rods are installed, make sure that the driven electrode is free to travel when the carbon rods are evaporated. Wire Evaporation (Platinum-Palladium) 1. Ball up 1.5 cm of platinum-palladium wire with a pair of coarse-tipped forceps. 2. Place the wire into a tungsten basket attached to a set of electrodes. 3. Load the specimen and place a small piece of 3 X 5 in. (7.6 X 12.7 cm) card stock for monitoring coating thickness near the specimen. The card should have been dried previously in a resin poly­ merizing oven so that no residual atomospheric moisture remains, which would slow down the pumping speed of the vacuum evaporator. Pump down the vacuum system as specified in the vacuum evaporator instructions. 4. Once the vacuum evaporator has reached high vacuum (1.3 X 10-2 Pa or better), slowly tum up the electrode current with the electrode rheostat until the wire in the basket melts. It is important to do this slowly enough so that the wire fuses with the tungsten basket. If the current is raised too quickly without careful visual monitoring, the noble metal wire can simply be cut by the heated tungsten and fall in large lumps to the vacuum chamber floor, producing no coating on the specimen. Caution: Wear welder's goggles to prevent eye injury from the bright electrodes. Firing the Wire Electrodes 1. For a fixed-angle bum, once the platinum-palladium is welded to the tungsten basket, continue rais­ ing the electrode current slowly until the card stock begins to darken. When this is noted, do not increase the current any further. Observe the lumps of platinum-palladium welded to the basket. After they have evaporated, return the electrode rheostat to the off position. 2. For a rotary bum, the procedure is identical except that the rotary specimen holder should be turned on prior to the bum. The specimen should rotate at about 100 rpm. Firing the Carbon Electrodes 1. Carbon rods are fired alone to coat polymer film-coated grids or to produce pure carbon films as specimen supports. They are also fired after the platinum-palladium has been evaporated onto a specimen surface to add electron-transparent physical support to the metal coat. To fire the electrodes, switch the electrode control to the set bearing the carbon rods, and slowly tum up the electrode rheostat until the pointed carbon rod begins to evaporate. Some sparking during this process is acceptable, but excessive sparks indicate that large chunks of the carbon rod are being discharged, which can result in debris on the specimen. 2. When coating a plastic support film or evaporating carbon onto a metal layer previously evaporated onto the specimen surface, a very thin carbon coat (light brown) is adequate. If coating a mica sub­ strate to produce a carbon support film, a thicker (black) film is needed, so bum the electrodes until the sharpened tip has been consumed. 274 Chapter 8 4. Results Expected The evaporated metal coat from a fixed-direction shadowing run will have a fine grain and will produce a shadow where no metal has been deposited behind a specimen area with topography. A rotary coating run will produce a specimen with increased bulk but without much evident shadow. A carbon coating will not be noticeable under the electron beam, but will have contributed structural stability and electrical conductivity to the specimen. 5. Cautionary Statements Always protect your eyes with welder's goggles during electrode firing, as the high-intensity light from the electrodes can damage the retina. The use of card stock to monitor coat thickness works well in the absence of an expensive crystal film monitor that can actually record the number of nanometers of film being deposited during electrode firing. Some workers use immersion oil or diffusion oil drops on a piece of broken white porcelain crucible lid to monitor film thickness. The vacuum may cause vaporization of the oil, however, coating surfaces within the vacuum chamber, including the specimen, and leading to problems for developing a good vacuum over time and to poor coating of the specimen surface. If oil is used, diffusion oil is preferred, because it is already part of the pumping system and has a lower vapor pressure than immer­ sion oil. DNA (Plasmid) Preparation for TEM' 1. Application and Objectives This is a quick method for the preparation of purified short segments of nucleic acids (e.g., plasmids) for visualization in a transmission electron microscope. The technique confers sufficient bulk and contrast to isolated short pieces of nucleic acids for the purpose of measuring their length or assessing whether they are single-stranded or double-stranded utilizing a transmission electron microscope.