TEM preparations

Microtomed sections, which contain polymer embedded in a resin, are usually directly supported on the grid. Very small sections or sections that break apart may require a support to hold them onto the grid. Particles, crystals, emulsions, and other fine materials are placed on an electron transparent support film on the TEM grid. The preparation of such support films will be described below. 

Plastic, carbon, and metal films (see Section 4.7) are used as specimen supports on TEM grids. There are two plastic support materials in general use collodion (0.5% solution of nitrocellulose in amyl acetate) and formvar (0.25% polyvinyl formal in ethylene dichloride). These polymers are available as powders, solutions, or prepared films on TEM grids. (The suppliers provide information on handling for hazardous materials.) Formvar films, especially holey ones, are used as substrates for the formation of holey carbon films. Collodion is less commonly used as it is not as stable in the electron beam as formvar or carbon films. 

Premixed solutions of formvar and collodion are recommended for high quality film supports as the powders take several days to dissolve and are less uniform. Collodion is generally film cast on a water surface. A large Petri dish filled 

Support films generally used for microscopy above 50,000x magnification are either perforated, holey, carbon coated plastic, or carbon or holey carbon films for highest resolution. In the case of holey plastic supports, the presence of moisture or some other immiscible liquid in the solution used for coating will provide holes in the films. A fuller discussion on this topic is found in the specimen preparation text by Goodhew [7]. In our experience, moisture from the air or from one s breath on the slide 

Powders are suspended in a fluid, mixed, and dropped onto a plastic or carbon coated support grid. If the powders do not disperse by this 

Specimen preparation for TEM generally involves the preparation or formation of a thin film of the material less than 100 nm thick. The methods used for this preparation depend upon the nature of the polymer and its physical form. In the case of thick or bulk specimens, microtomy is generally used. In the case of solutions, powders or particulates, simpler methods can provide a thin, dispersed form of the material. Three types of simple preparations will be described later in this section dispersion, disintegration and film casting. The more complex methods such as microtomy, replication, etching and staining will be described in other sections of this chapter. 

Formvar solutions are placed in any tall glass container that has a lid. Clean glass slides, pretreated with a detergent to aid release of the cast film, are dipped into the solution and then quickly lifted up above the solution, covered and permitted to dry slowly in the solvent vapor. To free the film, the glass slide is scored around the perimeter with a razor, scalpel or needle. The film may then be floated onto a water surface and the grids placed on it and picked up, as for col- 

Dilute latex emulsions, with a glass transition above room temperature, can be atomized or sprayed using a fine pipette attached to a standard can of Freon gas. Difficulties arise if the latex is not dilute enough, as clumps of particles are then observed. With low glass transition latexes, cryogenic or chemical hardening methods must be used to ensure the mechanical stability of the polymer particles. 

Samples for transmission electron microscopy must fit onto a specimen support known as a grid or screen. This grid is a metal mesh screen, generally 2-3 mm in diameter, which fits into the specimen holder of the microscope. Grids come 

Support films generally used for microscopy above 50,000 x magnification are either perforated, holey, carbon coated plastic, or holey carbon films for highest resolution. In the case of holey plastic supports, the presence of moisture 

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S5mthetic resin-embedded alfalfa samples were oven dried at 65 °C for 24 h and cut at 40-90 nm. The slides were analyzed using a JEOL 2010-F TEM prepared with field emission gun, EDS, and a high angle annular dark field detector for the analysis of solitary nanoparticles. 

III. Transmission electron microscopy of radish seeds Transmission electron microscopy (TEM) of radish seeds was done as listed below For TEM preparations, the specimens after fixation and dehydration, were embedded in Epon 812 resin (Luft, 1961). Thick sections (ca. 1mm each) were stained with 0.1% toluidine blue and observed with a Zeiss light photomicroscope. Thin sections, obtained with a diamond knife on a Supernova microtome, were sequentially stained at room temperature with 2% uranyle acetate (aqueous) for 5 min and by lead citrate for 10 min (Reynolds, 1963). Ultrastructural studies were made using a Philips CM12 transmission electrone microscope (TEM) operated at 80 KV. 

Negative staining is, perhaps, the most commonly used TEM preparation technique for biochemical research. The technique derives its name from the specimen s characteristic in which it is surrounded by a contrasting medium. If this medium is more than twice the density of the specimen, the image of the specimen remains electron lucent while the surrounding area is dark (Fig. 5a). Negative stains are salts of heavy metals that exist in a disassociated form in an aqueous medium and dry down to form a fine-grained film. 

At both routes, the determination of the size and shape of the nanoparticles is a prerequisite for description of the optical properties. Besides atomic force microscopy (AFM) and scanning electron microscopy (SEM), transmission electron microscopy (TEM) is the most powerful method to determine size and shape distributions of the nanoparticle assemblies. However, extensive sample preparation that is often required can cause preparation effects, and the TEM micrographs sometimes are not representative of the whole nanoparticle-containing insulating material. Therefore, an experimental material is required which can be investigated very easily without extensive TEM preparation. 

In other work relating to the protofibrillar concept in keratin filaments, Dobb [138] used ultrasonication techniques to isolate filaments with diameters of 2.0-2.5 nm from chemically treated keratinous materials. In negatively stained TEM preparations, he further observed an axial repeat of 20-nm spacing in the filaments. Filaments with diameters of approximately 2nm were also observed by Rogers and Clark [139] and Johnson and Speakman [140,141] in similar preparations. 

As an example of SPM using a TEM preparation method, the friction force micrograph in Fig. 4.1B is of a solution grown PE lamella... 

Generally, microtomy refers to the preparation of thin slices of material by sectioning for observation in an optical microscope by transmitted light. Microtomed sections are cut with steel or glass knives to about 1 to 40/tm thickness. Ultramicrotomy methods involve the preparation of ultrathin sections of material for observation in an electron microscope. Ultramicrotome sections are cut with glass or diamond knives to a thickness ca. 30-100 nm. If imaging is to be done via many techniques, the TEM preparation method can be utilized to prepare thin sections for OM, TEM, and AFM, and the flat block face is used for SEM and/or SPM. 

In addition to labeling NMJs, we have included procedures for transmission electron microscopy (TEM) preparation of NMJs in embryos and first-, second-, and third-instar larvae (see Protocol 11.5). These protocols are not described in detail elsewhere, and the ultrastructure of the NMJ can provide very valuable information about, for example, the number and morphology of active zones, size and number of synaptic vesicles, size of boutons, and alignment of pre- and postsynaptic densities. This information cannot be obtained in any other fashion than by TEM. 

The overall procedure for TEM preparation of embryos is similar to that of third-instar... 

Vieweg BP, Butz B, Peukert W, Klupp Taylor RN, Spiecker E TEM preparation method for site- and orientation-specific sectioning of individual anisotropic nanoparticles based on shadow-FIB geometry. Ultramicroscopy 113 165-170, 2012. 

Traditionally, most TEM preparation was by first mechanically polishing thin sections down to optical translucency, then cutting discs, which were subsequently thinned to allow electron transmission by ion milling. New equipment, such as the Tripod polishing method, allows easier preparation before ion beam milling. More recently FIB techniques have opened up new possibilities for more precise sample preparation. The examples here come from the recent studies of Bazzoni (2014) and Rossen (2014). 

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