Transmission electron staining, methods

J du Plessis, LR Tiedt, AF Kotze, CJ van Wyk, C Ackerman. A transmission electron microscope method for determination of droplet size in parenteral fat emulsions using negative staining. Int J Pharm 46, 1988. 

Draget KI, Myhre S, Ostgaard K. Plant protoplast immobilized in calcium alginate. A simple method of preparing fragile cells for transmission electron microscopy. Stain Technol 1988 63 159-163. 

Manara, G C, Ferrari, C, Torresani, C., Sansoni, P, and De Panfilis, G. (1990) The immunogold-silver staining approach in the study of lymphocyte subpopula-tions in transmission electron microscopy. J Immunol Methods 128, 59-63... 

Transmission electron micrography has, remarkably, been successfully used to image micelles formed by block copolymers in dilute solutions. Price and coworkers used two preparation methods. In the first method (Price and Woods 1973), f reeze etching, a drop of solution was rapidly frozen by quenching in liquid nitrogen. Solvent was then allowed to evaporate from a freshly microtomed surface of the droplet. Finally, a replica was made of collapsed micelles raised proud from the frozen surface. In the second method (Booth et al. 1978), a drop of micellar solution was allowed to spread and evaporate on a carbon substrate, and 0s04 was used to selectively stain one of the blocks. 

Transmission Electron Microscopy. Films of all samples designated R were obtained by evaporation of toluene from solutions of the block copolymers and were observed without staining using a Hitachi Hu-125 or a JEOL JEM 100 S electron microscope. Methods of preparing the films have been described previously (24). So far, we have obtained evidence for microphase separation in only the four highest molecular-weight samples by TEM. We have not obtained continuous films of the lower molecular-weight samples we plan to examine sections of these samples later. Because of the very small compatibility of styrene and polydimethylsiloxane, however, we expect phase separation in all of these samples. 

The ESCR performance of a resin is not easily modeled. A laboratory technique for the preparation of thin films of HIPS materials for the study of deformation processes by microscopy allows the deformation process to be better understood. The transmission electron microscope (TEM) allows direct visualization of the crazes themselves in thin films. For good contrast between the crazes and the bulk polystyrene, thin, cast films from 0.5 to 2 p,m are required, and also staining of the rubber phase with a heavy atomic species to provide contrast between the rubber and the polystyrene. Another intricacy of this method requires a solution of the HIPS material in a 65 35 methyl ethyl ketone-toluene solution to prevent significant swelling of the rubber particles during the preparation process. 

To examine craze microstructure, and to study the effect of molecular variables on craze morphology, the method described by Kramer was followed. Samples of polymers were cast in the form of thin films, strained in tension while bonded to carbon-coated grids, and examined in the transmission electron microscope either before or after staining. The TEM observations were made with an Hitachi HU-11 A unit or with a JEOL JEM-IOOCX unit, operating usually at 75-80 kV. Fracture surfaces of many bulk samples were coated with a thin layer of gold-palladium and examined by an Etec scanning electron microscope. 

Jackson, C.L. Chanzy, H.D. Booy, E.P. Drake, B.J. Tomalia, D.A. Bauer, B.J. Amis, E.J. Visualization of dendrimer molecules by transmission electron microscopy (TEM) staining methods and Cryo-TEM of vitrified solutions. Macromolecules 1998, 31, 6259-6265. 

Kakugo, M. Sadatoshi, H. Yokoyama, M. Kojima, K. Transmission electron microscopic investigation of nascent polypropylene particles using a new staining method. Macromolecules... 

Transmission Electron Microscopy (TEM) is perhaps the most used visualization method for studying polymer aggregates. Additionally, negative staining permits very high resolution imaging of surface details. Cryo-TEM offers another advantage since specimens are rapidly frozen and viewed in a natural hydrated state... 

Electron microscopic images of lipoproteins show predominantly spherical shapes. Only nascent HDL appear as stacks of discs by negative-stain transmission electron microscopy. The stacks are artifacts of the method, because in solution nascent HDL... 

Since the dimensions to be probed are of the order of few nanometers, the most useful microscopic method would be that of the transmission electron microscopy (TEM). Using this technique Fayt et al. [1986] observed location of P(S-b-HB) compatibilizer in a PE/PS blends. The authors inserted a short sequence of isoprene between the styrenic and hydrogenated butadiene blocks. After staining the isoprene double bonds with OsO, the authors were able to observe presence of the copolymer at the interface between the matrix and dispersed phase. Thickness of the interphase could then be measured. The experiments also demonstrated presence of the added compatibilizer as dispersed micelles inside the PE phase. This technique is applicable, however, only when selective staining affects only the compatibilizer. 

A powerful method of examining the morphology of many multicomponent polymer materials utilizes transmission electron microscopy [Woodward, 1989]. If the two phases are nearly equal in electron density, staining with osmium tetroxide or other agents can be used. For more detailed discussion on the methods of morphology characterization, see Chapter 8. 

Figure 40. Representative negative-stain transmission electron miCTographs of spontaneously made vesicles of L20 [( ), magnification, x 10000] its Cu(II) inclusion complex [(b), magnification, X12000] micrographs of vesicles made by extrusion method of L20 [( ), magnification, x 20000] its Cu(II) inclusion complex [(d), magnification, x20000].

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