Replica and shadowing techniques

In order to determine surface characteristics and particle thickness, it is usual to deposit obliquely a film of heavy metal on to a specimen or its replica. The metal is applied by deposition in a hard vacuum by a small source in order that a nearly parallel beam may reach the specimen. The technique was originated by Williams and Wyckoflf [175] in 1946 and has been used extensively since. 

Surface topography can also be studied using 3-D imaging. Two sueeessive photographs of the same field are made with the specimen tilted by 11° to 14° between photographs. This stereo-pair can then be viewed with stereo viewing lenses for a remarkable sense of the third dimension. 

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D.E. Bradley, Replica and Shadowing Techniques , Techniques of Electron Microscopy, Blackwell Scientific Publ, London (1961)... 

Electron microscopy is a powerful direct experimental technique. Using electron microscopy information can be obtained on the presence of inhomogeneities, and on their shapes, sizes, size dispersion and number density. The experimental and theoretical aspects of this technique have been reviewed by Hirsh a/. (1965). There are two methods of observation. In the first, the topography of the sample surface is replicated and it is this replica, and not the sample which is then examined in the electron microscope. Generally carbon is used as the replicating material and shadowing at an angle with heavy elements (Pt) is used to accentuate the surface reUef. The resolution limit is about 50 A due to a microstructure in the rephca. As the sample itself is not examined, a diffraction pattern is not obtained. The sample surface can be either etched or unetched. An unetched surface wiU reveal cracks, voids, and polyphase microstructures if the various phases... 

Because of the low penetrating power of electrons, specimens for examination in transmission have to be very thin, depending on the circumstances tens or hundreds of nanometres in thickness. Surface features on thick specimens can be examined by making a thin replica. A standard technique is to evaporate a thin film of carbon on to the surface of interest, to shadow it by depositing an even thinner layer of platinum at a different angle, and then to remove the deposited replica, for example, by dissolution of the original specimen. 

The samples of BR-reconstituted vesicle (100 pg BR/1.5 mM lipid) were quick-frozen using the technique of Heuser [23], and fractured in a Balzers BAF 400D freeze-fracture apparatus (Balzers, Liechtenstein). The replicas were obtained by rotary shadowing with platinum/carbon of ca. 7 nm thick and carbon of ca. 25 nm, and then examined in a Philips CM200 Ultra Twin electron microscope at 200 kV. 

Three different techniques, namely FFEM [20, 22], Cryo-Direct Imaging (Cryo-DI) [104] and freeze-fracture direct imaging (FFDI) [105], can be used to visualise the structure of micro emulsions. In FFEM the samples are prepared in a protected fashion in a sandwich. They are then rapidly frozen, fractured, shadowed with metal, and replicated with a thin carbon film. The replica of the fractured surface, the morphology of which is controlled by the sample s microstructure, is then studied by a TEM. In contrast to FFEM, in Cryo-DI thin films of the sample are rapidly frozen but immediately, without replication, trans-... 

J. H. M. Willison and A. J. Rowe, Replica, Shadowing and Freeze-etching Techniques , in Practical Methods in Electron Microscopy, Vol. 8 (ed. A. M. Glauert), North-Holland, Amsterdam, 1980, pp. 31-55. 

It had been anticipated that there might be a difference in the fine structure of the replica in the area of the adsorbed acid clusters compared to that of the bare glass substrate. Enlarged prints of the best micrographs of this series showed no variation of texture between these areas. When replicas of cleaved mica, which is thought to be structureless, were prepared by the technique used here, they had a similar fine texture this suggests that the fine structure results from the shadowed replica itself and not from the actual surfaces of the monolayered slides. 

In FFTEM, the sample is a replica of the microstructure revealed by a fractured plane and possibly enhanced by shadowing. The contrast comes from mass-thickness variations almost exclusively, and interpretation is more straightforward. Also, there may be interfacial effects between the sample and the Pt/C coating material during sample preparation, thereby giving so-called phase detection effects [21] that permit identification of water and oil phases. However, in all replication techniques, the resolution is limited by the grain size of the replica as well as by inadvertent decoration effects that may result in loss of resolution. 

J. H. M. Willison and A. J. Rowe, Replica, Shadowing and Freeze-etching Techniques , in Practical 264. 

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