Coating techniques, microscopic samples

The two main techniques commonly discussed in the literature are known as direct observation (or frozen hydrated observation) and the observation of replicas. Both techniques involve the fast freezing of the sample in a cryogen such as liquid nitrogen, propane, or freon. The frozen sample is then fractured to reveal the interior features. This fractured surface can be coated with a metal film or observed directly. Often, the metal film is removed from the sample and observed as a replica. This type of procedure allows the creation of a permanent archive of the samples prepared, and the observation is the same as with any other electron microscope sample with no concern about contamination of the microscope or beam damage to the sample. 

Freeze-Fracture Method A sample preparation technique used in electron microscopy in which specimens are quickly frozen in a cryogen and then cleaved to expose interior surfaces. In some techniques, the sample is then observed directly in an electron microscope equipped with a cryogenic stage in other cases, the cleaved sample is coated with a metal coating to produce a replica, which is observed in the electron microscope. See also Electron Microscopy. 

At the end of the 1930s, adsorption chromatography in columns as introduced by Tswett had become a powerful separation technique for plant extracts and natural products. Simultaneously, the need for a more rapid alternative suitable for identification of separated substances led to the invention of an open chromatographic system. In 1938, Izmailov and Shraiber reported the separation of belladonna alkaloids on a thin adsorbent layer, coated onto microscopic slides. Development of circular chromatograms was achieved by placing small amounts of various solvents to the center of samples previously applied as spots onto the layer. This method was an extremely rapid microtechnique requiring only small amounts of stationary and mobile phases. 

A Fourier transform infrared (FTIR) speetrometer equipped with an attenuated total refleetanee (ATR) aeeessory is used for the study of surfaees and coatings. A microscope attachment is useful for identifying particulate impurities. Through the technique of computerized spectral subtraction, many sample mixtures may be identified by FT-IR without prior physical separation and, thus, the technique lends itself to compositional analysis. Typical applications of FT-IR are the study of surfaces of polymers by ATR, identification of samples isolated by thin-layer chromatography or other preparatory chromatographic techniques, identification of impurities in polymer and polymer blends, product characterization and product formulations by spectral subtraction, as well as routine analysis of solid and liquid materials. 

After quick freezing in liquid nitrogen and fracture, another etching method was used at — 90 °C for less than 1 min in a 1.33 x 10-4 Pa chamber. The etched sample was cooled to — 130°C and coated with platinum and carbon in the same vacuum chamber and transferred to the scanning electron microscope at — 130 °C. The observed structure was closer to reality than that obtained by the method previously described. This is called the cryo-SEM technique [32]. CryoSEM images are shown in Fig. 4 which presents the structural change by stretching [16]. 

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