Cryo-electron microscopy Freeze-fracture technique

To prove the formation of vesicles a number of indirect techniques can be used such as dynamic light scattering, the use of fluorescent probes and pulsed field gradient NMR self-diffusion measurements. Some more direct techniques such as freeze-fracture and negative staining electron microscopy are less biased by the interpretation of the scientist, but also these methods have their limitations. Cryo-electron microscopy, as introduced by Dubochet in the 80s, is the method of choice when it comes to visualization of small colloidal structures. Recent developments in the vitrification of specimens make it now possible to observe vesicles and other aggregated structures artifact free. 

Microemulsion structure can be observed by electron microscopy (EM). Jahn and Strey (4) were the first to publish reliable electron microscopy images of microemulsions. Their systematic study involved both droplet and bicontinuous structures, using the so-called freeze-fracture technique where a replica of the sample is made which is then monitored by the electron microscope. Another technique, Cryo transmission electron microscopy (Cryo-TEM), is probably less suitable for microemulsions with larger oil contents. In this technique, a thin slice of the sample is directly monitored... 

Various electron microscopy techniques have been used to study the structures of whippable emulsions such as normal and cryo-scanning electron microscopy or transmission electron microscopy using various preparation methods such as freeze fracturing, freeze etching, etc. The literature is quite extensive, and only a few important papers will be discussed in this chapter. 

The interior of bulk samples can be revealed by fracture, freeze fracture, or (cryo-/ultra)microtoming. These techniques are well established in electron microscopy and require substantial training to ensure that the knives used do not introduce scratches and other artefacts. Unlike in SEM or TEM analysis, however, it is very well possible to analyze the trimmed specimen instead of the very thin sections removed (cryofacing). This loosens the constraint of ultrathin sections in many applications. Care has to be taken that the sample to be imaged is not significantly thicker or thinner than the calibration grating used for scanner calibration (see Sect. 2.2.5)... 

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