Electron microscopy freeze-fracture technique

With the freeze-fracture technique, the fracture plane passes through liposomes which are randomly positioned in the frozen sample. Some liposomes will be cut far from their midplane sections, others through their midplane section. Therefore, the analysis of freeze-fracture pictures requires corrections for nonequatorial fracture. Besides, corrections have to be made for the size-dependent probability of a vesicle being in the fracture plane (Jousma et al., 1987 Guiot et al., 1980). Recently, results with a new technique based on electron microscopy was discussed this technique allows analysis not only of liposome size, but also of the number of bilayers (Lauten-schlager et al., 1988). 

Using the freeze fracture technique, electron microscopy and laser scanning confocal microscopy, it became obvious that these gap junctional channels are arranged as a cluster of channels with about 50 channels within one disk as stated by Gourdie et al. [1990]. 

Integral proteins are usually free to move in the plane of the bilayer by lateral and rotational movement, but are not able to flip from one side of the membrane to the other (transverse movement). Immunofluorescence microscopy may be used to follow the movement of two proteins from different cells following fusion of the cells to form a hybrid heterokaryon. Immediately after fusion the two integral proteins are found segregated at either end of the heterokaryon but with time diffuse to all areas of the cell surface. The distribution of integral proteins within the membrane can be studied by electron microscopy using the freeze-fracture technique in which membranes are fractured along the interface between the inner and outer leaflets. 

The distribution of proteins in membranes can be revealed by electron microscopy using the freeze-fracture technique (Fig. 3b). In this technique, a membrane specimen is rapidly frozen to the temperature of liquid nitrogen (-196°C) and then fractured by a sharp blow. The bilayer often splits into mono-layers, revealing the interior. The exposed surface is then coated with a film of carbon and shadowed with platinum in order for the surface to be viewed in the electron microscope (see Topic A3). The fractured surface of the membrane is revealed to have numerous randomly distributed protuberances that correspond to integral membrane proteins. 

The arrangement of proteins within biological membranes can be directly observed by using freeze-fracture electron microscopy. In this technique, a rapidly frozen membrane is struck with a microtome knife. (A microtome is an instrument used for cutting thin sections of biological specimens for microscopic study.) The membrane often splits along the inner surfaces of the two lipid layers. In preparation for viewing in the electron microscope, the delicate membrane s inner surfaces are shadowed with a thin layer... 

For these and other reasons the manner in which the NPC is distributed in the NE has been a point of interest for many years. The freeze-fracture technique provided the possibility to expose parts of the nuclear envelope and to visualize the NPC distribution by electron microscopy. The outcome of such studies has been summarized by Maul (1977), who eventually concluded that a random distribution (of the NPC) has never been proved, and alt investigated cases seem to have non-random distribution. We may add that the number of investigated cases is small and that the claims of nonrandom distributions are not always convincing. 

Freeze-fracturing Technique in which a cell is first frozen and then broken with a knife so that the fracture reveals structures inside the cell when observed by electron microscopy. 

The structure of the final cream is rather complex and it can be investigated using various techniques polarizing optical microscopy, freeze-fracture electron microscopy, differential scanning calorimetry (DSC), low-angle X-ray and NMR. Figure 13.29 gives a schematic picture of the structure of the cream (a) and (b) fl-lustrate an example of a cavity in the continuous gel phase whereas part (c) shows the interfacial area between dispersed phase and continuous phase on a molecular scale. 

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... 

GSL clustering has been observed in classic studies by scaiming electron microscopy (EM) with freeze-fracture technique using ferritin-labeled or gold sol-labeled anti-GSL mAbs [6-8], and more recently by transmission EM using gold sol-labeled anti-GSL mAbs [9, 10]. Results indicate that ... 

Cationic quaternary ammonium compounds such as distearyldimethylammonium-chloride (DSDMAC) used as a softener and as an antistatic, form hydrated particles in a dispersed phase having a similar structure to that of the multilayered liposomes or vesicles of phospholipids 77,79). This liposome-like structure could be made visible by electron microscopy using the freeze-fracture replica technique as shown by Okumura et al. 79). The concentric circles observed should be bimolecular lamellar layers with the sandwiched parts being the entrapped water. In addition, the longest spacings of the small angle X-ray diffraction pattern can be attributed to the inter-lamellar distances. These liposome structures are formed by the hydrated detergent not only in the gel state but also at relatively low concentrations. 

The terminal complex hypothesis proposes that structural manifestations of the cellulose synthase enzyme complex can be visualized with the freeze fracture specimen preparation technique for electron microscopy. These... 

To obtain more detailed information on the ultrastructure of lipid dispersions and the morphology of the particles, electron microscopy is usually performed on replicas of freeze fractured or on frozen hydrated samples. These techniques aim to preserve the liquid-like state of the sample and the organization of the dispersed structures during preparation. By using special devices, the sample is frozen so quickly that all liquid structures, including the dispersion medium, solidify in an amorphous state. 

Through the use of freeze-fracture and freezeetching techniques of electron microscopy, it is possible to see, embeddded in the thylakoid membranes, particles which may represent individual photosyn-thetic units (also called quantosomes).227 256-258 They are about 20 nm in diameter, and at least many of them presumably contain a reaction center surrounded by light-collecting chlorophyll-protein complexes. Others may represent the cytochrome b6f complex and... 

Pinto da Silva, P., Barbosa, M. L. F, and Aguas, A P. (1986) A guide to fracture label, cytochemical labeling of freeze-fractured cells, in Advanced Techniques m Biological Electron Microscopy (Koehler, J K, ed), Springer-Verlag, Berlin, pp 201-227... 

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. 

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