Freeze fracture techniques

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). 

M Hirsch, G Renard, JP Faure, Y Pouliquen. (1977). Study of the ultrastructure of the rabbit corneal endothelium by the freeze-fracture technique Apical and lateral junctions. Exp Eye Res 25 277-288. 

The process of infection of lupine nodule cells by Rhizobia was examined by the thin-section electron microscopic technique, as well as the freeze-fracture technique. Different membranes such as infection thread membranes, peribacterioid membranes, plasma membranes, membranes of cytoplasmic vesicles, and membranes of the Golgi bodies and ER were stained with uranium-lead, silver, phosphotungstic acid, and ZIO (31). ZIO stained the membranes of the proximal face of the Golgi bodies and endoplasmic reticulum. ZIO staining has given good contrast in thick sections such as a cotyledon cell, a root cell, and an aleurone layer for ER, dictyosomes cisternae, mitochondria, and nuclear envelopes (17,32-37). 

Though cellulose is one of the most important biopolymers, it has not yet been possible to completely elucidate its biosynthetic pathway, or establish exactly the cell organellae involved in its synthesis. However, during the last decade, the freeze fracture technique has been applied to investigate cell wall formation, and this has produced much information on the site where cellulose synthesis occurs. It is now generally accepted that both terminal and rosette complexes are responsible for cellulose synthesis (21). Our results (19,22) support that view. In a TEM-autoradiographic investiga-... 

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. 

FIGURE 8.19 Replica of a freeze-fractured membrane. In the freeze-fracture technique, the lipid bilayer is split parallel to the surface of the membrane. The hydrocarbon tails of the two layers are separated from each other, and the proteins can be seen as hills in the replica shown. In the other layer, seen edge on, there are valleys where the proteins were. (From Singer, S.J, in G. Weissman and R. Claiborne, Eds., Cell Membranes Biochemistry, Cell Biology, and Pathology, New York HP Pub., 1975, p. 37.)... 

Microscopic observations were done on a Phillips High Resolution Scanning Electron Microscope. To reveal solid material inside the microcolumn, a freeze-fracture technique was used after samples were washed with methanol and dried under normal conditions. 

Luckham PF, Vincent B, McMahon J, Tadros TF (1983) The controlled flocculation of particulate dispersions using small particles of opposite charge. II. Investigation of floe structure using a freeze-fracture technique. CoUoids Surf 6(l) 83-95... 

FFTEM samples can also be prepared [29] by a double-replica freeze-fracture technique, in which a drop of sample is deposited on a metal disk. A second disk is then placed on top, and the two disks are centered. A further drop is placed in the central bore of the specimen sandwich, any excess sample being removed with filter paper. The sample is plunged into liquid cryogen and transferred to the cleavage unit of the microscope, also maintained under cold conditions. The sample is subsequently cleaved, etched, coated, and imaged. 

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. 

The PSII- complexes were incorporated into phospholipid liposomes and analysed by freeze-fracture techniques.The freeze-fracture particles were randomly distributed and their diameter was about 10.3 nm on the concave and convex fracture faces. Some particles showed a central furrow, that was also present in the EF-particles "in situ" side by side aggregations of two particles were observed too. With a height of 13-16 nm the particles span the liposome membrane. 

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. 

However, we had to overcome two major difficulties before applying the freeze-fracture technique coupled with immunogold labeling. First, it has been believed that conventional freeze-fracture technique does not allow for immunogold labeling. This is because the replica membrane made of platinum and carbon does not show any reaction with the antibody. This difficulty was overcome by utilizing... 

---