Freeze Fracture Replicas

Fig. XV-11. Electron micrograph of a freeze fracture replica of a region inside a mul-tivesicular liposome. Note the tetrahedral coordination nearly every vertex has three edges, and each face is connected to three others. The average number of edges per face is 5.1. (From Ref. 77.)...

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. 

Guiot, P., Baudhuin, P., and Gotfredsen, C. (1980). Morphological characterization of liposome suspensions by stereological analysis of freeze fracture replicas from spray frozen samples, J. Microsc., 120, 159-174. 

Figure 25. Freeze fractured replica. P-fracture face of the plasma membrane in 15h aplanospore of Valonia veniricosa. The clustered TC s are shown, suggesting the build-up of new axis for microfibril orientation.

Fig. 13. Freeze-fracture replicas of rhodopsin-polyl5/DOPC membranes in 30% glycerol/water frozen from room temperature. The particles are morphological manifestations of the protein rhodopsin. The nonrandom distribution of particles indicates the presence of enriched domains of lipid and of protein. The particle-free domains constitute about 30% of the surface area.

Conventional freeze-fracture replicas revealed the presence of the two membranes enveloping the hydrogenosomes presenting a different number and distribution of intramembranous particles (Fig. 7a-c) (Benchimol et al. 1996a Benchimol 2001). Four fracture faces were identified two concave faces representing the P faces of the outer and the inner membranes and two con-... 

Figure 1. Top Structures and CPK models of regioisomeric phospholipid analogues. Bottom (a, b) TEM micrographs of aggregates of 2. (a) Right-handed helices, (b) Superhelices formed by further assembly of the helices shown in (a). Inset freeze-fractured replica of the superhelix, (c) Schematic representation of the superhelical winding.

The result obtained by the transmission electron microscope observations of the freeze-fracture replica on trehalose solutionssuggested that the ice-crystal nucleation is enhanced in the solution with higher trehalose concentration. Based on these experimental results and assuming a similar nucleation process between ice and clathrate hydrates, we can speculate that trehalose, as a kinetic inhibitor, may not inhibit the nucleation but may work as an anti-agglomerant, keeping small hydrate particles dispersed as they form. 

Fig. 2. TEM image of a Freeze Fracture replica of the system O.IM AOT/ n-heptane / water (w=20)...

Figure 2 Sample holder for preparation of freeze-fracture replicas. Microemulsion is injected through the hole in the plate. The TEM grid tabs are folded over the copper plates and glued down. This ensures that the fracture propagates between the two grids and provides both cohesive and adhesive fracture surfaces. (From Ref 10.)...

Freeze-fracture replicas revealed the presence of parallel rows of particles on the EF face (Figs 7,8) of the thylakoids. These rows of EF particles are observed in several species of red and blue-green algae the particles are believed to be part of PSII. Regular arrays of particles were also seen on the PF face of the thylakoid (Fig. 8). These particles were more numerous and slightly smaller than the EF particles. 

Figs 7 8. Freeze-fracture replicas of the thylakoid membranes. Fig. 7 shows the parallel rows of particles on the EF face while Fig. 8 shows regular arrays of particles on the PF face as well as the rows of EF particles. 

Decrease in variable fluorescence in the presence of FeCN indicates a net loss of PSII-Qg-nonreducing centers at RT during high light exposure of the leaves. Concomitantly there occurred a decrease in the density of EFu particles in freeze fracture replicas of the thylakoids. These observations are consistent with the hypothesis that PSIIB-Qg-nonreducing centers from stroma thylakoid membranes are used to replace the photoinhibited PSII centers in grana partitions (1). 

Figure 4 Schematic representation of the TEM specimen preparation by freeze-fracture replica method.

Cryogenic transmission microscopy (Cryo-TEM) is an excellent method, by using which the objects in solution can be visualized without fixing and staining. As mentioned above, negative staining is a convenient method for the preparation of TEM specimen however, the object can be visualized only in dry state, which is sometimes much different from the structme in solution. In addition, using Cryo-TEM the structmal information inside the object can be obtained in contrast to freeze-fracture replica method. 

No mention of labelling of freeze-fracture replicas has been made here and the interested reader could consult the excellent book on the topic by Severs and Shotton (13). 

It is well known that the rosette and linear terminal complexes (TCs) can be observed by the freeze-fracture replication technique. The structures revealed by this technique are known as putative cellulose-synthesizing TCs. Kimura et al. (1999) demonstrated that TCs in vascular plants contain cellulose synthases using a novel technique of sodium dodecyl sulfate (SDS)-solubilized freeze fracture replica labeling (SDS-FRL). The localization of the cellulose synthase to the TC was accomplished almost 40 years after the hypothesis of Roelofsen (1958) in which he stated that enzyme complexes could be involved in cellulose biosynthesis. It has been more than 30 years since the discovery of the first TC by Brown, Jr. and Montezinos (1976) and in particular, 26 years after the discovery of rosette TCs in plants by Mueller and Brown, Jr. (1980). 

Fujimoto K. 1995. Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins. J Cell Sci 108 3443-3449. 

Fujimoto K., Umeda M., and Fujimoto T. 1996. Transmembrane phospholipid distribution revealed by freeze-fracture replica labeling. J Cell Sci 109 2453-2460. 

Figure 1. Electron micrograf of freeze>fracture replicas jM epared frwn diloroplasts saiq ended in 20mM Hq>es buffer (pH7.6) isolated frc n (A) wild-type Arabidopsis thaliana and (B) LK3 mutant strain thermally equilibrated and quaidied fr

SAXS was measured at 25 °C by a 6 m point focussing SAXS camera at the High-Intensity X-ray Laboratory in Kyoto University. TEM observation was carried out on a Hitachi H-800 electron microscope. A Hitachi H5001-C cold stage was used for the cryo method. Freeze-fracture replica film was prepared by using a balzers BAF 400 freeze-fracture device. 

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