Freeze-fracturing

Fig, XIV-12. Freeze-fracture transmission electron micrographs of a bicontinuous microemulsion consisting of 37.2% n-octane, 55.8% water, and the surfactant pentaethy-lene glycol dodecyl ether. In both cases 1 cm 2000 A (for purposes of microscopy, a system producing relatively coarse structures has been chosen), [(a) Courtesy of P. K. Vinson, W. G. Miller, L. E. Scriven, and H. T. Davis—see Ref. 110 (b) courtesy of R. Strey—see Ref. 111.]... 

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.)...

Knoll G 1995 Time resolved analysis of rapid events Rapid Freezing, Freeze-fracture and Deep Etching ed N Sievers and D Shotton (New York Wiley-Lyss) p 105... 

B. Donnio, D. N. Bruce, H. Delacroix, T. Gulik-Krzywicki. Freeze-fracture electron microscopy of thermotropic cubic and columnar mesophases. Liq... 

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. 

Although freeze-fracture experiments have demonstrated that monomers are assembled into stable tetramers in the membranes, radiation inactivation studies and, later, expression studies revealed that each monomer is a functional water channel (Fig. lc). 

Structured laundry liquids are currently available in Europe and were recently introduced in the United States [50,51]. These products typically contain high levels of surfactants and builder salts, as well as enzymes and other additives. In the presence of high ionic strength, the combination of certain anionic and nonionic surfactants form lamellar liquid crystals. Under the microscope (electron microscope, freeze fracturing) these appear as round droplets with an onion-like, multilayered structure. Formation of these droplets or sperulites permits the incorporation of high levels of surfactants and builders in a pourable liquid form. Stability of the dispersion is enhanced by the addition of polymers that absorb onto the droplet surface to reduce aggregation. 

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). 

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. 

The dimer chains of Ca -ATPase can also be observed by freeze-fracture electron microscopy [119,165,166,172-174], forming regular arrays of oblique parallel ridges on the concave P fracture faces of the membrane, with complementary grooves or furrows on the convex E fracture faces. Resolution of the surface projections of individual Ca -ATPase molecules within the crystalline arrays has also been achieved on freeze-dried rotary shadowed preparations of vanadate treated rabbit sarcoplasmic reticulum [163,166,173,175]. The unit cell dimensions derived from these preparations are a = 6.5 nm b = 10.7 nm and 7 = 85.5° [175], in reasonable agreement with earlier estimates on negatively stained preparations [88]. 

Analysis of the lanthanide-induced crystalline arrays by negative staining (Fig. 5) or freeze-fracture electron microscopy reveals obliquely oriented rows of particles, corresponding to individual Ca -ATPase molecules [119]. The unit cell dimensions for the gadolinium-induced Ca -ATPase crystals are a = 6. l A, b = 54.4 A and y = 111°. Similar cell constants were obtained for the crystals induced by lanthanum, praseodymium and calcium. The unit cell dimensions of the Ei crystals are consistent with a single Ca -ATPase monomer per unit cell. The space group of the Eptype crystals is PI [119], while that of the E2 crystals is P2 [88,90]. 

Two distinct patterns of repeats were observed by electron microscopy of sectioned, negatively stained, frozen-hyd rated, or freeze-fractured specimens of Ca -ATPase crystals that represent different projections of the same structure... 

Many structural components of the tight junctions (TJs) have been defined since 1992 [85-97]. Lutz and Siahaan [95] reviewed the protein structural components of the TJ. Figure 2.7 depicts the occludin protein complex that makes the water pores so restrictive. Freeze-fracture electronmicrographs of the constrictive region of the TJ show net-like arrays of strands (made partly of the cytoskeleton) circumscribing the cell, forming a division between the apical and the basolateral... 

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. 

BJ McLaughlin, RB Caldwell, Y Sasaki, T Wood. (1985). Freeze-fracture quantitative comparison of rabbit corneal epithelial and endothelial membranes. Curr Eye Res 4 951-961. 

A section of the film was removed from the bag and was immersed in liquid nitrogen. The section was then freeze-fractured and mounted on the SEM stage with the outside surface of the bag section facing downward. The sample was sputter coated prior to SEM/EDS analysis. Sputtering was performed to deposit approximately 20 nm of gold/palladium onto the sample. Double-sided conductive tape was used to ensure that the sample was sufficiently grounded to... 

Figure 21 (a) SEM cross-sectional image of the film sample after freeze fracturing. The... 

Figures 21(a) and 21(b) show the SEM micrographs of the freeze-fractured cross-section of the film used in the construction of the bag. There are two distinct layers and possibly a third very much thinner tie layer. The outside layer is a layer of nominal thickness 13 pm. The inside layer is much thicker and is approximately 70 pm thick. At the interface between the outer and inner layers the apparent very thin tie layer is about 1 pm thick. This is too thin to be identified by FUR microscopy on a cross-section of the sample, since the technique is diffraction-limited, which means that layers of about 10 pm thickness or greater can only be readily identified [1]. The tie layer thickness is also probably too thin for fingerprinting by Raman microspectroscopy on a cross-section the lateral spatial resolution of Raman microspectroscopy is about 1-2 pm.

In another catheter failure study, two samples of multi-layer catheters were tested. One catheter was breaking while the other was known to be a "good" sample. The catheters were freeze-fractured in order to obtain a cross-section view for SEM analysis, which was used to determine the construction of the catheter layers. ATR-FTIR spectroscopy was undertaken to identify the polymers used in the multi-layer catheter samples. 

The number of layers and the approximate thickness of each layer were determined from SEM images of freeze-fractured cross-sections of the films (Figure 65). A diagram of the apparent film construction is presented in Figure 66. The composition and thickness of each layer are shown in Table 15. The center opaque polyethylene layer and the EVA heat seal layer could not be differentiated in the SEM images of the freeze-fractured cross-section of the... 

Sternberg, B., Sorgi, F.L., and Huang, L., New structures in complex formation between DNA and cationic liposomes visualized by freeze-fracture electron microscopy, FEBS Letters, 1994, 356, 361-366. 

Freeze fracture electron microscopy has revealed the distribution of the preceding three components as... 

Incorporated amount of PS II and freeze-fracture electron microscopy images of PS II reconstituted vesicles. 

The samples of BR-reconstituted vesicle (100 pg BR/1.5 mM lipid) were quick-frozen using the technique of Heuser [23], and fractured in a Balzers BAF 400D freeze-fracture apparatus (Balzers, Liechtenstein). The replicas were obtained by rotary shadowing with platinum/carbon of ca. 7 nm thick and carbon of ca. 25 nm, and then examined in a Philips CM200 Ultra Twin electron microscope at 200 kV. 

Figure 5.9 Transmission electron microscopy (TEM) photographs of 3 wt % fluorinated glu-cophospholipid (13) dispersion at room temperature (a) cryo TEM (b) freeze-fracture TEM. Reprinted from Ref. 50 with permission of Academic Press.

Figure 5.38 (a) Negative-stained transmission electron micrograph of nanotubules formed from equimolar mixture of DCg PC and DNPC (2 mM total lipid concentration) stored at 4°C just prior to deposition, (b) Freeze-fracture electron micrograph of twisted ribbons at 27°C. Bars = 100 nm. Reprinted with permission from Ref. 153. Copyright 2001 by the American Chemical Society. 

Somljo It is within the measurement errors. There is a fenestration of the SR sheet, and sticking out come the caveolae. No one has really measured accurately this distance, or the distance between the caveolae and SR on top. The surface coupling space is pretty consistent. With regard to what is different in smooth muscle, if you are talking about the SR at the junction having Ca-ATPase or not, we don t know. What we do know from freeze-fracture studies of striated muscle is that the Ca-ATPase does not seem to be at the SR terminal cisternae. We don t know the answer in smooth muscle, but if there is Ca-ATPase at the junctional surface itself, this is different from what one sees in striated muscle. 

Prior to its cloning, GLT-1 was reconstituted and purihed and shown to have a relative molecular mass of 64 kDa, which agrees well both with the value of 65 kDa of the purihed and deglycosylated transporter (28,37) and its 573 amino acids determined from its nucleotide sequence (25). Reconstituted GLT-1 has been shown to form dimers, trimers, and higher molecular-weight homomers in the absence of reducing agents (38). The possibility of homomeric assemblies is corroborated by a recent freeze fracture, which shows pentameric assemblies (39). 

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