Sample freeze-fracture

Figure 4 SEM photographs of fractured surfaces of PEI-TLCP blend fibers at the draw ratio of 1 (x 3000). The samples were fractured after freezing in liquid nitrogen. The amount of PEsl in the blends are (A) 0 phr, (B) 0.75 phr, (C) 1.5 phr, (D) 2.25 phr, (E) 3.75 phr, and (F) 7.5 phr. Source Ref. 11.

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. 

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 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. 

TEM has been used to determine the shape and particle size of nanoparticles [27, 33]. Samples are prepared by placing a drop of preparation on copper grids, followed by negative staining with an aqueous solution of sodium phosphotungstate, phosphotungstic acid, or uranyl acetate [27, 163, 164]. Freeze fracturing with TEM has been... 

For freeze-fracture, a drop of the formulation containing 30% glycerol was deposited on a thin copper planchet and rapidly frozen in liquid propane. Fracturing and shadowing using Pt-C were performed in a Balzers BAF 310 freeze-etch unit. Other samples were simply deposited on a freshly cleaved mica plate and air-dried before shadowing as above. Replicas were examined with a Philips 410 electron microscope. 

The frozen sample is transferred into a freeze-fracture apparatus and processed at — 100°C at a vacuum between 10 and 5 X 10 bar. Within a homogeneous material the fracture happens randomly because all structural elements have equal probabilities for fracture. However, even a homogeneous material often consists of more or less polar areas. Within the polar areas, stronger interactions via hydrogen bonds prevent the fracture, which is thus less probable than the fracture within apolar areas. Therefore, the sample profile obtained after fracturing represents the microstructure of the sample only qualitatively but not quantitatively. 

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. 

Figure 5 presents the results of tensile tests for the HPC/OSL blends prepared by solvent-casting and extrusion. All of the fabrication methods result in a tremendous increase in modulus up to a lignin content of ca. 15 wt.%. This can be attributed to the Tg elevation of the amorphous HPC/OSL phase leading to increasingly glassy response. Of particular interest is the tensile strength of these materials. As is shown, there is essentially no improvement in this parameter for the solvent cast blends, but a tremendous increase is observed for the injection molded blend. Qualitatively, this behavior is best modeled by the presence of oriented chains, or mesophase superstructure, dispersed in an amorphous matrix comprised of the compatible HPC/OSL component. The presence of this fibrous structure in the injection molded samples is confirmed by SEM analysis of the freeze-fracture surface (Figure 6). This structure is not present in the solvent cast blends, although evidence of globular domains remain in both of these blends appearing somewhat more coalesced in the pyridine cast material.

FIGURE 20-30 Rosettes. The outside surface of the plant plasma membrane in a freeze-fractured sample, viewed here with electron microscopy, contains many hexagonal arrays of particles about 10 nm in diameter, believed to be composed of cellulose synthase molecules and associated enzymes. 

Samples in the SEM can be examined "as is" for general morphology, as freeze fractured surfaces or as microtome blocks of solid bulk samples. Contrast is achieved by any one or combination of the following methods ... 

High resolution SEM images of the freeze-fractured cross section of the RH-cycled samples were obtained. Many craze-like defects were observed on the cross section of samples that had been cycled from 80 to 120% RH, as shown in Fig. 16. In comparison,... 

Fig. 12. Electron micrographs of bovine casein micelles obtained by different techniques, (a) Freeze fractured and etched (L. K. Creamer and D. M. Hall, unpublished). (b) Fixed and rotary shadowed (Kalab et al., 1982). (c) Unstained, embedded thin section [reproduced from Knoop et al. (1979), by permission of the publishers, Cambridge University Press), (d). Unstained, unfixed, hydrated sample (van Bruggen et al., 1986).

The surface layer of the fat globule may act as a catalytic impurity (e.g., when it contains mono-glycerides or di-glycerides with long-chain fatty acid residues) however, there is still some uncertainty as to whether this process actually occurs (see Walstra, 1995). Although concentric layers of apparently crystalline fat have been observed in electron micrographs of freeze-etched or freeze-fractured milk or cream samples (Buchheim, 1970 Henson et al., 1971), these observations could not be confirmed by other microscopy techniques. Noda and Yamamoto (1994) reported that it is thermodynamically more favorable for fat crystals to be located at the oil/water interface, rather than in the interior of the droplet, which may explain the presence of fat crystals at the membrane. 

Although freeze-fracture TEM provides direct visualization of ME structures, it is not currently in wide use probably due to the experimental difficulties associated with the technique. The points to consider when preparing conventional TEM replicas are the physical and chemical sample properties, freezing, cleaving, etching, replication, cleaning, and mounting steps of the procedure. 

In this chapter, the results of past research are expanded because fiber cross sections were examined, rather than longitudinal views of fibers, and distributions of elements were obtained in addition to overall elemental spectra. Because the X-ray beam penetrates only a small distance into the surface of a sample (approximately 8-10 xm for a 25-kV excitation ), examination of a longitudinally mounted fiber produces elemental spectra of surface layers only. Such spectra may not be representative of the bulk of the fiber. In addition, this work improves upon past research in that the freeze-fracturing-freeze-drying EDS technique is suited to very small, fragile fiber samples (whether single fibers or small yam pieces), and is limited in size only in the operators ability to see and handle the samples. By using this procedure, compression of the fiber cross section and elemental redistribution are avoided. 

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