Electron microscopy freeze fracture

Pitkanen et al. [51] reported the isolated bovine RPE-choroid was up to 20 times more permeable to lipophilic than hydrophilic beta-blockers. Furthermore, the in vitro permeability of bovine RPE-choroid to hydrophilic compounds and macromolecules was 10 to 100 times less compared to sclera, whereas the permeability for lipophilic molecules was in the same range for both tissues. The isolated bovine RPE-choroid also exhibited differential permeation by molecular weight and Stokes radius. The permeation rate of 4, 10, and 20 kDa FITC dextrans was moderate compared to a good permeation rate for the 376 Da carboxyfluor-escein and a poor penetration rate for 40 and 80 kDa FITC-dextrans. The permeability to carboxyfluorescein was 35 times more than to 80 kDa FITC-dextran [51]. In a study on the permeability of the human ciliary epithelium to a horseradish peroxidase, Tonjum and Pedersen [52] reported that ciliary and iridial epithelium contained a system of paracellular zonulae occludentes. Peroxidase was applied on the stromal side of ciliary body and iris specimens obtained from freshly enucleated eyes. The 40 kDa peroxidase was blocked apically in the lateral intercellular spaces of the CNPE whereas in the iris the progression of peroxidase was blocked apically in the lateral intercellular spaces of the IPE. Freddo [53] studied the intercellular junctions in the posterior IPE cells of the rhesus monkey by electron microscopy, freeze-fracture, and horseradish peroxidase. Intravenously injected horseradish... 

Scanning electron microscopy Freeze-fracture electron microscopy... 

The final evidence for the formation of an Abrikosov flux lattice of screw dislocations in liquid crystals was achieved by Zasadzinski et al. [39] via the visualization of the screw dislocations of (R)- and (S-)l-methylheptyl 4 -(4-n-tetradecyloxyphenylpropioloyloxy)-biphenyl-4-carboxylates using freeze-fracture transmission electron microscopy. Freeze-fracture transmission microscopy (TEM) is an essential tool for visualizing the TGBA phase at sufficient resolution in order to resolve the molecular organization. 

A further point in favour of such a model is provided by preliminary electron microscopy freeze-fracture studies, which show that lipid vesicles, comprised of the same lipid mixture and incubated with the photopigment, contain aggregates, embedded in the lipid matrix, whose linear dimensions are of the order of 100 A. However, as already stated, current fluctuations in the dark did not show square-shaped current trnaistions, providing a direct measure of channel conductance. Conversely, square-shaped fluctuations were observed in the light. The observed transitions (cf. Fig. 5) are similar in magnitude of those induced by molecules (like e.g. alamethicin, hemocyanin) which are knoxm to form dynamic channels in the membrane (Ehrenstein and Lecar, 1977). Such values... 

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. 

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.]... 

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. 

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). 

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... 

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. 

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.

Epi-illumination Subcellular imaging structures Freeze fracture Preparation of cellular ultrastructures in frozen-hydrated and living state for electron microscopy macromolecular organization of bilayer membranes... 

Subsequent to possible solubilization of membrane-bound proteins, solubilization must be verified. The criteria listed in Table 2 are relevant in assessing whether solubilization has been accomplished. To ascertain whether the solubilized protein has retained biological activity, membrane reconstitution (28) is attempted subsequent to detergent removal (24). Reconstitution is often visualized by electron microscopy employing either negative staining or freeze fracture. 

Modification of the ratio of the two phospholipids yielded different structures in freeze-fracture electron microscopy. Without DMPG (molar ratio DMPC/DMPG/AmB 10/0/5), large lamellar structure were observed. The presence of DMPG favored the formation of shorter lamellar structures, both stacked and fused in places or a predominantly disc morphology. 

Figure 3 Electron microscopy of lipid complex of amphotericin B. (A) Air-drying and shadowing, bar = 200 nm (B) freeze-fracture, bar = 200 nm. Source From Ref. 1.

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