Freeze fracture direct imaging: a new freeze fracture method for specimen preparation in cryo-transmission electron microscopy

In this paper, we present a new freeze fracture method for specimen preparation for transmission electron microscopy frozen samples. We call it freeze fracture direct imaging (FFDI) because it is a hybrid of conventional freeze fracture electron microscopy (FFEM) and cryo-transmission electron microscopy (cryo-TEM), combining elements of the fracture technique with direct imaging. Like in FFEM, the sandwich method is used to prepare the sample in a protected fashion. However, after the sample is vitrified and fractured, it is not shadowed but directly imaged. The new technique avoids some experimental artifacts produced by the blotting procedure in conventional cryo-TEM. It relies, though, on occasional fractures transparent to the electrons. The advantageous features are demonstrated by a comparison between conventional cryo-TEM and FFDI micrographs of vesicular solutions. The second outstanding advantage over conventional cryo-TEM is the fact that it is now possible for the very first time to directly image oil-rich mixtures films which normally would dissolve in the cryo-medium ethane. Micrographs of pure oil and of oil-rich microemulsions clearly prove the reliability of the FFDI technique as well as its enormous potential.     

Cryo-field emission scanning electron microscopy imaging of a rigid surfactant mesophase

The aerosol OT/ L-alpha-phosphatidylcholine/isooctane/water system forms a rigid mesophase that transitions from reverse hexagonal to multilamellar in structure at specific water contents. This study shows that characteristics of ordered liquid-crystalline mesophases can be distinguished and imaged in high clarity using cryo-field emission scanning electron microscopy (cryo-FESEM). The reverse hexagonal phase consists of bundles of long cylinders, some with length scales of over 2 microm, that are randomly oriented as part of a larger domain. Cryo-imaging allows the visualization of the intercylinder spacings and the details of transitions from one domain to another. The multilamellar structured mesophase consists of spherical vesicles of 100 nm to 10 microm in diameter, with intervening noncrystalline isotropic regions. Coexistence regions containing both the reverse hexagonal and lamellar structures are also observed in the transition from the reverse hexagonal to the lamellar phase. These results complement and qualitatively verify our earlier studies with small-angle neutron scattering, high-field nuclear magnetic resonance spectroscopy, and freeze-fracture direct imaging transmission electron microscopy. The information is useful in understanding materials templating in these rigid systems.     

Densely stacked multilamellar and oligovesicular vesicles, bilayer cylinders, and tubes joining with vesicles of a salt-free catanionic extractant and surfactant system

In the phase diagram of an excellent extractant of rare earth metal ions, di(2-ethylhexyl) phosphate (HDEHP, commercial name P204), mixing with a cationic trimethyltetradecylammonium hydroxide (TTAOH) in water, a birefringent Lalpha phase was found, which consists of densely stacked multilamellar vesicles. The densely stacked multilamellar vesicles are remarkably deformed, as observed by means of cryotransmission electron microscopy (cryo-TEM). Further, self-assembled structures-oligovesicular vesicles, bilayer cylinders, and tubes joining with vesicles-were also observed. The self-assembled phase is transparent, anisotropic, and highly viscous, possessing elastic properties determined by rheological measurements. This is the first time that birefringent Lalpha phase with remarkably deformed amphiphilic bilayer membranes has been constructed through combining a hydrophobic organic extractant having double chains with a water-soluble surfactant having a single chain, which may direct primarily toward acquiring an understanding of the mechanism of salt-free catanionic vesicles and secondarily to determine if vesicle-extraction technology utilizing extractants is possible.     

Cryo-fracture TEM: direct imaging of a random mesh phase

A novel and a simple method that allows direct imaging of viscous samples by cryo-TEM (cryo-transmission electron microscopy) is presented. A fracture on the vitrified sample is created in a controlled way. In the fracture, some edges are thin enough to allow direct imaging in transmission mode. The method was used to directly image a nonionic surfactant lamellar phase where a random mesh structure is formed at lower temperatures (<10 degrees C). A so-called random mesh phase, characterized by the presence of perforated surfactant bilayers, is imaged here for the first time. Images from the mesh structure are compared with images from the classical lamellar structure formed at room temperature.     

Resolving intermediate solution structures during the formation of mesoporous SBA-15

The evolution of the solution microstructures during the formation of the hexagonal mesoporous material SBA-15 was studied by direct imaging and freeze-fracture replication cryo-TEM. A reaction mixture was sampled at different times after the addition of tetramethoxyorthosilane (TMOS) to an acidic solution of Pluronic P123 held at 50 degrees C. Solution microstructures were detected by direct imaging cryo-TEM in the time window of 6.5-40 min after the addition of the TMOS (t = 0). The micrographs revealed that the initial spheroidal micelles evolve into threadlike micelles, which become longer and straighter with time. Then bundles with the dimensions similar to those found in the final material appeared, although there was no sign of a hexagonal arrangement up to 40 min. Due to the appearance of a precipitate at 40 min the sample became too viscous, preventing clear observation of its content. To observe the structures present after 40 min, freeze-fracture replication was carried out as well. Such samples were collected also at 22 min and showed the presence of threadlike micelles in agreement with the direct imaging cryo-TEM micrographs. The 2 h samples showed some areas of hexagonal ordered structures, which become very clear at 2 h 50 min. The cryo-TEM measurements were carried out under the same reaction conditions used in earlier in situ EPR experiments, thus allowing us to correlate molecular level events with the microstructure shape evolutions. This showed that the elongation of the micelles is a consequence of a reduction of the polarity and the water content within the micelles due to silicate adsorption and polymerization. Similar experiments were carried out also on SBA-15 prepared with HCl and TMOS at 35 degrees C. The appearance of threadlike micelles, followed by clustering of the TLMs, was observed under these conditions as well, but the reaction rate was faster. This suggests that the observed mechanism for the formation of SBA-15 is general.     

Retinol fluorescence: a simple method to differentiate different bilayer morphologies

The fluorescence spectra of retinol obtained in bilayer structures of two different systems with dodecyl tetraethylenglycol ether are shown. A correlation between the fluorescence intensity of retinol and the different topologies of bilayers has been found. We have tested this correlation with the C₁₂E₄/benzyl alcohol/water system, and we have also applied this idea to the study of the lamellar phases of the C₁₂E₄/PEG/water system. The highest fluorescence intensity of retinol corresponds to unilamellar vesicles, while the lowest is observed for multilamellar vesicles. The kinetic study of the degradation of vitamin A in these media is also related to the different microstructures of the bilayers.     

Phase behavior of the lecithin/water/isooctane and lecithin/water/decane systems

The isothermal pseudo-ternary-phase diagram was determined at 25 degrees C for systems composed oflecithin, water, and, as oil, either isooctane or decane. This was accomplished by a combination of polarizing microscopy, small-angle X-ray scattering, and NMR techniques. The lecithin-rich region of the phase diagram is dominated by a lamellar liquid-crystalline phase (Lalpha). For lecithin contents less than 60% and low hydration (mole ratio water/lecithin = W0 < 5.5), the system forms a viscous gel of branched cylindrical reverse micelles. With increase in the water content, the system phase separates into two phases, which is either gel in equilibrium with essentially pure isooctane (for lecithin < 25%) or a gel in equilibrium with Lalpha (for lecithin > 25%). These two-phase regions are very thin with respect to water dilution. For 8 < W0 < 54 very stable water-in-oil emulsions form. It is only after ripening for more than 1 year that the large region occupied by the emulsion reveals a complex pattern of stable phases. Moving along water dilution lines, one finds (i) the coexistence of gel, isooctane and Lalpha, (ii) equilibrium between reverse micelles and spherulites, and, finally, (iii) disconnected reverse micelles that fail to solubilize water for W0 > 54. This results in a Winsor II phase equilibrium at low lecithin content, while for lecithin > 20% the neat water is in equilibrium with a reverse hexagonal phase and an isotropic liquid-crystalline phase. The use of the decane as oil does not change the main features of the phase behavior.     

AEOT反胶束中脂肪酶的催化活性

反胶束已广泛应用于膜模拟化学和蛋白质的液 液萃取中[1～ 3] ,反胶束酶反应作为实现有机相酶催化的方法之一 ,具有许多独特的优点 ,反胶束独特的结构特征使表面活性剂分子组成的膜将油水相隔开 ,从而有利于保持酶的活性和稳定性。酶在反胶束的微水环境中比在水溶液中更接近天然的细胞内环境 ,在这里酶和底物分子均可得到有效的分散 ,接触几率大大提高 ,因而催化效率也得到很大提高。反胶束可以适用于各种类型的 (亲水的、疏水的和双亲的 )底物[4] ,已逐步形成“胶束酶学”的研究分支 ,研究胶束酶学的Martinek等[3] 曾预言 :反胶束体系有可…     

Glycol-modified silanes as versatile precursors in the synthesis of thin periodically organized silica films

Optically transparent, hexagonally organized mesoporous silica films with variable thickness have been synthesized from aqueous solutions of tetrakis(2-hydroxyethyl)orthosilicate (EGMS) in the presence of an amphiphilic structure-directing agent (Pluronic P123). Two different methods for film formation have been compared: spin coating from a highly concentrated and viscous precursor solution comprising EGMS, P123 and water/HCl via a direct templating of a preformed LC phase and spin coating from diluted solutions in tetrahydrofuran, thus following a cooperative self-organization process induced by solvent-evaporation. The influence of processing parameters such as pH-value, concentration of the precursors as well as aging of the sol on the periodic mesostructure is investigated.     

Swelling of Lalpha-phases by matching the refractive index of the water-glycerol mixed solvent and that of the bilayers in the block copolymer system of (EO)15-(PDMS)15-(EO)15

The swelling of Lalpha-phases from the block copolymer polyethylenoxide-b-polydimethylsiloxane-polyethylenoxide (EO)15-(PDMS)15-(EO)15 in water/glycerol mixtures is reported. At low and medium polymer concentrations (<60%), the block copolymer forms a turbid vesicular dispersion in water. With time, the small unilamellar vesicles (SUV) and the large multilamellar vesicles (MLV) separate into a two phase L1/Lalpha-system. The turbid dispersions of the Lalpha-phase below 60% of the compound become more and more transparent with increasing glycerol and at 60% of glycerol become completely clear. Replacement of water by the solvent glycerol thus lowers the turbidity of the dispersion and swells the interlamellar distance between the bilayers. A 20% aqueous L1/Lalpha-dispersion can thus be transformed into a single birefringent transparent Lalpha-phase. The swelling of the Lalpha-phase in water and the decrease of the turbidity of the dispersion by the addition of glycerol is explained by the matching of the refractive index of the solvent to the refractive index of the bilayers of the block copolymer. The matching of a refractive index lowers the Hamaker constant in the DLVO theory between the bilayers and therefore decreases the attraction between the bilayers what allows them to swell to a larger separation. The microstructures in the phases were determined by cryo- and FFR-TEM. The interlamellar distance between the bilayers was determined by SAXS measurements. The viscous properties of the Lalpha-phases were determined by oscillatory rheological measurements. In comparison to other Lalpha-phases from normal surfactants, the Lalpha-phases from the block copolymer (EO)15-(PDMS)15-(EO)15 have low shear moduli. This is probably due to the high flexibility of the poly dimethylsiloxane block in the bilayers what can be recognized on the non-spherical shapes of the SUV's.     

Lipase-immobilized biocatalytic membranes for enzymatic esterification: Comparison of various approaches to membrane preparation

In this paper, lipase-immobilized membranes were prepared by both non-covalent and covalent immobilization methods using (i) lipase adsorption on membranes, (ii) inclusion of enzyme in membrane structure by filtration and (iii) covalent attachment of lipase to membrane. The catalytic properties of these membranes have been studied in reaction of butyloleate synthesis through esterification of oleic acid with n-butanol in isooctane. Ultrafiltration membranes made of regenerated cellulose (C030F) and polyethersulphone (PM30) were used for lipase immobilization. It was found that the lipase inclusion in the wide porous supporting layer of membrane was the most efficient method in preparing highly effective biocatalytic membranes. The degree of oleic acid conversion using these membranes was about 70–72% with a reaction time of 8 h. It was shown that the distribution profile of the lipase in the membrane was important for the effective enzyme utilization.The profile imaging atomic force microscopy (AFM) technique was used to visualize surfaces of lipase-immobilized biocatalytic membranes. AFM has also been used to directly quantify interactions between lipase-coated tip and membrane surfaces. It was concluded that the direct measurements of the interaction force between the enzyme-coated tip and the membrane surface would be a useful and practical approach for the choice of membranes as porous polymeric support for lipase immobilization through adsorption.     

Trace detection of hormones and sulfonamides in viscous cosmetic products by neutral desorption extractive electrospray ionization tandem mass spectrometry

A sensitive method based on a geometry-independent neutral desorption (GIND) in combination with extractive electrospray ionization mass spectrometry (EESI-MS) has been developed for fast detection of illicit additives such as sulfonamides and hormones in highly viscous cosmetic products. The method gave a low limit of detection (LOD) (in the range of 0.001-1 ng/g), acceptable relative standard deviation (RSD=6.8-11.4%) and reasonable recovery (87-116%) for direct measuring of nine types of hormones and sulfonamides in the cosmetic products. The average measurement time for two types of samples was less than 1 min. Trace amounts of analytes in commercial cosmetic products have been quantitatively detected, without any sample pretreatment. The experimental results showed that non-volatile illicit additives such as sulfonamides and hormones could be sensitively liberated using the GIND device for quantitative detection from the highly viscous cosmetic products, demonstrating that GIND-EESI-MS is a promising tool for high throughput, sensitive and quantitative analysis of highly complex viscous samples.     

The effect of electrolyte on the encapsulation efficiency of vesicles formed by the nonionic surfactant, 2C18E12

Encapsulation efficiencies of vesicles formed by the nonionic surfactant 1,2-dioctadecyl-rac-glycerol-3-omega-methoxydodecylethylene glycol (abbreviated as 2C18E12) and its phospholipid counterpart, distearoylphosphatidylcholine (DSPC) at 298 K, were determined by the entrapment of the water-soluble dye, carboxyfluorescein (CF) to be 0.045+/-0.001 and 0.03+/-0.04 L mol(-1) for 2C18E12 vesicles prepared using low osmolarity (270 m Osm) Krebs-Henseleit (K-H) buffer and a modified 'high salt' (1600 m Osm) variant of K-H buffer, respectively, and 0.64+/-0.01 and 0.31+/-0.04 Lmol(-1) for DSPC vesicles prepared under the same conditions and in the same buffers. Freeze fracture electron microscopy studies confirmed the presence of vesicles when 2C18E12 and DSPC were dispersed in water and both buffer solutions. Small angle neutron scattering (SANS) studies, using D2O in place of H2O, showed that when 2C18E12 vesicles were prepared in the 'high salt' variant of K-H buffer as opposed to K-H buffer or water, a higher proportion of multilamellar vesicles (MLV) were formed. Furthermore when prepared in the 'high salt' variant of K-H buffer, the 2C18E12 bilayers were thinner, and when present in the form of MLV exhibited a smaller layer of water separating the bilayers. However, even in the absence of electrolyte, 2C18E12 formed surprisingly thin bilayers due to the penetration of the polyoxyethylene chains into the hydrophobic chain region of the bilayer. Due to the dehydrating effect of the high concentration of electrolyte present in the 'high salt' variant of K-H, the polyoxyethylene head groups penetrated further into the hydrophobic region of the bilayer making the bilayer even thinner. In the case of the DSPC vesicles, although the SANS study showed an increase in the relative proportion of multilamellar to unilamellar vesicles when samples were prepared in the 'high salt' variant of K-H buffer, no differences were observed in the thickness and the d-spacing of the vesicle bilayers. Variable temperature turbidity measurements of 2C18E12, and DSPC vesicles prepared in water indicated phase changes at 320+/-0.5 and 327+/-0.5 K, respectively, and were unchanged when the 'high salt' variant of K-H buffer was used as hydrating medium. Taken together, these results suggest that a low phase transition temperature was not the reason for the poor entrapment efficiency of 2C18E12 vesicles but rather the very 'thin' hydrophobic barrier formed by the penetration of the polyoxyethylene chains into the hydrophobic region of the bilayer.     

Solvolysis of benzoyl halides in water/NH4DEHP/isooctane microemulsions

A study was carried out on the solvolysis reactions of different benzoyl halides in microemulsions of water/NH4DEHP/isooctane, where NH4DEHP is ammonium bis(2-ethylhexyl) phosphate. Because of the low solubility of benzoyl halides in water, they are distributed between the continuous medium and the interface of the microemulsion, where the reaction takes place. The application of the pseudophase model has allowed us to obtain the distribution constants and the rate constants at the interface for the benzoyl halides. Reaction mechanisms and the changes in these mechanisms in terms of the water content of the microemulsion have been determined on the basis of kinetic data. The influence of the substituent and the leaving group on the reaction rate has been investigated. A comparison of kinetic results with those previously obtained in water/AOT/isooctane microemulsions allows a kinetic evaluation of the change in the microemulsion properties with the surfactant.     

Spectrometric study of AOT-hydrolysis reaction in water/AOT/isooctane microemulsions using phenolphthalein as a chemical probe

The kinetics of the alkaline hydrolysis of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in water/AOT/isooctane microemulsions has been studied by monitoring the absorbance change of the phenolphthalein in the system with time. The apparent first-order rate constant k(obs) has been obtained and found to be dependent on both the molar ratio of water to AOT ω and the temperature. The dependences of k(obs) on ω have been analyzed by a pseudophase model which gives the true rate constants k(i) of the AOT-hydrolysis reaction on the interface and the partition coefficients K(wi) for the distribution of OH(-) between aqueous and interface pseudophases at various temperatures; the latter is almost independent of the temperature and ω. The temperature dependences of the reaction rate constants k(obs) and k(i) have been analyzed to obtain enthalpy ΔH(≠), entropy ΔS(≠), and energy E(a) of activation, which indicate that the distribution of OH(-) between aqueous and interface pseudophases increases ΔS(≠) but makes no contribution to E(a) and ΔH(≠). The influence of the overall concentration of AOT in the system on the rate constant has been examined and found to be negligible. It contradicts with what was reported by García-Río et al. (1) but confirms that the first-order reaction of the AOT-hydrolysis takes place on the surfactant interface. The study of the influence of AOT-hydrolysis on the kinetics of the alkaline fading of crystal violet or phenolphthalein in the water/AOT/isooctane microemulsions suggests that corrections for the AOT-hydrolysis in these reactions are required.     

Entrance effects at nanopores of nanocapsules functionalized with poly(ethylene glycol) and their flow through nanochannels

We studied the effect of poly(ethylene glycol) (PEG) on the extrusion of large, multilamellar nanocapsules (also called liposomes or vesicles) through nanochannels with a length of 6 microm. For the generation of the nanocapsules, we used a lipid mixture with lecithin consisting of saturated and unsaturated fatty acids (dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylcholine (DOPC)), cholesterol, and 2-8 mol % PEG linked to a lipid anchor (distearoylphosphatidylethanolamine (DSPE)) or the plain lipid anchor without PEG. An increase in PEG leads to a decrease of the critical tension for nanocapsule rupture (lysis tension) between 20-30%, whereas the pure lipid anchor does not produce any differences. We interpret these findings to be produced by a partial intrusion of the polymeric chain into the phospholipid bilayer of the nanocapsule which weakens its tensile strength. We evaluate statistically the discrepancies of lysis tensions found for different channels widths (50-100 nm) and two or four channels in series. Comparing our results on the flow resistance of either nanocapsules or pure water with lubrication theory, we find that the calculated viscous forces are not sufficient to account for the measured friction of nanocapsules. This shows that the nanocapsules are decelerated in the nanochannels by van der Waals interactions between channel and capsule walls and the intermediate water layer. The strength of these forces is 24 times higher for PEG and 94 times higher for the pure lipid anchor than the respective calculated viscous forces alone, showing that nanocapsule flow in nanochannels cannot be considered under the classical continuum assumption of the intermediate water layer.     

Characterizing interfacial friction in bis(2-ethylhexyl) sodium sulfosuccinate reverse micelles from photoisomerization studies of carbocyanine derivatives

Photoisomerization of two carbocyanine derivatives has been examined in bis(2-ethylhexyl) sodium sulfosuccinate (AOT) reverse micelles to understand the factors that govern this process in the interfacial region of organized assemblies. To this effect, fluorescence lifetimes and quantum yields of 3,3(')-diethyloxadicarbocyanine iodide and merocyanine 540 have been measured in AOT∕isooctane∕water and AOT∕cyclohexane∕water reverse micellar systems as a function of the mole ratio of water to the surfactant, W. The nonradiative rate constants, which have been identified as the rates of photoisomerization for these solutes, were obtained from the experimentally measured parameters. The steady rise and subsequent saturation observed in the nonradiative rate constants upon increasing W has been rationalized in terms of micellar packing. An inverse correlation has been obtained between the nonradiative rate constants and the critical packing parameter, indicating that the interfacial friction experienced by the solute molecule is essentially described by this parameter.     

Water dynamics in small reverse micelles in two solvents: two-dimensional infrared vibrational echoes with two-dimensional background subtraction

Water dynamics as reflected by the spectral diffusion of the water hydroxyl stretch were measured in w(0) = 2 (1.7 nm diameter) Aerosol-OT (AOT)/water reverse micelles in carbon tetrachloride and in isooctane solvents using ultrafast 2D IR vibrational echo spectroscopy. Orientational relaxation and population relaxation are observed for w(0) = 2, 4, and 7.5 in both solvents using IR pump-probe measurements. It is found that the pump-probe observables are sensitive to w(0), but not to the solvent. However, initial analysis of the vibrational echo data from the water nanopool in the reverse micelles in the isooctane solvent seems to yield different dynamics than the CCl(4) system in spite of the fact that the spectra, vibrational lifetimes, and orientational relaxation are the same in the two systems. It is found that there are beat patterns in the interferograms with isooctane as the solvent. The beats are observed from a signal generated by the AOT/isooctane system even when there is no water in the system. A beat subtraction data processing procedure does a reasonable job of removing the distortions in the isooctane data, showing that the reverse micelle dynamics are the same within experimental error regardless of whether isooctane or carbon tetrachloride is used as the organic phase. Two time scales are observed in the vibrational echo data, ~1 and ~10 ps. The slower component contains a significant amount of the total inhomogeneous broadening. Physical arguments indicate that there is a much slower component of spectral diffusion that is too slow to observe within the experimental window, which is limited by the OD stretch vibrational lifetime.     

Molecular conformation and craze fracture

A craze may fracture either by the breaking of covalent bonds or by a process of “viscous rupture” involving the movement of bulk material in the craze. In the latter case it is necessary that the majority of the chain ends of the polymer molecules passing through the craze-matrix interface terminate within the craze. We have therefore calculated the probability of a polystrene macromolecule spanning a thin craze and shown that for viscous rupture a craze in a normal commercial polystyrene must be something more than 40 nm thick. As the measured craze thicknesses have generally exceeded 100 nm, a viscous fracture process is clearly possible, though, of course, chain rupture is not excluded by the argument. More difficulties arise when fracture occurs within a specified region of the craze and the possibility of a bond fracture under these circumstances is briefly discussed.     

1H NMR Relaxation Studies on Glycerine-Water and Dioxan-Water with Paramagnetic Ions

Abstract

The Proton magnetic Resonance (PMR) spin-lattice and spin-spin relaxation times (T ₁ and T ₂) were measured in highly viscous (glycerine - water) and less viscous (dioxan-water) systems at different temperatures. The values of relaxation times increase with increasing the temperature. This result is interpreted as due to the combined effect of viscosity and temperature in these solutions. The relaxation times were also measured in these solutions containing paramagnetic ions(PMI). The results indicate that the possibility of an anti-parallel bonding of the paramagnetic ions is higher in highly viscous solutions as compared to low viscous systems and the association in the above mixtures appears to be weak.