Freeze-fracture Transmission Electron Microscopy Research Articles

Self-assembled structural transition from vesicle phase to sponge phase and emulsifying properties in mixtures of arginine and fatty acids

Abstract Two fatty acids, octanoic acid (OA) and decanoic acid (DA) were separately mixed with a natural biological molecule, l -arginine (Arg), in aqueous solutions for comparative investigation. Self-assembled structures including micelles, vesicles, sponge structures, were observed by varying the compositions and chain length of fatty acids. The structures of vesicles and sponge phase were determined by freeze fracture-transmission electron microscope (FF-TEM). The transition of self-assembled structures, especially from vesicles to sponge structure, was significantly reflected in the change of rheological properties. The double hydrogen bonding formed between the guanidyl group of Arg and the carboxyl group of fatty acids is considered to be the crucial force, which drives the self-assembly process combining with other non-covalent interaction, electrostatic interaction and hydrophobic interaction. Depending on the nature of the fatty acid, the systems exhibit good emulsion stability.

Ionogels of Sugar Surfactant in Ethylammonium Nitrate: Phase Transition from Closely Packed Bilayers to Right-Handed Twisted Ribbons.

In the simplest ionic liquid, ethylammonium nitrate (EAN), ionogels with high mechanical strength were prepared from a surfactant with a disaccharide polar head. Phase structures from closely packed bilayers to right-handed twisted ribbons were determined via freeze-fracture transmission electron microscopy (FF-TEM) observations. The phase transition mechanism was investigated deeply and systematically. The temperature contributes to suitable tail chain conformations of surfactant molecules for adapting to different self-assembled structures including right-handed twisted ribbons and bilayers. Two different arrays were revealed for different bilayers by the small-angle X-ray scattering (SAXS) measurements. The rheological and tribological properties of the ionogels were investigated. The better lubricating property and antiwear capability of the ionogels compared to the EAN may be attributed to the structure characteristics and the good thixotropic properties.

Self-Assembly and Rheological Properties of a Pseudogemini Surfactant Formed in a Salt-Free Catanionic Surfactant Mixture in Water

The surface and bulk properties of bola-type dicarboxylic acid (sebacic acid, SA) and zwitterionic surfactant tetradecyldimethylamine oxide (C14DMAO) mixtures in aqueous solutions were studied. Surface tension measurements indicate a pronounced synergistic effect between SA and C14DMAO. In bulk aqueous solutions, rich phase behavior was observed with a varied SA-to-C14DMAO ratio (ρ) and a total surfactant concentration. Typically at ρ = 0.5, a novel pseudogemini surfactant (C14-S-C14) forms, driven by electrostatic interaction and hydrogen bonding. The C14-S-C14/H2O system exhibits rich phase behavior induced by the transition of aggregates. With increasing concentration of C14-S-C14, one can observe a viscous L1 phase, an L1/Lα two-phase region where a birefringent Lα phase is on the top of an L1 phase, a single Lα phase, and finally a mixture of an Lα phase and a precipitate. Microstructures formed in the Lα phases were determined by freeze-fracture transmission electron microscopy (FF-TEM) and cryogenic-transmission electron microscopy (cryo-TEM) observations. Polymorphic aggregation behavior was observed with the formation of a variety of bilayer structures including unilamellar vesicles, onions, and open and hyperbranched bilayers. Rheological measurements showed that the Lα phases are viscoelastic and sensitive to temperature where a quick loss of viscoelasticity was observed at elevated temperature.

Aquaporin-Based Biomimetic Polymeric Membranes: Approaches and Challenges.

In recent years, aquaporin biomimetic membranes (ABMs) for water separation have gained considerable interest. Although the first ABMs are commercially available, there are still many challenges associated with further ABM development. Here, we discuss the interplay of the main components of ABMs: aquaporin proteins (AQPs), block copolymers for AQP reconstitution, and polymer-based supporting structures. First, we briefly cover challenges and review recent developments in understanding the interplay between AQP and block copolymers. Second, we review some experimental characterization methods for investigating AQP incorporation including freeze-fracture transmission electron microscopy, fluorescence correlation spectroscopy, stopped-flow light scattering, and small-angle X-ray scattering. Third, we focus on recent efforts in embedding reconstituted AQPs in membrane designs that are based on conventional thin film interfacial polymerization techniques. Finally, we describe some new developments in interfacial polymerization using polyhedral oligomeric silsesquioxane cages for increasing the physical and chemical durability of thin film composite membranes.

Chiroptical vesicles and disks that originated from achiral molecules.

We report a chiral gel of vesicles and disklike micelles that originated from achiral molecules. The supramolecular chirality was obtained via regulating pH, in which a sol-gel-sol transition in a colloidal system consisting of a gelator, 4,4-di(2,3-dicarboxylphenoxyl)azobenzene (AzoNa4), and a zwitterionic surfactant, tetradecyldimethylamine oxide (C14DMAO), happened. The supramolecular chirality was related to the state of aggregation, i.e., only the condensed gels show chiral sense and sols are chiral-silent. The coexistence of vesicles and disklike micelles was captured for the first time in supramolecular chiral hydrogels by cryo- and freeze-fracture transmission electron microscopy (cryo- and FF-TEM) observations. Ascribed to the photoisomerization of the azobenzene units, upon alternative UV/visible light irradiation, the gel chirality can be switched reversibly with the macroscopic changes between vesicles/disks and wormlike micelles. A pH- and light-dual-responsive chiroptical switch can be constructed, which may require understanding the regulating membrane permeability and reagent release of structural transformation through photoisomerization and also require understanding the origin of gelation-induced supramolecular chirality completely based on achiral molecules.

Synthesis of Silica Nanotube Using Myelin Figure as Template and their Formation Mechanism.

Silica nanotubes are synthesized through a sol-gel reaction of tetraethyl orthosilicate (TEOS) using myelin figures of Pluronic P123 as the structure-directing agent. The simultaneous progression of the formation of molecular assemblies that act as templates and the formation of silica frameworks though a sol-gel reaction of the silica precursor is a characteristic of this reaction system. The synthesized silica nanotubes were characterized using transmission electron microscopy (TEM), nitrogen adsorption/desorption measurements, and Fourier-transform infrared spectroscopy (FT-IR). The silica nanotubes were unilamellar with diameters of approximately 30 nm, membrane thicknesses of approximately 10 nm, and lengths exceeding a few hundred nanometers. The Brunauer-Emmett-Teller (BET) specific surface area was 589.46 m(2)/g. Silica nanotubes can also be obtained using other Pluronic surfactants that can form myelin figures. In this work, we also investigated the formation mechanism of the silica nanotubes. The typical diameter of a myelin figure is a few tens of micrometers. However, myelin figures with diameters of approximately 10 µm can form in systems with TEOS because bifurcation is induced by minute silica nuclei that form during the initial reaction between TEOS and water. Freeze fracture TEM (FF-TEM) observations revealed the existence of myelin figures with diameters of 20 to 30 nm, which are the same size and shape as the synthesized silica nanotubes. These results indicate that bifurcation of the myelin figures is induced by the silica nuclei that form via the initial reaction of TEOS, which result in the formation of bifurcated myelin figures with diameters of ~10 µm. Myelin figures with diameters of 20 to 30 nm form on the surface, and they become templates where the reaction of TEOS progresses to form silica nanotubes with diameters of approximately 30 nm.

Faceted fatty acid vesicles formed from single-tailed perfluorinated surfactants.

The aggregation behavior and rheological properties of two mixtures of perfluorononanoic acid (PFNA)/NaOH and perfluorodecanoic acid (PFDA)/NaOH were investigated in aqueous solutions. Interestingly, pH-sensitive polyhedral fatty acid vesicles were spontaneously formed in both systems, which were determined by freeze-fracture transmission electron microscopy (FF-TEM) measurements. Especially, a phase transition from faceted vesicles to the L3 phase with the increase of pH was observed in the PFNA/NaOH system while it was not observed in the PFDA/NaOH system. Differential scanning calorimetry (DSC) and wide angle X-ray scattering (WAXS) measurements confirmed that the bilayers of the faceted vesicles were in the crystalline station indicating that the crystallization of fluorocarbon chains was the main driving force for their formation. Besides, the two systems of faceted perfluorofatty acid vesicles exhibit interesting rheological properties, for instance, they showed high viscoelasticity and shear-thinning behaviour, and the elastic modulus (G') and viscous modulus (G'') of PFDA/NaOH vesicles were much higher than those of PFNA/NaOH vesicles. Conversely, the solution of the L3 phase with fluid bilayers did not present viscoelastic properties. Therefore, the viscoelastic properties of vesicles resulted from the crystalline fluorinated alkyl chains with high rigidity at room temperature and the dense packing of vesicles. As far as we know, such faceted fatty acid vesicles formed from single-tailed perfluorinated surfactants have been rarely reported. Our work successfully constructs polyhedral fatty acid vesicles and proposes their formation mechanism, which should be a great advance in the fundamental research of fatty acid vesicles.

Interfacial and emulsifying properties of soybean peptides with different degrees of hydrolysis.

In this study, the effects of the degree of hydrolysis on the interfacial and emulsifying properties of soybean peptides were evaluated based on surface and interfacial tension, dynamic light scattering (DLS), and freeze-fracture transmission electron microscopy (FF-TEM) analyses. Of the five evaluated soybean peptides (SP95, SP87, SP75, SP49, and SP23), those with higher degrees of hydrolysis (SP95 and SP87) did not exhibit noticeable surface-active properties in water, whereas those with relatively low degrees of hydrolysis (SP75, SP49, and SP23) exhibited remarkable surface tension-lowering activity. The latter set (SP75, SP49, and SP23) also formed giant associates with average sizes ranging from 64.5 nm to 82.6 nm above their critical association concentration (CAC). Moreover, SP23 with the lowest degree of hydrolysis exhibited excellent emulsifying activity for soybean oil, and FF-TEM analysis demonstrated that the emulsions were stabilized by a lamella-like multilayer peptide structure on the oil droplets that prevented coagulation. The peptide with the lowest degree of hydrolysis (SP23) was effective not only for soybean oil emulsification, but also for the emulsification of liquid paraffin and silicon oil that are generally difficult to emulsify.

Concentration and temperature induced dual-responsive wormlike micelle to hydrogel transition in ionic liquid-type surfactant [C₁₆imC₉]Br aqueous solution without additives.

A highly viscoelastic fluid formed by the ionic liquid-type surfactant 1-hexadecyl-3-nonyl imidazolium bromide ([C16imC9]Br) in water in the absence of any additive was studied. The phase behavior and morphology of aggregates were studied by a combination of rheological techniques, small-angle X-ray scattering (SAXS), cryo-etch-scanning electron microscopy (cryo-etch-SEM) and freeze-fractured transmission electron microscopy (FF-TEM). [C16imC9]Br aqueous solutions showed interesting rheological behavior as a function of both concentration and temperature, which invoked a transition between wormlike micelles and hydrogels. With the increase in [C16imC9]Br concentration, the aqueous solution could form viscoelastic wormlike micelles (50-80 mM), hydrogels (90-110 mM) and wormlike micelles (120-180 mM). As the temperature increased, the hydrogels (90-110 mM) could also transit to wormlike micelles. The unusual phase transition between wormlike micelles and elastic hydrogels was postulated to be the change of the average micellar length.

Transition of Phase Structures in Mixtures of Lysine and Fatty Acids.

Aggregation behaviors of the mixtures of lysine and fatty acids (FAs) with different chain lengths in aqueous solutions were investigated, and the self-assembled structural transition was determined in detail. Aggregates including micelles, vesicles, sponge structures, and fibers were observed by varying the compositions and the chain length of fatty acids. The sponge phase found in mixtures of octanoic acid and lysine was determined by freeze fracture-transmission electron microscope (FF-TEM). Circular dichroism (CD) signals were detected in the self-assembled structures due to the chirality of lysine molecules. The rheological properties of samples consisting of different aggregates formed by mixtures of lysine and fatty acids were measured, which provided the controlling factor of the chain length. The combined effect of noncovalent interactions including electrostatic interactions, hydrogen bonding, and hydrophobicity is supposed to be responsible for the aggregation behaviors, in which the hydrogen bonding acts as the main driving force in the self-assembled process.

Nanostructure and dielectric properties of a twist-bend nematic liquid crystal mixture

We present freeze-fracture transmission electron microscopy (FF-TEM), dielectric spectroscopy and electro-optic measurements on a dimeric liquid crystal mixture, which previously was proposed to form the twist-bend nematic (Ntb) phase. Our FF-TEM studies provide a direct image of a 10.5 nm periodic structure, consistent with the expected nanoscale, heliconical twist-bend modulation of the molecular orientation. Dielectric measurements in the 100 Hz to 10 MHz range reveal three nearly Debye-type dispersion processes in the nematic and the twist-bend phase. Low frequency 8 V/µm electric fields applied on planar cells cause the optical-scale stripe texture (another characteristic feature of the Ntb phase) to disappear. Higher (>16 V/µm) fields gradually realign the heliconical axis along the electric field; it relaxes back after the field removal.

Phase behavior and phase structure of 1:1 salt-free catanionic surfactant dodecyltrimethylammonium decanoate

The salt-free catanionic surfactant dodecyltrimethylammonium decanoate (DTAD) was synthesized by a novel method. Phase behavior and phase structure of DTAD with water at full concentration range were studied at 20–45°C, using differential scanning calorimetry (DSC), freeze-fracture transmission electron microscopy (FF-TEM), polarization microscopy, and small-angle X-ray diffraction (XRD) techniques. The DTAD–water system formed a large aqueous multi-phase region, a large lamellar liquid crystal (Lα phase) region, and a hydrated crystal phase. The densely packed vesicles and the lamellar structure induced the formation of aqueous multi-phase region. With increasing temperature, the multi-phase region and the Lα phase region reduced, and the isotropic solution phase (L1 phase) appeared.

Regional changes of AQP0-dependent square array junction and gap junction associated with cortical cataract formation in the Emory mutant mouse

The Emory mutant mouse has been widely used as an animal model for human senile cataract since it develops late-onset hereditary cataract. Here, we focus on the regional changes of aquaporin-0 (AQP0) and connexins that are associated with the cortical cataract formation in the Emory mutant mice. Emory mutant and CFW wild-type mice at age 1–16 months were used in this study. By using an established photography system with dissecting microscopy, the opacities were first detected at the anterior or posterior lens center surface in Emory mice at age 7 months, and gradually extended toward the equator during the 16 months examined. Scanning EM verified that disorganized and fragmented fiber cells were associated with the areas of opacities within approximately 200 μm from the lens surface, indicating that Emory mouse cataracts belong to the cortical cataracts. Freeze-fracture TEM further confirmed that cortical cataracts exhibited extensive wavy square array junctions, small gap junctions and globules. Immunofluorescence analysis showed that in contrast to the high labeling intensity of AQP0-loop antibody, the labeling of AQP0 C-terminus antibody was decreased considerably in superficial fibers in Emory cataracts. Similarly, a significant decrease in the labeling of the antibody against Cx50 C-terminus, but not Cx46 C-terminus, occurred in superficial and outer cortical fibers in Emory cataracts. Western blotting further revealed that the C-termini of both AQP0 and Cx50 in Emory cataracts were decreased to over 50% to that of the wild-type. Thus, this systematic study concludes that the Emory mouse cataract belongs to the cortical cataract which is due to regional breakdown of superficial fibers associated with formation of AQP0-dependent wavy square array junctions, small gap junctions and globules. The marked decreases of the C-termini of both AQP0 and Cx50 in the superficial fibers may disturb the needed interaction between these two proteins during fiber cell differentiation and thus play a role in the cortical cataract formation in Emory mutant mice.

Interaction between zero-charged catanionic vesicles and PEO–PPO–PEO triblock copolymers

We investigate the interaction between zero-charged catanionic vesicles and PEO–PPO–PEO (poly(ethylene oxide–poly(propylene oxide)–poly(ethylene oxide)) triblock copolymers. The 25-mg mL−1 aqueous solution of tetradecyltrimethylammonium laurate (TTAL) contains closely packed uni- and multi-lamellar vesicles and shows viscoelastic properties with a dominant elastic modulus (G′) over a viscous modulus (G″). When a small amount of F127 ((EO)97(PO)69(EO)97) or F68 ((EO)76(PO)29(EO)76) was added, an improvement of the viscoelasticity was observed at suitable polymer concentrations. Freeze–fracture transmission electron microscopy (FF-TEM) observations on an F68-containing system revealed interesting aggregate transition from vesicles to flexible tubules and back to vesicles. The improvement of the viscoelasticity of the vesicular solution containing F68 or F127 can be explained by the formation of tubule and polymer–vesicle associates, while no such phenomenon was noticed for P123 ((EO)19(PO)69(EO)19) which has the highest propylene oxide (PO) content and the strongest ability to self-associate in aqueous solution. In all the cases, vesicles will be destroyed and phase separation can be observed at high polymer contents (>5-mg mL−1).

Direct observation of liquid crystals using cryo-TEM: specimen preparation and low-dose imaging.

Liquid crystals (LCs) represent a challenging group of materials for direct transmission electron microscopy (TEM) studies due to the complications in specimen preparation and the severe radiation damage. In this paper, we summarize a series of specimen preparation methods, including thin film and cryo-sectioning approaches, as a comprehensive toolset enabling high-resolution direct cryo-TEM observation of a broad range of LCs. We also present comparative analysis using cryo-TEM and replica freeze-fracture TEM on both thermotropic and lyotropic LCs. In addition to the revisits of previous practices, some new concepts are introduced, e.g., suspended thermotropic LC thin films, combined high-pressure freezing and cryo-sectioning of lyotropic LCs, and the complementary applications of direct TEM and indirect replica TEM techniques. The significance of subnanometer resolution cryo-TEM observation is demonstrated in a few important issues in LC studies, including providing direct evidences for the existence of nanoscale smectic domains in nematic bent-core thermotropic LCs, comprehensive understanding of the twist-bend nematic phase, and probing the packing of columnar aggregates in lyotropic chromonic LCs. Direct TEM observation opens ways to a variety of TEM techniques, suggesting that TEM (replica, cryo, and in situ techniques), in general, may be a promising part of the solution to the lack of effective structural probe at the molecular scale in LC studies.

Spontaneous vesicle phase formation by pseudogemini surfactants in aqueous solutions.

The phase behavior of a kind of pseudogemini surfactant in aqueous solutions, formed by the mixture of sodium dodecyl benzene sulfonate (SDBS) and butane-1,4-bis (methylimidazolium bromide) ([mim-C4-mim]Br2) or butane-1,4-bis(methylpyrrolidinium bromide) ([mpy-C4-mpy]Br2) in a molar ratio of 2 : 1, is reported in the present work. When [mim-C4-mim]Br2 or [mpy-C4-mpy]Br2 is mixed with SDBS in aqueous solutions, one cationic [mim-C4-mim]Br2 or [mpy-C4-mpy]Br2 molecule "bridges" two SDBS molecules by noncovalent interactions (e.g. electrostatic, π-π stacking, and σ-π interactions), behaving like a pseudogemini surfactant. Vesicles can be formed by this kind of pseudogemini surfactant, determined by freeze-fracture transmission electron microscopy (FF-TEM) or cryogenic-transmission electron microscopy (cryo-TEM) and dynamic light scattering (DLS). The mixed system of sodium dodecyl sulfate (SDS) with [mim-C4-mim]Br2 or [mpy-C4-mpy]Br2 was also constructed, and only micelles were observed. We infer that a pseudogemini surfactant is formed under the synergic effect of electrostatic, π-π stacking, and σ-π interactions in the SDBS/[mim-C4-mim]Br2/H2O system, while electrostatic attraction and hydrophobic interactions may provide the directional force for vesicle formation in the SDBS/[mpy-C4-mpy]Br2/H2O system.

Sponge phase producing porous CeO2 for catalytic oxidation of CO.

The aggregation behavior of mixtures of the alkaline amino acid L-Arginine (L-Arg) and bis(2-ethylhexyl)phosphoric acid (DEHPA) in water was studied in detail. At a fixed L-Arg concentration, a phase sequence of micellar phase (L1 phase), vesicle phase (Lαv phase), planar lamellar phase (Lαl phase), and sponge phase (L3 phase) was obtained with increasing DEHPA concentration due to changes in the packing parameter. The phase transition of the lamellar structures was determined by freeze-fracture TEM and (2)H NMR spectroscopy. Rheological measurements reflected the phase transition through significant variations of both the elastic modulus and the viscous modulus. Porous CeO2 materials were produced by utilizing the L3 phase as template, and the porous CeO2 exhibited excellent catalytic oxidation activity toward CO due to its high surface area, which provides more active sites for CO conversion.

Massive formation of square array junctions dramatically alters cell shape but does not cause lens opacity in the cav1-KO mice

The wavy square array junctions are composed of truncated aquaporin-0 (AQP0) proteins typically distributed in the deep cortical and nuclear fibers in wild-type lenses. These junctions may help maintain the narrowed extracellular spaces between fiber cells to minimize light scattering. Herein, we investigate the impact of the cell shape changes, due to abnormal formation of extensive square array junctions, on the lens opacification in the caveolin-1 knockout mice. The cav1-KO and wild-type mice at age 1–22 months were used. By light microscopy examinations, cav1-KO lenses at age 1–18 months were transparent in both cortical and nuclear regions, whereas some lenses older than 18 months old exhibited nuclear cataracts. Scanning EM consistently observed the massive formation of ridge-and-valley membrane surfaces in young fibers at approximately 150 μm deep in all cav1-KO lenses studied. In contrast, the typical ridge-and-valleys were only seen in mature fibers deeper than 400 μm in wild-type lenses. The resulting extensive ridge-and-valleys dramatically altered the overall cell shape in cav1-KO lenses. Remarkably, despite dramatic shape changes, these deformed fiber cells remained intact and made close contact with their neighboring cells. By freeze-fracture TEM, ridge-and-valleys exhibited the typical orthogonal arrangement of 6.6 nm square array intramembrane particles and displayed the narrowed extracellular spaces. Immunofluorescence analysis showed that AQP0 C-terminus labeling was significantly decreased in outer cortical fibers in cav1-KO lenses. However, freeze-fracture immunogold labeling showed that the AQP0 C-terminus antibody was sparsely distributed on the wavy square array junctions, suggesting that the cleavage of AQP0 C-termini might not yet be complete. The cav1-KO lenses with nuclear cataracts showed complete cellular breakdown and large globule formation in the lens nucleus. This study suggests that despite dramatic cell shape changes, the massive formation of wavy square array junctions in intact fibers may provide additional adhesive support for maintaining the narrowed extracellular spaces that are crucial for the transparency of cav1-KO lenses.

Molecular modeling and imaging of initial stages of cellulose fibril assembly: evidence for a disordered intermediate stage.

The remarkable mechanical strength of cellulose reflects the arrangement of multiple β-1,4-linked glucan chains in a para-crystalline fibril. During plant cellulose biosynthesis, a multimeric cellulose synthesis complex (CSC) moves within the plane of the plasma membrane as many glucan chains are synthesized from the same end and in close proximity. Many questions remain about the mechanism of cellulose fibril assembly, for example must multiple catalytic subunits within one CSC polymerize cellulose at the same rate? How does the cellulose fibril bend to align horizontally with the cell wall? Here we used mathematical modeling to investigate the interactions between glucan chains immediately after extrusion on the plasma membrane surface. Molecular dynamics simulations on groups of six glucans, each originating from a position approximating its extrusion site, revealed initial formation of an uncrystallized aggregate of chains from which a protofibril arose spontaneously through a ratchet mechanism involving hydrogen bonds and van der Waals interactions between glucose monomers. Consistent with the predictions from the model, freeze-fracture transmission electron microscopy using improved methods revealed a hemispherical accumulation of material at points of origination of apparent cellulose fibrils on the external surface of the plasma membrane where rosette-type CSCs were also observed. Together the data support the possibility that a zone of uncrystallized chains on the plasma membrane surface buffers the predicted variable rates of cellulose polymerization from multiple catalytic subunits within the CSC and acts as a flexible hinge allowing the horizontal alignment of the crystalline cellulose fibrils relative to the cell wall.

Local Orientational Analysis of Helical Filaments and Nematic Director in a Nanoscale Phase Separation Composed of Rod-Like and Bent-Core Liquid Crystals Using Small- and Wide-Angle X-ray Microbeam Scattering

We analyzed the local nanostructure in binary mixtures of rod- and bent-shaped molecules, n-pentyl-4-cyanobiphenyl (5CB) and 1,3-phenylene bis[4-(4-n-octyloxyphenyliminomethyl) benzoates] (P-8-OPIMB), respectively, using small- and wide-angle X-ray microbeam and macrobeam scattering. From the orientational X-ray scattering patterns, we concluded that the nematic director of 5CB is almost parallel to the smectic layers dominated by bent-core molecules in Bx. Moreover, we observed oriented small-angle diffraction peaks (about 300 Å), which is close to the spacing of 5-7 layers, and also consistent with the width of a helical nanofilament textures as observed by freeze-fracture transmission electron microscopy. The kinetics in B4 was also discussed based on the contact experimental method.