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Stromal interacting molecule (STIM) and Orai proteins constitute the core machinery of store-operated calcium entry. We used transmission and freeze-fracture electron microscopy to visualize STIM1 and Orai1 at endoplasmic reticulum (ER)-plasma membrane (PM) junctions in HEK 293 cells. Compared with control cells, thin sections of STIM1-transfected cells possessed far more ER elements, which took the form of complex stackable cisternae and labyrinthine structures adjoining the PM at junctional couplings (JCs). JC formation required STIM1 expression but not store depletion, induced here by thapsigargin (TG). Extended molecules, indicative of STIM1, decorated the cytoplasmic surface of ER, bridged a 12-nm ER-PM gap, and showed clear rearrangement into small clusters following TG treatment. Freeze-fracture replicas of the PM of Orai1-transfected cells showed extensive domains packed with characteristic "particles"; TG treatment led to aggregation of these particles into sharply delimited "puncta" positioned upon raised membrane subdomains. The size and spacing of Orai1 channels were consistent with the Orai crystal structure, and stoichiometry was unchanged by store depletion, coexpression with STIM1, or an Orai1 mutation (L273D) affecting STIM1 association. Although the arrangement of Orai1 channels in puncta was substantially unstructured, a portion of channels were spaced at ∼15 nm. Monte Carlo analysis supported a nonrandom distribution for a portion of channels spaced at ∼15 nm. These images offer dramatic, direct views of STIM1 aggregation and Orai1 clustering in store-depleted cells and provide evidence for the interaction of a single Orai1 channel with small clusters of STIM1 molecules.

For the very first time, atomic force microscope images of lamellar phases were observed combined with a freeze fracture technique that does not involve the use of replicas. Samples are rapidly frozen, fractured, and scanned directly with atomic force microscopy, at liquid nitrogen temperature and in high vacuum. This procedure can be used to investigate micro-structured liquids. The lamellar phases in Sodium bis(2-ethylhexyl) sulfosuccinate (AOT)/water and in C12E5/water systems were used to asses this new technique. Our observations were compared with x-ray diffraction measurements and with other freeze fracture methods reported in the literature. Our results show that this technique is useful to image lyotropic lamellar phases and the estimated repeat distances for lamellar periodicity are consistent with those obtained by x-ray diffraction. [ABSTRACT FROM AUTHOR]

Cell membranes are composed of a large variety of lipids and proteins. While the localization and function of membrane proteins have been extensively investigated, the distribution of membrane lipids, especially in the non-cytoplasmic leaflet of organelle membranes, remains largely unknown. Fluorescent biosensors have been widely used to study membrane lipid distribution; however, they have some limitations. By utilizing the quick-freezing and freeze-fracture replica labeling electron microscopy technique, we can uncover the precise distribution of membrane lipids within cells and assess the function of lipid-transporting proteins. In this review, I summarize recent progress in analyzing intracellular lipid distribution by utilizing this method., (© 2023. The Author(s), under exclusive licence to Japanese Association of Anatomists.)

Electrophysiological and morphological methods were used to study connexin50 (Cx50) expressed in Xenopus laevis oocytes. Oocytes expressing Cx50 exhibited a new population of intramembrane particles (9.0 nm in diameter) in the plasma membrane. The particles represented hemichannels (connexin hexamers) because (a) their cross-sectional area could accommodate 24 +/- 3 helices, (b) when their density reached 300-400/microm2, they formed complete channels (dodecamers) in single oocytes, and assembled into plaques, and (c) their appearance in the plasma membrane was associated with a whole-cell current, which was activated at low external Ca2+ concentration ([Ca2+]o), and was blocked by octanol and by intracellular acidification. The Cx50 hemichannel density was directly proportional to the magnitude of the Cx50 Ca2+-sensitive current. Measurements of hemichannel density and the Ca2+-sensitive current in the same oocytes suggested that at physiological [Ca2+]o (1-2 mM), hemichannels rarely open. In the cytoplasm, hemichannels were present in approximately 0.1-microm diameter "coated" and in larger 0.2-0.5-microm diameter vesicles. The smaller coated vesicles contained endogenous plasma membrane proteins of the oocyte intermingled with 5-40 Cx50 hemichannels, and were observed to fuse with the plasma membrane. The larger vesicles, which contained Cx50 hemichannels, gap junction channels, and endogenous membrane proteins, originated from invaginations of the plasma membrane, as their lumen was labeled with the extracellular marker peroxidase. The insertion rate of hemichannels into the plasma membrane (80, 000/s), suggested that an average of 4,000 small coated vesicles were inserted every second. However, insertion of hemichannels occurred at a constant plasma membrane area, indicating that insertion by vesicle exocytosis (60-500 microm2 membranes/s) was balanced by plasma membrane endocytosis. These exocytotic and endocytotic rates suggest that the entire plasma membrane of the oocyte is replaced in approximately 24 h.

Abstract Saturation mechanisms are the cause considerable strength reduction and swelling enhancement in clayey soils. To overcome this phenomenon, stabilization of the weak layers could be useful when any chemical additives which are calcium based are added. In the presence of considerable amounts of sulfate ions, the conventional additive like lime leads to large heaving due to swelling. This paper presents the changes in the amount of California Bearing Ratio (CBR) of soaked samples and swelling index of Najmabad clayey soil after being stabilized with Colloidal Silica (CS). These tests were performed at different curing times of 3, 7, and 28 days. Moreover, CBR tests were performed after Freeze-Thaw (F-T) conditions under 1, 2, 4, 8, and 12 cycles in 3 selected samples which satisfied the required degree of improvement. In addition, free swelling in all the states was measured and controlled. The results revealed fulfilling CBR criterion of samples subjected to F-T cycles and significant reduction of swelling percent in samples with 1% Nano Silica (NS) particles after 28 days curing time and 2% NS particles after 7 days curing time. Also, Scanning Electron Microscopy (SEM) revealed structural changes and an integrated network for soil improved with CS. [ABSTRACT FROM AUTHOR]

Abstract Porous fibrous materials (PFMs) with three-dimensional networks were prepared by mullite fibers as skeleton and silica sols as binder via pressure and freeze-casting method. Scanning electron microscopy (SEM), X-ray diffraction (XRD) and mercury intrusion porosimetry (MIP) were used to investigate the microstructures, phase and porous characteristics. The density of PFMs can be controlled by adjusting the pressure and sintering during the synthetic process. With the increase of density, the mechanical and thermal conduction properties were increased due to densification of fibers. Based on their lightweight and low thermal conductivity, the PFMs can be used in the field of high temperature thermal insulation. [ABSTRACT FROM AUTHOR]

Detergent-solubilized plasma membrane protein of either adult bovine or calf lens and high-performance liquid chromatography-purified major intrinsic protein (MIP) of the lens were reconstituted into unilamellar vesicles and planar lipid bilayers. Freeze-fracture studies showed that the density of intramembrane particles in the vesicles was proportional to the protein/lipid ratio. At high ratios, these particles crystallized into tetragonal arrays as does MIP in lens fibers. Channels induced by either purified MIP or detergent-solubilized protein had essentially identical properties. The conductance of multichannel membranes was maximal near 0 mV and decreased to 0.49 +/- 0.08 of the maximum value at voltages greater than 80 mV. The dependence of the conductance on voltage was well fit by a two-state Boltzmann distribution. Voltage steps greater than 30 mV elicited an ohmic current step followed by a slow (seconds) biexponential decrease. The amplitudes and time constants depended on the magnitude but not the sign of the voltage. Steps from 100 mV to voltages less than 30 mV caused the channels to open exponentially with a millisecond time constant. Analysis of latency to first closure after a voltage step gave nearly the same time constants as multichannel kinetics. Single-channel conductance is proportional to salt concentration from 0.1 to 1.0 M in KCl. In 0.1M KCl, the channel had two preferred conductance states with amplitudes of 380 and 160 pS, as well as three additional substates. Multi- and single-channel data suggest that the channel has two kinetically important open states. The channel is slightly anion selective. The properties of the channel do not vary appreciably from pH 7.4 to 5.8 or from pCa 7 to 2. We propose that a channel with these properties could contribute to maintenance of lens transparency and fluid balance.

Abstract Biofilms have been intensively investigated over the past decades. Bacillus subtilis is able to form highly structured colony biofilms, and as one of the most studied Gram-positive model organisms, has helped to decipher the complex genetic regulation of biofilms. Several methods have been developed to analyze the architecture of biofilms. In this paper, we describe a method which allows the analysis of the internal structures of biofilms by scanning electron microscopy (SEM). The method uses a modified freeze-fracturing of chemically fixed biofilm to generate defined, well-preserved fractures at millimeter-scale which allows to analyze systematically the internal biofilm structure from macro- to nano-scale. Highlights • Simple and reproducible method for biofilm cross-section analysis • Osmium fixation and dehydration of biofilms allows freeze-fractures. • Clean, non-frayed cross-sections by tearing the sample using forceps [ABSTRACT FROM AUTHOR]

Water containing ultra-fine bubbles (UFB) may promote plant growth. But, as UFBs are too small to distinguish from other impurities in a nutrient solution, it is not known if UFBs survive transport from the water source to the rhizosphere. Here we use the freeze-fracture replica method and a transmission electron microscope (TEM) to observe UFBs in the nutrient solutions used in a crop-growing system known as a plant factory. In this factory, TEM images taken from various points in the supply line indicate that the concentration of UFBs in the nutrient solution is conserved, starting from their addition to the nutrient solution in the buffer tank, through the peat-moss layer, all the way to the rhizosphere. Measurements also show that a thin film formed on the surface of UFBs in the nutrient solution, with greater film thickness at the rhizosphere. This film is considered to be made from the accumulation of impurities coming from solute and the peat-moss layer. [ABSTRACT FROM AUTHOR]

Fracking to approach permeability enhancement is indispensable to enhance coalbed methane recovery, which can significantly reduce greenhouse gas emissions and produce substantial clean energy. To access efficient fracking, samples of Permian low permeable coal reservoir were cracked into different sizes or scales of blocks and particles with liquid nitrogen cryogenic freeze fracturing (FF) by maximizing the underlying heterogeneity of coal. To investigate the crack efficacy, this study systematically examined the permeability evolution and mesoscopic cracking behaviors of coal with different water contents and cleat–fracture systems in the context of cryogenic FF. Results showed that the permeability enhancement and microcracking tended to occur with increasing water content of coal sample; and the efficacy of cryogenic FF on the tighter coal sample was more remarkable. However, the permeability does not strictly increase with the cycles of cryogenic FF and has a close relationship with water content, structural plane direction, efficacy of cryogenic FF, and porosity compaction. The mesoscopic cracking behaviors indicate that numerous smaller pores are iteratively, not strictly sequentially, cracked and become connective in this process. The permeability evolution of coal sample is identified as significantly associated with the mesoscopic cracking behaviors. Notably, the first cycle of cryogenic FF acted on the detected several scales of pores and micro fissures, and partially caused these structures to be opened and interconnected to be permeable. Two main changes were observed in the microcracking of the coal samples in this study: 1) nonuniform shrinkage deformation and micro fissure expansion; and 2) pores opening mostly in the macropore and mesopore scale. It is reasoned that thermal cracking and intermittent opening of seepage pores due to the phase transition of free water in pores or micro fissures ultimately contribute to the permeability enhancement in low permeable and heterogeneous coal. [ABSTRACT FROM AUTHOR]

The ultrastructural analysis of biological membranes by freeze fracture has a 60-year tradition. In this review, we summarize the benefits of the freeze-fracture technique and review special structures analyzed by freeze fracture and by combined freeze-fracture replica immunogold labeling (FRIL) of cell cultures. In principle, every cellular membrane whether of cell suspensions, mono- or bilayers of cell cultures can be analyzed in freeze fracture. The combination of freeze fracture and immunogold labeling of the replica allows the ultrastructural identification of protein assemblies in combination with the molecular identification of their constituent proteins using specific antibodies. The analysis of fractured and labeled intramembrane particles enables determination of the arrangement and organization of proteins within the membrane due to the high resolution of the transmission electron microscope. Because of cell-specific ultrastructural features such as square arrays, identification of cell types can be performed in parallel. This review is aimed at presenting the possibilities of freeze fracture and FRIL in the high-resolution ultrastructural analysis of membrane proteins and their assembly in naïve, transfected or otherwise treated cultured cells. At the interface of molecular approaches and morphology, the application of FRIL in genetically modified cells provides a novel and intriguing aspect for their analysis. [ABSTRACT FROM AUTHOR]

Mitochondria are dynamic intracellular organelles with diverse roles in tissue- and cell type-specific processes, extending beyond bioenergetics. In keeping with this array of functions, mitochondria are described as heterogeneous both between and within tissue types based on multiple parameters, including a broad spectrum of morphological features, and new research points toward a need for the evaluation of mitochondria as isolated organelles. Although transmission electron microscopy (TEM) is commonly used for the evaluation of mitochondria in tissues and renders mitochondrial structures in ultra-thin sections in two-dimensions, additional information regarding complex features within these organelles can be ascertained using scanning electron microscopy (SEM), which allows for analysis of phenotypic differences in three-dimensions. One challenge in producing mitochondrial images for evaluation of ultrastructure using SEM has been the ability to reliably visualize important intramitochondrial features, the inner membrane and cristae structures, on a large-scale ( e.g. multiple mitochondria) within a sample preparation, as mitochondria are enclosed within a double membrane. This can be overcome using a ‘freeze-fracture’ technique in which mitochondrial preparations are snap-frozen followed by application of intense pressure to break open the organelles, revealing the intact components within. Previously published reports using freeze-fracture strategies for mitochondrial SEM have demonstrated feasibility in whole tissue specimens, but a detailed methodology for SEM analysis on isolated mitochondrial fractions has not been reported. By combining previously reported tissue freeze-fracture strategies, along with utilizing the depth of field created by SEM, herein we present a complete method reliant on the freeze-fracture of mitochondrial fractions prepared by differential centrifugation to produce a comprehensive and direct evaluation of three-dimensional mitochondrial ultrastructure by SEM. Image analysis of internal mitochondrial features demonstrates heterogeneity in mitochondrial ultrastructure from a single sample preparation. [ABSTRACT FROM AUTHOR]

Whereas asymmetric transbilayer lipid distribution in the plasma membrane is well recognized, methods to examine the precise localization of lipids are limited. In this review, we critically evaluate the methods that are applied to study transbilayer asymmetry of lipids, summarizing the factors that influence the measurement. Although none of the present methods is perfect, the current application of immunoelectron microscopy-based technique provides a new picture of lipid asymmetry. Next, we summarize the transbilayer distribution of individual lipid in both erythrocytes and nucleated cells. Finally we discuss the concept of the interbilayer communication of lipids. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Sodium dodecyl sulfate-treated freeze-fracture replica labeling (SDS-FRL) is an electron microscopic (EM) method that can define the two-dimensional distribution of membrane proteins and lipids in a quantitative manner. Despite its unsurpassed merit, SDS-FRL has been adopted in a limited number of labs, probably because it requires a laborious labeling process as well as equipment and technique for freeze-fracture. Here, we present a method that reduces the manual labor significantly by mounting freeze-fracture replicas on EM grids prior to labeling. This was made possible by the discovery that freeze-fracture replicas invariably adhere to the carbon-coated formvar membrane with their platinum-carbon side, ensuring that the membrane molecules retained in replicas are accessible to labeling solutions. The replicas mounted on EM grids can be stored dry until labeling, checked by light microscopy before labeling and labeled in the same manner as tissue sections. This on-grid method will make SDS-FRL easier to access for many researchers., (© The Author(s) 2022. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Glycolipids are mainly distributed in the outer leaflet of the plasma membrane and are involved in cellular signaling by modulating the activity of cell surface receptor proteins. Glycolipids themselves also work as cell surface receptors of bacterial toxins. Anti-glycolipid antibodies are associated with various pathological conditions. The cellular distribution of glycolipids has been studied using specific toxins or antibodies. However, these proteins are multivalent and thus potentially induce the artificial aggregation of glycolipids. Since chemical fixative such as paraformaldehyde does not fix glycolipids, an alternative methodology is required to localize glycolipids with multivalent probes. Sodium dodecyl sulfate-digested freeze-fracture replica labeling (SDS-FRL) physically fixes glycolipids on the cast after quick freezing. Thus, SDS-FRL provides the opportunity to observe the natural distribution of glycolipids using multivalent probes. Here, we describe the application of SDS-FRL on the cell surface distribution of phosphatidylglucoside., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

A novel water-based freeze-casting technique was used to prepare porous yttrium silicate (Y 2 SiO 5 ) ceramics. The formation mechanism of lamellar channels of porous ceramics that were arranged centrosymmetrically along the radial axis is discussed in this study. The effect of solid content on the pore structure, porosity, dielectric properties, and mechanical properties of porous Y 2 SiO 5 ceramics were investigated. The increase in solid content reduced the porosity, decreased the pore size, and changed the pore structure. Upon increasing the solid content from 15 vol% to 30 vol%, the compressive strength and dielectric constant of the porous Y 2 SiO 5 ceramics increased from 1.87 to 8.42 MPa and from 1.86 to 2.85, respectively. Porous Y 2 SiO 5 ceramics with these properties can be used in ultra-high temperature broadband radome structures. [ABSTRACT FROM AUTHOR]

The goal of this study was to investigate the influence of an incorporated model drug on the skin permeation of the vehicle itself as it may affect the microstructure and properties of the applied formulation via molecular interactions. For this purpose, we performed skin permeation studies using liposomes prepared with F-DPPC, a monofluorinated analog of dipalmitoylphosphatidylcholine (DPPC), with and without sodium fluorescein (SoFl) serving as model drug. Interestingly, the liposome preparation with F-DPPC yielded semi-solid opalescent systems. Hence, a thorough characterization was accomplished beforehand by electron microscopy imaging, rheological and thermoanalytical experiments. Freeze-fracture electron microscopy images confirmed the existence of globular shaped vesicles in the F-DPPC preparations and oscillatory rheological measurements proved the viscoelastic properties of F-DPPC and F-DPPC+SoFl liposomes in contrast to the viscous characteristics of DPPC liposomes. Thermoanalytical measurements revealed an increased phase transition temperature T m of about 50 °C for F-DPPC and F-DPPC+SoFl liposomes compared to pure DPPC liposomes with a T m of about 43 °C. The similar T m of F-DPPC+SoFl and F-DPPC liposomes as well as the similar skin permeation of the vehicle compound F-DPPC compared to its drug-free counterpart suggest an incorporation of sodium fluorescein into the aqueous core of F-DPPC liposomes. [ABSTRACT FROM AUTHOR]

The nanostructured heliconical twist-bend nematic (Ntb) phase is a new condensed phase of matter with unique properties. Here we present the first example of an achiral hybrid bent-core liquid crystal trimer that at temperatures below the conventional N phase exhibits the Ntb phase from approximately 80 °C down through room temperature. The Ntb phase has a helical structure with a period of ~19 nm. [ABSTRACT FROM AUTHOR]

We present a mathematical model of the fracturing of water-saturated rocks and other porous materials in cold climates. Ice growing inside porous rocks causes large pressures to develop that can significantly damage the rock. We study the growth of ice inside a penny-shaped cavity in a water-saturated porous rock and the consequent fracturing of the medium. Premelting of the ice against the rock, which results in thin films of unfrozen water forming between the ice and the rock, is one of the dominant processes of rock fracturing. We find that the fracture toughness of the rock, the size of pre-existing faults and the undercooling of the environment are the main parameters determining the susceptibility of a medium to fracturing. We also explore the dependence of the growth rates on the permeability and elasticity of the medium. Thin and fast-fracturing cracks are found for many types of rocks. We consider how the growth rate can be limited by the existence of pore ice, which decreases the permeability of a medium, and propose an expression for the effective 'frozen' permeability. [ABSTRACT FROM AUTHOR]

Claudins are one of the major components of tight junctions that play a key role in the formation and maintenance of the epithelial barrier function. Tight junction strands are dynamic and capable of adapting their structure in response to large-scale tissue rearrangement and cellular movement. Here, we present molecular dynamics simulations of claudin-15 strands of up to 225 nm in length in two parallel lipid membranes and characterize their mechanical properties. The persistence length of claudin-15 strands is comparable with those obtained from analyses of freeze-fracture electron microscopy. Our results indicate that lateral flexibility of claudin strands is due to an interplay of three sets of interfacial interaction networks between two antiparallel double rows of claudins in the membranes. In this model, claudins are assembled into interlocking tetrameric ion channels along the strand that slide with respect to each other as the strands curve over submicrometer-length scales. These results suggest a novel molecular mechanism underlying claudin-15 strand flexibility. It also sheds light on intermolecular interactions and their role in maintaining epithelial barrier function., (© 2022 Fuladi et al.)

ABSTRACT The urothelium forms the blood-urine barrier, which depends on the complex organization of transmembrane proteins, uroplakins, in the apical plasma membrane of umbrella cells. Uroplakins compose 16 nm intramembrane particles, which are assembled into urothelial plaques. Here we present an integrated survey on the molecular ultrastructure of urothelial plaques in normal umbrella cells with advanced microscopic techniques. We analyzed the ultrastructure and performed measurements of urothelial plaques in the normal mouse urothelium. We used field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), transmission electron microscopy (TEM) on immunolabeled ultrathin sections (immuno-TEM), and freeze-fracture replicas (FRIL). We performed immunolabeling of uroplakins for scanning electron microscopy (immuno-FESEM). All microscopic techniques revealed a variability of urothelial plaque diameters ranging from 332 to 1179 nm. All immunolabeling techniques confirmed the presence of uroplakins in urothelial plaques. FRIL showed the association of uroplakins with 16 nm intramembrane particles and their organization into plaques. Using different microscopic techniques and applied qualitative and quantitative evaluation, new insights into the urothelial apical surface molecular ultrastructure have emerged and may hopefully provide a timely impulse for many ongoing studies. The combination of various microscopic techniques used in this study shows how these techniques complement one another. The described advantages and disadvantages of each technique should be considered for future studies of molecular and structural membrane specializations in other cells and tissues. Microsc. Res. Tech. 77:896-901, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Weinvestigate the adsorption of submicrometer bulk-synthesizedpolymer dumbbells to oil–water interfaces using freeze-fracture,shadow-casting (FreSCa) cryo-scanning electron microscopy. We findthat the dumbbells are amphiphilic and adsorb to the interface witha preferred orientation. Most particles adsorb in a tilted configuration,with the polar and apolar lobes intersecting the interface and pointingtoward the water and oil, respectively. Some particles adsorb withonly one lobe attached to the interface. Moreover, we find that eachlobe has a preferred angle of contact with the interface, identicalin all observed configurations. A simple geometrical calculation usingthese contact angles accurately predicts the dominant configurationof particles at the interface. This calculation provides insight intohow the shape and composition of dumbbells can be tuned to stand uprightand pack efficiently on curved interfaces. [ABSTRACT FROM AUTHOR]

We have performed freeze-fracture replica immunogold labelling of endotoxin preparations (lipid A and deep rough mutant LPS Re from Salmonella enterica sv. Minnesota), i.e. adding the endotoxins to human monocytes, labelling with monoclonal Abs recognizing either lipid A or LPS Re (A6 and A20 respectively), and fixing with immunogold secondary Ab. We have found that the endotoxins intercalated into the cell membranes with subsequent internalization by the cells. Surprisingly, membrane uptake took place only in the inner, plasmic leaflet of the plasma membrane, but there was no uptake of the outer leaflet for both compounds. Remarkable labelling could be also found for the two membranes of the nuclear envelope—in the case of lipid A only at the plasmic leaflet, but in the case of LPS Re on both leaflets. Isothermal calorimetric titration of the AB A20 with LPS and phospholipids showed that the Ab may bind not only to LPS but also to negatively charged phosphatidylserine. These results are discussed in the frame of the published concepts of cell activation induced by the endotoxins, i.e. how they are able to cause a conformational change of signalling proteins, such as the TLR4/MD2 complex. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are membrane-limited structures released from the cells into the extracellular space and are implicated in intercellular communication. EVs consist of three populations of vesicles, namely microvesicles (MVs), exosomes, and apoptotic bodies. The limiting membrane of EVs is crucially involved in the interactions with the recipient cells, which could lead to the transfer of biologically active molecules to the recipient cells and, consequently, affect their behavior. The freeze-fracture electron microscopy technique is used to study the internal organization of biological membranes. Here, we present a protocol for MV isolation from cultured cancerous urothelial cells and the freeze-fracture of MVs in the steps of rapid freezing, fracturing, making and cleaning the replicas, and analyzing them with transmission electron microscopy. The results show that the protocol for isolation yields a homogenous population of EVs, which correspond to the shape and size of MVs. Intramembrane particles are found mainly in the protoplasmic face of the limiting membrane. Hence, freeze-fracture is the technique of choice to characterize the MVs' diameter, shape, and distribution of membrane proteins. The presented protocol is applicable to other populations of EVs.

Abstract: The mechanical and structural properties of the sublayers of osteonal lamellae were studied. Young’s modulus (E) of adjacent individual lamellae was measured by nanoindentation of parallel slices every 1–3μm, in planes parallel and perpendicular to the osteon axis (OA). In planes parallel to the OA, the modulus of a lamella could vary significantly between sequential slices. Significant modulus variations were also sometimes found on opposing sides of the osteonal canal for the same lamella. These results are rationalized by considerations involving the microstructural organization of the collagen fibrils in the lamellae. Scanning electron microscope imaging of freeze fractured surfaces revealed that the substructure of a single lamella can vary significantly on the opposing sides of the osteonal axis. Using a serial surface view method, parallel planes were exposed every 8–10nm using a dual-beam microscope. Analysis of the orientations of fibrils revealed that the structure is rotated plywood like, consisting of unidirectional sublayers of fibrils of several orientations, with occasional randomly oriented sublayers. The dependence of the measured mechanical properties of the lamellae on the indentation location may be explained by the observed structure, as well as by the curvature of the osteonal lamellae through simple geometrical-structural considerations. Mechanical advantages arising from the curved laminate structure are discussed. [Copyright &y& Elsevier]

Abstract: We examined the structure and biomineralization of prismatic magnetosomes in the magnetotactic marine vibrio Magnetovibrio blakemorei strain MV-1 and a non-magnetotactic mutant derived from it, using a combination of cryo-electron tomography and freeze-fracture. The vesicles enveloping the Magnetovibrio magnetosomes were elongated and detached from the cell membrane. Magnetosome crystal formation appeared to be initiated at a nucleation site on the membrane inner surface. Interestingly, while scattered filaments were observed in the surrounding cytoplasm, their association with the magnetosome chains could not be unequivocally established. Our data suggest fundamental differences between prismatic and octahedral magnetosomes in their mechanisms of nucleation and crystal growth as well as in their structural relationships with the cytoplasm and plasma membrane. [Copyright &y& Elsevier]

Freeze fracturing is applied to make the wetting behaviorof artificiallynanopatterned Si surfaces directly visible. For this purpose, almosthexagonally arranged nanopillars of fixed areal density (127 μm–2) and diameters (35 nm) but varying heights (40–150nm) were fabricated on silicon. Measurement of contact angles (CAs)including hysteresis allowed to distinguish between the Wenzel (W)and the Cassie–Baxter (CB) states with droplets completelywetting the pillars or residing on top of them, respectively. Providingadditional depth contrast by evaporating the ice replica with thincarbon and (typically 3 nm) platinum layers under 45° allowedresolving 3D features of 5 nm within the ice replica. In this way,laterally sharp transitions from CB- to W-states could be revealed,indicating the formation of zero-curvature water surfaces even onthe nanoscale. [ABSTRACT FROM AUTHOR]

Abstract: The evolution of soft templates in the synthesis of ZnSe nanocrystals realized through a surfactant/cosurfactant system was investigated and a micelle formation process model was proposed. Through freeze-fracture electron microscopy, it was proven that template micelles were formed in the zinc precursors. Furthermore, it was found that a long stirring period was essential for achieving the lowest energy state of the soft templates which were used for synthesizing monodisperse ZnSe quantum dots. [Copyright &y& Elsevier]

The structure of a complex between hydrated DNA and a non-cationic lipid is studied, including its phase diagram. The complex is spontaneously formed by adding DNA fragments (ca. 150 base pairs in length) to non-cationic lipids and water. The self-assembly process often leads to highly ordered structures. The structures were studied by combining X-ray scattering, fluorescence and polarized microscopy, as well as freeze-fracture experiments with transmission electron microscopy. We observe a significant increase of the smectic order as DNA is incorporated into the water layers of the lamellar host phase, and stabilization of single phase domains for large amounts of DNA. The effect of confinement on DNA ordering is investigated by varying the water content, following three dilution lines. A rich polymorphism is found, ranging from weakly correlated DNA-DNA in-plane organizations to highly ordered structures, where transmembrane correlations lead to the formation of columnar rectangular and columnar hexagonal superlattices of nucleotides embedded between lipid lamellae. From these observations, we suggest that addition of DNA to the lamellar phase significantly restricts membrane fluctuations above a certain concentration and helps the formation of the lipoplex. The alteration of membrane steric interactions, together with the appearance of interfacial interactions between membranes and DNA molecules may be a relevant mechanism for the emergence of highly ordered structures in the concentrated regime. [ABSTRACT FROM AUTHOR]

Unsaturated fatty acid vesicles (ufasomes) are suspensions of closed lipid bilayers that are composed of fatty acids, and their ionized species (soap) which are restricted to narrow pH range from 7 to 9. In ufasomes, fatty acid molecules are oriented in such a way that their hydrocarbon tails are directed toward the membrane interior and the carboxyl groups are in contact with water. Stable ufasome formulation critically depends on proper selection of fatty acid, amount of cholesterol, buffer, pH range, amount of lipoxygenase, and the presence of divalent cations. Recent innovations can provide opportunity to formulate ufasomes with tailorable features such as extension of pH range, insensitivity toward divalent cations, and enhancement of stability. This article describes method of ufasome preparation, key issues in ufasome manufacturing, recent innovations in ufasomes, dynamicity, stability, and microscopic characterization of ufasomes. Later part of this article deals with comparison of ufasomes with thoroughly studied liposomes. [ABSTRACT FROM AUTHOR]

This critical reviewprovides a critical discussion of the current state of knowledge of the key factors influencing the solubility of gases in ionic liquids (ILs), including sample purity, experimental methodology, “molecular” characteristics of ILs, temperature and pressure. The review starts with a brief introduction to the current developments and the existing problems in the studies of the gas solubility in ILs. Then, the experimental, computational and theoretical developments in conformational equilibria of ions, in nanosegregated polarand nonpolardomains in ILs, and in the mechanisms for dissolution of gases in ILs are discussed and subsequently collaborated together with our freeze-fracture transmission electron microscopic and Raman measurements to propose the new microscopic mechanism for dissolving the gases in ILs. Next, a critical and quantitative analysis of the influences of the sample purity and the experimental methodology on the gas solubility is made so that the “real” relationships between structure and solubility property can be revealed. In addition, a systematic and deeper understanding of how the “molecular” features of the ILs, the temperature, and the pressure influence the gas solubility is provided at the molecular level. In the section of concluding remarks, the comments are made on the molecular criteria for the future design of the ILs to enhance the gas solubility by specifically optimizing the molecular characteristics of the ILs (265 references). [ABSTRACT FROM AUTHOR]

Abstract: The characterization of different bicellar aggregates and the effects of these systems on the stratum corneum (SC) microstructure have been studied. Dynamic light scattering (DLS) and freeze fracture electron microscopy (FFEM) techniques showed that both of the systems studied, dimyristoyl-phosphatidilcholine/dihexanoyl-phosphocholine (DMPC/DHPC) and dipalmitoyl-phosphocholine (DPPC)/DHPC, were formed by small discoidal aggregates at room temperature (20°C). Treating skin with DMPC/DHPC bicelles does not affect the SC lipid microstructure, whereas bicellar systems formed by DPPC and DHPC can promote the formation of new structures in the SC lipid domains. This indicates the passage of lipids from bicelles through the SC layers and also a possible interaction of these lipids with the SC lipids. Given the absence of surfactant in the bicellar composition and the small size of these structures, the use of these smart nano-systems offers great advantages over other lipid systems for dermatological purposes. Bicelles could be promising applications as drug carriers through the skin. This contribution, based on the new biological use of bicelles, may be useful to scientists engaged in colloid science and offers a new tool for different applications in skin and cosmetic research. [Copyright &y& Elsevier]

We report on the structures exhibited by two different diblock poly(styrene)-b-poly(acrylic acid) (PS-b-PAA) copolymers in water, a selective solvent. Using a combination of X-ray scattering and freeze fracture-transmission electron microscopy (FF-TEM), we show that these structures can be widely swollen while retaining their initial morphology and a high degree of long-range order. The analysis of the FF-TEM pictures also evidences the presence of water crystallites of regular size and shape within the confined water domains. We relate the growth of these crystallites to the high local ionic strength of the water swelling the PAA brushes. Moreover, the confinement of the crystallites growth shows that the swollen phases have a very robust structure, potentially useful for confining colloidal particles. [ABSTRACT FROM AUTHOR]

Abstract: The synthesis of high-strength, completely dense nanograined hydroxyapatite (bioceramic) monoliths is a challenge as high temperatures or long sintering times are often required. In this study, nanorods of hydroxyapatite (HA) and calcium-deficient HA (made using a novel continuous hydrothermal flow synthesis method) were consolidated using spark plasma sintering (SPS) up to full theoretical density in ∼5min at temperatures up to 1000°C. After significant optimization of the SPS heating and loading cycles, fully dense HA discs were obtained which were translucent, suggesting very high densities. Significantly high three-point flexural strength values for such materials (up to 158MPa) were measured. Freeze-fracturing of disks followed by scanning electron microscopy investigation revealed selected samples possessed sub-200nm sized grains and no visible pores, suggesting they were fully dense. [Copyright &y& Elsevier]