Cryo-field Emission Scanning Electron Research Articles

Monitoring Freeze-Damage in Grapefruit by Electric Bioimpedance Spectroscopy and Electric Equivalent Models

Grapefruit is a cold-sensitive citrus fruit, and freezing can spoil the harvest when the fruit is still on the tree and even later during manufacturing and transport due to inappropriate postharvest management. This study performed a specific Electric Impedance Spectroscopy (EIS) analysis and statistical data treatment to obtain an EIS and Artificial Neural Networks (ANN)-based model for early freeze-damage detection in grapefruit showing a Correct Correlation Rate of 100%. Additionally, Cryo-Field Emission Scanning Electron Microscopy observations were conducted on both fresh and frozen/thawed samples, analyzing the different impedance responses in order to understand the biological changes in the tissue. Finally, a modified Hayden electric equivalent model was parameterized to simulate the impedance response electrically and link the electric behavior of biological tissue to the change in its properties due to freezing. The developed technique is introduced as an alternative to the traditional ones, as it is fast, economic, and easy to carry out.

Providing Stability to High Internal Phase Emulsion Gels Using Brewery Industry By-Products as Stabilizers.

The modern brewing industry generates high amounts of solid wastes containing biopolymers—proteins and polysaccharides—with interesting technological and functional properties. The novelty of this study was to use raw by-product from the brewing industry in the development of high internal phase emulsion (HIPE) gels. Thus, the influence of the emulsion’s aqueous phase pH and the by-product’s concentration on structural and physical stability of the emulsions was studied. The microstructure was analyzed using cryo-field emission scanning electron microscopy. To evaluate the rheological behavior, oscillatory tests (amplitude and frequency) and flow curves were conducted. Moreover, the physical stability of the emulsions and the color were also studied. The increase in by-product concentration and the pH of the aqueous phase allowed development of HIPE gels with homogeneously distributed oil droplets of regular size and polyhedral structure. The data from the rheology tests showed a more stable structure at higher pH and higher by-product concentration. This study widens the possibilities of valorizing the brewing industry’s by-products as stabilizers when designing emulsions.

Nuclear envelope impairment is facilitated by the herpes simplex virus 1 Us3 kinase

Background: Capsids of herpes simplex virus 1 (HSV-1) are assembled in the nucleus, translocated either to the perinuclear space by budding at the inner nuclear membrane acquiring tegument and envelope, or released to the cytosol in a “naked” state via impaired nuclear pores that finally results in impairment of the nuclear envelope. The Us3 gene encodes a protein acting as a kinase, which is responsible for phosphorylation of numerous viral and cellular substrates. The Us3 kinase plays a crucial role in nucleus to cytoplasm capsid translocation. We thus investigate the nuclear surface in order to evaluate the significance of Us3 in maintenance of the nuclear envelope during HSV-1 infection. Methods: To address alterations of the nuclear envelope and capsid nucleus to cytoplasm translocation related to the function of the Us3 kinase we investigated cells infected with wild type HSV-1 or the Us3 deletion mutant R7041(∆Us3) by transmission electron microscopy, focused ion-beam electron scanning microscopy, cryo-field emission scanning electron microscopy, confocal super resolution light microscopy, and polyacrylamide gel electrophoresis. Results: Confocal super resolution microscopy and cryo-field emission scanning electron microscopy revealed decrement in pore numbers in infected cells. Number and degree of pore impairment was significantly reduced after infection with R7041(∆Us3) compared to infection with wild type HSV-1. The nuclear surface was significantly enlarged in cells infected with any of the viruses. Morphometric analysis revealed that additional nuclear membranes were produced forming multiple folds and caveolae, in which virions accumulated as documented by three-dimensional reconstruction after ion-beam scanning electron microscopy. Finally, significantly more R7041(∆Us3) capsids were retained in the nucleus than wild-type capsids whereas the number of R7041(∆Us3) capsids in the cytosol was significantly lower. Conclusions: The data indicate that Us3 kinase is involved in facilitation of nuclear pore impairment and, concomitantly, in capsid release through impaired nuclear envelope.

Nuclear envelope impairment is facilitated by the herpes simplex virus 1 Us3 kinase.

Background: Capsids of herpes simplex virus 1 (HSV-1) are assembled in the nucleus, translocated either to the perinuclear space by budding at the inner nuclear membrane acquiring tegument and envelope, or released to the cytosol in a "naked" state via impaired nuclear pores that finally results in impairment of the nuclear envelope. The Us3 gene encodes a protein acting as a kinase, which is responsible for phosphorylation of numerous viral and cellular substrates. The Us3 kinase plays a crucial role in nucleus to cytoplasm capsid translocation. We thus investigate the nuclear surface in order to evaluate the significance of Us3 in maintenance of the nuclear envelope during HSV-1 infection. Methods: To address alterations of the nuclear envelope and capsid nucleus to cytoplasm translocation related to the function of the Us3 kinase we investigated cells infected with wild type HSV-1 or the Us3 deletion mutant R7041(∆Us3) by transmission electron microscopy, focused ion-beam electron scanning microscopy, cryo-field emission scanning electron microscopy, confocal super resolution light microscopy, and polyacrylamide gel electrophoresis. Results: Confocal super resolution microscopy and cryo-field emission scanning electron microscopy revealed decrement in pore numbers in infected cells. Number and degree of pore impairment was significantly reduced after infection with R7041(∆Us3) compared to infection with wild type HSV-1. The nuclear surface was significantly enlarged in cells infected with any of the viruses. Morphometric analysis revealed that additional nuclear membranes were produced forming multiple folds and caveolae, in which virions accumulated as documented by three-dimensional reconstruction after ion-beam scanning electron microscopy. Finally, significantly more R7041(∆Us3) capsids were retained in the nucleus than wild-type capsids whereas the number of R7041(∆Us3) capsids in the cytosol was significantly lower. Conclusions: The data indicate that Us3 kinase is involved in facilitation of nuclear pore impairment and, concomitantly, in capsid release through impaired nuclear envelope.

Anisamide-Anchored Lyotropic Nano-Liquid Crystalline Particles with AIE Effect: A Smart Optical Beacon for Tumor Imaging and Therapy.

The prospective design of nanocarriers for personalized oncotherapy should be an ensemble of targeting, imaging, and noninvasive therapeutic capabilities. Herein, we report the development of the inverse hexagonal nano-liquid crystalline (NLC) particles that are able to host formononetin (FMN), a phytoestrogen with known anticancer activity, and tetraphenylethene (TPE), an iconic optical beacon with aggregation-induced emission (AIE) signature, simultaneously. Ordered three-dimensional mesoporous internal structure and high-lipid-volume fraction of NLC nanoparticles (NLC NPs) frame the outer compartment for the better settlement of payloads. Embellishment of these nanoparticles by anisamide (AA), a novel sigma receptor targeting ligand using carbodiimide coupling chemistry ensured NLC's as an outstanding vehicle for possible utility in surveillance of tumor location as well as the FMN delivery through active AIE imaging. The size and structural integrity of nanoparticles were evaluated by quasi-elastic light scattering, cryo field emission scanning electron microscopy small-angle X-ray scattering. The existence of AIE effect in the nanoparticles was evidenced through the photophysical studies that advocate the application of NLC NPs in fluorescence-based bioimaging. Moreover, confocal microscopy illustrated the single living cell imaging ability endowed by the NLC NPs. In vitro and in vivo studies supported the enhanced efficacy of targeted nanoparticles (AA-NLC-TF) in comparison to nontargeted nanoparticles (NLC-TF) and free drug. Apparently, this critically designed multimodal NLC NPs may establish a promising platform for targeted and image-guided chemotherapy for breast cancer.

Rheology of novel self-thickening cationic gemini surfactant solutions

ABSTRACTA new cationic Gemini surfactant (25-HP-25) was synthesized with eruamidopropyl dimethylamine and epichlorohydrin, and its solution is self-thickening. The rheological properties of 25-HP-25 and 3-chloro-2-hydroxypropyl eruamidopropyl dimethyl ammonium acetate (EDAA) solutions were compared. The solution of EDAA behaves as Newtonian fluid (1 s−1 < γ < 1000s−1). Whereas the 25-HP-25 micelle solution (2 wt%) exhibits higher viscosity, which is about three hundred times than that of EDAA solution at low shear rate (γ < 1 s−1). Especially, the 25-HP-25 solution shows obvious thixotropy, and with the increase of concentration, the viscoelasticity becomes better. Furthermore, the compaction of network structures has been confirmed by cryo-field emission scanning electron microscopy (cryo-FESEM) micrographs. This work is expected to enrich the research field of self-thickening surfactant.

Solid state characterization of azelnidipine–oxalic acid co-crystal and co-amorphous complexes: The effect of different azelnidipine polymorphs

In present study, based on the two polymorphs (α and β form) of azelnidipine (AZE), 12 complexes of AZE and oxalic acid (OXA) were prepared by solvent-assisted grinding (SG) and neat powder grinding (NG) methods at the AZE/OXA molar ratios of 2:1, 1:1, and 1:2. The effect of the different polymorphs of AZE on the micro-structure of the complexes were investigated by powder X-ray diffraction (PXRD), tempreture modulated differential scanning calorimetry and thermogravimetric analysis, cryo-field emission scanning electron microscope system, fourier transform infrared (FTIR), and solid-state nuclear magnetic resonance spectroscopy. β-AZE-OXA co-crystal was produced at β-AZE/OXA molar ratio of 2:1 when SG method was used; while α-AZE was used to produce α-AZE-OXA co-crystal at same condition. However, the other 10 combinations were in co-amorphous forms, including the NG samples with α (or β)-AZE/OXA molar ratios of 2:1, 1:1 (SG and NG), and 1:2 (SG and NG). Although the XRD pattern and IR spectra of the two co-crystals showed no difference, the melting enthalpy and specific heat cp of the β-AZE-OXA co-crystal was higher than that of the α-AZE-OXA co-crystal, indicating that the numbers of solvent molecules which entered the two co-crystal lattices were different. Interestingly, obvious difference occurred in the IR spectra between the α-AZE-OXA and β-AZE-OXA co-amorphous systems. 1745cm−1 wave-numbers, which were assigned to the free CO groups, appeared in the α-AZE-OXA co-amorphous systems even when just a small amount of OXA was introduced, thereby indicating the presence of different intermolecular forces in the two series of co-amorphous forms. The solubility in different media and the dissolution rate in 0.1molL−1 HCl of the 12 complexes were determined. The dramatically improved dissolution rates of the α- and β-AZE-OXA 1:2 (NG) combinations in vitro showed potential in improving the physicochemical properties of AZE by co-amorphous complex formation.

Cubosome nanoparticles potentiate immune properties of immunostimulants.

Cubosomes have been explored as drug and antigen carriers in the past few years. A few reports have described that cubosomes can enhance the ability of immunostimulants to generate strong immune responses. Polysaccharide (PS), an immunostimulant, has been reported to be a promising adjuvant for vaccines. Herein, we incorporated PS into cubosomes to generate PS–cubosome (Cub-PS) nanoparticles, and Cub-PS was characterized by small-angle X-ray scattering scattering and cryo-field emission scanning electron microscopy. The immunological activity of Cub-PS was compared with that of Cub and PS. The results demonstrated that Cub-PS elicited more potent immune responses than Cub or PS alone. The enhanced immune responses might be attributed to the promotion of antigen transport into draining lymph nodes and efficient dendritic cell activation and memory T-helper cell differentiation in draining lymph nodes. Overall, these findings indicate that cubosomes have the potential to enhance the ability of immunostimulants to generate an immune response.

Time-resolved GISAXS and cryo-microscopy characterization of block copolymer membrane formation

Time-resolved grazing-incidence small-angle X-ray scattering (GISAXS) and cryo-microscopy were used for the first time to understand the pore evolution by copolymer assembly, leading to the formation of isoporous membranes with exceptional porosity and regularity. The formation of copolymer micelle strings in solution (in DMF/DOX/THF and DMF/DOX) was confirmed by cryo field emission scanning electron microscopy (cryo-FESEM) with a distance of 72 nm between centers of micelles placed in different strings. SAXS measurement of block copolymer solutions in DMF/DOX indicated hexagonal assembly with micelle-to-micelle distance of 84–87 nm for 14–20 wt% copolymer solutions. GISAXS in-plane peaks were detected, revealing order close to hexagonal. The d-spacing corresponding to the first peak in this case was 100–130 nm (lattice constant 115–150 nm) for 17 wt% copolymer solutions evaporating up to 100 s. Time-resolved cryo-FESEM showed the formation of incipient pores on the film surface after 4 s copolymer solution casting with distances between void centers of 125 nm.

Preparation and characterization of paclitaxel-loaded DSPE-PEG-liquid crystalline nanoparticles (LCNPs) for improved bioavailability

Lipid-based liquid crystalline nanoparticles (LCNPs) have attracted growing interest as a new drug nanocarrier system for improving bioavailability for both hydrophilic and hydrophobic drugs. In this study, self-assembled LCNPs based on soy phosphatidyl choline and glycerol dioleate, which was known possessing low toxicity and negligible hemolysis, were prepared using poly(ethylene glycol)-grafted 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE-PEG) as the dispersing agent. Paclitaxel (PTX) was used as a model hydrophobic drug. The particle size of the optimized DSPE-PEG-LCNPs and PTX-loaded DSPE-PEG-LCNPs were around 70nm. Crossed polarized light microscopy was used to characterize the phase behavior of liquid crystalline (LC) matrices, which showed a fan-like birefringent texture in dark background indicating the coexistence of reversed cubic and hexagonal phase in the optimized LC matrix. Transmission electron microscopy and cryo-field emission scanning electron microscopy revealed its internal water channel and “twig-like” surface morphology. PTX-loaded DSPE-PEG-LCNPs exhibited a biphasic drug sustained release pattern with a relatively fast release at the initial stage and a sustained release afterwards. PTX-loaded DSPE-PEG-LCNPs presented higher AUC (410.942±72.522μg/Lh) when compared with commercial product Taxol (212.670±41.396μg/Lh). These results indicated that DSPE-PEG-LCNPs might serve as a potential sustained release system for poorly water-soluble agents.

Lipid-based liquid crystalline nanoparticles as oral drug delivery vehicles for poorly water-soluble drugs: cellular interaction and in vivo absorption

BackgroundLipid-based liquid crystalline nanoparticles (LCNPs) have attracted growing interest as novel drug-delivery systems for improving the bioavailability of both hydrophilic and hydrophobic drugs. However, their cellular interaction and in vivo behavior have not been fully developed and characterized.MethodsIn this study, self-assembled LCNPs prepared from soy phosphatidylcholine and glycerol dioleate were developed as a platform for oral delivery of paclitaxel. The particle size of empty LCNPs and paclitaxel-loaded LCNPs was around 80 nm. The phase behavior of the liquid crystalline matrix was characterized using crossed polarized light microscopy and small-angle X-ray scattering, and showed both reversed cubic and hexagonal phase in the liquid crystalline matrix. Transmission electron microscopy and cryofield emission scanning electron microscopy analysis revealed an inner winding water channel in LCNPs and a “ ball-like”/“hexagonal” morphology.ResultsCellular uptake of LCNPs in Caco-2 cells was found to be concentration-dependent and time-dependent, with involvement of both clathrin and caveolae/lipid raft-mediated endocytosis. Under confocal laser scanning microscopy, soy phosphatidylcholine was observed to segregate from the internalized LCNPs and to fuse with the cell membrane. An in vivo pharmacokinetic study showed that the oral bioavailability of paclitaxel-loaded LCNPs (13.16%) was 2.1 times that of Taxol® (the commercial formulation of paclitaxel, 6.39%).ConclusionThe findings of this study suggest that this LCNP delivery system may be a promising candidate for improving the oral bioavailability of poorly water-soluble agents.

Supramolecular assembled nanogel made of mannan

The supramolecular assembly of amphiphilic mannan, synthesized by the Michael addition of hydrophobic 1-hexadecanethiol to vinyl methacrylated mannan, originates in aqueous medium the formation of a nanogel, stabilized by hydrophobic interactions among alkyl chains. The critical aggregation concentration, calculated by fluorescence spectroscopy ranged between 0.002 and 0.01mg/mL, depending on the polymer degree of substitution. The cryo-field emission scanning electron microscopy showed spherical macromolecular micelles with diameters between 100 and 500nm. The dynamic light scattering analysis revealed a polydisperse colloidal system, with mean hydrodynamic diameter between 50 and 140nm, depending on the polymer degree of substitution. The nanogel is negatively charged, stable over a 6months storage period, and stable at pH 3–8, salt or urea solutions. Bovine serum albumin and curcumin were spontaneously incorporated in the nanogel, being stabilized by the hydrophobic domains, opening the possibility for future applications as potential delivery systems for therapeutic molecules. In vitro assays were carried out to characterize the biocompatibility of the nanogel. A toxic effect of mannan-C16 was observed, specific to mouse macrophage-like cell line J774, not affecting mouse embryo fibroblast cell line 3T3 viability.

Switchable pH-Responsive Polymeric Membranes Prepared via Block Copolymer Micelle Assembly

A process is described to manufacture monodisperse asymmetric pH-responsive nanochannels with very high densities (pore density >2 × 10(14) pores per m(2)), reproducible in m(2) scale. Cylindric pores with diameters in the sub-10 nm range and lengths in the 400 nm range were formed by self-assembly of metal-block copolymer complexes and nonsolvent-induced phase separation. The film morphology was tailored by taking into account the stability constants for a series of metal-polymer complexes and confirmed by AFM. The distribution of metal-copolymer micelles was imaged by transmission electron microscopy tomography. The pH response of the polymer nanochannels is the strongest reported with synthetic pores in the nm range (reversible flux increase of more than 2 orders of magnitude when switching the pH from 2 to 8) and could be demonstrated by cryo-field emission scanning electron microscopy, SAXS, and ultra/nanofiltration experiments.

Synthesis and Characterization of Self-Assembled Nanogels Made of Pullulan.

Self-assembled nanogels made of hydrophobized pullulan were obtained using a versatile, simple, reproducible and low-cost method. In a first reaction pullulan was modified with hydroxyethyl methacrylate or vinyl methacrylate, further modified in the second step with hydrophobic 1-hexadecanethiol, resulting as an amphiphilic material, which self-assembles in water via the hydrophobic interaction among alkyl chains. Structural features, size, shape, surface charge and stability of the nanogels were studied using hydrogen nuclear magnetic resonance, fluorescence spectroscopy, cryo-field emission scanning electron microscopy and dynamic light scattering. Above the critical aggregation concentration spherical polydisperse macromolecular micelles revealed long-term colloidal stability in aqueous medium, with a nearly neutral negative surface charge and mean hydrodynamic diameter in the range 100–400 nm, depending on the polymer degree of substitution. Good size stability was observed when nanogels were exposed to potential destabilizing pH conditions. While the size stability of the nanogel made of pullulan with vinyl methacrylate and more hydrophobic chains grafted was affected by the ionic strength and urea, nanogel made of pullulan with hydroxyethyl methacrylate and fewer hydrophobic chains grafted remained stable.

Synthesis and Self‐Assembly of New Double‐Crystalline Amphiphilic Polyethylene‐block‐Poly[oligo(ethylene glycol) Methyl Ether Methacrylate] Coil‐Brush Diblock Copolymer

AbstractWe report on the synthesis and self‐assembly of a novel well‐defined coil‐brush diblock copolymer with linear crystalline polyethylene (PE) as the coil block and hydrophilic poly [oligo(ethylene glycol) methyl ether methacrylate] (POEGMA) as rod brush via a combination of chain shuttling ethylene polymerization (CSEP) and atom transfer radical polymerization (ATRP). Initially, bromine end‐terminated PE macroinitiator (PE–Br) was synthesized through the esterification of 2‐bromo‐2‐methylpropionyl bromide with monohydroxyl‐terminated PE (PE–OH) which was prepared by means of CSEP with 2,6‐bis[1‐(2,6‐dimethylphenyl)imino ethyl] pyridine iron (II) dichloride/methylaluminoxane (MAO)/ZnEt2 and subsequent in situ oxidation with oxygen. The resultant PE‐b‐POEGMA coil‐brush diblock copolymer was synthesized by ATRP of monomethoxy‐capped oligo(ethylene glycol)methacrylate (OEGMA) using PE–Br as macroinitiator. The self‐assembly of the double‐crystalline PE‐b‐POEGMA in aqueous solution was investigated by dynamic light scattering, transmission electron microscopy and cryofield emission scanning electron microscopy. It was found that, in water, a solvent selectively good for the POEGMA brush, PE‐b‐POEGMA chains could self‐assemble to form sandwich‐like micelles with the insoluble and crystallized PE blocks as the interlayer cores and the soluble and swollen POEGMA brush as the outer‐layer shell. The crystallization of both PE and POEGMA blocks in self‐assembled structure formed from aqueous solution was investigated by differential scanning calorimetry.magnified image

Self-Assembled Nanogel Made of Mannan: Synthesis and Characterization

Amphiphilic mannan (mannan-C(16)) was synthesized by the Michael addition of hydrophobic 1-hexadecanethiol (C(16)) to hydroxyethyl methacrylated mannan (mannan-HEMA). Mannan-C(16) formed nanosized aggregates in water by self-assembly via the hydrophobic interaction among C(16) molecules as confirmed by hydrogen nuclear magnetic resonance ((1)H NMR), fluorescence spectroscopy, cryo-field emission scanning electron microscopy (cryo-FESEM), and dynamic light scattering (DLS). The mannan-C(16) critical aggregation concentration (cac), calculated by fluorescence spectroscopy with Nile red and pyrene, ranged between 0.04 and 0.02 mg/mL depending on the polymer degree of substitution of C(16) relative to methacrylated groups. Cryo-FESEM micrographs revealed that mannan-C(16) formed irregular spherical macromolecular micelles, in this work designated as nanogels, with diameters ranging between 100 and 500 nm. The influence of the polymer degree of substitution, DS(HEMA) and DS(C(16)), on the nanogel size and zeta potential was studied by DLS at different pH values and ionic strength and as a function of mannan-C(16) and urea concentrations. Under all tested conditions, the nanogel was negatively charged with a zeta potential close to zero. Mannan-C(16) with higher DS(HEMA) and DS(C(16)) values formed larger nanogels and were also less stable over a 6 month storage period and at concentrations close to the cac. When exposed to solutions of different pH and aggressive conditions of ionic strength and urea concentration, the size of mannan-C(16) varied to some extent but was always in the nanoscale range.

Biocompatible and degradable nanogels via oxidation reactions of synthetic thiomers in inverse miniemulsion

We present the preparation of degradable and biocompatible nanohydrogels from thiol-functional macromers. Linear poly(glycidol) and star-shaped poly(ethylene oxide-stat-propylene oxide) with molecular weights below 15 kDa were functionalized with thiol groups by polymer-analogous reaction and crosslinked in inverse miniemulsion. Particle size was determined by dynamic light scattering, cryo-field emission scanning electron microscopy and scanning force microscopy. The disulfide crosslinked particles readily degrade on addition of 10 mM aqueous glutathione solution what resembles cytosolic conditions. Biocompatibility has been proven by incubation of L929 fibroblasts with nanogels for 24 and 72 h followed by life-dead staining. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Three‐dimensional reconstruction of biological macromolecular complexes from in‐lens scanning electron micrographs

Two helical samples: F-actin and the bacteriophage T4 tail sheath were reconstructed in three dimensions from contrast enhanced (rotational shadowing and negatively stained) in-lens cryo-field emission scanning electron micrographs, using the iterative real-space helical reconstruction method. The F-actin--and bacteriophage T4 reconstructions compare favourably to an atomic model refined against fibre diffraction data and a cryo-electron microscopy reconstruction, respectively. These results show that single-particle methods, developed for macromolecules imaged in the transmission electron microscope can be applied to cryo-field emission scanning electron micrographs data with appropriate symmetry.

Exploring the nuclear envelope of herpes simplex virus 1-infected cells by high-resolution microscopy.

Herpesviruses are composed of capsid, tegument, and envelope. Capsids assemble in the nucleus and exit the nucleus by budding at the inner nuclear membrane, acquiring tegument and the envelope. This study focuses on the changes of the nuclear envelope during herpes simplex virus 1 (HSV-1) infection in HeLa and Vero cells by employing preparation techniques at ambient and low temperatures for high-resolution scanning and transmission electron microscopy and confocal laser scanning microscopy. Cryo-field emission scanning electron microscopy of freeze-fractured cells showed for the first time budding of capsids at the nuclear envelope at the third dimension with high activity at 10 h and low activity at 15 h of incubation. The mean number of pores was significantly lower, and the mean interpore distance and the mean interpore area were significantly larger than those for mock-infected cells 15 h after inoculation. Forty-five percent of nuclear pores in HSV-1-infected cells were dilated to more than 140 nm. Nuclear material containing capsids protrude through them into the cytoplasm. Examination of in situ preparations after dry fracturing revealed significant enlargements of the nuclear pore diameter and of the nuclear pore central channel in HSV-1-infected cells compared to mock-infected cells. The demonstration of nucleoporins by confocal microscopy also revealed fewer pores but focal enhancement of fluorescence signals in HSV-1-infected cells, whereas Western blots showed no loss of nucleoporins from cells. The data suggest that infection with HSV-1 alters the number, size, and architecture of nuclear pores without a loss of nucleoporins from altered nuclear pore complexes.

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.