Direct visualisation of lipid bilayer cubic phases using Atomic Force Microscopy

Inverse bicontinuous cubic (Q(II)) phases are nanostructured materials formed by lipid self-assembly. We have successfully imaged thin films of hydrated Q(II) phases from two different systems using AFM. The images show periodic arrays of water channels with spacing and symmetry consistent with published SAXS data on the bulk materials.

Coupling thin layer electrochemistry with epifluorescence microscopy: an expedient way of investigating electrofluorochromism of organic dyes

The first example of thin layer electrochemistry coupled to epifluorescence microscopy in the total internal reflectance mode is described and applied to the investigation of electrochemically modulated fluorescence of an organic dye (chloromethoxytetrazine) in solution. This technique allows to generate full redox switch of fluorescence when converting reversibly the dye into its anion radical, as well as to record the spectral features of the electrogenerated species. Recording simultaneously fluorescence intensity and lifetime along with coulombic charge as a function of the electrode potential will lead to a deep insight into the redox quenching mechanism.

Fluorescence lifetime imaging microscopy (FLIM) to demonstrate the nuclear binding of flavanols and (--epigallocatechin gallate

The use of light microscopy and DMACA staining strongly suggested that plant and animal cell nuclei act as sinks for flavanols [1, 2]. Detailed uv-vis spectroscopic titration experiments indicated that histone proteins are the likely binding sites in the nucleus [2]. Here we report the development of a multi-photon excitation microscopy technique combined with fluorescent lifetime measurements of flavanols. Using this technique, (+) catechin, (-) epicatechin and (-) epigallocatechin gallate (EGCG) showed strikingly different excited state lifetimes in solution. Interaction of histone proteins with flavanols was indicated by the appearance of a significant τ2-component of 1.7 to 4.0ns. Tryptophan interference could be circumvented in the in vivo fluorescence lifetime imaging microscopy (FLIM) experiments with 2-photon excitation at 630nm. This enabled visualisation and semi-quantitative measurements that demonstrated unequivocally the absorption of (+)catechin, (-)epicatechin and EGCG by nuclei of onion cells. 3D FLIM revealed for the first time that externally added EGCG penetrated the whole nucleus in onion cells. The relative proportions of EGCG in cytoplasm: nucleus: nucleoli were ca. 1:10:100. FLIM experiments may therefore facilitate probing the health effects of EGCG, which is the major constituent of green tea.

A transmission electron microscopy study of polymer-stabilised liquid crystal structure

A method has been established for observing the internal structure of the network component of polymer-stabilised liquid crystals. In situ photopolymerisation of a mesogenic diacrylate monomer using ultraviolet light leads to a sparse network (∼1 wt%) within a nematic host. Following polymerisation, the host was removed through dissolution in heptane, revealing the network. In order to observe a cross-section through the network, it was embedded in a resin and then sectioned using an ultramicrotome. However, imaging of the network was not possible due to poor contrast. To improve this, several reagents were used for network staining, but only one was successful: bromine. The use of a Melinex-resin composite for sectioning was also found to be advantageous. Imaging of the network using transmission electron microscopy revealed solid “droplets” of width 0.07–0.20 μm, possessing an open, yet homogeneous structure, with no evidence for any large-scale internal structures.

Anodic polarization curves revisited

An experiment published in this Journal has been revisited and it is found that the curve pattern of the anodic polarization curve for iron repeats itself successively when the potential scan is repeated. It is surprising that this observation has not been reported previously in the literature because it immediately brings into question the long accepted and well-known explanations involving a passive film. A qualitative and plausible explanation is provided from surprisingly simple principles for this new finding. Some important pedagogic conclusions have been derived from this work. It is noteworthy that the somewhat complicated phenomenon can be simply explained, thus providing two important lessons to students. First, even well-accepted scientific work studying simple processes may be incomplete and worthy of further study, and second, such processes may be explained simply at the undergraduate level. The contents of the paper also confirm that presenting curricular contents in a new and more correct manner is beneficial, interesting, and that research in curricular contents represents one of the important forms of educational research.

Fusion of infrared polarization and intensity images using support value transform and fuzzy combination rules

Infrared polarization and intensity imagery provide complementary and discriminative information in image understanding and interpretation. In this paper, a novel fusion method is proposed by effectively merging the information with various combination rules. It makes use of both low-frequency and highfrequency images components from support value transform (SVT), and applies fuzzy logic in the combination process. Images (both infrared polarization and intensity images) to be fused are firstly decomposed into low-frequency component images and support value image sequences by the SVT. Then the low-frequency component images are combined using a fuzzy combination rule blending three sub-combination methods of (1) region feature maximum, (2) region feature weighting average, and (3) pixel value maximum; and the support value image sequences are merged using a fuzzy combination rule fusing two sub-combination methods of (1) pixel energy maximum and (2) region feature weighting. With the variables of two newly defined features, i.e. the low-frequency difference feature for low-frequency component images and the support-value difference feature for support value image sequences, trapezoidal membership functions are proposed and developed in tuning the fuzzy fusion process. Finally the fused image is obtained by inverse SVT operations. Experimental results of visual inspection and quantitative evaluation both indicate the superiority of the proposed method to its counterparts in image fusion of infrared polarization and intensity images.

Scanning tunneling microscopy and molecular dynamics study of the Li2TiO3(001) surface

We have investigated the (001) surface structure of lithium titanate (Li2TiO3) using auger electron spectroscopy (AES), low-energy electron diffraction (LEED), and scanning tunneling microscopy (STM). Li2TiO3 is a potential fusion reactor blanket material. After annealing at 1200 K, LEED demonstrated that the Li2TiO3(001) surface was well ordered and not reconstructed. STM imaging showed that terraces are separated in height by about 0.3 nm suggesting a single termination layer. Moreover, hexagonal patterns with a periodicity of ∼0.4 nm are observed. On the basis of molecular dynamics (MD) simulations, these are interpreted as a dynamic arrangement of Li atoms.

On the barrier properties of the cornea: a microscopy study of the penetration of fluorescently labeled nanoparticles, polymers, and sodium fluorescein

Overcoming the natural defensive barrier functions of the eye remains one of the greatest challenges of ocular drug delivery. Cornea is a chemical and mechanical barrier preventing the passage of any foreign bodies including drugs into the eye, but the factors limiting penetration of permeants and nanoparticulate drug delivery systems through the cornea are still not fully understood. In this study, we investigate these barrier properties of the cornea using thiolated and PEGylated (750 and 5000 Da) nanoparticles, sodium fluorescein, and two linear polymers (dextran and polyethylene glycol). Experiments used intact bovine cornea in addition to bovine cornea de-epithelialized or tissues pretreated with cyclodextrin. It was shown that corneal epithelium is the major barrier for permeation; pretreatment of the cornea with β-cyclodextrin provides higher permeation of low molecular weight compounds, such as sodium fluorescein, but does not enhance penetration of nanoparticles and larger molecules. Studying penetration of thiolated and PEGylated (750 and 5000 Da) nanoparticles into the de-epithelialized ocular tissue revealed that interactions between corneal surface and thiol groups of nanoparticles were more significant determinants of penetration than particle size (for the sizes used here). PEGylation with polyethylene glycol of a higher molecular weight (5000 Da) allows penetration of nanoparticles into the stroma, which proceeds gradually, after an initial 1 h lag phase.

Spin polarization, orbital occupation and band gap opening in vanadium dioxide: the effect of screened Hartree–Fock exchange

The metal–insulator transition of VO2 so far has evaded an accurate description by density functional theory. The screened hybrid functional of Heyd, Scuseria and Ernzerhof leads to reasonable solutions for both the low-temperature monoclinic and high-temperature rutile phases only if spin polarization is excluded from the calculations. We explore whether a satisfactory agreement with experiment can be achieved by tuning the fraction of Hartree Fock exchange (a) in the density functional. It is found that two branches of locally stable solutions exist for the rutile phase for 12:5% 6 a 6 20%. One is metallic and has the correct stability as compared to the monoclinic phase, the other is insulating with lower energy than the metallic branch. We discuss these observations based on the V 3d orbital occupations and conclude that a ¼ 10% is the best possible choice for spin-polarized VO2 calculations.

Quantitative single-molecule localization microscopy combined with rule-based modeling reveals ligand-induced TNF-R1 reorganization toward higher-order oligomers

We report on the assembly of tumor necrosis factor receptor 1 (TNF-R1) prior to ligand activation and its ligand-induced reorganization at the cell membrane. We apply single-molecule localization microscopy to obtain quantitative information on receptor cluster sizes and copy numbers. Our data suggest a dimeric pre-assembly of TNF-R1, as well as receptor reorganization toward higher oligomeric states with stable populations comprising three to six TNF-R1. Our experimental results directly serve as input parameters for computational modeling of the ligand-receptor interaction. Simulations corroborate the experimental finding of higher-order oligomeric states. This work is a first demonstration how quantitative, super-resolution and advanced microscopy can be used for systems biology approaches at the single-molecule and single-cell level.

Helium ion microscopy visualizes lipid nanodomains in mammalian cells

Cell membranes are composed of two-dimensional bilayers of amphipathic lipids, which allow a lateral movement of the respective membrane components. These components are arranged in an inhomogeneous manner as transient micro- and nanodomains, which are believed to be crucially involved in the regulation of signal transduction pathways in mammalian cells. Because of their small size (diameter 10-200 nm), membrane nanodomains cannot be directly imaged using conventional light microscopy. Here, we present direct visualization of cell membrane nanodomains by helium ion microscopy (HIM). We show that HIM is capable to image biological specimens without any conductive coating, and that HIM images clearly allow the identification of nanodomains in the ultrastructure of membranes with 1.5 nm resolution. The shape of these nanodomains is preserved by fixation of the surrounding unsaturated fatty acids while saturated fatty acids inside the nanodomains are selectively removed. Atomic force microscopy, fluorescence microscopy, 3D structured illumination microscopy and direct stochastic optical reconstruction microscopy provide additional evidence that the structures in the HIM images of cell membranes originate from membrane nanodomains. The nanodomains observed by HIM have an average diameter of 20 nm and are densely arranged with a minimal nearest neighbor distance of ~15 nm.