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.

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.

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.

A scanning electron microscopy study of diseased root surfaces conditioned with EDTA gel plus Cetavlon after scaling and root planing

In the present investigation, a scanning electron microscopy analysis was performed to evaluate the effects of the topical application of ethylenediaminetetraacetic acid (EDTA) gel associated with Cetavlon (EDTAC) in removing the smear layer and exposing collagen fibers following root surface instrumentation. Twenty-eight teeth from adult humans, single rooted and scheduled for extraction due to periodontal reasons, were selected. Each tooth was submitted to manual (scaling and root planing) instrumentation alone or combined with ultrasonic instruments, with or without etching using a 24% EDTAC gel. Following extraction, specimens were processed and examined under a scanning electron microscope. A comparative morphological semi-quantitative analysis was performed; the intensity of the smear layer and the decalcification of cementum and dentinal surfaces were graded in 12 sets using an arbitrary scale ranging from 1 (area covered by a smear layer) to 4 (no smear layer). Root debridement with hand instruments alone or combined with ultrasonic instruments resulted in a similar smear layer covering the root surfaces. The smear layer was successfully removed from the surfaces treated with EDTAC, which exhibited numerous exposed dentinal tubules and collagen fibers. This study supports the hypothesis that manual instrumentation alone or instrumentation combined with ultrasonic instrumentation is unable to remove the smear layer, whereas the subsequent topical application of EDTAC gel effectively removes the smear layer, uncovers dentinal openings and exposes collagen fibers.

Light, electron microscopy and histopathology of Myxobolus salminus n. sp., a parasite of Salminus brasiliensis from the Brazilian Pantanal

In this report, we describe the morphology and histopathology of Myxobolus salminus n. sp., a parasite of the gill filaments of wild Salminus brasiliensis (dourado) from the Brazilian Pantanal. The small polysporic plasmodia were similar to 100 mu m in diameter and the development was asynchronous. The mature spores were oval to pear shaped and had a smooth wall. The spore measurements were (mean +/- S.D., with range in parentheses): length 10.1 +/- 0.4 mu m (9.6-10.5), width 6.1 +/- 0.4 mu m (5.8-6.6) and thickness 5.0 +/- 0.6 mu m (4.7-5.3). The polar capsules were elongated and of equal size: length 4.6 +/- 0.2 mu m (4.3-4.8) and width 1.7 +/- 0.1 mu m (1.5-1.9). The histological analysis revealed numerous plasmodia in the blood vessels of the gill filaments. The site of parasite development was the wall of the large-caliber blood vessel of the gill filament, with progressive growth towards the lumen, resulting in the obstruction of blood flow, congestion and perivascular edema. The ultrastructural study revealed that the plasmodial wall was composed of two membranes, had numerous pinocytic canals and was in direct contact with the basement membrane of the vessel. The development of the parasite was asynchronous, with mature spores, immature spores and young developmental stages randomly distributed throughout the plasmodium. The prevalence of the parasite was 4.4%. with male and female fish being infected. (C) 2009 Elsevier B.V. All rights reserved.

Light and scanning electron microscopy of Henneguya arapaima n. sp (Myxozoa: Myxobolidae) and histology of infected sites in pirarucu (Arapaima gigas: Pisces: Arapaimidae) from the Araguaia River, Brazil

In this report, we describe Henneguya arapaima n. sp., a parasite of the gill arch and gall bladder of Arapaima gigas (pirarucu) collected in the Araguaia River, in the municipality of Nova Crixas, Goias State, central Brazil. The plasmodia were white, round or ellipsoidal and measured 200-600 mu m. Parasite development was asynchronous and the mature spores were fusifonn and had smooth wall. The spores measurements were (range, with means +/- S.D. in parentheses): total length-48.4-53.1 mu m (51.6 +/- 3.4 mu m), body length-13.5-15.2 mu m (14.2 +/- 0.8 mu m), body width-5.1-6.1 mu m (5.7 +/- 0.5 mu m), body thickness-4.7-5.3 mu m (4.9 +/- 0.2 mu m) and caudal process length-38.0-41.2 mu m (38.3 +/- 2.9 mu m). The polar capsules were elongated and of unequal size, with lengths of 6.3-6.8 mu m (6.5 +/- 0.2) and 6.2-6.6 mu m (6.3 +/- 0.1) for the longest and shortest axes, respectively. Capsule width was 1.4-1.6 mu m (1.5 +/- 0.1). Histological analysis showed that the plasmodia occurred in the tunica adventitia of the gall bladder and were delimited by a thin capsule of connective tissue. In the gill arch, the plasmodia were also surrounded by connective tissue similar to the endomesium, of striated skeletal muscle cells. Sixty-five juvenile specimens of A. gigas weighing 1.0-25.0 kg were examined, 17 (26.1%) of which were infected. Of these, 14 (82.3%) had cysts in the gall bladder, two (11.7%) had cysts in the gill arch and only one (5.9%) had cysts in both organs. When the fish were grouped by weight, the prevalence of infection in fish weighing up to 10.0 kg (20.7%) was significantly lower than in fish weighing 10.1-25.0 kg (50%) (G = 3.93; d.f. = 1; p < 0.05). (C) 2008 Elsevier B.V. All rights reserved.

Efficient but destructive: a test of the dental wash technique using secondary electron microscopy

The Boyadjian et al dental wash technique provides, in certain contexts, the only chance to analyze and quantify the use of plants by past populations and is therefore an important milestone for the reconstruction of paleodiet. With this paper we present recent investigations and results upon the influence of this method on teeth. A series of six teeth from a three thousand years old Brazilian shellmound (Jabuticabeira II) was examined before and after dental wash. The main focus was documenting the alteration of the surfaces and microstructures. The status of all teeth were documented using macrophotography, optical light microscopy, and atmospheric Secondary Electron Microscopy (aSEM) prior and after applying the dental wash technique. The comparison of pictures taken before and after dental wash showed the different degrees of variation and damage done to the teeth but, also, provided additional information about microstructures, which have not been visible before. Consequently we suggest that dental wash should only be carried out, if absolutely necessary, after dental pathology, dental morphology and microwear studies have been accomplished. (C) 2010 Elsevier Ltd. All rights reserved.