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Amorphization of cellulose by swelling in ionic liquid (IL)/water mixtures at room temperature is a suitable alternative to the dissolution-precipitation pretreatment known to facilitate enzymatic digestion. When soaking microcrystalline cellulose in the IL 1-ethyl-3-methylimidazolium acetate containing 20 wt % water, the crystallinity of the cellulose sample is strongly reduced. As less than 4 % of the cellulose dissolves in this mixture, this swelling method makes a precipitation step and subsequent energy-intensive IL purification redundant. Second-harmonic generation (SHG) microscopy is used as a structure-sensitive technique for in situ monitoring of the changes in cellulose crystallinity. Combined optical and SHG observations confirm that in the pure IL complete dissolution takes place, while swelling without dissolution in the optimal IL/water mixture yields a solid cellulose with a significantly reduced crystallinity in a single step.

One of the fundamental problems in the analysis of single particle tracking data is the detection of individual particle positions from microscopy images. Distinguishing true particles from noise with a minimum of false positives and false negatives is an important step that will have substantial impact on all further analysis of the data. A common approach is to obtain a plausible set of particles from a larger set of candidate particles by filtering using manually selected threshold values for intensity, size, shape, and other parameters describing a particle. This introduces subjectivity into the analysis and hinders reproducibility. In this paper, we introduce a method for automatic selection of these threshold values based on maximizing temporal correlations in particle count time series. We use Markov Chain Monte Carlo to find the threshold values corresponding to the maximum correlation, and we study several experimental data sets to assess the performance of the method in practice by comparing manually selected threshold values from several independent experts with automatically selected threshold values. We conclude that the method produces useful results, reducing subjectivity and the need for manual intervention, a great benefit being its easy integratability into many already existing particle detection algorithms.

Probe DNA, consisting of a 36-mer fragment was covalently immobilized on nanocrystalline chemical vapour deposition (CVD) diamond electrodes and hybridized with a 29-mer target DNA. In this paper, we report on the label-free real-time electronic monitoring of DNA denaturation upon exposure to NaOH at different flow rates and molarities, using electrochemical impedance spectroscopy as readout technology. The impedance response was separated into a denaturation time constant and a medium exchange time constant by means of a double exponential fit. It was observed that the denaturation time is dependent on the flow rate as well as on the molarity of the NaOH. Surprisingly, it was observed that at low molarities (0.05 M) the DNA does not fully denature at low flow rates. Only after flushing the flow cell a second time with 0.05 M NaOH, complete denaturation was achieved. Confocal images were obtained and plotted in 3D graphs to confirm the results. This paper provides a systematic overview of measured denaturation times for different flow rates and at different molarities of NaOH. Optimization of these parameters can be a valuable asset in the field of mutation analysis.

The study of protein adsorption on solid surfaces is interesting for theoretical and practical bio-analytical sensing applications. In this work we combine electrochemical impedance spectroscopy, enzyme linked immunosorbent assay, and fluorescence microscopy with thin boron doped nanocrystalline diamond films to address and study the adsorption behavior of globular proteins (antibodies) on hydrophobic and hydrophilic diamond surfaces. A powerful combination of time resolved impedance spectroscopy and data modeling with equivalent circuits allow a detailed insight in the protein behavior at an interface. It is found that hydrogenated diamond is greatly favorable for impedimetric read-out but causes slight conformational loss of the protein structure and therefore also its biological activity. The oxidized surface allows faster adsorption and a high biological activity but results in smaller impedimetric response. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. ispartof: Physica Status Solidi A, Applications and Materials Research vol:208 issue:9 pages:2093-2098 status: published

A visible-light-excitable, ratiometric, brightly fluorescent pH indicator for measurements in the pH range 5-7 has been designed and synthesized by conjugatively linking the BODIPY fluorophore at the 3-position to the pH-sensitive ligand imidazole through an ethenyl bridge. The probe is available as cell membrane permeable methyl ester 8-(4-carbomethoxyphenyl)-4,4-difluoro-3-[2-(1H-imidazol-4-yl)ethenyl]-1,5,7-trimethyl-3a,4a-diaza-4-bora-s-indacene (I) and corresponding water-soluble sodium carboxylate, sodium 8-(4-carboxylatophenyl)-4,4-difluoro-3-[2-(1H-imidazol-4-yl)ethenyl]-1,5,7-trimethyl-3a,4a-diaza-4-bora-s-indacene (II). The fluorescence quantum yield Φ(f) of ester I is very high (0.8-1.0) in the organic solvents tested. The fluorescence lifetime (ca. 4 ns) of I in organic solvents with varying polarity/polarizability (from cyclohexane to acetonitrile) is independent of the solvent with a fluorescence rate constant k(f) of 2.4×10(8) s(-1). Probe I is readily loaded in the cytosol of live cells, where its high fluorescence intensity remains nearly constant over an extended time period. Water-soluble indicator II exhibits two acid-base equilibria in aqueous solution, characterized by pK(a) values of 6.0 and 12.6. The Φ(f) value of II in aqueous solution is high: 0.6 for the cationic and anionic forms of the imidazole ligand, and 0.8 for neutral imidazole. On protonation-deprotonation in the near-neutral pH range, UV/Vis absorption and fluorescence spectral shifts along with isosbestic and pseudo-isoemissive points are observed. This dual-excitation and dual-emission pH indicator emits intense green-yellow fluorescence at lower pH and intense orange fluorescence at higher pH. The influence of ionic strength and buffer concentration on the absorbance and steady-state fluorescence of II has also been investigated. The apparent pK(a) of the near-neutral acid-base equilibrium determined by spectrophotometric and fluorometric titration is nearly independent of the added buffer and salt concentration. In aqueous solution in the absence of buffer and in the pH range 5.20-7.45, dual exponential fluorescence decays are obtained with decay time τ(1)=4.3 ns for the cationic and τ(2)=3.3 ns for the neutral form of II. The excited-state proton exchange of II at near-neutral pH becomes reversible on addition of phosphate (H(2)PO(4)(-)/HPO(4)(2-)) buffer, and a pH-dependent change of the fluorescence decay times is induced. Global compartmental analysis of fluorescence decay traces collected as a function of pH and phosphate buffer concentration was used to recover values of the deactivation rate constants of the excited cationic (k(01)=2.4×10(8) s(-1)) and neutral (k(02)=3.0×10(8) s(-1)) forms of II.

Statins have attracted interest as a treatment option for multiple sclerosis (MS) because of their pleiotropic antiinflammatory and immunomodulatory effects. However, contradictory results have been described when they are applied to oligodendrocytes (OLGs), the cell type predominantly affected in MS. In this study we focus on the in vitro effect of statins on process outgrowth in OLN-93 cells, a well-characterized OLG-derived cell line, and primary cultures of neonatal rat OLGs. Application of the lipophilic simvastatin, as low as 0.1-1 μM, disturbs process formation of both cell types, leading to less ramified cells. We show that both protein isoprenylation and cholesterol synthesis are required for the normal differentiation of OLGs. It is further demonstrated that the expression of 2',3'-cyclic-nucleotide-3' phosphodiesterase (CNP) and tubulin is lowered, concomitant with a reduction of membrane-bound CNP as well as tubulin. Therefore, we propose that lack of isoprenylation of CNP could help to explain the altered morphological and biochemical differentiation state of treated OLGs. Moreover, expression of specific myelin markers, such as myelin basic protein, myelin-associated glycoprotein, and myelin oligodendrocyte glycoprotein, was compromised after treatment. We conclude that simvastatin treatment has detrimental effects on OLG process outgrowth, the prior step in (re)myelination, thereby mortgaging long-term healing of MS lesions.

The authors thank Mrs. R. Beenaerts, Mr. J. Janssen, Mr. P. Pirotte, Mr. J. Soogen, Mr. R. van Werde for expert laboratory assistance, Prof. Dr. I. Lambrichts and Mr.M. Jans for logistics and fixation procedures, Mr. K. Vanstreels and Mr. B. Ruttens for assistance with the scanning electron microscope, Prof. Dr. R. Valcke for the use of his spectrophotometers, Mr. J. Duchateau, Mr. F. Horemans, Mrs. V. Vrindts for cleaning the diamond substrates, Ir. L. Naelaerts (KHLIM) for lending stereomicroscopes. S. W. is a post-doctoral research fellow of the Research Foundation – Flanders (FWO-Vlaanderen). This work was funded by the Research Council of the UHasselt, tUL, the K.U. Leuven (GOA/2006/02) and by a PhD grant of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen). Support by IAP P6/27 Functional Supramolecular Systems (BELSPO) and by the FWOonderzoeksgemeenschap “Scanning andWide Field Microscopy of (Bio)-organic Systems” is gratefully acknowledged.

In the framework of developing a fast and label-free immunosensor for C-reactive protein (CRP) detection, H-terminated nanocrystalline diamond (NCD) was functionalised with anti-CRP antibodies that were physically adsorbed to the surface. Impedance spectroscopy was used to electronically detect real-time CRP recognition. Different impedance behaviours were observed after CRP addition as compared to after FITC-labelled ssDNA addition at low (100 Hz) as well as at high frequencies (1 MHz). Physical interpretations of the observed impedance changes were obtained by fitting the data to an equivalent electrical circuit. Concentrations of 1 μM CRP were recognised with a reaction time of 30 minutes. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

This article reviews the Current state-of-the art of diamond-based DNA sensors. Some general concepts involved in biosensors are introduced and applied to DNA sensors. The properties of chemically vapor deposited (CVD) diamond relevant for this application are summed up, with special attention for the stability and bio-compatibility of the material. Several routes to functionalize the diamond Surface are considered. The physical properties of the obtained DNA layers are discussed in terms of surface density and molecular conformation. Possible read-out strategies are evaluated, including optical and electronic sensing. With diamond-based DNA sensors, real-time and label-free sensing is achieved.

The SAC8.5, a low-cost Peltier-cooled black and white 8-bit CCD camera for astronomy, was evaluated for its use in imaging microscopy. Two camera-microscope configurations were used: an epifluorescence microscope (Nikon Eclipse TE2000-U) and a bottom port laser scanning confocal microscope system (Zeiss LSCM 510 META). Main advantages of the CCD camera over the currently used photomultiplier detection in the scanning setup are fast image capturing, stable background, an improved signal-to-noise ratio and good linearity. Based on DAPI-labelled Chinese Hamster Ovarian cells, the signal-to-noise ratio was estimated to be 4 times higher with respect to the currently used confocal photomultiplier detector. A linear relationship between the fluorescence signal and the FITC-inulin concentrations ranging from 0.05 to 1.8 mg mL(-1) could be established. With the SAC8.5 CCD camera and using DAPI, calcein-AM and propidium iodide we could also distinguish between viable, apoptotic and necrotic cells: exposure to CdCl(2) caused necrosis in A6 cells. Additional examples include the observation of wire-like mitochondrial networks in Mito Tracker Green-loaded Madin-Darby canine kidney cells. Furthermore, it is straightforward to interface the SAC8.5 with automated shutters to prevent rapid fluorophore photobleaching via easy to use astrovideo software. In this study, we demonstrate that the SAC8.5 black and white CCD camera is an easy-to-implement and cost-conscious addition to quantitative fluorescence microfluorimetry on living tissues and is suitable for teaching laboratories.

A biochemical method to immobilize DNA on synthetic diamond for biosensor applications is developed. Nanocrystalline diamond is grown using microwave plasma-enhanced chemical vapour deposition. On the hydrogen-terminated surface 10-undecenoic acid is tethered photochemically under 254 nm illumination, followed by 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide crosslinker-mediated attachment of amino modified DNA. The attachment is functionally confirmed by comparison of supernatant fluorescence and gel electrophoresis. The linking procedure allowed for 35 denaturation and rehybridisation steps. (© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Cytokines, such as interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), can initiate dual effects resulting in either cell growth or cell death. In this study, the human oligodendroglial cell lines HOG and MO3.13 were used as a model to study the molecular mechanisms of cytokine-induced cell death in human oligodendrocytes. We have previously shown that TNF-alpha and IFN-gamma induce apoptosis in both oligodendroglial cell lines within 72 hr. In the present study, the cell death pathways operating within these cells were further investigated at the gene expression level. Both cell lines express a broad repertoire of caspases and apoptosis-related genes. Some of these genes are specifically up-regulated by cytokine treatment; e.g., caspase-1 is up-regulated by IFN-gamma. In addition to direct cytotoxic effects, IFN-gamma and TNF-alpha also enhance the expression of Fas, TNFR1, and MHC class I molecules in both cell lines. This suggests that cytokines can make oligodendrocytes more vulnerable to different cell death pathways in an inflammatory environment. cDNA microarray analysis of the HOG cell line revealed that TNF-alpha induces genes that regulate apoptosis, survival, inflammation, cell metabolism, and cell signaling. The data suggest that oligodendroglial cells activate both death and survival pathways upon cytokine challenges. However, the survival pathways seem to be unable to compete with the death signal after more than 24 hr of cytokine treatment. These results may contribute to the development of therapeutic strategies aimed at interfering with cytokine-induced cell death of oligodendrocytes in patients with multiple sclerosis.

Multiple sclerosis is a chronic inflammatory disease of the central nervous system. Myelin and oligodendrocytes are considered the major targets of injury caused by a cell-mediated immune response. There is circumstantial evidence that proinflammatory cytokines like tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) could have disease-promoting roles in multiple sclerosis (MS). In the present study, the cytotoxic effects of IFN-gamma and TNF-alpha on the human oligodendroglial cell lines human oligodendroglioma (HOG) and MO3.13 were analyzed. When the oligodendroglial cell lines were cultured in the presence of IFN-gamma or TNF-alpha, apoptotic cell death was observed in both cell lines after >24 hr incubation. Apoptosis was evidenced by a decrease in cell viability, apoptotic changes in cell and nucleus morphology, and disruption of the membrane asymmetry. Our data show that TNF-alpha and IFN-gamma induce apoptosis in a dose-dependent fashion in both oligodendroglial cell lines and that their synergistic effect results in enhanced cell death. Understanding the regulation of cell death pathways in oligodendrocytes is critical for protecting myelin-producing cells and their associated axons during injury in patients with MS.

Limburgs Univ Centrum, Mat Res Inst, B-3590 Diepenbeek, Belgium. Linburgs Univ Centrum, Biomed Res Inst, B-3590 Diepenbeek, Belgium.Wenmackers, S, Limburgs Univ Centrum, Mat Res Inst, Wetenschapspk 1, B-3590 Diepenbeek, Belgium.

The main objective of this review is to show how the concepts of compartmental modeling can be introduced and applied in photophysics. The term “compartment” in a photophysical context is defined as a subsystem composed of a distinct type of species that acts kinetically in a unique way. Compartments can be divided into ground-state and excited-state compartments, depending on the state of the composing species. In photophysics, a compartmental system is perturbed by a light pulse (photo-excitation), and its dynamics is followed via fluorescence within the time range from picoseconds to several hundred nanoseconds. In this review, we present the fluorescence δ-response functions for compartmental systems consisting of one excited-state compartment, two reversibly interconnected excited-state compartments, and their corresponding ground-state compartments. In deterministic identifiability, one investigates whether the parameters of a specific model can be uniquely defined, assuming perfect time-resolved fluorescence data. The identifiability is presented for the model with one excited-state compartment and three models of reversible intermolecular two-state excited-state processes in isotropic environments: (i) without external quencher, (ii) with added quencher, and (iii) with coupled species-dependent rotational diffusion described by Brownian reorientation. The parameters that have to be identified are time-invariant rate constants and parameters related to excitation and emission. The conditions under which the relevant parameters can be identified are discussed. For all models, the explicit relationships between the true and alternative model parameters are shown. © 2005 Wiley Periodicals, Inc. Int J Quantum Chem, 2006

Presentation d'un spectrometre de fluorescence a resolution temporelle par impulsion laser et detection par comptage de photon a correlation temporelle. Discussion du calcul des parametres de fluorescence a partir des resultats experimentaux et les methodes de correction d'erreur dans le comptage de photon. Liste des applications possibles a l'etude des polymeres