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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.

Z -scan fluorescence correlation spectroscopy (FCS) uses multiple focus planes to determine diffusion coefficients in supported phospholipid bilayers (SPBs) without the need for extrinsic calibration. The approach can be used to discriminate diffusion modes. In this work, Z -scan FCS was applied to study the diffusion of the fluorescent lipid analogue DiI–C 18 (5) in the top membrane of giant unilamellar vesicles (GUVs) in the electroformation chamber. In contrast to SPBs, the intrinsic estimate of the radial waist might differ from the one obtained by extrinsic calibration. We describe a pragmatic approach to verify the validity of the Z -scan approach under these conditions.

The covalent attachment method for DNA on nanocrystalline diamond (NCD), involving the introduction of COOH functionalities on the surface by photoattachment of 10-undecenoic acid (10-UDA), followed by the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)-mediated coupling to NH 2-labeled ssDNA, is evaluated in terms of stability, density, and functionality of the resulting biological interface. This is of crucial importance in DNA biosensor development. The covalent nature of DNA attachment will infer the necessary stability and favorable orientation to the ssDNA probe molecules. Using confocal fluorescence microscopy, the influence of buffer type for the removal of excess 10-UDA and ssDNA, the probe ssDNA length, the probe ssDNA concentration, and the presence of the COOH-linker on the density and functionality of the ssDNA probe layer were investigated. It was determined that the most homogeneously dense and functional DNA layer was obtained when 300 pmol of short ssDNA was applied to COOH-modified NCD samples, while H-terminated NCD was resistant for DNA attachment. Exploiting this surface functionality dependence of the DNA attachment efficiency, a shadow mask was applied during the photochemical introduction of the COOH-functionalities, leaving certain regions on the NCD H-terminated. The subsequent DNA attachment resulted in a fluorescence pattern corresponding to the negative of the shadow mask. Finally, NCD surfaces covered with mixtures of the 10-UDA linker molecule and a similar molecule lacking the COOH functionality, functioning as a lateral spacer, were examined for their suitability in preventing nonspecific adsorption to the surface and in decreasing steric hindrance. However, purely COOH-modified NCD samples, patterned with H-terminated regions and treated with a controlled amount of probe DNA, proved the most efficient in fulfilling these tasks.

Mimicking the selectivity and sensitivity of biological systems for sensor devices is of increasing interest in biomedical, environmental and chemical analysis. Synthetic materials with imprinted nanocavities, acting as highly selective artificial receptors, are a tailor-made solution in obtaining such a sensor. Incorporation of such molecularly imprinted polymers (MIPs) in a platform suitable for electrochemical measurements, can offer high sensitivity together with device miniaturization and an electronic read-out. As a proof of principle, a MIP-based sensor for l -nicotine has been developed. To this end, the molecular structure of l -nicotine was imprinted in a polymer matrix of polymethacrylic acid (PMAA). Subsequently, microparticles of the imprinted polymer were immobilized on thin films of the conjugated polymer OC1C10-PPV. These films were incorporated in an impedimetric sensing device. Using electrochemical impedance spectroscopy, the real part of the impedance was monitored for various concentrations. This setup allows for the detection of l -nicotine from 1 to 10 nM and is insensitive for the resembling molecule l -cotinine.

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.

Background: Several apical ammonium (NH4+/NH3) transport pathways have been described in medullary thick ascending limb (MTAL) cells. The exact nature and importance of some of these pathways remain controversial. Methods: Ammonium transport in primary cultured rabbit MTAL cells was investigated by measuring intracellular pH (pHi). Results: To create physiological conditions, experiments were performed in the symmetrical presence of NH4Cl, which acidified the cells to pHi 6.89. When blockers of apical NH4+ transport were used, the cells alkalinized due to a decreased NH4+ loading. The following values (pH units) were observed: bumetanide, +0.05; verapamil, +0.04; Ba2+ and Cs+, +0.19; tertiapin, +0.09. Tetraethylammonium had no effect. Depolarizing the cells by increasing the K+ concentration alkalinized the cells by 0.16 pH units. Because NH4+ might enter through nonspecific channels, ammonium pulse experiments were performed: an NH4Cl pulse acidified controls as well as depolarized cells. In contrast, when Ba2+, Cs+ or tertiapin were present, an NH4Cl pulse alkalinized the cells. The pharmacological profile of this apical NH4+ transport pathway correlates with the renal outer medullary K+ (ROMK) channel. Indirect immunofluorescence showed the presence of the ROMK protein. Conclusion: In these MTAL cells the Ba2+-sensitive component of NH4+ transport is predominant and consists of permeation of NH4+ through an apical ROMK-related channel.

A deterministic identifiability analysis of kinetic models of a reversible intramolecular excited-state process involving species-dependent rotational Brownian diffusion is performed for spherical and cylindrically symmetric rotors. It is assumed that the axes of rotation do not change upon interconversion, and that the rate constants are independent of orientation. Use of three quencher concentrations allows the determination of two internally consistent sets of quenching rate constants, combinations of excited-state deactivation/exchange rate constants, individual rotational diffusion coefficients, relative orientations of transition moments, and restricted spectral information. Assignment of each set obtained to a particular species is not possible without additional prior information.

[Fanton, Yanick; Rummens, Jean-Luc; Daniels, Annick; Windmolders, Severina; Willems, Leen; Declercq, Jeroen; Hensen, Karen; Koninckx, Remco] Jessa Hosp, Lab Expt Hematol, Hasselt, Belgium. [Fanton, Yanick; Robic, Boris; Rummens, Jean-Luc; Windmolders, Severina; Willems, Leen; Notelaers, Kristof; Paesen, Rik; Ameloot, Marcel; Bito, Virginie; Declercq, Jeroen; Hensen, Karen; Koninckx, Remco; Hendrikx, Marc] Hasselt Univ, Fac Med & Life Sci, Hasselt, Belgium. [Robic, Boris; Mees, Urbain; Hendrikx, Marc] Jessa Hosp, Dept Cardiothorac Surg, Hasselt, Belgium. [Jamaer, Luc; Dubois, Jasperina] Jessa Hosp, Dept Cardiac Anesthesia, Hasselt, Belgium. [Bijnens, Eric; Heuts, Nic] Jessa Hosp, Dept Radiol, MRI Unit, Hasselt, Belgium. [Notelaers, Kristof; Paesen, Rik; Ameloot, Marcel; Bito, Virginie] Hasselt Univ, Biomed Res Inst, Hasselt, Belgium.

Fluorescence recovery after photobleaching (FRAP) is a versatile tool for determining diffusion and interaction/binding properties in biological and material sciences. An understanding of the mechanisms controlling the diffusion requires a deep understanding of structure–interaction–diffusion relationships. In cell biology, for instance, this applies to the movement of proteins and lipids in the plasma membrane, cytoplasm and nucleus. In industrial applications related to pharmaceutics, foods, textiles, hygiene products and cosmetics, the diffusion of solutes and solvent molecules contributes strongly to the properties and functionality of the final product. All these systems are heterogeneous, and accurate quantification of the mass transport processes at the local level is therefore essential to the understanding of the properties of soft (bio)materials. FRAP is a commonly used fluorescence microscopy-based technique to determine local molecular transport at the micrometer scale. A brief high-intensity laser pulse is locally applied to the sample, causing substantial photobleaching of the fluorescent molecules within the illuminated area. This causes a local concentration gradient of fluorescent molecules, leading to diffusional influx of intact fluorophores from the local surroundings into the bleached area. Quantitative information on the molecular transport can be extracted from the time evolution of the fluorescence recovery in the bleached area using a suitable model. A multitude of FRAP models has been developed over the years, each based on specific assumptions. This makes it challenging for the non-specialist to decide which model is best suited for a particular application. Furthermore, there are many subtleties in performing accurate FRAP experiments. For these reasons, this review aims to provide an extensive tutorial covering the essential theoretical and practical aspects so as to enable accurate quantitative FRAP experiments for molecular transport measurements in soft (bio)materials.

The study of cell-nanoparticle interactions is an important aspect for understanding drug delivery using nanocarriers. In this regard, advances in fluorescence based microscopy are useful for the investigation of temporal and spatial behavior of nanoparticles (NPs) within the intracellular environment. In this work, we focus on the delivery of the naturally-occurring hydrophobic photosensitizer Hypericin in human lung carcinoma A549 cells by using biodegradable poly L-lactic acid NPs. For the first time, Hypericin containing NPs are prepared by combining the miniemulsion technique with the solvent evaporation method. This approach yields an efficient loading of the NPs with Hypericin and allows for additional cargo molecules. To monitor the release of Hypercin from the NPs, an additional fluorescent lipophilic dye Coumarin-6 is incorporated in the NPs. Temporal and spatiotemporal image correlation spectroscopy is used to determine the fate of the NPs carrying the potential cargo. Both directed and non-directed motions are detected. By using image cross-correlation spectroscopy and specific fluorescent labeling of endosomes, lysosomes and mitochondria, the dynamics of the cargo loaded NPs in association with the organelles is studied.

The zebrafish, a model organism for which a plethora of molecular and genetic techniques exists, has a lifelong replacing dentition of 22 pharyngeal teeth. This is in contrast to the mouse, which is the key organism in dental research but whose teeth are never replaced. Employing the zebrafish as the main organism to elucidate the mechanisms of continuous tooth replacement, however, poses at least one major problem, related to the fact that all teeth are located deep inside the body. Investigating tooth replacement thus relies on conventional histological methods, which are often laborious, time-consuming and can cause tissue deformations. In this review, we investigate the advantages and limitations of adapting current visualization techniques to dental research in zebrafish. We discuss techniques for fast sectioning, such as vibratome sectioning and high-resolution episcopic microscopy, and methods for in toto visualization, such as Alizarin red staining, micro-computed tomography, and optical projection tomography. Techniques for in vivo imaging, such as two-photon excitation fluorescence and second harmonic generation microscopy, are also covered. Finally, the possibilities of light sheet microscopy are addressed.

Recent research highlighted the impact of ROS as upstream regulators of tissue regeneration. We investigated their role and targeted processes during the regeneration of different body structures using the planarian Schmidtea mediterranea, an organism capable of regenerating its entire body, including its brain. The amputation of head and tail compartments induces a ROS burst at the wound site independently of the orientation. Inhibition of ROS production by diphenyleneiodonium (DPI) or apocynin (APO) causes regeneration defaults at both the anterior and posterior wound sites, resulting in reduced regeneration sites (blastemas) and improper tissue homeostasis. ROS signaling is necessary for early differentiation and inhibition of the ROS burst results in defects on the regeneration of the nervous system and on the patterning process. Stem cell proliferation was not affected, as indicated by histone H3-P immunostaining, fluorescence-activated cell sorting (FACS), in situ hybridization of smedwi-1, and transcript levels of proliferation-related genes. We showed for the first time that ROS modulate both anterior and posterior regeneration in a context where regeneration is not limited to certain body structures. Our results indicate that ROS are key players in neuroregeneration through interference with the differentiation and patterning processes. Bijzonder Onderzoeks- Fonds of Hasselt University (BOF08G01. Hasselt University tUL-impulsfinanciering (project toxicology). Grant BFU2012-31701 (Spain). Agency for Innovation by Science and Technology (IWT)

We investigated the possibilities of second harmonic imaging of microtubules clarifying the determinants of the second harmonic generation in live neurons. We used nonlinear microscopy correlated with electron microscopy to evaluate microtubule organization, dynamics and possible artifacts.

Using two-photon luminescence (TPL) scanning laser microscopy and FDTD simulations, we investigate the TPL process of Au nanoantennas beyond the fundamental mode. Enhanced two-photon absorption dominates the process and a clear signature of the mode parity is observed.

Recently, a supramolecular model was developed for predicting striated skeletal muscle intensity profiles obtained by label-free second harmonic generation (SHG) microscopy. This model allows for a quantitative determination of the length of the thick filament antiparallel range or M-band (M), and results in M ¼ 0.12 μm for single-band intensity profiles when fixing the A-band length (A) to A ¼ 1.6 μm, a value originating from electron microscopy (EM) observations. Using simulations and experimental data sets, we showed that the objective numerical aperture (NA) and the refractive index (RI) mismatch (Δn ¼ n2ω − nω) between the illumination wave (ω) and the second harmonic wave (2ω) severely affect the simulated sarcomere intensity profiles. Therefore, our recovered filament lengths did not match with those observed by EM. For an RI mismatch of Δn ¼ 0.02 and a moderate illumination NA of 0.8, analysis of single-band SHG intensity profiles with freely adjustable A- and M-band sizes yielded A ¼ 1.40 0.04 μm and M ¼ 0.07 0.05 μm for skeletal muscle. These lower than expected values were rationalized in terms of the myosin density distribution along the myosin thick filament axis. Our data provided new and practical insights for the application of the supramolecular model to study SHG intensity profiles in striated muscle. This research is part of the Interreg EMR IV-A project BioMiMedics (www.biomimedics.org) and is cofinanced by the European Union, local governments, research institutes and SMEs. Support by the BELSPO funded IAP-PAI network P7/05 on Functional Supramolecular Systems (FS2) is acknowledged. The Province of Limburg (Belgium) is acknowledged for financial support within the tUL IMPULS FASE II program, allowing for the upgrading of the laser source used in this work.

The plasma membrane of eukaryotic cells exhibits lateral inhomogeneities, mainly containing cholesterol and sphingomyelin, which provide liquid-ordered microdomains (lipid "rafts") that segregate membrane components. Rafts are thought to modulate the biological functions of molecules that become associated with them, and as such, they appear to be involved in a variety of processes, including signal transduction, membrane sorting, cell adhesion and pathogen entry. Although still a matter of ongoing debate, evidence in favor of the presence of these microdomains is gradually accumulating but a consensus on issues like their size, lifetime, composition, and biological significance has yet to be reached. Here, we provide an overview of the evidence supporting the presence of rafts in oligodendrocytes, the myelin-producing cells of the central nervous system, and discuss their functional significance. the myelin membrane differs fundamentally from the plasma membrane, both in lipid and protein composition. Moreover, since myelin membranes are unusually enriched in glycosphingolipids, questions concerning the biogenesis and functional relevance of microdomains thus appear of special interest in oligodendrocytes. The current picture of rafts in oligodendrocytes is mainly based on detergent methods. The robustness of such data is discussed and alternative methods that may provide complementary data are indicated. (c) 2006 Wiley-Liss, Inc.

In ischemic or hypoxic tissues, elevated cytosolic calcium levels can induce lethal processes. Mitochondria, besides the endoplasmic reticulum, play a key role in clearing excessive cytosolic Ca2+. In a previous study, it was suggested that the clearance of cytosolic Ca2+, after approximately 18 min of metabolic inhibition (MI) in renal epithelial cells, occurs via the reverse action of the mitochondrial Na+/Ca2+ exchanger (NCX). For further investigating the underlying mechanism, changes in the mitochondrial Na+ concentration ([Na+](m)) were monitored in metabolically inhibited MDCK cells. CoroNa Red, a sodium-sensitive fluorescence probe, was used to monitor [Na+]m. In the first 15 min of MI, a twofold increase of [Na+]m was observed reaching 113 +/- 7 mM, whereas the cytosolic Na+ concentration ([Na+]c) elevated threefold, to a level of 65 +/- 6 mM. In the next 45 min of MI, [Na+]m dropped to 91 +/- 7 mM, whereas [Na+]c further increased to 91 +/- 4 mM. The striking rise in [Na+]m is likely sufficient to sustain the driving force for mitochondrial Ca2+ uptake via the NCX. Furthermore, when CGP-37157, a specific inhibitor of the mitochondrial NCX, was applied during MI, the second-phase drop of [Na+]m was completely abolished. The obtained results support the hypothesis that the mitochondrial NCX reverses after approximately 15 min of MI. Moreover, because the cellular homeostasis can recover after MI, the mitochondria likely protect MDCK cells from injury during MI by the reversal of the mitochondrial NCX. This study is the first to report [Na+]m measurements in nonpermeabilized living cells.

The parameters describing the kinetics of excited-state processes can possibly be recovered by analysis of the fluorescence decay surface measured as a function of the experimental variables. The identifiability analysis of a photophysical model assuming errorless time-resolved fluorescence data can verify whether the model parameters can be determined and may lead to the minimal experimental conditions under which this is possible. In this work, we used the method of similarity transformation to investigate the identifiability of three kinetic models utilized to describe the time-resolved fluorescence of reversible intramolecular two-state excited-state processes in isotropic environments: (1) model without added quencher, (2) model with added quencher, (3) model with added quencher coupled with species-dependent rotational diffusion described by Brownian reorientation. Without a priori information, model 1 is not identifiable. For model 2, two sets of quenching rate constants and combinations of excited-state deactivation/exchange rate constants are possible, but they cannot be allocated to a specific excited-state species. For both sets, upper and lower limits on the excited-state deactivation/exchange rate constants can be obtained. For model 3, both spherically and cylindrically symmetric rotors, with no change in the principal axes of rotation in the latter, are considered. The fluorescence delta-response functions I(parallel)(t) and I(perpendicular)(t), for fluorescence polarized parallel and perpendicular, respectively, to the electric vector of linearly polarized excitation, are used to define the sum S(t) identically equal to I(parallel)(t) + 2 I(perpendicular)(t) and the difference D(t) identically equal to I(parallel)(t) - I(perpendicular)(t). The identifiability analysis is performed using the S(t) and D(t) functions. Also for model 3, two sets of kinetic parameters (i.e., quenching rate constants, combinations of deactivation/exchange rate constants, and rotational diffusion coefficients) exist, but these parameters cannot be assigned unequivocally to a specific species. For the three models, an infinite number of alternative spectroscopic parameters associated with excitation and emission are found.

The sensitivity of store-operated Ca(2+)-entry to changes in the extra- and intracellular pH (pH(o) and pH(i), respectively) was investigated in SH-SY5Y human neuroblastoma cells. The intracellular Ca(2+)-stores were depleted either with 1 mM carbachol (CCH) or with 2 microM thapsigargin (TG). Extracellular acidification suppressed both the CCH- and TG-mediated Ca(2+)-entry while external alkalinization augmented both the CCH- and the TG-induced Ca(2+)-influx. Mn(2+)-quenching experiments revealed that the rates of Ca(2+)-entry at the thapsigargin- or carbachol-induced plateau were both accelerated at pH(o) 8.2 and slowed down at pH(o) 6.8 with respect to the control at pH(o) 7.4. Alteration of pH(o) between 6.8 and 8.2 did not have any significant prompt effect on pH(i) and changes in pH(i) left the CCH-induced Ca(2+)-entry unaffected. These findings demonstrate that physiologically relevant changes in pH(o) affect the store-operated Ca(2+)-entry in SH-SY5Y cells and suggest that endogenous pH(o) shifts may regulate cell activity in situ via modulating the store-operated Ca(2+)-entry.

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)

Time-resolved fluorescence spectroscopy and microscopy in both time and frequency domains provide very useful and accurate information on dynamic processes. Good quality data are essential in obtaining reliable parameter estimates. Distortions of the fluorescence response due to artifacts may have disastrous consequences. We provide here a concise overview of potential difficulties encountered under daily laboratory circumstances in the use of time- and frequency-domain equipment as well as practical remedies against common error conditions, elucidated with several graphs to aid the researcher in visual inspection and quality-control of collected data. A range of artifacts due to sample preparation or to optical and electronic pitfalls are discussed, as are remedies against them. Also recommended data analysis strategies are described.

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.

In renal ischemia, tubular obstruction induced by swelling of epithelial cells might be an important mechanism for reduction of the glomerular filtration rate. We investigated ischemic cell swelling by examining volume regulation of A6 cells during metabolic inhibition (MI) induced by cyanide and 2-deoxyglucose. Changes in cell volume were monitored by recording cell thickness ( Tc). Intracellular pH (pHc) measurements were performed with the pH-sensitive probe 5-chloromethyl-fluoresceine diacetate. Tcmeasurements showed that MI increases cell volume. Cell swelling during MI is proportional to the rate of Na+transport and is not followed by a volume regulatory response. Furthermore, MI prevents the regulatory volume decrease (RVD) elicited by a hyposmotic shock. MI induces a pronounced intracellular acidification that is conserved during a subsequent hypotonic shock. A transient acidification induced by a NH4Cl prepulse causes a marked delay of the RVD in response to a hypotonic shock. On the other hand, acute lowering of external pH to 5, simultaneously with the hypotonic shock, allowed the onset of RVD. However, this RVD was completely arrested ∼10 min after the initiation of the hyposmotic challenge. The inhibition of RVD appears to be related to the pronounced acidification that occurred within this time period. In contrast, when external pH was lowered 20 min before the hyposmotic shock, RVD was absent. These data suggest that internal acidification inhibits cellular volume regulation in A6 cells. Therefore, the intracellular acidification associated with MI might at least partly account for the failure of volume regulation in swollen epithelial cells.

A straightforward protocol for the covalent functionalization of boron-doped diamond electrodes with either ferrocene or single-stranded deoxyribonucleic acid (DNA) is reported. The functionalization method is based on a combination of diazonium salt electrografting and click chemistry. An azide-terminated organic layer is first electrografted onto the diamond surface by electrochemical reduction of 4-azidophenyldiazonium chloride. The azidophenyl-modified surface then reacts rapidly and efficiently with molecules bearing a terminal alkyne moiety by means of Cu-1-catalyzed alkyne-azide cycloaddition. Covalent attachment of ferrocene moieties was analyzed by X-ray photoelectron spectroscopy and cyclic voltammetry, whereas impedance spectroscopy was applied for the characterization of the immobilized DNA. This work was financially supported by the Special Research Fund of Hasselt University, the Research Foundation Flanders (FWO) (G.0555.10N and G.0829.09N), and the EU FP7 Collaborative Project MOLESOL (No. 256617). We thank H. Penxten for the CV measurements, B. Ruttens for the SEM images, and P. Robaeys for the four-point probe measurements.

Diamond nanoparticles (DNPs) are very attractive for biomedical applications, particularly for bioimaging. The aim of this study was to evaluate the impact of DNPs on neural cancer cells and thus to assess the possible application of DNPs for these cells imaging. For this purpose, the neuroblastoma SH-SY5Y cell line was chosen. Cells were cultured in medium with different concentrations (15, 50, 100 and 150 μg/ml) of DNPs. After 48 h of incubation, cell metabolic activity was evaluated by the XTT assay. For assessment of cellular metabolic activity, cells were also cultured on differently terminated nanocrystalline diamond (NCD) coatings in medium with 150 μg/ml of DNPs. Cell adhesion and morphology were evaluated by brightfield microscopy. Diamond nanoparticle internalization was determined by confocal microscopy. The obtained results showed that low concentrations (15, 50 and 100 μg/ml) of nanoparticles did not significantly affect the SH-SY5Y cell metabolic activity. However, a higher concentration (150 μg/ml) of DNPs statistically significantly reduced SH-SY5Y cell metabolic activity. After 48 h incubation with 150 μg/ml DNPs, cell metabolic activity was 23% lower than in medium without DNPs on standard tissue culture polystyrene.

A microcavity-based deoxyribonucleic acid (DNA) optical biosensor is demonstrated for the first time using synthetic sapphire for the optical cavity. Transmitted and elastic scattering intensity at 1510 nm are analyzed from a sapphire microsphere (radius 500 µm, refractive index 1.77) on an optical fiber half coupler. The 0.43 nm angular mode spacing of the resonances correlates well with the optical size of the sapphire sphere. Probe DNA consisting of a 36-mer fragment was covalently immobilized on a sapphire microsphere and hybridized with a 29-mer target DNA. Whispering gallery modes (WGMs) were monitored before the sapphire was functionalized with DNA and after it was functionalized with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). The shift in WGMs from the surface modification with DNA was measured and correlated well with the estimated thickness of the add-on DNA layer. It is shown that ssDNA is more uniformly oriented on the sapphire surface than dsDNA. In addition, it is shown that functionalization of the sapphire spherical surface with DNA does not affect the quality factor (Q . ≈ 04) of the sapphire microspheres. The use of sapphire is especially interesting because this material is chemically resilient, biocompatible, and widely used for medical implants. ispartof: Journal of Biomedical Optics vol:19 issue:9 ispartof: location:United States status: published

Single particle tracking (SPT) of transmembrane receptors in the plasma membrane often reveals heterogeneous diffusion. A thorough interpretation of the displacements requires an extensive analysis suited for discrimination of differentmotion types present in the data. Here the diffusion pattern of the homomeric α3-containing glycine receptor (GlyR) is analyzed in themembrane of HEK 293 cells. More specifically, the influence of the α3 RNA splice variants α3K and α3L on lateralmembrane diffusion of the receptor is revealed in detail. Using a combination of ensemble and local SPT analysis, free and anomalous diffusion parameters are determined. The GlyR α3 free diffusion coefficient is found to be 0.13 ± 0.01 μm2/s and both receptor variants display confined motion. The confinement probability level and residence time are significantly elevated for the α3L variant compared to the α3K variant. Furthermore, for the α3L GlyR, the presence of directed motion was also established, with a velocity matching that of saltatory vesicular transport. These findings reveal that α3 GlyRs are prone to different types of anomalous diffusion and reinforce the role of RNA splicing in determining lateral membrane trafficking. This work was funded by the Research Council of the UHasselt, tUL. GlyR expression constructs were obtained thanks to funding by the Helmholtz Association (VH-NG-246 to J.C.M.) and the Bundesministerium fur Bildung und Forschung BMBF (Era-Net NEURON II CIPRESS to J.C.M.). The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Program (FP7/2007-2013)/ERC Grant Agreement no 291593 FLUOROCODE), from the Flemish government in the form of long-term structural funding "Methusalem" grant METH/08/04 CASAS, from the 'Fonds voor Wetenschappelijk Onderzoek Vlaanderen' (FWO grants G.0197.11; G0484.12,) and from the Hercules Foundation (HER/08/021). S.R., J.H. and M.A. thank the Federal Science Policy of Belgium (IAP-VI/27). The support by the FWO-onderzoeksgemeenschap "Scanning and Wide Field Microscopy of (Bio)-organic Systems" is gratefully acknowledged by J.H. and M.A.

In this study, the effect of glycine receptor (GlyR) ?3 alternative RNA splicing on the distribution of receptors in the membrane of human embryonic kidney 293 cells is investigated using optical super-resolution microscopy. Direct stochastic optical reconstruction microscopy is used to image both ?3K and ?3L splice variants individually and together using single- and dual-color imaging. Pair correlation analysis is used to extract quantitative measures from the resulting images. Autocorrelation analysis of the individually expressed variants reveals clustering of both variants, yet with differing properties. The cluster size is increased for ?3L compared to ?3K (mean radius 92 ? 4 and 56 ? 3 nm, respectively), yet an even bigger difference is found in the cluster density (9,870 ? 1,433 and 1,747 ? 200 ?m(-2), respectively). Furthermore, cross-correlation analysis revealed that upon co-expression, clusters colocalize on the same spatial scales as for individually expressed receptors (mean co-cluster radius 94 ? 6 nm). These results demonstrate that RNA splicing determines GlyR ?3 membrane distribution, which has consequences for neuronal GlyR physiology and function.

A model of reversible diffusion-mediated excited-state association is shown to be identifiable from four decay traces at two emission wavelengths and two concentrations, when only the dissociating species is excited, and the decays of the excited-state species can be followed separately.

The modeling of excited-state processes in photophysics can conveniently be done within the framework of compartmental analysis. In compartmental analysis substantial attention has been devoted to the study of deterministic identifiability, which verifies whether it is possible to determine the parameters of the compartmental model from error-free data. In this paper the similarity transformation approach is applied to the identifiability problem of the photophysical model for reversible intermolecular two-state excited-state processes. This method provides straightforward relations between the true and alternative sets of the system parameters. This allows one to explore directly the parameter space for identifiability. Since absolute values for the spectral parameters associated with excitation and emission are not available from time-resolved fluorescence experiments, the original similarity transformation approach to the identifiability problem was reformulated in terms of normalized spectral parameters, which are experimentally accessible. It is shown that six decay traces—measured at two coreactant concentrations and three emission wavelengths—are required for the model to be locally identifiable. Two sets of rate constants and associated spectral parameters may be found under these conditions. Enclosure in the analysis of the monoexponential decay at very low coreactant concentration results in global identifiability. The non-negativity requirement of the spectral parameters also can lead to the unique solution. If the fluorescence decays are independent of the emission wavelength, additional information about the photophysical system is necessary for identifiability.

The behavior of the sodium indicator sodium-binding benzofuran isophthalate (SBFI) is investigated in HeLa cells by time-resolved fluorescence microscopy. The fluorescence relaxation of SBFI in HeLa cells can be described by a triexponential for intracellular sodium concentration ([Na(+)](i)) between 0 and 90 mM. Changes in [Na(+)](i) affect neither the fluorescence relaxation times (0.21, 0. 60, and 2.7 ns) nor the average decay time (2.2 ns). The preexponential factor of the shortest decay time is negative. However, the ratio of the fluorescence excitation signal at 340 nm to that at 380 nm increases with [Na(+)](i). To elucidate the behavior of SBFI in cells, experiments are performed on SBFI in buffer at various concentrations of sodium, potassium, and bovine serum albumin (BSA) and at various viscosities. The fluorescence decay is triexponential only in the presence of BSA. The relaxation times are independent of [Na(+)] and [BSA]. The preexponential factor of the shortest decay time is negative from a certain [BSA] on, which depends on [Na(+)]. The data indicate that interactions with intracellular components rather than microviscosity influence the SBFI behavior in cells. A model is suggested in which the fluorescence intensities are mainly determined by the signals from the Na(+) subsetSBFI and SBFI subsetprotein complexes.

The photophysics of the polypeptide hormone glucagon in aqueous phosphate buffer (λex = 295 nm, pH 6.7, 17.2 °C) in the presence of the added quencher sodium iodide is investigated using standard s...

SBFO is a commercially available fluorescent indicator for the determination of Na+ concentrations. To investigate the reversible reaction of Na+ with this indicator in the ground and excited states, steady-state and time-resolved fluorescence measurements were performed. Global compartmental analysis was used to describe the fluorescence decay surfaces yielding the following values for the rate constants at room temperature in aqueous solution at pH 7.2: k01 = 5 × 108 s-1, 2 × 107 M-1 s-1 < k21 < 3 × 108 M-1 s-1, k02 = 5 × 108 s-1, k12 = 1 × 108 s-1. k01 and k02 denote the respective deactivation rate constants of the Na+ free and bound forms of the indicator in the excited state. k21 represents the second-order rate constant of association of Na+ with SBFO in the excited state, whereas k12 is the first-order rate constant of dissociation of the excited Na+−indicator complex. The effect of the reversible excited-state reaction between Na+ and the indicator on the fluorimetric determination of Kd can be ...

The identifiability of excited-state processes in the presence of transient effects is studied. The Smoluchowski and Collins-Kimball models of diffusion-mediated association are considered. The kinetic parameters of single-species quenching and related kinetic schemes (double-species quenching without excited-state interchange, and irreversible association with separated excited-species spectra) can be uniquely recovered from the same or a smaller number of decay traces than are necessary within classical kinetics where time-invariant rate constants are used. For single-species quenching in low dimensions, 1D and 2D, the system parameters can be uniquely recovered from a single decay trace at one nonzero quencher concentration. In three dimensions, decays traces measured at two concentrations are necessary and sufficient to recover the parameters. For double-species quenching without interchange of the excited states, decay traces at two wavelengths collected in the absence and presence of quencher guaran...

Although reversible intramolecular two-state excited-state processes without a priori information are unidentifiable, bounds on the excited-state rate constants kij can be specified when a quencher is used [Van Dommelen et al. J. Phys. Chem. 1993, 97, 11738]. These limits can be obtained by a scanning procedure consisting of a series of global compartmental analyses of a fluorescence decay surface in which one of the rate constants kij is held fixed at different preset values. The theory requires that the rate constants of quenching (kQ1, kQ2) for the two excited species have different values. In the present paper, computer-generated fluorescence decay surfaces are used to investigate the criteria under which reliable estimates of the bounds on the rate constants kij can be obtained. If the values of kQ1 and kQ2 are substantially different, reliable estimates are obtained. If kQ1 and kQ2 are nearly equal in value, the quality of the estimates of the bounds depends on the combinations of the values of the ...

The identifiability of two-state excited-state processes is studied when transient effects are present. When the emission bands of the excited species are well separated, the deactivation rate constants of the two excited species to their respective ground states, k 01 and k 02 , can be determined from two decay traces of the individual excited species at one concentration. Thus, two decay traces are sufficient for the recovery of k 01 and k 02 for inter- and intra-molecular two-state excited-state processes. When transient effects are absent more kinetic information is needed for the recovery of k 01 and k 02 .

The aim of this study was to assess the impact of nanocrystalline diamond (NCD) thin coatings on neural cell adhesion and proliferation. NCD was fabricated on fused silica substrates by microwave plasma chemical vapor deposition (MPCVD) method. Different surface terminations were performed through exposure to reactive hydrogen and by UV induced oxidation during ozone treatment. Boron doped NCD coatings were also prepared and investigated. NCD surface wettability was determined by contact angle measurement. To assess biocompatibility of the NCD coatings, the neuroblastoma SH-SY5Y cell line was used. Cells were plated directly onto diamond surfaces and cultured in medium with or without fetal bovine serum (FBS), in order to evaluate the ability of cells to adhere and to proliferate. The obtained results showed that these cells adhered and proliferated better on NCD surfaces than on the bare fused silica. The cell proliferation on NCD in medium with and without FBS after 48h from plating was on average, respectively, 20 and 58% higher than that on fused silica, irrespective of NCD surface modification. Our results showed that the hydrogenated, oxygenated and boron-doped NCD coatings can be used for biomedical purposes, especially where good optical transparency is required.

Fluorescence recovery after photobleaching (FRAP) is a common technique to probe mobility of fluorescently labeled proteins in biological membranes by monitoring the time-dependence of the spatially integrated fluorescence signals after a bleaching pulse. Discrimination by FRAP between free diffusion with an immobile fraction (FDIM) and the phenomenological model for anomalous diffusion based on the time-dependent diffusion coefficient (TDDC) is a challenging problem, requiring extremely long observation times for differentiation. Recently, rectangular FRAP (rFRAP) has been introduced for normal diffusion by considering not only the temporal but also spatial information, taking the effective point spread function of the optical system into account. In this work we provide an extension of rFRAP toward anomalous diffusion according to the continuous time random walk (CTRW). We explore whether the spatial information in rFRAP allows for enhanced discrimination between FDIM, TDDC, and CTRW in a single experiment within a feasible time window. Simulations indicate that rFRAP can indeed differentiate the different models by evaluating the spatial autocorrelation of the differences between the measured and fitted pixel values. Hence, rFRAP offers a tool that is capable of discriminating different types of diffusion at shorter time scales than in the case where spatial information is discarded.

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.

Second harmonic generation microscopy has recently become an important tool for studying materials. In this article, we use a recently developed analytical method, for second-harmonic generation microscopy, to determine the point group symmetry of micro crystals of enantiomerically pure 1,1’-bi-2-naphtol. ispartof: pages:87701- ispartof: 17TH INTERNATIONAL SCHOOL ON QUANTUM ELECTRONICS: LASER PHYSICS AND APPLICATIONS vol:8770 pages:87701- ispartof: 17th International School on Quantum Electronics - Laser Physics and Applications (ISQE) location:Nessebar: BULGARIA date:24 Sep - 28 Sep 2012 status: published

Aim: To develop a robust assay to evaluate and compare the intravitreal mobility of nanoparticles in the intact vitreous body. Materials & methods: Excised bovine eyes were prepared to preserve the fragile structure of the vitreous humor, while permitting high-resolution fluorescence microscopy and single-particle tracking analysis of intravitreally injected nanoparticles. This assay was validated by analyzing polystyrene beads and further employed to evaluate gene nanomedicines composed of poly(amido amine)s and plasmid DNA. Results: The assay was able to distinguish immobilized cationic nanoparticles from mobile PEGylated nanoparticles. PEGylation of the polyplexes resulted in a drastic improvement of their mobility. Conclusion: An ex vivo eye model is presented for studying nanoparticle mobility in intact vitreous humor by single-particle tracking microscopy. These results give important guidelines for developing gene- and drug-delivery nanomedicines that are compatible with intravitreal administration. Original submitted 20 April 2012; Revised submitted 22 November 2012

International audience; Microglia are the immune cells of the central nervous system. They are suspected to play important roles in adult synaptogenesis and in the development of the neuronal network. Microglial cells originate from progenitors in the yolk sac. Although it was suggested that they invade the cortex at early developmental stages in the embryo, their invasion pattern remains largely unknown. To address this issue we analyzed the pattern of cortical invasion by microglial cells in mouse embryos at the onset of neuronal cell migration using in vivo immunohistochemistry and ex vivo time-lapse analysis of microglial cells. Microglial cells begin to invade the cortex at 11.5 days of embryonic age (E11.5). They first accumulate at the pial surface and within the lateral ventricles, after which they spread throughout the cortical wall, avoiding the cortical plate region in later embryonic ages. The invasion of the cortical parenchyma occurs in different phases. First, there is a gradual increase of microglial cells between E10.5 and E14.5. From E14.5 to E15.5 there is a rapid phase with a massive increase in microglia, followed by a slow phase again from E15.5 until E17.5. At early stages, many peripheral microglia are actively proliferating before entering the parenchyma. Remarkably, activated microglia accumulate in the choroid plexus primordium, where they are in the proximity of dying cells. Time-lapse analysis shows that embryonic microglia are highly dynamic cells.

20 pags, 2 tabs, After recalling the basic relations relevant to both steady-state and time-resolved fluorescence polarization, it is shown how the values of steady-state polarized intensities recorded experimentally usually need to be corrected for systematic effects and errors, caused by instrumentation and sample properties. A list of selected reference values of steady-state fluorescence anisotropy and polarization is given. Attention is also paid to analysis of time-resolved fluorescence anisotropy data obtained by pulse fluorometry or phase and modulation fluorometry techniques. Recommendations for checking the accuracy of measure ments are provided together with a list of selected time-resolved fluorescence anisotropy data as reported in the literature. © 2013 IUPAC., Support by the Spanish Ministry of Science and Innovation 2010CTQ16457 (AUA) and the IAP P6/27 (BELSPO) Functional Supramolecular Systems (MvdV) is thankfully acknowledged.

The photophysical properties of 2,9-di- n -pentyl-5,6,11,12-tetraazo-5,6,11,12-tetrahydrocoronene-5,6,11,12-tetracarboxy-bis- n -heptadecylimide (P5–17) in solution are investigated as a function of concentration. In 2-methyltetrahydrofuran, above 2 × 10 −5 M, a new absorption band with a maximum at 688 nm is observed indicating the formation of an aggregate. Upon excitation in this absorption band, emission from the aggregate with a maximum at 710 nm is observed. This emission coincides with that of the liquid crystalline discotic phase. The rotational dynamics of P5–17 and its aggregate are investigated using steady-state fluorescence measurements and time-resolved fluorescence anisotropy. From the global analysis of the polarised fluorescence decay traces, it is found that for both monomer and aggregate, the anisotropy decay can be described by a monoexponential decay law. The rotational correlation time for the monomer is linked over two excitation wavelengths corresponding to S 0 → S 1 and S 0 → S n transitions. For the aggregate, the excitation and emission dipoles are perpendicular and a fast fluorescence depolarization was observed.

A previous study of the performance of the global, i.e., simultaneous, analysis of unmatched polarized decay traces [Crutzen, M.; et al. J. Phys. Chem. 1993, 97, 8133] is extended for the time-dependent anisotropy r(t), given by r(t) = β exp(−t/Φ) + r∞. The evaluation was done with computer-generated and experimentally collected data. Two different schemes of analysis of unmatched polarized decay traces were considered. In scheme A the decay curves measured with the analyzer set at 0°, 54.7°, and 90° with respect to the polarization of the incident light were globally analyzed, while only the traces at 0° and 90° were taken into account in scheme B. Data were simulated for various Φ/τ ratios (0.1, 1, 3, and 10). The value obtained for the fluorescence lifetime τ was accurate and precise for all cases considered. For Φ/τ = 0.1, 1, and 3 (τ = 5 ns) the parameters β and Φ were well recovered. The accuracy and precision were very similar to the results obtained with matched decay traces. The recovery of r∞ wa...

The fluorescence anisotropy of two neutral and nonpolar molecules, 2,5,8,11-tetra-tert-butylperylene (TP) and 2,5,10,13-tetra-tert-butylterrylene (TT), is studied in a series of n-alcohols and alkanes. Both probes show an absorption band in the UV region and one in the visible region. The polarized fluorescence decay traces are globally analyzed over different excitation wavelengths. TP and TT can be modeled as oblate ellipsoids with essentially identical rotational correlation times. The anisotropy decay can well be approximated by a monoexponential model for both solvent series. The rotational correlation times (φ) vary linearly with viscosity (η) for both solvent series. Higher values for φ/η are reached in alkanes than in alcohols, the effect being more pronounced for TP than for TT. The predictions of the quasihydrodynamic models of Gierer−Wirtz and of Dote−Kivelson−Schwartz (DKS) are compared to the experimental data. Only the DKS model can predict qualitatively the experimental observations. The di...

The photophysics of the complex forming reaction of Ca2+ and Fura-2 are investigated using steady-state and time-resolved fluorescence measurements. The fluorescence decay traces were analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution with BAPTA as Ca2+ buffer: k01 = 1.2 x 10(9)s-1, k21 = 1.0 x 10(11) M-1 s-1, k02 = 5.5 x 10(8) s-1, k12 = 2.2 x 10(7) s-1, and with EGTA as Ca2+ buffer: k01 = 1.4 x 10(9) s-1, k21 = 5.0 x 10(10) M-1 s-1, k02 = 5.5 x 10(8) s-1, k12 = 3.2 x 10(7) s-1. k01 and k02 denote the respective deactivation rate constants of the Ca2+ free and bound forms of Fura-2 in the excited state. k21 represents the second-order rate constant of binding of Ca2+ and Fura-2 in the excited state, whereas k12 is the first-order rate constant of dissociation of the excited Ca2+:Fura-2 complex. The ionic strength of the solution was shown not to influence the recovered values of the rate constants. From the estimated values of k12 and k21, the dissociation constant K*d in the excited state was calculated. It was found that in EGTA Ca2+ buffer pK*d (3.2) is smaller than pKd (6.9) and that there is negligible interference of the excited-state reaction with the determination of Kd and [Ca2+] from fluorimetric titration curves. Hence, Fura-2 can be safely used as an Ca2+ indicator. From the obtained fluorescence decay parameters and the steady-state excitation spectra, the species-associated excitation spectra of the Ca2+ free and bound forms of Fura-2 were calculated at intermediate Ca2+ concentrations.