Fluorescence recovery after photobleaching on the confocal laser-scanning microscope: generalized model without restriction on the size of the photobleached disk

Fluorescence recovery after photobleaching (FRAP) carried out on a confocal laser-scanning microscope (CLSM) performs well for photobleached disks that are large compared to the resolution of the bleaching beam. For smaller disks approaching this resolution, current FRAP models providing a closed-form solution do not allow one to extract the diffusion coefficient accurately. The new generalized disk model we present addresses this shortcoming by bringing into account the bleaching resolution and the total confocal imaging resolution. A closed-form solution is obtained under the assumption of linear photobleaching. Furthermore, simultaneous analysis of FRAP data collected at various disk sizes allows for the intrinsic determination of the instrumental resolution parameters, thereby obviating the need for an extrinsic calibration. A new method to estimate the variance of FRAP data is introduced to allow for proper weighting in this global analysis approach by nonlinear least squares. Experiments are performed on two independent CLSMs on homogeneous samples providing validation over a large range of diffusion coefficients. (C) 2011 Society of Photo-Optical Instrumentation Engineers (SPIE). [DOI: 10.1117/1.3569620

The peroxidation of leukocytes index ratio reveals the prooxidant effect of green tea extract

Despite tea increased plasma nonenzymatic antioxidant capacity, the European Food Safety Administration (EFSA) denied claims related to tea and its protection from oxidative damage. Furthermore, the Supplement Information Expert Committee (DSI EC) expressed some doubts on the safety of green tea extract (GTE). We performed a pilot study in order to evaluate the effect of a single dose of two capsules of a GTE supplement (200mg x 2) on the peroxidation of leukocytes index ratio (PLIR) in relation to uric acid (UA) and ferric reducing antioxidant potential (FRAP), as well as the sample size to reach statistical significance. GTE induced a prooxidant effect on leukocytes, whereas FRAP did not change, in agreement with the EFSA and the DSI EC conclusions. Besides, our results confirm the primary role of UA in the antioxidant defences. The ratio based calculation of the PLIR reduced the sample size to reach statistical significance, compared to the resistance to an exogenous oxidative stress and to the functional capacity of oxidative burst. Therefore, PLIR could be a sensitive marker of redox status

Differences in protein mobility between pioneer versus follower growth cones

Navigating growth cones need to integrate, process and respond to guidance signals, requiring dynamic information transfer within and between different compartments. Studies have shown that, faced with different navigation challenges, growth cones display dynamic changes in growth kinetics and morphologies. However, it remains unknown whether these are paralleled by differences in their internal molecular dynamics. To examine whether there are protein mobility differences during guidance, we developed multiphoton fluorescence recovery after photobleaching methods to determine molecular diffusion rates in pathfinding growth cones in vivo. Actively navigating growth cones (leaders) have consistently longer recovery times than growth cones that are fasciculated and less actively navigating (followers). Pharmacological perturbations of the cytoskeleton point to actin as the primary modulator of diffusion in differently behaving growth cones. This approach provides a powerful means to quantify mobility of specific proteins in neurons in vivo and reveals that diffusion is important during axon navigation

A FRAP model to investigate reaction-diffusion of proteins within a bounded domain: a theoretical approach

Temporally and spatially resolved measurements of protein transport inside cells provide important clues to the functional architecture and dynamics of biological systems. Fluorescence Recovery After Photobleaching (FRAP) technique has been used over the past three decades to measure the mobility of macromolecules and protein transport and interaction with immobile structures inside the cell nucleus. A theoretical model is presented that aims to describe protein transport inside the nucleus, a process which is influenced by the presence of a boundary (i.e. membrane). A set of reaction-diffusion equations is employed to model both the diffusion of proteins and their interaction with immobile binding sites. The proposed model has been designed to be applied to biological samples with a Confocal Laser Scanning Microscope (CLSM) equipped with the feature to bleach regions characterised by a scanning beam that has a radially Gaussian distributed profile. The proposed model leads to FRAP curves that depend on the on- and off-rates. Semi-analytical expressions are used to define the boundaries of on- (off-) rate parameter space in simplified cases when molecules move within a bounded domain. The theoretical model can be used in conjunction to experimental data acquired by CLSM to investigate the biophysical properties of proteins in living cells.Comment: 25 pages. Abstracts Proceedings, The American Society for Cell Biology, 46th Annual Meeting, December 9-13, 2006, San Dieg

Advanced Fluorescence Microscopy Techniques-FRAP, FLIP, FLAP, FRET and FLIM

Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen. The techniques described here are fluorescence recovery after photobleaching (FRAP), the related fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), Forster or fluorescence resonance energy transfer (FRET) and the different ways how to measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and fluorescence lifetime imaging microscopy (FLIM). First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and fluorescence, confocal, and multiphoton microscopy is given. Subsequently, these advanced microscopy techniques are introduced in more detail, with a description of how these techniques are performed, what needs to be considered, and what practical advantages they can bring to cell biological research

Effect of native gastric mucus on in vivo hybridization therapies directed at Helicobacter pylori

Helicobacter pylori infects more than 50% of the worldwide population. It is mostly found deep in the gastric mucus lining of the stomach, being a major cause of peptic ulcers and gastric adenocarcinoma. To face the increasing resistance of H. pylori to antibiotics, antimicrobial nucleic acid mimics are a promising alternative. In particular, locked nucleic acids (LNA)/2'-OMethyl RNA (2'OMe) have shown to specifically target H. pylori, as evidenced by in situ hybridization. The success of in vivo hybridization depends on the ability of these nucleic acids to penetrate the major physical barriers-the highly viscoelastic gastric mucus and the bacterial cell envelope. We found that LNA/2'OMe is capable of diffusing rapidly through native, undiluted, gastric mucus isolated from porcine stomachs, without degradation. Moreover, although LNA/2'OMe hybridization was still successful without permeabilization and fixation of the bacteria, which is normally part of in vitro studies, the ability of LNA/2'OMe to efficiently hybridize with H. pylori was hampered by the presence of mucus. Future research should focus on developing nanocarriers that shield LNA/2'OMe from components in the gastric mucus, while remaining capable of diffusing through the mucus and delivering these nucleic acid mimics directly into the bacteria

Efecto de las condiciones de almacenamiento refrigerado sobre el estrés oxidativo en raquis de uva de mesa, cv. Red Globe

Indexación: Web of Science; ScieloTable grape (Vitis vinifera L.) quality includes the condition of both the berries and the rachis. In the present report, physiological parameters of Red Globe rachises from fully elongated inflorescences (RFEI) and from mature clusters were studied after storage at 0 or 20 °C for different durations. To understand changes in rachis physiology as a result of changes in temperature conditions and storage time, the activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) were measured. In addition, hydrogen peroxide content, membrane lipoperoxidation (TBARS), total phenolic compounds and antioxidant capacity (FRAP) were assayed. TBARS was higher in mature rachises than in RFEI. This parameter remained constant throughout storage, indicating a change presumably associated with ontogeny or senescence processes. Short-term storage (096 h) increased SOD, CAT and APX activities in RFEI, while in mature rachises, no changes were observed in enzyme activities or in hydrogen peroxide content. Longer cold storage (25 or 53 days at 0 °C) of mature rachises reduced CAT activity, but SOD and APX activities did not change under these conditions. At 0 h, the FRAP and total phenolic contents of mature rachises were three and 20 times higher than in immature rachises, respectively.La calidad de uva de mesa (Vitis vinifera L.) involucra tanto la condición de bayas y el raquis. En el presente trabajo, los parámetros fisiológicos de raquis de 'Red Globe' de inflorescencias completamente elongadas (RFEI) y de racimos maduros fueron estudiados después de almacenamientos por diferentes tiempos a 0 ó 20 °C. Para entender los cambios en la fisiología del raquis debido a variaciones de temperatura y condiciones de almacenamiento, se midieron las actividades de superoxido dismutasa (SOD), catalasa (CAT), ascorbato peroxidasa (APX). Además fueron analizados el contenido de peróxido de hidrógeno, lipoperoxidación de membranas (TBARS), compuestos fenólicos totales y capacidad antioxidante (FRAP). TBARS de los raquis maduros a la cosecha fue mayor que el de inflorescencias completamente elongadas (RFEI). Este parámetro permaneció constante a través del almacenamiento, indicando cambios presuntamente asociados a ontogenia o procesos de senescencia. Almacenamiento cortos (0-96h) incrementaron la actividad de SOD, CAT y APX en RFEI, sin embargo no se observaron cambios en la actividad de estas enzimas o contenido de peróxido de hidrogeno en raquis maduros. Almacenamiento refrigerado prolongado (25 ó 30 días 0 °C) de raquis maduros redujo la actividad de CAT, pero SOD y APX no mostraron cambios bajo estas condiciones. A 0 h el contenido de FRAP y fenólicos totales de raquis maduros fueron tres y 20 veces mayores que en raquis inmaduro respectivamente.http://ref.scielo.org/m3mg4