Silicon nanoparticles with strong blue photoluminescence were synthesized by electrochemical etching of silicon wafers and ultrasonically removed under N2 atmosphere in organic solvents to produce colloids. Thermal treatment leads to the formation of colloidal Si particles of 3 ± 1 nm diameter, which upon excitation with 340 – 380 nm light exhibited room temperature luminescence in the range from 400 to 500 nm. The emission and the one- and two-photon excitation spectra of the particles are not sensitive to surface functionalization with methyl 2-methylprop-2-enoate. However, the derivatized particles show higher emission quantum yields in air-saturated suspensions (44%) than the underivatized particles (27%), as well as higher stability of its dispersions. FTIR and XPS spectra indicate a significant surface oxidation of the particles. The Si:O:C ratio at the surface of the derivatized particles estimated from XPS is Si3O6(C5O2Hy)1, with y = 7 - 8. Vibronic spacing is observed in both the emission and excitation spectra. The information obtained from one-photon excitation experiments (emission and excitation spectra, photoluminescence quantum yields, luminescence decay lifetimes and anisotropy correlation lifetimes), as well as from two-photon excitation fluorescence correlation spectroscopy (brightness and diffusion coefficients) and TEM indicate that the blue-emitting particles are monodisperse and ball-shaped. Particle size clearly determines the emission and excitation spectral region, as expected from quantum confinement, but the presence and extent of Si-O species on the silicon networks seem crucial for determining the spectrum features and intensity of emission. The nanoparticles could hold great potential as quantum dots for applications as luminescence sensors in biology and environmental science.

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Neurite outgrowth is a critical event in neuronal development, formation, and remodeling of synapses, response to injury, and regeneration. We examined the effects of 2,4-dinitrophenol (DNP), a recently described blocker of the aggregation and neurotoxicity of the beta-amyloid peptide, on neurite elongation of central neurons. Morphometric analysis of rat embryo hippocampal and cortical neuronal cultures showed that neurite outgrowth was stimulated by DNP. This effect was accompanied by increases in the neuronal levels of the microtubule-associated protein tau and of cyclic adenosine 3',5' monophosphate (cAMP). DNP also promoted cAMP accumulation, increased tau level, neurite outgrowth, and neuronal differentiation in the mouse neuroblastoma cell line N2A. We show that DNP-induced differentiation requires activation of the extracellular signal-regulated kinase (ERK). The finding that DNP promotes neuritogenesis and neuronal differentiation suggests that, in addition to its anti-amyloidogenic actions, it may be a useful lead compound in the development of novel therapeutic approaches targeting neurite dystrophy and synaptic dysfunction in neurodegenerative pathologies such as Alzheimer's disease.

Photon counting detectors currently used in fluorescence lifetime microscopy have a number of deficiencies that result in less-than-ideal signal-to-noise ratio of the lifetimes obtained: either the quantum efficiency is unsatisfactory or the active area is too small, and afterpulsing or tails in the temporal response contribute to overall timing inaccuracy. We have therefore developed a new FLIM detector based on a GaAsP hybrid photomultiplier. Compared with conventional PMTs and SPADs, GaAsP hybrid detectors have a number of advantages: The detection quantum efficiency reaches or surpasses the efficiency of fast SPADs, and the active area is on the order of 5 mm², compared with 2.5 10⁻³ mm² for a SPAD. The TCSPC response is clean, without the bumps and the diffusion tails typical for PMTs and SPADs. Most important, the hybrid detector is intrinsically free of afterpulsing. FLIM results are therefore free of signal-dependent background, and FCS curves are free of the known afterpulsing peak. We demonstrate the performance of the new detector for multiphoton NDD FLIM and for FCS.

As recently demonstrated by our group (da-Silva, W. S., Gómez-Puyou, A., Gómez-Puyou, M. T., Moreno-Sanchez, R., De Felice, F. G., de Meis, L., Oliveira, M. F., and Galina, A. (2004) J. Biol. Chem. 279, 39846 -39855) mitochondrial hexokinase activity (mt-HK) plays a preventive antioxidant role because of steady-state ADP re-cycling through the inner mitochondrial membrane in rat brain. In the present work we show that ADP re-cycling accomplished by the mitochondrial creatine kinase (mt-CK) regulates reactive oxygen species (ROS) generation, particularly in high glucose concentrations. Activation of mt-CK by creatine (Cr) and ATP or ADP, induced a state 3-like respiration in isolated brain mitochondria and prevention of H 2 O 2 production obeyed the steady-state kinetics of the enzyme to phosphorylate Cr. The extension of the preventive antioxidant role of mt-CK depended on the phosphocreatine (PCr)/Cr ratio. Rat liver mitochondria, which lack mt-CK activity, only reduced state 4-induced H 2 O 2 generation when 1 order of magnitude more exogenous CK activity was added to the medium. Simulation of hyperglycemic conditions, by the inclusion of glucose 6-phosphate in mitochondria performing 2-deoxyglucose phosphorylation via mt-HK, induced H 2 O 2 production in a Crsensitive manner. Simulation of hyperglycemia in embryonic rat brain cortical neurons increased both ⌬⌿ m and ROS production and both parameters were decreased by the previous inclusion of Cr. Taken together, the results presented here indicate that mitochondrial kinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.Mitochondrial electron transport chain is the major and continuous source of cellular reactive oxygen species (ROS), 5 which are involved in several conditions, such as apoptosis, ischemia-reperfusion injury, neurodegenerative diseases, and toxicity induced by hyperglycemia (1-5). Electron leakage at the complexes I (6, 7) and III (6, 8 -10) are the main sites for the monoelectronic reduction of oxygen, which results in superoxide (O 2 . ) radical production in the respiratory chain. The rate of mitochondrial ROS production is highly dependent on the mitochondrial membrane potential (⌬⌿ m ) (9, 11) and evidence supporting these observations have long demonstrated (9) that pharmacological uncoupling of oxidative phosphorylation caused a drastic reduction in mitochondrial H 2 O 2 formation. Similarly, activation of oxidative phosphorylation by ADP can also reduce the ⌬ m and ROS formation through activation of F 1 F 0 -ATP synthase complex by using the energy of the ⌬⌿ m to drive ATP synthesis (9, 11). On the other hand, when mitochondrial ADP levels drop, the respiratory rate is reduced, increasing the ⌬⌿ m levels, which ultimately leads to ROS generation. There is a clear link between the increased levels of oxidative stress markers and several neuropathies such as amyotrophic lateral sclerosis, Parkinson and Alzheimer disease and h...

SummaryFluorescence lifetime-resolved images of chlorophyll fluorescence were acquired at the maximum P-level and during the slower transient (up to 250 s, including P-S-M-T) in the green photosynthetic alga Chlamydomonas reinhardtii. At the P-level, wild type and the violaxanthin-accumulating mutant npq1 show similar fluorescence intensity and fluorescence lifetime-resolved images. The zeaxanthinaccumulating mutant npq2 displays reduced fluorescence intensity at the P-level (about 25-35% less) and corresponding lifetime-resolved frequency domain phase and modulation values compared to wild type/npq1. A two-component analysis of possible lifetime compositions shows that the reduction of the fluorescence intensity can be interpreted as an increase in the fraction of a short lifetime component. This supports the important photoprotection function of zeaxanthin in photosynthetic samples, and is consistent with the notion of a 'dimmer switch'. Similar, but quantitatively different, behaviour was observed in the intensity and Correspondence to: Robert Clegg. Tel: 1-217-244-8143; fax: 1-217244-7187; e-mail: rclegg@uiuc.edu fluorescence lifetime-resolved imaging measurements for cells that were treated with the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, the efficient PSI electron acceptor methyl viologen and the protonophore nigericin and. Lower fluorescence intensities and lifetimes were observed for all npq2 mutant samples at the P-level and during the slow fluorescence transient, compared to wild type and the npq1 mutant.The fluorescence lifetime-resolved measurements during the slow fluorescence changes after the P level up to 250 s for the wild type and the two mutants, in the presence and absence of the above inhibitors, were analyzed with a graphical procedure (polar plots) to determine lifetime compositions. At higher illumination intensity, wild type and npq1 cells show a rise in fluorescence intensity and corresponding rise in the species concentration of the slow lifetime component after the initial decrease following the P level. This reversal is absent in the npq2 mutant, and for all samples in the presence of the inhibitors. Lifetime heterogeneities were observed in experiments averaged over multiple cells as well as within single cells, and these were followed over time. Cells in the resting state (induced by several hours of darkness), instead of the normal swimming state, show shortened lifetimes. The aboveresults are discussed in terms of a superposition of effects on electron transfer and protonation rates, on the so-called 'State Transitions', and on non-photochemical quenching. Our data indicate two major populations of chlorophyll a molecules, defined by two 'lifetime pools' centred on slower and faster fluorescence lifetimes.

We present a lifetime imaging technique that simultaneously records fluorescence and phosphorescence lifetime images in laser scanning systems. It is based on modulating a high-frequency pulsed laser by a signal synchronous with the pixel clock of the scanner, and recording the fluorescence and phosphorescence signals by multi-dimensional TCSPC. Fluorescence is recorded during the on-phase of the laser, phosphorescence during the off-phase. The technique does not require a reduction of the laser pulse repetition rate by a pulse picker, and eliminates the need of using excessively high pulse power for phosphorescence excitation. Laser modulation is achieved either by electrically modulating picosecond diode lasers, or be controlling the lasers via the AOM of a standard confocal or multiphoton laser scanning microscope. Downloaded From: http://proceedings.spiedigitallibrary.org/ on 05/19/2015 Terms of Use: http://spiedl.org/terms Proc. of SPIE Vol. 7903 790320-5 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 05/19/2015 Terms of Use: http://spiedl.org/terms Proc. of SPIE Vol. 7903 790320-6 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 05/19/2015 Terms of Use: http://spiedl.org/terms