Quantitative real-time imaging of intracellular FRET biosensor dynamics using rapid multi-beam confocal FLIM

Fluorescence lifetime imaging (FLIM) is a quantitative, intensity-independent microscopical method for measurement of diverse biochemical and physical properties in cell biology. It is a highly effective method for measurements of Förster resonance energy transfer (FRET), and for quantification of protein-protein interactions in cells. Time-domain FLIM-FRET measurements of these dynamic interactions are particularly challenging, since the technique requires excellent photon statistics to derive experimental parameters from the complex decay kinetics often observed from fluorophores in living cells. Here we present a new time-domain multi-confocal FLIM instrument with an array of 64 visible beamlets to achieve parallelised excitation and detection with average excitation powers of ~ 1–2 μW per beamlet. We exemplify this instrument with up to 0.5 frames per second time-lapse FLIM measurements of cAMP levels using an Epac-based fluorescent biosensor in live HeLa cells with nanometer spatial and picosecond temporal resolution. We demonstrate the use of time-dependent phasor plots to determine parameterisation for multi-exponential decay fitting to monitor the fractional contribution of the activated conformation of the biosensor. Our parallelised confocal approach avoids having to compromise on speed, noise, accuracy in lifetime measurements and provides powerful means to quantify biochemical dynamics in living cells

Pt nanoparticles on tin oxide based support as a beneficial catalyst for oxygen reduction in alkaline solutions

A platinum nanocatalyst on Sb doped tin oxide support (Sb-SnO2) was synthesized and characterized as a catalyst for oxygen reduction reaction in 0.1 mol dm(-3) NaOH solution at 25 degrees C. Sb (5%) doped tin oxide support was synthesized by a modified hydrazine reduction procedure. The platinum nanocatalyst (20% Pt) on Sb-SnO2 support was synthesized by a borohydride reduction method. The synthesized support and catalyst were characterized by high resolution transmission electron microscopy (HRTEM) and X-ray photoelectron spectroscopy (XPS) and X-ray diffraction technique (XRD). X-ray photoelectron spectroscopy was applied to characterize the chemical status of elements before and after Pt-treatment. XPS spectra of Sn 3d, Pt 4f, Sb 3d and O 1s revealed that the Pt-deposition on Sb-SnO2 support induced the reduction of the Sn(4+) oxidation state to Sn(2+) and Sn(0) states, while Pt remained in the metallic state and Sb was in the (3+) oxidation state. Homogenous Pt nanoparticle distribution over the support, without pronounced particle agglomeration, was confirmed by HRTEM technique. The average Pt particle size was 2.9 nm. The electrochemically active Pt surface area of the catalyst was determined by the integration of the cyclic voltammetry curve in the potential region of underpotential deposition of hydrogen, after double layer charge correction, taking into account the reference value of 210 mu C cm(-2) for full monolayer coverage. This calculation gave the value of 51 m(2) g(-1). The kinetics of the oxygen reduction reaction with Pt/[Sb-SnO2 catalyst was studied by cyclic voltammetry and linear sweep voltammetry using a rotating gold disc electrode. Two different Tafel slopes were observed: one close to 60 mV dec(-1) in the low current density region, and another at similar to 120 mV dec(-1) in the higher current densities region, as was already referred in previous reports for the oxygen reduction reaction with polycrystalline Pt, as well as with different Pt based nanocatalysts. The specific activities for oxygen reduction, expressed in terms of kinetic current densities per electrochemically Pt active surface area, as well as per mass of Pt loaded, at the constant potential of practical interest (0.85 V and 0.90 V vs. RHE), were compared to a carbon supported (Vulcan XC-72) catalyst. The Pt/[Sb-SnO2 catalyst exhibited similar catalytic activity for oxygen reduction reaction like carbon supported one. The advantages of the carbon free support application in terms of the durability and stability of the catalysts were proved by accelerated stability tests

Electrodeposition and characterization of Fe–Mo alloys as cathodes for hydrogen evolution in the process of chlorate

Fe–Mo alloys were electrodeposited from a pyrophosphate bath using a single diode rectified AC current. Their composition and morphology were investigated by SEM, optical microscopy and EDS, in order to determine the influence of the deposition conditions on the morphology and composition of these alloys. It was shown that the electrodeposition parameters, such as: chemical bath composition and current density, influenced both the composition of the Fe–Mo alloys and the current efficiency for their deposition, while the micro and macro-morphology did not change significantly with changing conditions of alloy electrodeposition. It was found that the electrodeposited Fe–Mo alloys possessed a 0.15 V to 0.30 V lower overvoltage than mild steel for hydrogen evolution in an electrolyte commonly used in commercial chlorate production, depending on the alloy composition, i.e., the conditions of alloy electrodeposition

Synthesis, characterization and electrocatalytical behavior of Nb-TiO2/Pt nanocatalyst for oxygen reduction reaction

In order to point out the effect of the support to the catalyst for oxygen reduction reaction nano-crystalline Nb-doped TiO2 was synthesized through a modified sol-gel route procedure. The specific surface area of the support, S-BET, and pore size distribution, were calculated from the adsorption isotherms using the gravimetric McBain method. The support was characterized by X-ray diffraction (XRD) technique. The borohydride reduction method was used to prepare Nb-TiO2 supported Pt (20 wt.%) catalyst. The synthesized catalyst was analyzed by TEM technique. Finally, the catalytic activity of this new catalyst for oxygen reduction reaction was investigated in acid solution, in the absence and the presence of methanol, and its activity was compared towards the results on C/Pt catalysts. Kinetic analysis reveals that the oxygen reduction reaction on Nb-TiO2/Pt catalyst follows four-electron process leading to water, as in the case of C/Pt electrode, but the Tafel plots normalized to the electrochemically active surface area show very remarkable enhancement in activity of Nb-TiO2/Pt expressed through the value of the current density at the constant potential. Moreover, Nb-TiO2/Pt catalyst exhibits higher methanol tolerance during the oxygen reduction reaction than the C/Pt catalyst. The enhancement in the activity of Nb-TiO2/Pt is consequence of both: the interactions of Pt nanoparticles with the support and the energy shift of the surface d-states with respect to the Fermi level what changes the surface reactivity. (C) 2010 Elsevier B.V. All rights reserved

Specificity of the UPD of H to the structure of highly dispersed Pt on carbon support

Home-made carbon cryogel synthesized by sol-gel polycondensation and freeze-drying is used as support for preparation of highly dispersed Pt catalyst that is made by a modified polyol synthesis method in an ethylene glycol (EG) solution. Specific surface area of carbon support and Pt/C catalyst is determined from nitrogen adsorption isotherm. The adsorption isotherm demonstrates a significant mesoporosity of carbon support. Specific surface area of the carbon support, calculated by the BET equation, is found to be 573 m(2) g(-1). X-ray diffraction (XRD) results demonstrate a successful reduction of the Pt precursor to its metallic form, and transmission electron microscopy (TEM) images show very uniform Pt particle size distribution with mean particle size of about 2.7 +/- 0.7 nm of the catalyst. Potentiodynamic studies of the underpotential deposition of hydrogen (H-upd) on Pt/C electrode in 0.5 mol dm(-3) HClO4 aqueous solution in the temperature range from 274 to 318 K are made, and thermodynamic state functions for the hydrogen adsorption are determined. The experimental results are analyzed assuming linear variation of the Gibbs energy of adsorption versus theta H-upd on the basis of the surface heterogeneity. The increase of Delta G(Hupd)(theta) with the surface coverage indicates the repulsive interactions between H-upd adatoms. From the temperature dependence of the Gibbs energy of adsorption, the enthalpy and the entropy of adsorption are calculated. The values of these functions are determined to be Delta H-Hupd(theta=0) = -5.6 kJ mol(-1) and Delta S-Hupd(theta=0) = 69.1 J mol(-1) K-1 The value of Delta H-Hupd(theta) allows determinations of the bond energy between electrode surface and H-upd that is found to be Ept-H = 223 kJ mol(-1) for theta = 0. (c) 2006 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved

Electrooxidation of hydrogen on nanostructured Pt/C catalysts for polymer electrolyte fuel cells

Mesoporous carbon cryogel synthesized by sol-gel polycondensation and freeze-drying with specific surface area (BET) of 517 m(2) g(-1) was used as a catalyst support. Pt/C catalysts were prepared by a modified ethylene glycol method (EG). Transmission electron microscopy (TEM) images show that the dispersion of the catalyst is very uniform with a mean particle size of about 2.65 nm. Hydrogen oxidation reaction (HOR) was studied on Pt/C catalyst in 0.5 mol dm(-3) HClO4 acid solution. It has been found that HOR appears as a reversible two-electron direct discharged reaction (Tafel slope for this reaction is approximate to 30mV dec(-1)) and that Pt/C catalyst exhibits a very high catalytic activity. However, the corresponding value of the exchange current density obtained by dividing the exchange current by the active surface area of Pt particles has the same order of magnitude as those for the HOR in acidic solution at single crystal and polycrystalline Pt.Recent Developments in Advanced Materials and Processes, 7th Conference of the Yugoslav-Materials-Research-Society (Yu-MRS), Sep 12-16, 2005, Herceg Novi, Montenegr

Synthesis and Characterization of Pt Catalysts on SnO2 Based Supports for Oxygen Reduction Reaction

The oxygen reduction reaction was studied at Pt nanocatalysts on two different tin oxide based supports, Sb-SnO2 and Ru-SnO2, in acid solution. Tin oxide based supports were synthesized by hydrazine reduction method. Physical characterization of the supports was performed by BET, X-ray diffraction and TEM techniques. SnO2 belonging peaks were detected in Sb-SnO2 powder, while Ru-SnO2 XRD diffraction patterns contained peaks of RuO2 and SnO2. The average crystallite sizes, determined by Scherrer equation, were 3 nm and 4 nm for Sb-SnO2 and Ru-SnO2, respectively. Pt catalysts on Sb-SnO2 and Ru-SnO2 supports were synthesized by borohydride reduction method. TEM analysis revealed homogeneous particle size distribution, with average particle size of 2.9 and 5.4 nm, for Sb-SnO2 and Ru-SnO2, respectively. Electrocatalytic activity and stability of these catalysts for oxygen reduction were studied by cyclic voltammetry and linear sweep voltammetry at rotating disk electrode (RDE). Pt catalysts on Sb and Ru doped SnO2 support exhibited catalytic activities comparable to Pt on commercial carbon based support. Stability tests were also performed. Determined small loss of electrochemical active surface area of the Pt catalyst on Sb doped tin oxide support, after repetitive cycling, indicated high stability and durability of this cathode for-prospective fuel cells application. (C) 2013 The Electrochemical Society. All rights reserved

Novel Pt catalyst on ruthenium doped TiO2 support for oxygen reduction reaction

Ruthenium doped titanium oxide support was synthesized. The support was characterized by BET (Brunauer, Emmett, Teller) and X-ray diffraction techniques (XRD). Determined specific surface area was 41 m(2) g(-1). XRD revealed presence mainly TiO2 anatase phase and some peaks belonging to rutile phase. No Ru compounds have been detected. Platinum based catalyst on this support was prepared by borohydride reduction method. The catalyst was characterized by scanning transmission electron microscopy (STEM, HAADF) and electron energy loss spectroscopy (EELS). Homogenous Pt particle distribution over the support, with average Pt nanoparticle diameter of 3 nm was found. This novel catalyst was tested for oxygen reduction in acid solution. It exhibited remarkable higher catalytic activity in comparison with Pt/C, as well as with Pt nanocatalysts at titanium oxide based supports, reported in literature. (C) 2013 Elsevier B.V. All rights reserved

Characterization of carbon cryogel synthesized by sol-gel polycondensation and freeze-drying

Resorcinol-formaldehyde (RF) cryogels were synthesized by sol-gel polycondensation of resorcinol with formaldehyde and freeze-drying was carried out with t-butanol. Carbon cryogels were obtained by pyrolyzing RF cryogels in an inert atmosphere. Characterization by nitrogen adsorption showed that carbon cryogels were micro and mesoporous materials with high surface areas (500 m(2)/g LT S-BET LT 750 m(2)/g). Cyclic voltammetry experiments at various scan rates (2-200 mV s(-1)) have been performed to study the electrical double-layer charging of carbon cryogel electrodes in 0.5 mol dm(-3) HClO4 solution. It has been demonstrated that it is possible to divide further total specific capacitance into mesoporous and microporous specific capacitance by analyzing the linear dependence of the specific capacitance (C) on the reciprocal of the square root of the potential scan rate (v(-1/2)), and linear dependence of the reciprocal specific charge (1/C) on the square root of the potential scan rate (v(1/2)). (C) 2004 Elsevier Ltd. All rights reserved

Preparation and characterization TiOx-Pt/C catalyst for hydrogen oxidation reaction

The hydrogen oxidation reaction (HOR) was studied at the home made TiOx-Pt/C nanocatalysts in 0.5 mol dm(-3) HClO4 at 25 degrees C. Pt/C catalyst was first synthesized by modified ethylene glycol method (EG) on commercially used carbon support (Vulcan XC-72). Then TiOx-Pt/C catalyst was prepared by the polyole method followed by TiOx post-deposition. The synthesized catalyst was characterized by XRD, TEM and EDX techniques. It was found that Pt/C catalyst nanoparticles were homogenously distributed over carbon support with the mean particle size of about 2.4 nm. The quite similar, homogenous distribution and particle size were obtained for Pt/C doped by TiOx catalyst which was the confirmation that TiOx post-deposition did not lead to significant growth of the Pt nanoparticles. The electrochemically active surface area of the catalyst was determined by using the cyclic voltammetry technique. The kinetics of hydrogen oxidation was investigated by the linear sweep voltammetry technique at the rotating disc electrode (RDE). The kinetic equations used for the analysis were derived considering the reversible or irreversible nature of the kinetics of the HOR. It was found that the hydrogen oxidation reaction for an investigated catalyst proceeded as an electrochemically reversible reaction. The values determined for the kinetic parameters-Tafel slope of 28 mV dec(-1) and exchange current density about 0.4 mA cm(Pt)(-2) are in good agreement with usually reported values for a hydrogen oxidation reaction with platinum catalysts in acid solutions