Photophysical aspects of single-molecule detection by two-photon excitation with consideration of sequential pulsed illumination.

An important goal in single molecule fluorescence correlation spectroscopy is the theoretical simulation of the fluorescence signal stemming from individual molecules and its autocorrelation function. The simulation approaches developed up to now are based exclusively on continuous-wave (cw) illumination and consequently on cw-excitation. However, this approximation is no longer valid in the case of two-photon excitation, for which pulsed illumination is usually employed. We present a novel theoretical model for the simulation of the fluorescence signal of single molecules and its autocorrelation function with consideration of the time dependence of the excitation flux and thus of all illumination-dependent photoprocesses: two-photon excitation, induced emission and photobleaching. Further important characteristics of our approach are the consideration of the dependence of the photobleaching rate on illumination and the low intersystem-crossing rates of the studied coumarins. Moreover, using our approach, we can predict quantitatively the effect of the laser pulse width on the fluorescence signal of a molecule, that is, the contributions of the photobleaching and saturation effects, and thus we can calculate the optimal laser pulse width. The theoretical autocorrelation functions were fitted to the experimental data, and we could ascertain a good agreement between the resulting and the expected parameters. The most important parameter is the photobleaching constant sigma, the cross section of the transition Sn<--S1, which characterises the photostability of the molecules independent of the experimental conditions. Its value is 1.7 x 10(-23) cm2 for coumarin 153 and 5 x 10(-23) cm2 for coumarin 314.     

Parameterization of daily solar global ultraviolet irradiation

Daily values of solar global ultraviolet (UV) B and UVA irradiation as well as erythemal irradiation have been parameterized to be estimated from pyranometer measurements of daily global and diffuse irradiation as well as from atmospheric column ozone. Data recorded at the Meteorological Observatory Potsdam (52 degrees N, 107 m asl) in Germany over the time period 1997-2000 have been used to derive sets of regression coefficients. The validation of the method against independent data sets of measured UV irradiation shows that the parameterization provides a gain of information for UVB, UVA and erythemal irradiation referring to their averages. A comparison between parameterized daily UV irradiation and independent values of UV irradiation measured at a mountain station in southern Germany (Meteorological Observatory Hohenpeissenberg at 48 degrees N, 977 m asl) indicates that the parameterization also holds even under completely different climatic conditions. On a long-term average (1953-2000), parameterized annual UV irradiation values are 15% and 21% higher for UVA and UVB, respectively, at Hohenpeissenberg than they are at Potsdam. Daily global and diffuse irradiation measured at 28 weather stations of the Deutscher Wetterdienst German Radiation Network and grid values of column ozone from the EPTOMS satellite experiment served as inputs to calculate the estimates of the spatial distribution of daily and annual values of UV irradiation across Germany. Using daily values of global and diffuse irradiation recorded at Potsdam since 1937 as well as atmospheric column ozone measured since 1964 at the same site, estimates of daily and annual UV irradiation have been derived for this site over the period from 1937 through 2000, which include the effects of changes in cloudiness, in aerosols and, at least for the period of ozone measurements from 1964 to 2000, in atmospheric ozone. It is shown that the extremely low ozone values observed mainly after the eruption of Mt. Pinatubo in 1991 have substantially enhanced UVB irradiation in the first half of the 1990s. According to the measurements and calculations, the nonlinear long-term changes observed between 1968 and 2000 amount to +4%, ..., +5% for annual global irradiation and UVA irradiation mainly because of changing cloudiness and + 14%, ..., +15% for UVB and erythemal irradiation because of both changing cloudiness and decreasing column ozone. At the mountain site, Hohenpeissenberg, measured global irradiation and parameterized UVA irradiation decreased during the same time period by -3%, ..., -4%, probably because of the enhanced occurrence and increasing optical thickness of clouds, whereas UVB and erythemal irradiation derived by the parameterization have increased by +3%, ..., +4% because of the combined effect of clouds and decreasing ozone. The parameterizations described here should be applicable to other regions with similar atmospheric and geographic conditions, whereas for regions with significantly different climatic conditions, such as high mountainous areas and arctic or tropical regions, the representativeness of the regression coefficients would have to be approved. It is emphasized here that parameterizations, as the one described in this article, cannot replace measurements of solar UV radiation, but they can use existing measurements of solar global and diffuse radiation as well as data on atmospheric ozone to provide estimates of UV irradiation in regions and over time periods for which UV measurements are not available.     

Inverted and anti-inverted populations of a doublets of chemically produced OH

With the aid of nanosecond laser flash photolysis OH is chemically produced and observed “collision free”. The rotational states show inverted population in the A doublets J = $\text{3}\text{2}$, $\text{5}\text{2}$, $\text{7}\text{2}$, and $\text{9}\text{2}$ of ²Π_{$\text{3}\text{2}$} and anti-i higher rotational states. The intensities of the main and satellite absorption lines further indicate an inversion in special hyperfine states. Application to interstellar OH maser emission is considered.     

Noniterative biexponential fluorescence lifetime imaging in the investigation of cellular metabolism by means of NAD(P)H autofluorescence.

The cofactors NADH and NADPH, hereafter NAD(P)H [NAD(P)= nicotinamide adenine dinucleotide (phosphate)], belong to the principal endogenous indicators of energetic cellular metabolism. Since the metabolic activity of cells is given by the ratio between the concentrations of free and protein-bound NAD(P)H, the development of autofluorescence techniques which accurately measure the modifications to this ratio is particularly significant. Hitherto the methods applied in the monitoring of cellular metabolism have provided either imprecise results, due to interference of the NAD(P)H signal by perturbing factors, or they have required a complicated internal calibration. We employ biexponential fluorescence lifetime imaging (FLIM) in order to discriminate between the free and protein-bound NAD(P)H without any previous calibration. Thus, we have obtained directly, and for the first time, a high-resolution map of cellular metabolism, that is, an image of the contribution of the protein-bound NAD(P)H to the cumulative NAD(P)H fluorescence signal. Moreover, we demonstrate that protein-NAD(P)H complexes characterised by different fluorescence lifetimes are not uniformly distributed all over the cell, as assumed until now, but are concentrated in certain cellular regions. The different fluorescence lifetimes indicate either different protein-NAD(P)H complexes or different bond strengths between NAD(P)H and the protein in these complexes. Since an important aspect in biological applications is to monitor the dynamics of the relevant processes (such as cellular metabolism), rapid dynamical techniques, for example, rapid biexponential fluorescence lifetime imaging, are needed. Furthermore, it is necessary to reduce the evaluation effort as much as possible. Most of the evaluation techniques in multiexponential FLIM are time-expensive iterative methods. The few exceptions are connected with a loss of information, for example, global analysis; or a loss in accuracy, for example, the rapid evaluation technique (RLD). We implement for the first time in FLIM a noniterative, nonrestrictive method originally developed by Prony for approximations of multiexponential decays. The accuracy of this method is verified in biexponential FLIM experiments in time-domain on mixtures of two chromophores both in homogenous and in heterogeneous media. The resulting fluorescence lifetimes agree (within error margins) with the lifetimes of the pure substances determined in monoexponential FLIM experiments. The rapidity of our evaluation method as compared to iterative pixel-by-pixel methods is evidenced by a reduction of the evaluation time by more than one order of magnitude. Furthermore, the applicability of this method for the biosciences is demonstrated in the investigation of cellular metabolism by means of NAD(P)H endogenous fluorescence.     

Preparation and Characterisation of a Bis-μ-Hydroxo-NiIII2 Complex

Hydroxide-bridged high-valent oxidants have been implicated as the active oxidants in methane monooxygenases and other oxidases that employ bimetallic clusters in their active site. To understand the properties of such species, bis-μ-hydroxo-Ni^II₂ complex ( 1 ) supported by a new dicarboxamidate ligand (N,N′-bis(2,6-dimethyl-phenyl)-2,2-dimethylmalonamide) was prepared. Complex 1 contained a diamond core made up of two Ni^II ions and two bridging hydroxide ligands. Titration of the 1 e⁻ oxidant (NH₄)₂[Ce^IV(NO₃)₆] with 1 at −45 °C showed the formation of the high-valent species 2 and 3 , containing Ni^IINi^III and Ni^III₂ diamond cores, respectively, maintaining the bis-μ-hydroxide core. Both complexes were characterised using electron paramagnetic resonance, X-ray absorption, and electronic absorption spectroscopies. Density functional theory computations supported the spectroscopic assignments. Oxidation reactivity studies showed that bis-μ-hydroxide-Ni^III₂ 3 was capable of oxidizing substrates at −45 °C at rates greater than that of the most reactive bis-μ-oxo-Ni^III complexes reported to date.     

Polyphenolics with Strong Antioxidant Activity from Acacia nilotica Ameliorate Some Biochemical Signs of Arsenic-Induced Neurotoxicity and Oxidative Stress in Mice

Neurotoxicity is a serious health problem of patients chronically exposed to arsenic. There is no specific treatment of this problem. Oxidative stress has been implicated in the pathological process of neurotoxicity. Polyphenolics have proven antioxidant activity, thereby offering protection against oxidative stress. In this study, we have isolated the polyphenolics from Acacia nilotica and investigated its effect against arsenic-induced neurotoxicity and oxidative stress in mice. Acacia nilotica polyphenolics prepared from column chromatography of the crude methanol extract using diaion resin contained a phenolic content of 452.185 ± 7.879 mg gallic acid equivalent/gm of sample and flavonoid content of 200.075 ± 0.755 mg catechin equivalent/gm of sample. The polyphenolics exhibited potent antioxidant activity with respect to free radical scavenging ability, total antioxidant activity and inhibition of lipid peroxidation. Administration of arsenic in mice showed a reduction of acetylcholinesterase activity in the brain which was counteracted by Acacia nilotica polyphenolics. Similarly, elevation of lipid peroxidation and depletion of glutathione in the brain of mice was effectively restored to normal level by Acacia nilotica polyphenolics. Gallic acid methyl ester, catechin and catechin-7-gallate were identified in the polyphenolics as the major active compounds. These results suggest that Acacia nilotica polyphenolics due to its strong antioxidant potential might be effective in the management of arsenic induced neurotoxicity.