CONTRIBUTION OF RESONANCE RAMAN EXCITATION SPECTROSCOPY FOR PROBING ELECTRONICALLY EXCITED-STATES - NATURE OF A PORPHYRIN-DNA EXCIPLEX

The resonance Raman spectra of a water-soluble metalloporphyrin Cu(TMpy-P4), complexed with a synthetic nucleic acid, poly(dA-dT), were measured by using excitation wavelengths located within the B (Soret) transition of the porphyrin (417-470 nm), while its excited state was synchronously pumped with 545-nm pulsed excitation corresponding to the Q transition. In the presence of pump pulses, the aqueous solution of the Cu(TMpy-P4).poly(dA-dT) complex exhibits resonance Raman bands at 1558 and 1353 cm-1 that are not observed in the absence of pump pulses. These new features were previously assigned to electronically excited Cu(TMpy-P4), stabilized by forming an exciplex with the A-T sites of the nucleic acid. Here we present resonance Raman excitation profiles (RREP) of both the excited and ground states of the complex, and we experimentally confirm the very short lifetime of the exciplex. To our knowledge this is the first time that a RREP of a very short lived (ca. 20 ps) intermediate excited state has been obtained with a two-color experiment. We use this to help to characterize the nature of the porphyrin-AT specific complex formed in the porphyrin excited state.

Effect of excitation wavelength on the Raman spectroscopy of the porcine photoreceptor layer from the area centralis.

Raman microscopy, based upon the inelastic scattering (Raman) of light by molecular species, has been applied as a specific structural probe in a wide range of biomedical samples. The purpose of the present investigation was to assess the potential of the technique for spectral characterization of the porcine outer retina derived from the area centralis, which contains the highest proportion of cone:rod cell ratio in the pig retina. METHODS: Retinal cross-sections, immersion-fixed in 4% (w/v) PFA and cryoprotected, were placed on salinized slides and air-dried prior to direct Raman microscopic analysis at three excitation wavelengths, 785 nm, 633 nm, and 514 nm. RESULTS: Raman spectra of each of the photoreceptor inner and outer segments (PIS, POS) and of the outer nuclear layer (ONL) of the retina acquired at 785 nm were dominated by vibrational features characteristic of proteins and lipids. There was a clear difference between the inner and outer domains in the spectroscopic regions, amide I and III, known to be sensitive to protein conformation. The spectra recorded with 633 nm excitation mirrored those observed at 785 nm excitation for the amide I region, but with an additional pattern of bands in the spectra of the PIS region, attributed to cytochrome c. The same features were even more enhanced in spectra recorded with 514 nm excitation. A significant nucleotide contribution was observed in the spectra recorded for the ONL at all three excitation wavelengths. A Raman map was constructed of the major spectral components found in the retinal outer segments, as predicted by principal component analysis of the data acquired using 633 nm excitation. Comparison of the Raman map with its histological counterpart revealed a strong correlation between the two images. CONCLUSIONS: It has been demonstrated that Raman spectroscopy offers a unique insight into the biochemical composition of the light-sensing cells of the retina following the application of standard histological protocols. The present study points to the considerable promise of Raman microscopy as a component-specific probe of retinal tissue.

Absolute Calibration of Atomic Density Measurements by Laser-Induced Fluorescence Spectroscopy with Two-Photon Excitation (TALIF)

The two-photon resonances of atomic hydrogen (? = 2 × 205.1 nm), atomic nitrogen (? = 2 × 206.6 nm) and atomic oxygen (? = 2 × 225.6 nm) are investigated together with two selected transitions in krypton (? = 2×204.2 nm) and xenon (? = 2×225.5 nm). The natural lifetimes of the excited states, quenching coefficients for the most important collisions partners, and the relevant ratios of the two-photon excitation cross sections are measured. These data can be applied to provide a calibration for two-photon laser-induced fluorescence measurements based on comparisons with spectrally neighbouring noble gas resonances.

Negative photoion spectroscopy of freon molecules in the vicinity of the Cl 2p edge

Polar photodissociation of CFnCl4-n (n=0-2) has been studied using synchrotron radiation within the energy range 195-217 eV. The first observations of negative photoion fragments from these molecules after core excitation are reported. In addition to observing a number of previously known resonances two additional resonant states, just above the Cl 2p ionization limit, are observed and play an important role in the polar photodissociation process. The difficulties in identifying these above threshold spin-split features using negative photoion spectroscopy are discussed.

Correlated electron-ion dynamics: the excitation of atomic motion by energetic electrons

Correlated electron-ion dynamics (CEID) is an extension of molecular dynamics that allows us to introduce in a correct manner the exchange of energy between electrons and ions. The formalism is based on a systematic approximation: small amplitude moment expansion. This formalism is extended here to include the explicit quantum spread of the ions and a generalization of the Hartree-Fock approximation for incoherent sums of Slater determinants. We demonstrate that the resultant dynamical equations reproduce analytically the selection rules for inelastic electron-phonon scattering from perturbation theory, which control the mutually driven excitations of the two interacting subsystems. We then use CEID to make direct numerical simulations of inelastic current-voltage spectroscopy in atomic wires, and to exhibit the crossover from ionic cooling to heating as a function of the relative degree of excitation of the electronic and ionic subsystems.

Phase-resolved emission spectroscopy of a hydrogen rf discharge for the determination of quenching coefficients

Collisional effects can have strong influences on the population densities of excited states in gas discharges at elevated pressure. The knowledge of the pertinent collisional coefficient describing the depopulation of a specific level (quenching coefficient) is, therefore, important for plasma diagnostics and simulations. Phase resolved optical emission spectroscopy (PROES) applied to a capacitively coupled rf discharge excited with a frequency of 13.56 MHz in hydrogen allows the measurement of quenching coefficients for emitting states of various species, particularly of noble gases, with molecular hydrogen as a collision partner. Quenching coefficients can be determined subsequent to electron-impact excitation during the short field reversal phase within the sheath region from the time behavior of the fluorescence. The PROES technique based on electron-impact excitation is not limited â?? in contrast to laser techniques â?? by optical selection rules and the energy gap between the ground state and the upper level of the observed transition. Measurements of quenching coefficients and natural fluorescence lifetimes are presented for several helium (3 1S,4 1S,3 3S,3 3P,4 3S), neon (2p1 ,2p2 ,2p4 ,2p6), argon (3d2 ,3d4 ,3d18 and 3d3), and krypton (2p1 ,2p5) states as well as for some states of the triplet system of molecular hydrogen.

Prospects of phase resolved optical emission spectroscopy as a powerful diagnostic tool for RF-discharges

Phase resolved optical emission spectroscopy (PROES) bears considerable potential for diagnostics of RF discharges that give detailed insight of spatial and temporal variations of excitation processes. Based on phase and space resolved measurements of the population dynamics of excited states several diagnostic techniques have been developed. Results for a hydrogen capacitively coupled RF (CCRF) discharge are discussed as an example. The gas temperature, the degree of dissociation and the temporally and spatially resolved electron energy distribution function (EEDF) of energetic electrons (>12eV) are measured. Furthermore, the pulsed electron impact excitation during the field reversal phase, typical for hydrogen CCRF discharges, is exploited for measurements of atomic and molecular data like lifetimes of excited states, coefficients for radiationless collisional de-excitation (quenching coefficients), and cascading processes from higher electronic states.

Determination of quenching coefficients in a hydrogen RF discharge by time-resolved optical emission spectroscopy

In gas discharges at elevated pressure, radiation-less collisional de-excitation (quenching) has a strong influence on the population of excited states. The knowledge of quenching coefficients is therefore important for plasma diagnostics and simulations. A novel time-resolved optical emission spectroscopic (OES) technique allows the measurement of quenching coefficients for emission lines of various species, particularly of noble gases, with molecular hydrogen as collision partner. The technique exploits the short electron impact excitation during the field reversal phase within the sheath region of a hydrogen capacitively coupled RF discharge at 13.56 MHz. Quenching coefficients can be determined subsequent to this excitation from the effective lifetime of the fluorescence decay at various hydrogen pressures. The measured quenching coefficients agree very well with results obtained by means of laser excitation. The time-resolved OES technique based on electron impact excitation is not limited - in contrast to laser techniques - by optical selection rules and the energy gap between the ground state and the observed excited level.

Absolute Atomic Oxygen Density Measurements by Two-Photon Absorption Laser-induced Fluorescence Spectroscopy in an RF-Excited Atmospheric Pressure Plasma Jet

The atmospheric pressure plasma jet is a capacitively coupled radio frequency discharge (13.56 MHz) running with a high helium flux (2m3 h-1) between concentric electrodes. Small amounts (0.5%) of admixed molecular oxygen do not disturb the homogeneous plasma discharge. The jet effluent leaving the discharge through the ring-shaped nozzle contains high concentrations of radicals at a low gas temperature—the key property for a variety of applications aiming at treatment of thermally sensitive surfaces. We report on absolute atomic oxygen density measurements by two-photon absorption laser-induced fluorescence (TALIF) spectroscopy in the jet effluent. Calibration is performed with the aid of a comparative TALIF measurement with xenon. An excitation scheme (different from the one earlier published) providing spectral matching of both the two-photon resonances and the fluorescence transitions is applied.

Measurement of Quenching Coefficients and Developement of Calibration Methods for Quantitative Spectroscopy of Plasma at Elevated Pressures

Measurements of collisional de-excitation (quenching) coefficients required for the interpretation of emission and fluorescence spectroscopic measurements are reported. Particular attention is turned on argon transitions which are of interest for actinometric determinations of atomic ground state populations and on fluorescence lines originating from excited atoms and noble gases in connection with two-photon excitation (TALIF) of atomic radicals. A novel method is described which allows to infer quenching coefficients for collisions with molecular hydrogen of noble gas states in the energy range up to 24 eV. The excitation is performed in these experiments by collisions of energetic electrons in the sheath of an RF excited hydrogen plasma during the field reversal phase which lasts about 10 ns. We describe in addition a calibration method - including quenching effects - for the determination by TALIF of absolute atomic radical densities of hydrogen, nitrogen and oxygen using two-photon resonances in noble gases close by the resonances of the species mentioned. The paper closes with first ideas on a novel technique to bypass quenching effects in TALIF by introducing an additional, controllable loss by photoionization that will allow quenching-free determination of absolute atomic densities with prevalent nanosecond laser systems in situations where collisional de-excitation dominates over spontaneous emission.

Composition profiling of seized ecstasy tablets by Raman spectroscopy

Raman spectroscopy with far-red excitation has been investigated as a simple and rapid technique for composition profiling of seized ecstasy (MDMA, N-methyl-3,4-methylenedioxyamphetamine) tablets. The spectra obtained are rich in vibrational bands and allow the active drug and excipient used to bulk the tablets to be identified. Relative band heights can be used to determine drug/excipient ratios and the degree of hydration of the drug while the fact that 50 tablets per hour can be analysed allows large numbers of spectra to be recorded. The ability of Raman spectroscopy to distinguish between ecstasy tablets on the basis of their chemical composition is illustrated here by a sample set of 400 tablets taken from a large seizure of > 50000 tablets that were found in eight large bags. The tablets are all similar in appearance and carry the same logo. Conventional analysis by GC-MS showed they contained MDMA. Initial Raman studies of samples from each of the eight bags showed that despite some tablet-to-tablet variation within each bag the contents could be classified on the basis of the excipients used. The tablets in five of the bags were sorbitol-based, two were cellulose-based and one bag contained tablets with a glucose excipient. More extensive analysis of 50 tablets from each of a representative series of sample bags gave distribution profiles that showed the contents of each bag were approximately normally distributed about a mean value, rather than being mixtures of several discrete types. Two of the sorbitol-containing sample sets were indistinguishable while a third was similar but not identical to these, in that it contained the same excipient and MDMA with the same degree of hydration but had a slightly different MDMA/sorbitol ratio. The cellulose-based samples were badly manufactured and showed considerable tablet-to-tablet variation in their drug/excipient ratio while the glucose-based tablets had a tight distribution in their drug/excipient ratios. The degree of hydration in the MDMA feedstocks used to manufacture the cellulose-, glucose- and sorbitol-based tablets were all different from each other. This study, because it centres on a single seizure of physically similar tablets with the same active drug, highlights the fact that simple physical descriptions coupled with active drug content do not in themselves fully characterize the nature of the seized materials. There is considerable variation in the composition of the tablets within this single seizure and the fact that this variation can be detected from Raman spectra demonstrates that the potential benefits of obtaining highly detailed spectra can indeed translate into information that is not readily available from other methods but would be useful for tracing of drug distribution networks.

Rapid analysis of ecstasy and related phenethylamines in seized tablets by Raman spectroscopy

Raman spectroscopy with far-red excitation has been used to study seized, tableted samples of MDMA (N-methyl-3,4-methylenedioxyamphetamine) and related compounds (MDA, MDEA, MBDB, 2C-B and amphetamine sulfate), as well as pure standards of these drugs. We have found that by using far-red (785 nm) excitation the level of fluorescence background even in untreated seized samples is sufficiently low that there is little difficulty in obtaining good quality data with moderate 2 min data accumulation times. The spectra can be used to distinguish between even chemically-similar substances, such as the geometrical isomers MDEA and MBDB, and between different polymorphic/hydrated forms of the same drug. Moreover, these differences can be found even in directly recorded spectra of seized samples which have been bulked with other materials, giving a rapid and non-destructive method for drug identification. The spectra can be processed to give unambiguous identification of both drug and excipients (even when more than one compound has been used as the bulking agent) and the relative intensities of drug and excipient bands can be used for quantitative or at least semi-quantitative analysis. Finally, the simple nature of the measurements lends itself to automatic sample handling so that sample throughputs of 20 samples per hour can be achieved with no real difficulty.

Analysis of luminescent samples using subtracted shifted Raman spectroscopy

A novel method of obtaining high-quality Raman spectra of luminescent samples was tested using cyclohexane solutions which had been treated with a fluorescent dye. The method involves removing the fixed pattern irregularity found in the spectra taken with CCD detectors by subtracting spectra taken at several different, closely spaced spectrometer positions. It is conceptually similar to SERDS (shifted excitation Raman difference spectroscopy) but has the distinct experimental advantage that it does not require a tunable laser source. The subtracted spectra obtained as the raw data are converted into a more recognisable and conventional form by iterative fitting of appropriate double Lorentzian functions whose peak parameters are then used to 'reconstruct' a conventional representation of the spectrum. Importantly, it is shown that the degree of uncertainty in the resultant 'reconstructed' spectra can be gauged reliably by comparing reconstructed spectra obtained at two different spectrometer shifts (delta and 2 delta), The method was illustrated and validated using a solvent (cyclohexane) the spectrum of which is well known and which contains both regions with complex overlapping bands and regions with isolated bands, Possible sources of error are discussed and it is shown that, provided the degree of uncertainty in the data is correctly characterised, it is completely valid to draw conclusions about the spectra of the sample on the basis of the reconstructed data. The acronym SSRS (subtracted shifted Raman spectroscopy; pronounced scissors) is proposed for this method, to distinguish it from the SERDS technique.

TIME-RESOLVED RESONANCE RAMAN-SPECTROSCOPY OF TRIPLET-STATE METALATED AND FREE-BASE TETRAARYLPORPHYRINS

Resonance Raman spectra of the T-1 excited states of Zn and free-base tetra-4-sulfonatophenylporphyrin (TPPS) have been recorded at room temperature in aqueous solution using two-colour time-resolved methods. The spectra of both sulfonated molecules are very similar to their tetraphenylporphyrin (TPP) analogues, which have been recorded in THF solution using the same pump-probe conditions, but they have higher signal-to-noise ratios because interference from strong solvent bands is reduced. Although two different T-1 spectra of Zn(TPP) have been reported these spectra differ slightly from each other and from the spectrum reported here, which has band positions very close (+/-6 cm(-1)) to those of Zn(TPPS). The high S/N ratios obtainable for the water-soluble porphyrins have allowed reliable polarization data to be recorded for their S-0 and T-1 states. This data set allows a realistic comparison of the changes in bonding associated with excitation of both free-base and Zn tetraarylporphyrins to the T-1 state.