Remote sensing in an optically dense environment by using two-photon excited fluorescence and a single multimode fiber optic

The feasibility of remote sensing by means of two-photon excited molecular fluorescence was investigated by using a single 115-m multimode optical fibr.Because of modal dispersion and reflecton of Rayleigh scatter, time-resolved detection ws required to resolve the weak fluorescence from the relatively intense laser background. The bare-ended probe, which both delivered the excitation and collected the fluorescence emission, was employed to detect an analyte in an optically dense environment. For a fixed amount of fluorophore, the solvent was continuously changed from 90% (w/w) ethanol (non-absorbing) to 100% acetone (absorbing). The result was a flurorescence signal constant to within 5%. Quantitation via one-photon excitatation using a 50-cm optical fiber demonstrated the characteristic inner filter roll-off of optically dense systems. Because of th elarge attenuation of the waveguide in the ultraviolet range, remote sensing via one-photon excited fluorescence proved impossible. However, linear calibration plots for the nonlinear excitation process were obtained from micromolar to near saturation levels. Sources of interference were isolated, and the extension to a single-mode fiber was made.     

The symmetry of two-photon excited states as determined by time-resolved fluorescence depolarization experiments

A new experimental and theoretical approach is presented for the quantitative determination and assignment of the two-photon absorption tensor of fluorophores dissolved in liquid solutions. Two linearly independent time-resolved fluorescence anisotropies and the two-photon polarization ratio were determined from experiments based on using the time-correlated single photon counting technique. The data were analyzed in a global manner under the assumption of prevailing diffusive molecular reorientations and when accounting for the influence of rapid unresolved reorientations. The method has been applied in fluorescence studies of perylene, two-photon excited at 800 nm. The analysis suggests that the two-photon transition is mediated via vibronic coupling including at least two vibrations of different symmetry, and also that the first singlet excited electronic state acts as a dominating intermediate state.     

Strong modulation of two-photon excited fluorescence of quadripolar dyes by (de)protonation

Two quadripolar dyes have been designed and synthesized that present large cross sections for two-photon excitation and whose fluorescence responds strongly to (de)protonation. These dyes are considered as prototypes of molecular pH probes for multiphoton fluorescence microscopy.     

Coupling micelle-mediated extraction using mixtures of surfactants and fluorescence measurements with a fiber-optic for the screening of PAHs in seawater

The screening of PAHs from seawater samples using cloud-point extraction (CPE) as a step prior to their determination by fluorescence measurements with a fiber-optic is proposed. The CPE is carried out with the nonionic surfactants mixture POLE and Brij 30. The fluorescence measurement parameters were optimized, allowing selection of benzo(a)pyrene (B(a)Py) and benzo(k)fluoranthene (B(k)Ft) as the target analytes for the screening. The reproducibility of the whole screening system, expressed as relative standard deviation, was 9.0% for B(a)Py and 12.1% for B(k)Ft (both for n = 7). The reliability of the method was established at five concentrations for B(a)Py (between 0.5 and 3.3 times the detection limit: 0.31 ng ml(-1)) and at three concentrations for B(k)Ft (between 0.6 and 2.5 times the detection limit: 0.56 ng ml(-1)). The resolution of binary mixtures of these PAHs at different levels of concentration, and a study of the interferences with the rest of the PAHs were also carried out.     

Trivalent boron as an acceptor in donor-pi-acceptor-type compounds for single- and two-photon excited fluorescence

The synthesis, structure, and fluorescence properties of a series of new donor-pi-acceptor (D-pi-A) type compounds, with a trivalent boron, protected by two mesityl groups, as acceptor, and with various typical donors and different pi-conjugated bridges, are reported. All these stable organoboron compounds show intense single-photon excited fluorescence (SPEF) and two-photon excited fluorescence (TPEF) in a wide spectral range from blue to green, with the spectral peak position of the SPEF being basically the same as that of the TPEF. The remarkably strong Cbond;B(mesityl)(2) bonding, and the well-conjugated pi-system, shown in X-ray crystal structures of two compounds, indicate some charge transfer features of the ground state. Meanwhile, spectral data indicate that the charge transfer from donor to acceptor is greatly enhanced in the excited states. Based on typical structural data and comprehensive spectral data, the following structure-property relationships can be drawn: 1) the moderate arylamino donor can more effectively enhance the SPEF and TPEF intensities than can the strong alkylamino donor; 2) stilbene is a better pi-bridge than styrylthiophene for its capability of enhancing and blue-shifting the SPEF and TPEF of the corresponding D-pi-A compounds; and 3) when compared to its boron-free precursors and other analogues, -B(mesityl)(2) invariably and consistently acts as an effective SPEF and TPEF fluorophore in all this series of organoboron compounds, which may result from its strong pi-electron-withdrawing and charge transfer-inducing nature in the ground-state and, more dominantly, in the excited-state. Combining all the above positive structure factors, trans-4'-N,N-diphenylamino-4-dimesitylborylstilbene (compound 3) stands out as the optimized green SPEF and TPEF emitter. This compound exhibits an SPEF quantum yield Phi of 0.91 at 522 nm in THF, a TPEF cross-section sigma' that is an order of magnitude larger than that of its boron-free precursor upon excitation by 800 nm femto-second laser pulses, and a two-photon absorption section sigma of 3.0 x 10(-48) cm(4) s. In the blue light region, trans-4'-N-carbazolyl-4-dimesitylboryl-stilbene (compound 4) shows significant SPEF and TPEF properties, with Phi=0.79 at 464 nm in THF and a large sigma' value, which is five times that of fluorescein upon excitation by 740 nm femto-second laser pulses.     

Determination of urea in serum by a fiber-optic fluorescence biosensor

A new fiber-optic biosensor for urea has been developed, based on immobilized urease coupled to a fluorescence ammonia sensor. The enzymatically generated ammonia diffuses through the membrane into a solution of the fluorescent pH indicator trisodium 8-hydroxypyrene-1,3,6-trisulfonate. The sensor has been successfully used for the determination of urea in serum samples, with results in good agreement with those reported by a local hospital. The proposed sensor is reversible and selective to urea. The ease of construction of the sensor tip offers the possibility of designing disposable tips for use in clinical applications.     

Photochemical reaction kinetics in optically dense media: The influence of thermal reactions

The kinetics of photochemical reactions in optically dense media essentially free from diffusion was considered. The photochromic isomerization A ↔ B was studied as an example. If thermal isomerization is possible, a stationary state is achieved in time determined by rate constants for the thermal reactions. The concentration wave profile is changed during the photochemical reaction propagation. Low values of thermal reaction constants and decrease in sample optical density during photochemical isomerization were found to be essential for maximal wave penetration into the sample. Sharp concentration gradients of A and B can be observed when both the optical density is increased during photochemical isomerization and the quantum yield of the direct photochemical reaction A → B is higher than that of the reverse photochemical reaction B → A.     

Lifetime measurements of two-photon excited vibronic levels in the low pressure limit: naphthalene

Single vibronic lifetimes of two-photon excited states are measured in the low pressure limit for naphthalene. The two-photon spectrum, besides yielding new states in S₁, is also free of the interference from the neighborhood of the S₂, known from one-photon experiments, and hence unperturbed lifetimes can be measured to higher excess energies. The overall pattern of lifetimes with excess energy including both one-photon and two-photon states shows that the vibronic structure is subordinate to an overall monotonic decrease, much as found earlier for β-naphthylamine. This indicates, at least for naphthalene, that radiationless processes into triplets are dominated by Franck—Condon factors and not by vibronic inductions and promoting modes.     

Simulation of x-ray absorption near-edge spectra and x-ray fluorescence spectra of optically excited molecules

The x-ray absorption near-edge spectra (XANES) and fluorescence spectra of molecules in the ground state and optically excited states are computed using time-dependent density functional theory and time-dependent Hartree-Fock theory. The calculated XANES spectra of optically excited methanol, benzonitrile, hydrogen sulphide, and titanium tetrachloride and the fluorescence spectra of optically excited methanol can be used to simulate ultrafast optical pump/x-ray probe experiments.     

Viscosity measurements using a two-photon ratiometric fluorescent sensor with two rotors

A new ratiometric two-photon fluorescence sensor, Qca-Cy2, comprised of a hybrid cyanine-carbazole platform with two rotating sites is described. Qca-Cy2 can report on mitochondrial viscosity within live cells, as well as in living tissues at depths of 50–130?μm by ratiometric fluorescence imaging using two-photon microscopy.     

Triaryl boron-based A-pi-A vs triaryl nitrogen-based D-pi-D quadrupolar compounds for single- and two-photon excited fluorescence

Three new A-pi-A-type compounds with trivalent boron, protected by two mesityl groups, as electron acceptor have been synthesized and investigated together with their two diphenylamino-ended D-pi-D analogues. These boranes exhibit large two-photon absorption cross sections and high fluorescence quantum yields.     

Synchrotron radiation excited X-ray fluorescence analysis using a graphite monochromator

Summary X-ray fluorescence analysis with monochromatic synchrotron radiation enables the direct analysis even of samples with a very strong matrix, while other methods require already a chemical pretreatment or preconcentration step. Because of free tunable excitation energy special elements or element groups of interest can be selectively excitated. The use of a graphite crystal for monochromatizing leads to a sufficient fluorescence intensity even for thin samples, because of a larger band width compared to other monochromator crystals. Nevertheless, a sufficient selectivity of the excitation will be achieved. Using a curved crystal focussing the beam in the horizontal plane of the storage ring, the intensity can be improved by a factor of about 20. The limits of detection for realistic samples (in this case aerosol samples are analysed) are in the range of 40 ng/ g. The results of the direct analysis of arsenic in aerosol samples are discussed and compared with an interlaboratory test.

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Röntgenfluorescenzanalyse mit Synchrotronstrahlung unter Verwendung eines Graphitmonochromators

Zusammenfassung Röntgenfluorescenzanalyse mit monochromatisierter Synchrotronstrahlung erlaubt die Direktanalyse auch von stark matrixbeladenen Proben, bei denen bei anderen Methoden schon eine chemische Vorbehandlung oder Anreicherung notwendig wird. Die frei wählbare Monochromatorenergie erlaubt die selektive Anregung bestimmter Elemente oder Elementgruppen. Die Verwendung eines Graphitkristalls zur Monochromatisierung ergibt aufgrund der höheren Bandbreite gegenüber anderen Monochromatorkristallen auch bei dünnen Proben eine ausreichende Fluorescenzintensität bei gleichzeitig genügender Selektivität. Durch Verwendung eines gebogenen Graphitkristalls zur Fokussierung des Strahls in der horizontalen Ebene kann die Intensität noch einmal um den Faktor 20 gesteigert werden. Die Nachweisgrenzen von mit Aerosolen beaufschlagten Filtern liegen im Bereich von 40 ng/g. Die Ergebnisse der Analyse von Arsen in Aerosolproben werden diskutiert und mit den Ergebnissen einer Ringanalyse verglichen.

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Dedicated to Prof. Dr. K. H. Lieser on the occasion of his 65th birthday     

Fusing three perylenebisimide branches and a truxene core into a star-shaped chromophore with strong two-photon excited fluorescence and high photostability

A novel star-shaped chromophore, Tr-PBI, was constructed by fusing three perylenebisimide branches and a truxene core. Tr-PBI exhibits high photostability and excellent two-photon properties: the maximum of δ(TPA) is 11,000 GM at 990 nm and fluorescence quantum efficiency Φ is 0.40 in THF.     

Simultaneous two-photon excited fluorescence and one-photon excited phosphorescence from single molecules of an organometallic complex Ir(ppy)3

We report a study of excited-state lifetimes of the phosphorescent organometallic complex Ir(ppy)3 on ensemble and a single-molecule level. With decreasing concentration, an increasingly intense fast component of 3 ns lifetime appears next to the 1.1 mus phosphorescence. Experimental evidence suggests that strong two-photon absorption followed by fluorescence is responsible for the fast lifetime component.     

Construction of a molecular beacon based on two-photon excited fluorescence resonance energy transfer with quantum dot as donor

A new molecular beacon (MB) driven by two-photon excitation (TPE) using quantum dots as energy donor is constructed, which provides reduced direct excitation of acceptor and is free of interferences from autofluorescence or scattering light in a complicated biological matrix.     

Determination of warfarin by sensitized fluorescence using organized media

The effect of surfactant micelles and albumin on the fluorescence warfarin of and the fluorescence of Eu³⁺ and Tb³⁺ sensitized by warfarin and the second ligand is studied. It was shown that sensitized fluorescence in the system Eu³⁺-tenoyltrifluoroacetone-warfarin allows a 3800-fold reduction of the detection limit for warfarin. The developed procedure was used to determine warfarin in soil.     

Photobleaching kinetics of Verteporfin and Lemuteporfin in cells and optically trapped multilamellar vesicles using two-photon excitation

Verteporfin and Lemuteporfin are compared to examine the effect of their functional groups and therefore the localization in two-photon excitation (TPE) photodynamic therapy (PDT). We used singlet oxygen-related photobleaching of the sensitizers to assess TPE-induced singlet oxygen generation in multilamellar vesicles (MLVs) and U343 glioma cells under a variety of conditions. It was found that Lemuteporfin photobleached at a faster rate than Verteporfin in the majority of environments. Also, Verteporfin and Lemuteporfin exhibited different behaviors when in hypoxic environments relative to those in oxygenated MLVs. These differences are attributed to the sensitizer location in the membrane and their relative mobilities throughout membranes and cells.     

Dispersed fluorescence spectroscopy and fluorescence lifetime measurements of excited vibrational levels of CdCH3

Laser-induced excitation and dispersed fluorescence spectra (originating from the 0⁰, 2¹ and 3¹ vibrational levels of the electronic state) of jet-cooled CdCH₃ are presented and assigned. The observation of emission from these levels is a result having important implications for the dynamics of photodissociation of Cd(CH₃)₂. Fluorescence lifetimes for emission from the 0⁰, 2¹ and 3¹ vibrational levels are also presented.     

Non-linear absorption and fluorescence of chlorophyll a excited by a ruby laser

Excitation of chlorophyll a in solution by a Q-switched ruby laser permits the determination of the two-photon and excited singlet state cross sections from fluorescence and transmission measurements.