Laser-induced population depletion effects in double resonance excitation with Hg vapors

By monitoring the green fluorescence transition of mercury vapor 7 ³S₁→6 ³P⁰₂ (546.074 nm) excited by two pulsed dye lasers tuned at two connected atomic resonant frequencies, i.e. 6 ¹S₀→6 ³P⁰₁ (253.652 nm) and 6 ³P⁰₁→ 7 ³S₁ (435.835 nm), a decrease in the green fluorescence yield is experimentally observed when the intensity of the 435.835-nm excitation transition exceeds 5 kW/cm². A similar result is obtained at the yellow fluorescence transition 6 ³D₁→6 ¹P⁰₁ (578.967 nm) when the second step is tuned to the 6 ³P⁰₁→6 ³D₁ transition (313.159 nm). At the same time, an increase in the transmittance of the ground state transition (253.652 nm) is observed. It is speculated that this effect, which occurs only when both laser pulses are temporally coincident, and is therefore not due to photoionization, can be ascribed to the existence of laser induced effects, such as a.c. Stark splitting of levels and possibly electromagnetically-induced transparency (EIT). Our experiment does not allow us to distinguish between these two effects, nor their quantitative evaluation. However, it is stressed that one cannot overlook them in those atomic multi-step excitation experiments in low collisional environments where a depletion of an intermediate level is involved, as for example in the case of atomic fluorescence dip spectroscopy or atomic multistep and multiphoton resonance ionization spectroscopy.     

Computer modeling of fluorescence dip spectroscopy with pulsed laser excitation

This article is an electronic publication in Spectrochimica Acta Electronica (SAE), the electronic section of Spectrochimica Acta Part B (SAB). The hard copy text is accompanied by a disk with a demonstration program, block libraries, demonstration files, and ancillary files. The article discusses the computer modeling of fluorescence dip spectroscopy, a relatively new and powerful atomic diagnostic technique. The models were found to agree essentially perfectly with previously published photostationary results and to extend those results to the more realistic laser temporal profiles encountered in actual experiments.

The fluorescence dip parameters were found to be robust so long as the system under study approached saturation conditions. Short, noisy laser pulses were found to be acceptable, for determination of the fluorescence dip parameters, provided the laser pulses were sufficiently intense to cause near saturation of the three level system. Conversely, steady state excitation was not found to be crucial to the determination of accurate fluorescence dip parameters. For a five level model of the silver atomic system, “negative fluorescence dips” were readily found in the simulations, even though some of the relevant parameters were rough estimates of the actual, unknown parameters.     

Quenching and blinking of fluorescence of a single dye molecule bound to gold nanoparticles

A fluorescein derivative (SAMSA) bound to gold nanoparticles of different diameters is investigated by time-resolved fluorescence at the single molecule level in a wide dynamic range, from nanosecond to second time scale. The significant decrease of both SAMSA excited state lifetime and fluorescence quantum yield observed upon binding to gold nanoparticles can be essentially traced back to an increase of the nonradiative deactivation rate, probably due to energy transfer, that depends on the nanoparticle size. A slow single molecule fluorescence blinking, in the ms time scale, has a marked dependence on the excitation intensity both under single and under two photon excitation. The blinking dynamics is limited by a low probability nonlinear excitation to a high energy state from which a transition to a dark state occurs. The results point out a strong coupling between the vibro-electronic configuration of the dye and the plasmonic features of the metal nanoparticles that provide dye radiationless deactivation channels on a wide dynamic range.     

The effects of saturation and velocity selective population in two-step 6S1/2 → 6P3/2 → 6D5/2 laser excitation in cesium

Excited states population distributions created by two-step 6S_1/2 → 6P_3/2 → 6D_5/2 laser excitation in room temperature cesium vapor were quantitatively analyzed applying absorption and saturation spectroscopy. A simple method for the determination of the excited state population in a single excitation step that is based on the measurements of the saturated and unsaturated absorption coefficients was proposed and tested. It was shown that only ≈ 2% of the ground state population could be transferred to the first excited state by pumping the Doppler broadened line with a single-mode narrow-line laser. With complete saturation of the second excitation step, the population amounting to only ≈ 1% of the ground state can be eventually created in the 6D_5/2 state. The fluorescence intensity emerging at 7P_3/2 → 6S_1/2 transition, subsequent to the radiative decay of 6D_5/2 population to the 7P_3/2 state, was used to assess the efficiency of the population transfer in the chosen two-step excitation scheme. The limitations imposed on the sensitivity of such resonance fluorescence detector caused by velocity-selective excitation in the first excitation step were pointed out and the way to overcome this obstacle is proposed.     

3,5-Diformylboron dipyrromethenes as fluorescent pH sensors

A series of boron dipyrromethene (BODIPY) dyes containing two aldehyde functional groups at the 3 and 5 positions have been synthesized in low-to-decent yields in two steps. In the first step, the meso-aryl dipyrromethanes were treated with POCl(3) in N,N-dimethylformamide to afford 1,9-diformylated dipyrromethanes. In the second step, the diformylated dipyrromethanes were first in situ oxidized with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone and then reacted with BF(3)·OEt(2) to afford 3,5-diformylboron dipyrromethenes. The X-ray structural analysis indicated that the aldehyde groups are involved in intramolecular hydrogen bonding with fluoride atoms, which may be responsible for the stability of the diformylated BODIPY compounds. The presence of two formyl groups significantly alters the electronic properties, which is clearly evident in downfield shifts in the (1)H and (19)F NMR spectra, bathochromic shifts in the absorption and fluorescence spectra, better quantum yields, and increased lifetimes compared to 3,5-unsubstituted BODIPYs. Furthermore, 3,5-diformylboron dipyrromethenes are highly electron-deficient and undergo facile reductions compared to unsubstituted BODIPYs. These compounds exhibit pH-dependent on/off fluorescence and thus act as fluorescent pH sensors.     

Enhanced melanin fluorescence by stepwise three-photon excitation

The fluorescence of eumelanin (from Sepia officinalis and black human hair) was activated and enhanced by almost three orders of magnitude by exposure to near-infrared radiation. No activation or enhanced emission was observed when the samples were heated up to 100°C. The near-infrared irradiation caused obvious changes to the eumelanin and could be seen by fluorescence and bright field imaging. The area of enhanced emission appeared to originate from a region with changes in the morphology of the eumelanin's granule and increased with exposure time. At least two different components with enhanced fluorescence were activated and could be distinguished by their excitation properties. One component could be excited efficiently with wavelengths in the visible region and exhibited linear absorption dependence with respect to the laser power level. The second component could be excited efficiently using near-infrared wavelengths by a nonlinear process and exhibited a third-order dependence on the excitation. The third-order dependence is explained by a step-wise excited-state absorption process since the same third-order dependence was present when either continuous wave or femtosecond pulsed laser, with similar average-power levels, was used.     

Relationship between steps in 8-anilino-1-naphthalene sulfonate (ANS) fluorescence and changes in the energized membrane state and in intracellular and extracellular adenosine 5'-triphosphate (ATP) levels following bacteriophage T5 infection of Escherichia coli.

The addition of bacteriophage T5 to anaerobic, fermenting cells of Escherichia coli B or K-12 in the presence of 8-anilino-1-naphthalene sulfonate (ANS), N-phenylnaphthyl-1-amine (NPN), or dansyl ethylamine causes the fluorescence of these probes to rise in two steps, the first occurring immediately upon addition, the second delayed by 6 min. The conditions necessary for observing this phenomenon are defined (cell density, probe concentration, substrate, absence of an electron acceptor, multiplicity of infection, growth, and harvesting conditions). The magnitudes of the first and second steps in fluorescence are dependent upon the multiplicity of infection; the timing of the steps is not. The first step correlates with a breakdown in the potassium or rubidium permeability barrier of the cells, and it occurs either aerobically or anaerobically, with fermentable or nonfermentable substrates. The second step occurs only with cells that are without an available electron acceptor, are fermenting, and which have a functional membrane-bound, Ca2+-dependent adenosine triphosphatase (ATPase). The results are consistent with disturbance of energization of the cell membrane by the membrane-bound ATPase at the time of the second step in fluorescence. No changes in the intracellular level of adenosine 5'-triphosphate (ATP) was seen, whereas the extracellular level increased sharply, starting 3--6 min after phage addition. The quantity of ATP found in the medium by 30 min after infection amounted to about four times the amount present inside the cells at the time of infection. The quantity and rate of efflux of ATP was similar under aerobic and anaerobic conditions.     

Observation of slow charge redistribution preceding excited-state proton transfer

The photoacid 8-hydroxy-N,N,N',N',N',N'-hexamethylpyrene-1,3,6-trisulfonamide (HPTA) and related compounds are used to investigate the steps involved in excited-state deprotonation in polar solvents using pump-probe spectroscopy and time correlated single photon counting fluorescence spectroscopy. The dynamics show a clear two-step process leading to excited-state proton transfer. The first step after electronic excitation is charge redistribution occurring on a tens of picoseconds time scale followed by proton transfer on a nanosecond time scale. The three states observed in the experiments (initial excited state, charge redistributed state, and proton transfer state) are recognized by distinct features in the time dependence of the pump-probe spectrum and fluorescence spectra. In the charge redistributed state, charge density has transferred from the hydroxyl oxygen to the pyrene ring, but the OH sigma bond is still intact. The experiments indicate that the charge redistribution step is controlled by a specific hydrogen bond donation from HPTA to the accepting base molecule. The second step is the full deprotonation of the photoacid. The full deprotonation is clearly marked by the growth of stimulated emission spectral band in the pump-probe spectrum that is identical to the fluorescence spectrum of the anion.     

Confocal microscopic X-ray fluorescence at the HASYLAB microfocus beamline: characteristics and possibilities

The possibilities of performing non-destructive elemental analysis in three dimensions on a variety of heterogeneous materials by means of an innovative variation of the microscopic X-ray fluorescence analysis (μ-XRF) method are described. Next to employing focusing optics for concentration of the primary beam of X-rays, a second optical element between the sample and the energy-dispersive detector is used in confocal μ-XRF. Thus, only X-ray fluorescence signals from a cube-like volume (within certain limits imposed by the absorption of the radiation in the sample) can be arbitrarily positioned within the sample. The distribution of major, minor and trace elements (down to the sub-ppm concentration level in some matrices) along lines and planes within the sample can be visualized with a spatial resolution of the order of 15–40 μm. The lowest detectable amounts in confocal mode using pink-beam excitation are situated at the sub-femtogram level.     

DensMat: fully time-resolved simulation of two-step pulsed laser excitation of atoms in highly collisional media

A program that simulates and displays the level populations of atomic systems exposed to dual-wavelength (i.e. two-colour) pulsed laser excitation in highly collisional media (such as flames and plasmas) has been developed. The program is based upon a previously published fully time-dependent density-matrix model that describes step-wise excitations of atoms with degenerate states under collision-dominated conditions, and which thus goes beyond the rate-equations formalism. This model can predict such phenomena as Rabi flopping and a.c.-Stark splitting, shifting and broadening. The program can be used as a prediction tool for laser-enhanced ionization, laser-induced fluorescence, fluorescence dip spectroscopy and other two-colour laser-based spectroscopic experiments. The program provides the user with a flexible four-level atomic system, configurable as a one- or two-step excitation ladder, along with an ionization continuum and non-laser-connected level(s) that may act as trap(s) or metastable level(s). Parameters such as level degeneracy, collisional rates and laser pulse widths, shapes, wavelengths, intensities and bandwidths are accessible to the user. The program can display both the time development of the level populations and also level populations versus laser wavelength. This article is an electronic publication in Spectrochimica Acta Electronica (SAE), the electronic section of Spectrochimica Acta Part B (SAB). The hardcopy text is accompanied by a disk containing the program DensMat, an associated on-line help file and manual, an installation program, and data files pertaining to the examples illustrated in this article. The program runs under Windows 3.1 on an IBM-compatible computer.     

One-, two-, and three-photon absorption induced fluorescence of a novel chromophore in chloroform solution

One-, two-, and three-photon absorption induced fluorescence intensities of a novel nonlinear optical chromophore have been measured by using a tunable femtosecond pulsed laser as the excitation. Four resonance peaks are observed as the excitation wavelength is tuned from 600 to 2000 nm. These peaks correspond to the one-, two- and three-photon fluorescence resonance. Except for intensity difference, the lifetime and the fluorescence spectrum are found to be the same for the one-, two-, or three-photon resonance, hence suggesting that the same excited energy level is involved in emitting the fluorescence intensity. A three-level model is developed to account for the incident excitation laser intensity dependence of the one-photon and multiphoton fluorescence intensity. The model allows the multiphoton absorption cross sections to be extracted; it can also account for the deviation observed in the linear, square, and cubic intensity dependence of the one-, two-, and three-photon fluorescence intensity, respectively. To determine the absorption cross sections, the present method does not require the fluorescence quantum efficiency data, needed in the low intensity technique.     

Electron Transfer and Electron Excitation Processes in 2,5-Diaminoterephthalate Derivatives with Broad Scope for Functionalization

Derivatives of 2,5-diaminoterephthalate (DAT) are efficient fluorescence dyes that are also redox-active, thus allowing for the electrochemical manipulation of spectral properties. The electrochemical behaviour of seven DAT derivatives was studied by cyclic voltammetry in dichloromethane. In the absence of a proton donor, DATs should be oxidized in two one-electron steps. The first step is usually quasi-reversible while the second step is either quasi-reversible or irreversible. Some electrochemical properties such as the formal potentials and the ratio between the anodic and the cathodic current were determined from the cyclic voltammograms. Correlation between the formal potential of first oxidation and the absorption or the fluorescence emission wavelengths are established for this specific type of dyes. These correlations were confirmed with density functional theory calculations.     

Fluorescence spectroscopy of jet-cooled chiral (+/-)-indan-1-ol and its cluster with (+/-)-methyl- and ethyl-lactate

The laser-induced fluorescence excitation, dispersed fluorescence, and IR-UV double resonance spectra of chiral (+/-)-indan-1-ol have been measured in a supersonic expansion. Three low energy conformers of the molecule have been identified, and the ground state vibrational modes of each conformer are tentatively assigned with the aid of quantum chemistry calculations. The frequencies of the nu(OH) and nu(CH) stretch modes of the two most abundant conformers have been measured by fluorescence dip IR spectroscopy and have been used for their assignment. The dispersed fluorescence spectra clearly indicate the coupling of low-frequency modes, as was seen in other substituted indanes such as 1-aminoindan and 1-amino-2-indanol. (R)- and (S)-indan-1-ol distinctly form different types of clusters with (R)- and (S)- methyl- and ethyl-lactate. Both hetero- and homochiral clusters are characterized by complex spectra which exhibit a progression built on low-frequency intermolecular modes.     

CHLOROPHYLL a FLUORESCENCE OF GONYAULAX POLYEDRA GROWN ON A LIGHT-DARK CYCLE AND AFTER TRANSFER TO CONSTANT LIGHT

Abstract— The chlorophyll a fluorescence properties of Gonyaulax polyedra cells before and after transfer from a lightdark cycle (LD) to constant dim light (LL) were investigated. The latter display a faster fluorescence transient from the level ‘I’ (intermediary peak) to ‘D’ (dip) to ‘P’ (peak) than the former (3 s as compared to 10 s), and a different pattern of decline in fluorescence from ‘I’ to ‘D’ and from ‘P’ to the steady state level with no clearly separable second wave of slow fluorescence change, referred to as ‘s' (quasi steady state)→‘M’ (maximum) →‘T’ (terminal steady state). The above differences are constant features of cells in LD and LL, and are not dependent on the time of day. They are interpreted as evidence for a greater ratio of photosystem II/photosystem I activity in cells in LL. After an initial photoadaptive response following transfer from LD to LL, the cell absorbance at room temperature and fluorescence emission spectra at 77 K for cells in LL and LD are comparable. The major emission peak is at 685–688 nm (from an antenna Chl a 680, perhaps Chl a-c complex), but, unlike higher plants and other algae, the emission bands at 696–698 nm (from Chl a_II complex, Chl a 685, close to reaction center II) and 710–720 nm (from Chl a₁, complexes, Chl a 695, close to reaction center I) are very minor and could be observed only in the fluorescence emission difference spectra of LL minus LD cells and in the ratio spectra of DCMU-treated to non-treated cells. Comparison of emission spectra of cells in LL and LD suggested that, in LL, there is a slightly greater net excitation energy transfer from the light-harvesting peridinin-Chl a (Chl a 670) complex, fluorescing at 675 nm, to the other antenna chlorophyll a complex fluorescing at 685–688 nm, and from the Chl a., complex to the reaction center II. Comparison of excitation spectra of fluorescence of LL and LD cells, in the presence of DCMU, confirmed that cells in LL transfer energy more extensively from the peridinin-Chl a complex to other Chl a complexes than do cells in LD.     

Lyman-α excitation spectra in the photodissociation of the doubly excited states of H2

A Lyman-α excitation spectrum has been observed using synchrotron radiation in the energy region corresponding to the double electron excitation of H₂. There exist in the spectrum three thresholds at 26.6±0.5 eV, 29.2±0.7 eV and 30.9±0.6 eV, and a dip at 34.1±0.5 eV. A Lyman-α excitation spectrum in the energy region corresponding to the single electron excitation has been also observed using a detection system which works as a band pass filter for detecting of Lyman-α fluorescence. The cross section of Lyman-α fluorescence in the photodissociation of the doubly excited states is very small, e.g., in the order of 10^?20 cm² at 30 eV, in comparison with that from the single electron excitation.     

Short-wavelength fluorescence of 1,12-benzperylene

Two relatively strong bands, subband 1 and subband 2, observed in the short-wavelength region of the fluorescence spectrum of 1,12-benzperylene have been studied by varying the excitation wavelength and temperature and by adding n-hexane gas. In solution, subband 1 was observed at higher temperatures and can be attributed to an emission from a thermally-populated vibronic level of the first excited singlet state. In the gas phase, however, it is attributed to a υυ ? 1 resonance fluorescence. Subband 2 is attributed to fluorescence from the second excited singlet state.     

In vivo fluorescence imaging using two excitation and/or emission wavelengths for image contrast enhancement

Steady-state fluorescence imaging can be used in conjunction with selective exogenous or endogenous fluorescent compounds for the diagnosis of skin lesions, for example cancer. Depending on the excitation and emission properties of the fluorescent compound used, various excitation and/or emission wavelengths can be chosen in order to allow fluorescence imaging. Unwanted background signals such as autofluorescence and scattering can decrease the image quality and, hence, the diagnosis potential of this imaging method. We have used an inexpensive dual excitation and/or emission wavelength approach in order to suppress the unwanted background signal and allow contrast enhanced fluorescence imaging. One excitation and/or emission wavelength is at the corresponding maximum of the fluorescent compound, while the second is at a nearby excitation/emission minimum. The first image contains the emission from the fluorescent compound used combined with the signal from the unwanted background. The second image provides an image of just the unwanted background signal. The difference of both images taken, thus gives a contrast enhanced image of the skin lesion. The method relies on the assumption that the background signal does not change significantly due to the small changes in wavelength for excitation or emission. Image ratio methods have already been applied towards diagnosis of basal cell carcinomas after administration of aminolevulinic acid-induced protoporphyin IX. In this study, we describe in vivo measurements in mice where the second image, usually the background signal only, contains new unwanted image data. This simple method can successfully resolve the desired image, thus demonstrating the versatility of the image processing procedure.     

Two-photon uncaging with fluorescence reporting: evaluation of the o-hydroxycinnamic platform

This paper evaluates the o-hydroxycinnamic platform for designing efficient caging groups with fluorescence reporting upon one- and two-photon excitation. The model cinnamates are easily prepared in one step by coupling commercial or readily available synthons. They exhibit a large one-photon absorption that can be tuned in the near-UV range. Uncaging after one-photon excitation was investigated by 1H NMR, UV-vis absorption, and steady-state fluorescence emission. In the whole investigated series, the caged substrate is quantitatively released upon photolysis. At the same time, uncaging releases a strongly fluorescent coproduct that can be used as a reporter for quantitative substrate delivery. The quantum yield of double bond photoisomerization leading to uncaging after one-photon absorption mostly lies in the 10% range. Taking advantage of the favorable photophysical properties of the uncaging coproduct, we use a series of techniques based on fluorescence emission to measure the action uncaging cross sections with two-photon excitation of the present cinnamates. Exhibiting values in the 1-10 GM range at 750 nm, they satisfactorily compare with the most efficient caging groups reported to date. Noticeably, the uncaging behavior with two-photon excitation is retained in vivo as suggested by the results observed in living zebrafish embryos. Reliable structure property relationships were extracted from analysis of the present collected data. In particular, the careful kinetic analysis allows us to discuss the relevance of the o-hydroxycinnamic platform for diverse caging applications with one- and two-photon excitation.     

New Anthracene Derivatives as Triplet Acceptors for Efficient Green‐to‐Blue Low‐Power Upconversion

Three new anthracene derivatives [2‐chloro‐9,10‐dip‐tolylanthracene (DTACl), 9,10‐dip‐tolylanthracene‐2‐carbonitrile (DTACN), and 9,10‐di(naphthalen‐1‐yl)anthracene‐2‐carbonitrile (DNACN)] were synthesized as triplet acceptors for low‐power upconversion. Their linear absorption, single‐photon‐excited fluorescence, and upconversion fluorescence properties were studied. The acceptors exhibit high fluorescence yields in DMF. Selective excitation of the sensitizer Pd^IIoctaethylporphyrin (PdOEP) in solution containing DTACl, DTACN, or DNA‐CN at 532 nm with an ultralow excitation power density of 0.5 W cm^?2 results in anti‐Stokes blue emission. The maximum upconversion quantum yield (Φ_UC=17.4 %) was obtained for the couple PdOEP/DTACl. In addition, the efficiency of the triplet–triplet energy transfer process was quantitatively studied by quenching experiments. Experimental results revealed that a highly effective acceptor for upconversion should combine high fluorescence quantum yields with efficient quenching of the sensitizer triplet.     

Solid-surface derivative room-temperature luminescence spectrometry of mixtures

Derivative solid-surface room-temperature fluorescence and phosphorescence were combined for the identification of components in binary and ternary mixtures of nanogram amounts of nitrogen heterocycles. Direct, and first and second derivative room-temperature phosphorescence excitation, room-temperature fluorescence and room-temperature phosphorescence spectra were used in the identification of the components in the mixtures by matching spectral wavelengths from mixtures with spectral wavelengths from standards. The criteria for matching wavelengths, subtracting background signals and obtaining derivative spectra at the 0.5-ng level are discussed.