Two-photon and multi-photon excitation of naphthalene in the vapor phase,studied by excitation and fluorescence spectra

Naphthalene vapor is irradiated by μsec dye laser pulses of 150 kW peak power and a spectral bandwidth of 0.3 nm. A two-photon excitation spectrum is detected by monitoring the near UV fluorescence as a function of laser wavelength which is tuned between 570 and 610 nm. The fluorescence obtained by irradiation into the strongest band of the two-photon spectrum could be spectroscopically resolved using a bandwidth of 80 cm^?1. The spectrum exhibits vibrational structure which lies on a strong non-resolved background. From information in both spectra it can be definitely concluded that vibronic levels of B_3u × b_3u species in the lowest singlet state are predominantly excited in a two-photon process. The non-resolved background in the fluorescence spectrum is attributed to subsequent excitation of the two-photon state by a third photon. Further stepwise excitation in the strong radiation field of the laser is also taken into account.     

FLUORESCENCE RELAXATION KINETICS AND QUANTUM YIELD FROM THE PHYCOBILISOMES OF THE BLUE-GREEN ALGA NOSTOC SP. MEASURED AS A FUNCTION OF SINGLE PICOSECOND PULSE INTENSITY*

Abstract— A detailed experimental study of the effect of intensity of a 6 ps excitation pulse on the decay kinetics and yield from phycobilisomes (PBsomes) is presented. The fluorescence from the c-phycoerythrin (PE) emission from PBsomes was found to decay as a single exponential with a time of 31 ± 4ps for an excitation intensity <10¹⁴ photons/cm² per pulse. The risetime of the c-phycocyanin (PC) and allophycocyanin (APC) emission from PBsomes was found to be 34 ± 13 ps. Therefore, at low excitation intensities, the energy transfer time between the constituent phycobiliproteins, PE and PC, is measured to be 34 ± 13ps from the fluorescence decay time of PE and the fluorescence risetime of the PC and APC emission. The fluorescence yield from the PE emission component in PBsomes was found to be intensity dependent for excitation intensities >10¹⁴ photons/cm². The decrease in yield with increased intensity in this case occurred at a higher intensity than in the isolated phycobiliprotein PE. The fluorescence yield of the PC and APC emission component was also found to decrease markedly with increasing excitation intensity. This is in contrast to the case of the isolated phycobiliprotein APC which showed only a slight quenching of the fluorescence. The higher quenching observed for the APC emission in the PBsome evidences the higher effective absorption of APC via energy transfer from PE to PC and APC.     

Two-photon laser-induced fluorescence in oxygen and nitrogen atoms

Two-photon absorption is used to populate the 3p ³P state of O and the 2s²2p²3p⁴D^o state of N in a flow discharge. Exciting photons are produced by anti-Stokes Raman frequency conversion of tunable UV laser radiation; the resulting near IR fluorescence from the excited state yielded lifetimes, quenching rates, and relative two-photon transition probabilities.     

Two-photon absorption spectrum of (2,2)-paracyclophane

The two-photon absorption spectrum of crystalline (2,2)-paracyclophane was measured in the regions of 31200–33800 cm^?1, 34700–36500 cm^?1, and 40800–48600 cm^?1 by monitoring the fluorescence intensity using a tunable dye laser as a two-photon excitation source. Two absorption bands in the region 34700–36500 cm^?1 were assigned to the two even-parity allowed ¹B_1g ← ¹A_1g and ¹B_2g ← ¹A_1g transitions.     

Two-color two-photon excitation using femtosecond laser pulses

The use of two-color two-photon (2c2p) excitation easily extends the wavelength range of Ti:sapphire lasers to the UV, widening the scope of its applications especially in biological sciences. We report observation of 2c2p excitation fluorescence of p-terphenyl (PTP), 2-methyl-5-t-butyl-p-quaterphenyl (DMQ) and tryptophan upon excitation with 400 and 800 nm wavelengths using the second harmonic and fundamental wavelength of a mode-locked Ti:sapphire femtosecond laser. This excitation is energetically equivalent to a one-photon excitation wavelength at 266 nm. The fluorescence signal is observed only when both wavelengths are spatially and temporally overlapping. Adjustment of the relative delay of the two laser pulses renders a cross correlation curve which is in good agreement with the pulse width of our laser. The fluorescence signal is linearly dependent on the intensity of each of the two colors but quadratically on the total incident illumination power of both colors. In fluorescence microscopy, the use of a combination of intense IR and low-intensity blue light as a substitute for UV light for excitation can have numerous advantages. Additionally, the effect of differently polarized excitation photons relative to each other is demonstrated. This offers information about different transition symmetries and yields deeper insight into the two-photon excitation process.     

Simultaneous Two-Photon Activation of Type-I Photodynamic Therapy Agents

The excitation and emission properties of several psoralen derivatives are compared using conventional single-photon excitation and simultaneous two-photon excitation (TPE). Two-photon excitation is effected using the output of a mode-locked titanium: sapphire laser, the near infrared output of which is used to promote non-resonant TPE directly. Specifically, the excitation spectra and excited-state properties of 8-methoxypsoralen and 4′-aminomethyl-4,5,8-trimethylpsoralen are shown to be equivalent using both modes of excitation. Further, in vitro feasibility of two-photon photodynamic therapy (PDT) is demonstrated using Salmonella typhimurium. Two-photon excitation may be beneficial in the practice of PDT because it would allow replacement of visible or UV excitation light with highly penetrating, nondamag-ing near infrared light and could provide a means for improving localization of therapy. Comparison of possible laser excitation sources for PDT reveals the titanium: sapphire laser to be exceptionally well suited for nonlinear excitation of PDT agents in biological systems due to its extremely short pulse width and high repetition rate that together provide efficient PDT activation and greatly reduced potential for biological damage     

Two-photon absorption coefficient in p-terphenyl crystal determined through three-photon carrier generation

Charge carrier generation in p-terphenyl crystal is studied employing pulsed excitation with a dye laser. The number of photogenerated cariers is found to be proportional to I³, where I is the intensity of the exciting light at 590 nm. The fluorescence intensity is proportional to I² under the same conditions. The results are interpreted in terms of the photoionization of two-photon excited singlet excitons. A coefficient of (2.1 ± 0.8) × 10^?50 cm⁴ s/photon for the two-photon absorption at 5 90 nm in p-terphenyl crystal is obtained.     

Two-photon spectroscopic behaviors and photodynamic effect on the BEL-7402 cancer cells of the new chlorophyll photosensitizer

The spectroscopic properties of a new chlorophyll derivate photosensitizer(CDP) are studied under the excitation wavelengths at 800 and 400 nm using femtosecond pulses from a Ti:sapphire laser.The damaging effect of CDP on the BEL-7402 cancer cells is also investigated upon two-photon illumination at 800 nm.The normalized fluorescence spectra of CDP in tetrahydrofuran(THF) show that two-photon and one-photon spectra have the same distributions and the same emission bands(675 nm).The life-times of two-and one-photon induced fluorescence of this molecule are of the order of 5.0 ns.By comparing the data it is shown that there is some difference between the two lifetimes,but the differ-ence is less than one nanosecond.The two-photon absorption cross section of the molecule is also measured at 800 nm and estimated as about σ′2 ≈ 31.5×10-50 cm4·s·photon-1.The results of two-photon photodynamic therapy(TPPDT) tests show that CDP can kill all of the tested cancer cells according to the usual Eosine assessment.Our results indicate that the two-photon-induced photophysical,photo-chemical and photosensitizing processes of CDP may be basically similar to those of one-photon ex-citation.These behaviors of the sample suggest that one may find other possible methods to estimate some photosensitizers' effects in details such as their distribution in cells and the reactive targets of the sub-cellular parts of some tumor cells via two-photon excitation techniques.     

The two-photon excitation cross section of 6MAP, a fluorescent adenine analogue

6MAP is a fluorescent analogue of adenine that undergoes Watson-Crick base pairing and base stacking in double-stranded DNA. The one-photon absorption spectrum of 6MAP is characterized by a maximum around 330 nm with moderate quantum yield fluorescence centered at about 420 nm. To take advantage of this probe for confocal and single-molecule microscopy, it would be advantageous to be able to excite the analogue via two photons. We report the first determination of the two-photon excitation cross section and spectrum for 6MAP from 614 to 700 nm. The power dependence of the fluorescence indicates that emission results from the absorption of two photons. The one-photon and two-photon emission line shapes are identical within experimental error. A study of the concentration dependence of the fluorescence yield for one-photon excitation shows no measurable quenching up to about 5 microM. The maximum in the two-photon excitation spectrum gives a two-photon cross section, delta(TPE), of 3.4 +/- 0.1 Goeppert-Mayer (G.M.) at 659 nm, which correlates well with the one-photon absorption maximum. This compares quite favorably with cross sections of various naturally fluorescent biological molecules such as flavins and nicotiamide. In addition, we have also obtained the two-photon-induced fluorescence emission spectrum of quinine sulfate. It is approximately the same as that for one-photon excitation, suggesting that two-photon excitation of quinine sulfate may be used for calibration purposes.     

Visible fluorescence spectrometry using second harmonic emission self-induced in semiconductor laser as a light source

Second harmonic emission (416 or 453 nm) self-induced in a nearinfrared semiconductor laser (832 or 906 nm) is used as a light source for excitation of the fluorescent molecules which have absorption bands in the visible region. The conversion efficiency from fundamental to second harmonic emission is 1.7 × 10^–11 (0.5 pW) for a continuous wave (CW) laser, when it is operated at 30 mW. This value is further improved for a pulsed laser operated at a peak power of 10 W. Perylene is used as a standard sample for construction of an analytical curve. The detection limit is 10^–6 M for CW laser excitation. The present fluorimetric system is used for measurements of pH dependence of the fluorescence intensity for 8-hydroxy-1,3,6-pyrenetrisulfonic acid (HPTS). Neutralization titration is demonstrated by using HPTS as a pH indicator.     

Determination of resveratrol in wine by photochemically induced second-derivative fluorescence coupled with liquid–liquid extraction

Basic studies on the photochemical behaviour of trans-resveratrol and its photoproduct are reported. Photometrically and fluorimetrically calculated acidity constants of the former were determined. The usefulness of the determination of resveratrol by photochemically induced fluorescence and second-derivative photochemically induced fluorescence was also examined. The very weakly fluorescent trans-resveratrol is converted into a highly fluorescent photoproduct by irradiating hydroethanolic solutions of trans-resveratrol containing 40% v/v of ethanol for 60 s with intense UV radiation. The photoproduct presents excitation and emission maxima centred at 260 nm, and 364 and 382 nm, respectively. Under these conditions, a linear relationship between fluorescence intensity and trans-resveratrol concentration was found between 6.6 and 66 ng mL⁻¹. Optimum conditions for the extraction of trans-resveratrol from an aqueous phase at pH 5.0 with diethylether were a phase ratio (aqueous/organic) of 2, a shaking time of 60 s and a buffer concentration of 0.15 mol L⁻¹. An extraction recovery of 100% was reached under these conditions. The optimized extraction procedure was applied to the analysis of resveratrol in wine samples, employing the amplitude between 356 and 364 nm of the second-derivative photoinduced emission spectrum as analytical signal. It was found that there is not matrix effect and recoveries around 100% were obtained at different fortification levels.     

两种吩噻嗪衍生物的合成，光学性质及其电化学性质

The synthesis, luminescence and electrochemical properties of two novel phenothiazine derivatives were presented, which exhibit good one-photon fluorescence emission. The quantum yields and solvent effects of them were studied in detail. Compound 3,7-bis（2-4＇-imidazolylbenzylidenehydrazonoethyl）-（0-ethylphenothiazine） （5） was used as an effective initiator, of which two-photon fluorescence spectra were investigated under 800 nm fs laser pulse and the measured two-photon absorption cross-section was 18 × 10^-50 cm^4·s per photon. Two-photon initiated polymerization microfabrication experiments were carried out. X-ray diffraction analyses revealed that the shape of the compound 3,7-bis（2-pyrid-4＇-ylmethylidenehydrazonoethyl）-（0-ethylphenothiazine） （4） looks like a butterfly with nearly planar wings. The dihedral angle of the two benzene rings is 37.6° and there is an obvious π-π stacking interaction between the molecules in the crystal.     

Upconverting‐Nanoparticle‐Assisted Photochemistry Induced by Low‐Intensity Near‐Infrared Light: How Low Can We Go?

Upconverting nanoparticles (UCNPs) convert near‐infrared (NIR) light into UV or visible light that can trigger photoreactions of photosensitive compounds. In this paper, we demonstrate how to reduce the intensity of NIR light for UCNP‐assisted photochemistry. We synthesized two types of UCNPs with different emission bands and five photosensitive compounds with different absorption bands. A λ=974 nm laser was used to induce photoreactions in all of the investigated photosensitive compounds in the presence of the UCNPs. The excitation thresholds of the photoreactions induced by λ=974 nm light were measured. The lowest threshold was 0.5 W cm^?2, which is lower than the maximum permissible exposure of skin (0.726 W cm^?2). We demonstrate that low‐intensity NIR light can induce photoreactions after passing through a piece of tissue without damaging the tissue. Our results indicate that the threshold for UCNP‐ assisted photochemistry can be reduced by using highly photosensitive compounds that absorb upconverted visible light. Low excitation intensity in UCNP‐assisted photochemistry is important for biomedical applications because it minimizes the overheating problems of NIR light and causes less photodamage to biomaterials.     

Structure elucidation of laser dye coumarin-540A by joint application of X-ray diffraction, X-ray fluorescence, prompt fluorescence, UV and visible spectroscopy

X-ray, ultraviolet, and visible light induced photophysical changes of coumarin-540A in ethanol have been studied by the joint applications of X-ray, ultraviolet, and visible spectroscopy. Some impurities were found by X-ray fluorescence measurements. During the high power optical pumping, coumarin showed photochemical changes. Photoproduct emission spectra characteristics showed that photoproduct molecules can also be used as a laser dye at a different emission frequency in the ultraviolet region.     

Identification and reactivity of the triplet excited state of 5-hydroxytryptophan

Both the neurotransmitter serotonin and the unnatural amino acid 5-hydroxytryptophan (5HT), contain the 5-hydroxyindole chromophore. The photochemistry of 5HT is being investigated in relation to the multiphoton excitation of this chromophore to produce a characteristic photoproduct with green fluorescence ('hyperluminescence'). Laser flash photolysis (308 nm) of 5HT in aqueous solution at neutral pH produces both the neutral 5-indoloxyl radical (lambda(max) 400-420 nm) and another transient absorption with lambda(max) 480 nm and lifetime of 2 micros in deaerated solutions. Based on quenching by oxygen and beta-carotene, the species at 480 nm is identified as the triplet excited state of 5HT. In acidic solution a new oxygen-insensitive intermediate with lambda(max) 460 is assigned to the radical cation of 5HT. Time-resolved measurements of luminescence at 1270 nm have shown that the triplet state of 5HT is able to react with oxygen to form singlet excited oxygen (1O2*) with a quantum yield of approximately 0.1. However, 5HT has also been found to be an effective quencher of singlet oxygen with a second order rate constant of 1.3 x 10(8) dm3 mol(-1) s(-1). The results are discussed in the light of recent observations on the multiphoton-excited photochemistry of serotonin.     

Measurement of uranium isotope ratios in solid samples using laser ablation and diode laser-excited atomic fluorescence spectrometry

A diode laser is used for the selective excitation of ²³⁵U and ²³⁸U in a laser-induced plasma applying Nd:YAG laser pulses to UO₂ samples. The diode laser is rapidly scanned immediately following each laser sampling and the resonance atomic fluorescence spectrum for both isotopes is obtained on a pulse-to-pulse basis. Time-integrated measurements, with the diode laser fixed at either isotope, were also made. Optimum signal-to-noise was obtained at a distance of 0.8 cm from the sample surface, a pressure of 0.9 mbar and a Nd:YAG laser pulse energy of 0.5 mJ (880 MW cm⁻²). Three samples with 0.204, 0.407 and 0.714% ²³⁵U were measured. For example, for the UO₂ pellet with the natural uranium isotopic composition (99.281% ²³⁸U and 0.714% ²³⁵U), the accuracy and precision were 7% and 5% (460 shots), respectively, limited by the continuum emission background from the laser-induced plasma.     

BiF<Subscript>3</Subscript>:Ho<Superscript>3+</Superscript> system for upconversion of 2-μm laser radiation into visible emission

Ho³⁺-doped bismuth(III) fluoride was suggested for upconversion of 2-μm laser radiation (1.9–2.1 µm) into visible emission. The process is possible owing to direct excitation of the ⁵I₇ level, followed by the excitation of the ⁵F₅ level with the emission threshold of 1.4 W.     

Two-photon absorption chromophores with a tunable [2,2′]bithiophene core

Sonogashira coupling between 3,5,3′,5′-tetrabromo-[2,2′]bithiophene and various terminal alkynes provides two-photon absorption (TPA) chromophores 1-6, which possess electron donor (D) and/or acceptor (A) alkynyl substituents at 3(3′) and 5(5′) sites of the bithiophene core. The up-converted fluorescence emission excited at 800 nm (Ti:sapphire femtosecond laser, ∼100 fs pulses) was used to determine the two-photon absorption cross-sections (σ) of these compounds. The corresponding TPA cross-section (σ) values ranging from 132 to 1120 GM (10⁻⁵⁰ cm⁴ s photon⁻¹) can be fine-tuned by the substitutents. The quadrupolar-type (A-π-D-π-A) chromophore 5 exhibits the largest σ value (1120 GM) in CH₂Cl₂ upon 800 nm excitation.     

NIR-Ⅱ Excitation and NIR-I Emission Based Two-photon Fluorescence Lifetime Microscopic Imaging Using Aggregation-induced Emission Dots

Near-infrared(NIR)lights are powerful tools to conduct deep-tissue imaging since NIR-Ⅰ wavelengths hold less photon absorption and NIR-Ⅱ wavelengths serve low photon scattering in the biological tissues compared with visible lights.Two-photon fluorescence lifetime microscopy(2PFLM)can utilize NIR-Ⅱ excitation and NIR-Ⅰ emission at the same time with the assistance of a well-designed fluorescent agent.Aggregation induced emission(AIE)dyes are famous for unique optical properties and could serve a large two-photon absorption(2PA)cross-section as aggregated dots.Herein,we report two-photon fluorescence lifetime microscopic imaging with NIR-Ⅱ excitation and NIR-Ⅰ emission using a novel deep-red AIE dye.The AIE-gens held a 2PA cross-section as large as 1.61×10⁴GM at 1040 nm.Prepared AIE dots had a two-photon fluorescence peak at 790 nm and a stable lifetime of 2.2 ns under the excitation of 1040 nm femtosecond laser.The brain vessels of a living mouse were vividly reconstructed with the two-photon fluorescence lifetime information obtained by our home-made 2PFLM system.Abundant vessels as small as 3.17μm were still observed with a nice signal-background ratio at the depth of 750μm.Our work will inspire more insight into the improvement of the working wavelength of fluorescent agents and traditional 2PFLM.     

Two-photon excitation induced fluorescence of a trifluorophore-labeled DNA

Two-photon excitation of a trifluorophore (6-carboxyfluorescein, N,N,N',N'-tetramethyl-6-carboxyrhodamine and cyanine-5 monofunctional dye) labeled DNA, which has a scaffold of 26 nucleotides, was achieved using focused laser light of a Q-switched Nd-YAG laser (1064 nm). The observed fluorescence signature (emission ratio from the three fluorophores) of the labeled DNA after two-photon excitation is very different from the fluorescence signatures produced by one-photon excitation at different wavelength. The additional fluorescence signatures produced by two-photon excitation of the fluorescent oligonucleotides will facilitate their use as combinatorial fluorescence energy transfer tags for multiplex genetic analysis.