Fluorescence resonance energy transfer in a novel two-photon absorbing system

A novel fluorescence resonance energy transfer (FRET) system containing a two-photon absorbing dye and a nile red chromophore has been synthesized. Upon two-photon excitation by laser at 815 nm this molecule displays efficient energy transfer from the two-photon absorbing dye to the nile red moiety, with an 8-fold increase in emission compared to the model compound. Similarly, single-photon excitation of the two-photon absorbing moiety at 405 nm results in >99% energy-transfer efficiency, along with a 3.4-fold increase in nile red emission compared to direct excitation of the nile red chromophore at 540 nm. This system provides an effective way to use IR radiation to excite molecules that, by themselves, have little or no two-photon absorption.     

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.     

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.     

A femtosecond study of excitation wavelength dependence of solvation dynamics in a PEO-PPO-PEO triblock copolymer micelle

Excitation wavelength (lambdaex) dependence of solvation dynamics of coumarin 480 (C480) in the micellar core of a water soluble triblock copolymer, PEO20-PPO70-PEO20 (Pluronic P123), is studied by femtosecond and picosecond time resolved emission spectroscopies. In the P123 micelle, the width of the emission spectrum of C480 is found to be much larger than that in bulk water. This suggests that the P123 micelle is more heterogeneous than bulk water. The steady state emission maximum of C480 in P123 micelle shows a significant red edge excitation shift by 25 nm from 453 nm at lambdaex=345 nm to 478 nm at lambdaex=435 nm. The solvation dynamics in the interior of the triblock copolymer micelle is found to depend strongly on the excitation wavelength. The excitation wavelength dependence is ascribed to a wide distribution of locations of C480 molecules in the P123 micelle with two extreme environments-a bulklike peripheral region with very fast solvent response and a very slow core region. With increase in lambdaex, contribution of the bulklike region having an ultrafast component (< or =2 ps) increases from 7% at lambdaex=375 nm to 78% at lambda(ex)=425 nm while the contribution of the ultraslow component (4500 ps) decreases from 79% to 17%.     

Red to blue tunable upconversion in Tm3+-doped ZrO2 nanocrystals

The effect of dopant concentration on the blue upconversion (UPC) emission of Tm(3+) -doped ZrO(2) nanocrystals under different excitation wavelengths in the red region is reported. The UPC emissions are due to the f-f electronic transitions from excited states (1)G(4) and (1)D(2) of Tm(3+). We observed a chromatic change in the UPC with tuning the excitation wavelength. The UPC emission bands at 475, 488, and 501 nm are observed under excitation at 649 nm, but bands centered at 454 and 460 nm are observed when the excitation wavelength is tuned to 655 nm. The UPC emission could be tuned from 501 to 454 nm ( approximately 47 nm) by changing the excitation wavelength from 649 to 655 nm ( approximately 6 nm). The pump power dependence of the emission bands at 475, 488, and 501 nm were investigated on excitation intensity at 649 nm, and the emission bands at 454 and 460 nm are investigated on excitation intensity at 655 nm, which confirms that all of these UPC emission lines are a two-photon absorption process.     

Two-photon absorption cross section determination for fluorene derivatives: analysis of the methodology and elucidation of the origin of the absorption processes

A comprehensive analysis of the well-known open aperture Z-scan method, using a modified equation for the change in transmittance, is presented and accounts for discrepancies in two-photon absorption (2PA) cross sections between picosecond and femtosecond excitation. This new approach takes into account excited-state absorption and stimulated emission of the molecules studied. The two-photon absorption cross-section spectra of a series of six fluorene-based derivatives, determined using picosecond pulses, over a broad spectral range (500-900 nm), and this approach using a modified fitting procedure in the open aperture Z-scan is reported. We demonstrate that the fluorene derivatives exhibit two-photon absorption cross-section values between 700 and 5000 GM, when excited into the two-photon allowed electronic state. Excitation anisotropy spectra, measured to investigate the nature of the observed linear and nonlinear absorption bands, are presented and provide insight into the 2PA process.     

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.     

Two-photon Lysotrackers for in vivo imaging

We report two-photon Lysotrackers (CLT-blue and CLT-yellow) that can be excited by 750-840 nm femtosecond laser pulses and emit at 470 and 550 nm, respectively. They can be easily loaded into cells and tissue slices for visualization of lysosomes in live cells and tissues for a long period of time through two-photon microscopy. When combined with appropriate two-photon probes for other biological targets, these novel probes would greatly facilitate the two-photon microscopy colocalization experiments.     

A two-photon antenna for photochemical delivery of nitric oxide from a water-soluble, dye-derivatized iron nitrosyl complex using NIR light

The experiments described here demonstrate the use of two-photon excitation (TPE) to sensitize nitric oxide (NO) release from a dye-derivatized iron/sulfur/nitrosyl cluster Fe2(mu-RS)2(NO)4 (Fluor-RSE, RS = 2-thioethyl ester of fluorescein) with near-infrared (NIR) light in the form of femtosecond pulses from a Ti:sapphire laser. TPE at 800 nm leads both to weak fluorescence from the organic chromophore at lambda(max) = 532 nm and to NO labilization from the cluster. Since the emission from the reference compound Fluor-Et (the ethyl ester of fluorescein) under identical conditions (50/50 CH3CN/phosphate buffer (1 mM) at pH 7.4) is considerably more intense, the weaker emission from Fluor-RSE and the NO generation indicate that the fluorescein excited states initially formed by TPE are largely quenched by energy transfer to the cluster core. The two-photon absorption (TPA) cross section of Fluor-RSE at 800 nm was determined to be delta = 63 +/- 7 GM via the TPA photoluminescence technique. This can be compared to the TPA cross section of 36 GM reported for fluorescein dye in pH 11 aqueous solution and of 32 +/- 3 GM for Fluor-Et measured under conditions comparable to those used for Fluor-RSE. Pulse intensity dependence studies showed that the quantity of NO released from the latter as the result of NIR photoexcitation follows a quadratic relationship to excitation intensity, consistent with the expectation for a TPE process. These studies demonstrate the potential utility of a two-photon antenna for sensitization of the photochemical release of an active agent (in this case, NO) from a photoactive pro-drug.     

HYDRATED ELECTRON FORMATION IN NANOSECOND and PICOSECOND LASER FLASH PHOTOLYSIS OF HEMATOPORPHYRIN IN AQUEOUS SOLUTION

Nanosecond (lambda exc = 266, 355 and 532 nm) and picosecond (lambda exc = 355 nm) laser flash photolysis of hematoporphyrin (Hp) was performed in neutral (pH 7.4) and alkaline (pH 12) aqueous solution, as well as in the presence of 0.1% Triton X-100. The dependence of the yield of photoproduced hydrated electrons (e-aq) on laser pulse energy was studied over a wide range of energies (0.2 to greater than 1000 mJ cm-2). The results show that e-aq are predominantly formed in a two-photon process at lambda exc = 266 and 355 nm. One-photon quantum yields are higher at lambda exc = 266 nm than at lambda exc = 355 nm. Both one-photon and two-photon pathways are less efficient at higher Hp concentration, reflecting the influence of Hp self-aggregation. Two-photon e-aq formation is more efficient when 30 ps pulses are used for excitation, as compared to 10 ns pulses. No e-aq could be detected at lambda exc = 532 nm. Nanosecond pulse-induced transient spectra obtained at pH 7.4 are also discussed.     

Molecular photobleaching kinetics of Rhodamine 6G by one- and two-photon induced confocal fluorescence microscopy.

Under high-excitation irradiance conditions in one- and two-photon induced fluorescence microscopy, the photostability of fluorescent dyes is of crucial importance for the detection sensitivity of single molecules and for the contrast in fluorescence imaging. Herein, we report on the dependence of photobleaching on the excitation conditions, using the dye Rhodamine 6G as a typical example. The different excitation modes investigated include 1) one-photon excitation into the first-excited singlet state in the range of 500 to 528 nm by continuous wave and picosecond-pulsed lasers and 2) two- and one-photon excitation to higher-excited singlet states at 800 and 350 nm, respectively, by femtosecond pulses. Experimental strategies are presented, which allow resolving the photophysics. From single-molecule trajectories and fluorescence correlation spectroscopy, as well as with a simple theoretical model based on steady-state solutions of molecular rate equation analysis, we determined the underlying photobleaching mechanisms and quantified the photokinetic parameters describing the dependence of the fluorescence signal on the excitation irradiance. The comparison with experimental data and an exact theoretical model show that only minor deviations between the different theoretical approaches can be observed for high-pulsed excitation irradiances. It is shown that fluorescence excitation is in all cases limited by photolysis from higher-excited electronic states. In contrast to picosecond-pulsed excitation, this is extremely severe for both one- and two-photon excitation with femtosecond pulses. Furthermore, the photostability of the higher-excited electronic states is strongly influenced by environmental conditions, such as polarity and temperature.     

Examination of the Spatial Distribution of Dyes and Polymers in Thin Films by Two-Photon Microscopy

 Two-photon absorption induced fluorescence microscopy was used as a tool for the examination of the spatial distribution of a thin dye film. The two-photon absorption induced fluorescence signal is essentially the same as that produced by excitation with a single photon of equivalent energy. When femtosecond pulses are focused into a sample there is an intrinsic spatial selectivity of the two-photon emission signal, since it is dependent upon the square of the light intensity. This has tremendous implications in fluorescence microscopy. Since two-photon absorption is confined in a small region at the focal waist of an objective lens, photodamage and photobleaching of the sample are significantly reduced. In addition, the two-photon signal has inherent z-axis spatial resolution, which facilitates the construction of 3-D images. In the present work an application of this technique to a thin film of a dye is presented. The method can generally be applied to thin films made from photonic polymers.     

A two-photon fluorescent probe for ratiometric imaging of hydrogen peroxide in live tissue

We report a two-photon fluorescent probe (PN1) that can be excited by 750 nm femto-second pulses, shows high photostability and negligible toxicity, and can visualize H(2)O(2) distribution in live cells and tissue by two-photon microscopy.     

RAPID COMMUNICATION

Abstract— We show that the calcium fiuorophore Indo-1 can be excited by simultaneous absorption of three-photons at 885 nra, a wavelength readily available from Ti:sapphire lasers. Three-photon excitation was demonstrated by the emission intensity of Indo-1 which depended on the cube of the laser power, and by a higher anisotropy than was observed for two-photon excitation. Excitation of Indo-1 becomes a two-photon process when the wavelength is decreased to 820 nm. Three-photon excitation was accomplished at a low 17μ concentration of Indo-1. Examination of the spatial profile of the excited Indo-1 showed a smaller volume for three- versus two-photon excitation. These results suggest that three-photon excitation may be useful in fluorescence microscopy using the long wavelength output of Tksapphire lasers, and may provide higher spatial resolution than available using two-photon excitation.     

Tracking of mercury ions in living cells with a fluorescent chemodosimeter under single- or two-photon excitation

Tracking of Hg²⁺ in solutions as well as in living cells was conducted with a fluorescent chemodosimeter by measuring the spectral shift of its fluorescence under single- or two-photon excitation. The spectral hypsochromic shifts of this chemodosimeter when reacting with Hg²⁺ were found to be about 50 nm in acetonitrile/water solutions and 32 nm in Euglena gracilis 277 living cells. This chemodosimeter shows high sensitivity and selectivity, and is not influenced by the pH values. It can signal Hg²⁺ in solutions down to the ppb range under either single-photon excitation (SPE) at 405 nm or two-photon excitation (TPE) at 800 nm. However, with low cellular chemodosimeter concentrations, the SPE spectra were disturbed by the auto-fluorescence from the native fluorophore in the cell, while the TPE spectra were still of high quality since the two-photon absorption cross section of this chemodosimeter is much larger than that of the native fluorophores in the cell.     

Photophysical properties of blue - emitting silicon nanoparticles

Silicon nanoparticles with strong blue photoluminescence were synthesized by electrochemical etching of silicon wafers and ultrasonically removed under N(2) atmosphere in organic solvents to produce colloids. Thermal treatment leads to the formation of colloidal Si particles of 3 ± 1 nm diameter, which upon excitation with 340 - 380 nm light exhibited room temperature luminescence in the range from 400 to 500 nm. The emission and the one- and two-photon excitation spectra of the particles are not sensitive to surface functionalization with methyl 2-methylprop-2-enoate. However, the derivatized particles show higher emission quantum yields in air-saturated suspensions (44%) than the underivatized particles (27%), as well as higher stability of its dispersions.FTIR and XPS spectra indicate a significant surface oxidation of the particles. The Si:O:C ratio at the surface of the derivatized particles estimated from XPS is Si(3)O(6)(C(5)O(2)H(y))(1), with y = 7 - 8. Vibronic spacing is observed in both the emission and excitation spectra. The information obtained from one-photon excitation experiments (emission and excitation spectra, photoluminescence quantum yields, luminescence decay lifetimes and anisotropy correlation lifetimes), as well as from two-photon excitation fluorescence correlation spectroscopy (brightness and diffusion coefficients) and TEM indicate that the blue-emitting particles are monodisperse and ball-shaped. Particle size clearly determines the emission and excitation spectral region, as expected from quantum confinement, but the presence and extent of Si-O species on the silicon networks seem crucial for determining the spectrum features and intensity of emission. The nanoparticles could hold great potential as quantum dots for applications as luminescence sensors in biology and environmental science.     

Examination of the Spatial Distribution of Dyes and Polymers in Thin Films by Two-Photon Microscopy

Summary.  Two-photon absorption induced fluorescence microscopy was used as a tool for the examination of the spatial distribution of a thin dye film. The two-photon absorption induced fluorescence signal is essentially the same as that produced by excitation with a single photon of equivalent energy. When femtosecond pulses are focused into a sample there is an intrinsic spatial selectivity of the two-photon emission signal, since it is dependent upon the square of the light intensity. This has tremendous implications in fluorescence microscopy. Since two-photon absorption is confined in a small region at the focal waist of an objective lens, photodamage and photobleaching of the sample are significantly reduced. In addition, the two-photon signal has inherent z-axis spatial resolution, which facilitates the construction of 3-D images. In the present work an application of this technique to a thin film of a dye is presented. The method can generally be applied to thin films made from photonic polymers. Received June 23, 2000. Accepted (revised) July 31, 2000     

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.     

Enhanced two-photon singlet oxygen generation by photosensitizer-doped conjugated polymer nanoparticles

We have prepared photosensitizer-doped conjugated polymer nanoparticles by using a reprecipitation method. The conjugated polymer, poly[9,9-dibromohexylfluorene-2,7-ylenethylene-alt-1,4-(2,5-dimethoxy)phenylene] (PFEMO), was used as the host matrix to disperse tetraphenylporphyrin (TPP). These TPP-doped PFEMO nanoparticles are stable and have a uniform size of ～50 nm. Efficient intraparticle energy transfer from PFEMO to TPP has been observed. The TPP emission of the nanoparticles was found to be enhanced by 21-fold by PFEMO under two-photon excitation. Enhanced two-photon excitation singlet oxygen generation efficiency in the TPP-doped PFEMO nanoparticles has been demonstrated. Our results suggest that these photosensitizer-doped conjugated polymer nanoparticles can act as novel photosensitizing agents for two-photon photodynamic therapy and related applications.     

Synthesis, structural characterization, photophysics, and broadband nonlinear absorption of a platinum(II) complex with the 6-(7-benzothiazol-2'-yl-9,9-diethyl-9 H-fluoren-2-yl)-2,2'-bipyridinyl ligand

A platinum complex with the 6-(7-benzothiazol-2'-yl-9,9-diethyl-9H-fluoren-2-yl)-2,2'-bipyridinyl ligand (1) was synthesized and the crystal structure was determined. UV/Vis absorption, emission, and transient difference absorption of 1 were systematically investigated. DFT calculations were carried out on 1 to characterize the electronic ground state and aid in the understanding of the nature of low-lying excited electronic states. Complex 1 exhibits intense structured (1)π-π* absorption at λ(abs)<440?nm, and a broad, moderate (1)MLCT/(1)LLCT transition at 440-520?nm in CH(2)Cl(2) solution. A structured (3)π-π*/(3)MLCT emission at about 590?nm was observed at room temperature and at 77?K. Complex 1 exhibits both singlet and triplet excited-state absorption from 450?nm to 750?nm, which are tentatively attributed to the (1)π-π* and (3)π-π* excited states of the 6-(7-benzothiazol-2'-yl-9,9-diethyl-9H-fluoren-2-yl)-2,2'-bipyridine ligand, respectively. Z-scan experiments were conducted by using ns and ps pulses at 532?nm, and ps pulses at a variety of visible and near-IR wavelengths. The experimental data were fitted by a five-level model by using the excited-state parameters obtained from the photophysical study to deduce the effective singlet and triplet excited-state absorption cross sections in the visible spectral region and the effective two-photon absorption cross sections in the near-IR region. Our results demonstrate that 1 possesses large ratios of excited-state absorption cross sections relative to that of the ground-state in the visible spectral region; this results in a remarkable degree of reverse saturable absorption from 1 in CH(2)Cl(2) solution illuminated by ns laser pulses at 532?nm. The two-photon absorption cross sections in the near-IR region for 1 are among the largest values reported for platinum complexes. Therefore, 1 is an excellent, broadband, nonlinear absorbing material that exhibits strong reverse saturable absorption in the visible spectral region and large two-photon-assisted excited-state absorption in the near-IR region.