CALCIUM-DEPENDENT FLUORESCENCE LIFETIMES OF INDO-1 FOR ONE- AND TWO-PHOTON EXCITATION OF FLUORESCENCE

Abstract— We characterized the fluorescence intensity decays of Indo-1, which is commonly used as an emission wavelength-ratiometric calcium probe. The apparent lifetime of the long-wavelength side of the emission of Indo-1 is dependent on Ca²⁺. This long-wavelength emission displays the characteristics of an excited-state reaction, that is, a negative preexponential component in thc multiexponential analysis. The emission spectra and lifetime of Indo-1 appear to be identical for one-photon and two-photon excitation at 351 and 702 mn, respectively, suggesting that the relative one- and two-photon cross sections are similar for the calcium-free and calcium-bound forms of Indo-1. Also, the two-photon cross section of Indo-1 is relatively high, about 4 × 10⁻⁴⁹ cm⁴ s/photon molecule at 690 nm for both the calcium-free and calcium-bound forms. Hence, Indo-1 can be used for calcium imaging based on one- or two-photon excitation, using either emission wavelength ratios or lifetime imaging methods.     

FLUORESCENCE QUANTUM YIELDS OF 124-kDa PHYTOCHROME FROM OAT UPON EXCITATION WITHIN DIFFERENT ABSORPTION BANDS

Abstract— A fluorescence quantum yield (emission at650–850 nm) of π= (2.3 ± 0.3)10⁻³ was measured for the red-absorbing form (Pr) of 124-kDa phytochrome from etiolated oat seedlings ( Avena sativa ) upon excitation in the Soret band at Λ^exc= 380 nm. The small difference between this value and the previously determined quantum yield with Λ^exc= 640 nm, π= (3.5 ± 0.4)10⁻³is attributed to a blue-absorbing emitter responsible for the "anomalous" or "blue" emission of the chromoprotein in the region from ca. 400 to 550 nm. The absorption of Pr at 380 nm is consequently somewhat lower than that measured directly from the spectrum. Processes from upper excited states of the Pr phytochromobilin-derived chromophore other than rapid relaxation to the emitting state are not important. A quantum yield of Φ ' 1.2 times 10⁻³ is estimated for the blue fluorescence. The proportion of the blue emitters relative to Pr appears to be relatively high.     

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.     

Multiphoton-Excited Visible Emission by Serotonin Solutions

Nonlinear excitation of the neurotransmitter serotonin (5HT) in aqueous solution is shown to generate a blue-green-emitting photoproduct in addition to UV fluorescence characteristic of native 5HT. The visible emission rate in diffusional steady-state measurements scales as the sixth power of excitation intensity, demonstrating that absorption of six near-IR photons is required to generate emission of one visible photon. Transient measurements reveal that this process is composed of two sequential nonlinear steps, the first excited by four photons and the second by two photons. These results, in combination with measurements of multiphoton-excited serotonin UV fluorescence, support a model in which 5HT is photochemically transformed as a consequence of four-photon absorption (E_tot?6 eV) to a photoproduct that then emits in the visible region via two-photon excitation. A minimum bound of ?10^-51 cm⁴ s photon^-1 is observed for the two-photon emission action cross section at 830 nm. Photoionization, rather than reaction with a dissolved oxygen species, appears to be the primary mechanism for generation of the blue-green-emitting photoproduct. The peak intensities required to generate significant blue-green emission (?5 times 10¹¹ W cm^-2 from 80 MHz 150 fs titanium: sapphire laser pulses) are approximately five-fold higher than are typically used in two-photon laser scanning microscopy but are still substantially lower than the estimated intensity needed to induce dielectric breakdown of water.     

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.     

Irradiation of DNA with 193 nm Light Yields Formamidopyrimidine-DNA Glycosylase (Fpg) Protein-Sensitive Lesions

Abstract— Irradiation of aqueous solutions of plasmid DNA (pUC18) at pH 7.6 with 193 nm laser light results in low yields of prompt single strand breakage (air-saturated sample φ_ssb= [1.5 ± 0.1] ± 10⁻⁴, argon-saturated sample φ_ssb= [0.9 ± 0.1] ± 10⁻⁴). Treatment of the irradiated DNA samples with Escherichia coli formamidopyrimi-dine-DNA glycosylase (Fpg) protein results in an approximate 20-fold increase in the yield of single strand breakage (air-saturated sample φ_fpg= [33.1 ± 3.1] ± 10⁻⁴, argon-saturated sample φ_fpg= [23.8 ± 2.6] × 10 ⁴). This result indicates that 193 nm light induces other modification) (most likely of the purine moieties) that are 20 times more abundant than prompt strand breakage within the DNA matrix.     

LASER PHOTOLYSIS STUDIES OF SUPEROXIDE ADDUCTS OF COBALT(II) AND ZINC(II) PORPHYRINS IN DIMETHYLFORMAMIDE

Abstract— Photochemistry of superoxide adducts of cobalt(II) and zinc(II) porphyrins has been studied by laser photolysis. It was found that the former in dimethlformamide photodissociates the superoxide anion radical, O₂^-, with the quantum yield of 0.5 ± 0.05 at the excitation wavelenths 355 and 532 nm, and the latter gives flurescence and the triple state without giving rise to the photodissociation of O₂^-     

Lutetium Texaphyrin (PCI-0123): A Near-Infrared, Water-Soluble Photosensitizer

Lutetium texaphyrin, PCI-0123, is a pure, water-soluble photosensitizer with a large broad absorption band centered at 732 nm. The compound was tested for photodynamic therapy (PDT) effectiveness in a murine mammary cancer model. The texaphyrin macrocycle as illustrated by magnetic resonance imaging and ¹⁴C-radiolabeled texaphyrin studies was shown to be tumor selective; a tumor-to-muscle ratio of 10.55 was seen after 5 h. Lutetium texaphyrin, at a drug dose of 20 μmol/kg with irradiation 5 h postinjection at 150 J/cm² and 150 mW/cm², had significant efficacy (P < 0.0001) in treating neoplasms of moderate size (40 ± 14 mm³) and also had significant efficacy ( P < 0.0001) in treating larger neoplasms (147 ± 65 mm³). The PDT efficacy was correlated with the time interval between PCI-0123 administration and light exposure. A 100% cure rate was achieved when photoirradiation took place 3 h postinjection compared to 50% for 5 h using 10 μmol/kg and 150 J/cm² at 150 mW/cm². The PDT efficacy was attributable to the selective uptakehetention of the texaphyrin photosensitizer in addition to the depth of light penetration achievable at the 732 nm laser irradiation.     

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.     

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 studies of hypocrellins for photodynamic therapy

The photophysical and photochemical properties of hypocrellins (HA and HB) are examined with two-photon excitations at 800 nm using femtosecond pulses from a Ti:sapphire laser. The two-photon excited fluorescence spectra of HA and HB are very similar to those obtained by one-photon excitation, which may indicate that the two-photon induced photodynamic processes of hypocrellins are similar to one-photon induced photodynamic processes. The two-photon excitation cross sections of HA and HB are measured at 800 nm as about 34.8 x 10(-50) cm(4) s/photon and 21.3 x 10(-50) cm(4) s/photon, respectively. The large two-photon cross sections of both HA and HB, suggest that the hypocrellins can be potential two-photon phototherapeutic agents. As an example for two-photon photodynamic therapy of hypocrellins, we also further examine the cell-damaging effects of HA upon two-photon illumination. Our preliminary results of cell viability test indicate hypocrellins can effectively damage the Hela cells under two-photon illumination.     

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.     

双光子聚合引发剂BVPDA的合成、结构及非线性光学性质

合成了双光子聚合引发剂{4-[2-(4-溴苯基)-乙烯基]苯基}-二苯基胺(BVPDA),并测定了其晶体结构.结果表明,BVPDA为三斜晶系,P1空间群,a=1.0834(3)_nm,b=1.5625(2)_nm,c=1.9640(2)_nm,α=92.807(8)°,β=103.647(10)°,γ=106.676(13)°,V=3.0705(10)_nm³,Z=6,T=293(2)K,Dc=1.383g/cm³,R₁=0.0735,wR=0.1063.用¹HNMR谱、¹³CNMR谱及元素分析进行了表征.测试了紫外吸收光谱、单光子荧光光谱、单光子荧光寿命和双光子荧光光谱.在760nm的飞秒脉冲激光激发下,BVP-DA发出较强的上转换荧光,荧光峰位于462nm.以BVPDA作引发剂,加入丙烯酸酯型齐聚物(CN120C80),用Ti:sapphire飞秒激光器作光源,制作了一个三维周期性微结构.     

A PHYTOCHROME STUDY USING TWO-LASER/TWO-COLOR FLASH PHOTOLYSIS: I700 IS A MANDATORY INTERMEDIATE IN THE PrPfr PHOTOTRANSFORMATION

Abstract— The phototransformation of native (124 kDa)oat phytochrome, P_r P_fr, Has been studied at 10C by two laser/ two-color flash photolysis. the overall P_rP_fr reaction yield did not vary with temperature within the range4–21C. Foloeing the excitation of P_r with a single 15 ns laser flash at 650nm, the formation of P_fr was quantitavely measured in a time-resolved experiment in the presence of a second 8 ns laser flash at 710 nm delayed from the initial flash. the second laser flash causes at 1.0 s after the initial laser flash a depletion of the uintermediate I₇₀₀ as welll as a reduction of the P_fr absorption at 730 nm. The depletion of I₇₀₀ correlates quantitavely with the reduction of P_fr formation. The absorpton spectra of I₇₀₀ and of the following intermendiate, I_bi, were calculated assuming that the amount of P_r, which is photoconverted by a single laser, equals the amount of P_fr formed.     

RESOLVED FLUORESCENCE EMISSION SPECTRA OF IRON-FREE CYTOCHROME c

Abstract The fluorescence emission of iron-free cytochrome c (⁰Cyt c ) in a glassy matrix was investigated under conditions of very low temperature (4.2 K.) and narrow bandwidth laser excitation. Excitation into the vibronic band, Q _x (1,0) resulted in highly resolved emission spectra of linewidth 10-20 cm⁻¹. Using the model of selective excitation developed by Abram el al. (1975) and McColgan et al. (1978), the emission spectra of vibronic excitation afforded a method to investigate excited state vibrational structure. Furthermore, emission profiles have shown that in ⁰Cyt c , the site distribution (inhomogeneous broadening) has a width in the order of 200cm⁻¹.     

Generation of DNA photolesions by two-photon absorption of a frequency-doubled Ti:sapphire laser

The formation of spatially localized regions of DNA damage by multiphoton absorption of light is an attractive tool for investigating DNA repair. Although this method has been applied in cells, little information is available about the formation of lesions by multiphoton absorption in the absence of exogenous or endogenous sensitizing agents. Therefore, we have investigated DNA damage induced in vitro by direct two-photon absorption of frequency-doubled femtosecond pulses from a Ti:sapphire laser. We first developed a quantitative polymerase chain reaction assay to measure DNA damage, and determined that the quantum yield of lesions formed by one-photon absorption of 254 nm light is 7.86×10(-4). We then measured the yield of lesions resulting from exposure to the visible femtosecond laser pulses, which exhibited a quadratic intensity dependence. The two-photon absorption cross section of DNA has a value (per nucleotide) of 2.6 GM at 425 nm, 2.4 GM at 450 nm, and 1.9 GM at 475 nm. A comparison of these in vitro results to several in vivo studies of multiphoton photodamage indicates that the onset of DNA damage occurs at lower intensities in vivo; we suggest possible explanations for this discrepancy.     

AGREGATION DES CHLOROPHYLLES IN VITRO-II. PHOTODECOLORATION DE LA CHLOROPHYLLE a ADSORBEE SUR DES PARTICULES DE LIPOPROTEINES EXTRAITES DU LAIT

Abstract— Chlorophyll a (chl a ) adsorbed on milk proteins or lipoproteins has absorption maxima at 437 ± 1 nm and 671 ± 1 nm, whatever its concentration. A 750-nm-absorbing form appears when (chl/proteins) > 2 × 10^-2 mg chl/mg proteins, in the case of lipoproteins; or 4.5 × 10^-3 mg chl/mg proteins, in the case of proteins. In both cases, the apparent molar extinction coefficient at 671 nm is the same (3 × 10⁴ liter mole^-1 cm^-1) at the highest (chl/proteins) ratio for which no 750 nm absorbancy appears. It is shown that adsorbed chl a undergoes irreversible (in the present conditions) photo-oxidation by light in the presence of oxygen. The reaction is second order, with similar rate constants for chl a adsorbed to proteins or to lipoproteins.     

DETECTION OF THE EXCITED SINGLET STATE OF A DEPROTONATED, REDUCED FLAVIN

Abstract— The excited singlet state of a deprotonated, reduced flavin [1, 5-dihydro- N (3)-carboxymethyllumiflavin] in aqueous solution at pH 8 has been detected by laser flash photolysis. The broad absorption band maximized at ∼ 490 nm (ε= 9.9 × 10³ M ^-1 cm^-1). The lifetime of the transient was found to be 100 ± 15 ps. The lifetime was not affected by the presence of pyrimidine dimers, which would be monomerized under these conditions. A longer-lived transient, tentatively identified as the solvated electron, was also detected. The neutral reduced flavin did not give a detectable transient.     

Fluorescence spectral properties of the anticancer drug topotecan by steady-state and frequency domain fluorometry with one-photon and multi-photon excitation

Topotecan is an antitumor agent with activity against a variety of cancers. We examined the steady-state and time-resolved fluorescence spectral properties of topotecan with one- and two-photon excitation. Topotecan was found to display a high two-photon cross section near 20 GM for wavelengths within the fundamental output of a Ti:sapphire laser, 800-880 nm. In frozen solution the anisotropies of topotecan are near the theoretical maxima for one-photon and two-photon excitation with colinear electronic transitions. The intensity and anisotropy decays of topotecan fluorescence were found to be homogeneous (single exponentials) in phosphate-buffered saline and propylene glycol. The steady-state and time-resolved data indicate that topotecan binds to a double-helical DNA oligomer d(AT)10 resulting in increased anisotropies and multiexponential intensity and anisotropy decays. Subnanosecond components in the anisotropy decay of the DNA-topotecan complex suggest loose binding of the drug to DNA. Loose binding of topotecan to DNA is also revealed by accessibility of topotecan to collisional quenching by iodide.     

AUTOFLUORESCENCE SPECTROSCOPY OF OPTICALLY TRAPPED CELLS

Abstract— Cellular autofluorescence spectra were monitored in a single-beam gradient force optical trap ("optical tweezers") in order to probe the physiological effects of near infrared and UVA (320–400 nm) microirradiation. Prior to trapping, Chinese hamster ovary cells exhibited weak UVA-excited autofluorescence with maxima at 455 nm characteristic of β-nicotinamide adenine dinucleotide (phosphate) emission. No strong effect of a 1064 nm NIR microbeam on fluorescence intensity and spectral characteristics was found during trapping, even for power densities up to 70 MW/cm² and radiant exposures of 100 GJ/cm². In contrast to the 1064 nm trap, a 760 nm trapping beam caused a two-fold autofluorescence increase within 5 min (about 20 GJ/cm²). Exposure to 365 nm UVA (1 W/cm²) during 1064 nm trapping significantly altered cellular autofluorescence, causing, within 10 min, a five-fold increase and a 6 nm red shift versus initial levels. We conclude that 1064 nm microbeams can be applied for an extended period without producing autofluorescence changes characteristic of alterations in the cellular redox state. However, 760 nm effects may occur via a two-photon absorption mechanism, which, in a manner similar to UVA exposure, alters the redox balance and places the cell in a state of oxidative stress.