Effect of side groups on two-photon absorption of soluble polythiophenes

The effect of pendant side groups on two-photon absorption cross-section in a number of soluble fluorescent polythiophenes was investigated. Different side groups lead to different planarity and conformation of the polymer. As the side-group-induced planarity of the polymer chain increases, the two-photon absorption cross-section also increases. The increased delocalization of the π-electrons along the polymer backbone enhances the third-order optical nonlinearity. The aggregation of the polymer chain in solution lowers the quantum efficiency of fluorescence and decreases the two-photon absorption cross-section. This study demonstrates a novel method of enhancing the two-photon absorption cross-section by tuning the pendant side group.     

Linear and nonlinear optical properties of two-photon absorption dye doped linear copolymer

2-hydroxyethyl methacrylate (abbreviated as HEMA) and acrylonitrile have been used as the monomers to form a trans-4-[p-(N-hydroxyethyl-N-methylamino)styryl]-N-methylpyridinium p-toluene sulfonate (abbreviated as HMASPS) doped linear copolymer. The spectra of one and two-photon excited fluorescence and two-photon pumped superradiance and two-photon pumped lasing of the polymer all shifted to short wavelengths compared with the solution sample of the dye. The copolymer shows much longer one- and two-photon excited fluorescence lifetimes of nanoseconds. When pumped by the picosecond Nd:YAG laser, the superradiance and lasing can be simultaneously obtained. The maximum two-photon pumped lasing conversion efficiency is 3.3%. The place of the maximum upconversion efficiency of the copolymer does not coincide with that of the maximum nonlinear absorption.     

Two-photon fluorescence measurements in a hexagonal CdS crystal

Measurements of two-photon fluorescence (TPF) in a CdS crystal using a dye laser pumped by a Q-switched ruby laser are reported. The TPF is measured as a function of source intensity and wavelength. The total fluorescence shows linear and non-linear dependence on the source intensity; from these measurements the TPF part is determined. The measured transition of the crystal's fundamental gap is 2.5 ± 0.02 eV and another forbidden one-photon but allowed two-photon transition is found at 3.24 ± 0.02 eV.     

Polarization-induced control of two-photon excited fluorescence in a chiral polybinaphthyl

The fluorescence behavior of a chiral polybinaphthyl excited with 100 fs 800 nm laser pulses was investigated in tetrahydrofuran solution. The peak fluorescence intensity versus the input irradiance was measured to meet a square dependence, giving evidence for two-photon excited fluorescence (TPF). The variations of the TPF intensity were found to be strongly modulated by the different polarized incident lights and tightly depend on the linearly polarized component of the incident light. Furthermore, combining with the characteristics of chiral molecules, the two-photon polarization ratio was studied to reveal the symmetry of the involved excited states.     

DHL细胞中5-ALA代谢PpIX的双光子荧光光谱

双光子激发生物组织荧光,激发光仅作用于焦点区域,对生物样品的光漂白性和光毒性都很小,因而双光子荧光显微技术已成为细胞生物学研究的一种新技术。文章采用波长为820 nm飞秒激光激发孵育有5-ALA的DHL细胞,在激光扫描显微镜的Lambda模式中获得单个DHL细胞的双光子荧光光谱,并测量DHL细胞内积聚的卟啉九(PpIX)特征荧光值。获得了浓度分别为2, 4和10 mmol·L-1的5-ALA溶液中,细胞代谢的PpIX含量随孵育时间的变化情况。DHL细胞内积聚的PpIX处于动态变化过程,并呈现出两阶段性的特点：细胞内积聚的PpIX含量随着孵育时间增长而增加,在3 h附近达到最大值,随后随着孵育时间增长反而下降。结果表明,基于激光扫描显微的双光子荧光光谱可成为DHL细胞等白血病细胞摄取5-ALA并生成PpIX的动力学研究的有效方法。     

Absorption and Emission Properties of a New Two-photon Absorption Dye

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Absorption and Emission Properties of a New Two-photon Absorption Dye

The emission properties of a newly synthesized organic two-photon absorption dye, trans-4-[p-(N-ethyl-N-ethylamino)-styryl]-N-methyl-pyridinium tetraphenylborate, have been investigated. When pumped by infrared pulses from a picosecond Nd∶YAG laser, the dye exhibits intense upconverted fluorescence and strong superradiance properties. For comparison, the one-photon induced fluorescence and superradiance are also measured. The one- and two-photon excited fluorescent lifetimes are 86 and 64 ps, respectively. The maximum efficiency of the dye is measured to be 1.97% and the upconversion efficiencies at different pump wavelengths have also been investigated by the optical parametric amplifier.     

Effects of absorption saturation in colloidal quantum dots as fluorophores for multiphoton fluorescence microscopy

It is shown that colloidal semiconductor nanocrystals (quantum dots), which are promising fluorophores for multiphoton fluorescence microscopy, exhibit a two-photon absorption saturation effect at moderate powers (not exceeding 10 mW) of femtosecond pumping radiation. An analytical expression for the power of two-photon fluorescence of quantum dots as a function of the average pumping power is obtained. With this expression, the deviation of the found dependence from the quadratic law is explained by two factors, i.e., a large two-photon absorption cross section of quantum dots and their slow (compared to the typical pumping pulse-repetition period) relaxation to an unexcited state. Using an LSM 510 Carl Zeiss laser scanning microscope equipped with a Ti:Sa tunable femtosecond laser, a series of model experiments is performed to reveal the saturation effect in a solution of commercially available quantum dots. Good agreement is obtained between the measured dependence of the power of two-photon fluorescence on the average pump power and the theoretical calculation results. It is also experimentally demonstrated that, under fluorescence saturation conditions, the spatial resolution of the method of multiphoton fluorescence microscopy is lost; this effect is analyzed numerically.     

Biosensing based on two-photon fluorescence measurements through optical fibers

We have performed two-photon fluorescence detection in a new scheme in which femtosecond laser pulses were delivered thorugh an optical fiber for nonlinear excitation and the emitted fluorescence was collected through the same fiber. Single-mode fibers were determined to give higher detection efficiency than multimode fibers, consistent with theoretical considerations. The utility of fiber-optic sensing based on two-photon fluorescence detection was proved by an experiment that measured the uptake of a targeted drug delivery agent into cultured cancer cells.     

Three-dimensional reversible laser micromachining with subnanojoule femtosecond pulses based on two-photon photochromism

Three-dimensional reversible laser micromachining of polymer materials based on two-photon photochromism is demonstrated. Unamplified 60-fs, 0.5-nJ pulses of 790-nm Ti:sapphire laser radiation are used to induce a refractive-index change in a polymethyl methacrylate (PMMA) sample doped with spiropyran molecules through a two-photon absorption process. Waveguides are written in the bulk of spiropyran-doped PMMA samples by scanning these samples with respect to a tightly focused Ti:sapphire laser beam. Laser-induced fluorescence is used for on-line monitoring of the laser-micromachining process. The structures written in photochromic samples can be erased and reconfigured due to the reversibility of the photochromic effect. Received: 23 April 2003 / Published online: 6 June 2003 RID="*" ID="*"Corresponding author. Fax: +7-095/939-51-74, E-mail: zheltikov@top.phys.msu.su     

Fabrication of a micro-optical lens using femtosecond laser 3D micromachining for two-photon imaging of bio-tissues

We report, for the first time to our knowledge, on the application of a micro-optical lens fabricated by three-dimensional (3D) femtosecond laser direct writing for two-photon fluorescence imaging of biological tissues. We show that the two-photon fluorescence images of a plant leaf tissue acquired with the micro-optical lens are comparable to that of a 5× objective lens. Our result represents an important step towards the application of micro-optical components fabricated by femtosecond laser micromachining in miniaturized nonlinear fluorescence microscopy applications, such as two-photon endoscopy.     

The production and measurement of ultrashort pulses in a ruby layer

The two-photon flourescence from a whole pulse train is measured photoelectrically for a variety of laser conditions. A compound mode-locking solution containing DDI and DCI is found to reduce pulse lengths. An etalon which cancels gain-narrowing also has this effect. Intensity-dependent variations are found in the two-photon fluorescence profile which suggest a threshold for self-phase modulation.     

Acetylene-Substituted Two-Photon Absorbing Molecules With Rigid Elongated Pi-Conjugation: Synthesis,Spectroscopic Properties and Two-Photon Fluorescence Cell Imaging Applications

Two asymmetrical molecules with substituted acetylene as central rigid elongated conjugation are reported as potential chromophores for two-photon microscopic imaging. These molecules consist of a typical D–π–A structure, have different donors (D), the same π-conjugated center (π) and the same acceptor (A). Structural characterization and spectroscopic properties, including single-photon (linear) absorption, quantum yields, single-photon fluorescence, and two-photon absorption spectra, were studied in solvents with different polarity. These acetylene-substituted molecules were found to have high two-photon absorption cross-sections (for example, 690 GM for molecule 1 in toluene), which were determined by a two-photon induced fluorescence method using a femtosecond Ti: sapphire laser as excitation source. Single- and two-photon cellular imaging experiments demonstrate that the substituted acetylene derivatives could be one kind of promising two-photon fluorescence probes for cellular imaging.     

Two-photon fluorescence measurements of reversible photodegradation in a dye-doped polymer

We report on the dynamics of photodegradation and subsequent recovery of two-photon fluorescence in a dye-doped polymer. The energy dependence suggests that photodegradation is a linear process, while recovery is entropic. Such recovery could be useful to high-intensity devices such as two-photon absorbers, which can be used in many applications.     

Ultrafast nonlinear optical properties of dye-doped PMMA discs irradiated by 40 fs laser pulses

The two-photon absorption (TPA) characteristics of PMMA discs doped with three different dyes were studied using an fs-pulsed Ti-Sapphire laser as the pump source, and employing the open-aperture Z-scan technique. TPA cross-sections obtained for PMMA doped with the dyes PM597, DCM and rhodamine 6G–rhodamine B (co-doped) were found to be equal to 24.7, 33.3 and 32.3 GM, respectively (1 GM=10^?50 cm⁴ s phot^?1 mol^?1). Furthermore, two-photon fluorescence was measured for the samples containing DCM and rhodamine 6G–rhodamine B (co-doped). Compared to the one-photon fluorescence spectrum, the peaks in the two-photon fluorescence spectrum were red shifted and the extent of red shift increased with increasing doping concentration. We have also observed that the red shift in the two-photon fluorescence peak of the samples in the solid form is much larger than that in the solution state. This phenomenon could be explained by a twisted intra-molecular charge transfer model.     

双光子吸收材料二苯乙烯衍生物的合成及光谱性质

设计、合成并用红外光谱、1H NMR、元素分析表征了三种用于双光子吸收材料的二苯乙烯衍生物,4,4′-双(二苯氨基-反式-苯乙烯基)联苯(BPSBP),4,4′-双(二乙氨基-反式-苯乙烯基)联苯(BESBP)和4,4′-双(9-咔唑基-反式-苯乙烯基)联苯(BCSBP)。实验结果表明三者最强的单光子吸收出现在350～400 nm之间,且单光子吸收和荧光光谱中表现出明显的溶剂化显色效应,揭示了分子内对称电荷转移的本质,双光子荧光光谱则揭示了单光子和双光子吸收具有相同的发射机理。利用双光子上转换荧光法测试发现,三种双光子吸收材料在800 nm飞秒激光的激发下具有较大的吸收截面,分别为892,617和483 GM,这表明在双光子领域有潜在的应用价值。     

Two-photon induced optical-power limiting and upconverted superradiance properties of a new organic dye HEASPI

A new organic dye trans-4-[p-(N-ethyl-N-hydroxyethylamino)styryl]-N-methylpyridinium iodide (abbreviated as HEASPI hereafter) with large two-photon absorption (TPA) cross section and excellent upconverted superradiance properties was synthesized in our group recently. The TPA cross section was measured to be σ₂′=7.0×10⁻⁴⁸ cm⁴ s/photon by using an open aperture Z-scan system. Linear absorption, single-photon induced fluorescence, two-photon induced fluorescence and two-photon pumped (TPP) upconverted superradiance properties were systematically studied. The highest net upconversion efficiency from the absorbed pump energy to the output upconverted superradiance energy is as high as 19.6% at the pump energy of 2.07 mJ from a mode-locked Nd : YAG picosecond laser. The dye solution also shows a clear optical power limiting effect.     

Flow imaging by use of femtosecond-laser-induced two-photon fluorescence

A novel technique is demonstrated for the imaging of turbulent flows in which a single window to the flow is the only optical access required. A femtosecond laser is used to excite two-photon fluorescence in a disodium-fluorescein-seeded water jet. The fluorescence signal is generated at only the focal point of the laser because of the highly nonlinear nature of the two-photon absorption, and it is collected in a direction counterpropagating to the excitation beam. Tight focusing of the laser is used to limit the probe volume, and the two-dimensional mean and rms concentration images are collected by raster scanning the laser.     

Two-photon near- and far-field fluorescence microscopy with continuous-wave excitation

We report on scanning far- and near-field two-photon microscopy of cell nuclei stained with DAPI and bisbenzimidazole Hoechst 33342 (BBI-342) with the 647-nm laser line of a cw ArKr mixed-gas laser. Two-photon-excited fluorescence images are obtained for 50-200 mW of average power at the sample. A nearly quadratic dependence of fluorescence intensity on laser power confirmed the two-photon effect. The nonlinearity was further supported by evidence of three-dimensional sectioning in a scanning far-field microscope. We find that the cw two-photon irradiation sufficient for imaging within typically 5 s does not significantly impair cell cycling of BBI-342-labeled live cells. Finally, high-resolution imaging in scanning near-field microscopy with good contrast is demonstrated.     

Fluorescence Study of DNA–Dye Complexes Using One-Photon and Two-Photon Picosecond Excitation

Preliminary results of investigation of one-photon- and two-photon-induced fluorescence of acridine orange (AO), epirubicin (ER), hypericin (HYP), and ethidium bromide (EB) in complexes with DNA are presented. A spectrofluorometer based on a picosecond Nd:YAG laser was used for investigations of two-photon (1064-nm, 1-mJ, 40-ps) and one-photon (532- and 355-nm) dye excitation. The spectra of two-photon-induced fluorescence of dyes and their complexes with DNA as well as the kinetics of dyes' fluorescence intensification during their interactions with DNA in dependence on the biomacromolecule concentration were obtained. The intensities of AO, HYP, and EB fluorescence were increased 2.4, 3.2, and 8 times, respectively, after binding with DNA at two-photon excitation, while at one-photon excitation the corresponding values were 2.5, 3.7, and 10 times. The difference in fluorescence enhancement during DNA–dye complex formation at linear and nonlinear excitation may possibly be associated with the fact that the cross sections of one-photon and two-photon absorption, in general, change unequally during the binding of dyes to organic molecules and bathocromic shift of the electronic transitions. It was shown that the peak of AO fluorescence shifted to a longer wavelength on 10 nm after two-photon excitation at 1064 nm in comparison with one-photon excitation at 532 nm.