Picosescond fluorescence lifetime standards for frequency- and time-domain fluorescence

We characterized a series of dimethylamino-stilbene derivatives as standards for time-domain and frequency-domain lifetime measurements. The substances have reasonable quantum yields, are soluble in solvents available with a high purity, and do not show significant sensitivity to oxygen quenching. All the fluorophores displayed single exponential intensity decays, as characterized by frequency-domain measurements to 10 GHz. The decay times vary from 880 to 57 ps, depending on structure, solvent, and temperature, which is a useful range for modern picosecond time-domain or gigahertz frequency-domain instruments. These fluorophores may be used either to test an instrument or as reference compounds to eliminate color effects. We also characterized two-fluorophore mixtures, with the decay times spaced twofold (150 and 300 ps), with varying proportions. These mixtures are useful for testing the resolution of other time- and frequency-domain instrumentation. The excitation wavelength ranges from 260 to 430 nm, and the emission from 350 to 550 nm. The decay times are independent of the excitation and emission wavelengths.     

Light quenching of tetraphenylbutadiene fluorescence observed during two-photon excitation

We observed the steady-state and time-resolved emission of tetraphenylbutadiene (TPB) whea excited by simultaneous absorption of two photons (514 to 610 nm). The intensity initially increased quadratically with laser power, as expected for a two-photon process. At higher laser powers the intensity increases in TPB were subquadratic. The intensity and anisotropy decay times of TPB were unchanged under the locally intense illumination. Importantly, the time zero anisotropy of TPB was decreased under conditions where the intensity was subquadratic. Furthermore, the subquadratic dependence on incident power was not observed for two-photon excitation of 2,5-diphenyloxazole (PPO), for which the incident wavelength does not overlap with the emission spectrum. These results are consistent with stimulated emission (light quenching) of TPB at high laser intensities. The phenomenon of light quenching may be important for other fluorophores used in biochemical research, particularly for the high local intensities used for two-photon excitation.     

Anisotropy Spectra of the Solvent-Sensitive Fluorophore 4-Dimethylamino-4′-Cyanostilbene in the Presence of Light Quenching

We examined the emission wavelength-dependent anisotropies of the solvent-sensitive fluorophore 4-dimethylamino-4-cyanostilbene (DCS) under conditions of light quenching by polarized time-delayed quenching pulses. Illumination on the long-wavelength side of the emission spectrum with time-delayed light pulses resulted in a progressive decrease in the emission anisotropy as the observation wavelength increased toward the stimulating wavelength. The anisotropy changes of DCS were most wavelength dependent when spectral relaxation occurred during the excited-state lifetime. Light quenching of DCS in a low-viscosity solvent revealed no wavelength-dependent anisotropies. Control measurements using a solvent-insensitive fluorophore did not show any wavelength-dependent anisotropy with light quenching. The data for DCS can be explained by a model which allows wavelength-selective quenching of the long-wavelength emission formed by time-dependent spectral relaxation. These results indicate that polarized light quenching can be used to study systems which display multiple emissions and/or time-dependent spectral shifts.     

Introducing ‘Simple Variable Selection (SVS) Approach’ for Improving the Quantitative Accuracy of Chemometric Assisted Fluorimetric Estimations of Dilute Aqueous Mixtures

Excitation emission matrix fluorescence (EEMF) spectroscopy is a multiparametric fluorescence technique where the fluorescence intensity of a fluorophore is a function of excitation wavelength, emission wavelength and its concentration. The manual analysis of large volume of highly correlated EEMF data sets towards developing a calibration model for quantifying each fluorophores present in multifluorophoric mixtures is a difficult and time-consuming task. Over the years, Partial least square (PLS) algorithm has found its application towards providing swift and efficient analyses of large volumes of highly correlated spectral data sets. The PLS assisted EEMF spectroscopy has been successfully used towards quantifying the fluorophores in multifluorophoric mixtures without involving any pre-separation. However, the accuracy and robustness of developed calibration model can be significantly improved provided PLS analysis is carried out on the analytically relevant EEMF spectral variables. In the present work, a variable selection method baptized as simple variable selection (SVS) approach is introduced that provides a simple and computationally economical means of identifying the useful spectral variables for subsequent PLS analysis. The proposed SVS approach is successfully validated by analyzing the complex EEMF data sets of multifluorophoric mixtures of consisting of multifluorophoric mixtures of biological relevance. The proposed approach is found to provide a simple, swift and efficient means for developing a robust PLS assisted EEMF spectroscopy based calibration model for simultaneous quantification of various fluorophores present in multifluorophoric mixtures.     

Review of fluorescence anisotropy decay analysis by frequency-domain fluorescence spectroscopy

This didactic paper summarizes the mathematical expressions needed for analysis of fluorescence anisotropy decays from polarized frequency-domain fluorescence data. The observed values are the phase angle difference between the polarized components of the emission and the modulated anisotropy, which is the ratio of the polarized and amplitude-modulated components of the emission. This procedure requires a separate measurement of the intensity decay of the total emission. The expressions are suitable for any number of exponential components in both the intensity decay and the anisotropy decay. The formalism is generalized for global analysis of anisotropy decays measured at different excitation wavelengths and for different intensity decay times as the result of quenching. Additionally, we describe the expressions required for associated anisotropy decays, that is, anisotropy decays where each correlation time is associated with a decay time present in the anisotropy decay. And finally, we present expressions appropriate for distributions of correlation times. This article should serve as a reference for researchers using frequency-domain fluorometry.     

Quenching of fluorescence by light: A new method to control the excited-state lifetimes and orientations of fluorophores

We report steady-state and time-resolved studies of quenching of fluorescence by light i.e. light quenching. The dyes rhodamine B (RhB) and 4-dicyanomethylene-2-methyl-6(p-dimethamino)-4H-pyrane (DCM) were excited in the anti-Stokes region from 560 to 615 nm. At a high illumination power the intensities of DCM and RhB were sublinear with incident power, an effect we believe is due to stimulated emission, andnot ground-state depopulation. The extent of light quenching was proportional to the amplitude of the emission spectrum at the incident wavelength, as expected for light-stimulated decay from the excited state. Control measurements at a decreased average illumination power, and in solvents of various viscosities, indicated that the effect was not due to undesired photochemical processes. Importantly, the frequency-domain intensity decays remained single exponentials, and the lifetimes were unchanged with light quenching, which suggests that the effect was not due to heating or other photochemical effects. These results are consistent with a quenching process which occurs within the quenching pulse. Importantly, as expected for light quenching with a single pulsed laser beam, the time 0 anisotropies of RhB and DCM were decreased due to orientation-dependent quenching of the excited-state population. In closing we discuss some possible future applications of light quenching to studies of dynamic processes.This report is partially based on the experimental data published previously [1,2].     

Photophysical study on daunorubicin by fluorescence spectroscopy

Steady-state fluorescence and time-resolved fluorescence intensity decays of daunorubicin have been studied in polar solvents and in aqueous solution by a time-correlated single-photon counting technique. Daunorubicin, quinizarin, and 2,3-dimethylquinizarin show bi-exponential decay. The decay of daunorubicin becomes tri-exponential in the presence of adenosine 5′ monophosphate. The quenching of the fluorescence of daunorubicin by adenosine 5′ monophosphate exhibits downward deviation from the Stern-Volmer linearity, suggesting the existence of fluorophore in two conformers in the ground state differing only in the extent of hydrogen bonding.     

丹酰氯SiO2纳米发光标记物的制备

通过合成丹酰氯的荧光单体硅酯前驱物,采用油包水的反相微乳液法,以丹酰氯的荧光单体硅酯前驱物为核材料,成功地制备了丹酰氯的荧光纳米颗粒,克服了传统方法制备核壳荧光纳米颗粒中存在的荧光染料泄漏问题.通过透射电子显微镜表征该纳米粒子呈球形,大小均匀,直径为40nm左右.所制得纳米颗粒荧光性质稳定,受外界环境的影响小,且潜在生物亲和性,是一种新型的荧光标记物.     

Enhancing the Emission of Polydiacetylene Sensing Materials Through Fluorophore Addition and Energy Transfer

Enhancement of the environmentally responsive fluorescent properties of polydiacetylene (PDA) by combination with lipophilic fluorophores was demonstrated and properties of the PDA/fluorophore systems were explored. Liposomes containing PDA and fluorophores exhibited enhanced Stokes shift and increase in emission as a result of energy transfer from PDA to fluorophore. The effects of fluorophore variation, degree of PDA polymerization and diyne placement in the diacetylene lipid tails on the emission enhancement were studied. It was determined that signal generation was optimized at a relatively low extent of PDA polymerization with the optimal degree of polymerization dependent on the other parameters. Energy transfer was used as a tool to detect fluorophore exchange between polymerized and unpolymerized liposomes and to study the effects of fluorophore structure on exchange from unpolymerized to PDA liposomes. Fluorophores that locate at the aqueous interface with alkyl anchors were slow to transfer while fluorophores that partition into the alkyl regions of the liposomes transfer quickly.     

A Miniaturized XeCl Dielectric Barrier Discharge as a Source of Short Lived,Fast Decaying UV Radiation

A dielectric barrier discharge (DBD) of coaxial geometry has been examined as source of intensive, short‐lived UV radiation. A binary gas mixture consisting of 98% Xe + 2% Cl₂ and a ternary composition of 96% Ne + 6% Xe + 0.2% HCl were investigated in the pressure range between 10 and 750 mbar. The discharge was excited by unipolar high voltage square pulses with amplitudes of 3 to 7 kV at a repetition rate of 100 Hz. UV radiation intensity is increased by combining 5 square pulses with a period of 1 μs each in one train (burst). Radiation decay on three orders of magnitude within 10 μs was obtained for both gas mixtures in burst mode. Maximum energy of the light pulse was the same for both gas mixtures and estimated as 40 nJ. It is shown that the optimum gas pressure for highest radiation intensity depends not only on gas composition but also on applied voltage and number of pulses in the train. A variation of the spectral band shape of XeCl emission during the pulse is detected (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Origin of Tryptophan Fluorescence Lifetimes Part 1. Fluorescence Lifetimes Origin of Tryptophan Free in Solution

Fluorescence intensity decays of L-tryptophan free in polar, hydrophobic and mixture of polar-hydrophobic solvents were recorded along the emission spectrum (310–410 nm). Analysis of the data show that emission of tryptophan occurs with two lifetimes in 100 % polar and hydrophobic environments. The values of the two lifetimes are not the same in both environments while their populations (pre-exponentials values) are identical. Fluorescence lifetimes and pre-exponentials values do not change with the excitation wavelength and thus are independent of excitation energy. Our results indicate that tryptophan emission occurs from two specific sub-structures existing in the excited state. These sub-structures differ from those present in the ground states and characterize an internal property and/or organization of the tryptophan structure in the excited state. By sub-substructure, we mean here tryptophan backbone and its electronic cloud. In ethanol, three fluorescence lifetimes were measured; two lifetimes are very close to those observed in water (0.4–0.5 ns and 2–4 ns). Presence of a third lifetime for tryptophan in ethanol results from the interaction of both hydrophobic and hydrophilic dipoles or chemical functions of ethanol with the fluorophore.     

Investigation of Abnormal Long‐Wavelength Fluorescence Emissions Occurring in Binary Organic Nanoparticle Films

In some fluorophores with planar groups, a long‐wavelength emission band different from their native one can sometimes be observed. The main cause of this long‐wavelength band is excimer formation. It is generally accepted that once condensed in the solid state, whether fluorophores can exhibit excimer emission or not depends only on their molecular structure and packing. However, here it is shown that there are exceptions when fluorophores are present in nanoparticles (NPs), where excimer emission can be affected by the environment surrounding the NPs, even in the solid state. It is found that in some binary NP films consisting of fluorophore NPs and other NPs, unusual long‐wavelength bands ascribed to excimer emission can be activated, even though these bands are absent from the photoluminescence spectra of the pure fluorophore NP films. This finding is beneficial to better understand and control excimer emissions. In addition, such a binary NP system provides an ideal platform to investigate the interplay between two fluorophores, because it keeps them effectively separated while maintaining suitable spatial distances for exciton migration and dipole–dipole interactions. This work also provides evidence for the long‐debated origin of the green emission band (g‐band) of fluorene‐based fluorophores.     

Photoluminescence and time-resolved photoluminescence of star-shaped ZnO nanostructures

Optical properties of star-shaped ZnO nanostructures were studied. The temperature-dependent photoluminescence (PL) was examined up to fourth-order longitudinal optical (LO) phonon assisted emissions of free excitons and confirmed that the nature of the room temperature PL in ZnO is 1-LO phonon assisted emission of free excitons. Low threshold ultraviolet stimulated emissions (SE) were obtained for our powder samples at room temperature. Picosecond time-resolved PL measurements detected a bi-exponential decay behavior which is strongly dependent on the excitation intensity: the slow decay term decreased faster than the fast decay term as the excitation intensity increased and the emission decays were dominated by the fast one. We also found that the emission decays decreased super-linearly before the appearance of the SE. This behavior may be used to deduce the threshold of SE or lasing.     

Three-photon-induced fluorescence of diphenylhexatriene in solvents and lipid bilayers

We observed the emission of l,6-diphenyl-l,3,5-hexatriene (DPH) when excited with the fundamental output of a fs Ti:sapphire laser at 860 nm. The emission spectra of DPH were identical to that observed for one-photon excitation at 287 nm. The dependence of the DPH emission intensity on laser power was cubic, indicating three-photon excitation of DPH at 860 nm. At a shorter wavelength of 810 nm, the dependence on laser power was quadratic, indicating a two-photon process. At an intermediate wavelength of 830 nm the mode of excitation was a mixture of two- and three-photon excitation. At 830 nm the anisotropy is no longer a molecular parameter, and the mode of excitation and anisotropy of DPH depends on laser power. Frequency-domain anisotropy decays of DPH in triacetin revealed the same rotational correlation times for two- and three-photon excitation. However, the time 0 anisotropy of DPH was larger for three-photon excitation than for two-photon excitation. Steady-state anisotropy data for DPH-labeled membranes revealed the same transition temperature for one- and three-photon excitation. These anisotropy data indicate that membrane heating was not significant with three-photon excitation and that three-photon excitation may thus be of practical usefulness in fluorescence spectroscopy and microscopy of membranes.     

Fluorescence properties of biochemicals in dry NaCl composite aerosol particles and in solutions

Several fluorophores, such as tryptophan, NADH, NADPH, and riboflavin are found in airborne micro-organisms. In this work, the fluorescence properties of these biochemicals were studied both in dry NaCl composite aerosol particles and in saline solutions by means of laser-induced fluorescence. Fluorescence spectra were measured from individual, airborne aerosol particles and from solutions in cuvette. The excitation wavelength was varied in steps from 210 nm to 419 nm and the fluorescence was detected within a wavelength band of 310–670 nm. For each sample, the measured fluorescence emission spectra were combined into fluorescence maps. The fluorescence maximum of riboflavin in a dry NaCl composite particle is 20 nm red-shifted compared with the solution, whereas the maxima are blue-shifted by about 25 nm for tryptophan and 15 nm for NADH and NADPH. The molecular fluorescence cross sections have significant differences between the aerosol particles and the solutions, except for tryptophan. For NADH and NADPH the cross sections are over 20 times larger in the aerosol particles than in the solutions probably as a result of partial quenching of fluorescence in solution caused by the collision or stacking with the adenine moiety. The fluorescence cross section of riboflavin is almost 60 times larger in the solution than in the dry NaCl composite aerosol. This is probably caused by the different microenvironment around the fluorophore molecule and by the concentration quenching in the particles where the fluorescing molecules are relatively close to each other.     

Excitation wavelength dependent pump–probe signatures of molecular crystals

The photodynamics of single crystals of 4-(diisopropylamino)benzonitrile (DIABN) have been studied by femtosecond UV/Vis and mid-IR absorption spectroscopy. The observed spectroscopic response, especially in the UV/Vis, strongly depends on the optical excitation conditions. For the excitation light in resonance with the absorption of DIABN, broad and structureless transient spectra are observed, which decay non-exponentially within pico- to nanoseconds. For off-resonance excitation in the very red wing of the absorption band a stimulated emission signal is detected, which decays in ∼10 ps matching the known behavior of the non-interacting chromophore in solution. The 10 ps decay is due to an intramolecular charge transfer process which is supported by femtosecond IR spectroscopy. The observed dependence on the excitation wavelength is assigned to a change in the density of excited chromophores and therefore bears relevance for time resolved X-ray diffraction experiments.     

Behaviour of scintillators under XUV free electron laser radiation

Free electron lasers (FEL) are new generation accelerator-based short wavelength light sources providing high pulse intensity and femtosecond pulse duration, which enable investigation of interaction of elementary excitations in solids under extreme conditions. Using the FLASH facility of HASYLAB at DESY (Hamburg, Germany), we investigated the response of different materials with scintillating properties based on intrinsic emissions to the 25.6 and 13.8 nm FEL radiation by means of time-resolved luminescence spectroscopy. FLASH delivered single pulses of 25 fs duration having energy per pulse up to 30 μJ resulting in power densities of ～10¹² W/cm² on crystals. As a function of excitation density we observed the shortening of lifetime and non-exponential behaviour of emission decays in CaWO₄, while the emission spectra recorded are comparable to those obtained at conventional excitation sources.     

Yb :YAG晶体的闪烁特性

通过不同Yb³⁺掺杂浓度(5%~30%,原子数分数)的Yb:YAG晶体的阴极射线发光谱、衰减时间、光输出及其温度依赖关系的测量,研究了Yb:YAG晶体的闪烁性能.不同Yb³⁺掺杂浓度的Yb:YAG晶体具有不同的光输出和猝灭温度,光输出随Yb³⁺掺杂浓度的增大而降低,猝灭温度则随掺杂浓度的增大而升高.室温下Yb:YAG晶体的发光衰减时间较短,均小于50ns.Yb³⁺掺杂浓度为5%的Yb:YAG晶体具有较高的光输出和较低的猝灭温度.     

Fluorescence of horse liver alcohol dehydrogenase using one- and two-photon excitation

We examined the steady-state and time-resolved emission of liver alcohol dehydrogenase resulting from one-photon and two-photon excitation. Previous studies with one-photon excitation revealed that the two nonidentical tryptophan residues display different emission spectra and decay times. The use of two-photon excitation resulted in similar emission spectra, multiexponential intensity decays, time-resolved emission spectra, and anisotropy decays as was observed for one-photon excitation. These results suggest that both nonidentical tryptophan residues are excited to a similar extent for one- and two-photon excitation. However, the limiting anisotropy (r ₀) with two-photon excitation from 585 to 610 nm is below 0.1 and appears distinct from that observed previously forN-acetyl-l-tryptophanamide.Abbreviations LADH liver alcohol dehydrogenase - -NAD⁺ -nicotinamide adenine dinucleotide - OPE one-photon excitation - OPIF one-photon induced fluorescence - TPE two-photon excitation - TCSPC time-correlated single photon counting - TPIF two-photon induced fluorescence     

乙醚溶液荧光光谱特性及其机理研究

研究了低浓度乙醚水溶液在紫外光激励下的荧光光谱,以及其荧光特性随激发光波长和乙醚溶液浓度改变的变化规律,并对其产生机理和谱线特性进行了分析和探讨。结果显示,乙醚溶液在306 nm附近出现明显的荧光峰,其最佳激励波长为245 nm,且在292 nm处还有次峰出现。激发光波长改变时,相应的荧光峰值位置基本不变,且荧光峰的强度随激发光波长的变化呈高斯分布。荧光次峰和主峰的强度产生竞争。随浓度增加,306 nm处的荧光强度线性增强,当增至7%后,发生浓度猝灭,强度线性减弱。研究结果将为检测有毒、麻醉物质——乙醚的浓度及纯度等提供参考。