One- and Two-Photon Fluorescence Anisotropy of Selected Fluorene Derivatives

The steady-state excitation anisotropy spectra of fluorene derivatives were measured in viscous solvents, under the one- and two-photon excitation, over a broad spectral range (UV–Visible). The orientation of their absorption transition moments for the first, S₀S₁, and second, S₀S₂, excited states were determined. It was shown experimentally that a decrease in the angle between S₀S₁and S₀S₂ transitions corresponded to an increased value of two-photon absorption (2PA) cross section for these molecules. Two-photon excitation anisotropy was nearly constant over the spectral region investigated (in contrast to one-photon excitation anisotropy spectra) and can be roughly explained by a simple model of 2PA based on the single intermediate state approximation. For comparison, the same trend in two-photon excitation anisotropy was observed for Rhodamine B inglycerol.     

Improved Routine Bio-Medical and Bio-Analytical Online Fluorescence Measurements Using Fluorescence Lifetime Resolution

Fluorescence techniques are widely used as sensitive detection methods in bio-analytics. The use of the bio-physical parameter fluorescence lifetime additional to the spectral characteristics of fluorescence has the potential to improve fluorescence-related detection methods in terms of selectivity in signal recognition, robustness against disturbing influences, and the accessibility of novel bio-chemical process parameters. This article describes the technical set up of a time-resolving instrument with either a fixed time-gated detection principle for improved evaluation of tissue metabolism by an online monitoring of the tissue autofluorescence or a direct fluorescence lifetime detection principle for lifetime-based fluorescent assays.     

Effect of Polystyrene Microsphere Surface to Fluorescence Lifetime Under Two-Photon Excitation

Molecular assays such as immunoassays are often performed using solid carriers and fluorescent labels. In such an assay format a question can be raised on how much the fluorescence of the label is influenced by the bio-affinity binding events and the solid carrier surface. Since changes in fluorescence intensity as labels bind to surfaces are notoriously difficult to quantify other approaches are preferred. A good indicator, independent of the fluorescence intensity of the label, is the fluorescence lifetime of the marker fluorophore. Changes in fluorescence lifetime reliably indicate the presence of dynamic quenching, energy transfer or other de-excitation processes. A microsphere based assay system is studied under two-photon excitation. Changes in fluorescence lifetime are studied as labeled protein conjugates bind on microsphere surfaces – both direct on the surface and with a few nanometer distance from the surface. Fluorescence signal is measured from individual polystyrene microspheres and the fluorescence lifetime histogram is simultaneously recorded. The results indicate that self-quenching and quenching by the polystyrene surface are both present in such a system. However, the effect of the surface can be avoided by increasing the distance between the surface and the label. Typical distances achieved by a standard sandwich type of assay, are already sufficient to overcome the surface induced quenching in fluorescence detection.     

Steady-State and Time-Resolved Fluorescence Polarization Behavior of Acenaphthene

Steady-state and time-resolved fluorescence polarization studies have been carried out on acenaphthene (ACE) in low-temperature glass solutions and at room temperature. In the low-temperature glass the fluorescence polarization values vary considerably with both emission and excitation wavelength. There is a time dependence (on the nanosecond time scale) of the fluorescence anisotropy, r(t), at 77 K, which has a strong dependence upon the excitation and emission wavelengths. Under these conditions, the time-dependent decay of the anisotropy is not attributable to chromophoric motion. The observations are consistent with emission from two closely lying and interconverting excited states. Rate constants for the photophysical processes involved have been determined by fitting the data using a model proposed by Fleming et. al. The results are discussed with particular reference to the care required in using dynamic fluorescence polarization measurements to determine energy transfer rates in systems containing this chromophore.     

Steady-State and Time-Resolved Spectroscopic Characteristics of Mesoionic Oxazolones Solutions

Three new mesoionic oxazolo[3,2-b]pyridazin-2-one derivatives, in different solutions have been investigated by UV-Vis absorption, steady-state and time-resolved fluorescence methods. The effect of substituents on the extension of conjugation of the pi-electrons from mesoionic oxazolone has been evidenced by bathochromic shifts of the absorption and fluorescence maxima positions. The fluorescence decay data could be fitted to single-exponential or double-exponential function. The lifetime values are much higher in aprotic polar solvents and in the case of the derivatives that present an extension of the conjugation of pi-electrons. The properties of the compounds present a solvent dependence, being tested in micellar solutions as potential molecular probe "sensitive" to the environment polarity.     

用于双光子激发荧光寿命显微成像的高重复频率皮秒扫描相机

建立了一台基于新研制的高重复频率皮秒扫描相机的双光子激发荧光寿命显微成像系统,重点介绍所研制的高重复频率皮秒扫描相机。为了在高时间分辨力的同时扩大时间测量范围,实现大面积两维空间高时间分辨取样测量,从而提高采样速率和更有效地发挥扫描相机的作用,设计和研制了一种大面积、高时间分辨力扫描变像管和一种重复频率高达1MHz的斜坡电压扫描电路。基于上述关键部件所研制的扫描相机具有重复频率高、扫描速度可调、时间分辨力高、工作面积大、非线性低、触发晃动小等优点。用钛宝石飞秒激光器作为激光脉冲源,通过脉冲提取器将76MHz的高重复频率降低为1MHz,采用可调延时器和标准具对扫描相机的时间分辨力、扫描速度和非线性进行标定。该系统的时间分辨力达到6.5ps,非线性为2.60%,可测量的时间范围从十几皮秒到几十纳秒。测量了若丹明6G和香豆素314两种标准荧光染料的荧光寿命,取得了与参考文献一致的实验结果。     

时域两维荧光寿命显微测量研究

提出了一种新的时域两维荧光寿命显微测量技术,建立了一套荧光寿命成像显微系统,介绍了这种测量技术的数据处理方法。用标准样品对该系统进行了测试,实验表明,该系统的时间分辨率为2ps,在放大倍率为100倍的情况下,该系统的空间分辨率为8um。如果在现有的设备下采用更细的网格板和微位移系统,那么该系统的空间分辨率可小于1um.     

Tunable Two-Frequency Lasers for Lifetime Measurements

We built a compact two-tunable-frequency longitudinally pumped solid-state laser specially designed to measure atomic lifetimes. Weak coupling between the laser eigenstates permits the obtaining of a continuously tunable frequency difference between 0 and 10 GHz. Such a source provides either a linear polarization rotating at such frequencies, or a sinusoidally modulated intensity with a 100% modulation amplitude. As an example, the fluorescence lifetime of the first excited state of Cr⁴⁺ in YAG is measured in the zero-fluence limit using a two-frequency Nd:YAG laser at 1064 nm.     

Segmented Frequency-domain Fluorescence Lifetime Measurements: Minimizing the Effects of Photobleaching Within a Multi-component System

This study introduces a newly developed frequency segmentation and recombination method for frequency-domain fluorescence lifetime measurements to address the effects of changing fractional contributions over time and minimize the effects of photobleaching within multi-component systems. Frequency segmentation and recombination experiments were evaluated using a two component system consisting of fluorescein and rhodamine B. Comparison of experimental data collected in traditional and segmented fashion with simulated data, generated using different changing fractional contributions, demonstrated the validity of the technique. Frequency segmentation and recombination was also applied to a more complex system consisting of pyrene with Suwannee River fulvic acid reference and was shown to improve recovered lifetimes and fractional intensity contributions. It was observed that photobleaching in both systems led to errors in recovered lifetimes which can complicate the interpretation of lifetime results. Results showed clear evidence that the frequency segmentation and recombination method reduced errors resulting from a changing fractional contribution in a multi-component system, and allowed photobleaching issues to be addressed by commercially available instrumentation. Electronic supplementary material The online version of this article doi: contains supplementary material, which is available to authorized users.     

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 Excited Fluorescence and Molecular Reorientations in Liquid Solutions

Theoretical expressions are derived that relate the two-photon excited fluorescence depolarisation experiments to the molecular symmetry and the rotational motions of fluorescent molecules. Diffusive rotational motions in liquid solvents are considered, as well as the influence of fast unresolved motions (e.g. librations). The results obtained are compared with one-photon excited fluorescence depolarisation experiments. The derived theoretical expressions can be applied for detailed analyses of the molecular rotation in solvent. Several of the results are useful for determining and assigning the components of two-photon absorption tensors.     

稳态荧光探针法研究槐糖脂生物表面活性剂的胶束化行为

应用稳态荧光探针法测定了非离子生物表面活性剂槐糖内脂临界胶束浓度(CMC)和胶束聚集数(N_agg),并考察了添加无机电解质和短链脂肪醇对槐糖内酯CMC的影响。结果表明,槐糖内酯的CMC为1.3×10-4 mol·L-1,并随加入电解质浓度的升高而略有降低,外加短链脂肪则使其CMC值升高;在4~8倍的CMC浓度范围内,槐糖内酯溶液N_agg随其浓度增大而增大,但胶束聚集数较小,为4~8个;激光纳米粒径结果显示槐糖内酯胶束动态直径较大且分布较为集中,平均约为90 nm。槐糖内酯胶束具有聚集数小而直径大的特点,表明该生物表面活性剂所形成的胶束排列较为松弛。     

The Influence of Solvent Viscosity on the Fluorescence Decay and Time-Resolved Anisotropy of Green Fluorescent Protein

This report describes fluorescence decay and time-resolved anisotropy studies of green fluorescent protein (GFP) in various environments. The addition of glucose and fructose, NaCl, or polyethylene glycol changes the viscosity of the medium surrounding the GFP. Both the time-resolved anisotropy and the fluorescence decay of GFP are measured and it is shown that only the time-resolved anisotropy of GFP is affected by the viscosity, but not its fluorescence decay.     

激光诱导植物荧光寿命校正技术及其特性分析

激光诱导植物荧光寿命测量法是在植物荧光光谱分析法基础上开发的一种评估植物生长状况及环境监测的新技术。根据植物叶绿素荧光信号的物理特性,利用信息仿真技术开发了一种叶绿素荧光寿命校正方法,可提高植物叶绿素荧光寿命的测量精度。利用激光诱导叶绿素荧光寿命测量系统分别测得叶绿素荧光及其背景信号,先用解卷积法叶绿素荧光信号中分离出荧光衰减函数,可获取荧光寿命估计值。再结合叶绿素荧光寿命校正技术就能反演得到高精度的植物荧光寿命。仿真与实验结果表明：该方法可实现高精度的植物荧光寿命实时监测;并对不同含量的叶绿素提取液进行了测试,构建了植物荧光寿命与叶绿素含量的对应关系模型。未来该技术可用于遥感监测海洋、湖泊、河流中藻类植物的生物含量。     

硝基羟乙唑与溶菌酶反应机制的荧光光谱研究

分别在298,310,318 K温度下,利用荧光光谱法研究了pH=7.40生理条件下硝基羟乙唑(TRI)与溶菌酶(LYSO)的相互作用机理。结果表明,TRI与LYSO间通过静态猝灭方式相互作用。测定了LYSO与TRI反应的结合常数、结合位点数。利用反应过程的热力学参数,确定了LYSO-TRI体系的作用力类型;由Hill系数得出了LYSO或TRI的协同性;根据非辐射能量转移理论,确定了TRI到LYSO的结合距离,同时采用同步光谱法考察了TRI对LYSO构象的影响。     

Steady-State and Frequency-Domain Lifetime Measurements of an Activated Molecular Imprinted Polymer Imprinted to Dipicolinic Acid

We recently demonstrated the synthesis and fluorescence activity associated with an optical detector incorporating a molecular imprinted polymer (MIP). Steady-state and time-resolved (lifetime) fluorescence measurements were used to characterize the binding activity associated with MIP microparticles imprinted to dipicolinic acid (DPA). DPA is a unique biomarker associated with the sporulation phase of endospore-forming bacteria. Vinylic monomers were polymerized in a dimethylformamide solution containing DPA as a template. The resulting MIP was then pulverized and sorted into small microscale particles. Tests were conducted on replicate samples of biologically active cultures representing both vegetative stationary phase and sporulation phase of Bacillus subtilis in standard media. Samplers were adapted incorporating the MIP particles within a dialyzer cartridge (500 MW). The permeability of the dialyzer membrane permitted diffusion of lighter molecular weight constituents from microbial media effluents to enter the dialyzer chamber and come in contact with the MIP. Results showed dramatic (10-fold over background) steady-state fluorescence changes (as a function of excitation, emission and intensity) for samples associated with high endospore biomass (DPA), and a frequency-domain lifetime of 5.3 ns for the MIP-DPA complex.     

Nd:GGG晶体荧光寿命的测试

针对Nd:GGG激光器,介绍了荧光寿命测试原理,设计了低频窄脉宽荧光寿命测试系统.该系统由光源、光路系统、驱动电路和探测系统等几部分组成.利用脉冲取样技术测试了Nd:GGG晶体的荧光寿命约为250μs左右.     

应用小波边缘检测技术分析荧光寿命显微像

研究了小波图象边缘检测技术在荧光寿命显微像分析中的应用，利用小波分析的多尺度特性进行了荧光寿命显微像的边缘提取，结果表明，这种方法非常有效。     

Fluorescence lifetime-enhanced spectral characterization of human serum

Frequency-domain fluorescence lifetime techniques were used for the characterization of pooled human serum, including normal serum, hyperlipid serum, and sera that had been stripped of various components. Fluorescence lifetime measurements of normal human serum revealed lifetime components primarily in the regions of 10² ps, 1–2 ns, 4–7 ns, and 9–10 ns. Phase-resolved fluorescence spectroscopy (PRFS), a frequency-domain technique that combines spectral and lifetime information, in measurements of phase-resolved fluorescence intensity (PRFI), provided the basis for comparison of the various sera. Measurements of PRFI vs excitation wavelength and emission wavelength yield a phase-resolved excitation-emission matrix (PREEM) at a given modulation frequency. Multifrequency measurements yield a three-way excitation-emission-frequency array. The multifrequency PREEMs of the various sera were compared with each other and with the corresponding two-way excitation-emission matrices (EEMs) that are obtained using conventional, steady-state fluorescence spectroscopy. Application of matrix-based analysis techniques to the steady-state and PRFS data arrays allowed direct comparison between the two approaches. Results demonstrate the enhanced discrimination among samples that is achieved through the additional dimension of fluorescence lifetime in PRFS.