Analysis of Quantum Rod Diffusion by Polarized Fluorescence Correlation Spectroscopy

To measure the polarization dependence of fluorescent probes, a confocal-microscope-based polarized fluorescence correlation spectroscopy system was developed, and the polarization dependence on the rotational diffusion of well-defined quantum rods (Qrods) was investigated and characterized. The rotational diffusion region of the Qrods was observed over a time range of less than 10^?5 s in a water solution, and the rotational diffusion parameters were extracted using a rotational diffusion model in which the viscosity of the solution media was varied. Our work demonstrated that polarized fluorescence correlation spectroscopy (FCS) is useful for investigating both the rotational and translational diffusion of fluorescent probes.     

长时程双光子成像技术

双光子成像(Two-Photon Imaging)技术以其优越特性被广泛用于活细胞动态三维成像,但光功率极高的短脉冲光对焦平面荧光分子严重的光漂白极大地影响了双光子长时间成像的图像质量,针对双光子荧光漂白问题,本文提出一种优化光照的双光子(Optimized Lighting-Two Photon,OL-TP)成像技术。通过预扫描获取双光子图像分析高低阈值,以预设的高低阈值为标准优化一幅图像中不同区域的光照时长,利用扫描过程中记录的荧光信息和光照时间信息可以重建OL-TP图像,既保证信噪比又降低荧光漂白。重建的OL-TP图像与传统双光子图像基本一致,信噪比略有降低,但图像并未失真。对110 nm的荧光小球样本分别连续取30幅普通双光子和优化光照的双光子图像,到第30幅图时,重建后的优化光照双光子图像比普通双光子图像荧光漂白降低了28.86%。OL-TP通过优化光照时间大幅降低双光子成像的荧光漂白,使双光子荧光显微镜能够更好地对生物样本进行长时间观测。     

Remote Estimation of the Chlorophyll Concentration of Living Trees Using Laser-induced Fluorescence Imaging Lidar

Monthly variation in chlorophyll concentration of living ginko tree leaves 65 m away from a system was remotely estimated by a laser-induced fluorescence imaging lidar. The combination of a pulsed laser and a short-time gated CCD using an image intensifier made it possible to monitor the weak fluorescence signal from the ginko tree leaves as an image. By applying the experimental idea that a ratio of intensity of the chlorophyll fluorescence at 740 nm to that at 685 nm showed a linear correlation to the chlorophyll concentration, the fluorescence image of the ginko tree obtained by the lidar was converted to the chlorophyll concentration distribution image.     

Fluorescence and Rotational Dynamics of Dityrosine

We have examined the lifetimes and rotational correlation times of dityrosine emission by time-correlated single-photon counting. We first noticed dityrosine fluorescence in samples of tyrosine and tyrosine dipeptides by its characteristic red-shifted emission at 400 to 430 nm. The longer rotational correlation time relative to tyrosine proved that this fluorescence emanated from a distinct species. Comparison with the fluorescence properties of synthesized dityrosine established the identity of the emitting species. Fluorescence intensity decays of dityrosine are generally characterized by two decay components, one with a lifetime in the range of 150 to 800 ps and another between 2.5 and 4.5 ns. We found no evidence for an excited-state reaction, since a rising phase (negative-amplitude component) was not observed. In the pH range from 4 to 10, two ground-state species exist in equilibrium with pK _a 7. Both species exhibit two fluorescence decays. The average fluorescence lifetime increases gradually with pH over the pH range from 4 to 10 and decreases at pH 2. Anisotropy decays were measured for dityrosine and the alanine–dityrosine–alanine and leucine–dityrosine–leucine dipeptides. The rotational correlation times of dityrosine and dityrosine dipeptides increase linearly with van der Waals volumes. The slope indicates a stronger solute–solvent interaction than predicted with stick boundary conditions. It is suggested that these interactions result from the presence of two zwitterionic pairs.     

Study of Macromolecular Chain Dynamics in Polymer Complexes by Time-Resolved Fluorescence Spectroscopy

Poly(methyl methacrylate)s labeled with the anthracene fluorophore were prepared by free radical, anionic, and coordination polymerization yielding atactic and syndiotactic polymers. Unlabeled isotactic poly(methyl methacrylate) was prepared by anionic polymerization. Time-resolved fluorescence spectroscopy was used to study polymer association in solution. The time-dependent decays of fluorescence anisotropy show that stereocomplexation causes an increase in rotational correlation times of anthracene fluorophores both embedded in the polymer backbone and attached at the end of the side chain of polymer molecules. The rotational correlation time of anthracene fluorophore in dimethylformamide as a part of stereocomplex is 11.9 and 30 ns in the side chain and embedded in the polymer backbone, respectively, and shorter than 3 ns in noncomplexing solvent.     

Effects of Refractive Index and Viscosity on Fluorescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins

The fluorescence lifetime strongly depends on the immediate environment of the fluorophore. Time-resolved fluorescence measurements of the enhanced forms of ECFP and EYFP in water–glycerol mixtures were performed to quantify the effects of the refractive index and viscosity on the fluorescence lifetimes of these proteins. The experimental data show for ECFP and EYFP two fluorescence lifetime components: one short lifetime of about 1 ns and a longer lifetime of about 3.7 ns of ECFP and for EYFP 3.4. The fluorescence of ECFP is very heterogeneous, which can be explained by the presence of two populations: a conformation (67% present) where the fluorophore is less quenched than in the other conformation (33% present). The fluorescence decay of EYFP is much more homogeneous and the amplitude of the short fluorescence lifetime is about 5%. The fluorescence anisotropy decays show that the rotational correlation time of both proteins scales with increasing viscosity of the solvent similarly as shown earlier for GFP. The rotational correlation times are identical for ECFP and EYFP, which can be expected since both proteins have the same shape and size. The only difference observed is the slightly lower initial anisotropy for ECFP as compared to the one of EYFP.     

Tyrosyl Fluorescence Decays and Rotational Dynamics in Tyrosine Monomers and in Dipeptides

Picosecond time-correlated single-photon counting was used to measure fluorescence lifetimes and fluorescence anisotropy decays of tyrosine and the tyrosine–alanine and tyrosine–leucine dipeptides. After excitation of tyrosine at 287 nm two emitting species were observed, one at 303 nm with a lifetime of 3.3 ns and another at 340 nm with a lifetime of 360 ps. The rotational correlation time of tyrosine at 303 nm is 38 ps in water at pH 7 and depends linearly on viscosity with a slope of 44 ps/cP, consistent with Stokes–Einstein–Debye theory. We calculated a value of 45 ns for the radiative lifetime of tyrosine, yielding a fluorescence quantum yield of 0.07. The dipeptides Tyr–Ala and Tyr–Leu exhibit two- or three-exponential decays. The amplitudes of the decay components for three-exponential fits correlate closely with the populations of rotamers in these peptides as determined by NMR. The quenching of dipeptide fluorescence is shown to depend on the solvent polarity, strongly supporting the hypothesis that tyrosyl fluorescence in peptides is quenched by charge transfer. The rotational correlation times of tyrosine, Tyr–Ala, and Tyr–Leu increase linearly with the van der Waals volumes. However, rotational relaxation is somewhat faster than expected from Stokes–Einstein–Debye theory with stick boundary conditions.     

基于激光诱导叶绿素荧光寿命成像技术的植物荧光特性研究

针对植物荧光遥感探测中信号易受干扰的问题, 提出了一种用于评估植物生长状况及环境监测的荧光寿命成像技术. 采用凹透镜对355 nm波长的激光扩束, 再照射植物激发叶绿素荧光, 由增强型电荷耦合器件接收荧光信号. 采用时间分辨测量法, 连续用相同激光脉冲照射植物以激发相同的荧光信号, 同时不断改变激光脉冲触发探测器启动的延时时间, 从而能够得到完整的离散荧光信号分布图像. 对植物特定位置点产生的离散荧光信号进行拟合, 再运用一种改进型的迭代解卷积法可反演高精度的荧光寿命; 进而反演图像各点的荧光寿命以生成植物的荧光寿命分布图. 该方法所绘制的荧光寿命图比荧光强度图能更准确地反映植物内部的叶绿素含量, 并对活体植物叶绿素荧光寿命的物理特性进行了初步研究, 证明叶绿素荧光寿命与植物生理状态存在一定关联; 并且叶绿素荧光寿命与活体植物所处环境存在着复杂的关系. 未来将与生物物理学家们合作, 继续探寻叶绿素荧光寿命与植物生存环境的关系.     

A General Method for Constrained Analysis of Fluorescence Anisotropy Decay: Application of the Steady-State Anisotropy

Time-resolved fluorescence anisotropy is an invaluable method for investigating the internal and rotational dynamics of biomolecules. The range of rotational motions detectable by anisotropy decay is limited by the fluorescence lifetime; typically, a depolarizing motion may be resolved if the associated correlation time is between 0.1 and 10 times the intensity decay lifetime. To extend that range and to improve the recovery of anisotropy decay parameters, a general analytical method has been developed. This procedure utilizes a modification of Lagrange multiplier methods to constrain the values of the iterated kinetic parameters during nonlinear least-squares analysis of anisotropy decay data. The form of the constraint equation is derived from the classic relationship between the decay parameters and the steady-state anisotropy, which can be simply and accurately measured. Application of the constraint to analyses of synthetic data sets increased the accuracy of recovery by decreasing the uncertainty in the iterated parameters. The constraint also enabled the accurate recovery of correlation times that were a factor of 30 greater than the fluorescence lifetime, although it did not improve recovery of correlation times that were much shorter than the lifetime. Using this technique, it should now be possible to characterize the dynamics of larger macromolecules and assemblies than those that can currently be studied by fluorescence anisotropy decay.     

Translational and Rotational Motions of Albumin Sensed by a Non-Covalent Associated Porphyrin Under Physiological and Acidic Conditions: A Fluorescence Correlation Spectroscopy and Time Resolved Anisotropy Study

The interaction between a free-base, anionic water-soluble porphyrin, TSPP, and the drug carrier protein, bovine serum albumin (BSA) has been studied by time-resolved fluorescence anisotropy (TRFA) and fluorescence correlation spectroscopy (FCS) at two different pH-values. Both rotational correlation times and translational diffusion times of the fluorescent species indicate that TSPP binding to albumin induces very little conformational changes in the protein under physiological conditions. By contrast, at low pH, a bi-exponential decay is obtained where a short rotational correlation time (phi (int) = 1.2 ns) is obtained, which is likely associated to wobbling movement of the porphyrin in the protein binding site. These physical changes are corroborated by circular dichroism (CD) data which show a 37% loss in the protein helicity upon acidification of the medium. In the presence of excess porphyrin formation of porphyrin J-aggregates is induced, which can be detected by time-resolved fluorescence with short characteristic times. This is also reflected in FCS data by an increase in molecular brightness together with a decrease in the number of fluorescent molecules passing through the detection volume of the sample.     

Multipoint parallel excitation and CCD-based imaging system for high-throughput fluorescence detection of biochip micro-arrays

We report the development and the characterization of a multipoint parallel excitation and CCD-based imaging system for high-throughput fluorescence detection of biochip micro-arrays. A two-dimensional array of (19×19) points with uniform intensity distribution, generated by a holographic array generator, was used for parallel excitation of two-dimensional micro-arrays of fluorescence samples. A CCD-based imaging system was used for high-throughput parallel detection and quantitative analysis of the fluorescence output. Micro-array samples of cyanine (Cy5) dye dots on silicon wafers and on glass substrates with varying concentration were used to evaluate the performance of the system. Results of fluorescence intensity measurements with varying concentration of dye and with different image acquisition time are presented. We have demonstrated that this novel approach will, in general, outperform the conventional approach in the excitation efficiency, the signal-to-noise ratio, and the throughput. The limitations and the potential improvements of the present method are discussed.     

Determining if a System is Heterogeneous: The Analysis of Single Molecule Rotational Correlation Functions and Their Limitations

Single molecule spectroscopy can be utilized to measure distributions of individual molecular properties that may be averaged out in the ensemble measurement. For example, complex dynamics in disordered systems can be investigated by observing single molecule rotations via fluorescence spectroscopy. The rotational time of a single transient can be calculated from the correlation function of the reduced linear dichroism signal which fluctuates over time as the molecule reorients in its surroundings. Distributions of rotational time constants can be used to characterize the heterogeneity of molecular environments in the material. This paper reviews some theoretical studies on (1) the high numerical aperture effects on the final correlation function, and how it can be related to optical anisotropy decays in a bulk measurement; (2) the statistical errors resulting from the finite observation length that will propagate into distributions of rotational times. These lead to the discussions on how to interpret correctly the distribution of properties measured from a set of single molecule data, and to determine if in fact the system is heterogeneous.     

Narrow-linewidth passband filter for ultraviolet rotational Raman imaging

We present a narrow-passband spectral filter capable of frequency-resolved imaging of rotational Raman light scattering with strong spectral rejection of out-of-band Raman, Rayleigh, and Mie scattering. The filter is based on mercury-vapor absorption, and subsequent resonant fluorescence and has a passband of less than 1 cm(-1). It is paired with an injection-seeded, cavity-locked, frequency-tripled Ti:sapphire laser that produces >30 mJ/pulse of single-mode, tunable light in the vicinity of 253.7 nm. The laser and filter are combined to spectrally resolve scattering from individual rotational Raman lines of nitrogen and oxygen.     

Rotational correlation functions of single molecules

Single molecule rotational correlation functions are analyzed for several reorientation geometries. Even for the simplest model of isotropic rotational diffusion our findings predict nonexponential correlation functions to be observed by polarization sensitive single molecule fluorescence microscopy. This may have a deep impact on interpreting the results of molecular reorientation measurements in heterogeneous environments.     

Origins of nonexponential decay in single molecule measurements of rotational dynamics

Recent reports have demonstrated that the correlation function of the fluorescence dichroism signal, measured as a probe of single molecule rotational dynamics, should not manifest a single exponential decay even for isotropic diffusion. This has called into question the attribution of observed nonexponential behavior in supercooled fluids and polymer systems to dynamical heterogeneity. We show here that, for the case of a high numerical aperture objective, the dichroism decay becomes indistinguishable from a single exponential. As a consequence, observed nonexponential decays can be associated with complex rotational dynamics. These effects are illustrated via simulated rotational trajectories for isotropic diffusion of a dipole.     

25 nm resolution single molecular fluorescence imaging by scanning near-field optical/atomic force microscopy

High-resolution single molecular near-field fluorescence images were observed by scanning near-field optical/atomic force microscopy (SNOM/AFM). We modified the SNOM/AFM for both high-resolution fluorescence imaging and high-resolution topographic imaging. The imaged fluorophore, Alexa 532, is prepared with a poly-methyl-methacrylate (PMMA) film coating. A fluorescence resolution of 25 nm was obtained with a simultaneous topographic image of a flat surface. A sample prepared with a lower PMMA concentration exhibited a rough surface in the micro area. The results for the flat surface indicated that the fluorescence resolution is worst in the rough surface sample, that the maximum fluorescence intensities for the individual fluorophore are similar, and that the decay rate is faster. Thus, we concluded that the morphological effect is an important factor in fluorescence image resolution and the apparent lifetimes of the fluorescence molecules.     

Fluorescence ghost imaging with pseudothermal light

We extend classical light ghost imaging to the area of fluorescence imaging and propose a new fluorescence imaging method. For the first time, we demonstrate both theoretically and experimentally that fluorescence ghost imaging can be realized with pseudothermal light. Important factors influencing the visibility and resolution of the images are discussed to improve the quality of the fluorescence ghost imaging. We hope that this work may pave the road for ghost imaging to biomedical applications.     

赤潮生物监测荧光成像系统背景光源改进

介绍了一种海洋赤潮生物监测荧光成像系统,该系统的背景光源为海洋赤潮生物监测荧光成像系统的重要组成部分,赤潮生物监测荧光成像系统背景光源选择是否合理将直接影响着藻类成像清晰度,由于各种赤潮藻含有不同的特征纹理,其纹理特征将决定是否能准确识别出藻类并对其进行分类,进而影响到对赤潮的准确预报。为了解决赤潮藻图像的清晰度等问题,作者利用自行设计的荧光成像系统,对不同成像光源的成像效果进行了分析和比较。实验证明,近红外光源可以有效地获得清晰的藻类纹理信息,使得后继的图像处理工作更加容易,从而解决了藻类图像暗黑以至于图像处理起来较为困难等问题。     

Dynamics of tRNAtyr Probed with Long-Lifetime Metal-Ligand Complexes

The metal-ligand complexes, [Ru(bpy)₂(dppz)]²⁺ (bpy = 2,2??-bipyridine, dppz = dipyrido[3,2-a:2??,3??-c]phenazine) (RuBD) and [Ru(phen)₂(dppz)]²⁺ (phen = 1,10-phenanthroline) (RuPD), display favorable photophysical properties including long lifetime, polarized emission, and very little background fluorescence. To check if RuBD and RuPD reflect the overall rotational mobility of small nucleic acid, we measured the intensity and anisotropy decays of RuBD and RuPD when intercalated into tRNA^tyr using pBC SK(+) phagemid as a control. We used frequency-domain fluorometry with a blue light-emitting diode (LED) as the modulated light source. We observed shorter lifetimes for tRNA^tyr than those for the pBC SK(+) phagemid for both probes, however, RuPD showed much larger decrease in the mean lifetime values (64%). The slow rotational correlation time of RuBD (31.3 ns) and the fast rotational correlation time of RuPD (26.0 ns) reflected the overall rotational mobility of tRNA^tyr. In addition, the steady-state anisotropy and time-resolved anisotropy decay data showed a clear difference between tRNA^tyr and pBC SK(+) phagemid. This suggests the possibility of a homogeneous assay for identifying target nucleic acids and/or nucleic acid binding proteins.     

Cl+CHD3(v1=1)反应中的成对动态学:侦测激光波长的效应

用交叉分子束试验探讨氯原子与C?H 键激发的CHD3化学反应，并用(2+1)共振多光子电离及离子影像法来侦测CD3基态生成物．发现所得的影像对电离光子的波长极为敏感．这表明，与基态反应比较，C?H激发反应较易产生转动激发的CD3．实验结果也证明CD3D的转动与HCl的振动激发有相反的关联性．