Spatial Fourier-decomposition optical fluorescen tomography-theoretical investigation

A new three-dimensional (3D) optical fluorescent tomographic imaging scheme is proposed with structured illumination and spatial Fourierdomain decomposition methods for the first time. In this spatial Fourier-decomposition optical fluorescence tomography (SF-OFT), the intensity of focused excitation light from an objective lens is modulated to be a cosine function along the optical axis of the system. For a given position in a two-dimensional (2D) raster scanning process, the spatial frequency of the cosine function along the optical axis sweeps in a proper range while a series of fluorescence intensity are detected accordingly. By making an inverse discrete cosine transformation of these recorded intensity profiles, the distribution of fluorescent markers along the optical axis of a focused laser beam is obtained. A 3D optical fluorescent tomography can be achieved with this proposed SF-OFT technique with a simple 2D raster scanning process.     

An investigation into the role of the optical detection set-up in the recording of cardiac optical mapping signals: A Monte Carlo simulation study

Photon scattering is known to distort the fluorescence signals recorded from optically mapped cardiac tissue. However, the contribution of the parameters which define the optical detection set-up has not been assessed. In this study, Monte Carlo (MC) simulations of photon scattering within ventricular tissue are combined with a detailed model of a tandem-lens optical detection apparatus to characterise (i) the spatial origin upon emission of photons recorded in voltage-sensitive fluorescence measurements of cardiac electrical activity (using the fluorescent dye di-4-ANEPPS) and how this affects signal distortion, and (ii) the role the detector characteristics could play in modulating signal distortion during uniform illumination and photon emission from tissue depth. Results show that, for the particular excitation/emission wavelengths considered (488 nm and 669 nm, respectively), the dimensions of the scattering volume during uniform illumination extend around 3 times further in the surface recording plane than in depth. As a result, fluorescence recordings during electrical propagation are more distorted when transmembrane potential levels differ predominantly in the surface plane than in depth. In addition, MC simulation results show that the spatial accuracy of the fluorescence signal is significantly limited due to photon scattering, with only a small fraction of the recorded signal intensity originating from tissue beneath the pixel (approximately 11% for a 0.25×0.25 mm pixel). Increasing pixel size increases this fraction, however, it also results in an increase in the scattering volume dimensions, thus reducing the spatial resolution of the optical system, and increasing signal distortion. MC simulations also demonstrate that photon scattering in cardiac tissue limits the ability of optical detection system tuning in accurately locating fluorescent emission from depth. Specifically, our results prove that the focal plane depth that yields maximum signal intensity provides an underestimation of the emission depth. In conclusion, our study demonstrates the potential of MC simulations of photon scattering in guiding the design of optical mapping set-ups to optimise performance under diverse experimental conditions.     

Quantification of fluorescent molecules in heterogeneous media by use of the fluorescence decay amplitude analysis

In heterogeneous media, including biological objects, fluorescent molecules of one kind often exist as a mixture of species with different fluorescence parameters. Fractional concentrations of these species can be measured by analyzing their fluorescence decay amplitudes. The amplitudes are linear functions of concentrations of actually fluorescent molecules, i.e., molecules whose fluorescence decay can be measured. Other (quenched) molecules do not influence these amplitudes. The other parameter that has to be measured to calculate these concentrations is the radiative rate constant. The parameter can be excluded by comparison of decay amplitudes of the sample studied and a standard. The comparison should be made taking into account the dependence of the radiation rates on emision wavelength. The method has been tested in experiments with the fluorescent probe 3-methoxybenzanthrone (MBA) bound with phosphatidylcholine bilayer membranes. The probe has a complex fluorescence decay in these membranes. The decay can be described as two exponentials, with decay times of 2 and 12 ns and a blue-shifted fluorescence spectrum of the short-life component as compared with long-life one. The shift was used to correct calculated radiative rate values. After this, about 100% of the MBA molecules were found to be fluorescent in these membranes. Thus, this approach can be used to measure absolute concentrations of subpopulations of fluorescent molecules in heterogeneous biological objects.     

3D Particle Tracking on a Two-Photon Microscope

A 3D single-particle-tracking (SPT) system was developed based on two-photon excitation fluorescence microscopy that can track the motion of particles in three dimensions over a range of 100 μm and with a bandwidth up to 30 Hz. We have implemented two different techniques employing feedback control. The first technique scans a small volume around a particle to build up a volumetric image that is then used to determine the particle's position. The second technique scans only a single plane but utilizes optical aberrations that have been introduced into the optical system that break the axial symmetry of the point spread function and serve as an indicator of the particle's axial position. We verified the performance of the instrument by tracking particles in well-characterized models systems. We then studied the 3D viscoelastic mechanical response of 293 kidney cells using the techniques. Force was applied to the cells, by using a magnetic manipulator, onto the paramagnetic spheres attached to the cell via cellular integrin receptors. The deformation of the cytoskeleton was monitored by following the motion of nearby attached fluorescent polystyrene spheres. We showed that planar stress produces strain in all three dimensions, demonstrating that the 3D motion of the cell is required to fully model cellular mechanical responses.     

TbxTmyY1-x-yP5O14晶体中Tb3+和Tm3+间的能量转移

本文提出了Tb3+和Tm3+的交叉弛豫机理,计算了交叉弛豫几率,研究了Tm3+对Tb3+离子发光性质的影响,结果指出Tm3+对Tb3+离子的5D3能级的荧光有很强的猝灭作用,对5D4能级的荧光有增强作用,并且给出Tb的荧光强度和Tb3+,Tm3+离子浓度之间的依赖关系。     

超分辨率成像荧光探针材料应用进展

为了进一步认知复杂环境中的细胞生物学过程,研究人员发展了各种各样的生物成像技术。在这些技术中,生物荧光成像因简单的成像条件以及对生物样品的相容性而得到了广泛的发展。然而,传统的荧光成像技术受到了光学衍射极限的限制,无法分辨低于200 nm的空间结构,阻碍了对亚细胞结构的生物学过程研究。超分辨荧光显微镜技术突破了传统光学衍射对成像分辨率的限制,能够获取纳米尺度的细胞动态过程。除了对传统的宽场荧光显微镜框架的改进及升级改造之外,目前典型的超分辨成像显微镜技术通常依赖于荧光探针材料的光物理性质。常用的荧光探针材料包括荧光蛋白、有机荧光分子和纳米荧光材料等。本文介绍了几种主流的超分辨荧光显微成像技术并总结了已经成功应用到超分辨生物荧光成像中的荧光探针材料的应用进展。     

高压下Na5Tb(WO4)4单晶的光谱

研究了Na5Tb(WO4)4单晶的高压发光规律,化学计量的Na5Tb(WO4)4基质发光单晶的发光来源于基质中高浓度Tb3+.用金刚石对顶砧显微光谱系统在0-4GPa范围内研究了Na5Tb(WO4)4的室温高压光谱,确定了各发射谱线的高压移动率.对Tb3+的5D4→7Fj(j=0,1,2,3,4,5),测到18条谱线高压移动率中除一条(5D4→7F2,常压峰值15509cm-1)蓝移外,都红移,红移率最大为-19.5cm-1/GPa(对应谱线5D4→7F4,常压峰值16876cm-1),与Tb3+在其它基质中相比,此移动率很大.     

Novel fluorescent security marker. Part I: morphological and optical properties of 2-amino-6-ethoxy-4-[4-(4-morpholinyl)phenyl]-3,5-pyridinedicarbonitrile nanoparticles

Newly synthesized fluorescent nanoparticles of 2-amino-6-ethoxy-4-[4-(4-morpholinyl)phenyl]-3,5-pyridinedicarbonitrile have been developed and characterized for possible applications as security marker in paper documents. Nanoparticles have been prepared by reprecipitation in water under sonication. The size and the shape of these nanoparticles, characterized by light scattering and atomic force microscopy, have been found to be highly dependent on sonication power. Typical sizes range from tens to hundreds of nanometers. Furthermore, a remarkable increase in the fluorescence yield has been observed as nanoparticles sizes decrease. Finally, all of the above features, together with the striking stability of optical and mechanical properties over the course of months, allow for straightforward applications that rely on strong and stable fluorescence such as marking important or valuable documents.     

Fluorescent Aluminum Oxide Crystals for Volumetric Optical Data Storage and Imaging Applications

New fluorescent aluminum oxide crystals (Al₂O₃:C,Mg) for volumetric optical data storage and imaging applications were investigated. Magnesium impurity and double oxygen vacancy aggregate defects are responsible for the main optical properties of the new material. Spectroscopic investigation of these defects in different charge states was performed. Optical absorption, excitation-emission spectra and fluorescent lifetime of the new fluorescent medium were studied. Quantum yield of fluorescence from Al₂O₃:C,Mg crystals was measured in comparison with standard laser dyes.     

LiF∶F2晶体中实现三维光数据存储的实验研究

基于LiF∶F2晶体在近红外飞秒脉冲激光作用下产生F2色心向F+3色心的转变,以及两种色心荧光光谱的不同,实现了荧光反射共焦读出和多光子写入的三维光数据存储的原理性实验.钛宝石再生放大器输出的脉冲宽度100 fs、中心波长800 nm、重复频率1 kHz的超短脉冲激光束,用数值孔径0.68的显微物镜聚焦到LiF：F2晶体内部,通过移动晶体实现了三维逐位式数据写入；用405 nm的连续蓝光激发存储位,通过探测F+3色心产生的540 nm荧光,实现了对信息位进行快速非破坏性的反射共焦读出.与多光子三维存储的透射共焦散射读出和相衬读出相比,格式与现存光盘技术兼容,结构简单；更重要的是,存储信息位是依靠荧光光谱的变化,折射率变化很小,可以有效增加读出层数.     

Clustering study in Eu(DBM)3Phen-doped polymer optical fibers by optical properties and near-field scanning microscopy

The clusters of Eu3+ ion in Eu(DBM)3Phen-doped polymethyl methacrylate (PMMA) have been studied by three means. The relative fluorescence intensity ratio of the 5D0 → 7F2 to 5D0 → 7F1 transitions with different concentrations of Eu3+ in Eu(DBM)3Phen-doped PMMA and metastable-state (5D0) lifetime dependence on Eu3+ concentration are analyzed. The analysis indicates that there are no clustering effects in Eu3+ ions of Eu(DBM)3Phen-doped PMMA when the Eu3+ doping concentration is up to 1.0 wt.-%. At the same time, the clustering effect has not been observed by the scanning near-field optical microscopy (SNOM) in Eu(DBM)3Phen-doped PMMA with 1.0 wt.-% of Eu3+ ions. The analysis reveals that a high concentration of Eu3+ can be incorporated into polymer optical fiber (POF) without clustering effect.OCIS codes: 180.5810, 300.6280, 250.5460, 160.5690.     

小鼠卵母细胞染色体三维双光子荧光图像的轴向衰减

双光子荧光显微镜作为一种高分辨光学仪器,已经被广泛应用于生物样品的非侵入式三维光学成像中。相比共聚焦显微镜,双光子荧光显微镜拥有更深的探测深度。然而,即便如此,在对较厚的生物样品进行非侵入式光学三维成像时,样品的成像质量也往往会随着探测深度的增加而下降。在临床和生物学领域对研究母性遗传起重要作用的小鼠卵母细胞拥有较大的直径(80～100 μm),吸收和散射效应较为明显。本文研究小鼠卵母细胞染色体的三维双光子荧光图像随探测深度增加图像质量的衰减程度。通过对所得图像进行轴向衰减矫正,利用体积作为参数,将矫正前后小鼠卵母细胞内染色体三维双光子荧光图像进行对比。结果表明,由于吸收和散射效应,卵母细胞存在较严重的光学轴向衰减问题,因此,对用双光子荧光三维成像手段获得的小鼠卵母细胞图像进行衰减矫正是有必要的。这为进一步精确定量的研究卵母细胞内染色体的三维构像打下良好的基础。     

荧光波长对共焦显微镜成像特性的影响

导出了共焦显微镜中不同荧光波长情况下的荧光功率传输函数、三维脉冲响应函数（3D－PSF）和三维光学传递函数（3D－OTF）。结果表明，不同的荧光波长对共焦显微镜的空间截止频率、分辨率、光学传递函数存在明显的影响。当激发波长与荧光波长的比值下降到一定程度时，可以看到明显的失锥现象。     

Optical pumping and coherence effects in fluorescence from a four level system

An effective four-level system around the D2 line of ⁸⁵Rb at room temperature, is experimentally investigated by fluorescent studies under the action of two driving fields L1 and L2. This system exhibits unique features in fluorescence as a function of frequency separation between L1 and L2. In particular, at two-photon resonance, when the Rabi frequency of L1 exceeds that of L2, signatures of Electromagnetically Induced Transperancy effect (EIT) arising from the three-level Λ sub-system is present as a sub-natural dip in fluorescence from the fourth level. At comparable strengths of L1 and L2 the fluorescence features indicate a regime, where the effects arising from optical pumping and EIT effect due to ground hyperfine level coherence coexist. We see in the coexistence regime, saturation effects arising from difference frequency crossing (DFC) resonances and optical pumping around the EIT window. At low strengths of L1, all signs of coherence vanishes from the system and the fluorescent features result from incoherent optical pumping through the Autler-Townes split states of the excited state hyperfine levels, which are split due to the stronger L2 laser. The dominant role of the L1 laser in creating a robust transparency signal even in the presence of an off-resonant excitation is brought out. The results are supported by density matrix calculations.     

4-苯乙炔-1,8-萘酰亚胺荧光化合物的合成及电致发光性能

采用Pd(PPh₃)₂Cl₂-CuI为催化剂,Ph₃P为配体,4-溴-1,8-萘二酸酐为原料,在乙醇溶液中高收率地合成了4-苯乙炔-1,8-萘酰亚胺和4-对甲基苯乙炔-1,8-萘酰亚胺荧光新化合物。光谱研究表明,它们的最大紫外吸收波长(λ_UV,max)分别为374,380 nm,最大荧光发射波长(λ_FL,max)分别为446,462 nm,且有较高的荧光量子效率。电致发光性能测试表明,器件的最大发光亮度达到2250 cd/m²,是一类有潜在应用价值的小分子发光材料。     

二维相关光谱探测荧光能量转移现象

以EuCl₃和NdCl₃混合水溶液为研究对象,按正交浓度序列以浓度为外部扰动构建紫外可见-荧光二维相关光谱。在混合溶液的二维相关光谱中,观察到了Eu3+的荧光发射谱峰与Nd^{3+的吸收谱峰之间存在交叉峰。交叉峰的出现表明Eu3+和Nd3+的荧光发射与吸收之间存在能量传递。二维相关光谱中交叉峰的产生并非由于溶剂水分子与溶质(Eu3+或Nd3+)之间相互作用;若以单一溶质的EuCl₃和NdCl₃的水溶液构造模拟的“混合溶液”的拟合光谱构建二维紫外可见-荧光相关光谱,由于Eu3+和Nd3+在空间上相互分离,无相互作用发生,交叉峰并不存在。二维相关光谱的交叉峰可为从光谱学角度探测复杂体系能量传递及其相关机制提供一条新思路。}     

基于荧光淬灭原理的光纤氧传感器的实验研究

基于荧光淬灭原理的光纤氧传感器一直是许多研究工作的重点。介绍了一种制作简单、成本低的光纤氧传感器制造方法。该方法基于荧光淬灭原理，在光纤末端涂覆荧光材料铂八乙基卟啉(PtOEP)实现的。传感器中荧光材料被395nm的紫光激发，并由Y形光纤引导，使用广州犀谱光电USB2000+光谱仪记录荧光的发光强度时序图。最后得到的PtOEP的(I₀/I₁₀₀)-1的值为0.78，即光纤氧传感器的灵敏度为0.78，而且，斯特恩-沃尔默(Stern-Volmer)图显示出很好的线性特性。从氧气到空气环境的响应时间为24 s，从空气环境到氧气的响应时间是5 s。结果表明，基于荧光淬灭原理的光纤氧传感器具有较高的灵敏度和更快的响应时间。     

A comparison of experimental and simulated propagators in porous media using confocal laser scanning microscopy, lattice Boltzmann hydrodynamic simulations and nuclear magnetic resonance

Confocal laser scanning microscopy has been used to obtain 3D optical image stacks of packings of glass ballotini in various fluorescent dye-containing fluids inside a 3D micromodel. The fluids' refractive index was matched to that of the glass ballotini so that clear images at an appreciable depth (approximately 400 microm) inside the packings were obtained. The lattice Boltzmann method was then used to produce 3D velocity fields through the 3D image stacks of the packed ballotini. These have been used in conjunction with a stochastic random-walk algorithm to produce simulated displacement propagators, which have been shown to be in qualitative agreement with experimental propagators, obtained using nuclear magnetic resonance, of water flowing through the exact same micromodel.     

新型PVA荧光凝胶的制备及光物理性能研究

为了开发一系列能够在纯水介质中使用的荧光凝胶材料,以TbCl_3·6H_2O和乙酰丙酮(ACAC)为初始原料合成了铽配合物Tb(ACAC)_3·2H_2O,并对其结构进行了表征。然后将铽配合物Tb(ACAC)_3·2H_2O以不同的质量分数引入到PVA基质中,在交联剂硼酸的作用下形成荧光凝胶。利用FTIR、PL、DSC、TGA等对其结构、发光性能及其热性能进行了研究。FTIR结果表明,铽配合物成功地掺杂到了PVA凝胶体系中。荧光发射光谱表明,所有铽配合物的PVA荧光凝胶都具有很好的荧光性能,更重要的是掺杂了3%、5%、7%铽配合物的PVA荧光凝胶位于545 nm处的特征发射峰的发射强度相比于小分子铽配合物Tb(ACAC)_3·2H_2O的发射强度更高,分别是其2.15、3.27和5.65倍。表明PVA基质对铽配合物具有较好的敏化作用。此外,DSC和TGA研究结果表明,铽配合物的引入使得PVA凝胶的热稳定性略有下降,但影响不大。     

苯基羧甲基亚砜体系中高氯酸铥对高氯酸铽的荧光增强效应研究

合成了不同摩尔比的苯基羧甲基亚砜高氯酸铽、铥异核配合物 ,对配合物进行了组成分析 ,确定了配合物组成为 (Tb1 -xTmx) L2 (ClO4 ) ·2H2 O(x=0 0 0 0～ 0 2 0 0 ,L=C6 H5SOCH2 COO- )。通过IR光谱及摩尔电导的测定推测了配位情况 ,溶解性实验表明配合物在乙醇、丙酮等极性溶剂中具有很高的溶解性。荧光光谱实验表明 :当 x =0 0 0 1～ 0 10 0时铥对铽的荧光均产生敏化增强效应 ,当x =0 0 0 1时敏化强度最大 ,可使Tb3+ 荧光强度增加 32 6 %。