几十纳秒的荧光寿命的测定

利用光学多道分析仪(OMAⅢ)的选通功能,配合使 1302型快脉冲发生器,可以测 出大于100ns的荧光寿命,若荧光寿命可以与探测器门脉冲宽度(最小值为5ns)相比拟,测得荧光寿命与实际荧光寿命有很大出入,而且探测器的“延展效应”展宽了有效门脉冲宽度。本文提出一种方法解决了此问题。 利用OMAⅢ的内插板1303型脉冲发生器的递增延时功能,可以探测到光脉冲随时间变化的三维光谱图(图略),光谱的峰值包络线与时间亡的函数关系为F(t),其半宽     

人体食道和乳腺组织的时间分辨光谱研究

实验研究了在514nm波长激发下人体食道和乳腺组织，包括正常组织和癌变组织的时间分辨光谱。由时间分辨光谱拟合求得的特征峰荧光寿命结果表明：食道正常组织和鳞癌组织的荧光寿命分别为2.09ns和1.71ns，乳腺正常组织和腺癌组织的荧光寿命分别为2.05ns和1.43ns。正常组织的荧光寿命都大于癌变组织的荧光寿命，它们之间存在明显差异，这一方法将有助于结合稳态荧光光谱应用于早期癌变组织的精确诊断。     

血卟啉衍生物HpD激光3号不同浓度下的时间窗荧光光谱学

采用改进后的时间相关单光子计数系统研究了血卟啉衍生物HpD激光3号在磷酸盐缓冲液中浓度0.625μg/ml至10μg/ml的时间窗荧光谱,这是在不同延迟时间测量的一种光谱。根据测量的光谱积分荧光寿命分别为2ns和15ns,选择两个不同宽度的时间窗,分别延迟0和18ns,来分析样品中各个荧光组份对整个光谱的贡献。峰值波长615am的荧光主要是HpD中单体的贡献,具有较长的荧光寿命(～15ns),而波长630～660nm区的荧光则是荧光寿命约0.7ns的聚合物和荧光寿命约2.3ns的多聚物剩余部份与“自由生色团”相互作用的结果。所得结果还与国外类似样品DHE作了比较。     

激光染料的时间分辨光谱研究——香豆素系列荧光衰变过程的溶剂效应

采用时间分辨光谱技术(时间分辨率为0.15ns)系统地研究了两种典型香豆素激光染料(C311和 C102)的荧光衰变过程的溶剂效应。测量结果表明,这类香豆素激光染料的荧光寿命溶剂效应具有“反常”特性,即随着溶剂极性的增大其荧光寿命加大。基于香豆素激光染料处于激发态时发生电荷转移而使分子偶极矩有很大增加这一事实,作者认为处于激发态的染料分子将使溶剂分子进行重新排列,即发生了溶剂“结构化”过程,极性越大,“结构化”程度越高。这种“结构化”效应将阻止染料分子弛豫到基态,从而加大了荧光寿命。但对具有可能转动端基(CH_3)_2N—     

荧光相位成像及实验研究

荧光相位成像是最近发展起来的一项应用于生物的光子学技术．由于大多数有机物或生物组织在紫外或可见光激发下都会产生荧光，而荧光寿命通常能反映出细胞内某些成份（O2，K ，Ca2 ）的浓度变化，这对于研究细胞的功能及动力学过程具有十分重要的意义．荧光相位成像技术则通过相位与寿命之间的关系，可确定荧光寿命．通过下列表达式E（t）＝EO（1＋mesinωt）（1）tanθF＝ωT（2）可以简要阐明荧光相位成像的基本原理．式（1）中EO为激发光源功率，me为调制度，ω为调制频率，式（2）中θF为相位差，r为荧光寿命．当参考信号的相移为…     

量子点偶联RGD用于喉癌血管的靶向活体成像

研究了偶联环状精氨酸-甘氨酸-天冬氨酸-D-苯丙氨酸-赖氨酸[c(RGDfK)]肽段的CdSe/ZnS量子点(QDRGD)对喉癌血管靶向成像。利用羧基与氨基反应将c(RGDfK)肽段与QD偶联;采用荧光分光光度计对QD-RGD在RMPI1640培养基和小鼠血清溶液中的光谱稳定性进行了检测;利用荧光显微镜检测QD-RGD对Hep-2细胞和MCF-7细胞上整合素αvβ3的靶向性;最后将QD-RGD尾静脉注射到小鼠体内,检测了其对皮脊翼视窗中喉癌血管的靶向性。结果表明QD-RGD的发射光谱在RMPI1640培养基4h内没有明显变化,在小鼠血清中24h内发射光谱的荧光强度仅下降了20%;对细胞荧光成像表明QD-RGD能特异性与细胞表面的整合素αvβ3结合;血管成像表明QD-RGD在注射2h后聚集在喉癌局部血管,24h后QD-RGD从血管中移除。该研究表明QD-RGD能用于活体喉癌肿瘤血管靶向成像,这为喉癌的靶向诊断和靶向治疗研究提供了参考。     

双光子激发二氢卟吩衍生物的光物理特性

用飞秒脉冲激光,研究了二氢卟吩光敏剂CPD3分子在双光子激发(TPE)下的光物理过程。报道了该分子在四氢呋喃(THF)溶剂中的TPE荧光光谱及其寿命,以及在波长800nm处的双光子吸收(TPA)截面,其中,TPE与单光子激发(OPE)的荧光光谱形状一致,具有相同的荧光发射带,荧光寿命分别为5.1ns和5.7ns;在波长800nm处的TPA截面σ2≈12.5×10-22cm4/GW。本文分析表明:在TPE下,该分子跃迁到激发态S2,经历了无辐射弛豫到达OPE的同一荧光能级,呈现激发Q带所产生的正常的荧光发射;该分子具有大的TPA截面是起源于该分子的刚性平面共轭结构所固有的线性吸收特性和TPA共振增强;CPD3作为光动力治疗(PDT)的光敏剂,又具有长波长的荧光发射、ns级的荧光寿命和大的TPA截面特性,这些双重特性使其有可能成为双光子荧光分子探针,借助于双光子荧光显微和成像技术,在分子水平上揭开PDT光敏药物与细胞器的结合特性和作用靶点等深层次问题。     

利用条纹相机和锁模氩离子激光器测量染料寿命和时间分辨光谱

利用脉宽为200ps的主动锁模氩离子激光器在C1307—01条纹相机测量几种荧光寿命在12ns以内的染料的时间分辨光谱。     

单分子荧光成像技术下的运动员疲劳程度检测

无接触环境下对运动员疲劳程度进行检测是一项新型技术,提出基于单分子荧光成像技术的运动员疲劳程度检测方法,通过单分子荧光成像装置,获取运动员身体机能荧光信号,并计算荧光寿命数值,获取运动员心率、血氧饱和度、体温、运动能耗在不同运动强度下的变动值。将这4种身体机能荧光寿命变动值参数,带入BP神经网络模型进行迭代训练检测,获取运动员疲劳程度检测结果。实验结果显示,当20位运动员在8 km/h的长跑速度下,运动160 min后,其4种生理荧光寿命均逐渐变大,疲劳程度从10.55%提升至80.44%。经计算,该方法检测结果均方根误差较小,在50 dB背景噪声下,检测结果差值不超出所设定的允许误差范围。     

铯原子共振滤波器对XeCl/Pb蓝激光脉冲的吸收和响应

本文从理论上和实验上研究了充5Torr Ar气的铯原子共振滤波器对自由振荡XeCl/Pb蓝激光脉冲(40ns脉宽)的吸收和响应特性。从滤波器的侧向和纵向分别探测到脉宽为40ns和150ns的近红外荧光脉冲。     

Photophysics of Conjugated Oligoelectrolytes Relevant to Two-Photon Fluorescence-Lifetime Imaging Microscopy

Conjugated oligoelectrolytes (COEs) comprise a class of cell-membrane intercalating molecules that serve as effective optical reporters. However, little is known about the photophysical properties of COEs in biological environments such as buffers, cell membranes, and intracellular organelles, which is critical to optimize performance. Herein, how COE self-assembly depends on the dielectric environment (polarity and ion content) is explored based on the representative molecule 6-ring phenylenevinylene (PV) conjugated oligoelectrolyte (COE-S6), and its optical properties within mammalian cells are subsequently studied. Two-photon fluorescence lifetime imaging microscopy (FLIM), confocal laser scanning microscopy, and optical properties in solutions are brought together to obtain information about the location, accumulation, and characteristics of the local surroundings. FLIM imaging lifetime phasor plots, decays, and fluorescence spectra on stained mammalian cells provide evidence of successful COE-S6 internalization via endocytosis. The fluorescence lifetime of COE-S6 is identical when in A549 mammalian cells and in giant unilamellar vesicle model membranes, thereby providing a correlation between living system and artificial constructs.     

Tetrahydro[5]helicene‐Based Nanoparticles for Structure‐Dependent Cell Fluorescent Imaging

Four new fluorescent dyes containing tetrahydro[5]helicene moiety characterized by three‐primary emission colors (blue‐green‐red) are designed and synthesized, and their structures are characterized by NMR, MS, and single crystal X‐ray crystallography. Organic nanoparticles based on the fluorescent dyes are then prepared by re‐precipitation method, and their photophysical properties are investigated. These nanoparticles retain the strong emissions of the organic dyes, and multicolor nanoparticles were also prepared by simply tuning the ratios of the three‐primary colors dyes. These organic nanoparticles exhibit low cytotoxicity, good photostability, and high quantum yields. Moreover, the nanoparticles can also be applied in the cell fluorescence imaging. Especially, it is interestingly found that the stained regions of these nanoparticles from membrane to cytoplasm for HeLa cells show obvious structure‐dependent properties. This strategy provides a new perspective to fluorescence probe by molecular design for specific location imaging of living cells.     

Multicore Liquid Perfluorocarbon‐Loaded Multimodal Nanoparticles for Stable Ultrasound and 19F MRI Applied to In Vivo Cell Tracking

Ultrasound is the most commonly used clinical imaging modality. However, in applications requiring cell‐labeling, the large size and short active lifetime of ultrasound contrast agents limit their longitudinal use. Here, 100 nm radius, clinically applicable, polymeric nanoparticles containing a liquid perfluorocarbon, which enhance ultrasound contrast during repeated ultrasound imaging over the course of at least 48 h, are described. The perfluorocarbon enables monitoring the nanoparticles with quantitative ¹⁹F magnetic resonance imaging, making these particles effective multimodal imaging agents. Unlike typical core–shell perfluorocarbon‐based ultrasound contrast agents, these nanoparticles have an atypical fractal internal structure. The nonvaporizing highly hydrophobic perfluorocarbon forms multiple cores within the polymeric matrix and is, surprisingly, hydrated with water, as determined from small‐angle neutron scattering and nuclear magnetic resonance spectroscopy. Finally, the nanoparticles are used to image therapeutic dendritic cells with ultrasound in vivo, as well as with ¹⁹F MRI and fluorescence imaging, demonstrating their potential for long‐term in vivo multimodal imaging.     

Dopamine‐Modified Cationic Conjugated Polymer as a New Platform for pH Sensing and Autophagy Imaging

A dopamine‐modified conjugated polymer PFPDA is synthesized and characterized. At low pH, dopamine exists in its hydroquinone form and lacks the ability to quench fluorescence. At high pH, the proportion of the quinone form of dopamine increases due to its autooxidation, and efficient intramolecular electron transfer from the polymer main chain to quinone occurs, resulting in the quenching of the fluorescence of PFPDA. Thus, PFPDA exhibits a fluorescence “turn‐on” response at low pH. PFPDA possesses excellent photostability and exhibits no cytotoxicity, which makes it a good fluorescent material for pH sensing and cell imaging. A light‐induced hydroxyl anion emitter, MGCB, is also used to change the pH of the solution and thus regulate the fluorescence of PFPDA via remote control under light irradiation. Because the cytoplasm becomes acidic when cell autophagy occurs, PFPDA can also be used for autophagy imaging of HeLa cells with good selectivity.     

Interactions Between Amino Acid‐Tagged Naphthalenediimide and Single Walled Carbon Nanotubes for the Design and Construction of New Bioimaging Probes

A new synthetic route to functionalized single walled carbon nanotubes (SWNTs) via supramolecular interactions using a specifically designed naphthalenediimide (NDI) nanoreceptor is demonstrated. The tendency of the NDI to spontaneously form composites with carbon nanomaterials leads to fluorescent amino acid tagged SWNTs, which are dispersible in widely accessible organic solvents (CHCl₃, DMSO) as well as in biocompatible cell medium (EMEM, Eagle's modified essential medium). The X‐ray crystal structure of the first iodine‐tagged and amino acid‐functionalized NDI molecule, designed especially to facilitate the high resolution transmission electron microscopy (HR TEM) imaging whilst retaining its ability to self‐assemble into a nanodimensional receptor in weakly polar solvents, is also described. A new hybrid material, NDI@SWNT, was prepared and characterized as dispersed in organic solvents and aqueous media and in the solid state by HR TEM, tapping mode atomic force microscopy (TM AFM), scanning electron microscopy (SEM), circular dichroism, Raman and fluorescence spectroscopies (steady‐state single and two‐photon techniques). Combined microscopy techniques, density functional theory (DFT) calculations using the Spanish Initiative for Electronic Simulations with Thousands of Atoms (SIESTA) program and spectroscopic measurements in solution indicate that amino acid‐functionalized NDI interacts strongly with SWNTs and forms a donor‐acceptor complex. Density functional theory (DFT) calculations predicted the geometry and the binding energies of an NDI molecule loaded onto a SWNT strand and the possibility of charge transfer interactions within the hybrid. The NDI@SWNT composite translocates into cells (e.g. FEK‐4, HeLa, MCF‐7) as an intact object and localizes in the cells' cytoplasm and partially in the nucleus. The NDI coating enhances the biocompatibility of SWNTs and mediates its intracellular localization as shown by confocal fluorescence imaging and fluorescence lifetime imaging (FLIM) techniques. The excited state fluorescence lifetime of the probes in cells versus solution phase indicates that the probes remain unaffected by the change in their chemical environment within the experimental timescale (2 h).     

荧光寿命观测系统的设计

本文介绍了一种测量荧光寿命的新方法:利用荧光衰减曲线和RC电路衰减曲线合成李萨如图形来测量固体荧光材料的荧光寿命.文中阐述了荧光寿命的测试原理,对激励光源、供电的脉冲电源和光电转换器进行了分析,用所设计的荧光寿命观测系统对Nd:YAG晶体的荧光寿命进行了测量,得到的数值为230 m,与理论值相等.     

用时间分辨荧光光谱研究乙酸-水溶液的聚合特性

对284nm激发光照射不同体积百分比乙酸一水溶液的峰值荧光强度时间衰变曲线进行了实验测量,并检测了不同紫外光激励下334nm荧光强度随时间的衰变过程,将获得的荧光衰减动力学曲线采用指数拟合方法进行了解卷积处理,测试了相应的荧光寿命。分析了乙酸-水溶液中线状二聚物（LD）缔合结构的变化规律,计算了混合物质中不同组分的浓度...     

Motif‐Designed Peptide Nanofibers Decorated with Graphene Quantum Dots for Simultaneous Targeting and Imaging of Tumor Cells

Nanohybrids based on biomolecular nanostructures and graphene quantum dots (GQDs) have found wide application in the biological and biomedical fields. Herein, the design of a peptide with trifunctional motifs is reported as the precursor building block for constructing a novel multifunctional protein nanofiber (PNF), and further conjugated with highly fluorescent GQDs by noncovalent interactions. The physicochemical properties of these PNF–GQD nanohybrids are thoroughly characterized by a variety of spectroscopic and microscopic techniques, revealing that the GQDs essentially maintain their favorable optical properties in the nanohybrids. A good biocompatibility of the PNF–GQD nanohybrids is found with cell viability assays. With both, a recognition moiety (RGD) and an imaging probe (GQD), these PNF–GQD nanohybrids possess the capability of targeting and imaging tumor cells simultaneously. A potential application of these novel nanohybrids, i.e., fluorescence imaging of HeLa tumor cells, has been investigated by confocal fluorescence microscopy, which shows much enhanced labeling efficiency compared with GQDs only. Moreover, cellular internalization by nontumorous COS‐7 cells was much weaker than by HeLa cells. Our results show that GQD‐decorated PNF nanohybrids have great potential as multifunctional platforms for biomedical applications, particularly, where the capability of sensitive tracking and efficient labeling is appreciated.     

Multiple Nuclei Tracking Using Integer Programming for Quantitative Cancer Cell Cycle Analysis

Automated cell segmentation and tracking are critical for quantitative analysis of cell cycle behavior using time-lapse fluorescence microscopy. However, the complex, dynamic cell cycle behavior poses new challenges to the existing image segmentation and tracking methods. This paper presents a fully automated tracking method for quantitative cell cycle analysis. In the proposed tracking method, we introduce a neighboring graph to characterize the spatial distribution of neighboring nuclei, and a novel dissimilarity measure is designed based on the spatial distribution, nuclei morphological appearance, migration, and intensity information. Then, we employ the integer programming and division matching strategy, together with the novel dissimilarity measure, to track cell nuclei. We applied this new tracking method for the tracking of HeLa cancer cells over several cell cycles, and the validation results showed that the high accuracy for segmentation and tracking at 99.5% and 90.0%, respectively. The tracking method has been implemented in the cell–cycle analysis software package, DCELLIQ, which is freely available.      

Picosecond time-resolved fluorescence spectroscopy of hematoporphyrin derivative

The picosecond time-resolved fluorescence decaysI(t)and spectraI(lambda, t)for hematoporphyrin derivative (HPD) in a phosphate buffer saline aqueous solution at different concentrations (8.4 times 10^{-6}sim 8.4 times 10^{-3}M) are measured by a two-dimensional synchroscan streak camera with a mode-locked CW dye laser, and a new emission band (which we call theY-band) is found at high concentration. It is shown that the fluorescence decays composed of fast and slow components at high concentration are due to theY-band (120 ps lifetime) from head-to-tail aggregates including equilibrium dimer and stable dimer, and the usual band (3.6 ns lifetime) from monomer, respectively, and the latter band is dynamically quenched by the Förster type resonance energy transfer from the monomer to the aggregate. Furthermore, the measurement of static fluorescence spectra from human gastric cancers and the surrounding in vivo after HPD injection shows that a band corresponding to theY-band from the aggregate appears at only the cancerous cells.