活细胞内单个大分子的行为

以往细胞内大分子活动的研究,多数是许多分子活动的一个平均结果,即一种集群平均（ensemble averaging）的结果,随着各种技术,特别是光学及荧光检测技术的成熟,实时视见活细胞内单个大分子的时代已经到来.在现时条件下,有许多方法可供选择,如荧光共振能量转移、原子力显微镜、全内反射显微技术、荧光相关光谱法等等.“活细胞内单个大分子的行为”的研究有可能为了解活细胞内单个分子的活动带来完全新的认识,但目前还存在不少方法学上的局限性,有待进一步提高,如有效的特异标记物、细胞深部荧光的检测、新型显微镜的开发等.     

梯度场中荧光关联谱测量的Monte Carlo模拟

荧光关联谱（FCS）利用少量分子的荧光涨落获得分子运动和反应信息,是一种探测活细胞内生物过程的有力工具。本文利用Monte Carlo方法模拟激光梯度场下瑞利粒子的布郎运动,分析梯度场对FCS测量得到的扩散时间、粒子数等参数的影响,模拟与初步实验结果定性吻合。文中还讨论了Monte Carlo模拟预测的一些实验现象。     

人体血清中原卟啉荧光相关光谱分析方法研究

原卟啉(PPIX)是血清中内源性荧光物质之一,目前研究表明癌症病人血液中的原卟啉含量高于正常人水平。因此,测定血清样品中的PPIX含量对于癌症病人的早期诊断具有重要的意义。本文中以超灵敏的荧光相关光谱(FCS)单分子检测技术,建立了直接测定血清中PPIX的含量的新方法。通过对100例正常人血清样本和12例疑似癌症病人血清样本(甲胎蛋白AFP含量异常)中PPIX的FCS数据分析,发现相对于检测PPIX-HSA的单个分子荧光强度和总荧光强度,PPIX-HSA在病人血清中的浓度与正常人血清有着极显著差异。FCS方法具有样品需求量少、分析时间短、可实现自动化操作等优点,有望成为癌症临床诊断的一种新方法。     

Couette型液体粘滞系数测量公式修正

Searle型系统测量液体粘滞系数文献研究较多,而Couette型液体粘滞系数测量系统研究很少且已有的文献描述错误或者不完善。利用流体力学和粘性流体力学基本理论,结合Searle型内筒旋转式测量液体粘滞系数的研究方法,得到Couette型液体粘滞系数测量系统测量液体粘滞系数表达式,并通过实验测量25℃下蓖麻油的粘滞系数。结果表明:不同尺寸圆筒得到的粘滞系数分别为0.614、0.621、0.625、0.618,误差分别为1%,0.2%,0.8%,0.3%。     

光谱学 激光光谱学

O433．54 2005031677 饱和激发情况下的荧光关联谱测量=Fluorescence corre- lation spectroscopy with saturated excitation[刊,中]／丁建 英(清华大学物理系,原子分子纳米科学教育部重点实验 室．北京(100084)),陈波…∥物理学报．-2004,53 (8)．-2503-2508 研究了激发光谱增强时其对荧光关联谱(FCS)测量的 影响。强激发光产生的饱和激发会改变激光诱导荧光的 空间分布函数,从而影响FCS的测量结果。首先根据饱和 吸收的物理模型,推导出强激发光情况下有效探测区域变     

活细胞内的单分子荧光成像方法

文章介绍近年来新发展的几种重要的活细胞内单分子荧光成像方法,如转盘式共聚焦显微术、全内反射荧光显微术、荧光共振能量转移技术等。通过介绍它们的原理、特点和在活细胞内单分子行为研究中的应用实例,展示了这些新方法在生命科学领域广阔的应用前景。     

饱和激发情况下的荧光关联谱测量

研究了激发光增强时其对荧光关联谱(FCS)测量的影响. 强激发光产生的饱和激发会改 变激光诱导荧光的空间分布函数,从而影响FCS的测量结果. 首先根据饱和吸收的物理模型,推导出强激发光情况下有效探测区域变化的定量公式,获得了FCS测量所得到的粒子数和扩散时间与饱和激发光强和激光光强的函数关系,并用于消除饱和吸收造成的系统误差.采用Monte-Carlo模拟方法和实验,对理论分析结果进行了验证. 这将有助于完善FCS分析方法的理论模型,为高激发光强度下的FCS探测提供依据. 关键词： 荧光关联谱 饱和激发 Monte-Carlo模拟     

隧道电子激发的辛硫醇条纹相自组装膜上卟啉单分子荧光

利用扫描隧道显微镜诱导发光技术,对单个卟啉分子的电致荧光现象进行了研究. 为了避免金属衬底对单个卟啉分子的荧光淬灭,利用条纹状辛硫醇自组装膜作为脱耦合层,实现了单个中性卟啉分子的电致荧光,并且发现分子荧光的产生呈现双极性特征. 另外,分子瓣上的荧光强度要强于分子中心的荧光强度.     

新型快速显微多道分光光度技术对血红蛋白的光谱分析

用新型快速显微多道分光光度(MMSP)技术,在生理环境(pH、温度、氧分压和渗透压)改变下,对单个活态人血红细胞内血红蛋白分子的吸收光谱进行监测。发现生理环境的改变,引起血红蛋白吸收光谱曲线540,575 nm特征吸收峰的峰高、峰位和峰形发生了不同程度的变化。MMSP技术提示血红蛋白分子的结构、浓度和功能与上述生理环境密切相关。     

激光梯度场中荧光关联谱学的实验研究

研究了荧光关联谱(fluorescence correlation spectroscopy,简称FCS)在FCS测量中外加激光梯度场的影响.实验显示,微区内粒子的扩散时间随梯度场强度增加而增加,近似呈线性关系.同时,微区内平均粒子数目随梯度场相应地增加.粒子的极化率越大,相同条件下受到激光梯度场的影响就越大. 关键词： 荧光关联谱(FCS) 激光梯度场 扩散时间     

Characterization of a confocal microscope for time-resolved photon counting fluorescence

A confocal microscope setup is developed for time-resolved fluorescence measurements. It is added to a traditional cuvette time-resolved setup, with a pumped Ti-Sa light source. The temporal resolution of 37 ps (FWHM) is not degraded, in comparison with the cuvette setup also described. These setups allow both decay lifetime and anisotropy relaxation time determination. Fluorescence correlation spectroscopy (FCS) is used to determine the observation point size. When associated with the calcium probe calcium green, calcium concentration in single cells can be determined in 10 ms by simultaneous acquisition of early and late fluorescence photons.     

Fast, flexible algorithm for calculating photon correlations

We introduce a new algorithm for computing correlations of photon arrival time data acquired in single-molecule fluorescence spectroscopy and fluorescence correlation spectroscopy (FCS). The algorithm is based on rewriting the correlation as a counting operation on photon pairs and can be used with arbitrary bin widths and spacing. The flexibility of the algorithm is demonstrated by use of FCS simulations and single-molecule photon antibunching experiments. Execution speed is comparable to the commonly used multiple-tau correlation technique. Wide bin spacings are possible that allow for real-time software calculation of correlations, even for high count rates.     

Statistical Analysis of Diffusion Coefficient Determination by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) has become an important and widely used technique for many applications in physics, chemistry, and biology. The parameter most frequently addressed by FCS is the diffusion of molecules in solution. Due to the highly non-linear connection between the diffusion coefficient and a measured autocorrelation function, it is extremely difficult to analyse the accuracy of the diffusion-coefficient determination in a FCS experiment. Here, we present a simplified analysis based on some general maximum-likelihood considerations, and numerical result are given for the dependence of the accuracy of the diffusion-coefficient determination on sample concentration, brightness, and measurement time. Optimal concentration values for performing FCS are found.     

Coherence time measurements using a single detector with variable time resolution

We present a simple technique for measuring coherence times for stationary light fields using a single detector with tunable time resolution. By measuring the equal-time second-order correlation function at varying instrument response functions it is possible to determine the coherence time and also the shape of the temporal decay without the need to record time-resolved data. The technique is demonstrated for pseudothermal light. Possible applications for dynamic light scattering and photon statistics measurements are discussed.     

Fluorescence Correlation Spectroscopy with Photobleaching Correction in Slowly Diffusing Systems

Fluorescence correlation spectroscopy (FCS) is a powerful tool to quantitatively study the diffusion of fluorescently labeled molecules. It allows in principle important questions of macromolecular transport and supramolecular aggregation in living cells to be addressed. However, the crowded environment inside the cells slows diffusion and limits the reservoir of labeled molecules, causing artifacts that arise especially from photobleaching and limit the utility of FCS in these applications. We present a method to compute the time correlation function from weighted photon arrival times, which compensates computationally during the data analysis for the effect of photobleaching. We demonstrate the performance of this method using numerical simulations and experimental data from model solutions. Using this technique, we obtain correlation functions in which the effect of photobleaching has been removed and in turn recover quantitatively accurate mean-square displacements of the fluorophores, especially when deviations from an ideal Gaussian excitation volume are accounted for by using a reference calibration correlation function. This allows quantitative FCS studies of transport processes in challenging environments with substantial photobleaching like in living cells in the future.     

基于光场一阶关联的时域成像

与通常利用二阶强度关联测量实现时域鬼成像不同,本文利用时域热光源借助干涉仪通过一阶关联实现时域成像.基于空域光束的近轴衍射和时域窄带脉冲在色散介质中色散之间的空间-时间二象性,在时域脉冲响应函数的基础上得到了表征一阶关联时域成像的强度表达式,分析研究了光源脉冲宽度和相干时间对成像可见度和分辨率的影响.结果一方面表明基于热光场一阶关联的时域成像在不需要额外色散补偿或消除条件下可以实现时域物体信号的再现,另一方面表明当光源脉冲宽度一定时,成像可见度随光源脉冲相干时间的增加而增加,但是成像分辨率逐渐降低,其中当光源脉冲宽度约为100 ps,相干时间约为0.5 ps时,间隔为20 ps,宽度为8 ps的时域矩形波型物体的成像质量(兼顾可见度和分辨率)较好.该结果对于基于热光一阶关联的时域成像在时序信号测量中的应用具有重要意义.     

Fluorescence Correlation Spectroscopy (FCS): A Promising Tool for Biological Research

Abstract

Fluorescence correlation spectroscopy (FCS) is an important biophysical technique. FCS is currently being used in many areas of biology to solve several scientific problems. Its properties such as detection at the single molecular level, higher sensitivity, and use of lower sample volume make FCS a promising molecular diagnostic tool. The promising applications of FCS extend from DNA kinetics/dynamics studies to the comprehensive understanding of receptor–ligand interactions. In this article, we review various promising biological applications of FCS.     

Simultaneous Surface-Near and Solution Fluorescence Correlation Spectroscopy

We report the first simultaneous measurement of surface-confined and solution fluorescence correlation spectroscopy (FCS). We use an optical configuration for tightly focused excitation and separate detection of light emitted below (undercritical angle fluorescence, UAF) and above (supercritical angle fluorescence, SAF) the critical angle of total internal reflection of the coverslip/sample interface. This creates two laterally coincident detection volumes which differ in their axial extent. While detection of far-field UAF emission producesa standard confocal volume, near-field-mediated SAF produces a highly surface-confined detection volume at the coverslip/sample interface which extends only ~200 nm into the sample. A characterization of the two detection volumes by FCS of free diffusion is presented and compared with analytical models and simulations. The presented FCS technique allows to determine bulk solution concentrations and surface-near concentrations at the same time.     

Dynamics of DNA: Experimental controversies and theoretical insights

Recent experimental advances using fluorescence correlation spectroscopy (FCS) have given unprecedented information about the small-scale kinetics of large biopolymers in solution. However some of the first studies in this direction yielded conflicting results for the mean squared displacement of the tagged end-point of a DNA chain, deviating from traditional theories of polymer dynamics. Spurred by this controversy, we have developed a hydrodynamic mean-field theory for single semiflexible polymers which points to a resolution of the differing experimental observations. The theory precisely captures, without fitting parameters, one set of recent FCS results, reproducing the experimental dynamics over five decades in time and three decades of chain lengths. The success of the theory makes it an excellent candidate for a variety of biophysical contexts where the internal fluctuations of semiflexible polymers play a role.