单分子荧光检测在生命科学中的应用

文章对单分子荧光检测在分子马达、离子通道、信号分子、蛋白折叠、蛋白构象变化动力学、酶活性反应、细胞过程实时观察等生命科学领域中的应用进行了介绍.这些研究结果表明,单分子荧光检测在研究生物大分子的活动规律与机制方面不但有着无法替代的优越性,而且有着广阔的发展空间.     

基于单分子成像技术研究λ-DNA分子穿越微米通道端口的电动力学特性

操控单个DNA分子,将其有效引入、导出微纳通道是实现DNA生物芯片功能的前提条件.本文利用单分子荧光显微成像技术系统地实时观察分析λ-DNA单分子在电场力驱动下进入/穿出50μm通道端口处的电动力学特性及规律.研究发现:λ-DNA分子能够顺利进入trans端口并穿出cis端口,外加电场强度存在最大(E_max)和最小(E_min)阈值,只有场强E满足:E_min≤E≤E_max时, λ-DNA分子才能进入trans端口并顺利穿出cis端口;当电场强度小于最小阈值场强时, DNA分子不能进入trans端口;当电场强度大于最大阈值场强时, λ-DNA分子虽可能从trans端口进入通道内部,但不容易从cis端口穿出,而是在迁移至通道内cis端口附近时,运动方向反转、往复、甚至旋转等新现象,并且易于粘附到管壁上;随着场强增大,反转位置距cis端口越大.基于微流体电动力学理论,对λ-DNA分子在微通道端口的不同运动状态的物理机制进行了初步分析.本研究结果对研制基于微纳通道系统的基因芯片实验室及DNA分子传感器具有一定...     

单分子荧光过程的Monte-Carlo模拟

从单分子发光过程中电子在激发态上的停留时间t服从P(t)=γe^-γt分布出发,对单分子荧光过程进行M-C(Monte-Carlo)随机试验,得到了单分子的激发态相邻两次发光的时间间隔的统计结果以及单分子荧光强度的自相关函数,并对其进行了详细地讨论,这些结果与目前文献报道的实验结果相一致.     

基于ICCD的快速荧光显微成像技术及在活细胞研究中的初步应用

利用像增强型CCD、氩离子激光器和氙灯等建立了一套快速荧光显微成像系统,并初步应用于活细胞研究。实时观测和拍摄了大鼠脑微血管内皮细胞增殖分裂过程中细胞内钙敏感荧光探针Fluo-3标记的钙离子浓度分布快速变化的图像,并选取动态图像中四个典型的点给出荧光强度灰度值的变化曲线。该系统可用于高灵敏实时记录活细胞内基于荧光显微成像的快速变化过程,为活细胞的研究提供了一种很好的观察和分析手段。     

一种荧光单分子高精度快速跟踪方法的研究

论述了一种高精度快速的单分子实时跟踪方法,包括图像去噪处理与高斯掩模质心法相结合的定位算法和同时考虑等效光强和帧间最大运动位移的同分子概率链接算法。对不同信噪比(SNR)下的单分子荧光图像的仿真结果表明,该定位算法与被认为表现最好的二维直接高斯拟合相比,不仅运算时间缩短为原来的1/10,而且在整个测试信噪比范围内具有更低的偏差和更好的精度,甚至在低信噪比时也能得到可信的小偏差、高精度中心位置值。在普通个人计算机上利用该方法对单分子的百万像素级序列帧进行实时跟踪时,跟踪速率能达到4~5 frame/s。     

单分子的荧光特性及其在生物学上的应用

近年来,单分子探测在许多学科领域的研究取得了显著的进展。它为科学家提供了一种新的手段来研究这些领域的前沿课题。光学和光谱技术是单分子探测最常用的方法之一。单个分子的荧光强度的涨落及其荧光的偏振特性是单分子荧光的重要特征,在单分子探测的广泛应用中,人们正是利用这种单个分子的重要特征来研究和推测生物大分子的结构和功能。文章简要介绍了单分子的荧光特点、探测方法及其在生物学中的应用。     

荧光分子断层成像正向问题的并行计算

针对荧光分子断层成像中相应于激发光和发射光的两个正向方程必须串行求解的实际情况,提出了一种可同时对两个扩散方程进行求解的并行算法。其思想是通过引入乘子矩阵对耦合方程进行解耦来实现并行计算,并利用有限元方法进行了二维数值模拟,将算法求解所得结果与基于串行方法,以Ralf B．Schulz等提出的并行算法所得到的数值模拟结果进行了综合比较。实验表明,该算法一方面适合于任何大小的斯托克斯频移条件,具有更广泛的适应性;另一方面提高了荧光分子断层成像正向问题的求解速度和精度,从而有利于整个荧光分子断层成像的快速精确求解。     

多维度单分子成像研究进展

单分子成像方法被广泛应用于亚细胞结构的三维空间定位。点扩散函数是分析单分子信息的重要窗口,除了能反映空间坐标外还蕴含着丰富的额外信息。本文介绍了从点扩散函数中解析空间位置、荧光波长、偶极子朝向及干涉相位等多维度单分子成像研究进展,简要地概括了目前主流定位方法,并对该技术的发展方向进行了展望。     

原位成像检测活性酶的分子荧光探针研究进展

活性酶普遍存在于各种生命活动中,一些疾病与活性酶的异常表达息息相关,精确检测酶的表达水平以及原位成像,为相关疾病的诊断与治疗提供了有力的判断依据。至今,大量的检测技术已经开发出来,其中以分子荧光探针为代表的光学技术具有非侵袭性以及灵敏度高、检测限低、响应时间快和生物相容性好等优势,在检测活性酶上备受青睐。然而,在使用分子荧光探针检测时,由于小分子容易在酶活性位点发生扩散,无法定位,导致探针时空分辨率较差。因此,为提高成像检测的时空分辨率、降低背景干扰和假阳性,原位成像的设计理念随之提出,已成为生物光学成像的研究热点之一。目前,研究者已报道多种分子荧光探针用于酶的原位成像的设计并取得显著效果。本文将深入介绍用于活性酶检测的分子荧光探针的设计策略及其在原位成像中的研究进展,希望为该领域的研究者们提供一些启发。     

利用三通道实时荧光成像方法研究单个活细胞凋亡与胞浆pH值变化的关系

利用双波长分光器(Dual-View)和自制滤光片滑块,在基于像增强型电荷耦合器件(ICCD)的实时/快速荧光成像系统基础上,建立了一种基于单个ICCD的三通道实时荧光成像方法,同时发展了相应的图像校准处理方法用于消除光谱串扰的影响,并将该方法应用于单个活细胞的研究。利用Annexin V-FITC和SNARF-1两种荧光探针进行双标记,在单细胞水平上对亚硝基谷胱甘肽(GSNO)诱导小鼠胸腺细胞凋亡与其胞浆pH值变化的关系进行实时研究。结果揭示了GSNO诱导的细胞凋亡与细胞发生自发凋亡时胞浆pH值有不同的变化规律,为这种三通道实时荧光成像方法在生物医学领域的应用展示了广阔的前景。     

Hybrid Rayleigh,Raman and two‐photon excited fluorescence spectral confocal microscopy of living cells

A hybrid fluorescence–Raman confocal microscopy platform is presented, which integrates low‐wavenumber‐resolution Raman imaging, Rayleigh scatter imaging and two‐photon fluorescence (TPE) spectral imaging, fast ‘amplitude‐only’ TPE‐fluorescence imaging and high‐spectral‐resolution Raman imaging. This multi‐dimensional fluorescence–Raman microscopy platform enables rapid imaging along the fluorescence emission and/or Rayleigh scatter dimensions. It is shown that optical contrast in these images can be used to select an area of interest prior to subsequent investigation with high spatially and spectrally resolved Raman imaging. This new microscopy platform combines the strengths of Raman ‘chemical’ imaging with light scattering microscopy and fluorescence microscopy and provides new modes of correlative light microscopy. Simultaneous acquisition of TPE hyperspectral fluorescence imaging and Raman imaging illustrates spatial relationships of fluorophores, water, lipid and protein in cells. The fluorescence–Raman microscope is demonstrated in an application to living human bone marrow stromal stem cells. Copyright © 2009 John Wiley & Sons, Ltd.     

Theoretical simulation of the fluorescence spectrum of a single molecule

The possibility of computer simulation of the fluorescence spectrum of an actual system of single molecules, whose initial isomeric structure is photochemically transformed as a result of a radiationless resonance transition, is demonstrated.Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 71, No. 6, pp. 740–744, November–December, 2004.This revised version was published online in April 2005 with a corrected cover date.     

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.     

Calcium imaging using fluorescence lifetimes and long-wavelength probes

We describe imaging of calcium concentrations using the long-wavelength Ca²⁺ indicators, Calcium Green, Orange, and Crimson. The lifetimes of these probes were measured using the frequency-domain method and were found to increase from 50% to severalfold in response to calcium. The two-dimensional images of the calcium concentration were obtained using a new apparatus for fluorescence lifetime imaging (FLIM). We also describe procedures to correct for the position-dependent frequency response of the gain-modulated image intensifier used in the FLIM apparatus. Importantly, the FLIM method does not require the probe to display shifts in the excitation or emission spectra. Using the FLIM method, calcium imaging is possible using probes which display changes in lifetime in response to calcium. Consequently, calcium imaging is possible with excitation wavelengths ranging from 488 to as long as 620 nm, where autofluorescence and/or photochemical damage is minimal. These probes are also suitable for calcium measurements of single cells using lifetime-based flow cytometry.     

Interaction of a phosphatidylcholine derivative of 1,6-diphenyl-1,3,5-hexatriene (DPH) with intact living cells: Steady-state fluorescence polarization and phase fluorometry studies

The potential interest of DPH-PC was checked with a macrophagic cell line (P388D1). The uptake of DPH-PC was associated with a rapid increase in both fluorescence intensity and a slow decrease in anisotropy values. A flow cytometry comparative study with DPH revealed in both cases the existence of two cell subpopulations with different labeling levels. The analysis of fluorescence decay of DPH-PC showed two components. The fractional intensity of the main component (9.7 ns) is higher than 92%. The Lorentzian distribution of the main lifetime presents an important homogeneity. The observation that an increase in temperature induced a decrease in steady state anisotropy values but did not affect the lifetime suggests that the anisotropy variations effectively reflect modifications in the cohesion of probe micro-surroundings. A transmembrane diffusional phenomenon of a fraction of fluorescent phospholipids (205) was suggested by a study with a nonpermeant membrane quencher. The transmembrane diffusion was confirmed by extraction of the phospholipid analog with fatty acid free BSA. The use of inhibitors of endogenous phospholipase A2 showed a progressive hydrolysis of the fluorescent phospholipid. Nevertheless, the hydrolysis can be neglected in the case of short term interactions with cells (<30 min). Therefore, it can be assumed that DPH-PC can be used as a membrane probe.     

用荧光显微示踪方法研究RecA在DNA同源识别过程中的工作机理

RecA是原核生物体内参与DNA同源识别过程的一种关键蛋白,长期以来一直是同源重组相关课题的重要研宄对象.通过荧光显微示踪方法,发现在同源识别过程中RecA与单链DNA形成的核蛋白丝与模板DNA的结合是短时(τ=0.2 s)和短程(l=1.05μm)的,结合后搜寻模板DNA上的同源位点的过程可分为布朗运动和定向运动两种模式.结合时核蛋白丝并不是缠绕在模板DNA上,而是以一种更弱的方式结合在模板DNA外侧进行位点搜寻.如果在该过程中没有找到同源位点,核蛋白丝就会脱离模板DNA,并寻找下一次与模板DNA结合的机会,重复以上过程.     

Fluorescence lifetime imaging of oxygen in living cells

The usefulness of the fluorescent probe ruthenium tris(2,2′-dipyridyl) dichloride hydrate (RTDP) for the quantitative imaging of oxygen in single cells was investigated utilizing fluorescence lifetime imaging. The results indicate that the fluorescence behavior of RTDP in the presence of oxygen can be described by the Stem-Volmer equation. This shows that fluorescence quenching by oxygen is a dynamic quenching process. In addition, it was demonstrated that the fluorescence lifetime of RTDP is insensitive to pH, ion concentration, and cellular contents. This implies that a simple calibration procedure in buffers can be used to quantify oxygen concentrations within cells. First fluorescence imaging experiments on J774 macrophages show a nonuniform fluorescence intensity and a uniform fluorescence lifetime image. This indicates that the RTDP is heterogeneously partitioned throughout the cells, while the oxygen concentration is constant.     

结合虚拟单像素成像解卷积的双边照明光片荧光显微技术

光片荧光显微术(light-sheet fluorescence microscopy,LSFM)采用薄片光束从侧面激发样品,在垂直于光片方向上进行成像,具有成像速度快、光学层析能力强以及光漂白和光毒性低等优点,适用于对较大活体生物样品进行高质量、长时间三维动态观测.然而,传统高斯光束LSFM存在分辨率低和成像视场小的...     

应用于单分子测序的纳米微结构器件

基因测序技术是现代最为重要的生物医学研究手段之一,单分子测序技术作为最新一代测序技术被广泛研究,并形成了微纳制造、光电子、微流控和分子生物学等多学科的交叉探索和多种技术创新的有机结合.文章系统总结了应用于单分子测序的纳米微结构器件的原理和功能,重点阐述了零模波导器件和纳米孔器件在单分子测序中的作用以及制备工艺,从器件的角度提出了单分子测序技术所面临的挑战.     

An integrated phase-resolved fluorescence imaging system with sub-nanosecond lifetime resolution

In this paper, a novel configuration of an integrated phase-resolved (PR) fingerprint fluorescence imaging system is proposed and implemented. In this integrated PR imaging system, a current modulated 402 nm dual diode laser is proposed to be the light source, to obtain both high laser power and easy modulability. To estimate the lifetime resolution of this PR imaging system, a novel method of using distance-selective suppression of fluorescence signals from two identical fluorescing samples is proposed. Detailed theoretical and experimental analyses are presented. The experimental results demonstrate that this integrated PR imaging system has a lifetime resolution of 0.1 ns. Fingerprint detection experiments are also carried out using this system with latent fingerprints deposited on substrates of aluminum foil and currency.