荧光相关谱测量技术研究

荧光相关谱(fluorescence correlation spectroscopy,FCS)是对处于热平衡态条件下的荧光分子发出的荧光强度涨落进行时间相关处理的一种单分子检测方法,能够直接测量分子在溶液里的扩散系数和浓度.影响FCS测量扩散系数准确性的因素有分子量子效率,测量时间,样本折射率和温度偏差等.用FCS分别测量溶有荧光染料罗丹明6G(rhodamine 6G,Rh.6G)和青色素Cy5甘油水溶液的粘滞系数,实验结果表明:荧光分子的量子效率是影响测量准确性的重要因素,要求其每秒发射的光子数目(photon counts per second,cps)至少达到1 000(photons/s).     

基于双光子激发的荧光相关谱测量

本文利用多光子激发激光扫描显微镜的部分光路和探测器．建立了双光子荧光相关谱系统(Two-Photo Fluorescence Correlation Spectroscopy．简称TP-FCS)。利用TP-FCS系统观察到了“光子爆发”现象．实现了染料分子的双光子激发,测量出若丹明B染料分子在蔗糖溶液中的扩散系数。实验证明该系统具有操作简便、可靠性高,费用低廉等等点,可实现单分子检测。     

CorTectorTM SX100：一款桌面式荧光相关光谱仪的原理和应用

荧光相关光谱检测技术具有超灵敏(单分子)、快速(数秒至数分钟)和多功能(检测分子浓度、大小和相互作用)等技术优点,且无需反应物分离,因此有潜力成为一种新型均相、高敏荧光免疫检测技术,适用于在溶液中或单个活细胞内检测生物分子特性．本文首先介绍荧光相关光谱检测技术的原理和研究进展,然后结合项目团队自主研发的目前全球唯一一款可靠、易使用的桌面式荧光相关光谱仪,进一步探讨荧光相关光谱检测技术的具体实现和潜在应用．     

荧光单分子检测技术在生命科学中的应用

荧光单分子检测技术是用荧光标记来显示和追踪单个分子的构象变化、动力学,单分子之间的相互作用以及单分子操纵的研究。过去对于生命科学分子机制的研究,都是对分子群体进行研究,然后平均化来进行单分子估测。因此,单个分子的动态性和独立性也被平均化掉而无法表现出来。荧光单分子检测技术真正实现了对单个分子的实时观测,将过去被平均化并隐藏在群体测量中不能获得的信息显示出来。近几年来,荧光单分子检测技术的飞速发展,为生命科学的发展,开辟了全新的研究领域。现就荧光单分子检测技术在研究动力蛋白、DNA转录、酶反应、蛋白质动态性和细胞信号转导方面的应用进展作一综述。     

荧光相关光谱在生物化学领域中的应用

荧光相关光谱(fluorescence correlation spectroscopy,FCS)是一种通过监测荧光涨落从而获得单分子水平的分子扩散行为信息的技术。FCS高灵敏度的优点使得它已发展成为一种可以在活体外与活体内检测分子浓度、扩散系数、结合和解离常数等参数的有力工具。荧光互相关光谱(fluorescence cross-correlation spectroscopy,FCCS)是FCS技术的进一步发展,其大大扩展了FCS技术的应用范围。本文介绍了FCS及其衍生技术的原理及其在生物化学领域的应用。     

单分子荧光共振能量转移技术

单分子荧光共振能量转移技术（single molecule fluorescence resonance energy transfer,smFRET）通过检测单个分子内的荧光供体及受体间荧光能量转移的效率,来研究分子构象的变化。在单分子探测技术发展之前,大多数的分子实验是探测分子的综合平均效应（ensemble averages）,这一平均效应掩盖了许多特殊的信息。单分子探测可以对体系中的单个分子进行研究,得到某一分子特性的分布状况,也可研究生物分子的动力学反应。介绍了近来单分子荧光共振能量转移技术的进展。     

单分子荧光检测技术的发展及其在植物生物学研究中的应用

单分子荧光检测技术是利用荧光基团对目的分子标记后,在单分子水平成像并追踪分子的构象变化、动力学特征以及分子之间相互作用的研究方法.相较于传统分子生物学和遗传学的研究手段,单分子检测技术可以对单个分子的动态和特性进行分析,特别是瞬时或偶发性的事件,从而更加深入地挖掘在群体测量中被掩盖的信息.该技术已广泛应用于动物细胞生物...     

单分子荧光原位杂交（smFISH）技术及应用

单分子荧光原位杂交（single-molecule fluorescence in situ hybridization,smFISH）技术是一种通过用偶联荧光基团的寡核苷酸探针,对固定细胞或组织中单个mRNA分子进行成像的方法。smFISH可对RNA进行定位、定量,以此对目标转录本进行实时研究。smFISH适用于细胞、组织切片等多种类型生物样本。近年来,多种基于基础smFISH的改进技术被发明,进一步促进了该技术的实际应用。smFISH良好的RNA单分子可视化能力,使得其在发育生物学、神经生物学及肿瘤生物学等基础生物学科中得到了广泛的应用。本文综述了smFISH技术基本原理、smFISH技术的局限性、smFISH衍生技术方法、smFISH在不同生物学科中的应用进展,并对smFISH技术的发展前景做出展望。     

绿色荧光蛋白及其应用

绿色荧光蛋白是在水母中发现的新型报告分子,能在多种生物体内表达并发出荧光。对GFP中一些特定氨基酸进行突变可以产生多种类型的突变体,有利于研究蛋白之间或细胞器之间的相互作用。目前,GFP已经用于基因表达的报告、细胞动态的研究、活细胞内蛋白的定位及westernbloting检测中。GFP美好的应用前景也促进了有关GFP的研究,特别是寻找新的突变体并将之运用到细胞生物学和分子生物学的各个领域。     

单分子荧光共振能量转移技术在核糖体移位研究中的进展

核糖体是蛋白质的"合成工厂",也是临床上多种抗菌药物的作用靶点,因此,深入理解细菌核糖体的蛋白质翻译机制意义重大.蛋白质翻译是通过多步骤相互协调、多组分精细配合来实现高保真和精确调控.核糖体在mRNA上的移位作为翻译过程中最重要的事件之一,需要核糖体大规模的构象重排以及tRNA2-mRNA沿着核糖体的精确移动.在细菌中,移位是由延伸因子EF-G催化GTP水解来驱动的.近年来,单分子荧光共振能量技术(smFRET)的发展使得人们可以探究单个tRNA分子移位的动力学过程并实时观测核糖体的构象变化.本文首先介绍了smFRET技术的原理及特点,对其在核糖体结构动态及tRNA移位研究中的应用进行了较为系统的总结,并对其应用前景进行了展望.     

Fluorescence Correlation Spectroscopy Measures Molecular Transport in Cells

Fluorescence correlation spectroscopy (FCS) can measure dynamics of fluorescent molecules in cells. FCS measures the fluctuations in the number of fluorescent molecules in a small volume illuminated by a thin beam of excitation light. These fluctuations are processed statistically to yield an autocorrelation function from which rates of diffusion, convection, chemical reaction, and other processes can be extracted. The advantages of this approach include the ability to measure the mobility of a very small number of molecules, even down to the single molecule level, over a wide range of rates in very small regions of a cell. In addition to rates of diffusion and convection, FCS also provides unique information about the local concentration, states of aggregation and molecular interaction using fluctuation amplitude and cross-correlation methods. Recent advances in technology have rendered these once difficult measurements accessible to routine use in cell biology and biochemistry. This review provides a summary of the FCS method and describes current areas in which the FCS approach is being extended beyond its original scope.     

The presence of non‐native helical structure in the unfolding of a beta‐sheet protein MPT63

MPT63, a major secreted protein from Mycobacterium tuberculosis, has been shown to have immunogenic properties and has been implicated in virulence. MPT63 is a β‐sandwich protein containing 11 β strands and a very short stretch of 3₁₀ helix. The detailed experimental and computational study reported here investigates the equilibrium unfolding transition of MPT63. It is shown that in spite of being a complete β‐sheet protein, MPT63 has a strong propensity toward helix structures in its early intermediates. Far UV‐CD and FTIR spectra clearly suggest that the low‐pH intermediate of MTP63 has enhanced helical content, while fluorescence correlation spectroscopy suggests a significant contraction. Molecular dynamics simulation complements the experimental results indicating that the unfolded state of MPT63 traverses through intermediate forms with increased helical characteristics. It is found that this early intermediate contains exposed hydrophobic surface, and is aggregation prone. Although MPT63 is a complete β‐sheet protein in its native form, the present findings suggest that the secondary structure preferences of the local interactions in early folding pathway may not always follow the native conformation. Furthermore, the Gly25Ala mutant supports the proposed hypothesis by increasing the non‐native helical propensity of the protein structure.     

Determination of lipid raft partitioning of fluorescently-tagged probes in living cells by Fluorescence Correlation Spectroscopy (FCS)

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. They play a role in cellular physiological processes such as signalling, and trafficking but are also thought to be key players in several diseases including viral or bacterial infections and neurodegenerative diseases. Yet their existence is still a matter of controversy. Indeed, lipid raft size has been estimated to be around 20 nm, far under the resolution limit of conventional microscopy (around 200 nm), thus precluding their direct imaging. Up to now, the main techniques used to assess the partition of proteins of interest inside lipid rafts were Detergent Resistant Membranes (DRMs) isolation and co-patching with antibodies. Though widely used because of their rather easy implementation, these techniques were prone to artefacts and thus criticized. Technical improvements were therefore necessary to overcome these artefacts and to be able to probe lipid rafts partition in living cells. Here we present a method for the sensitive analysis of lipid rafts partition of fluorescently-tagged proteins or lipids in the plasma membrane of living cells. This method, termed Fluorescence Correlation Spectroscopy (FCS), relies on the disparity in diffusion times of fluorescent probes located inside or outside of lipid rafts. In fact, as evidenced in both artificial membranes and cell cultures, probes would diffuse much faster outside than inside dense lipid rafts. To determine diffusion times, minute fluorescence fluctuations are measured as a function of time in a focal volume (approximately 1 femtoliter), located at the plasma membrane of cells with a confocal microscope (Fig. 1). The auto-correlation curves can then be drawn from these fluctuations and fitted with appropriate mathematical diffusion models. FCS can be used to determine the lipid raft partitioning of various probes, as long as they are fluorescently tagged. Fluorescent tagging can be achieved by expression of fluorescent fusion proteins or by binding of fluorescent ligands. Moreover, FCS can be used not only in artificial membranes and cell lines but also in primary cultures, as described recently. It can also be used to follow the dynamics of lipid raft partitioning after drug addition or membrane lipid composition change.     

Single Pair Förster Resonance Energy Transfer: A Versatile Tool To Investigate Protein Conformational Dynamics

Conformational changes of proteins and other biomolecules play a fundamental role in their functional mechanism. Single pair Förster resonance energy transfer (spFRET) offers the possibility to detect these conformational changes and dynamics, and to characterize their underlying kinetics. Using spFRET on microscopes with different modes of detection, dynamic timescales ranging from nanoseconds to seconds can be quantified. Confocal microscopy can be used as a means to analyze dynamics in the range of nanoseconds to milliseconds, while total internal reflection fluorescence (TIRF) microscopy offers information about conformational changes on timescales of milliseconds to seconds. While the existence of dynamics can be directly inferred from the FRET efficiency time trace or the correlation of FRET efficiency and fluorescence lifetime, additional computational approaches are required to extract the kinetic rates of these dynamics, a short overview of which is given in this review.

     

Subtle Change in the Charge Distribution of Surface Residues May Affect the Secondary Functions of Cytochrome c

Although the primary function of cytochrome c (cyt c) is electron transfer, the protein caries out an additional secondary function involving its interaction with membrane cardiolipin (CDL), its peroxidase activity, and the initiation of apoptosis. Whereas the primary function of cyt c is essentially conserved, its secondary function varies depending on the source of the protein. We report here a detailed experimental and computational study, which aims to understand, at the molecular level, the difference in the secondary functions of cyt c obtained from horse heart (mammalian) and Saccharomyces cerevisiae (yeast). The conformational landscape of cyt c has been found to be heterogeneous, consisting of an equilibrium between the compact and extended conformers as well as the oligomeric species. Because the determination of relative populations of these conformers is difficult to obtain by ensemble measurements, we used fluorescence correlation spectroscopy (FCS), a method that offers single-molecule resolution. The population of different species is found to depend on multiple factors, including the protein source, the presence of CDL and urea, and their concentrations. The complex interplay between the conformational distribution and oligomerization plays a crucial role in the variation of the pre-apoptotic regulation of cyt c observed from different sources. Finally, computational studies reveal that the variation in the charge distribution at the surface and the charge reversal sites may be the key determinant of the conformational stability of cyt c.     

Single-Molecule Imaging and Spectroscopy Using Fluorescence and Surface-Enhanced Raman Scattering

We extended single molecule fluorescence imaging and time-resolved fluorometry from the green to the violet-excitation regime to find feasibility of detecting and identifying fluorescent analogs of nucleic-acid bases at the single-molecule level. Using violet excitation, we observed fluorescent spotsfrom single complexes composed of a nucleotide analogue and the Klenow fragmentof DNA polymerase I. Also, we implemented Raman imaging and spectroscopy of adenine molecules adsorbed on Ag colloidal nanoparticles to find feasibility of identifying nucleic-acid bases at the single-molecule level. Surface enhanced Raman scattering (SERS) of adenine molecules showed an intermittent on-and-off behavior called blinking. The observation of blinking provides substantial evidence for detecting single adenine molecules.