Single-molecule monitoring in living cells by use of fluorescence microscopy

Monitoring single molecules in living cells is becoming a powerful tool for study of the location, dynamics, and kinetics of individual biomolecules in real time. In recent decades, several optical imaging techniques, for example epi-fluorescence microscopy, total internal reflection fluorescence microscopy (TIRFM), confocal microscopy, quasi-TIRFM, and single-point edge excitation subdiffraction microscopy (SPEED), have been developed, and their capability of capturing single-molecule dynamics in living cells has been demonstrated. In this review, we briefly summarize recent advances in the use of these imaging techniques for monitoring single-molecules in living cells for a better understanding of important biological processes, and discuss future developments.     

Using single-molecule fluorescence spectroscopy to study electron transfer.

Techniques in single-molecule fluorescence spectroscopy now allow sophisticated studies of photophysical processes in single molecules. As interest grows in the possibilities of molecular electronics, researchers have begun to turn these techniques to the study of electron transfer. Electron-transfer reactions have now been detected and measured at the single-molecule level in a variety of systems and on a variety of timescales by adapting techniques from previous single-molecule fluorescence studies.     

Single-molecule studies on DNA and RNA.

DNA and RNA are the most individual molecules known. Therefore, single-molecule experiments with these nucleic acids are particularly useful. This review reports on recent experiments with single DNA and RNA molecules. First, techniques for their preparation and handling are summarised including the use of AFM nanotips and optical or magnetic tweezers. As important detection techniques, conventional and near-field microscopy as well as fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) are touched on briefly. The use of single-molecule techniques currently includes force measurements in stretched nucleic acids and in their complexes with binding partners, particularly proteins, and the analysis of DNA by restriction mapping, fragment sizing and single-molecule hybridisation. Also, the reactions of RNA polymerases and enzymes involved in DNA replication and repair are dealt with in some detail, followed by a discussion of the transport of individual nucleic acid molecules during the readout and use of genetic information and during the infection of cells by viruses. The final sections show how the enormous addressability in nucleic acid molecules can be exploited to construct a single-molecule field-effect transistor and a walking single-molecule robot, and how individual DNA molecules can be used to assemble a single-molecule DNA computer.     

单分子操作技术在核酸研究中的应用

单分子操作技术,如原子力显微镜技术、光镊技术和单分子荧光光谱技术,能够对单分子局部力进行测量,因而能在单分子水平上研究核酸的弹性性质和机械诱导的结构转变。单分子操作技术已越来越多地应用于相关的核酸研究中,如DNA的打开与修饰、DNA．蛋白质相互作用、DNA凝聚、复制和转录。与经典的分子生物学技术相比,单分子操作技术避免了从大量实验结果中取平均的需要,因而可以提供更为详细的生物信息。本文概述了单分子操作技术的原理及其在核酸研究中的应用。     

Electrochemistry probed one molecule at a time

Probing electrochemical reactions at the single-reaction level is the ultimate goal for electroanalytical chemistry. The development of electrical approaches and optical methods has enabled addressing the electrochemistry of individual molecules in various systems such as scanning probe microscopy, fixed nanogaps, nanopores, single-molecule fluorescence microscopy, and single-molecule electrochemiluminescence microscopy, which can bring new views on fundamental electrochemistry, electroanalytical applications, and electrochemical cells. We conclude with potential directions for further improving the spatial and temporal resolution and developing new techniques towards meeting the requirements for achieving the outlined goals.     

Oxygen imaging of living cells and tissues using luminescent molecular probes

Oxygen imaging of biological cells and tissues is becoming increasingly important in cell biology and in the pathophysiology of various hypoxia-related diseases. The optical oxygen-sensing method using luminescent probes provides very useful, high spatial resolution information regarding oxygen distribution in living cells and tissues. This review focuses on recent advances in biological oxygen measurements based on the phosphorescence quenching of probe molecules by oxygen, and on hypoxia-sensitive fluorescent probes. Special attention is devoted to metal complex probes, Pt(II)- and Pd(II)-porphyrins, Ru(II) complexes, and Ir(III) complexes. Current knowledge regarding the mechanism of phosphorescence quenching of metal complexes by oxygen is described in relation to the oxygen sensitivity of the probes, and recent advances in optical oxygen probes and detection techniques for intracellular and tissue oxygen measurements are reviewed, emphasizing the usefulness of chemical modifications for improving probe properties. Tissue oxygen imaging and hypoxic tumor imaging using these metal complex probes demonstrate the vast potential of optical oxygen-sensing methods using luminescent probes.     

Combining optical trapping, fluorescence microscopy and micro-fluidics for single molecule studies of DNA-protein interactions

Complexity and heterogeneity are common denominators of the many molecular events taking place inside the cell. Single-molecule techniques are important tools to quantify the actions of biomolecules. Heterogeneous interactions between multiple proteins, however, are difficult to study with these technologies. One solution is to integrate optical trapping with micro-fluidics and single-molecule fluorescence microscopy. This combination opens the possibility to study heterogeneous/complex protein interactions with unprecedented levels of precision and control. It is particularly powerful for the study of DNA-protein interactions as it allows manipulating the DNA while at the same time, individual proteins binding to it can be visualized. In this work, we aim to illustrate several published and unpublished key results employing the combination of fluorescence microscopy and optical tweezers. Examples are recent studies of the structural properties of DNA and DNA-protein complexes, the molecular mechanisms of nucleo-protein filament assembly on DNA and the motion of DNA-bound proteins. In addition, we present new results demonstrating that single, fluorescently labeled proteins bound to individual, optically trapped DNA molecules can already be tracked with localization accuracy in the sub-10 nm range at tensions above 1 pN. These experiments by us and others demonstrate the enormous potential of this combination of single-molecule techniques for the investigation of complex DNA-protein interactions.     

Recent advances in optical manipulation of cells and molecules for biological science

Noninvasive and nondestructive techniques for monitoring and manipulating cells or biomolecules are essential for understanding biological processes. Optical methodologies have been used for the noninvasive and nondestructive monitoring of intracellular molecules and manipulation of cellular activities to elucidate the localization and interactions of these biomolecules. Since the pioneering work of Ashkin, optical trapping has been used to study cellular elasticity and mechanical characteristics of intracellular molecules. In recent years, there has been a substantial amount of research on the optical manipulation of nanometer-sized objects, including the manipulation of the assembly of nanomaterials and the enhancement of optical forces with optical resonance effects. In the study of biomolecular manipulation by optical forces, the functions and roles of biomolecules have been clarified by analyzing the changes in cellular functions induced by manipulation. In this review, we focus on recent studies on optical trapping for the manipulation of living cells or biomolecules and introduce techniques for the manipulation of cellular functions using optical forces.     

单个等离子体纳米颗粒在生化分析和生物成像中的应用

等离子体纳米颗粒（PNPs）因其独特的物理、化学、光学和生物学特性而被广泛地应用于材料科学、生物学和医药学等研究领域。PNPs的光学性质是可以通过改变其组成、形状和大小来进行调控的,所以利用可控合成的方式能够筛选出适合的光散射探针。在单分子水平上实时研究PNPs的动态行为对于理解细胞及活体组织的生命活动机制、制备功能型纳米材料和开发新型化学生物传感器等有着重要的意义。基于传统的暗场显微镜（DFM）,通过对光源、检测器及其它光学元件的择优组装和调试,我们开发出了一系列具有高灵敏度、高时空分辨率和高通量的等离子体光散射成像技术,并将其应用于单分子检测、多颗粒传感、单细胞成像以及生物过程示踪等领域。基于具有光学各向异性的PNPs,我们还研制出了活细胞三维扫描成像系统和超连续激光光片成像与高速毛细管电泳联用系统,推进了单分子光谱方面的研究。本文将总结近十年来本课题组在PNP单颗粒分析及成像中的工作,并为该领域未来的发展提出一些新的思路。     

细胞膜上信号转导蛋白的单分子成像与分析

结合本课题组的研究工作, 介绍了单分子荧光成像原理、 荧光标记方法及数据分析方法, 并进一步综述了单分子荧光成像在几种重要的膜蛋白信号转导分子机制和相关药物研究中的进展.     

Novel fluorophores for single-molecule imaging

Nonlinear optical chromophores based on dicyanodihydrofuran acceptors paired with amine donors have been found to exhibit sufficiently large fluorescence quantum yields and stability to enable single-molecule detection in polymeric hosts. To illustrate the breadth of this class, six fluorophores are presented, spanning the emission range from 505 to 646 nm. In contrast to conventional single-molecule fluorophores, the new molecules feature sensitivity to local rigidity, large ground-state dipole moments, and large polarizability anisotropies, properties that can be used to design new reporter experiments at the single-molecule level.     

基于纳米粒子的光学细胞传感器在癌细胞研究中的应用

作为一种极为灵敏、快速的新型生物检测技术,光学细胞传感器在生物医学领域的研究应用备受关注,成为当今生物分析化学领域的研究前沿和热点.它是以细胞作为传感元件来研究信号识别、传导和指示的过程,在毒性物质、病原体等外界条件作用下,研究细胞中活性分子及其生理条件的变化,通过光学信号的变化定量分析细胞膜表面分子、胞内酶分子及微环...     

Light-activated functional mesostructured silica

Mesostructured silica thin films and particles provide highly versatile supports or frameworks for functional materials where a desired function (such as energy transfer, electron transfer, or molecular machines) is induced by molecules deliberately placed in specific regions of the structure. The relatively gentle templated sol–gel synthesis methods allow a wide variety of molecules to be used, and the optical transparency of the framework is very suitable for studies of light-induced functionality. In this paper, three types of functionality are used to obtain fundamental understanding of the materials themselves and to develop active materials that can trap and release molecules from the pores upon command. Photo-induced energy transfer is used to verify that molecules can be placed in specific spatially separated regions of the framework; fluorescence resonance energy transfer is used as a molecular ruler to measure quantitatively the distance between pairs of molecules. Secondly, photo-induced electron transfer is used to obtain fundamental information about the electrical insulating properties of the framework. Finally, two types of molecular machines, a light-driven impeller and a light activated nanovalve, are described. Both machines contain moving parts attached to solid supports and do useful work. The valves trap and release molecules from the mesopores, and the impellers expel molecules from the pores. Applications of the materials to drug delivery and the release of drug molecules inside living cells is described.     

Diffusion of lipid-like single-molecule fluorophores in the cell membrane

The dicyanomethylenedihydrofuran (DCDHF) class of single-molecule fluorophores contains an amine donor and a dicyanomethylenedihydrofuran acceptor linked by a conjugated unit (benzene, naphthalene, or styrene). Molecules in this class have a number of useful properties in addition to those usually required for single-molecule studies (such as high fluorescence quantum yield and photostability), including second-order optical nonlinearity, large ground-state dipole moment, and sensitivity to local environment. Moreover, most DCDHF molecules have amphiphilic structures, with a polar dicyanomethylenedihydrofuran headgroup and nonpolar hydrocarbon tails on the amine or furan ring, and can be used as fluorescent lipid analogues for live cell imaging. Here we demonstrate that individual molecules of several different DCDHF lipid analogues can be observed diffusing in the plasma membrane of Chinese hamster ovary cells. The photophysical and diffusive behaviors of the DCDHF lipid analogues in membranes are described and are found to be competitive with the well-known lipid probe N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine.     

基于裂结技术的单分子尺度化学反应研究进展

分子电子学是研究单分子器件的构筑、性质以及功能调控的一门新兴学科。其中,金属/分子/金属结的构筑和表征是现阶段分子电子学的主要研究内容。裂结技术是当前分子电子学研究的主要实验方法,主要包括机械可控裂结技术和扫描隧道显微镜裂结技术。本文对裂结技术进行了介绍,并对近年来利用这些技术,在单分子尺度化学反应的检测和动力学研究,以及将这些技术与溶液环境、静电场、电化学门控等方法相结合,调控单分子器件的电输运性质等方面所取得的进展进行了概述。     

Monitoring intracellular proteins using fluorescence techniques: from protein synthesis and localization to activity

The recent breakthroughs in genomics and proteomics and improvements of optical methods have made it possible to obtain localized, real-time information on intracellular proteins dynamics, through dynamic three-dimensional (3D) maps of the living cell with nanometric resolution of individual molecules. On one side, brighter variants of the Green Fluorescence Protein (GFP) have been engineered that have different excitation and/or emission spectra that better match available light sources. Like their parent molecule, these variants retain their fluorescence when fused to heterologous proteins on the N- and C- terminals, and this binding generally does not affect the functionality of the tagged protein leading the way to their use as an intracellular reporter. On the other side, optical methods have been improved to allow reaching the level of single-molecule detection inside living cells. Nevertheless some limitations exist for the use of GFP variants for probing 3D conformational changes of proteins. First, these variants are fused to the N and/or C terminals of the studied protein, which are generally not the best location to detect conformational changes resulting from the binding to other proteins or enzyme substrates. Then their own relatively large size makes them unusable for tagging small proteins. These limitations suggest that new tagging processes, permitting the location of the right fluorescent markers at the right places, must be found to built up inter- and/or intra-molecular rulers allowing one to monitor conformational changes resulting from intracellular protein-protein, protein-membrane, and enzyme-substrate binding. These specific locations can be obtained from in vitro studies of 3D conformational changes that occur during protein docking.     

Tools for visualizing multidimensional images from living specimens

Over the last 50 years modern cell biology has been driven by the development of powerful imaging techniques. In particular, new developments in light microscopy that provide the potential to image the dynamics of biological events have had significant impact. Optical sectioning techniques allow three-dimensional information to be obtained from living specimens noninvasively. When used with multimodal fluorescence microscopy, advanced optical sectioning techniques provide multidimensional image data that can reveal information not only about the changing cytoarchitecture of a cell but also about its physiology. These additional dimensions of information, although providing powerful tools, also pose significant visualization challenges to the investigator. Particularly in the current postgenomic era there is a greater need than ever for the development of effective tools for image visualization and management. In this review we discuss the visualization challenges presented by multidimensional imaging and describe three open-source software programs being developed to help address these challenges: ImageJ, the Open Microscopy Environment, and VisBio.     

Nanosensing at the single cell level

This article presents an overview of the development, operation, and applications of optical nanobiosensors for use in in vivo detection of biotargets in individual living cells. The nanobiosensors are equipped with immobilized bioreceptor probes (e.g., antibodies, enzyme substrate) selective to specific molecular targets. Laser excitation is transmitted into the fiber producing an evanescent field at the tip of the fiber in order to excite target molecules bound to the bioreceptors immobilized at the fiber tips. A photometric system detects the optical signal (e.g., fluorescence) originated from the analyte molecules or from the analyte–bioreceptor reaction. Examples of detection of biospecies and molecular signaling pathways of apoptosis in a living cell are discussed to illustrate the potential of the nanobiosensor technology for single cell analysis.     

From Langmuir–Blodgett films to single molecules

The author recollects his time in the group of D. Möbius in 1985–1986, within the department headed by Hans Kuhn, and the influence of Kuhn's concept of molecular assembly in the gestation of the first single-molecule experiments. He then briefly surveys the recent applications of single-molecule spectroscopy to complex and heterogeneous systems. In particular the field of single-molecule microscopy in membranes and at biological interfaces is now strongly growing, closing the circle back to Langmuir films, the systems which led to the development of single-molecule optical techniques.     

Energy transfer in single-molecule photonic wires.

Molecular photonics is a new emerging field of research around the premise that it is possible to develop optical devices using single molecules as building blocks. Truly technological impact in the field requires focussed efforts on designing functional molecular devices as well as having access to their photonic properties on an individual basis. In this Minireview we discuss our approach towards the design and single-molecule investigation of one-dimensional multimolecular arrays intended to work as molecular photonic wires. Three different schemes have been explored: a) perylene-based dimer and trimer arrays displaying coherent exciton delocalisation at room temperature; b) DNA-based unidirectional molecular wires containing up to five different chromophores and exhibiting weak excitonic interactions between neighbouring dyes; and c) one-dimensional multichromophoric polymers based on perylene polyisocyanides showing excimerlike emission. As a whole, our single-molecule data show the importance of well-defined close packing of chromophores for obtaining optimal excitonic behaviour at room temperature. Further improvement on (bio)chemical synthesis, together with the use of single-molecule techniques, should lead in the near future to efficient and reliable photonic wires with true device functionality.