Photobleaching in Two‐Photon Scanning Fluorescence Correlation Spectroscopy

Two‐photon excitation in fluorescence correlation spectroscopy (FCS) is often preferred to one‐photon excitation because of reduced bulk photobleaching and photodamage, and deeper penetration into scattering media, such as thick biological specimens. Two‐photon FCS, however, suffers from lower signal‐to‐noise ratios which are directly related to the lower molecular brightness achieved. We compare standard FCS with a fixed measurement volume with scanning FCS, where the measurement volume is scanned along a circular path. The experimental results show that photobleaching is the dominant cause of the effects observed at the high excitation powers necessary for good signal‐to‐noise ratios. Theoretical calculations assuming a nonuniform excitation intensity profile, and using the concept of generalized volume contrast, provide an explanation for the photobleaching effects commonly observed in two‐photon FCS at high excitation intensities, without having to assume optical saturation. Scanning alleviates these effects by spreading the photobleaching dose over a larger area, thereby reducing the depletion of fluorescent molecules in the measurement volume. These results, which facilitate understanding of the photobleaching in FCS and of the positive effects of scanning, are particularly important in studies involving the autocorrelation amplitude g(0), such as concentration measurements or binding studies using fluorescence cross‐correlation between two labeled species.     

二维相关荧光光谱技术

从发展历史、计算方程、一般规则和特有性质等方面系统地介绍了近年来在二维相关荧光光谱技术方面的方法探索和应用进展。以不同的外扰方式,如浓度、激发波长、猝灭以及其他如pH等分类,举例阐述了二维荧光相关光谱的可操作性及其应用,并与普通一维荧光光谱比较,说明了二维荧光相关光谱技术的优势。     

荧光相关光谱及其在单分子检测中的应用进展

单分子检测在生命科学、化学、物理学等领域具有重要的意义。荧光相关光谱是单分子检测的新技术,在生命科学领域有巨大的应用潜力。综述了荧光相关光谱单分子检测的原理、实验技术以及在生物分子相互作用、活细胞、核酸、疾病诊断、高通量筛选以及与毛细管电泳联用等领域的研究,并展望了其发展前景。     

Cross‐Reactive Sensor Arrays for the Detection of Peptides in Aqueous Solution by Fluorescence Spectroscopy

A simple but powerful method for the sensing of peptides in aqueous solution has been developed. The transition‐metal complexes [PdCl₂(en)], [{RhCl₂Cp*}₂], and [{RuCl₂(p‐cymene)}₂] were combined with six different fluorescent dyes to build a cross‐reactive sensor array. The fluorescence response of the individual sensor units was based on competitive complexation reactions between the peptide analytes and the fluorescent dyes. The collective response of the sensor array in a time‐resolved fashion was used as an input for multivariate analyses. A sensor array comprised of only six metal–dye combinations was able to differentiate ten different dipeptides in buffered aqueous solution at a concentration of 50 μM . Furthermore, the cross‐reactive sensor could be used to obtain information about the identity and the quantity of the pharmacologically interesting dipeptides carnosine and homocarnosine in a complex biological matrix, such as deproteinized human blood serum. The sensor array was also able to sense longer peptides, which was demonstrated by differentiating mixtures of the nonapeptide bradykinin and the decapeptide kallidin.     

Dynamics of Supramolecular Association Monitored by Fluorescence Correlation Spectroscopy

Supramolecular binding is a key process in many biological systems and in newly developed supramolecular assemblies. Most of the scientific work on these systems is focused on their structural properties and on the thermodynamics of the association process. However, the underlying dynamics are usually much less known, in spite of the great importance they have during the binding process in these highly dynamic systems. Understanding supramolecular binding in biological systems and controlling the functionality of new synthetic supramolecular systems can only be achieved through knowledge of the structure–dynamics relationship. There is a strong need for suitable techniques which cover the typically wide time interval of the association dynamics and which do not need a perturbation of the system. We briefly review high‐resolution fluorescence correlation spectroscopy (FCS) as a technique to monitor supramolecular dynamics and to give information on how structure determines the dynamics of host–guest association. The comparison of hosts and guests with different structures shows that geometrical and orientational requirements determine the association rate constant, whereas the dissociation is defined by the strength of specific interactions. As model hosts cyclodextrins and micelles are studied.     

Probing the Hydrodynamic Behavior of Drug-Induced Tubulin Rings by Fluorescence Correlation Spectroscopy

We describe fluorescence correlation spectroscopy measurements of tubulin polymers composed of tubulin and either of two cytotoxic peptides – dolastatin 10 and cryptophycin 1. These peptides inhibit tubulin polymerization into microtubules, a major component of cellular networks, and instead induce the formation of predominantly single-walled tubulin rings. We determine ratios of the hydrodynamic diameter of dolastatin-tubulin or cryptophycin-tubulin polymers to that of tubulin dimers, which are compared with corresponding ratios calculated for two simple models: 1) a circular ring made with N contiguous spherical beads and 2) a circular ring constructed with N non-spherical monomers, each monomer being represented by 21-minibeads. We find that the computed ratios from the 21-minibead representation agree well with the measured ones when N = 28 and N = 16 for dolastatin-tubulin and cryptophycin-tubulin ring polymers, respectively. That is, the rings are made with 14 and 8 tubulin dimers, confirming the numbers derived by other techniques. The present results further support the applicability of theories that have been developed for calculating hydrodynamic properties of supramolecular biological structures.     

Nanomolar Nitric Oxide Concentrations in Living Cells Measured by Means of Fluorescence Correlation Spectroscopy

Measurement of the nitric oxide (NO) concentration in living cells in the physiological nanomolar range is crucial in understanding NO biochemical functions, as well as in characterizing the efficiency and kinetics of NO delivery by NO-releasing drugs. Here, we show that fluorescence correlation spectroscopy (FCS) is perfectly suited for these purposes, due to its sensitivity, selectivity, and spatial resolution. Using the fluorescent indicators, diaminofluoresceins (DAFs), and FCS, we measured the NO concentrations in NO-producing living human primary endothelial cells, as well as NO delivery kinetics, by an external NO donor to the immortal human epithelial living cells. Due to the high spatial resolution of FCS, the NO concentration in different parts of the cells were also measured. The detection of nitric oxide by means of diaminofluoresceins is much more efficient and faster in living cells than in PBS solutions, even though the conversion to the fluorescent form is a multi-step reaction.     

Determining Metal Ion Complexation Kinetics with Fluorescent Ligands by Using Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) has been extensively used to measure equilibrium binding constants (K) or association and dissociation rates in many reversible chemical reactions across chemistry and biology. For the majority of investigated reactions, the binding constant was on the order of ∼100 M⁻¹, with dissociation constants faster or equal to 10³ s⁻¹, which ensured that enough association/dissociation events occur during the typical diffusion-determined transition time of molecules through the FCS detection volume. However, complexation reactions involving metal ions and chelating ligands exhibit equilibrium constants exceeding 10⁴ M⁻¹. In the present paper, we explore the applicability of FCS for measuring reaction rates of such complexation reactions, and apply it to binding of iron, europium and uranyl ions to a fluorescent chelating ligand, calcein. For this purpose, we exploit the fact that the ligand fluorescence becomes strongly quenched after binding a metal ion, which results in strong intensity fluctuations that lead to a partial correlation decay in FCS. We also present measurements for the strongly radioactive ions of ²⁴¹Am³⁺, where the extreme sensitivity of FCS allows us to work with sample concentrations and volumes that exhibit close to negligible radioactivity levels. A general discussion of the applicability of FCS to the investigation of metal-ligand binding reactions concludes our paper.     

Characterization of Diblock Copolymer Order–Order Transitions in Semidilute Aqueous Solution Using Fluorescence Correlation Spectroscopy

The temperature and pH‐dependent diffusion of poly(glycerol monomethacrylate)‐block‐poly(2‐hydroxypropyl methacrylate) nanoparticles prepared via polymerization‐induced self‐assembly in water is characterized using fluorescence correlation spectroscopy (FCS). Lowering the solution temperature or raising the solution pH induces a worm‐to‐sphere transition and hence an increase in diffusion coefficient by a factor of between four and eight. FCS enables morphological transitions to be monitored at relatively high copolymer concentrations (10% w/w) compared to those required for dynamic light scattering (0.1% w/w). This is important because such transitions are reversible at the former concentration, whereas they are irreversible at the latter. Furthermore, the FCS data suggest that the thermal transition takes place over a very narrow temperature range (less than 2 °C). These results demon­strate the application of FCS to characterize order–order transitions, as opposed to order–disorder transitions.

     

Comparative Photostability Studies of BODIPY and Fluorescein Dyes by Using Fluorescence Correlation Spectroscopy

In single‐molecule applications, the photostability of fluorescent molecules is a key parameter. We apply fluorescence correlation spectroscopy to compare the photostability of four fluorescein and four borondipyrromethene (BODIPY) dyes of similar structure but different triplet yields. The latter class of dyes are more stable. In the kinetic analysis the, diffusion and photobleaching are treated as competitive processes. Corrections, which account for saturation and for experimental artefacts, are achieved solely by using experimental data. Photobleaching is found to occur mainly through the first excited singlet state S₁, in contrast to previous findings.     

Monitoring the Dynamics of Phase Separation in a Polymer Blend by Confocal Imaging and Fluorescence Correlation Spectroscopy

The phase separation of the polymer blend polystyrene/poly(methyl phenyl siloxane) (PS/PMPS) is studied in situ by laser scanning confocal microscopy (LSCM) and by fluorescence correlation spectroscopy (FCS) at macroscopic and microscopic length scales, respectively. It is shown for the first time that FCS when combined with LSCM can provide independent information on the local concentration within the phase‐separated domains as well as the interfacial width.     

Calibration and Limits of Camera‐Based Fluorescence Correlation Spectroscopy: A Supported Lipid Bilayer Study

Camera‐based fluorescence correlation spectroscopy (FCS) approaches allow the measurement of thousands of contiguous points yielding excellent statistics and details of sample structure. Imaging total internal reflection FCS (ITIR‐FCS) provides these measurements on lipid membranes. Herein, we determine the influence of the point spread function (PSF) of the optical system, the laser power used, and the time resolution of the camera on the accuracy of diffusion coefficient and concentration measurements. We demonstrate that the PSF can be accurately determined by ITIR‐FCS and that the laser power and time resolution can be varied over a wide range with limited influence on the measurement of the diffusion coefficient whereas the concentration measurements are sensitive to changes in the measurement parameters. One advantage of ITIR‐FCS is that the measurement of the PSF has to be performed only once for a given optical setup, in contrast to confocal FCS in which calibrations have to be performed at least once per measurement day. Using optimized experimental conditions we provide diffusion coefficients for over ten different lipid membranes consisting of one, two and three constituents, measured in over 200000 individual correlation functions. Using software binning and thus the inherent advantage of ITIR‐FCS of providing multiple observation areas in a single measurement we test the FCS diffusion law and show how they can be complemented by the local information provided by the difference in cross‐correlation functions (ΔCCF). With the determination of the PSF by ITIR‐FCS and the optimization of measurement conditions ITIR‐FCS becomes a calibration‐free method. This allows us to provide measurements of absolute diffusion coefficients for bilayers with different compositions, which were stable over many different bilayer preparations over a time of at least one year, using a single PSF calibration.     

Fluorescence Correlation Spectroscopy Study of Probe Diffusion in Poly(vinyl alcohol) Solutions and Gels

In this study, we demonstrate how the diffusion of probe particles in aqueous poly(vinyl alcohol) (PVA) solutions and gels is affected by: (i) the presence of cross-links, (ii) the cross-link density, (iii) the polymer concentration. We apply fluorescence correlation spectroscopy (FCS) to measure the diffusion time of a rhodamine-based fluorescent particle (TAMRA) and TAMRA-labeled dextran in PVA solutions and gels prepared at various polymer concentrations (1% to 8.6% w/v) and cross-link densities (1/400 to 1/50 cross-link monomers per PVA monomers). The measurements indicate that the probe particles are slowed down with increasing polymer concentration and with increasing cross-link density. Also, FCS can detect differences in the diffusion times measured in “fresh” and “aged” PVA solutions. We find that FCS provides a quantitative measure of network inhomogeneities.     

二维相关振动光谱技术

从发展历史、计算方程、性质规则等方面系统地介绍了近年来发展起来的二维相关光谱技术.结合各种常见的一维振动光谱, 如红外、拉曼、荧光、近红外-红外等光谱举例阐述了二维振动光谱的优势及其普适性.介绍了在广义二维相关光谱理论上最新延伸发展起来的二维样品-样品相关技术和二维杂化相关技术的基本理论, 并将之与传统的二维变量-变量相关技术(广义二维相关光谱)进行了比较.     

荧光相关光谱单分子检测系统的研究

基于激光共焦构型,构建了一套高灵敏度和高稳定性的荧光相关光谱单分子检测系统。采用高数值孔径物镜使激光束高度聚焦,并用同一物镜收集样品的荧光,单光子计数器用于实时记录荧光信号。优化系统保持物镜焦点、针孔以及单光子计数器光敏区高度同轴。采用60×1.2(NA)的水浸物镜及直径为160μm的针孔得到的检测系统结构参数为ω0和z0分别为0.42μm和1.33μm,照射微区的体积为1.3fL。单个RhodamineGreen分子可获得13倍的信噪比。用该系统获得了小分子和生物大分子(DNA片段和蛋白质)的高质量荧光相关图谱,成功实现了单个分子的检测。     

Combination of Micro-fluidic Chip with Fluorescence Correlation Spectroscopy for Single Molecule Detection

A single molecule detection technique was developed by the combination of a single channel poly (dimethylsiloxane)/glass micro-fluidic chip and fluorescence correlation spectroscopy (FCS). This method was successfully used to determine the proportion of two model components in the mixture containing fluorescein and the rhodamine-green succinimidyl ester.     

Differences in Human Plasma Protein Interactions between Various Polymersomes and Stealth Liposomes as Observed by Fluorescence Correlation Spectroscopy

A significant factor hindering the clinical translation of polymersomes as vesicular nanocarriers is the limited availability of comparative studies detailing their interaction with blood plasma proteins compared to liposomes. Here, polymersomes are self-assembled via film rehydration, solvent exchange, and polymerization-induced self-assembly using five different block copolymers. The hydrophilic blocks are composed of anti-fouling polymers, poly(ethylene glycol) (PEG) or poly(2-methyl-2-oxazoline) (PMOXA), and all the data is benchmarked to PEGylated “stealth” liposomes. High colloidal stability in human plasma (HP) is confirmed for all but two tested nanovesicles. In situ fluorescence correlation spectroscopy measurements are then performed after incubating unlabeled nanovesicles with fluorescently labeled HP or the specific labeled plasma proteins, human serum albumin, and clusterin (apolipoprotein J). The binding of HP to PMOXA-polymersomes could explain their relatively short circulation times found previously. In contrast, PEGylated liposomes also interact with HP but accumulate high levels of clusterin, providing them with their known prolonged circulation time. The absence of significant protein binding for most PEG-polymersomes indicates mechanistic differences in protein interactions and associated downstream effects, such as cell uptake and circulation time, compared to PEGylated liposomes. These are key observations for bringing polymersomes closer to clinical translation and highlighting the importance of such comparative studies.     

广义二维相关光谱学进展

介绍了一种重要的广义二维相关光谱方法,对其基本原理、获取谱图的方法、数学计算过程、谱图的性质及其解释规则等作了详细的介绍,并介绍了其在化学、物理、生物、医药等领域中的应用。