Optical saturation in fluorescence correlation spectroscopy under continuous-wave and pulsed excitation.

A detailed theoretical and experimental study of the dependence of fluorescence correlation measurements on optical excitation power due to optical saturation effects is presented. It is shown that the sensitivity of a fluorescence correlation measurement on excitation power becomes increasingly stronger for decreasing excitation power. This makes exact measurements or diffusion coefficients with fluorescence correlation spectroscopy rather difficult. A strong difference of this behavior for continuous-wave and pulsed excitation is found.     

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.     

Free energy and dimensions of macromolecules under confinement in layered assemblies

The changes in the free energy ΔA accompanying penetration of polymer solutions from bulk into slit-like cavities were determined by lattice simulations. In dilute solutions the thermodynamics of penetration is controlled mainly by the parameter ϵ_w specifying interaction between polymer and walls of repulsive or adsorptive cavities. However, the magnitude of |ΔA| is substantially reduced by increasing concentration ∅︁ in bulk solution. Furthermore, compression of chains by concentration in good solvents and adsorptive cavities was found to be larger in the slit then in the bulk. At intermediate confinement, a region of a minimum coil size was observed at all concentrations and attraction strengths, where molecules are squeezed along all three axes.     

Dynamics of Biological Polyelectrolytes

Summary: Biological polymers and structures, including proteins and DNAs, can be made in essentially monodisperse form. Proteins usually have well-defined shapes. Duplex oligonucleotides are rigid and rodlike, and longer DNAs are semiflexible coils. The DNAs also constitute a homologous series. The dynamics of both proteins and DNAs can be studied by readily available techniques such as dynamic light scattering (DLS) and fluorescence correlation spectroscopy (FCS). These systems can thus be used as model systems to elucidate elusive charge effects on the dynamics of macromolecules in solution (polyelectrolyte effects) for both rigid and semiflexible polymers. We present here as examples the results of measurements of mutual and self-diffusion coefficients dynamics of a rodlike oligonucleotide as functions of polymer concentration and the concentration of added salt (which screens the charges).     

Kinetics of Photoinduced Reactions at the Single-Molecule Level: The KACB Method

The kinetics of photoinduced reactions can be approached by laser flash photolysis techniques. Although such techniques allow for a detailed understanding of the important photophysics of molecules, they normally require a substantial amount of sample for measurements (>1 nmol), and thus, they are difficult to apply to analytical and diagnostic applications. The photophysics of a fluorescent molecule can be accessed by monitoring the kinetics of the fluctuation of fluorescence, which is called blinking. Blinking is a phenomenon that can be monitored only if molecules are observed at the single-molecule level. In bulk solution, blinking kinetics can be measured by using fluorescence correlation spectroscopy (FCS), which normally requires more than 10⁵ times less sample than that required for laser flash photolysis. Blinking is controlled to extract fruitful microenvironmental information around a fluorescent molecule, by using a method named kinetic analysis based on the control of fluorescence blinking (KACB). This Concept highlights the adaption of the KACB method to investigate the local conformation of DNA with less than 1 pmol of DNA sample.     

氧化锡和氧化锌双层薄膜的XPS研究

用XPS法研究了SnO2 /ZnO及ZnO/SnO2 双层膜中锌和锡的扩散情况 ,结果表明 :锌在SnO2中的扩散比锡在ZnO中的扩散更容易。讨论了锌在SnO2 层中的扩散方式和扩散的结果。在ZnO层留下了较多的氧 ,并使部分的SnO2 还原为SnO。     

Polymer gels and brushes at surfaces

We discuss the adsorption of polymer gels on flat surfaces. Even in cases of complete wetting, where the spreading power S is positive and where an equivalent liquid would spread, the elastic stresses due to the gel deformation upon adsorption oppose spreading. The competition between elasticity characterized by the bulk shear modulus G and capillarity, characterized by the spreading power S, defines a typical length scale 1 = S/G for the deformation in the gel. Macroscopic gels larger than 1 deform only at their edges over a region of size 1. Microscopic gels show a finite deformation despite the elastic stresses. These results can be compared to confined polymer brushes.     

Model Biological Membranes and Possibilities of Application of Electrochemical Impedance Spectroscopy for their Characterization

Biological membranes are essential parts of living systems. They represent an interface between intracellular and extracellular space. Depending on their structure, they often perform very complex functions and play an important role in the transport of both charged and uncharged particles in any organism. Structure of the biological membranes, which play very important role in electrochemical processes inside living organisms, is very complicated and still not precisely defined and explained. Model lipid membranes are used to gain detail information about properties of real biological membranes and about associated electrochemical processes. Electrochemistry, especially electrochemical impedance spectroscopy (EIS), can play a useful role in the characterization of properties of model lipid membranes (planar and supported lipid bilayers, tethered lipid membranes, liposomes, etc.). This review is focused on model biological membranes and the possibilities and limitations of electrochemical methods and namely of EIS in this field.     

Correcting for Spectral Cross‐Talk in Dual‐Color Fluorescence Cross‐Correlation Spectroscopy

Dual‐color fluorescence cross‐correlation spectroscopy (dcFCCS) allows one to quantitatively assess the interactions of mobile molecules labeled with distinct fluorophores. The technique is widely applied to both reconstituted and live‐cell biological systems. A major drawback of dcFCCS is the risk of an artifactual false‐positive or overestimated cross‐correlation amplitude arising from spectral cross‐talk. Cross‐talk can be reduced or prevented by fast alternating excitation, but the technology is not easily implemented in standard commercial setups. An experimental strategy is devised that does not require specialized hardware and software for recognizing and correcting for cross‐talk in standard dcFCCS. The dependence of the cross‐talk on particle concentrations and brightnesses is quantitatively confirmed. Moreover, it is straightforward to quantitatively correct for cross‐talk using quickly accessible parameters, that is, the measured (apparent) fluorescence count rates and correlation amplitudes. Only the bleed‐through ratio needs to be determined in a calibration measurement. Finally, the limitations of cross‐talk correction and its influence on experimental error are explored.     

Structure and growth of oxides on polycrystalline nickel surfaces

The oxidation of polycrystalline nickel (Ni) metal surfaces after exposure to oxygen gas (O₂) at 25 and 300 °C and pressures near 130 Pa, was studied using X‐ray photoelectron spectroscopy (XPS). Oxide structures involving both divalent (Ni²⁺) and trivalent (Ni³⁺) species could be distinguished using Ni 2p spectra, while surface adsorbed O₂ and atomic oxygen (O) species could be differentiated from bulk oxide (O^2?) using O 1s spectra. Oxide thicknesses and distributions were determined using QUASES^?, and the average oxide thickness was verified using the Strohmeier formula. The reaction kinetics for oxide films grown at 300 °C followed a parabolic mechanism, with an oxide thickness of greater than 4 nm having formed after 60 min. Exposure at 25 °C followed a direct logarithmic mechanism with an oxide growth rate about four to five times slower than at 300 °C. Reaction of a Ni (100) single crystal under comparable conditions showed much slower reaction rates compared to polycrystalline specimens. The higher reaction rate of the polycrystalline materials is attributed to grain boundary transport of Ni cations. Oxide thickness was measured on a microscopic scale for polycrystalline Ni exposed to large doses of O₂ at 25 and 300 °C. The thickness of oxide was not strongly localized on this scale. However, the QUASES^? analysis suggests that there is localized growth on a nanometric scale—the result of island formation. Copyright © 2007 John Wiley & Sons, Ltd.     

Functional macromolecules from single-walled carbon nanotubes: synthesis and photophysical properties of short single-walled carbon nanotubes functionalised with 9,10-diphenylanthracene

9,10-Diphenylanthracene (DPA), a well-studied organic chromophore (Phi(fl) = 0.98) that exhibits electroluminescence, has been covalently bound through 2-(ethylthio)ethylamido linkers to the carboxylic acid groups of short, soluble single-walled carbon nanotubes (sSWNTs) of 1 microm average length, and the resulting DPA-functionalised sSWNT (DPA- sSWNT) macromolecular adducts (4.6 wt % DPA content) characterised by solution (1)H NMR, Raman and IR spectroscopy and thermogravimetric analysis. Comparison of the quenching of DPA fluorescence (steady-state and time-resolved) and of the transient optical spectra of sSWNTs and DPA-sSWNTs show that the covalent linkage boosts the interaction between the DPA and the sSWNT units. DPA-sSWNTs exhibit emission in the near-IR region from 1100-1400 nm with an enhanced quantum yield (Phi = 5.7x10(-3)) compared with sSWNTs (Phi = 3.9x10(-3)).     

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.     

Single‐Molecule 3D Orientation Imaging Reveals Nanoscale Compositional Heterogeneity in Lipid Membranes

In soft matter, thermal energy causes molecules to continuously translate and rotate, even in crowded environments, thereby impacting the spatial organization and function of most molecular assemblies, such as lipid membranes. Directly measuring the orientation and spatial organization of large collections (>3000 molecules μm^?2) of single molecules with nanoscale resolution remains elusive. In this paper, we utilize SMOLM, single‐molecule orientation localization microscopy, to directly measure the orientation spectra (3D orientation plus “wobble”) of lipophilic probes transiently bound to lipid membranes, revealing that Nile red's (NR) orientation spectra are extremely sensitive to membrane chemical composition. SMOLM images resolve nanodomains and enzyme‐induced compositional heterogeneity within membranes, where NR within liquid‐ordered vs. liquid‐disordered domains shows a ≈4° difference in polar angle and a ≈0.3π sr difference in wobble angle. As a new type of imaging spectroscopy, SMOLM exposes the organizational and functional dynamics of lipid‐lipid, lipid‐protein, and lipid‐dye interactions with single‐molecule, nanoscale resolution.     

Single-Molecule 3D Orientation Imaging Reveals Nanoscale Compositional Heterogeneity in Lipid Membranes

In soft matter, thermal energy causes molecules to continuously translate and rotate, even in crowded environments, thereby impacting the spatial organization and function of most molecular assemblies, such as lipid membranes. Directly measuring the orientation and spatial organization of large collections (>3000 molecules μm⁻²) of single molecules with nanoscale resolution remains elusive. In this paper, we utilize SMOLM, single-molecule orientation localization microscopy, to directly measure the orientation spectra (3D orientation plus “wobble”) of lipophilic probes transiently bound to lipid membranes, revealing that Nile red's (NR) orientation spectra are extremely sensitive to membrane chemical composition. SMOLM images resolve nanodomains and enzyme-induced compositional heterogeneity within membranes, where NR within liquid-ordered vs. liquid-disordered domains shows a ≈4° difference in polar angle and a ≈0.3π sr difference in wobble angle. As a new type of imaging spectroscopy, SMOLM exposes the organizational and functional dynamics of lipid-lipid, lipid-protein, and lipid-dye interactions with single-molecule, nanoscale resolution.     

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。