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2007 FCS papers

2006 FCS papers

2005 FCS papers

2004 FCS papers

Avaltroni, F., M. Seijo, S. Ulrich, S. Stoll and J. Wilkinson Kevin. (2007) Conformational changes and aggregation of alginic acid as determined by fluorescence correlation spectroscopy. Biomacromolecules 8(1):106-12.

Insight into the conformations and aggregation of alginic acid was gained by measuring its diffusion coefficient at very dilute concentrations using fluorescence correlation spectroscopy. Both the pH and ionic strength (I) had an important influence on the diffusion coefficient of the polysaccharide. For pH, three effects were isolated: (i) below pH 4, the charge density decreased causing increased aggregation; (ii) between pH 4 and 8, a molecular expansion was observed with increasing pH, whereas (iii) above pH 8 some dissociation of the polymer was observed. Increasing I from 0.001 to 0.1 M resulted in a ca. 20% increase in the diffusion coefficient. By coupling these measurements to molar mass determinations obtained by size exclusion chromatography and monomer size estimations determined from ab initio calculations, it was possible to determine the radii of gyration via de Gennes renormalization theory. From diffusion coefficients and radii of gyration obtained as a function of ionic strength, persistence lengths (total, electrostatic, and intrinsic) were calculated from the Benoit-Doty relationship.

Ball David, A., G. Shen and M. Davis Lloyd. (2007) Single-molecule detection with axial flow into a micrometer-sized capillary. Appl Opt 46(7):1157-64.

We characterize a new geometry for single-molecule detection with flow for use with a submilliliter drop of sample on an inverted confocal microscope. The solution is sucked into a glass capillary positioned above the ellipsoidal confocal volume so that molecules traverse the longest axis of the ellipsoid for greatest photon yield. Decreased spacing between the capillary tip and laser focus gives increased flow speed, as measured by fluorescence correlation spectroscopy, but also increased background from capillary autofluorescence. Flow can alleviate localized triplet and photobleaching effects and speed single-molecule sampling rates for fluorescence fluctuation spectroscopy determinations of slowly diffusing biomolecules in pharmaceutical drug discovery research.

Birkmann, E., F. Henke, N. Weinmann, C. Dumpitak, M. Groschup, A. Funke, D. Willbold and D. Riesner. (2007) Counting of single prion particles bound to a capture-antibody surface (surface-FIDA). Veterinary microbiology 123(4):294-304.

Hitherto accredited prion tests use the PK resistance of PrP(Sc), the pathogenic isoform of the prion protein, as a marker for the disease. Because of variations in the amount of disease-related aggregated PrP, which is not PK-resistant, these prion tests offer only limited sensitivity. Therefore, a prion detection method that does not rely on PK digestion would allow for the detection of both PK-resistant as well as PK-sensitive PrP(Sc). Furthermore, single particle counting is more sensitive than methods measuring an integrated signal. Our new test system is based on dual-colour fluorescence correlation spectroscopy (FCS). This method quantifies the number of protein aggregates that have been simultaneously labelled with two different antibodies using dual-colour fluorescence intensity distribution analysis (2D-FIDA). This only counts PrP aggregates, and not PrP monomers. To increase the sensitivity, PrP(Sc) was concentrated in a two-dimensional space by immobilizing it so that the antibodies could be captured on the surface of the slide (surface-FIDA). When the surface was systematically scanned, even single prion particles were detected. Using this new technique, the sensitivity to identify samples from scrapie-infected hamster as well as BSE-infected cattle can be dramatically increased in comparison with identification using FIDA in solution.

Boukari, H., L. Sackett Dan, P. Schuck and J. Nossal Ralph. (2007) Single-walled tubulin ring polymers. Biopolymers 86(5-6):424-36.

An unusual class of nanoscopic, ring-shaped, single-walled biopolymers arises when alphabeta-tubulin is mixed with certain small peptides obtained from various marine organisms and cyanobacteria. The single-ring structures, whose mean molecular weight depends on the specific peptide added to the reaction mixture, usually have sharp mass distributions corresponding, e.g., to rings containing eight tubulin dimers (when the added peptide is cryptophycin) and 14 dimers (e.g., with dolastatin). Although the ring-forming peptides have been shown to possess antimitotic properties when tested with cultured eukaryotic cells (and thus have generated considerable interest as possible agents to be used in the treatment of cancer), it is not our intention to extensively discuss the potential pharmacological properties of the peptides. Rather, we will review the polymeric structures that form and illustrate how certain physical techniques can be used to characterize their properties and interactions. The nanoscopic size and particular geometry of the individual rings make them appropriate targets for scattering and hydrodynamic techniques that provide details about their structure in solution, but it is necessary to relate measured data to postulated structures by nontrivial, albeit straight-forward, mathematical, and computational means. We will discuss how this is done when one uses such methods as small angle neutron scattering, dynamic light scattering, fluorescence correlation spectroscopy, and sedimentation velocity measurements. Moreover, we show that, by using several techniques, one can eliminate degeneracy to provide better discrimination between model structures.

Braet, C., H. Stephan, M. Dobbie Ian, M. Togashi Denisio, G. Ryder Alan, Z. Foldes-Papp, N. Lowndes and P. Nasheuer Heinz. (2007) Mobility and distribution of replication protein A in living cells using fluorescence correlation spectroscopy. Exp Mol Pathol 82(2):156-62.

Replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA) binding protein, is essential for all pathways of DNA metabolism. To study the function of RPA in living cells the second largest RPA subunit and an N-terminal deletion mutant thereof were fused to green fluorescent protein (GFP; GFP-RPA2 and GFP-RPA2deltaN, respectively) in a controlled, molecular biological way. These proteins were expressed in HeLa cells under the control of the inducible tetracycline expression system. GFP-RPA2 and GFP-RPA2deltaN are predominately nuclear proteins as determined by confocal laser scanning microscopy. Low basal expression of GFP-RPA2deltaN allowed the measurement of kinetic parameters of RPA. Using fluorescence correlation spectroscopy (FCS) two populations--a fast and a slow moving species--were detected in the nucleus and the cytosol of human cells. The translational diffusion rates of these two RPA populations were approximately 15 microm2/s and 1.8 microm2/s. This new finding reveals the existence of different multiprotein and ssDNA-protein complexes of RPA in both cellular compartments and opens the possibility for their analyses.

Bulseco Dylan, A. and E. Wolf David. (2007) Fluorescence correlation spectroscopy: molecular complexing in solution and in living cells. Methods in cell biology 81:525-59.

Chen, H., E. Rhoades, S. Butler James, N. Loh Stewart and W. Webb Watt. (2007) Dynamics of equilibrium structural fluctuations of apomyoglobin measured by fluorescence correlation spectroscopy. Proc Natl Acad Sci U S A 104(25):10459-64.

The spectra of equilibrium chain conformation fluctuations of apomyoglobin (apoMb) as a function of folding, from the acid-denatured state at pH 2.6 through the stable molten globule state pH approximately 4.1 to the folded state at pH 6.3, are reported, as measured by fluorescence correlation spectroscopy. The conformational fluctuations, which are detected by quenching of an N-terminal fluorescent label by contact with various amino acids, can be represented by superpositions of decaying exponentials with time scales ranging from approximately 3 to approximately 200 micros. Both the time scales and amplitudes of the fluctuations increase with the degree of acid denaturation, with principal shifts associated with the transition across the molten globule state. Measurements of the diffusion of apoMb confirm theoretical values showing a approximately 40% increase in the hydrodynamic radius upon acid denaturation. This study uses the model protein apoMb to illustrate the complex scope of folding associated structural dynamics.

Chiantia, S., N. Kahya and P. Schwille. (2007) Raft domain reorganization driven by short- and long-chain ceramide: a combined AFM and FCS study. Langmuir 23(14):7659-65.

Naturally occurring long-chain ceramides (Cer) are known to alter the lateral organization of biological membranes. In particular, they produce alterations of microdomains that are involved in several cellular processes, ranging from apoptosis to immune response. In order to induce similar biological effects, short-chain Cer are extensively used in in vivo experiments to replace their long-chain analogues. In this work, we used the combined approach of atomic force microscopy (AFM) and fluorescence correlation spectroscopy (FCS) to investigate the effect of Cer chain length in lipid bilayers composed of sphingomyelin, dioleoyl-phosphatidylcholine, and cholesterol. Our results show that only long-chain Cer, like C18 and C16, are able to segregate from the liquid-ordered phase, forming separate Cer-enriched domains. Conversely, short-chain Cer do not form a separate phase but alter the physical properties of the liquid-ordered domains, decreasing their stability and viscosity and perturbing the lipid packing. These differences may contribute to the explanation of the different physiological effects that are often observed for the long- and short-chain Cer.

Chowdhury, P., W. Wang, S. Lavender, R. Bunagan Michelle, W. Klemke Jason, J. Tang, G. Saven Jeffrey, S. Cooperman Barry and F. Gai. (2007) Fluorescence correlation spectroscopic study of serpin depolymerization by computationally designed peptides. J Mol Biol 369(2):462-73.

Members of the serine proteinase inhibitor (serpin) family play important roles in the inflammatory and coagulation cascades. Interaction of a serpin with its target proteinase induces a large conformational change, resulting in insertion of its reactive center loop (RCL) into the main body of the protein as a new strand within beta-sheet A. Intermolecular insertion of the RCL of one serpin molecule into the beta-sheet A of another leads to polymerization, a widespread phenomenon associated with a general class of diseases known as serpinopathies. Small peptides are known to modulate the polymerization process by binding within beta-sheet A. Here, we use fluorescence correlation spectroscopy (FCS) to probe the mechanism of peptide modulation of alpha(1)-antitrypsin (alpha(1)-AT) polymerization and depolymerization, and employ a statistical computationally-assisted design strategy (SCADS) to identify new tetrapeptides that modulate polymerization. Our results demonstrate that peptide-induced depolymerization takes place via a heterogeneous, multi-step process that begins with internal fragmentation of the polymer chain. One of the designed tetrapeptides is the most potent antitrypsin depolymerizer yet found.

Culbertson Michael, J. and L. Burden Daniel. (2007) A distributed algorithm for multi-tau autocorrelation. The Review of scientific instruments 78(4):044102.

Network data-transfer times in distributed simulation environments can be reduced by performing data analysis at the remote source, if the analytical technique does not require the entire set of data at once. This novel multi-tau autocorrelation algorithm allows time-domain data records to be processed in discrete, distributed segments and combined at a later point in time. The new approach agrees with autocorrelation results performed by concatenating the discrete segments before correlation, but it operates with significantly shortened processing times. The multi-tau algorithm also benefits from reduced memory requirements since it does not require access to the entire data record at once, and from improved scalability since the multi-tau algorithm has order O(N), while fast Fourier transform autocorrelation algorithms have order O(N log N). This distributed algorithm has particular utility in simulations of fluorescence correlation spectroscopy or photon correlation spectroscopy.

Culbertson Michael, J., T. B. Williams Joshua, W. L. Cheng Wayland, A. Stults Dee, R. Wiebracht Emily, J. Kasianowicz John and L. Burden Daniel. (2007) Numerical fluorescence correlation spectroscopy for the analysis of molecular dynamics under nonstandard conditions. Anal Chem 79(11):4031-9.

The suitability of mathematical models used to extract kinetic information from correlated data constitutes a significant issue in fluorescence correlation spectroscopy (FCS). Standard FCS equations are derived from a simple Gaussian approximation of the optical detection volume, but some investigations have suggested this traditional practice can lead to inaccurate and misleading conclusions under many experimental circumstances, particularly those encountered in one-photon confocal measurements. Furthermore, analytical models cannot be derived for all measurement scenarios. We describe a novel numerical approach to FCS that circumvents conventional analytical models, enabling meaningful analyses even under extraordinarily unusual measurement conditions. Numerical fluorescence correlation spectroscopy (NFCS) involves quantitatively matching experimental correlation curves with synthetic curves generated via diffusion simulation or direct calculation based on an experimentally determined 3D map of the detection volume. Model parameters are adjusted iteratively to minimize the residual differences between synthetic and experimental correlation curves. In order to reduce analysis time, we distribute calculations across a network of processors. As an example of this new approach, we demonstrate that synthetic autocorrelation curves correspond well with experimental data and that NFCS diffusion measurements of Rhodamine B remain constant, regardless of the distortion present in a confocal detection volume.

Dertinger, T., V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor and J. Enderlein. (2007) Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements. Chemphyschem 8(3):433-43.

We present a new method to measure absolute diffusion coefficients at nanomolar concentrations with high precision. Based on a modified fluorescence correlation spectroscopy (FCS)-setup, this method is improved by introducing an external ruler for measuring the diffusion time by generating two laterally shifted and overlapping laser foci at a fixed and known distance. Data fitting is facilitated by a new two-parameter model to describe the molecule detection function (MDF). We present a recorded MDF and show the excellent agreement with the fitting model. We measure the diffusion coefficient of the red fluorescent dye Atto655 under various conditions and compare these values with a value achieved by gradient pulsed field NMR (GPF NMR). From these measurements we conclude, that the new measurement scheme is robust against optical and photophysical artefacts which are inherent to standard FCS. With two-focus-FCS, the diffusion coefficient of 4.26 x 10(-6) cm2s(-1) for Atto655 in water at 25 degrees C compares well with the GPF NMR value of 4.28 x 10(-6) cm2s(-1).

Donsmark, J. and C. Rischel. (2007) Fluorescence correlation spectroscopy at the oil-water interface: hard disk diffusion behavior in dilute beta-lactoglobulin layers precedes monolayer formation. Langmuir 23(12):6614-23.

We have performed a thorough characterization of fluorescence correlations spectroscopy (FCS) applied to oil-water interfaces of viscous oil droplets in aqueous solution, including numerical wave-optical calculations of the detection geometry and regularized multicomponent analysis of sample data. It is shown how significant errors in the estimation of the surface concentration can be avoided when FCS is applied to an interface region. We present data on the adsorption dynamics of beta-lactoglobulin (BLG), a well-studied model system. It is found that electrostatic repulsion slows the adsorption process and reduces the initial saturation density far below the monolayer concentration. During the first stages of adsorption, the diffusion coefficients of adsorbed protein closely follow the 2D hard disk model of Lahtinen et al.1 in response to increased surface concentration, which suggests that protein-protein interactions are limited to long-range Coulombic interactions at this stage.

Doose, S., H. Barsch and M. Sauer. (2007) Polymer properties of polythymine as revealed by translational diffusion. Biophys J 93(4):1224-34.

Biopolymers, such as single-stranded DNA (ssDNA), are often described as semiflexible polymers or wormlike chains. We investigated the length dependence of diffusional properties of homogeneous ssDNA (polythymine) with up to 100 nucleotides using fluorescence correlation spectroscopy. We found that the hydrodynamic radius R(h) scales according to a power law, with an exponent between 0.5 and 0.7 depending on ionic strength I. With R(h) being proportional to the square root of the persistence length L(p), we found that L(p) approximately I(m), with m = -0.22 +/- 0.01 for polythymine with 100 residues. For comparison, we performed molecular dynamics (MD) simulations with a force field that accounts for short-range interactions in vacuum, and determined the characteristic polymer properties end-to-end distance R, radius of gyration S, and persistence length L(p) of various labeled and nonlabeled polythymine derivatives. We found excellent agreement for the length dependence of simulated S and experimental R(h) measured at 100 mM NaCl, revealing that electrostatic interactions are completely shielded in aqueous solution at such ionic strength. MD simulations further showed that polythymine with > approximately 30 residues can be described as a semiflexible polymer with negligible influence of the fluorescent label; and that static flexibility is limited by geometrical and steric constraints as expressed by an intrinsic persistence length of approximately 1.7 nm. These results provide a benchmark for theories and MD simulations describing the influence of electrostatic interactions on polyelectrolyte properties, and thus help to develop a complete and accurate description of ssDNA.

Foldes-Papp, Z. (2007) 'True' single-molecule molecule observations by fluorescence correlation spectroscopy and two-color fluorescence cross-correlation spectroscopy. Exp Mol Pathol 82(2):147-55.

Fluorescence correlation spectroscopy (FCS) and two-color fluorescence cross-correlation spectroscopy (FCCS) are a measure of fluctuations of detected light as a fluorescence molecule diffuses through a femtoliter detection volume caused by a tightly focused laser and confocal optics. Fluorescence from a single molecule can easily be distinguished from the slight background associated with a femtoliter of solvent. At a solution concentration of about 1 nM, the probability that there is an analyte molecule in the probe volume is less than one. Although fluorescence from individual molecules is collected, the data are analyzed by autocorrelation or two-color cross-correlation functions that are the average of thousands of molecules. Properties of single molecules are not obtained. I have been working on problems and opportunities associated with very dilute solutions. The molecule in the confocal probe volume is most probably the molecule that just diffused out, turned around, and diffused back in, i.e., reentered. For the first time, some theoretical results of the novel theory of the meaningful time are presented that enable study of just one single molecule over extended periods of times without immobilization or hydrodynamic focusing. Reentries that may also be called reoccurrences or encounters of a single molecule are significant because during measurement times they give rise to fluctuation phenomena such as molecule number fluctuations. Likewise, four criteria have been developed that can be used to verify that there is only one "selfsame" molecule in the laser probe volume during the experiment: (Foldes-Papp, Z., 2006. What it means to measure a single molecule in a solution by fluorescence fluctuation spectroscopy. Exp. Mol. Pathol. 80 (3) 209-218).

Garai, K., R. Sureka and S. Maiti. (2007) Detecting amyloid-beta aggregation with fiber-based fluorescence correlation spectroscopy. Biophys J 92(7):L55-7.

Soluble aggregates critically influence the chemical and biological aspects of amyloid protein aggregation, but their population is difficult to measure, especially in vivo. We take an optical fiber-based fluorescence correlation spectroscopy (FCS) approach to characterize a solution of aggregating amyloid-beta molecules. We find that this technique can easily resolve aggregate particles of size 100 nm or greater in vitro, and the size distribution of these particles agrees well with that obtained by conventional FCS techniques. We propose fiber FCS as a tool for studying aggregation in vivo.

Garcia-Saez Ana, J. and P. Schwille. (2007) Single molecule techniques for the study of membrane proteins. Applied microbiology and biotechnology 76(2):257-66.

Single molecule techniques promise novel information about the properties and behavior of individual particles, thus enabling access to molecular heterogeneities in biological systems. Their recent developments to accommodate membrane studies have significantly deepened the understanding of membrane proteins. In this short review, we will describe the basics of the three most common single-molecule techniques used on membrane proteins: fluorescence correlation spectroscopy, single particle tracking, and atomic force microscopy. We will discuss the most relevant findings made during the recent years and their contribution to the membrane protein field.

Grabowski Christopher, A. and A. Mukhopadhyay. (2007) Contraction and reswelling of a polymer chain near the critical point of a binary liquid mixture. Phys Rev Lett 98(20):207801.

We have studied the conformation change of a flexible linear polymer chain near the critical point of a binary liquid mixture using fluorescence correlation spectroscopy, which measured the hydrodynamic radius of the chains. Our results indicate that as the critical temperature (Tc) is approached, the chain size decreases. The polymer attains its most compact conformation when the correlation length of the critical fluctuations becomes comparable to the coil size. At very close to Tc, the polymer reexpands dramatically. To our knowledge, this is the first experimental evidence supporting the prediction of Brochard and de Gennes that a polymer chain will collapse and subsequently reswell on approaching Tc.

Guigas, G., C. Kalla and M. Weiss. (2007) Probing the nanoscale viscoelasticity of intracellular fluids in living cells. Biophys J 93(1):316-23.

We have used fluorescence correlation spectroscopy to determine the anomalous diffusion properties of fluorescently tagged gold beads in the cytoplasm and the nucleus of living cells. From the extracted mean-square displacement v(tau) approximately tau(alpha), we have determined the complex shear modulus G(omega) approximately omega(alpha) for both compartments. Without treatment, all tested cell lines showed a strong viscoelastic behavior of the cytoplasm and the nucleoplasm, highlighting the crowdedness of these intracellular fluids. We also found a similar viscoelastic response in frog egg extract, which tended toward a solely viscous behavior upon dilution. When cells were osmotically stressed, the diffusion became less anomalous and the viscoelastic response changed. In particular, the anomality changed from alpha approximately 0.55 to alpha approximately 0.66, which indicates that the Zimm model for polymer solutions under varying solvent conditions is a good empirical description of the material properties of the cytoplasm and the nucleoplasm. Since osmotic stress may eventually trigger cell death, we propose, on the basis of our observations, that intracellular fluids are maintained in a state similar to crowded polymer solutions under good solvent conditions to keep the cell viable.

Gullapalli Ramachandra, R., T. Tabouillot, R. Mathura, H. Dangaria Jhanvi and J. Butler Peter. (2007) Integrated multimodal microscopy, time-resolved fluorescence, and optical-trap rheometry: toward single molecule mechanobiology. J Biomed Opt 12(1):014012.

Cells respond to forces through coordinated biochemical signaling cascades that originate from changes in single-molecule structure and dynamics and proceed to large-scale changes in cellular morphology and protein expression. To enable experiments that determine the molecular basis of mechanotransduction over these large time and length scales, we construct a confocal molecular dynamics microscope (CMDM). This system integrates total-internal-reflection fluorescence (TIRF), epifluorescence, differential interference contrast (DIC), and 3-D deconvolution imaging modalities with time-correlated single-photon counting (TCSPC) instrumentation and an optical trap. Some of the structures hypothesized to be involved in mechanotransduction are the glycocalyx, plasma membrane, actin cytoskeleton, focal adhesions, and cell-cell junctions. Through analysis of fluorescence fluctuations, single-molecule spectroscopic measurements [e.g., fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence] can be correlated with these subcellular structures in adherent endothelial cells subjected to well-defined forces. We describe the construction of our multimodal microscope in detail and the calibrations necessary to define molecular dynamics in cell and model membranes. Finally, we discuss the potential applications of the system and its implications for the field of mechanotransduction.

Hanson Kerry, M., K. Davis Sara and J. Bardeen Christopher. (2007) Two-photon standing-wave fluorescence correlation spectroscopy. Optics letters 32(15):2121-3.

A fluorescence correlation spectroscopy experiment that combines two-photon excitation and a standing-wave interference pattern is presented. The experimental correlation function can be analyzed using a simple expression involving (1) an exponential decay with time constant tau(f), which reflects diffusion across the interference fringes, and (2) a longer-lived decay with time constant tau(omega), which reflects diffusion in and out of the focal spot. The diffusion of Rhodamine 110 in water and ethylene glycol is measured using this method. The ability to simultaneously measure diffusion on two different time and lengthscales makes this experiment especially useful in environments where anomalous diffusion is suspected.

Haustein, E. and P. Schwille. (2007) Fluorescence correlation spectroscopy: novel variations of an established technique. Annual review of biophysics and biomolecular structure 36:151-69.

Fluorescence correlation spectroscopy (FCS) is one of the major biophysical techniques used for unraveling molecular interactions in vitro and in vivo. It allows minimally invasive study of dynamic processes in biological specimens with extremely high temporal and spatial resolution. By recording and correlating the fluorescence fluctuations of single labeled molecules through the exciting laser beam, FCS gives information on molecular mobility and photophysical and photochemical reactions. By using dual-color fluorescence cross-correlation, highly specific binding studies can be performed. These have been extended to four reaction partners accessible by multicolor applications. Alternative detection schemes shift accessible time frames to slower processes (e.g., scanning FCS) or higher concentrations (e.g., TIR-FCS). Despite its long tradition, FCS is by no means dated. Rather, it has proven to be a highly versatile technique that can easily be adapted to solve specific biological questions, and it continues to find exciting applications in biology and medicine.

Hayek, A., S. Ercelen, X. Zhang, F. Bolze, J.-F. Nicoud, E. Schaub, L. Baldeck Patrice and Y. Mely. (2007) Conjugation of a new two-photon fluorophore to poly(ethylenimine) for gene delivery imaging. Bioconjugate chemistry 18(3):844-51.

We report herein the molecular engineering of an efficient two-photon absorbing (TPA) chromophore based on a donor-donor bis-stilbenyl entity to allow conjugation with biologically relevant molecules. The dye has been functionalized using an isothiocyanate moiety to conjugate it with the amine functions of poly(ethylenimine) (PEI), which is a cationic polymer commonly used for nonviral gene delivery. Upon conjugation, the basic architecture and photophysical properties of the active TPA chromophore remain unchanged. At the usual N/P ratio (ratio of the PEI positive charges to the DNA negative charges) of 10 used for transfection, the transfection efficiency and cytotoxicity of the labeled PEI/DNA complexes were found to be comparable to those of the unlabeled PEI/DNA complexes. Moreover, when used in combination with unlabeled PEI (at a ratio of 1 labeled PEI to 3 unlabeled PEI), the labeled PEI does not affect the size of the complexes with DNA. The labeled PEI was successfully used in two-photon fluorescence correlation spectroscopy measurements, showing that at N/P = 10 most PEI molecules are free and the diffusion coefficient of the complexes is consistent with the 360 nm size measured by quasielastic light scattering. Finally, two-photon images of the labeled PEI/DNA complexes confirmed that the complexes enter into the cytoplasm of HeLa cells by endocytosis and hardly escape from the endosomes. As a consequence, the functionalized TPA chromophore appears to be an adequate tool to label the numerous polyamines used in nonviral gene delivery and characterize their complexes with DNA in two-photon applications.

Heuff Romey, F., L. Swift Jody and T. Cramb David. (2007) Fluorescence correlation spectroscopy using quantum dots: advances, challenges and opportunities. Physical chemistry chemical physics : PCCP 9(16):1870-80.

Semiconductor nanocrystals (quantum dots) have been increasingly employed in measuring the dynamic behavior of biomacromolecules using fluorescence correlation spectroscopy. This poses a challenge, because quantum dots display their own dynamic behavior in the form of intermittent photoluminescence, also known as blinking. In this review, the manifestation of blinking in correlation spectroscopy will be explored, preceded by an examination of quantum dot blinking in general.

Hoshina, Y., Y. Yamada, H. Tanaka, T. Doi and T. Takahashi. (2007) Synthesis of fluorescent-labeled aeruginosin derivatives for high-throughput fluorescence correlation spectroscopy assays. Bioorganic & medicinal chemistry letters 17(10):2904-7.

The design and solid-phase synthesis of effective fluorescent-labeled aeruginosin derivatives and their application to the fluorescence correlation spectroscopy (FCS)-based competitive binding assay of an aeruginosin library are described. The phenolic hydroxyl group on the (R)-3-(4-hydroxyphenyl)lactic acid (d-Hpla) residue was observed to be suitable for connecting Rhodamine green derivative with minimum loss of biological activity. In addition, the FCS-based binding assay of the library using fluorescent-labeled chemical probes was also achieved.

Hossain, S., M. Grande, G. Ahmadkhanov and A. Pramanik. (2007) Binding of the Alzheimer amyloid beta-peptide to neuronal cell membranes by fluorescence correlation spectroscopy. Exp Mol Pathol 82(2):169-74.

The deposition of the Alzheimer amyloid beta-peptide (Abeta) fibrils in brain is a key step in Alzheimer's disease. The aggregated Abeta is found to be toxic to neurons since cells die when the aggregated Abeta is added to the cell culture medium. However, target of action of Abeta to cells is unknown. We have applied the fluorescence correlation spectroscopy (FCS) technique to study the existence of a receptor or target molecule for the Alzheimer amyloid beta-peptide (Abeta) in cultured human cerebral cortical neurons. FCS measurement of the fluorophore rhodamine-labeled Abeta (Rh-Abeta) shows diffusion times: 0.1 ms, 1.1 ms and 5.9 ms. Thus, 0.1 ms corresponds to the unbound Rh-Abeta, and 1.1 ms and 5.9 ms correspond to slowly diffusing complexes of Rh-Abeta bound to a kind of receptor or target molecule for Abeta. Addition of excess non-labeled Abeta is accompanied by a competitive displacement, showing that the Abeta binding is specific. Full saturation of the Abeta binding is obtained at nanomolar concentrations, indicating that the Abeta binding is of high affinity. The notion that using FCS we have found a kind of receptor or target molecule for Abeta makes an important point that Abeta kills cells possibly by affecting cell membranes via a receptor or target molecule. This study is of highly significance since it suggests that Abeta possibly affects neuronal cell membranes of Alzheimer patients via a receptor or target molecule.

Hunke, C., W.-J. Chen, H.-J. Schafer and G. Gruber. (2007) Cloning, purification, and nucleotide-binding traits of the catalytic subunit A of the V1VO ATPase from Aedes albopictus. Protein expression and purification 53(2):378-83.

The Asian tiger mosquito, Aedes albopictus, is commonly infected by the gregarine parasite Ascogregarina taiwanensis, which develops extracellularly in the midgut of infected larvae. The intracellular trophozoites are usually confined within a parasitophorous vacuole, whose acidification is generated and controlled by the V(1)V(O) ATPase. This proton pump is driven by ATP hydrolysis, catalyzed inside the major subunit A. The subunit A encoding gene of the Aedes albopictus V(1)V(O) ATPase was cloned in pET9d1-His(3) and the recombinant protein, expressed in the Escherichia coli Rosetta 2 (DE3) strain, purified by immobilized metal affinity- and ion-exchange chromatography. The purified protein was soluble and properly folded. Analysis of secondary structure by circular dichroism spectroscopy showed that subunit A comprises 43% alpha-helix, 25% beta-sheet and 40% random coil content. The ability of subunit A of eukaryotic V-ATPases to bind ATP and/or ADP is demonstrated by photoaffinity labeling and fluorescence correlation spectroscopy (FCS). Quantitation of the FCS data indicates that the ADP-analogues bind slightly weaker to subunit A than the ATP-analogues. Tryptophan fluorescence quenching of subunit A after binding of different nucleotides provides evidence for secondary structural alterations in this subunit caused by nucleotide-binding.

Ito, S., T. Sugiyama, N. Toitani, G. Katayama and H. Miyasaka. (2007) Application of fluorescence correlation spectroscopy to the measurement of local temperature in solutions under optical trapping condition. The journal of physical chemistry. B 111(9):2365-71.

Fluorescence correlation spectroscopy (FCS) was applied to the quantitative evaluation of the local heating in small domains <1 microm in solutions under the laser trapping condition in the presence of a near-infrared (NIR) laser beam at 1064 nm. On the basis of the translational diffusion coefficient of fluorescent molecules obtained by FCS, the relationship between temperature rise and the incident NIR laser power, DeltaT/DeltaP, were determined to be 62 +/- 6, 49 +/- 7, and 23 +/- 1 deg K/W in ethylene glycol, ethanol, and water, respectively, while no remarkable temperature increase was observed for deuterated water. The value of DeltaT/DeltaP linearly increased as a function of alpha/lambda (alpha is the extinction coefficient of solvent at the wavelength and lambda is the thermal conductivity of the medium). The validity and the applicability of the present method for the measurement of the local temperature increase were discussed by comparing the present results with previous ones by other various methods.

Kapusta, P., M. Wahl, A. Benda, M. Hof and J. Enderlein. (2007) Fluorescence lifetime correlation spectroscopy. J Fluoresc 17(1):43-8.

This article explains the basic principles of FLCS, a genuine fusion of Time-Correlated Single Photon Counting (TCSPC) and Fluorescence Correlation Spectroscopy (FCS), using common terms and minimum mathematics. The usefulness of the method is demonstrated on simple FCS experiments. The method makes possible to separate the autocorrelation function of individual components of a mixture of fluorophores, as well as purging the result from parasitic contributions like scattered light or detector afterpulsing.

Kii, H., T. Takagi, A. Sasaki, T. Okajima and M. Kinjo. (2007) DNA microstructure based on self-assembly of 4-sticky-end Holliday junctions in aqueous solution. Journal of nanoscience and nanotechnology 7(3):726-9.

Liverwort-like DNA microscale structures consist of 4-sticky-end Holiday junctions as DNA bricks that can be used in nanotechnology and nanobiotechnology to direct the self-assembly of nanomachines as well as DNA assembly. Previously it has not been possible to obtain such DNA microscale structural forms, but herein we report construction of a mesh-like material made up of 4 strands of 40-base DNA. Advanced bioimaging techniques such as fluorescence correlation spectroscopy (FCS), laser scanning microscopy (LSM), and atomic force microscopy (AFM) help us as ultrasensitive detection tools for examing structures in solutions. Combinations of these techniques allow us to survey various chemical conditions of materials and solutions.

Kitajima, I. (2007) Development of molecular pathology analysis testing which can be practiced in the hospital laboratory. The clinical application of a transcription factor activation test. Rinsho byori. The Japanese journal of clinical pathology 55(3):262-71.

The nuclear factor-kappa B (NF-kappaB) family of transcription factors is known to play an important role in the regulation of the immune system. NF-kappaB is activated by bacterial and viral antigens, which lead to the production of proinflammatory cytokines and chemokines. The rapid detection of activated NF-kappaB by systemic inflammatory response syndrome (SIRS) is considered to be crucial for the treatment of patients with septicemia. The aim of the present study was to evaluate the sensitivity of two methods, electrophoretic mobility shift assay (EMSA) and transcription factor enzyme-linked immunoassay (TF-ELISA). TF-ELISA detected 25ng of recombinant human NF-kappaB p50 (rhp50) and 5 microg of TNFalpha-stimulated HeLa nuclear protein, while EMSA detected approximately 100 ng of rhp50 and 10 microg of HeLa nuclear protein. We found that TF-ELISA was more sensitive than EMSA in detecting NF-kappaB; however, it was judged that the 3-6 hour measuring time required in TF-ELISA was excessively long for patients with SIRS. Therefore, the development of new analytical methods with improved sensitivity and measurement time was necessary for the detection and quantification of activated NF-kappaB protein in the hospital laboratory. Consequently, we have developed a new NF-kappaB analyzer based on surface plasmon resonance (SPR), which is recognized as one of the most sensitive direct optical detection methods. This method can detect nanomolar concentrations of NF-kappaB within 15 minutes. In addition, we have developed new experimental apparatus for the detection of NF-kappaB based on fluorescence correlation spectroscopy (FCS), which is able to analyze binding between DNA and protein in the liquid phase. At present, we are carrying out clinical trials using this new transcription factor analysis apparatus for SIRS patients.

Koopman Werner, J. H., A. Hink Mark, S. Verkaart, H.-J. Visch, A. M. Smeitink Jan and H. G. M. Willems Peter. (2007) Partial complex I inhibition decreases mitochondrial motility and increases matrix protein diffusion as revealed by fluorescence correlation spectroscopy. Biochimica et biophysica acta 1767(7):940-7.

We previously reported that inhibition of mitochondrial complex I (CI) by rotenone induces marked increases in mitochondrial length and degree of branching, thus revealing a relationship between mitochondrial function and shape. We here describe the first time use of fluorescence correlation spectroscopy (FCS) to simultaneously probe mitochondrial mobility and intra-matrix protein diffusion, with the aim to investigate the effects of chronic CI inhibition on the latter two parameters. To this end, EYFP was expressed in the mitochondrial matrix of human skin fibroblasts (mitoEYFP) using baculoviral transduction and its diffusion monitored by FCS. This approach revealed the coexistence of moving and stationary mitochondria within the same cell and enabled simultaneous quantification of mitochondrial velocity and mitoEYFP diffusion. When CI activity was chronically reduced by 80% using rotenone treatment, the percentage of moving mitochondria and their velocity decreased by 30%. MitoEYFP diffusion did not differ between moving and stationary mitochondria but was increased 2-fold in both groups of mitochondria following rotenone treatment. We propose that the increase in matrix protein diffusion together with the increase in mitochondrial length and degree of branching constitutes part of an adaptive response which serves to compensate for the reduction in CI activity and mitochondrial motility.

Koynov, K., G. Mihov, M. Mondeshki, C. Moon, H. W. Spiess, K. Mullen, H. J. Butt and G. Floudas. (2007) Diffusion and conformation of peptide-functionalized polyphenylene dendrimers studied by fluorescence correlation and 13C NMR spectroscopy. Biomacromolecules 8(5):1745-50.

We report on the combined use of fluorescence correlation spectroscopy (FCS) and 1H and 13C NMR spectroscopy to detect the size and type of peptide secondary structures in a series of poly-Z-L-lysine functionalized polyphenylene dendrimers bearing the fluorescent perylenediimide core in solution. In dilute solution, the size of the molecule as detected from FCS and 1H NMR diffusion measurements matches nicely. We show that FCS is a sensitive probe of the core size as well as of the change in the peptide secondary structure. However, FCS is less sensitive to functionality. A change in the peptide secondary conformation from beta-sheets to alpha-helices detected by 13C NMR spectroscopy gives rise to a steep increase in the hydrodynamic radii for number of residues n > or = 16. Nevertheless, helices are objects of low persistence.

Kudryavtsev, V., S. Felekyan, K. Wozniak Anna, M. Konig, C. Sandhagen, R. Kuhnemuth, A. M. Seidel Claus and F. Oesterhelt. (2007) Monitoring dynamic systems with multiparameter fluorescence imaging. Analytical and bioanalytical chemistry 387(1):71-82.

A new general strategy based on the use of multiparameter fluorescence detection (MFD) to register and quantitatively analyse fluorescence images is introduced. Multiparameter fluorescence imaging (MFDi) uses pulsed excitation, time-correlated single-photon counting and a special pixel clock to simultaneously monitor the changes in the eight-dimensional fluorescence information (fundamental anisotropy, fluorescence lifetime, fluorescence intensity, time, excitation spectrum, fluorescence spectrum, fluorescence quantum yield, distance between fluorophores) in real time. The three spatial coordinates are also stored. The most statistically efficient techniques known from single-molecule spectroscopy are used to estimate fluorescence parameters of interest for all pixels, not just for the regions of interest. Their statistical significance is judged from a stack of two-dimensional histograms. In this way, specific pixels can be selected for subsequent pixel-based subensemble analysis in order to improve the statistical accuracy of the parameters estimated. MFDi avoids the need for sequential measurements, because the registered data allow one to perform many analysis techniques, such as fluorescence-intensity distribution analysis (FIDA) and fluorescence correlation spectroscopy (FCS), in an off-line mode. The limitations of FCS for counting molecules and monitoring dynamics are discussed. To demonstrate the ability of our technique, we analysed two systems: (i) interactions of the fluorescent dye Rhodamine 110 inside and outside of a glutathione sepharose bead, and (ii) microtubule dynamics in live yeast cells of Schizosaccharomyces pombe using a fusion protein of Green Fluorescent Protein (GFP) with Minichromosome Altered Loss Protein 3 (Mal3), which is involved in the dynamic cycle of polymerising and depolymerising microtubules.

Kuroki, K., S. Kobayashi, M. Shiroishi, M. Kajikawa, N. Okamoto, D. Kohda and K. Maenaka. (2007) Detection of weak ligand interactions of leukocyte Ig-like receptor B1 by fluorescence correlation spectroscopy. Journal of immunological methods 320(1-2):172-6.

Fluorescence correlation spectroscopy (FCS) can directly and quickly detect the translational diffusion of individual fluorescence-labeled molecules in solutions. Although FCS analyses for protein-protein interactions have been performed, the very weak interactions generally observed in cell-cell recognition of the immune system have not been examined in detail. Here, we report the FCS analysis for low-affinity and fast-kinetic binding (K(d) greater than muM range) of the human inhibitory immune cell surface receptor, leukocyte immunoglobulin-like receptor B1 (LILRB1), to its ligands, MHC (major histocompatibility complex) class I molecules (MHCIs) by using the single-molecule FCS detection system which requires only a small amount of sample. Since the random labeling technique for LILRB1 disturbed the MHCI binding, we performed site-specific labeling of LILRB1 by introducing a cysteine residue at the C-terminus, which could be covalently attached with the fluorescence reagent, Alexa647. This technique can be applied to other type I membrane receptors. The low-affinity binding of LILRB1-Alexa647 to MHCIs (HLA-Cw4, and -G1) was detected by FCS, even though non-labeled MHCIs were only twice as big as the labeled LILRB1. Their dissociation constants (7.5 muM (HLA-Cw4) and 5.7 muM (HLA-G1)) could be determined and were consistent with surface plasmon resonance (SPR) data. These results indicate that the single-molecule FCS detection system is capable of analyzing the binding characteristics of immune cell surface receptors even in difficult cases such as (1) small amount of protein samples, (2) small difference in molecular weight and (3) weak affinity. Therefore, it is a powerful tool for characterization and high throughput inhibitor screening of a wide variety of cell-cell recognition receptors involved in immunologically relevant events.

Kyoung, M., K. Karunwi and E. D. Sheets. (2007) A versatile multimode microscope to probe and manipulate nanoparticles and biomolecules. Journal of microscopy 225(Pt 2):137-46.

We describe a flexible, multifaceted optical setup that allows quantitative measurement and manipulation of biomolecules and nanoparticles in biomimetic and cellular systems. We have implemented integrated biophotonics techniques (i.e. differential interference contrast, wide-field fluorescence, prism- and objective-based total internal reflection excitation, single particle tracking, fluorescence correlation spectroscopy and dynamic holographic optical trapping) on a single platform. The adaptability of this versatile, custom-designed system allows us to simultaneously monitor cell morphology, while measuring lateral diffusion of biomolecules or controlling their cellular location or interaction partners.

Labille, J., N. Fatin-Rouge and J. Buffle. (2007) Local and average diffusion of nanosolutes in agarose gel: the effect of the gel/solution interface structure. Langmuir 23(4):2083-90.

Fluorescence correlation spectroscopy (FCS) has been used to study the diffusion of nanometric solutes in agarose gel, at microscopic and macroscopic scales. Agarose gel was prepared and put in contact with aqueous solution. Several factors were studied: (i) the role of gel relaxation after its preparation, (ii) the specific structure of the interfacial zone and its role on the local diffusion coefficient of solutes, and (iii) the comparison between the local diffusion coefficient and the average diffusion coefficient in the gel. Fluorescent dyes and labeled biomolecules were used to cover a size range of solutes of 1.5 to 15 nm. Their transport through the interface from the solution toward the gel was modeled by the first Fick's law based on either average diffusion coefficients or the knowledge of local diffusion coefficients in the system. Experimental results have shown that, at the liquid/gel interface, a gel layer with a thickness of 120 microm is formed with characteristics significantly different from the bulk gel. In particular, in this layer, the porosity of agarose fiber network is significantly lower than in the bulk gel. The diffusion coefficient of solutes in this layer is consequently decreased for steric reasons. Modeling of solute transport shows that, in the bulk gel, macroscopic diffusion satisfactorily follows the classical Fick's diffusion laws. For the tested solutes, the local diffusion coefficients in the bulk gel, measured at microscopic scale by FCS, were equal, within experimental errors, to the average diffusion coefficients applicable at macroscopic scales (>or=mm). This confirms that anomalous diffusion applies only to solutes with sizes close to the gel pore size and at short time (<or=min) and spatial scales (<or=1 microm).

Laurence Ted, A., Y. Kwon, E. Yin, W. Hollars Christopher, A. Camarero Julio and D. Barsky. (2007) Correlation spectroscopy of minor fluorescent species: signal purification and distribution analysis. Biophys J 92(6):2184-98.

We are performing experiments that use fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) to monitor the movement of an individual donor-labeled sliding clamp protein molecule along acceptor-labeled DNA. In addition to the FRET signal sought from the sliding clamp-DNA complexes, the detection channel for FRET contains undesirable signal from free sliding clamp and free DNA. When multiple fluorescent species contribute to a correlation signal, it is difficult or impossible to distinguish between contributions from individual species. As a remedy, we introduce "purified FCS", which uses single molecule burst analysis to select a species of interest and extract the correlation signal for further analysis. We show that by expanding the correlation region around a burst, the correlated signal is retained and the functional forms of FCS fitting equations remain valid. We demonstrate the use of purified FCS in experiments with DNA sliding clamps. We also introduce "single-molecule FCS", which obtains diffusion time estimates for each burst using expanded correlation regions. By monitoring the detachment of weakly-bound 30-mer DNA oligomers from a single-stranded DNA plasmid, we show that single-molecule FCS can distinguish between bursts from species that differ by a factor of 5 in diffusion constant.

Lee, T.-H., J. Lapidus Lisa, W. Zhao, J. Travers Kevin, D. Herschlag and S. Chu. (2007) Measuring the folding transition time of single RNA molecules. Biophys J 92(9):3275-83.

We describe a new, time-apertured photon correlation method for resolving the transition time between two states of RNA in folding--i.e., the time of the transition between states rather than the time spent in each state. Single molecule fluorescence resonance energy transfer and fluorescence correlation spectroscopy are used to obtain these measurements. Individual RNA molecules are labeled with fluorophores such as Cy3 and Cy5. Those molecules are then immobilized on a surface and observed for many seconds during which time the molecules spontaneously switch between two conformational states with different levels of flourescence resonance energy transfer efficiency. Single photons are counted from each fluorophore and cross correlated in a small window around a transition. The average of over 1000 cross correlations can be fit to a polynomial, which can determine transition times as short as the average photon emission interval. We applied the method to the P4-P6 domain of the Tetrahymena group I self-splicing intron to yield the folding transition time of 240 micros. The unfolding time is found to be too short to measure with this method.

Michelman-Ribeiro, A., F. Horkay, R. Nossal and H. Boukari. (2007) Probe diffusion in aqueous poly(vinyl alcohol) solutions studied by fluorescence correlation spectroscopy. Biomacromolecules 8(5):1595-600.

We report fluorescence correlation spectroscopy measurements of the translational diffusion coefficient of various probe particles in dilute and semidilute aqueous poly(vinyl alcohol) solutions. The range of sizes of the particles (fluorescent molecules, proteins, and polymers) was chosen to explore various length scales of the polymer solutions as defined by the polymer-polymer correlation length. For particles larger than the correlation length, we find that the diffusion coefficient, D, decreases exponentially with the polymer concentration. This can be explained by an exponential increase in the solution viscosity, consistent with the Stokes-Einstein equation. For probes on the order of the correlation length, the decrease of the diffusion coefficient cannot be accounted for by the Stokes-Einstein equation, but can be fit by a stretched exponential, D approximately exp(-alphacn), where we find n = 0.73-0.84 and alpha is related to the probe size. These results are in accord with a diffusion model of Langevin and Rondelez (Polymer 1978, 19, 1875), where these values of n indicate a good solvent quality.

Middleton Richard, J., J. Briddon Stephen, Y. Cordeaux, S. Yates Andrew, L. Dale Clare, W. George Michael, G. Baker Jillian, J. Hill Stephen and B. Kellam. (2007) New fluorescent adenosine A1-receptor agonists that allow quantification of ligand-receptor interactions in microdomains of single living cells. Journal of medicinal chemistry 50(4):782-93.

Fluorescence spectroscopy is becoming a valuable addition to the array of techniques available for scrutinizing ligand-receptor interactions in biological systems. In particular, scanning confocal microscopy and fluorescence correlation spectroscopy (FCS) allow the noninvasive imaging and quantification of these interactions in single living cells. To address the emerging need for fluorescently labeled ligands to support these technologies, we have developed a series of red-emitting agonists for the human adenosine A1-receptor that, collectively, are N6-aminoalkyl derivatives of adenosine or adenosine 5'-N-ethyl carboxamide. The agonists, which incorporate the commercially available fluorophore BODIPY [630/650], retain potent and efficacious agonist activity, as demonstrated by their ability to inhibit cAMP accumulation in chinese hamster ovary cells expressing the human adenosine A1-receptor. Visualization and confirmation of ligand-receptor interactions at the cell membrane were accomplished using confocal microscopy, and their suitability for use in FCS was demonstrated by quantification of agonist binding in small areas of cell membrane.

Mikuni, S., M. Tamura and M. Kinjo. (2007) Analysis of intranuclear binding process of glucocorticoid receptor using fluorescence correlation spectroscopy. FEBS letters 581(3):389-93.

The diffusion properties of EGFP-hGRalpha and mutants C421G, A458T and I566 in living cells were analyzed. The wild type and mutants C421G and A458T translocated from the cytoplasm to the nucleus after addition of Dex; however, the Brownian motions of the proteins were different. The diffusion constant of wild-type GRalpha after addition of Dex slowed to 15.6% of that in the absence of Dex, whereas those of A458T and C421G slowed to 34.8% and 61.7%, respectively. This is the first report that dimer formation is less important than the binding activity of GRalpha to GRE in the living cell.

Mukhopadhyay, S., R. Krishnan, A. Lemke Edward, S. Lindquist and A. Deniz Ashok. (2007) A natively unfolded yeast prion monomer adopts an ensemble of collapsed and rapidly fluctuating structures. Proc Natl Acad Sci U S A 104(8):2649-54.

The yeast prion protein Sup35 is a translation termination factor, whose activity is modulated by sequestration into a self-perpetuating amyloid. The prion-determining domain, NM, consists of two distinct regions: an amyloidogenic N terminus domain (N) and a charged solubilizing middle region (M). To gain insight into prion conversion, we used single-molecule fluorescence resonance energy transfer (SM-FRET) and fluorescence correlation spectroscopy to investigate the structure and dynamics of monomeric NM. Low protein concentrations in these experiments prevented the formation of obligate on-pathway oligomers, allowing us to study early folding intermediates in isolation from higher-order species. SM-FRET experiments on a dual-labeled amyloid core variant (N21C/S121C, retaining wild-type prion behavior) indicated that the N region of NM adopts a collapsed form similar to "burst-phase" intermediates formed during the folding of many globular proteins, even though it lacks a typical hydrophobic core. The mean distance between residues 21 and 121 was approximately equal to 43 A. This increased with denaturant in a noncooperative fashion to approximately equal to 63 A, suggesting a multitude of interconverting species rather than a small number of discrete monomeric conformers. Fluorescence correlation spectroscopy analysis of singly labeled NM revealed fast conformational fluctuations on the 20- to 300-ns time scale. Quenching from proximal and distal tyrosines resulted in distinct fast and slower fluctuations. Our results indicate that native monomeric NM is composed of an ensemble of structures, having a collapsed and rapidly fluctuating N region juxtaposed with a more extended M region. The stability of such ensembles is likely to play a key role in prion conversion.

Munishkina Larissa, A. and L. Fink Anthony. (2007) Fluorescence as a method to reveal structures and membrane-interactions of amyloidogenic proteins. Biochimica et biophysica acta 1768(8):1862-85.

Amyloidogenesis is a characteristic feature of the 40 or so known protein deposition diseases, and accumulating evidence strongly suggests that self-association of misfolded proteins into either fibrils, protofibrils, or soluble oligomeric species is cytotoxic. The most likely mechanism for toxicity is through perturbation of membrane structure, leading to increased membrane permeability and eventual cell death. There have been a rather limited number of investigations of the interactions of amyloidogenic polypeptides and their aggregated states with membranes; these are briefly reviewed here. Amyloidogenic proteins discussed include A-beta from Alzheimer's disease, the prion protein, alpha-synuclein from Parkinson's disease, transthyretin (FAP, SSA amyloidosis), immunoglobulin light chains (primary (AL) amyloidosis), serum amyloid A (secondary (AA) amyloidosis), amylin or IAPP (Type 2 diabetes) and apolipoproteins. This review highlights the significant role played by fluorescence techniques in unraveling the nature of amyloid fibrils and their interactions and effects on membranes. Fluorescence spectroscopy is a valuable and versatile method for studying the complex mechanisms of protein aggregation, amyloid fibril formation and the interactions of amyloidogenic proteins with membranes. Commonly used fluorescent techniques include intrinsic and extrinsic fluorophores, fluorescent probes incorporated in the membrane, steady-state and lifetime measurements of fluorescence emission, fluorescence correlation spectroscopy, fluorescence anisotropy and polarization, fluorescence resonance energy transfer (FRET), fluorescence quenching, and fluorescence microscopy.

Neuweiler, H., M. Lollmann, S. Doose and M. Sauer. (2007) Dynamics of unfolded polypeptide chains in crowded environment studied by fluorescence correlation spectroscopy. J Mol Biol 365(3):856-69.

Proteins have evolved to fold and function within a cellular environment that is characterized by high macromolecular content. The earliest step of protein folding represents intrachain contact formation of amino acid residues within an unfolded polypeptide chain. It has been proposed that macromolecular crowding can have significant effects on rates and equilibria of biomolecular processes. However, the kinetic consequences on intrachain diffusion of polypeptides have not been tested experimentally, yet. Here, we demonstrate that selective fluorescence quenching of the oxazine fluorophore MR121 by the amino acid tryptophan (Trp) in combination with fast fluorescence correlation spectroscopy (FCS) can be used to monitor end-to-end contact formation rates of unfolded polypeptide chains. MR121 and Trp were incorporated at the terminal ends of polypeptides consisting of repetitive units of glycine (G) and serine (S) residues. End-to-end contact formation and dissociation result in "off" and "on" switching of MR121 fluorescence and underlying kinetics can be revealed in FCS experiments with nanosecond time resolution. We revisit previous experimental studies concerning the dependence of end-to-end contact formation rates on polypeptide chain length, showing that kinetics can be described by Gaussian chain theory. We further investigate effects of solvent viscosity and temperature on contact formation rates demonstrating that intrachain diffusion represents a purely diffusive, entropy-controlled process. Finally, we study the influence of macromolecular crowding on polypeptide chain dynamics. The data presented demonstrate that intrachain diffusion is fast in spite of hindered diffusion caused by repulsive interactions with macromolecules. Findings can be explained by effects of excluded volume reducing chain entropy and therefore accelerating the loop search process. Our results suggest that within a cellular environment the early formation of structural elements in unfolded proteins can still proceed quite efficiently in spite of hindered diffusion caused by high macromolecular content.

Nishimura, G., C.-G. Pack and M. Tamura. (2007) Phosphorescence decay time measurements using intensity correlation spectroscopy. Exp Mol Pathol 82(2):175-83.

In this paper, we report on phosphorescence measurements for oxygen dynamics in cells by means of a correlation method, which is an expansion of the fluorescence correlation spectroscopy. The intensity correlation function of the emission excited by a pulsed light source was measured. With changing the pulse timing, both the fluorescence correlation function and the decay time of phosphorescence could be analyzed. This method was applied for the analysis of the oxygen dynamics in HeLa cells stained by Pd(II)-porphine. The decay function consisted of two exponential components, which might be attributed to free and protein-bound forms of Pd(II)-porphine in the cell, respectively. The relative change of the oxygen concentration under normal and uncoupled respiration conditions was also measured. The simplicity of this method is a great advantage in the biological applications. Although the current system we used was limited in the temporal resolution, the method is in principle applicable to faster decay time measurements down to the nano-second range of the fluorescence decay times.

Novo, M., S. Felekyan, A. M. Seidel Claus and W. Al-Soufi. (2007) Dye-exchange dynamics in micellar solutions studied by fluorescence correlation spectroscopy. The journal of physical chemistry. B 111(14):3614-24.

We investigated the dye-exchange dynamics between rhodamine 123 (R123), a mitochondrial fluorescent dye, and micelles as membrane mimetic systems. In the presence of neutral micelles (Triton X-100 and Brij 35) R123 partitions between the aqueous solution and the micellar pseudo-phase, undergoing red shift of the absorption and the emission spectra. Fluorescence correlation spectroscopy (FCS) was used to study the dynamics of these systems over an extremely wide time range and at the single-molecule level, yielding information in one and the same experiment about the diffusional dynamics of free and bound rhodamine and about the dye-exchange dynamics as well as several photophysical properties of the rhodamine bound to the micelles. It was found that the entry rate constants are diffusion-controlled, indicating that there are no geometric or orientational requirements for the association of the dye with the micelle. With respect to the dye-exchange dynamics, micelles are found to behave as soft supramolecular cages in contrast to other rigid supramolecular cavities, such as cyclodextrins. The exit rate constants depend on the surfactant and determine the stability of the binding. Single-molecule multiparameter fluorescence detection (MFD) was used to examine the fluorescence properties of individual molecules in comparison to ensembles of molecules. The MFD histograms confirm the fast dye-exchange dynamics observed by FCS and yield mean values of fluorescence lifetimes and anisotropies in agreement with those obtained in bulk measurements.

Pan, X., W. Foo, W. Lim, H. Y. Fok Marcus, P. Liu, H. Yu, I. Maruyama and T. Wohland. (2007) Multifunctional fluorescence correlation microscope for intracellular and microfluidic measurements. The Review of scientific instruments 78(5):053711.

A modified fluorescence correlation microscope (FCM) was built on a commercial confocal laser scanning microscope (CLSM) by adding two sensitive detectors to perform fluorescence correlation spectroscopy (FCS). A single pinhole for both imaging and spectroscopy and a simple slider switch between the two modes thus facilitate the accurate positioning of the FCS observation volume after the confocal image acquisition. Due to the use of a single pinhole for CLSM and FCS the identity of imaged and spectroscopically observed positions is guaranteed. The presented FCM system has the capability to position the FCS observation volume at any point within the inner 30% of the field of view without loss in performance and in the inner 60% of the field of view with changes of FCS parameters of less than 10%. A single pinhole scheme for spatial fluorescence cross correlation spectroscopy performed on the FCM system is proposed to determine microfluidic flow angles. To show the applicability and versatility of the system, we measured the translational diffusion coefficients on the upper and lower membranes of Chinese hamster ovary cells. Two-photon excitation FCS was also realized by coupling a pulsed Ti: sapphire laser into the microscope and used for flow direction characterization in microchannels.

Pan, X., H. Yu, X. Shi, V. Korzh and T. Wohland. (2007) Characterization of flow direction in microchannels and zebrafish blood vessels by scanning fluorescence correlation spectroscopy. J Biomed Opt 12(1):014034.

The investigation of flow profiles in microstructures and tissues by fluorescence correlation spectroscopy (FCS) has been a challenging topic in the past decade. Due to its inherent optical configuration, a circular focused laser beam, FCS is unable to resolve microfluidic flow directions. Earlier schemes reported the use of two laser beams or the use of nonsymmetrical laser foci to break the symmetry of the measurement system. This, however, is difficult to combine with confocal systems since it would require modifications that interfere with the imaging capabilities. We propose a method called line-scan FCS to measure different flow angles in microchannels and tissues. This method is implemented on a combined laser scanning confocal microscopy (LSCM) and FCS system that enables uncompromised imaging and spectroscopy measurements. We demonstrate that by scanning the laser beam with a defined speed and direction we can measure flow direction with the current system at an optimal resolution of at least 3 microm. The combination system is assessed by measuring flow profiles in a microchannel with and without obstruction. To extend the technique to live tissue measurements we demonstrate that line-scan FCS can determine the flow direction in zebrafish small blood vessels in a label-free approach.

Paradise, A., K. Levin Mikhail, G. Korza and H. Carson John. (2007) Significant proportions of nuclear transport proteins with reduced intracellular mobilities resolved by fluorescence correlation spectroscopy. J Mol Biol 365(1):50-65.

Nuclear transport requires freely diffusing nuclear transport proteins to facilitate movement of cargo molecules through the nuclear pore. We analyzed dynamic properties of importin alpha, importin beta, Ran and NTF2 in nucleus, cytoplasm and at the nuclear pore of neuroblastoma cells using fluorescence correlation spectroscopy. Mobile components were quantified by global fitting of autocorrelation data from multiple cells. Immobile components were quantified by analysis of photobleaching kinetics. Wild-type Ran was compared to various mutant Ran proteins to identify components representing GTP or GDP forms of Ran. Untreated cells were compared to cells treated with nocodazole or latrunculin to identify components associated with cytoskeletal elements. The results indicate that freely diffusing importin alpha, importin beta, Ran and NTF2 are in dynamic equilibrium with larger pools associated with immobile binding partners such as microtubules in the cytoplasm. These findings suggest that formation of freely diffusing nuclear transport intermediates is in competition with binding to immobile partners. Variation in concentrations of freely diffusing nuclear transport intermediates among cells indicates that the nuclear transport system is sufficiently robust to function over a wide range of conditions.

Patil, S., G. Matei, C. A. Grabowski, P. M. Hoffmann and A. Mukhopadhyay. (2007) Combined atomic force microscopy and fluorescence correlation spectroscopy measurements to study the dynamical structure of interfacial fluids. Langmuir 23(9):4988-92.

We have studied the dynamic structure of thin (approximately a few nanometers) liquid films of a nearly spherical, nonpolar molecule tetrakis(2-ethylhexoxy)silane (TEHOS) by using a combination of atomic force microscopy (AFM) and fluorescence correlation spectroscopy (FCS). Ultra-sensitive interferometer-based AFM was used to determine the stiffness (force gradient) and the damping coefficient of the liquid film. The experiments show oscillations in the damping coefficient with a period of approximately 1 nm, which is consistent with the molecular dimension of TEHOS as well as previous X-ray reflectivity measurements. Additionally, we performed FCS experiments for direct determination of the molecular dynamics within the liquid film. From the fluctuation autocorrelation curve, we measured the translational diffusion of the probe molecule embedded within the fluid film formed on a solid substrate. The autocorrelation function was best fitted with two components, which indicate that the dynamics are heterogeneous in nature. However, the heterogeneity is not as pronounced as had been previously observed for molecularly thin liquid films sandwiched between two solid substrates.

Petrasek, Z., M. Krishnan, I. Monch and P. Schwille. (2007) Simultaneous two-photon fluorescence correlation spectroscopy and lifetime imaging of dye molecules in submicrometer fluidic structures. Microsc Res Tech 70(5):459-66.

Fluorescence correlation spectroscopy (FCS) is a very sensitive technique that can be used, e.g., for the measurement of low concentrations and for the investigation of transport of fluorescent molecules. Fluorescence lifetime imaging (FLIM) provides spatially resolved information about molecular fluorescence lifetimes reflecting the interactions of the molecules with their environment. We have applied simultaneous two-photon FCS and FLIM to probe the behavior of fluorescent molecules diffusing in submicrometer silicon oxide channels. Our measurements reveal differences in fluorescence lifetimes compared to bulk solution that result from the effects of confinement and the presence of interfaces. Confinement also affects diffusional characteristics of fluorophores as reflected in fluorescence autocorrelation functions. These possible consequences of both spatial confinement and the presence of interfaces between media with different refractive indices on the diffusion and fluorescence lifetime of molecules in nanostructures are discussed in general. Copyright 2007 Wiley-Liss, Inc.

Philip, F., P. Sengupta and S. Scarlata. (2007) Signaling through a G Protein-coupled receptor and its corresponding G protein follows a stoichiometrically limited model. The Journal of biological chemistry 282(26):19203-16.

The bradykinin receptor is a G protein-coupled receptor (GPCR) that is coupled to the Galpha(q) family of heterotrimeric G proteins. In general, a GPCR can exert intracellular signals either by transiently associating with multiple diffusing G protein subunits or by activating a G protein that is stably bound to the receptor, thus generating a signal that is limited by the stoichiometry of the complex. Here we have distinguished between these models by monitoring the association of type 2 bradykinin receptor (B(2)R) and the Galpha(q)/Gbetagamma heterotrimer in living human embryonic kidney 293 cells expressing fluorescent-tagged proteins. Stable B(2)R-Galpha(q) x Gbetagamma complexes are observed in resting cells by fluorescence resonance energy transfer from either Galpha(q)-eCFP or eCFP-Gbetagamma to B(2)R-eYFP. Stimulating the cells with bradykinin causes detachment of B(2)R from the G protein subunits as the receptor internalizes into early endosomes, with a corresponding elimination of B(2)R-G protein fluorescence resonance energy transfer because Galpha(q) and its associated Gbetagamma remain on the plasma membrane. Single point and scanning fluorescence correlation spectroscopy measurements show that a portion of B(2)R molecules diffuses with a mobility corresponding to dimers or small oligomers, whereas a second fraction diffuses in higher order molecular assemblies. Our studies support a model in which receptors are pre-coupled with their corresponding G proteins in the basal state of cells thereby limiting the response to an external signal to a defined stoichiometry that allows for a rapid and directed cellular response.

Prenner, L., A. Sieben, K. Zeller, D. Weiser and H. Haberlein. (2007) Reduction of high-affinity beta2-adrenergic receptor binding by hyperforin and hyperoside on rat C6 glioblastoma cells measured by fluorescence correlation spectroscopy. Biochemistry 46(17):5106-13.

Beta-adrenergic receptors (beta-AR) are potential targets for antidepressants. Desensitization and downregulation of beta-AR are discussed as possible modes of action for antidepressants. We have investigated the effects of hyperforin and hyperoside, compounds with potentially antidepressant activity from St. John's Wort, on the binding behavior and dynamics of beta2-AR in living rat C6 glioblastoma cells, compared to desipramine (desmethylimipramine; DMI) by means of fluorescence correlation spectroscopy (FCS) and fluorescence microscopy. FCS-binding studies with the fluorescently labeled ligand Alexa532-noradrenaline (Alexa532-NA) binding to beta2-AR of C6 cells showed a significant reduction in total beta2-AR binding after preincubation with hyperforin and hyperoside for 3 days, respectively, which was also found for DMI. This was mainly observed in high-affinity receptor-ligand complexes with hindered lateral mobility (D2 = 1.1 (+/-0.4) microm2/s) in the biomembrane. However, internalization of beta2-AR was found neither in z-scans of these C6 cells nor in HEK 293 cells stably transfected with GFP-tagged beta2-adrenergic receptors (beta2AR-GFP) after incubation up to 6 days with either DMI, hyperforin, or hyperoside. Thus, under these conditions reduction of beta2-AR binding was not mediated by receptor internalization. Additionally, preincubation of C6 cells with DMI, hyperforin, and hyperoside led to a loss of second messenger cAMP after beta2-adrenergic stimulating conditions with terbutaline. Our current results indicate that hyperforin and hyperoside from St. John's Wort, as well as DMI, reduce beta2-adrenergic sensitivity in C6 cells, emphasizing the potential usefulness of St. John's Wort dry extracts in clinical treatment of depressive symptoms.

Remaut, K., B. Lucas, K. Raemdonck, K. Braeckmans, J. Demeester and S. C. De Smedt. (2007) Can we better understand the intracellular behavior of DNA nanoparticles by fluorescence correlation spectroscopy? Journal of controlled release : official journal of the Controlled Release Society 121(1-2):49-63.

The use of non-viral gene carriers to deliver small nucleic acids like antisense oligonucleotides (ODNs) and small interfering RNA (siRNA) remains an attractive but challenging goal in antisense therapy. Indeed, different barriers need to be overcome in the delivery process before a therapeutic effect can be obtained. One promising technique which we have been evaluating to improve our understanding of the intracellular behavior of nucleic acids/carrier complexes is Fluorescence Correlation Spectroscopy (FCS). In particular, we have used FCS for studying the protection of the nucleic acids against enzymatic degradation, and the association and dissociation of the nucleic acids with their carrier, both in buffer and in living cells. In this report, we will review our experience and findings on the use of FCS for that purpose and discuss the strengths and weaknesses of this interesting technique.

Remaut, K., B. Lucas, K. Raemdonck, K. Braeckmans, J. Demeester and S. C. De Smedt. (2007) Protection of oligonucleotides against enzymatic degradation by pegylated and nonpegylated branched polyethyleneimine. Biomacromolecules 8(4):1333-40.

Among the cationic polymers, polyethyleneimine (PEI) is a promising candidate for delivery of oligodeoxynucleotides (ODNs). In this study, we wondered whether pegylation of PEI influences the complexation with ODNs. We especially aimed to investigate whether ODNs are differently protected against enzymatic degradation in PEI and polyethylene glycol-polyethyleneimine (PEG-PEI) polyplexes. Using fluorescence resonance energy transfer combined with fluorescence correlation spectroscopy, we found that PEI/ODN polyplexes remain to protect the ODNs they carry over a prolonged period of time while in PEG-PEI/ODN polyplexes the degradation of the ODNs slowly proceeds. We attribute this to the fact that PEI seems to compact the ODNs more firmly in the polyplexes' core than PEG-PEI, which apparently also results in a better protection against enzymatic degradation. These observations may also influence the efficiency of PEI-based ODN delivery in vivo, where pegylation is an attractive strategy to enhance the stability of the polyplexes in the blood stream.

Reuter, A., W. U. Dittmer and F. C. Simmel. (2007) Kinetics of protein-release by an aptamer-based DNA nanodevice. The European physical journal. E, Soft matter 22(1):33-40.

A recently introduced DNA nanodevice can be used to selectively bind or release the protein thrombin triggered by DNA effector strands. The release process is not well described by simple first or second order reaction kinetics. Here, fluorescence resonance energy transfer and fluorescence correlation spectroscopy experiments are used to explore the kinetics of the release process in detail. To this end the influence of concentration variations and also of temperature is determined. The relevant kinetic parameters are extracted from these experiments and the kinetic behavior of the system is simulated numerically using a set of rate equations. The hydrodynamic radii of the aptamer device alone and bound to thrombin are determined as well as the dissociation constant for the aptamer device-thrombin complex. The results from the experiments and a numerical simulation support the view that the DNA effector strand first binds to the aptamer device followed by the displacement of the protein.

Romantsov, T., I. Fishov and O. Krichevsky. (2007) Internal structure and dynamics of isolated Escherichia coli nucleoids assessed by fluorescence correlation spectroscopy. Biophys J 92(8):2875-84.

The morphology and dynamics of DNA in a bacterial nucleoid affects the kinetics of such major processes as DNA replication, gene expression. and chromosome segregation. In this work, we have applied fluorescence correlation spectroscopy to assess the structure and internal dynamics of isolated Escherichia coli nucleoids. We show that structural information can be extracted from the amplitude of fluorescence correlation spectroscopy correlation functions of randomly labeled nucleoids. Based on the developed formalism we estimate the characteristic size of nucleoid structural units for native, relaxed, and positively supercoiled nucleoids. The degree of supercoiling was varied using the intercalating agent chloroquine and evaluated from fluorescence microscopy images. The relaxation of superhelicity was accompanied by 15-fold decrease in the length of nucleoid units (from approximately 50 kbp to approximately 3 kbp).

Sabanayagam Chandran, R., M. Oram, R. Lakowicz Joseph and W. Black Lindsay. (2007) Viral DNA packaging studied by fluorescence correlation spectroscopy. Biophys J 93(4):L17-9.

The DNA packaging machinery of bacteriophage T4 was studied in vitro using fluorescence correlation spectroscopy. The ATP-dependent translocation kinetics of labeled DNA from the bulk solution, to the phage interior, was measured by monitoring the accompanied decrease in DNA diffusibility. It was found that multiple short DNA fragments (100 basepairs) can be sequentially packaged by an individual phage prohead. Fluorescence resonance energy transfer between green fluorescent protein donors within the phage interior and acceptor-labeled DNA was used to confirm DNA packaging. Without ATP, no packaging was observed, and there was no evidence of substrate association with the prohead.

Saffarian, S., Y. Li, L. Elson Elliot and J. Pike Linda. (2007) Oligomerization of the EGF receptor investigated by live cell fluorescence intensity distribution analysis. Biophys J 93(3):1021-31.

Recent evidence suggests that the EGF receptor oligomerizes or clusters in cells even in the absence of agonist ligand. To assess the status of EGF receptors in live cells, an EGF receptor fused to eGFP was stably expressed in CHO cells and studied using fluorescence correlation spectroscopy and fluorescent brightness analysis. By modifying FIDA for use in a two-dimensional system with quantal brightnesses, a method was developed to quantify the degree of clustering of the receptors on the cell surface. The analysis demonstrates that under physiological conditions, the EGF receptor exists in a complex equilibrium involving single molecules and clusters of two or more receptors. Acute depletion of cellular cholesterol enhanced EGF receptor clustering whereas cholesterol loading decreased receptor clustering, indicating that receptor aggregation is sensitive to the lipid composition of the membrane.

Sakudo, A., I. Nakamura, K. Ikuta and T. Onodera. (2007) Recent developments in prion disease research: diagnostic tools and in vitro cell culture models. The Journal of veterinary medical science / the Japanese Society of Veterinary Science 69(4):329-37.

After prion infection, an abnormal isoform of prion protein (PrP(Sc)) converts the cellular isoform of prion protein (PrP(C)) into PrP(Sc). PrP(C)-to-PrP(Sc) conversion leads to PrP(Sc) accumulation and PrP(C) deficiency, contributing etiologically to induction of prion diseases. Presently, most of the diagnostic methods for prion diseases are dependent on PrP(Sc) detection. Highly sensitive/accurate specific detection of PrP(Sc) in many different samples is a prerequisite for attempts to develop reliable detection methods. Towards this goal, several methods have recently been developed to facilitate sensitive and precise detection of PrP(Sc), namely, protein misfolding cyclic amplification, conformation-dependent immunoassay, dissociation-enhanced lanthanide fluorescent immunoassay, capillary gel electrophoresis, fluorescence correlation spectroscopy, flow microbead immunoassay, etc. Additionally, functionally relevant prion-susceptible cell culture models that recognize the complexity of the mechanisms of prion infection have also been pursued, not only in relation to diagnosis, but also in relation to prion biology. Prion protein (PrP) gene-deficient neuronal cell lines that can clearly elucidate PrP(C) functions would contribute to understanding of the prion infection mechanism. In this review, we describe the trend in recent development of diagnostic methods and cell culture models for prion diseases and their potential applications in prion biology.

Sanabria, H., Y. Kubota and M. N. Waxham. (2007) Multiple diffusion mechanisms due to nanostructuring in crowded environments. Biophys J 92(1):313-22.

One of the key questions regarding intracellular diffusion is how the environment affects molecular mobility. Mostly, intracellular diffusion has been described as hindered, and the physical reasons for this behavior are: immobile barriers, molecular crowding, and binding interactions with immobile or mobile molecules. Using results from multi-photon fluorescence correlation spectroscopy, we describe how immobile barriers and crowding agents affect translational mobility. To study the hindrance produced by immobile barriers, we used sol-gels (silica nanostructures) that consist of a continuous solid phase and aqueous phase in which fluorescently tagged molecules diffuse. In the case of molecular crowding, translational mobility was assessed in increasing concentrations of 500 kDa dextran solutions. Diffusion of fluorescent tracers in both sol-gels and dextran solutions shows clear evidence of anomalous subdiffusion. In addition, data from the autocorrelation function were analyzed using the maximum entropy method as adapted to fluorescence correlation spectroscopy data and compared with the standard model that incorporates anomalous diffusion. The maximum entropy method revealed evidence of different diffusion mechanisms that had not been revealed using the anomalous diffusion model. These mechanisms likely correspond to nanostructuring in crowded environments and to the relative dimensions of the crowding agent with respect to the tracer molecule. Analysis with the maximum entropy method also revealed information about the degree of heterogeneity in the environment as reported by the behavior of diffusive molecules.

Sanchez Susana, A., A. Tricerri Maria and E. Gratton. (2007) Interaction of high density lipoprotein particles with membranes containing cholesterol. Journal of lipid research 48(8):1689-700.

In this study, free cholesterol (FC) efflux mediated by human HDL was investigated using fluorescence methodologies. The accessibility of FC to HDL may depend on whether it is located in regions rich in unsaturated phospholipids or in domains containing high levels of FC and sphingomyelin, known as "lipid rafts." Laurdan generalized polarization and two-photon microscopy were used to quantify FC removal from different pools in the bilayer of giant unilamellar vesicles (GUVs). GUVs made of POPC and FC were observed after incubation with reconstituted particles containing apolipoprotein A-I and POPC [78A diameter reconstituted high density lipoprotein (rHDL)]. Fluorescence correlation spectroscopy data show an increase in rHDL size during the incubation period. GUVs made of two "raft-like" mixtures [DOPC/DPPC/FC (1:1:1) and POPC/SPM/FC (6:1:1)] were used to model liquid-ordered/liquid-disordered phase coexistence. Through these experiments, we conclude that rHDL preferentially removes cholesterol from the more fluid phases. These data, and their extrapolation to in vivo systems, show the significant role that phase separation plays in the regulation of cholesterol homeostasis.

Sasaki, K., T. Ose, N. Okamoto, K. Maenaka, T. Tanaka, H. Masai, M. Saito, T. Shirai and D. Kohda. (2007) Structural basis of the 3'-end recognition of a leading strand in stalled replication forks by PriA. The EMBO journal 26(10):2584-93.

In eubacteria, PriA helicase detects the stalled DNA replication forks. This critical role of PriA is ascribed to its ability to bind to the 3' end of a nascent leading DNA strand in the stalled replication forks. The crystal structures in complexes with oligonucleotides and the combination of fluorescence correlation spectroscopy and mutagenesis reveal that the N-terminal domain of PriA possesses a binding pocket for the 3'-terminal nucleotide residue of DNA. The interaction with the deoxyribose 3'-OH is essential for the 3'-terminal recognition. In contrast, the direct interaction with 3'-end nucleobase is unexpected, considering the same affinity for oligonucleotides carrying the four bases at the 3' end. Thus, the N-terminal domain of PriA recognizes the 3'-end base in a base-non-selective manner, in addition to the deoxyribose and 5'-side phosphodiester group, of the 3'-terminal nucleotide to acquire both sufficient affinity and non-selectivity to find all of the stalled replication forks generated during DNA duplication. This unique feature is prerequisite for the proper positioning of the helicase domain of PriA on the unreplicated double-stranded DNA.

Schmitt, M., J. Wagner, G. Jung and R. Hempelmann. (2007) Functionalized polymer colloids bearing primary amino groups. J Colloid Interface Sci 311(2):425-9.

Polymer colloids are prepared via radicalic emulsion polymerisation of butylacrylate. Functionalization with amino groups is achieved by copolymerisation of 2-amino-ethylmethacrylates. In order to over-compensate the positive surface charges resulting from the amino groups additionally vinylbenzenesulfonic acid is copolymerized. The size of the resulting particles is controlled by the molar ratio of amino to sulfonic acid groups. The suitability of amino groups for coupling reactions is demonstrated by electrophilic addition of fluorescein-5-isothiocyanate. The resulting particles are characterized by dynamic light scattering and zeta potential measurements as well as by optical spectroscopy. The suitability of labelled particles for optical tracer experiments is demonstrated by fluorescence correlation spectroscopy.

Shimizu, M., S. Sasaki and M. Kinjo. (2007) Triplet fraction buildup effect of the DNA-YOYO complex studied with fluorescence correlation spectroscopy. Anal Biochem 366(1):87-92.

DNA fragments of various lengths and YOYO-1 iodide (YOYO) were mixed at various ratios, and fluorescence was measured using fluorescence correlation spectroscopy. The number of substantially emitting YOYO molecules binding to the DNA and the binding intervals between the YOYO molecules were estimated for DNA-YOYO complexes of various lengths. In the present study, we found an interesting phenomenon: triplet buildup. Because fluorophores that fall into the triplet state do not emit fluorescence, a part of the dark period can be recovered by emitting photons from other excited YOYO molecules in the same DNA strings in the confocal elements. The remaining dark period can be considered to be the total miss-emission rate. Estimates of the total miss-emission rate are important for calculation of the length and amount of DNA.

Stevens Andrew, P., V. Hlady and O. Dull Randal. (2007) Fluorescence correlation spectroscopy can probe albumin dynamics inside lung endothelial glycocalyx. American journal of physiology. Lung cellular and molecular physiology 293(2):L328-35.

The endothelial glycocalyx is believed to play a major role in capillary permeability by functioning as a macromolecular barrier overlying the intercellular junction. Little is known about the functional attributes of the glycocalyx (i.e., porosity and permeability) or which constituents contribute to its overall structure-function relationship. In this report, we demonstrate the utility of fluorescence correlation spectroscopy (FCS) to measure albumin diffusion rates and concentration profiles above the cell surface and overlying the intercellular junctions of lung capillary endothelial cells. Albumin diffusion rates and concentration profiles were obtained before and after enzymatic digestion of the glycocalyx with pronase, heparanase, or hyaluronidase. The results suggest a structure interacting with albumin located from 1.0 to 2.0 mum above the cell membrane capable of reducing albumin diffusion by 30% while simultaneously increasing albumin concentration fivefold. Digestion of the glycocalyx with pronase or heparanase resulted in only modest changes in albumin diffusion and concentration profiles. Hyaluronidase digestion completely eliminated albumin-glycocalyx interactions. These data also suggest that hyaluronan is a major determinant for albumin interactions with the lung endothelial glycocalyx. Confocal images of heparan sulfate and hyaluronan confirm a cell-surface layer 2-3 mum in thickness, thus supporting FCS measurements. In summary, we report the first use of FCS to probe extracellular structures and further our understanding of the structure-function relationship of the lung microvascular endothelial glycocalyx.

Sutter, M., S. Oliveira, N. Sanders Niek, B. Lucas, A. van Hoek, A. Hink Mark, J. W. G. Visser Antonie, C. De Smedt Stefaan, E. Hennink Wim and W. Jiskoot. (2007) Sensitive spectroscopic detection of large and denatured protein aggregates in solution by use of the fluorescent dye Nile red. J Fluoresc 17(2):181-92.

The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein beta-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of beta-galactosidase below and above the protein's unfolding temperature of 57.4 degrees C, and the presence of aggregates in heated solutions was confirmed by static light scattering. Interaction of Nile red with beta-galactosidase aggregates led to a shift of the emission maximum (lambda (max)) from 660 to 611 nm, and to an increase of fluorescence intensity. Time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) measurements showed that Nile red detected large aggregates with hydrodynamic radii around 130 nm. By steady-state fluorescence measurements, it was possible to detect 1 nM of denatured and aggregated beta-galactosidase in solution. The comparison with size exclusion chromatography (SEC) showed that native beta-galactosidase and small aggregates thereof had no substantial effect on the fluorescence of Nile red. Large aggregates were not detected by SEC, because they were excluded from the column. The results with beta-galactosidase demonstrate the potential of Nile red for developing complementary analytical methods that overcome the size limitations of SEC, and can detect the formation of large protein aggregates at early stages.

Szymanski, J., E. Pobozy, M. Trojanowicz, A. Wilk, P. Garstecki and R. Holyst. (2007) Net charge and electrophoretic mobility of lysozyme charge ladders in solutions of nonionic surfactant. The journal of physical chemistry. B 111(19):5503-10.

We report on the electrophoretic mobility and on the thermal diffusion of lysozyme proteins dissolved in aqueous solutions of a nonionic surfactant (C12E6) at a wide range of concentrations of the surfactant (0-20% by weight). We want to estimate the influence of a dense network of elongated micelles of C12E6 on the effective charge of the proteins as observed in the capillary electrophoresis experiments. The possible mechanism leading to the change in the effective charge of protein could involve the deformation of the cloud of counterions around the protein when it squeezes through the narrow (of the order of a protein diameter) aqueous channels formed in the solution of elongated micelles. The combination of independent measurements of the electrophoretic mobility of a family of modified proteins (lysozyme charge ladder [Colton et al. J. Am. Chem. Soc. 1997, 119, 12701]), of the microviscosity of the solutions of surfactant (obtained via fluorescence correlation spectroscopy), and of the hydrodynamic radius of the proteins (photon correlation spectroscopy) allow us to conclude that the effective charge of the proteins is not affected by the presence of surfactant, even at high concentrations.

Thompson Nancy, L. and L. Steele Bridgett. (2007) Total internal reflection with fluorescence correlation spectroscopy. Nature protocols 2(4):878-90.

Total internal reflection-fluorescence correlation spectroscopy (TIR-FCS) is an emerging technique that is used to measure events at or near an interface, including local fluorophore concentrations, local translational mobilities and the kinetic rate constants that describe the association and dissociation of fluorophores at the interface. TIR-FCS is also an extremely promising method for studying dynamics at or near the basal membranes of living cells. This protocol gives a general overview of the steps necessary to construct and test a TIR-FCS system using either through-prism or through-objective internal reflection geometry adapted for FCS. The expected forms of the autocorrelation function are discussed for the cases in which fluorescent molecules in solution diffuse through the depth of the evanescent field, but do not bind to the surface of interest, and in which reversible binding to the surface also occurs.

Torres, T. and M. Levitus. (2007) Measuring conformational dynamics: a new FCS-FRET approach. The journal of physical chemistry. B 111(25):7392-400.

Fluorescence correlation spectroscopy (FCS) has recently emerged as a powerful technique to study conformational dynamics of biomolecules, but often its applicability is limited by the difficulty of separating the contributions of kinetics from those due to diffusion. We present a new approach based on the simultaneous analysis of the auto- and cross-correlation functions of the intensities measured in two independent detectors for a donor-acceptor labeled biomolecule. Fluctuations in fluorescence intensity are a consequence of diffusion and the variations in fluorescence resonance energy transfer (FRET) efficiency due to changes in donor-acceptor distance. Although the complete description of the correlation functions requires that diffusion is well-characterized, the ratio of any two correlation functions depends on kinetic parameters only. This provides a means by which kinetic information can be obtained independently of the diffusion contributions. As proof of principle, we reanalyze data obtained in previous work with nucleosomes. In contrast to our previous work, where a donor-only sample was used to characterize diffusion, we now show that the same kinetic information can be obtained from a single experiment with a double-labeled biomolecule. This eliminates not only the need of a reference, but also artifacts associated with changes in the observation volume between measurements.

Tudor, C., N. Feige Jerome, H. Pingali, B. Lohray Vidya, W. Wahli, B. Desvergne, Y. Engelborghs and L. Gelman. (2007) Association with coregulators is the major determinant governing peroxisome proliferator-activated receptor mobility in living cells. The Journal of biological chemistry 282(7):4417-26.

The nucleus is an extremely dynamic compartment, and protein mobility represents a key factor in transcriptional regulation. We showed in a previous study that the diffusion of peroxisome proliferator-activated receptors (PPARs), a family of nuclear receptors regulating major cellular and metabolic functions, is modulated by ligand binding. In this study, we combine fluorescence correlation spectroscopy, dual color fluorescence cross-correlation microscopy, and fluorescence resonance energy transfer to dissect the molecular mechanisms controlling PPAR mobility and transcriptional activity in living cells. First, we bring new evidence that in vivo a high percentage of PPARs and retinoid X receptors is associated even in the absence of ligand. Second, we demonstrate that coregulator recruitment (and not DNA binding) plays a crucial role in receptor mobility, suggesting that transcriptional complexes are formed prior to promoter binding. In addition, association with coactivators in the absence of a ligand in living cells, both through the N-terminal AB domain and the AF-2 function of the ligand binding domain, provides a molecular basis to explain PPAR constitutive activity.

Turner Nicholas, W., E. Wright Bryon, V. Hlady and W. Britt David. (2007) Formation of protein molecular imprints within Langmuir monolayers: a quartz crystal microbalance study. J Colloid Interface Sci 308(1):71-80.

Protein imprinting leading to enhanced rebinding of ferritin to ternary lipid monolayers is demonstrated using a quartz crystal microbalance. Monolayers consisting of cationic dioctadecyldimethylammonium bromide, non-ionic methyl stearate, and poly(ethylene glycol) bearing phospholipids were imprinted with ferritin at the air/water interface of a Langmuir-Blodgett trough and transferred hydrated to hydrophobic substrates for study. This immobilization was shown by fluorescence correlation spectroscopy to significantly hinder any further diffusion of lipids, while rebinding studies demonstrated up to a six-fold increase in ferritin adsorption to imprinted versus control monolayers. A diminished rebinding of ferritin to its imprint was observed through pH reduction to below the protein isoelectric point, demonstrating the electrostatic nature of the interaction. Rebinding to films where imprint pockets remained occupied by the template protein was also minimal. Studies with a smaller acidic protein revealed the importance of the steric influence of poly(ethylene glycol) in forming the protein binding pockets, as albumin-imprinted monolayers showed low binding of ferritin, while ferritin-imprinted monolayers readily accommodated albumin. The controllable structure-function relationship and limitations of this system are discussed with respect to the application of protein imprinting in sensor development as well as fundamental studies of proteins at dynamic interfaces.

Varriale, A., M. Rossi, M. Staiano, E. Terpetschnig, B. Barbieri, M. Rossi and S. D'Auria. (2007) Fluorescence correlation spectroscopy assay for gliadin in food. Anal Chem 79(12):4687-9.

Gliadin proteins are primarily responsible for celiac disease. As gliadin is a complex mixture of proteins difficult to solubilize and to extract from food, it is difficult to develop an assay capable of accurate quantization of gliadin in food for celiac patients. In this work, we present an advanced fluorescence assay for the detection of traces of gliadin in food. The described assay is based on measurement of the fluctuations of fluorescein-labeled gliadin peptides (GP) in a focused laser beam in the absence and in the presence of anti-GP antibodies. A competitive assay based on the utilization of unlabeled GP was developed. The obtained results indicate that the combination of high-avidity IgG antibodies together with the innovative fluorescence immunoassay strategy resulted in a gluten detection limit of 0.006 ppm, which it is much lower than the values reported in the literature.

Verma, S., Y. Xiong, M. U. Mayer and C. Squier Thomas. (2007) Remodeling of the bacterial RNA polymerase supramolecular complex in response to environmental conditions. Biochemistry 46(11):3023-35.

Directed binding of RNA polymerase to distinct promoter elements controls transcription and promotes adaptive responses to changing environmental conditions. To identify proteins that modulate transcription, we have expressed a tagged alpha-subunit of RNA polymerase in Shewanella oneidensis under controlled growth conditions, isolated the protein complex using newly developed multiuse affinity probes, and used LC-MS/MS to identify proteins in the complex. Complementary fluorescence correlation spectroscopy measurements were used to determine the average size of the RNA polymerase complex in cellular lysates. We find that RNA polymerase exists as a large supramolecular complex with an apparent mass in excess of 1.4 MDa, whose protein composition substantially changes in response to growth conditions. Enzymes that copurify with RNA polymerase include those associated with tRNA processing, nucleotide metabolism, and energy biosynthesis, which we propose to be necessary for optimal transcriptional rates.

Visser Nina, V., D. Wang, A. Stanley Will, R. Groves Matthew, M. Wilmanns, M. Veenhuis and J. van der Klei Ida. (2007) Octameric alcohol oxidase dissociates into stable, soluble monomers upon incubation with dimethylsulfoxide. Archives of biochemistry and biophysics 459(2):208-13.

Alcohol oxidase (AO) is a peroxisomal, homo-octameric flavoenzyme, which catalyzes methanol oxidation in methylotrophic yeast. Here, we report on the generation of soluble, FAD-lacking AO monomers. Using steady-state fluorescence, fluorescence correlation spectroscopy, circular dichroism and static light scattering approaches, we demonstrate that FAD-lacking AO monomers are formed upon incubation of purified, native octameric AO in a solution containing 50% dimethylsulfoxide (DMSO). Upon removal of DMSO the protein remained monomeric and soluble and did not contain FAD. Binding experiments revealed that the AO monomers bind to purified pyruvate carboxylase, a protein that plays a role in the formation of enzymatically active AO octamers in vivo.

von der Hocht, I. and J. Enderlein. (2007) Fluorescence correlation spectroscopy in cells: confinement and excluded volume effects. Exp Mol Pathol 82(2):142-6.

Fluorescence correlation spectroscopy (FCS) has become an important technique in biophysical research, which is also used for in vivo studies of molecular mobilities in cells. We theoretically study how confinement or exclusion of the diffusing fluorescent molecules by a spherical region influences the measured autocorrelation function in an FCS experiment. It is shown that close to the boundary of the spherical region the diffusion time can be significantly changed due to the geometric restriction of the detection volume. This is important when quantitatively evaluating and interpreting FCS measurements in cells.

Wang, S. and J. Zhao. (2007) First-order conformation transition of single poly(2-vinylpyridine) molecules in aqueous solutions. The Journal of chemical physics 126(9):091104.

By measuring diffusion rate, the conformation change of single poly(2-vinylpyridine) chain in aqueous solution was studied by fluorescence correlation spectroscopy. The data showed a stepwise change of hydrodynamic radius when pH value was tuned, reflecting a sign of first-order conformation transition, and a continuous change was found at varying salt concentration.

Wenger, J., F. Conchonaud, J. Dintinger, L. Wawrezinieck, W. Ebbesen Thomas, H. Rigneault, D. Marguet and P.-F. Lenne. (2007) Diffusion analysis within single nanometric apertures reveals the ultrafine cell membrane organization. Biophys J 92(3):913-9.

We describe the development of a new methodology to probe the plasma membrane organization of living cells at the nanometric scale. Single nanometric apertures in a metallic film limit the observed membrane area below the optical diffraction barrier. The new approach performs fluorescence correlation spectroscopy with increasing aperture sizes and extracts information on the diffusion process from the whole set of data. In particular, transient diffusion regimes are clearly observed when the probed area comes close to the size of the confining structures. First, this strategy allows identification of the mechanism controlling the diffusion of various fluorescent lipid analogs and green fluorescent protein-tagged proteins. Second, it gives an estimate of the characteristic size of the nanometric membrane heterogeneities, allowing a quantitative study of membrane domains such as lipid rafts. Compared to other optical techniques, this method combines the advantages of high spatio-temporal resolution and direct statistical analysis.

Widengren, J., A. Chmyrov, C. Eggeling, P.-A. Lofdahl and A. M. Seidel Claus. (2007) Strategies to improve photostabilities in ultrasensitive fluorescence spectroscopy. The journal of physical chemistry. A 111(3):429-40.

Given the particular importance of dye photostability for single-molecule and fluorescence fluctuation spectroscopy investigations, refined strategies were explored for how to chemically retard dye photobleaching. These strategies will be useful for fluorescence correlation spectroscopy (FCS), fluorescence-based confocal single-molecule detection (SMD) and related techniques. In particular, the effects on the addition of two main categories of antifading compounds, antioxidants (n-propyl gallate, nPG, ascorbic acid, AA) and triplet state quenchers (mercaptoethylamine, MEA, cyclo-octatetraene, COT), were investigated, and the relevant rate parameters involved were determined for the dye Rhodamine 6G. Addition of each of the compound categories resulted in significant improvements in the fluorescence brightness of the monitored fluorescent molecules in FCS measurements. For antioxidants, we identify the balance between reduction of photoionized fluorophores on the one hand and that of intact fluorophores on the other as an important guideline for what concentrations to be added for optimal fluorescence generation in FCS and SMD experiments. For nPG/AA, this optimal concentration was found to be in the lower micromolar range, which is considerably less than what has previously been suggested. Also, for MEA, which is a compound known as a triplet state quencher, it is eventually its antioxidative properties and the balance between reduction of fluorophore cation radicals and that of intact fluorophores that defines the optimal added concentration. Interestingly, in this optimal concentration range the triplet state quenching is still far from sufficient to fully minimize the triplet populations. We identify photoionization as the main mechanism of photobleaching within typical transit times of fluorescent molecules through the detection volume in a confocal FCS or SMD instrument (<1-20 ms), and demonstrate its generation via both one- and multistep excitation processes. Apart from reflecting a major pathway for photobleaching, our results also suggest the exploitation of the photoinduced ionization and the subsequent reduction by antioxidants for biomolecular monitoring purposes and as a possible switching mechanism with applications in high-resolution microscopy.

Winkler Roland, G. (2007) Diffusion and segmental dynamics of rodlike molecules by fluorescence correlation spectroscopy. The Journal of chemical physics 127(5):054904.

The dynamics of weakly bending polymers is analyzed on the basis of a Gaussian semiflexible chain model and the fluorescence correlation spectroscopy (FCS) correlation function is determined. Particular attention is paid to the influence of the rotational motion on the decay of the FCS correlation function. An analytical expression for the correlation function is derived, from which the averaged segmental mean square displacement can be determined independent of any specific model for the polymer dynamcis. The theoretical analysis exhibits a strong dependence of the correlation function on the rotational motion for semiflexible polymers with typical lengths and persistence lengths of actin filaments or fd viruses. Hence, FCS allows for a measurement of the rotational motion of such semiflexible polymers. The theoretical results agree well with experimental measurements on actin filaments and confirm the importance of large relaxation times.

Wong Felix, H. C., S. Ng-Kamstra Joshua, L. H. Chen Nelson and C. Fradin. (2007) Localized photodamage of the human erythrocyte membrane causes an invagination as a precursor of photohaemolysis. Journal of microscopy 226(Pt 1):6-17.

Fluorescence excitation can result in the formation of reactive oxygen species and free radicals damaging to live cells. In the case of erythrocytes, reaction of these reactive oxygen species with membrane components causes large-scale morphological changes followed by cell haemolysis. In an effort to understand the origin of these morphological changes, we have studied the consequences of localized photodamage on the erythrocyte membrane. For this, we irradiated a small area of the cell membrane using a focused laser beam in the presence of an external photosensitizer. We observed the rapid formation of an invagination (approximately 1 microm deep) at the laser focus, long before photohaemolysis occurred. We measured the rate of invagination formation and the rate of cell haemolysis, using a combination of fluorescence contrast imaging (to detect the membrane position) with fluorescence correlation spectroscopy (to measure photosensitizer concentration). We found that the kinetics of both processes depend in a similar manner on light energy flux, fluorophore concentration and the presence of oxygen scavenger. This leads us to the conclusion that the observed invagination is due to the photooxidation of membrane-associated proteins, representing a precursor of cellular photohaemolysis. We then discuss two different molecular mechanisms (conformational change of the protein band 3 and detachment of the spectrin cytoskeleton from the lipid membrane) that may explain how the photodamage of membrane-associated proteins can lead to a deformation of the lipid bilayer.

Wong John, E., B. Muller Claus, A. Laschewsky and W. Richtering. (2007) Direct Evidence of Layer-by-Layer Assembly of Polyelectrolyte Multilayers on Soft and Porous Temperature-Sensitive PNiPAM Microgel Using Fluorescence Correlation Spectroscopy. The journal of physical chemistry. B 111(29):8527-31.

We describe the layer-by-layer assembly of polyelectrolyte multilayers on soft and porous temperature-sensitive poly(N-isopropylacrylamide) (PNiPAM) microgel. Microgels are not hard and rigid but rather are soft and porous particles, and polyelectrolytes not only interdigitate with each other during multilayer formation but also with the microgel. Because of this difference, there could be concerns about the feasibility of the layer-by-layer technique on these systems. The argument is that the layer being deposited is stripping the underlying layer instead of anchoring to the latter, and common methods of characterizing film growth on particles such as zeta-potentials will still show "successful" charge reversal. To address this issue, we used two differently labeled polyelectrolytes during the deposition. Because of the small size of the microgel (400 nm) studied, we cannot distinguish between polyelectrolytes adsorbed on or in the microgel. However, with fluorescence correlation spectroscopy, we can clearly distinguish between free labeled polyelectrolytes and those that are bound to the microgel. Dual-color correlation confirms the presence of both polyelectrolytes bound to the same particle while fluorescence imaging (on a dry sample) provides the visual proof.

Wruss, J., D. Runzler, C. Steiger, P. Chiba, G. Kohler and D. Blaas. (2007) Attachment of VLDL receptors to an icosahedral virus along the 5-fold symmetry axis: multiple binding modes evidenced by fluorescence correlation spectroscopy. Biochemistry 46(21):6331-9.

Human rhinoviruses (HRVs) are composed of 60 identical subunits, each comprising one copy of the viral capsid proteins VP1, 2, 3, and 4. Consequently, 60 symmetry-related epitopes are available for binding of antibodies or receptors. The minor receptor group of HRVs uses members of the low-density lipoprotein receptor family for cell entry. The ligand binding domains of these receptors are composed of various numbers of ligand binding repeats, and several of these modules within a single molecule are believed to attach simultaneously to the star-shaped dome at the 5-fold symmetry axis of the virus. Using fluorescence correlation spectroscopy (FCS), we have now determined the equilibrium binding constants and the mode of attachment of recombinant concatemers of ligand binding module 3 of the human very-low-density lipoprotein receptor to HRV2. We demonstrate that the avidity of the interaction drastically increases with the number of concatenated modules. For the trimer, the binding isotherm was biphasic, indicating that attachment of two and of three modules within the same molecule was resolved. The receptor consisting of seven repeats was found to bind most strongly, but a complete binding isotherm could not be established due to cross-linking of virions. The values of the dissociation constants were about 1 order of magnitude higher than those previously determined by using surface plasmon resonance techniques reflecting the different presentation of the binding partners. As compared to the concatemers, the natural receptors are composed of similar but not identical repeats; thus, cooperativity and different specificity of the ligand-binding modules allow for recognition of many ligands and viral serotypes. Due to the low concentrations and amounts of sample required, FCS is ideally suited for the determination of receptor binding parameters of viruses difficult to produce in high quantities and/or concentrations.

Wu, J. and K. Berland. (2007) Fluorescence intensity is a poor predictor of saturation effects in two-photon microscopy: Artifacts in fluorescence correlation spectroscopy. Microsc Res Tech 70(8):682-6.

Fluorescence correlation spectroscopy (FCS) has become an increasingly important measurement tool for biological and biomedical investigations, with the capability to assay molecular dynamics and interactions both in vitro and within living cells. Information recovery in FCS requires an accurate characterization and calibration of the observation volume. A number of recent reports have demonstrated that the calibration of the observation volume is excitation power dependent, a complication that arises due to excitation saturation. While quantitative models are now available to account for these volume variations, many researchers attempt to avoid saturation issues by working with low nonsaturating excitation intensities. For two-photon excited fluorescence, this is typically thought to be achievable by working with excitation powers for which the total measured fluorescence signal maintains its quadratic dependence on excitation intensity. We demonstrate that observing only the power dependence of the fluorescence intensity will tend to underestimate the importance of saturation, and explain these findings in terms of basic physical models. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc.

Yaghootfam, A., T. Sorkalla, H. Haberlein, V. Gieselmann, J. Kappler and M. Eckhardt. (2007) Cerebroside sulfotransferase forms homodimers in living cells. Biochemistry 46(32):9260-9.

Cerebroside sulfotransferase (CST) catalyzes the 3'-sulfation of galactose residues in several glycolipids. Its major product in the mammalian brain is sulfatide, which is an essential myelin component. Using epitope-tagged variants, murine CST was found to localize to the Golgi apparatus, but in contrast to previous assumptions, not to the trans-Golgi network. An examination of enhanced green fluorescent protein (EGFP)-tagged CST suggests that CST forms homodimers and that dimerization is mediated by the lumenal domain of the enzyme, as shown by immunoprecipitation and density gradient centrifugation. In order to verify that dimerization of CST observed by biochemical methods reflects the behavior of the native protein within living cells, the mobility of CST-EGFP was examined using fluorescence correlation spectroscopy. These experiments confirmed the homodimerization of CST-EGFP fusion proteins in vivo. In contrast to full-length CST, a fusion protein of the amino-terminal 36 amino acids of CST fused to EGFP was exclusively found as a monomer but nevertheless showed Golgi localization.

Yeung, C., M. Shtrahman and X.-l. Wu. (2007) Stick-and-diffuse and caged diffusion: a comparison of two models of synaptic vesicle dynamics. Biophys J 92(7):2271-80.

Two models were recently proposed to enable us to understand the dynamics of synaptic vesicles in hippocampal neurons. In the caged diffusion model, the vesicles diffuse in small circular cages located randomly in the bouton, while in the stick-and-diffuse model the vesicles bind and release from a cellular cytomatrix. In this article, we obtain analytic expressions for the fluorescence correlation spectroscopy (FCS) autocorrelation function for the two models and test their predictions against our earlier FCS measurements of the vesicle dynamics. We find that the stick-and-diffuse model agrees much better with the experiment. We find also that, due to the slow dynamics of the vesicles, the finite experimental integration time has an important effect on the FCS autocorrelation function and demonstrate its effect for the different models. The two models of the dynamics are also relevant to other cellular environments where mobile species undergo slow diffusionlike motion in restricted spaces or bind and release from a stationary substrate.

Yokozeki, T., S. Wakatsuki, K. Hatsuzawa, A. Black Roy, I. Wada and A. Sehara-Fujisawa. (2007) Meltrin beta (ADAM19) mediates ectodomain shedding of Neuregulin beta1 in the Golgi apparatus: fluorescence correlation spectroscopic observation of the dynamics of ectodomain shedding in living cells. Genes to cells : devoted to molecular & cellular mechanisms 12(3):329-43.

Membrane-anchored Neuregulin beta1 sheds its ectodomain as soluble factors. Two proteases that belong to a disintegrin and metalloprotease (ADAM) family are known to cleave Neuregulin beta1. One is tumor necrosis factor-alpha converting enzyme (TACE/ADAM17). The other is Meltrin beta (ADAM19). Against our expectation that shedding by ADAM proteases occurs at the cell surface, here we found that Meltrin beta mediates the ectodomain shedding of Neuregulin beta1 in the Golgi apparatus. Meltrin beta was localized in and around the Golgi apparatus in developing sensory neurons. Subcellular fractionation revealed that Meltrin beta generated soluble Neuregulin beta1 in Golgi-enriched fractions while TACE-cleaved Neuregulin beta1 was recovered in lighter fractions. To examine whether Meltrin beta-mediated ectodomain shedding occurs in the Golgi apparatus in living cells, we took advantage of different diffusion properties of cleavage products from those of membrane-anchored precursor proteins. Fluorescence correlation spectroscopy (FCS) is the most sensitive method to determine milli approximately submillisecond diffusion in vivo. Protease-active Meltrin beta caused a shift in autocorrelation function in FCS of green fluorescent protein (GFP)-tagged Neuregulin beta1 in the Golgi apparatus, suggesting a conversion of Neuregulin beta1 molecules from membrane-anchored to soluble forms in that organelle. The Golgi apparatus is a site of processing Neuregulin beta1 by Meltrin beta.

Zorrilla, S., A. Hink Mark, J. W. G. Visser Antonie and M. P. Lillo. (2007) Translational and rotational motions of proteins in a protein crowded environment. Biophysical chemistry 125(2-3):298-305.

Fluorescence correlation spectroscopy (FCS) was used to measure the translational diffusion of labeled apomyoglobin (tracer) in concentrated solutions of ribonuclease A and human serum albumin (crowders), as a quantitative model system of protein diffusive motions in crowded physiological environments. The ratio of the diffusion coefficient of the tracer protein in the protein crowded solutions and its diffusion coefficient in aqueous solution has been interpreted in terms of local apparent viscosities, a molecular parameter characteristic for each tracer-crowder system. In all protein solutions studied in this work, local translational viscosity values were larger than the solution bulk viscosity, and larger than rotational viscosities estimated for apomyoglobin in the same crowding solutions. Here we propose a method to estimate local apparent viscosities for the tracer translational and rotational diffusion directly from the bulk viscosity of the concentrated protein solutions. As a result of this study, the identification of protein species and the study of hydrodynamic changes and interactions in model crowded protein solutions by means of FCS and time-resolved fluorescence depolarization techniques may be expected to be greatly simplified.