Fluorescence correlation spectroscopy, a tool to investigate supramolecular dynamics: inclusion complexes of pyronines with cy

2005 FCs papers

2004 FCS papers

Fluorescence correlation spectroscopy, a tool to investigate supramolecular dynamics: inclusion complexes of pyronines with cyclodextrin. Al-Soufi, W., Reija, B., Novo, M., Felekyan, S., Kuhnemuth, R., and Seidel, C.A., J Am Chem Soc, 2005. 127(24): pp. 8775-84

The control of supramolecular systems requires a thorough understanding of their dynamics on a molecular level. We present fluorescence correlation spectroscopy (FCS) as a powerful spectroscopic tool to study supramolecular dynamics with single molecule sensitivity. The formation of a supramolecular complex between beta-cyclodextrin (beta-CD) as host and pyronines Y (PY) and B (PB) as guests is studied by FCS. Global target analysis of full correlation curves with a newly derived theoretical model yields in a single experiment the fluorescence lifetimes and the diffusion coefficients of free and complexed guests and the rate constants describing the complexation dynamics. These data give insight into the recently published surprising fact that the association equilibrium constant of beta-CD with PY is much lower than that with the much bulkier guest PB. FCS shows that the stability of the complexes is dictated by the dissociation and not by the association process. The association rate constants are very similar for both guests and among the highest reported for this type of systems, although much lower than the diffusion-controlled collision rate constant. A two-step model including the formation of an encounter complex allows one to identify the unimolecular inclusion reaction as the rate-limiting step. Simulations indicate that this step may be controlled by geometrical and orientational requirements. These depend on critical molecular dimensions which are only weakly affected by the different alkyl substituents of PY and PB. Diffusion coefficients of PY and PB, of their complexes, and of rhodamine 110 are given and compared to those of similar molecules.

Evidence for major structural changes in subunit C of the vacuolar ATPase due to nucleotide binding. Armbruster, A., Hohn, C., Hermesdorf, A., Schumacher, K., Borsch, M., and Gruber, G., FEBS Lett, 2005. 579(9): pp. 1961-7

The ability of subunit C of eukaryotic V-ATPases to bind ADP and ATP is demonstrated by photoaffinity labeling and fluorescence correlation spectroscopy (FCS). Quantitation of the photoaffinity and the FCS data indicate that the ATP-analogues bind more weakly to subunit C than the ADP-analogues. Site-directed mutagenesis and N-terminal sequencing of subunit C from Arabidopsis (VHA-C) and yeast (Vma5p) have been used to map the C-terminal region of subunit C as the nucleotide-binding site. Tryptophan fluorescence quenching and decreased susceptibility to tryptic digestion of subunit C after binding of different nucleotides provides evidence for structural changes in this subunit caused by nucleotide-binding.

Quantitative measurement of the resolution and sensitivity of confocal microscopes using line-scanning fluorescence correlation spectroscopy. Balaji, J. and Maiti, S., Microsc Res Tech, 2005. 66(4): pp. 198-202

Spatial resolution and the sensitivity to detect a fluorophore are the two most important optical parameters that characterize a confocal microscope. However, these are rather difficult to estimate quantitatively. We show that fluorescence correlation spectroscopy (FCS) provides an easy and reliable measure of these quantities. We modify existing schemes for performing FCS on a commercial confocal microscope to carry out these measurements, and provide an analysis routine that can yield the relevant quantities. Our method does not require any modification of the confocal microscope, yet it yields a robust measure of the resolution and sensitivity of the instrument.

Anomalous diffusion of proteins due to molecular crowding. Banks, D.S. and Fradin, C., Biophys J, 2005. 89(5): pp. 2960-71

We have studied the diffusion of tracer proteins in highly concentrated random-coil polymer and globular protein solutions imitating the crowded conditions encountered in cellular environments. Using fluorescence correlation spectroscopy, we measured the anomalous diffusion exponent alpha characterizing the dependence of the mean-square displacement of the tracer proteins on time, r(2)(t) approximately t(alpha). We observed that the diffusion of proteins in dextran solutions with concentrations up to 400 g/l is subdiffusive (alpha < 1) even at low obstacle concentration. The anomalous diffusion exponent alpha decreases continuously with increasing obstacle concentration and molecular weight, but does not depend on buffer ionic strength, and neither does it depend strongly on solution temperature. At very high random-coil polymer concentrations, alpha reaches a limit value of alpha(l) approximately 3/4, which we take to be the signature of a coupling between the motions of the tracer proteins and the segments of the dextran chains. A similar, although less pronounced, subdiffusive behavior is observed for the diffusion of streptavidin in concentrated globular protein solutions. These observations indicate that protein diffusion in the cell cytoplasm and nucleus should be anomalous as well, with consequences for measurements of solute diffusion coefficients in cells and for the modeling of cellular processes relying on diffusion.

Fluorescence correlation spectroscopy for flow rate imaging and monitoring--optimization, limitations and artifacts. Brister, P.C., Kuricheti, K.K., Buschmann, V., and Weston, K.D., Lab Chip, 2005. 5(7): pp. 785-91

We recently demonstrated a new method for mapping fluid velocities in 3 dimensions and with exceptionally high spatial resolution for the characterization of flow in microfluidic devices. In the method, a colloidal suspension containing fluorescent tracer particles, dye doped polymer spheres, is pumped through a microchannel and confocal microscopy combined with fluorescence correlation spectroscopy is used to measure fluid velocities. In this report, we further characterize the technique and report on optimizations that allow a 5-fold increase in speed of single point velocity measurements. This increase in measurement speed will yield a 25 fold reduction in the time needed to collect a complete velocity image. The precision of measured velocities was characterized as a function of tracer particle concentration, measurement time, and fluid velocity. In addition, we confirm the linearity of the measurement method (velocity vs. applied pressure) over a range of velocities spanning four orders of magnitude. Furthermore, we demonstrate that an artifact in velocity measurements using fluorescence correlation spectroscopy (FCS) that was interpreted by others as being caused by optical trapping forces is actually an artifact caused by detector saturation and can be avoided by careful choice of experimental conditions.

Four-color fluorescence correlation spectroscopy realized in a grating-based detection platform. Burkhardt, M., Heinze, K.G., and Schwille, P., Opt Lett, 2005. 30(17): pp. 2266-8

We have developed a filterless multicolor detection unit for fluorescence correlation spectroscopy (FCS). This grating-based setup is continuously tunable for multicolor separation and is thus a powerful alternative to the classical cascade of dichroic mirrors and filters. Our tailored platform allows for accommodation of up to 15 detection channels covering the entire visible spectral range. As a proof of principle, we successfully demonstrate simultaneous FCS of four distinct fluorescent quantum dot species being mixed in aqueous solution. Grating-based detection allows for spectral high-resolution FCS in a stable and compact setup and is a feasible tool for quantitative investigation of complexbiomolecular dynamics on a single molecule level.

Local mobility in lipid domains of supported bilayers characterized by atomic force microscopy and fluorescence correlation spectroscopy. Burns, A.R., Frankel, D.J., and Buranda, T., Biophys J, 2005. 89(2): pp. 1081-93

Fluorescence correlation spectroscopy (FCS) is used to examine mobility of labeled probes at specific sites in supported bilayers consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid domains in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Those sites are mapped beforehand with simultaneous atomic force microscopy and submicron confocal fluorescence imaging, allowing characterization of probe partitioning between gel DPPC and disordered liquid DOPC domains with corresponding topography of domain structure. We thus examine the relative partitioning and mobility in gel and disordered liquid phases for headgroup- and tailgroup-labeled GM1 ganglioside probes and for headgroup- and tailgroup-labeled phospholipid probes. For the GM1 probes, large differences in mobility between fluid and gel domains are observed; whereas unexpected mobility is observed in submicron gel domains for the phospholipid probes. We attribute the latter to domain heterogeneities that could be induced by the probe. Furthermore, fits to the FCS data for the phospholipid probes in the DOPC fluid phase require two components (fast and slow). Although proximity to the glass substrate may be a factor, local distortion of the probe by the fluorophore could also be important. Overall, we observe nonideal aspects of phospholipid probe mobility and partitioning that may not be restricted to supported bilayers.

Reactant concentrations from fluorescence correlation spectroscopy with tailored fluorescent probes. An example of local calibration-free pH measurement. Charier, S., Meglio, A., Alcor, D., Cogne-Laage, E., Allemand, J.F., Jullien, L., and Lemarchand, A., J Am Chem Soc, 2005. 127(44): pp. 15491-505

The present account is concerned with the measurement of local reactant concentrations by observing specific fluorescent probes in fluorescence correlation spectroscopy (FCS). The Theoretical Analysis section revisits the photophysical, thermodynamic, and kinetic information that is contained in the corresponding FCS correlation curves. In particular, we examine the conditions under which FCS is revealed as a superior tool to measure concentrations of reactive species. Careful molecular engineering of the specific fluorescent probes that simultaneously integrates photophysical, thermodynamic, and kinetic constraints will be required to benefit most from FCS. We illustrate the FCS titration approach with a series of fluorescent probes that we tailored to measure pH at around 4-6 by FCS after two-photon excitation. We show that an optimal design allows one to access pH without any preliminary calibrations such as the determination of the protonation constant or the photophysical properties of the fluorescent probe.

The kinetics of conformational fluctuations in an unfolded protein measured by fluorescence methods. Chattopadhyay, K., Elson, E.L., and Frieden, C., Proc Natl Acad Sci U S A, 2005. 102(7): pp. 2385-9

The simplest dynamic model for an unfolded protein is a statistical coil that continually undergoes substantial conformational fluctuations. A growing number of studies indicate that the unfolded protein is not a simple random coil but rather forms transient structures. We have directly measured the rate of conformational fluctuations of unfolded intestinal fatty acid binding protein (131 aa, 15 kDa) by using fluorescence self-quenching in combination with fluorescence correlation spectroscopy. The conformational fluctuations in this state have an apparent relaxation time, tauR, of 1.6 microsec in 3 M guanidine-HCl at pH 7 and 20 degrees C. The value of tauR increases with increasing solution viscosity, suggesting a diffusive process. In the molten globule state at pH 2, tauR is 2.5 microsec, increasing further with the formation of salt-induced secondary structure. These measurements, which should be widely applicable to other systems, can provide important information about the still incompletely understood conformational properties of unfolded proteins and the mechanism of protein folding.

Measuring unfolding of proteins in the presence of denaturant using fluorescence correlation spectroscopy. Chattopadhyay, K., Saffarian, S., Elson, E.L., and Frieden, C., Biophys J, 2005. 88(2): pp. 1413-22

IFABP is a small (15 kDa) protein consisting mostly of antiparallel beta-strands that surround a large cavity into which ligands bind. We have previously used FCS to show that the native protein, labeled with fluorescein, exhibits dynamic fluctuation with a relaxation time of 35 micros. Here we report the use of FCS to study the unfolding of the protein induced by guanidine hydrochloride. Although the application of this technique to measure diffusion coefficients and molecular dynamics is straightforward, the FCS results need to be corrected for both viscosity and refractive index changes as the guanidine hydrochloride concentration increases. We present here a detailed study of the effects of viscosity and refractive index of guanidine hydrochloride solutions to calibrate FCS data. After correction, the increase in the diffusion time of IFABP corresponds well with the unfolding transition monitored by far ultraviolet circular dichroism. We also show that the magnitude of the 35 micros phase, reflecting the conformational fluctuation in the native state, decreases sharply as the concentration of denaturant increases and the protein unfolds. Although FCS experiments indicate that the unfolded state at pH 2 is rather compact and native-like, the radius in the presence of guanidine hydrochloride falls well within the range expected for a random coil.

Podophyllotoxin-loaded solid lipid nanoparticles for epidermal targeting. Chen, H., Chang, X., Du, D., Liu, W., Liu, J., Weng, T., Yang, Y., Xu, H., and Yang, X., J Control Release, 2006. 110(2): pp. 296-306

The purpose of this study was to evaluate solid lipid nanoparticles as the topical carrier for epidermal targeting of podophyllotoxin (POD). The high pressure homogenization was employed to prepare drug-loaded solid lipid nanoparticles. The POD-loaded SLN stabilized by 0.5% poloxamer 188 and 1.5% soybean lecithin (P-SLN) and 2% polysorbate 80 (T-SLN) was characterized by photon correlation spectroscopy (PCS). P-SLN showed an average diameter of 73.4 nm and a zeta potential of -48.36 mV. The imaging of AFM indicated that the P-SLN had a spherical shape. DSC and X-ray diffraction analysis showed that POD was dispersed in SLN in an amorphous state. The in vitro permeation study showed that P-SLN increased the accumulative amount of POD in porcine skin 3.48 times over 0.15% tincture. But T-SLN with a diameter of 123.1 nm and a zeta potential of -17.4 mV did not show a high accumulative amount of POD when compared with P-SLN, though both P-SLN and T-SLN could avoid the systemic uptake of POD. Because of the fluorescence property of POD, fluorescence microscopy imaging was employed to visualize the penetration of POD into skin from SLN. The penetration of POD from P-SLN seemed to follow two pathways along the stratum corneum and hair follicle route. The imaging revealed that P-SLN had a strong localization of POD within epidermis. The penetration of P-SLN with low particle size into stratum corneum along the skin surface 'furrow' and the consequent controlled release of POD might lead to the epidermal targeting. P-SLN provides a good epidermal targeting effect and may be a promising carrier for topical delivery of POD.

Dual-color photon-counting histogram. Chen, Y., Tekmen, M., Hillesheim, L., Skinner, J., Wu, B., and Muller, J.D., Biophys J, 2005. 88(3): pp. 2177-92

We report on the development of dual-color photon-counting histogram (PCH) analysis. Dual-color PCH is an extension of regular PCH and considers the photon counts received in two detection channels instead of one. Because each detection channel records a different color, dual-color PCH distinguishes fluorescent species not only by differences in their brightness, but also according to their color. The additional discrimination by color increases the sensitivity of PCH in resolving a mixture of species considerably. Most dual-color fluorescence fluctuation experiments are performed on fluorophores with overlapping emission spectra. This overlap results in spectral cross talk between the detector channels, which reduces resolvability. Here, we demonstrate that dual-color PCH is able to resolve binary dye mixtures in the presence of cross talk from a single measurement without any additional information about the sample. We discuss the effect of sampling time on the fit parameters of dual-color PCH. Differences between dual-color fluorescence correlation spectroscopy and dual-color PCH will also be addressed. We quantitatively resolve a mixture of the two fluorescent proteins CFP and YFP, which is challenging because of the strong spectral overlap of their emission spectra. Dichroic mirrors are needed to direct the light into the two detection channels. We quantify the influence of these filters on dual-color PCH analysis and determine the optimal transition wavelength of the dichroic mirror for the CFP-YFP pair.

Unraveling protein-protein interactions in living cells with fluorescence fluctuation brightness analysis. Chen, Y., Wei, L.N., and Muller, J.D., Biophys J, 2005. 88(6): pp. 4366-77

Fluorescence correlation spectroscopy is a potentially powerful tool for measuring protein-protein interactions directly in single living cells. We previously reported on the detection of homodimer formation in cells using molecular brightness analysis. Here, we extend the technique to detect binding between different proteins. Proteins are labeled with the fluorescent markers YFP and CFP. We first determine the coexpression ratio of both proteins by measuring the intensity ratio with a dual-color setup. The effect of fluorescence resonance energy transfer on the intensity ratio is explicitly taken into account. The brightness of cells coexpressing both proteins is measured in a single-color setup. Selecting the laser wavelength of the two-photon light source allows us to either coexcite both proteins or to selectively excite YFP-labeled proteins. This approach enables us to distinguish between homodimer and heterodimer formation. We first present the theory and then demonstrate experimental feasibility using the ligand binding domains of retinoic acid receptor (RARLBD) and of retinoid X receptor (RXRLBD). Both proteins form heterodimers, and RXRLBD also forms homodimers in the presence of its agonist. We explore binding between these proteins in the presence and absence of RXR agonist. Our results demonstrate that brightness analysis offers a quantitative method for determining protein interactions in cells.

Actin cytoskeleton as the principal determinant of size-dependent DNA mobility in cytoplasm: a new barrier for non-viral gene delivery. Dauty, E. and Verkman, A.S., J Biol Chem, 2005. 280(9): pp. 7823-8

The cytosol of mammalian cells is a crowded environment containing soluble proteins and a network of cytoskeletal filaments. Gene delivery by synthetic vectors involves the endocytosis of DNA-polycation complexes, escape from endosomes, and diffusion of non-complexed DNA through the cytosol to reach the nucleus. We found previously that the translational diffusion of large DNAs (>250 bp) in cytoplasm was greatly slowed compared with that of smaller DNAs (Lukacs, G. L., Haggie, P., Seksek, O., Lechardeur, D., Freedman, N., and Verkman, A. S. (2000) J. Biol. Chem. 275, 1625-1629). To determine the mechanisms responsible for size-dependent DNA diffusion, we used fluorescence correlation spectroscopy to measure the diffusion of single fluorophore-labeled DNAs in crowded solutions, cytosol extracts, actin network, and living cells. DNA diffusion (D) in solutions made crowded with Ficoll-70 (up to 40 weight percentage) or soluble cytosol extracts (up to 100 mg/ml) relative to diffusion of the same sized DNAs in saline (D/D(o)) was approximately independent of DNA size (20-4500 bp), quite different from the strong reduction in D/D(o) in the cytoplasm of living cells. However, the reduced D/D(o) with increasing DNA size was closely reproduced in solutions containing cross-linked actin filaments assembled with gelsolin, whereas soluble macromolecules of the same size and concentration did not reduce D/D(o). In intact cells microinjected with fluorescent DNAs and studied by fluorescence correlation spectroscopy or photobleaching methods, D/D(o) was reduced by 5-150-fold (20-6000 bp); however, the size-dependent reduction in D/D(o) was abolished after actin cytoskeleton disruption. Our results identify the actin cytoskeleton as a major barrier restricting cytoplasmic transport of non-complexed DNA in non-viral gene transfer.

Imaging molecular interactions in living cells. Day, R.N. and Schaufele, F., Mol Endocrinol, 2005. 19(7): pp. 1675-86

Hormones integrate the activities of their target cells through receptor-modulated cascades of protein interactions that ultimately lead to changes in cellular function. Understanding how the cell assembles these signaling protein complexes is critically important to unraveling disease processes, and to the design of therapeutic strategies. Recent advances in live-cell imaging technologies, combined with the use of genetically encoded fluorescent proteins, now allow the assembly of these signaling protein complexes to be tracked within the organized microenvironment of the living cell. Here, we review some of the recent developments in the application of imaging techniques to measure the dynamic behavior, colocalization, and spatial relationships between proteins in living cells. Where possible, we discuss the application of these different approaches in the context of hormone regulation of nuclear receptor localization, mobility, and interactions in different subcellular compartments. We discuss measurements that define the spatial relationships and dynamics between proteins in living cells including fluorescence colocalization, fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, fluorescence resonance energy transfer microscopy, and fluorescence lifetime imaging microscopy. These live-cell imaging tools provide an important complement to biochemical and structural biology studies, extending the analysis of protein-protein interactions, protein conformational changes, and the behavior of signaling molecules to their natural environment within the intact cell.

Studying biophysical barriers to DNA delivery by advanced light microscopy. De Smedt, S.C., Remaut, K., Lucas, B., Braeckmans, K., Sanders, N.N., and Demeester, J., Adv Drug Deliv Rev, 2005. 57(1): pp. 191-210

Advanced light microscopy (ALM) has been intensively employed by biophysicists to reveal cellular mechanisms. As described in this review, ALM clearly has potential to enhance our understanding of the mechanisms that affect macromolecular therapeutics or nanoscopic drug vectors in biological environments. However, while in recent years confocal microscopy and related techniques became rather routinely used in drug delivery it remains challenging to extract reliable information on the biophysical behaviour of drug delivery systems from ALM measurements. This review discusses studies in which confocal imaging, fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and fluorescence energy transfer were employed to reveal biophysical properties of DNA and DNA containing nanoparticles in extra- and intracellular media.

Measuring fast dynamics in solutions and cells with a laser scanning microscope. Digman, M.A., Brown, C.M., Sengupta, P., Wiseman, P.W., Horwitz, A.R., and Gratton, E., Biophys J, 2005. 89(2): pp. 1317-27

Single-point fluorescence correlation spectroscopy (FCS) allows measurements of fast diffusion and dynamic processes in the microsecond-to-millisecond time range. For measurements on living cells, image correlation spectroscopy (ICS) and temporal ICS extend the FCS approach to diffusion times as long as seconds to minutes and simultaneously provide spatially resolved dynamic information. However, ICS is limited to very slow dynamics due to the frame acquisition rate. Here we develop novel extensions to ICS that probe spatial correlations in previously inaccessible temporal windows. We show that using standard laser confocal imaging techniques (raster-scan mode) not only can we reach the temporal scales of single-point FCS, but also have the advantages of ICS in providing spatial information. This novel method, called raster image correlation spectroscopy (RICS), rapidly measures during the scan many focal points within the cell providing the same concentration and dynamic information of FCS as well as information on the spatial correlation between points along the scanning path. Longer time dynamics are recovered from the information in successive lines and frames. We exploit the hidden time structure of the scan method in which adjacent pixels are a few microseconds apart thereby accurately measuring dynamic processes such as molecular diffusion in the microseconds-to-seconds timescale. In conjunction with simulated data, we show that a wide range of diffusion coefficients and concentrations can be measured by RICS. We used RICS to determine for the first time spatially resolved diffusions of paxillin-EGFP stably expressed in CHOK1 cells. This new type of data analysis has a broad application in biology and it provides a powerful tool for measuring fast as well as slower dynamic processes in cellular systems using any standard laser confocal microscope.

Characterization of the photoconversion on reaction of the fluorescent protein Kaede on the single-molecule level. Dittrich, P.S., Schafer, S.P., and Schwille, P., Biophys J, 2005. 89(5): pp. 3446-55

Fluorescent proteins are now widely used in fluorescence microscopy as genetic tags to any protein of interest. Recently, a new fluorescent protein, Kaede, was introduced, which exhibits an irreversible color shift from green to red fluorescence after photoactivation with lambda = 350-410 nm and, thus, allows for specific cellular tracking of proteins before and after exposure to the illumination light. In this work, the dynamics of this photoconversion reaction of Kaede are studied by fluorescence techniques based on single-molecule spectroscopy. By fluorescence correlation spectroscopy, fast flickering dynamics of the chromophore group were revealed. Although these dynamics on a submillisecond timescale were found to be dependent on pH for the green fluorescent Kaede chromophore, the flickering timescale of the photoconverted red chromophore was constant over a large pH range but varied with intensity of the 488-nm excitation light. These findings suggest a comprehensive reorganization of the chromophore and its close environment caused by the photoconversion reaction. To study the photoconversion in more detail, we introduced a novel experimental arrangement to perform continuous flow experiments on a single-molecule scale in a microfluidic channel. Here, the reaction in the flowing sample was induced by the focused light of a diode laser (lambda = 405 nm). Original and photoconverted Kaede protein were differentiated by subsequent excitation at lambda = 488 nm. By variation of flow rate and intensity of the initiating laser we found a reaction rate of 38.6 s(-1) for the complete photoconversion, which is much slower than the internal dynamics of the chromophores. No fluorescent intermediate states could be revealed.

Distribution, lateral mobility and function of membrane proteins incorporated into giant unilamellar vesicles. Doeven, M.K., Folgering, J.H., Krasnikov, V., Geertsma, E.R., van den Bogaart, G., and Poolman, B., Biophys J, 2005. 88(2): pp. 1134-42

GUVs have been widely used for studies on lipid mobility, membrane dynamics and lipid domain (raft) formation, using single molecule techniques like fluorescence correlation spectroscopy. Reports on membrane protein dynamics in these types of model membranes are by far less advanced due to the difficulty of incorporating proteins into GUVs in a functional state. We have used sucrose to prevent four distinct membrane protein(s) (complexes) from inactivating during the dehydration step of the GUV-formation process. The amount of sucrose was optimized such that the proteins retained 100% biological activity, and many proteo-GUVs were obtained. Although GUVs could be formed by hydration of lipid mixtures composed of neutral and anionic lipids, an alternate current electric field was required for GUV formation from neutral lipids. Distribution, lateral mobility, and function of an ATP-binding cassette transport system, an ion-linked transporter, and a mechanosensitive channel in GUVs were determined by confocal imaging, fluorescence correlation spectroscopy, patch-clamp measurements, and biochemical techniques. In addition, we show that sucrose slows down the lateral mobility of fluorescent lipid analogs, possibly due to hydrogen-bonding with the lipid headgroups, leading to larger complexes with reduced mobility.

Kinetics of Insulin Adsorption at the Oil-Water Interface and Diffusion Properties of Adsorbed Layers Monitored Using Fluorescence Correlation Spectroscopy. Donsmark, J., Jorgensen, L., Mollmann, S., Frokjaer, S., and Rischel, C., Pharm Res, 2005

The adsorption of insulin at an oil-water interface was studied with fluorescence correlation spectroscopy (FCS). FCS is able to measure diffusion properties of insulin at nanomolar concentrations, making it possible to detect the very early steps in the adsorption process. Below 20 nM bulk insulin concentration, the insulin molecules adsorbed to the surface diffuse freely at all times during the experiment (a few hours). At higher concentrations, a surprisingly abrupt transition to a slow diffusion phase is observed. Based on the information about both diffusion times and molecular brightness derived from the FCS experiments, we suggest that the transition represents the formation of a fractal network. FCS may be a valuable tool in pharmaceutical formulation science, because it provides information about concentration buildup and phase changes at interfaces formed in drug delivery systems.

A close look at fluorescence quenching of organic dyes by tryptophan. Doose, S., Neuweiler, H., and Sauer, M., Chemphyschem, 2005. 6(11): pp. 2277-85

Understanding fluorescence quenching processes of organic dyes by biomolecular compounds is of fundamental importance for in-vitro and in-vivo fluorescence studies. It has been reported that the excited singlet state of some oxazine and rhodamine derivatives is efficiently and almost exclusively quenched by the amino acid tryptophan (Trp) and the DNA base guanine via photoinduced electron transfer (PET). We present a detailed analysis of the quenching interactions between the oxazine dye MR121 and Trp in aqueous buffer. Steady-state and time-resolved fluorescence spectroscopy, together with fluorescence correlation spectroscopy (FCS), reveal three contributing quenching mechanisms: 1) diffusion-limited dynamic quenching with a bimolecular quenching rate constant k(d) of 4.0 x 10(9) s(-1) M(-1), 2) static quenching with a bimolecular association constant K(s) of 61 M(-1), and 3) a sphere-of-action contribution to static quenching described by an exponential factor with a quenching constant lambda of 22 M(-1). The latter two are characterized as nonfluorescent complexes, formed with approximately 30 % efficiency upon encounter, that are stable for tens of nanoseconds. The measured binding energy of 20-30 kJ mol(-1) is consistent with previous estimates from molecular dynamics simulations that proposed stacked complexes due to hydrophobic forces. We further evaluate the influence of glycerol and denaturant (guanidine hydrochloride) on the formation and stability of quenched complexes. Comparative measurements performed with two other dyes, ATTO 655 and Rhodamine 6G show similar results and thus demonstrate the general applicability of utilizing PET between organic dyes and Trp for the study of conformational dynamics of biopolymers on sub-nanometer length and nanosecond time-scales.

Comparison of photophysical and colloidal properties of biocompatible semiconductor nanocrystals using fluorescence correlation spectroscopy. Doose, S., Tsay, J.M., Pinaud, F., and Weiss, S., Anal Chem, 2005. 77(7): pp. 2235-42

A number of different surface chemistries have been developed in recent years to render semiconductor nanocrystals (NCs) stable in water and biocompatible. However, most of these surface modifications affect NCs' photophysical properties, calling for a method to simultaneously monitor colloidal and fluorescence properties. Fluorescence correlation spectroscopy (FCS) combined with ensemble spectroscopic methods and Monte Carlo simulations were used to interpret and derive photophysical as well as colloidal properties of four different NC surface treatments. Using a novel FCS scheme with alternating laser excitation at two different intensities, we first ruled out influences from optical gradient forces (optical trapping). We then compared concentration of emitting particles, brightness per particle, saturation intensity, blinking (intermittency), hydrodynamic radius, and propensity for aggregation of the different bioconjugated NCs. This approach was successfully applied during the development and optimization of peptide-coated NCs.

Investigation by fluorescence correlation spectroscopy of the chaperoning interactions of HIV-1 nucleocapsid protein with the viral DNA initiation sequences. Egele, C., Schaub, E., Piemont, E., de Rocquigny, H., and Mely, Y., C R Biol, 2005. 328(12): pp. 1041-51

HIV-1 nucleocapsid protein (NC) exhibits nucleic acid chaperone properties that are important during reverse transcription. Herein, we review and extend our recent investigation by fluorescence correlation spectroscopy (FCS) of the NC chaperone activity on the primer binding site sequences (PBS) of the (-) and (+) DNA strands, which are involved in the second strand transfer during reverse transcription. In the absence of NC, the PBS stem-loops exhibited a fraying limited to the terminal G-C base pair. The kinetics of fraying were significantly activated by NC, a feature that may favour (-)PBS/(+)PBS annealing during the second strand transfer. In addition, NC was found to promote the formation of PBS kissing homodimers through interaction between the loops. These kissing complexes may favour secondary contacts between viral sequences and thus, promote recombination and viral diversity.

Comparison of different fluorescence fluctuation methods for their use in FRET assays: monitoring a protease reaction. Eggeling, C., Jager, S., Winkler, D., and Kask, P., Curr Pharm Biotechnol, 2005. 6(5): pp. 351-71

We compare the accuracy of a variety of Fluorescence Fluctuation Spectroscopy (FFS) methods for the study of Forster Resonance Energy Transfer (FRET) assays. As an example, the cleavage of a doubly labeled, FRET-active peptide substrate by the protease Trypsin is monitored and analyzed using methods based on fluorescence intensity, Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Intensity Distribution Analysis (FIDA). The presented fluorescence data are compared to High-Pressure Liquid Chromatography (HPLC) data obtained from the same assay. The HPLC analysis discloses general disadvantages of the FRET approach, such as incomplete labeling and the need for aliquots. However, the simultaneous use of two photon detectors monitoring the fluorescence signal of both labels significantly improves the analysis. In particular, the two global analysis tools Two-Dimensional Fluorescence Intensity Distribution Analysis (2D-FIDA) and Two-Color Global Fluorescence Correlation Spectroscopy (2CG-FCS) highlight the potential of a combination of FFS and FRET. While conventional FIDA and FCS auto- or cross-correlation analysis leaves the user with drawbacks inherent in two-color and FRET applications, these effects are overcome by the global analysis on the molecular level. Furthermore, it is advantageous to analyze the unnormalized as opposed to the normalized correlation data when combining any fluorescence correlation method with FRET, since the analysis of the unnormalized data introduces more accuracy and is less sensitive to the experimental drawbacks.

Rapid analysis of Forster resonance energy transfer by two-color global fluorescence correlation spectroscopy: trypsin proteinase reaction. Eggeling, C., Kask, P., Winkler, D., and Jager, S., Biophys J, 2005. 89(1): pp. 605-18

In this study we introduce the combination of two-color global fluorescence correlation spectroscopy (2CG-FCS) and Forster resonance energy transfer (FRET) as a very powerful combination for monitoring biochemical reactions on the basis of single molecule events. 2CG-FCS, which is a new variation emerging from the family of fluorescence correlation spectroscopy, globally analyzes the simultaneously recorded auto- and cross-correlation data from two photon detectors monitoring the fluorescence emission of different colors. Overcoming the limitations inherent in mere auto- and cross-correlation analysis, 2CG-FCS is sensitive in resolving and quantifying fluorescent species that differ in their diffusion characteristics and/or their molecular brightness either in one or both detection channels. It is able to account for effects that have often been considered as sources of severe artifacts in two-color and FRET measurements, the most prominent artifacts comprising photobleaching, cross talk, or concentration variations in sample preparation. Because of its very high statistical accuracy, the combination of FRET and 2CG-FCS is suited for high-throughput applications such as drug screening. Employing beam scanning during data acquisition even further enhances this capability and allows measurement times of <2 s. The improved performance in monitoring a FRET sample was verified by following the protease cleavage reaction of a FRET-active peptide. The FRET-inactive subpopulation of uncleaved substrate could be correctly assigned, revealing a substantial portion of inactive or missing acceptor label. The results were compared to those obtained by two-dimensional fluorescence intensity distribution analysis.

Molecular photobleaching kinetics of Rhodamine 6G by one- and two-photon induced confocal fluorescence microscopy. Eggeling, C., Volkmer, A., and Seidel, C.A., Chemphyschem, 2005. 6(5): pp. 791-804

Under high-excitation irradiance conditions in one- and two-photon induced fluorescence microscopy, the photostability of fluorescent dyes is of crucial importance for the detection sensitivity of single molecules and for the contrast in fluorescence imaging. Herein, we report on the dependence of photobleaching on the excitation conditions, using the dye Rhodamine 6G as a typical example. The different excitation modes investigated include 1) one-photon excitation into the first-excited singlet state in the range of 500 to 528 nm by continuous wave and picosecond-pulsed lasers and 2) two- and one-photon excitation to higher-excited singlet states at 800 and 350 nm, respectively, by femtosecond pulses. Experimental strategies are presented, which allow resolving the photophysics. From single-molecule trajectories and fluorescence correlation spectroscopy, as well as with a simple theoretical model based on steady-state solutions of molecular rate equation analysis, we determined the underlying photobleaching mechanisms and quantified the photokinetic parameters describing the dependence of the fluorescence signal on the excitation irradiance. The comparison with experimental data and an exact theoretical model show that only minor deviations between the different theoretical approaches can be observed for high-pulsed excitation irradiances. It is shown that fluorescence excitation is in all cases limited by photolysis from higher-excited electronic states. In contrast to picosecond-pulsed excitation, this is extremely severe for both one- and two-photon excitation with femtosecond pulses. Furthermore, the photostability of the higher-excited electronic states is strongly influenced by environmental conditions, such as polarity and temperature.

Performance of fluorescence correlation spectroscopy for measuring diffusion and concentration. Enderlein, J., Gregor, I., Patra, D., Dertinger, T., and Kaupp, U.B., Chemphyschem, 2005. 6(11): pp. 2324-36

Fluorescence correlation spectroscopy (FCS) has become an important tool for measuring diffusion, concentration, and molecular interactions of cellular components. The interpretation of FCS data critically depends on the measurement set-up. Here, we present a rigorous theory of FCS based on exact wave-optical calculations. Six of the most important optical and photophysical factors that influence FCS are studied: fluorescence anisotropy, cover-slide thickness, refractive index of the sample, laser-beam geometry, optical saturation, and pinhole adjustment. Our theoretical framework represents a general attempt to link all relevant parameters of the experimental set-up with the measured correlation function.

Statistical analysis of diffusion coefficient determination by fluorescence correlation spectroscopy. Enderlein, J., Gregor, I., Patra, D., and Fitter, J., J Fluoresc, 2005. 15(3): pp. 415-22

Fluorescence correlation spectroscopy (FCS) has become an important and widely used technique for many applications in physics, chemistry, and biology. The parameter most frequently addressed by FCS is the diffusion of molecules in solution. Due to the highly non-linear connection between the diffusion coefficient and a measured autocorrelation function, it is extremely difficult to analyse the accuracy of the diffusion-coefficient determination in a FCS experiment. Here, we present a simplified analysis based on some general maximum-likelihood considerations, and numerical result are given for the dependence of the accuracy of the diffusion-coefficient determination on sample concentration, brightness, and measurement time. Optimal concentration values for performing FCS are found.

Fluorescence imaging reveals the nuclear behavior of peroxisome proliferator-activated receptor/retinoid X receptor heterodimers in the absence and presence of ligand. Feige, J.N., Gelman, L., Tudor, C., Engelborghs, Y., Wahli, W., and Desvergne, B., J Biol Chem, 2005. 280(18): pp. 17880-90

In a global approach combining fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and fluorescence resonance energy transfer (FRET), we address the behavior in living cells of the peroxisome proliferator-activated receptors (PPARs), a family of nuclear receptors involved in lipid and glucose metabolism, inflammation control, and wound healing. We first demonstrate that unlike several other nuclear receptors, PPARs do not form speckles upon ligand activation. The subnuclear structures that may be observed under some experimental conditions result from overexpression of the protein and our immunolabeling experiments suggest that these structures are subjected to degradation by the proteasome. Interestingly and in contrast to a general assumption, PPARs readily heterodimerize with retinoid X receptor (RXR) in the absence of ligand in living cells. PPAR diffusion coefficients indicate that all the receptors are engaged in complexes of very high molecular masses and/or interact with relatively immobile nuclear components. PPARs are not immobilized by ligand binding. However, they exhibit a ligand-induced reduction of mobility, probably due to enhanced interactions with cofactors and/or chromatin. Our study draws attention to the limitations and pitfalls of fluorescent chimera imaging and demonstrates the usefulness of the combination of FCS, FRAP, and FRET to assess the behavior of nuclear receptors and their mode of action in living cells.

How the molecule number is correctly quantified in two-color fluorescence cross-correlation spectroscopy: corrections for cross-talk and quenching in experiments. Foldes-Papp, Z., Curr Pharm Biotechnol, 2005. 6(6): pp. 437-44

Fluorescence correlation spectroscopy (FCS) and two-color fluorescence cross-correlation spectroscopy (FCCS) are among the cutting-edge technologies for measuring molecule numbers at the single-molecule level in liquid phases. Yet, even after single molecule technologies caught up with theory, the techniques remained tools only for specialists able to navigate the formulas that give meaning to their observations. This original article aims at the derivations of relevant and useful quantification of molecule numbers for researchers with more diverse backgrounds who have begun probing questions previously unanswerable, except on the level of the molecule. The quantitation depends on the exact conditions of measurement. To some extent these are arbitrary, so that standard procedures are necessary in for valid comparisons of measurements among different data sets. To agree on and specify such procedures is one of the further aims here. No matter what fluorophores, which have, of course, to meet photophysical and photochemical requirements for FCS/FCCS, and optical setups/devices are used, the primary measurement signal arises from fluctuations of the mean molecule number in a confocal femtoliter or smaller probe region. Since FCS/FCCS relies on fluorescence emission measurements of rare events, one is looking for small signals on essentially zero background. Optical separation by FCCS setups is usually defined in terms of cross-talk and cross-excitation/cross-emission, respectively, which can be calculated and minimized by the experimenter from readily measurable quantities of the absorption/emission scenario for single labels and multiple labels n and m bound to or incorporated into the two-color molecules. Furthermore, this article derives relevant formulas for the quantification of molecule numbers under different experimental conditions with substantial quenching of the two-color molecules such as single labels and multiple labels n and m bound to or incorporated into the two-color molecules, high-density labeling of two-color molecules with multiple n green labels and one red label. Here, we summarize and extend the formulas to make them more generally applicable.

Dissociation of nuclear import cargo complexes by the protein Ran: a fluorescence correlation spectroscopy study. Fradin, C., Zbaida, D., and Elbaum, M., C R Biol, 2005. 328(12): pp. 1073-82

In nucleated cells, proteins designed for nuclear import form complexes with soluble nuclear transport receptors prior to translocation across the nuclear envelope. The directionality of transport is due to the asymmetric distribution of the protein Ran, which dissociates import cargo complexes only in its nuclear RanGTP form. Using fluorescence correlation spectroscopy, we have studied the stability of cargo complexes in solution in the presence and in the absence of RanGTP. We find that RanGTP has a higher affinity for the major import receptor, the importin alpha/beta heterodimer, when importin alpha does not carry a cargo, suggesting that some nuclear transport targets might be preferentially released.

CD8 kinetically promotes ligand binding to the T-cell antigen receptor. Gakamsky, D.M., Luescher, I.F., Pramanik, A., Kopito, R.B., Lemonnier, F., Vogel, H., Rigler, R., and Pecht, I., Biophys J, 2005. 89(3): pp. 2121-33

The mechanism of CD8 cooperation with the TCR in antigen recognition was studied on live T cells. Fluorescence correlation measurements yielded evidence of the presence of two TCR and CD8 subpopulations with different lateral diffusion rate constants. Independently, evidence for two subpopulations was derived from the experimentally observed two distinct association phases of cognate peptide bound to class I MHC (pMHC) tetramers and the T cells. The fast phase rate constant ((1.7 +/- 0.2) x 10(5) M(-1) s(-1)) was independent of examined cell type or MHC-bound peptides' structure. Its value was much faster than that of the association of soluble pMHC and TCR ((7.0 +/- 0.3) x 10(3) M(-1) s(-1)), and close to that of the association of soluble pMHC with CD8 ((1-2) x 10(5) M(-1) s(-1)). The fast binding phase disappeared when CD8-pMHC interaction was blocked by a CD8-specific mAb. The latter rate constant was slowed down approximately 10-fold after cells treatment with methyl-beta-cyclodextrin. These results suggest that the most efficient pMHC-cell association route corresponds to a fast tetramer binding to a colocalized CD8-TCR subpopulation, which apparently resides within membrane rafts: the reaction starts by pMHC association with the CD8. This markedly faster step significantly increases the probability of pMHC-TCR encounters and thereby promotes pMHC association with CD8-proximal TCR. The slow binding phase is assigned to pMHC association with a noncolocalized CD8-TCR subpopulation. Taken together with results of cytotoxicity assays, our data suggest that the colocalized, raft-associated CD8-TCR subpopulation is the one capable of inducing T-cell activation.

Diffusion behavior of gap junction hemichannels in living cells. Gerken, M., Thews, E., Tietz, C., Wrachtrup, J., and Eckert, R., Curr Pharm Biotechnol, 2005. 6(2): pp. 151-8

Due to its non-invasive character, fluorescence correlation spectroscopy (FCS) is particularly suited for the investigation of diffusion behavior of proteins in living cells. In this study we have investigated the diffusion properties of CFP-labeled gap junction hemichannels in the plasma membrane of living HeLa cells. Gap junction hemichannels or connexons are the precursors for the cell-cell- or gap junction channels that form large plaques at the contact areas between two adjacent cells. It has been proposed that new channels are recruited into a gap junction structure from a pool of hemichannels that can freely diffuse over the entire plasma membrane. The statistical approach shows that the geometry of the membrane within the focus is the most important property for the form of the autocorrelation curve and in turn for the determination of the diffusion coefficient. On the other hand binding-unbinding events which lead to anomalous diffusion have only a minor effect to the position and shape of the correlation curve compared to the geometry of the membrane.

Diffusion of sphingomyelin and myelin oligodendrocyte glycoprotein in the membrane of OLN-93 oligodendroglial cells studied by fluorescence correlation spectroscopy. Gielen, E., Vercammen, J., Sykora, J., Humpolickova, J., Vandeven, M., Benda, A., Hellings, N., Hof, M., Engelborghs, Y., Steels, P., and Ameloot, M., C R Biol, 2005. 328(12): pp. 1057-64

Evidence has been accumulated that the plasma membrane of various mammalian cell types is heterogeneous in structure and may contain lipid microdomains (lipid rafts). This study focuses on the membrane organization of living oligodendrocytes, which are the myelin-producing cells of the central nervous system. Fluorescence correlation spectroscopy (FCS) was used to monitor the lateral diffusion of a lipid and of a protein in the oligodendroglial cell line OLN-93. The lipid was fluorescently labelled sphingomyelin (Bodipy FL-C5 SM). The protein was the myelin oligodendrocyte glycoprotein (MOG). In order to monitor the lateral diffusion of MOG, OLN-93 cells were transfected with a MOG-EGFP (enhanced green fluorescent protein) fusion plasmid. The measurements were performed at room temperature. FCS data were analyzed for two-dimensional (2D) diffusion according to three models which all included a triplet fraction: (a) 2D 1 component (2D1C), (b) 2D anomalous diffusion (2D1Calpha), and (c) 2D 2 components (2D2C). Preliminary results indicate that for the lipid case, the best fits are obtained with 2D2C. In the case of MOG-EGFP, 2D2C and 2D1Calpha give fits of similar quality. The parameter estimates obtained with 2D1Calpha, however, have a lower standard deviation. The anomaly parameter for MOG-EGFP is 0.59+/-0.01.

Molecular and structural characterization of fluorescent human parvovirus B19 virus-like particles. Gilbert, L., Toivola, J., White, D., Ihalainen, T., Smith, W., Lindholm, L., Vuento, M., and Oker-Blom, C., Biochem Biophys Res Commun, 2005. 331(2): pp. 527-35

Although sharing a T=1 icosahedral symmetry with other members of the Parvoviridae family, it has been suggested that the fivefold channel of the human parvovirus B19 VP2 capsids is closed at its outside end. To investigate the possibility of placing a relatively large protein moiety at this site of B19, fluorescent virus-like particles (fVLPs) of B19 were developed. The enhanced green fluorescent protein (EGFP) was inserted at the N-terminus of the structural protein VP2 and assembly of fVLPs from this fusion protein was obtained. Electron microscopy revealed that these fluorescent protein complexes were very similar in size when compared to wild-type B19 virus. Further, fluorescence correlation spectroscopy showed that an average of nine EGFP domains were associated with these virus-like structures. Atomic force microscopy and immunoprecipitation studies showed that EGFP was displayed on the surface of these fVLPs. Confocal imaging indicated that these chimeric complexes were targeted to late endosomes when expressed in insect cells. The fVLPs were able to efficiently enter cancer cells and traffic to the nucleus via the microtubulus network. Finally, immunoglobulins present in human parvovirus B19 acute and past-immunity serum samples were able to detect antigenic epitopes present in these fVLPs. In summary, we have developed fluorescent virus-like nanoparticles displaying a large heterologous entity that should be of help to elucidate the mechanisms of infection and pathogenesis of human parvovirus B19. In addition, these B19 nanoparticles serve as a model in the development of targetable vehicles designed for delivery of biomolecules.

Characterization of interaction between cationic lipid-oligonucleotide complexes and cellular membrane lipids using confocal imaging and fluorescence correlation spectroscopy. Gordon, S.P., Berezhna, S., Scherfeld, D., Kahya, N., and Schwille, P., Biophys J, 2005. 88(1): pp. 305-16

Complexes formed by cationic liposomes and single-strand oligodeoxynucleotides (CL-ODN) are promising delivery systems for antisense therapy. ODN release from the complexes is an essential step for inhibiting activity of antisense drugs. We applied fluorescence correlation spectroscopy and confocal laser scanning microscopy to monitor CL-ODN complex interaction with membrane lipids leading to ODN release. To model cellular membranes we used giant unilamellar vesicles and investigated the transport of Cy-5-labeled ODNs across DiO-labeled membranes. For the first time, we directly observed that ODN molecules are transferred across the lipid bilayers and are kept inside the giant unilamellar vesicles after release from the carriers. ODN dissociation from the carrier was assessed by comparing diffusion constants of CL-ODN complexes and ODNs before complexation and after release. Freely diffusing Cy-5-labeled ODN (16-nt) has diffusion constant D(ODN) = 1.3 +/- 0.1 x 10(-6) cm2/s. Fluorescence correlation spectroscopy curves for CL-ODN complexes were fitted with two components, which both have significantly slower diffusion in the range of D(CL-ODN) = approximately 1.5 x 10(-8) cm2/s. Released ODN has the mean diffusion constant D = 1.1 +/- 0.2 x 10(-6) cm2/s, which signifies that ODN is dissociated from cationic lipids. In contrast to earlier studies, we report that phosphatidylethanolamine can trigger ODN release from the carrier in the full absence of anionic phosphatidylserine in the target membrane and that phosphatidylethanolamine-mediated release is as extensive as in the case of phosphatidylserine. The presented methodology provides an effective tool for probing a delivery potential of newly created lipid formulations of CL-ODN complexes for optimal design of carriers.

Fluorescence correlation spectroscopy of molecular motions and kinetics. Gosch, M. and Rigler, R., Adv Drug Deliv Rev, 2005. 57(1): pp. 169-90

The foundations for fluorescence correlation spectroscopy (FCS) were already laid in the early 1970s, but this technique did not become widely used until single-molecule detection was established almost 20 years later with the use of diffraction-limited confocal volume element. The analysis of molecular noise from the GHz- to the Hz-region facilitates measurements over a large dynamic range covering photophysics, conformational transitions and interactions as well as transport properties of fluorescent biomolecules. From the Poissonian nature of the noise spectrum the absolute number of molecules is obtainable. Originally used for the analysis of molecular interactions in solutions, the strength of FCS lies also in its applicability to molecular processes at either the surface or interior of single cells. Examples for the analysis of surface kinetics including on and off rates of ligand-receptor interactions will be given. The possibility of obtaining this type of information by FCS will be of particular interest for cell-based drug screening.

Optical saturation in fluorescence correlation spectroscopy under continuous-wave and pulsed excitation. Gregor, I., Patra, D., and Enderlein, J., Chemphyschem, 2005. 6(1): pp. 164-70

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

Single-molecule studies on DNA and RNA. Greulich, K.O., Chemphyschem, 2005. 6(12): pp. 2458-71

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

Diffusion and binding properties investigated by Fluorescence Correlation Spectroscopy (FCS). Grunwald, D., Cardoso, M.C., Leonhardt, H., and Buschmann, V., Curr Pharm Biotechnol, 2005. 6(5): pp. 381-6

During the last years, Fluorescence Correlation Spectroscopy (FCS) has proven to be a powerful tool for basic research in many applications. The combination of a minimal detection volume in the femtoliter range coupled with very high sensitivity extends the possibilities to design sensitive homogeneous tests. In this article we illustrate the analysis of binding processes with FCS based on the changes in diffusion characteristics of GFP upon binding to an antibody. Problems induced by highly heterogeneous samples are discussed and differences of GFP binding to a monoclonal and a polyclonal antibody are shown and analyzed. We stress data processing, limitations and useful approximations in FCS methodology. Basic ideas of data acquisition and processing as well as new developments and applications are presented.

Structural aspects of histone H1-DNA complexes and their relation to transfection efficiency. Haberland, A., Cartier, R., Heuer, D., Zaitsev, S., Paulke, B.R., Schafer-Korting, M., and Bottger, M., Biotechnol Appl Biochem, 2005. 42(Pt 2): pp. 107-17

During transfection, polycation-DNA complexes are normally diluted by the transfection medium, which often contains salt in the physiological concentration range and serum. It is not exactly known to what extent this dilution step influences the properties of the complexes, which in turn influence the transfection efficiency. In order to gain more insight into the size-structure-transfection activity relationship, we prepared histone H1-DNA complexes in NaCl solutions at various concentrations known to determine the size and structure of the resulting complexes. We characterized the complexes by physicochemical methods. Fluorescence correlation spectroscopy enabled relative measurements of complex sizes even under physiological conditions. The different appearances of the complexes were correlated with their transfection efficiency. When transfection was performed by dilution of the complexes in cell-cultivation media, the initial structure of H1-DNA complexes preformed under distinct salt conditions had no significant influence on the transfection efficiency. The dilution of the preformed complexes with cell-cultivation medium resulted in re-formation and aggregation of the complexes. The addition of the complexes to the cells without cell-cultivation medium, however, showed a direct correlation between the size of the complexes and the transfection efficiency (correlation coefficient 0.91). Small complexes did not contribute to the transfection.

Diffusion in two-component lipid membranes--a fluorescence correlation spectroscopy and monte carlo simulation study. Hac, A.E., Seeger, H.M., Fidorra, M., and Heimburg, T., Biophys J, 2005. 88(1): pp. 317-33

Using fluorescence correlation spectroscopy, calorimetry, and Monte Carlo simulations, we studied diffusion processes in two-component membranes close to the chain melting transition. The aim is to describe complex diffusion behavior in lipid systems in which gel and fluid domains coexist. Diffusion processes in gel membranes are significantly slower than in fluid membranes. Diffusion processes in mixed phase regions are therefore expected to be complex. Due to statistical fluctuations the gel-fluid domain patterns are not uniform in space and time. No models for such diffusion processes are available. In this article, which is both experimental and theoretical, we investigated the diffusion in DMPC-DSPC lipid mixtures as a function of temperature and composition. We then modeled the fluorescence correlation spectroscopy experiment using Monte Carlo simulations to analyze the diffusion process. It is shown that the simulations yield a very good description of the experimental diffusion processes, and that predicted autocorrelation profiles are superimposable with the experimental curves. We believe that this study adds to the discussion on the physical nature of rafts found in biomembranes.

High count rates with total internal reflection fluorescence correlation spectroscopy. Hassler, K., Anhut, T., Rigler, R., Gosch, M., and Lasser, T., Biophys J, 2005. 88(1): pp. L01-3

Application of fluorescence correlation spectroscopy to hapten-antibody binding. Hazlett, T.L., Ruan, Q., and Tetin, S.Y., Methods Mol Biol, 2005. 305: pp. 415-38

Two-photon fluorescence correlation spectroscopy 2P-FCS has received a large amount of attention over the past ten years as a technique that can monitor the concentration, the dynamics, and the interactions of molecules with single molecule sensitivity. In this chapter, we explain how 2P-FCS is carried out for a specific ligand-binding problem. We briefly outline considerations for proper instrument design and instrument calibration. General theory of autocorrelation analysis is explained and straightforward equations are given to analyze simple binding data. Specific concerns in the analytical methods related to IgG, such as the presence of two equivalent sites and fractional quenching of the bound hapten-fluorophore conjugate, are explored and equations are described to account for these issues. We apply these equations to data on two antibody-hapten pairs: antidigoxin IgG with fluorescein-digoxin and antidigitoxin IgG with Alexa488-digitoxin. Digoxin and digitoxin are important cardio glycoside drugs, toxic at higher levels, and their blood concentrations must be monitored carefully. Clearly, concentration assays based on IgG rely on accurate knowledge of the hapten-IgG binding strengths. The protocols for measuring and determining the dissociation constants for both IgG-hapten pairs are outlined and discussed.

Deficiency of disulfide bonds facilitating fibrillogenesis of endostatin. He, Y., Zhou, H., Tang, H., and Luo, Y., J Biol Chem, 2006. 281(2): pp. 1048-57

Endostatin is an endogenous inhibitor of tumor angiogenesis and tumor growth. It has two pairs of disulfide bonds in a unique nested pattern, which play a key role in its native conformation, stability, and activity. Here, we constructed a disulfide-deficient variant of endostatin, endo-all-Ala, to examine the effects of the two disulfide bonds on fibrillogenesis of endostatin under nondenaturing conditions. Based on thioflavin T fluorescence, atomic force microscopy, far-UV circular dichroism, and Fourier transform infrared spectroscopy, we found that endo-all-Ala, which has a higher alpha-helical content compared with wild type, is prone to forming fibrils in a pH-dependent manner. Subsequently, more hydrophobic patches with a lower stability of endo-all-Ala were observed when compared with wild type, which possibly contributes to the propensity of amyloid formation of endo-all-Ala. To our surprise, the significant increase of the alpha-helical content in endostatin induced by trifluoroethanol can also facilitate fibril formation. In addition, the cytotoxicity of fibrillar aggregates of endo-all-Ala, which were generated at different stages of the fibril formation process, was evaluated by cell viability assay. The results indicate that the cytotoxicity is not due to the fibrils but rather due to the granular aggregates of endo-all-Ala. Moreover, endostatin was interestingly found to be reduced by glutathione at physiological concentrations. Our present work not only elucidates the correlation between the existence of disulfide bonds and the fibril formation of endostatin but also may provide some insights into the structural and functional basis of endostatin in Alzheimer disease brains.

CK2 phosphorylation of eukaryotic translation initiation factor 5 potentiates cell cycle progression. Homma, M.K., Wada, I., Suzuki, T., Yamaki, J., Krebs, E.G., and Homma, Y., Proc Natl Acad Sci U S A, 2005. 102(43): pp. 15688-93

Casein kinase 2 (CK2) is a ubiquitous eukaryotic Ser/Thr protein kinase that plays an important role in cell cycle progression. Although its function in this process remains unclear, it is known to be required for the G(1) and G(2)/M phase transitions in yeast. Here, we show that CK2 activity changes notably during cell cycle progression and is increased within 3 h of serum stimulation of quiescent cells. During the time period in which it exhibits high enzymatic activity, CK2 associates with and phosphorylates a key molecule for translation initiation, eukaryotic translation initiation factor (eIF) 5. Using MS, we show that Ser-389 and -390 of eIF5 are major sites of phosphorylation by CK2. This is confirmed using eIF5 mutants that lack CK2 sites; the phosphorylation levels of mutant eIF5 proteins are significantly reduced, relative to WT eIF5, both in vitro and in vivo. Expression of these mutants reveals that they have a dominant-negative effect on phosphorylation of endogenous eIF5, and that they perturb synchronous progression of cells through S to M phase, resulting in a significant reduction in growth rate. Furthermore, the formation of mature eIF5/eIF2/eIF3 complex is reduced in these cells, and, in fact, restricted diffusional motion of WT eIF5 was almost abolished in a GFP-tagged eIF5 mutant lacking CK2 phosphorylation sites, as measured by fluorescence correlation spectroscopy. These results suggest that CK2 may be involved in the regulation of cell cycle progression by associating with and phosphorylating a key molecule for translation initiation.

Cluster formation of nanoparticles in an optical trap studied by fluorescence correlation spectroscopy. Hosokawa, C., Yoshikawa, H., and Masuhara, H., Phys Rev E Stat Nonlin Soft Matter Phys, 2005. 72(2 Pt 1): p. 021408

We report in situ observation of cluster growth of nanoparticles confined in an optical trapping potential by means of fluorescence correlation spectroscopy. When an optical trapping force caused by a highly focused laser beam acts on nanoparticle suspensions, the number of nanoparticles increases and an assembly can be formed at the focal spot. The decay times of fluorescence autocorrelation curves were investigated as a function of the irradiation time of the laser beam and the laser power. In the initial stage of the optical assembling, the decay time increases with the irradiation time of the laser beam. On the other hand, in the later stage, a decrease of the decay time was observed. This behavior is explained successfully by using two models of Brownian motion under weak and strong optical trapping. It was revealed that trapping and clustering of nanoparticles proceed simultaneously and clusters confined in the focal spot make larger aggregates spontaneously.

Potential application of synchronous fluorescence spectroscopy to determine benzo[a]pyrene in soil extracts. Hua, G., Killham, K., and Singleton, I., Environ Pollut, 2006. 139(2): pp. 272-8

Benzo[a]pyrene (BaP) is a significant environmental pollutant and rapid, accurate methods to quantify this compound in soil for both research and environmental investigation purposes are required. In this work, solvent extracts from five contrasting soils spiked with four different polycyclic aromatic hydrocarbons (PAHs) were rapidly analysed by using a synchronous fluorescence spectroscopy (SFS) method. The SFS method was validated using HPLC with ultraviolet detection. A good correlation for the quantification of BaP in soil extracts by the two methods was observed. The detection limit of the SFS method was 1.6x10(-9)g/ml in CTAB micellar medium (7.8mmol/l). The work demonstrates that SFS has potential as a sensitive, accurate, rapid, simple and economic methodology and an efficient alternative to HPLC for fast confirmation and quantification of BaP in complex soil extracts.

Diffusion-time distribution analysis reveals characteristic ligand-dependent interaction patterns of nuclear receptors in living cells. Jankevics, H., Prummer, M., Izewska, P., Pick, H., Leufgen, K., and Vogel, H., Biochemistry, 2005. 44(35): pp. 11676-83

Nuclear receptors initiate transcription, interact with regulatory proteins, and are influenced by hormones, drugs, and pollutants. Herein, we discover ligand-specific mobility patterns of human estrogen receptor-alpha (ER) in living cells using diffusion-time distribution analysis (DDA). This novel method, based on fluorescence correlation spectroscopy (FCS), is especially suited to unraveling multiple protein interactions in vivo at native expression levels. We found that ER forms a limited number of distinct complexes with a varying population by dynamic interaction with other nuclear components. Dose-response curves of different ligands could be obtained for each receptor interaction. The potential to identify interacting proteins was demonstrated by comparing DDA of the ER cofactor SRC-3 attached to yellow fluorescent protein (YFP) with those of YFP-ER. Our findings open up new routes to elucidating transcription regulation and to detecting and distinguishing pharmacologically and toxicologically active compounds in vivo. Moreover, DDA provides a general approach to monitoring biochemical networks in individual living cells.

Calixarene-coated water-soluble CdSe-ZnS semiconductor quantum dots that are highly fluorescent and stable in aqueous solution. Jin, T., Fujii, F., Sakata, H., Tamura, M., and Kinjo, M., Chem Commun (Camb), 2005(22): pp. 2829-31

A simple method for the preparation of highly fluorescent and stable, water-soluble CdSe-ZnS quantum dots is reported using calix[4]arene carboxylic acids as surface coating agents; the coating of the surface with the calixarene and the conjugation of antibodies to the quantum dots are confirmed by fluorescence correlation spectroscopy.

Raft partitioning and dynamic behavior of human placental alkaline phosphatase in giant unilamellar vesicles. Kahya, N., Brown, D.A., and Schwille, P., Biochemistry, 2005. 44(20): pp. 7479-89

Much attention has recently been drawn to the hypothesis that cellular membranes organize in functionalized platforms called rafts, enriched in sphingolipids and cholesterol. The notion that glycosylphosphatidylinositol (GPI)-anchored proteins are strongly associated with rafts is based on their insolubility in nonionic detergents. However, detergent-based methodologies for identifying raft association are indirect and potentially prone to artifacts. On the other hand, rafts have proven to be difficult to visualize and investigate in living cells. A number of studies have demonstrated that model membranes provide a valuable tool for elucidating some of the raft properties. Here, we present a model membrane system based on domain-forming giant unilamellar vesicles (GUVs), in which the GPI-anchored protein, human placental alkaline phosphatase (PLAP), has been functionally reconstituted. Raft morphology, protein raft partitioning, and dynamic behavior have been characterized by fluorescence confocal microscopy and fluorescence correlation spectroscopy (FCS). Approximately 20-30% of PLAP associate with sphingomyelin-enriched domains. The affinity of PLAP for the liquid-ordered (l(o)) phase is compared to that of a nonraft protein, bacteriorhodopsin. Next, detergent extraction was carried out on PLAP-containing GUVs as a function of temperature, to relate the lipid and protein organization in distinct phases of the GUVs to the composition of detergent resistant membranes (DRMs). Finally, antibody-mediated cross-linking of PLAP induces a shift of its partition coefficient in favor of the l(o) phase.

Differential lipid packing abilities and dynamics in giant unilamellar vesicles composed of short-chain saturated glycerol-phospholipids, sphingomyelin and cholesterol. Kahya, N., Scherfeld, D., and Schwille, P., Chem Phys Lipids, 2005. 135(2): pp. 169-80

The ability of membrane components to arrange themselves heterogeneously within the bilayer induces the formation of microdomains. Much work has been devoted to mimicking domain-assembly in artificial bilayers and characterizing their physico-chemical properties. Ternary lipid mixtures composed of unsaturated phospholipids, sphingomyelin and cholesterol give rise to large, round domains. Here, we replaced the unsaturated phospholipid in the ternary mixture with sphingomyelin and cholesterol by saturated glycero-phospholipids of different chain length and characterized the critical role of cholesterol in sorting these lipids by confocal imaging and fluorescence correlation spectroscopy (FCS). More cholesterol is needed to obtain phase segregation in ternary mixtures, in which the unsaturated phospholipid is replaced by a saturated one. Finally, lipid dynamics in distinct phases is very low and astonishingly similar, thereby suggesting the poor ability of cholesterol in sorting short-chain saturated glycero-phospholipids and sphingomyelin.

Diffusion of spheres in crowded suspensions of rods. Kang, K., Gapinski, J., Lettinga, M.P., Buitenhuis, J., Meier, G., Ratajczyk, M., Dhont, J.K., and Patkowski, A., J Chem Phys, 2005. 122(4): p. 44905

Translational tracer diffusion of spherical macromolecules in crowded suspensions of rodlike colloids is investigated. Experiments are done using several kinds of spherical tracers in fd-virus suspensions. A wide range of size ratios L/2a of the length L of the rods and the diameter 2a of the tracer sphere is covered by combining several experimental methods: fluorescence correlation spectroscopy for small tracer spheres, dynamic light scattering for intermediate sized spheres, and video microscopy for large spheres. Fluorescence correlation spectroscopy is shown to measure long-time diffusion only for relatively small tracer spheres. Scaling of diffusion coefficients with a/xi, predicted for static networks, is not found for our dynamical network of rods (with xi the mesh size of the network). Self-diffusion of tracer spheres in the dynamical network of freely suspended rods is thus fundamentally different as compared to cross-linked networks. A theory is developed for the rod-concentration dependence of the translational diffusion coefficient at low rod concentrations for freely suspended rods. The proposed theory is based on a variational solution of the appropriate Smoluchowski equation without hydrodynamic interactions. The theory can, in principle, be further developed to describe diffusion through dynamical networks at higher rod concentrations with the inclusion of hydrodynamic interactions. Quantitative agreement with the experiments is found for large tracer spheres, and qualitative agreement for smaller spheres. This is probably due to the increasing importance of hydrodynamic interactions as compared to direct interactions as the size of the tracer sphere decreases.

Fluorescence fluctuation spectroscopy in subdiffraction focal volumes. Kastrup, L., Blom, H., Eggeling, C., and Hell, S.W., Phys Rev Lett, 2005. 94(17): p. 178104

We establish fluorescence fluctuation spectroscopy (FFS) with nanoscale detection volumes generated by stimulated emission depletion. Our method applies fluorescence correlation spectroscopy and fluorescence intensity distribution analysis to extract molecular information about mobilities and fluorescence emission in solution. The combination of correlation analysis with that of photon intensity distributions reveals a fivefold squeezing of the detection volume over current diffraction-limited systems, which is in full agreement with the simultaneously demonstrated 25-fold reduction in (axial) focal transit time. Our method significantly extends the potential of far-field FFS, including for the noninvasive investigation of molecular reactions at higher concentrations.

Fluorescence correlation microscopy with real-time alignment readout. Kaushalya, S.K., Balaji, J., Garai, K., and Maiti, S., Appl Opt, 2005. 44(16): pp. 3262-5

In confocal fluorescence correlation microscopy (FCM) it is important to ensure that the correlation measurement is actually performed at the chosen location of the three-dimensional image of the specimen. We present a confocal FCM design that provides an automatic real-time readout of the location in the confocal microscopic image, which is aligned with the detector of the fluorescence correlation spectrometer. The design accomplishes this without using any special positioning device. The design is based on an apertured fluorescence detector placed close to the back aperture of the objective lens and can be easily incorporated into virtually any confocal microscope. We demonstrate the method by performing FCM measurements of a dye diffusing on a cell membrane.

The phosducin-like protein PhLP1 is essential for G{beta}{gamma} dimer formation in Dictyostelium discoideum. Knol, J.C., Engel, R., Blaauw, M., Visser, A.J., and van Haastert, P.J., Mol Cell Biol, 2005. 25(18): pp. 8393-400

Phosducin proteins are known to inhibit G protein-mediated signaling by sequestering Gbetagamma subunits. However, Dictyostelium discoideum cells lacking the phosducin-like protein PhLP1 display defective rather than enhanced G protein signaling. Here we show that green fluorescent protein (GFP)-tagged Gbeta (GFP-Gbeta) and GFP-Ggamma subunits exhibit drastically reduced steady-state levels and are absent from the plasma membrane in phlp1(-) cells. Triton X-114 partitioning suggests that lipid attachment to GFP-Ggamma occurs in wild-type cells but not in phlp1(-) and gbeta(-) cells. Moreover, Gbetagamma dimers could not be detected in vitro in coimmunoprecipitation assays with phlp1(-) cell lysates. Accordingly, in vivo diffusion measurements using fluorescence correlation spectroscopy showed that while GFP-Ggamma proteins are present in a complex in wild-type cells, they are free in phlp1(-) and gbeta(-) cells. Collectively, our data strongly suggest the absence of Gbetagamma dimer formation in Dictyostelium cells lacking PhLP1. We propose that PhLP1 serves as a cochaperone assisting the assembly of Gbeta and Ggamma into a functional Gbetagamma complex. Thus, phosducin family proteins may fulfill hitherto unsuspected biosynthetic functions.

Determining protease activity in vivo by fluorescence cross-correlation analysis. Kohl, T., Haustein, E., and Schwille, P., Biophys J, 2005. 89(4): pp. 2770-82

To date, most biochemical approaches to unravel protein function have focused on purified proteins in vitro. Whereas they analyze enzyme performance under assay conditions, they do not necessarily tell us what is relevant within a living cell. Ideally, cellular functions should be examined in situ. In particular, association/dissociation reactions are ubiquitous, but so far there is no standard technique permitting online analysis of these processes in vivo. Featuring single-molecule sensitivity combined with intrinsic averaging, fluorescence correlation spectroscopy is a minimally invasive technique ideally suited to monitor proteins. Moreover, endogenous fluorescence-based assays can be established by genetically encoding fusions of autofluorescent proteins and cellular proteins, thus avoiding the disadvantages of in vitro protein labeling and subsequent delivery to cells. Here, we present an in vivo protease assay as a model system: Green and red autofluorescent proteins were connected by Caspase-3- sensitive and insensitive protein linkers to create double-labeled protease substrates. Then, dual-color fluorescence cross-correlation spectroscopy was employed to study the protease reaction in situ. Allowing assessment of multiple dynamic parameters simultaneously, this method provided internal calibration and improved experimental resolution for quantifying protein stability. This approach, which is easily extended to reversible protein-protein interactions, seems very promising for elucidating intracellular protein functions.

Fluorescence correlation spectroscopy with autofluorescent proteins. Kohl, T. and Schwille, P., Adv Biochem Eng Biotechnol, 2005. 95: pp. 107-42

Fluorescence correlation spectroscopy (FCS) is a versatile technique operating at the single-molecule level, that successfully meets many challenges of modern biological research. Based on the detection of mobile fluorescent molecules diffusing in and out of a diffraction-limited laser focus, the method allows to resolve particle dynamics within cells and their compartments. Previous FCS studies have described various parameters of protein function, namely mobility, transport and localization phenomena, enzymatic turnovers of biochemical substrates and molecular association and dissociation reactions. Recent progress in the application of FCS to intracellular systems has particularly taken advantage of detecting autofluorescent proteins and their genetically encoded fusions to cellular proteins. This review discusses recent applications of FCS analysis with and on fluorescent proteins, particularly highlighting chemical and physical properties. Inherent limitations of the presented approaches are discussed in detail and strategies for optimisation of experimental systems outlined.

Detection of motional heterogeneities in lipid bilayer membranes by dual probe fluorescence correlation spectroscopy. Korlach, J., Baumgart, T., Webb, W.W., and Feigenson, G.W., Biochim Biophys Acta, 2005. 1668(2): pp. 158-63

We report the detection of heterogeneities in the diffusion of lipid molecules for the three-component mixture dipalmitoyl-PC/dilauroyl-PC/cholesterol, a chemically simple lipid model for the mammalian plasma membrane outer leaflet. Two-color fluorescence correlation spectroscopy (FCS) was performed on giant unilamellar vesicles (GUVs) using fluorescent probes that have differential lipid phase partition behavior--DiO-C18:2 favors disordered fluid lipid phases, whereas DiI-C20:0 prefers spatially ordered lipid phases. Simultaneously-obtained fluorescence autocorrelation functions from the same excitation volume for each dye showed that, depending on the lipid composition of this ternary mixture, the two dyes exhibited different lateral mobilities in regions of the phase diagram with previously proposed submicroscopic two-phase coexistence. In one-phase regions, both dyes reported identical diffusion coefficients. Two-color FCS thus may be detecting local membrane heterogeneities at size scales below the optical resolution limit, either due to short-range order in a single phase or due to submicroscopic phase separation.

Trapping, deformation, and rotation of giant unilamellar vesicles in octode dielectrophoretic field cages. Korlach, J., Reichle, C., Muller, T., Schnelle, T., and Webb, W.W., Biophys J, 2005. 89(1): pp. 554-62

The behavior of freestanding lipid bilayer membranes under the influence of dielectric force potentials was studied by trapping, holding, and rotating individual giant unilamellar vesicles (GUVs) inside dielectrophoretic microfield cages. Using laser scanning confocal microscopy and three-dimensional image reconstructions of GUVs labeled with fluorescent membrane probes, field strength and frequency-dependent vesicle deformations were observed which are explained by calculations of the dielectric force potentials inside the cage. Dynamical membrane properties under the influence of the field cage were studied by fluorescence correlation spectroscopy, circumventing potential artifacts associated with measurements involving GUV immobilization on support surfaces. Lipid transport could be accelerated markedly by the applied fields, aided by hydrodynamic fluid streaming which was also studied by fluorescence correlation spectroscopy.

Propidium iodide and PicoGreen as dyes for the DNA fluorescence correlation spectroscopy measurements. Kral, T., Widerak, K., Langner, M., and Hof, M., J Fluoresc, 2005. 15(2): pp. 179-83

Many experimental designs, in which nucleic acid conformational changes are of interest, require reliable fluorescence labeling. The appropriate fluorescence probe should have suitable optical properties and, more importantly, should not interfere with the investigated processes. In order to avoid chemical modifications the fluorescence label needs to be associated with nucleic acid via weak non-covalent interactions. There are a number of fluorescent probes that change their fluorescent properties (i.e. their quantum yield and/or spectral characteristics) upon association with nucleic acid. Such probes are frequently used to detect, visualize and follow processes involving nucleic acid and its conformational changes. In order to obtain reliable data regarding macromolecule or aggregate topology a detailed knowledge of probe-nucleic acid interactions on the molecular level is needed. In this paper we show that the association of propidium iodide with DNA alters its conformation and that it selectively labels plasmid fragments and/or its subpopulations in a concentration-dependent meaner. Another dye, PicoGreen, exhibits better properties. It labels nucleic acid uniformly and without any concentration-dependent artifacts.

Separating the contribution of translational and rotational diffusion to protein association. Kuttner, Y.Y., Kozer, N., Segal, E., Schreiber, G., and Haran, G., J Am Chem Soc, 2005. 127(43): pp. 15138-44

The association of two proteins is preceded by a mutual diffusional search in solution. The role of translational and rotational diffusion in this process has been studied theoretically for many years. However, systematic experimental verification of theoretical results is still lacking. We report here measurements of association rates of the proteins beta-lactamase (TEM) and beta-lactamase inhibitor protein (BLIP) in solutions of glycerol and poly(ethylene glycol) of increasing viscosity. We also measured translational and rotational diffusion in the same solutions, using fluorescence correlation spectroscopy and fluorescence anisotropy, respectively. It is found that in glycerol both translational and rotational diffusion rates are inversely dependent on viscosity, as predicted by the classical Stokes-Einstein relations, while the association rate depends nonlinearly on viscosity. In contrast, the association rate depends only weakly on the viscosity of the polymer solutions, which results in a similar weak dependence of k(on) on viscosity. The data are modeled using the theory of diffusion-limited association. Deviations from the theory are explained by a short-range solute-induced repulsion between the proteins in glycerol solution and an attractive depletion interaction generated by the polymers. These results open the way to the creation of a unified framework for all nonspecific effects involved in the protein association process, as well as to better theoretical understanding of these effects. Further, they reflect on the complex factors controlling protein association within the crowded environment of cells and suggest that a high concentration of macromolecules does not significantly impede protein association.

In situ measurements of viral particles diffusion inside mucoid biofilms. Lacroix-Gueu, P., Briandet, R., Leveque-Fort, S., Bellon-Fontaine, M.N., and Fontaine-Aupart, M.P., C R Biol, 2005. 328(12): pp. 1065-72

Fluorescence correlation spectroscopy (FCS) under two-photon excitation was used successfully to characterize the diffusion properties of model virus particles (bacteriophages) in bacterial biofilm of Stenotrophonas maltophilia. The results are compared to those obtained with fluorescent latex beads used as a reference. The FCS data clearly demonstrated the possibility for viral particles to penetrate inside the exopolymeric matrix of mucoid biofilms, and hence to benefit from its protective effect toward antimicrobials (antibiotics and biocides). Microbial biofilms should hence be considered as potential reservoirs of pathogenic viruses, and are probably responsible for numerous persistent viral infections.

Enhancing the sensitivity of fluorescence correlation spectroscopy by using time-correlated single photon counting. Lamb, D.C., Muller, B.K., and Brauchle, C., Curr Pharm Biotechnol, 2005. 6(5): pp. 405-14

Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) are methods that extract information about a sample from the influence of thermodynamic equilibrium fluctuations on the fluorescence intensity. This method allows dynamic information to be obtained from steady state equilibrium measurements and its popularity has dramatically increased in the last 10 years due to the development of high sensitivity detectors and its combination with confocal microscopy. Using time-correlated single-photon counting (TCSPC) detection and pulsed excitation, information over the duration of the excited state can be extracted and incorporated in the analysis. In this short review, we discuss new methodologies that have recently emerged which incorporated fluorescence lifetime information or TCSPC data in the FCS and FCCS analysis. Time-gated FCS discriminates between which photons are to be incorporated in the analysis dependent upon their arrival time after excitation. This allows for accurate FCS measurements in the presence of fluorescent background, determination of sample homogeneity, and the ability to distinguish between static and dynamic heterogeneities. A similar method, time-resolved FCS can be used to resolve the individual correlation functions from multiple fluorophores through the different fluorescence lifetimes. Pulsed interleaved excitation (PIE) encodes the excitation source into the TCSPC data. PIE can be used to perform dual-channel FCCS with a single detector and allows elimination of spectral cross-talk with dual-channel detection. For samples that undergo fluorescence resonance energy transfer (FRET), quantitative FCCS measurements can be performed in spite of the FRET and the static FRET efficiency can be determined.

Quantifying Pb and Cd complexation by alginates and the role of metal binding on macromolecular aggregation. Lamelas, C., Avaltroni, F., Benedetti, M., Wilkinson, K.J., and Slaveykova, V.I., Biomacromolecules, 2005. 6(5): pp. 2756-64

The Pb and Cd binding capacity of alginates were quantified by the determination of their complex stability constants and the concentration of complexing sites using H+, Pb2+, or Cd2+ selective electrodes in both static and dynamic titrations. Centrifugation filter devices (30 kDa filter cutoff), followed by inductively coupled plasma mass spectrometry (ICP-MS) measurements of lead or cadmium in the filtrates, were used to validate the results. The influence of ionic strength, pH, and the metal-to-alginate ratio was determined for a wide range of metal concentrations. Because of their polyelectrolytic properties, alginates may adopt different conformations depending on the physicochemistry of the medium, including the presence of metals. Therefore, molecular diffusion coefficients of the alginate were determined by fluorescence correlation spectroscopy under the same conditions of pH, ionic strength, and metal-to-alginate ratios that were used for the metal binding studies. The complexation and conformational properties of the alginate were related within the framework of the nonideal competitive adsorption isotherm (NICA) combined with a Donnan approach to account for both intrinsic and electrostatic contributions.

Temporally resolved interactions between antigen-stimulated IgE receptors and Lyn kinase on living cells. Larson, D.R., Gosse, J.A., Holowka, D.A., Baird, B.A., and Webb, W.W., J Cell Biol, 2005. 171(3): pp. 527-36

Upon cross-linking by antigen, the high affinity receptor for immunoglobulin E (IgE), FcepsilonRI, is phosphorylated by the Src family tyrosine kinase Lyn to initiate mast cell signaling, leading to degranulation. Using fluorescence correlation spectroscopy (FCS), we observe stimulation-dependent associations between fluorescently labeled IgE-FcepsilonRI and Lyn-EGFP on individual cells. We also simultaneously measure temporal variations in the lateral diffusion of these proteins. Antigen-stimulated interactions between these proteins detected subsequent to the initiation of receptor phosphorylation exhibit time-dependent changes, suggesting multiple associations between FcepsilonRI and Lyn-EGFP. During this period, we also observe a persistent decrease in Lyn-EGFP lateral diffusion that is dependent on Src family kinase activity. These stimulated interactions are not observed between FcepsilonRI and a chimeric EGFP that contains only the membrane-targeting sequence from Lyn. Our results reveal real-time interactions between Lyn and cross-linked FcepsilonRI implicated in downstream signaling events. They demonstrate the capacity of FCS cross-correlation analysis to investigate the mechanism of signaling-dependent protein-protein interactions in intact, living cells.

Real-time RNA profiling within a single bacterium. Le, T.T., Harlepp, S., Guet, C.C., Dittmar, K., Emonet, T., Pan, T., and Cluzel, P., Proc Natl Acad Sci U S A, 2005. 102(26): pp. 9160-4

Characterizing the dynamics of specific RNA levels requires real-time RNA profiling in a single cell. We show that the combination of a synthetic modular genetic system with fluorescence correlation spectroscopy allows us to directly measure in real time the activity of any specific promoter in prokaryotes. Using a simple inducible gene expression system, we found that induced RNA levels within a single bacterium of Escherichia coli exhibited a pulsating profile in response to a steady input of inducer. The genetic deletion of an efflux pump system, a key determinant of antibiotic resistance, altered the pulsating transcriptional dynamics and caused overexpression of induced RNA. In contrast with population measurements, real-time RNA profiling permits identifying relationships between genotypes and transcriptional dynamics that are accessible only at the level of the single cell.

Functional conformational motions in the turnover cycle of cholesterol oxidase. Lerch, H.P., Rigler, R., and Mikhailov, A.S., Proc Natl Acad Sci U S A, 2005. 102(31): pp. 10807-12

Reexamining experimental data of single-molecule fluorescence correlation spectroscopy for cholesterol oxidase, we find that the existing Michaelis-Menten models with dynamical disorder cannot explain strong correlations between subsequent turnover cycles revealed in the diagonal feature in the joint statistical distribution of adjacent "on" times of this enzyme. We suggest that functional conformational motions representing ordered sequences of transitions between a set of conformational substates are involved, along with equilibrium conformational fluctuations in the turnover cycle of cholesterol oxidase. A two-channel model of single-enzyme dynamics, including a slow functional conformational motion in one of the channels, is proposed that allows us to reproduce such strong correlations.

Albumin-protamine-oligonucleotide nanoparticles as a new antisense delivery system. Part 1: physicochemical characterization. Lochmann, D., Weyermann, J., Georgens, C., Prassl, R., and Zimmer, A., Eur J Pharm Biopharm, 2005. 59(3): pp. 419-29

In this paper, a ternary system of albumin-protamine-oligonucleotide nanoparticles (AlPrO-NP) recently developed by Vogel et al. [V. Vogel, D. Lochmann, J. Weyermann, G. Mayer, C. Tziatzios, J.A. van den Broek, W. Haase, D. Wouters, U.S. Schubert, J. Kreuter, A. Zimmer, D. Schubert, Oligonucleotide-protamine-albumin nanoparticles: preparation, physical properties and intracellular processing, J. Controlled Rel. (in press)] which could serve as a potential drug delivery system for antisense oligonucleotides. Former studies of binary protamine-oligonucleotide nanoparticles showed two main disadvantages: (i) aggregation of the particles within a few minutes in the presence of salt; (ii) low intracellular dissociation between protamine and oligonucleotide, especially phosphorothioates. To overcome these problems, human serum albumin (HSA) as a non-toxic, biodegradable macromolecule was introduced as protective colloid. The assembly process of AlPrO-NP was investigated by small angle X-ray scattering (SAXS), fluorescence correlation spectroscopy (FCS), photon correlation spectroscopy (PCS) measurements and scanning electron microscopy (SEM). 'Initial complexes' of HSA and protamine sulphate with a mean hydrodynamic diameter (dh) of about 10-14 nm were found. After adding oligonucleotides (unmodified, phosphorothioate DNA and small interfering RNA), nanoparticles (NPs) were assembled in water and in isotonic media with a dh in a range of 230-320 nm for most preparations. The chemical composition of the particles was investigated by high performance liquid chromatography and fluorescence spectrometry. The whole amount of oligonucleotides (30 microg) was entrapped into the particles at a 1:2 mass ratio (oligonucleotide/protamine). Approximately 7-10% (w/w) of the HSA was bound to the particles. The surface charge of the particles ranged from about +12 to -60 mV depending on the protamine concentration and the ionic conditions. The size and the molecular weight of the components, initial complexes and two model NP preparations were calculated from FCS data. These data verified the PCS, SEM and SAXS measurements.

Measuring protein-protein interactions inside living cells using single color fluorescence correlation spectroscopy. Application to human immunodeficiency virus type 1 integrase and LEDGF/p75. Maertens, G., Vercammen, J., Debyser, Z., and Engelborghs, Y., FASEB J, 2005. 19(8): pp. 1039-41

Recently we described the interaction of human immunodeficiency virus type 1 (HIV-1)1 integrase (IN) with a cellular protein, lens epithelium-derived growth factor/transcription co-activator p75 (LEDGF/p75). We now present the study of the diffusion behavior of the three independent domains of IN and LEDGF/p75 using fluorescence correlation microscopy (FCM). We show that diffusion in the cell of the different enhanced green fluorescent protein (EGFP) fusion proteins is described by two components with different fractions and that the average parameters in the nucleus are comparable with those in the cytoplasm. In addition, we demonstrate that specific interaction between EGFP-fused HIV-1 IN and LEDGF/p75 results in a shift in diffusion coefficient (D). The opposite shift was observed in an IN-deletion mutant that does not exhibit LEDGF/p75 binding or in a LEDGF/p75 knock-down experiment using siRNA. We thus demonstrate that protein-protein interactions can be studied in living cells, using single-color FCM (scFCM).

Membrane perturbation effects of peptides derived from the N-termini of unprocessed prion proteins. Magzoub, M., Oglecka, K., Pramanik, A., Goran Eriksson, L.E., and Graslund, A., Biochim Biophys Acta, 2005. 1716(2): pp. 126-36

Peptides derived from the unprocessed N-termini of mouse and bovine prion proteins (mPrPp and bPrPp, respectively), comprising hydrophobic signal sequences followed by charged domains (KKRPKP), function as cell-penetrating peptides (CPPs) with live cells, concomitantly causing toxicity. Using steady-state fluorescence techniques, including calcein leakage and polarization of a membrane probe (diphenylhexatriene, DPH), as well as circular dichroism, we studied the membrane interactions of the peptides with large unilamellar phospholipid vesicles (LUVs), generally with a 30% negative surface charged density, comparing the effects with those of the CPP penetratin (pAntp) and the pore-forming peptide melittin. The prion peptides caused significant calcein leakage from LUVs concomitant with increased membrane ordering. Fluorescence correlation spectroscopy (FCS) studies of either rhodamine-entrapping (REVs) or rhodamine-labeled (RLVs) vesicles, showed that addition of the prion peptides resulted in significant release of rhodamine from the REVs without affecting the overall integrity of the RLVs. The membrane leakage effects due to the peptides had the following order of potency: melittin>mPrPp>bPrPp>pAntp. The membrane perturbation effects of the N-terminal prion peptides suggest that they form transient pores (similar to melittin) causing toxicity in parallel with their cellular trafficking.

Modeling the endosomal escape of cell-penetrating peptides: transmembrane pH gradient driven translocation across phospholipid bilayers. Magzoub, M., Pramanik, A., and Graslund, A., Biochemistry, 2005. 44(45): pp. 14890-7

Cell-penetrating peptides (CPPs) are able to mediate the efficient cellular uptake of a wide range of cargoes. Internalization of a number of CPPs requires uptake by endocytosis, initiated by binding to anionic cell surface heparan sulfate (HS), followed by escape from endosomes. To elucidate the endosomal escape mechanism, we have modeled the process for two CPPs: penetratin (pAntp) and the N-terminal signal peptide of the unprocessed bovine prion protein (bPrPp). Large unilamellar phospholipid vesicles (LUVs) were produced encapsulating either peptide, and an ionophore, nigericin, was used to create a transmembrane pH gradient (DeltapH(mem), inside acidic) similar to the one arising in endosomes in vivo. In the absence of DeltapH(mem), no pAntp escape from the LUVs is observed, while a fraction of bPrPp escapes. In the presence of DeltapH(mem), a significant amount of pAntp escapes and an even higher degree of bPrPp escape takes place. These results, together with the differences in kinetics of escape, indicate different escape mechanisms for the two peptides. A minimum threshold peptide concentration exists for the escape of both peptides. Coupling of the peptides to a cargo reduces the fraction escaping, while complexation with HS significantly hinders the escape. Fluorescence correlation spectroscopy results show that during the escape process the LUVs are intact. Taken together, these results suggest a model for endosomal escape of CPPs: DeltapH(mem)-mediated mechanism, following dissociation from HS of the peptides, above a minimum threshold peptide concentration, in a process that does not involve lysis of the vesicles.

G-protein-coupled glucocorticoid receptors on the pituitary cell membrane. Maier, C., Runzler, D., Schindelar, J., Grabner, G., Waldhausl, W., Kohler, G., and Luger, A., J Cell Sci, 2005. 118(Pt 15): pp. 3353-61

Rapid, nongenomic actions of glucocorticoids (GCs) have been well documented, but information about putative membrane receptors that mediate them is scarce. We used fluorescence correlation spectroscopy to search for membrane GC-binding on the mouse pituitary cell line AtT-20. A slowly diffusing fraction (tau3; diffusion constant 3x10(-10) cm2 s-1) of fluorescein-labeled dexamethasone on the cell membrane corresponds to fluorescein-dexamethasone binding. Preincubation experiments were performed to test binding specificity: a 500-fold excess of unlabeled dexamethasone abolished subsequent fluorescein-dexamethasone membrane binding from 58+/-2 (control) to 8+/-1 (% of tau3, mean+/-s.e.m.), the natural ligand corticosterone prevented it partially (29+/-2), while the sex steroids estradiol (56+/-4) and progesterone (50+/-4) and the GC-receptor antagonist RU486 (56+/-2) had no effect. Preincubation with pertussis toxin resulted in disappearance of the slowest diffusion component (11+/-4) suggesting association of the receptor with a G-protein. Varying the concentration of fluorescein-dexamethasone showed that membrane binding is highly cooperative with an apparent Kd of 180 nM and Bmax of 230 nM. Taken together, these results demonstrate high-affinity GC-binding on the cell membrane of AtT-20 cells with characteristics distinct from intracellular binding.

Translocation of dynorphin neuropeptides across the plasma membrane. A putative mechanism of signal transmission. Marinova, Z., Vukojevic, V., Surcheva, S., Yakovleva, T., Cebers, G., Pasikova, N., Usynin, I., Hugonin, L., Fang, W., Hallberg, M., Hirschberg, D., Bergman, T., Langel, U., Hauser, K.F., Pramanik, A., Aldrich, J.V., Graslund, A., Terenius, L., and Bakalkin, G., J Biol Chem, 2005. 280(28): pp. 26360-70

Several peptides, including penetratin and Tat, are known to translocate across the plasma membrane. Dynorphin opioid peptides are similar to cell-penetrating peptides in a high content of basic and hydrophobic amino acid residues. We demonstrate that dynorphin A and big dynorphin, consisting of dynorphins A and B, can penetrate into neurons and non-neuronal cells using confocal fluorescence microscopy/immunolabeling. The peptide distribution was characterized by cytoplasmic labeling with minimal signal in the cell nucleus and on the plasma membrane. Translocated peptides were associated with the endoplasmic reticulum but not with the Golgi apparatus or clathrin-coated endocytotic vesicles. Rapid entry of dynorphin A into the cytoplasm of live cells was revealed by fluorescence correlation spectroscopy. The translocation potential of dynorphin A was comparable with that of transportan-10, a prototypical cell-penetrating peptide. A central big dynorphin fragment, which retains all basic amino acids, and dynorphin B did not enter the cells. The latter two peptides interacted with negatively charged phospholipid vesicles similarly to big dynorphin and dynorphin A, suggesting that interactions of these peptides with phospholipids in the plasma membrane are not impaired. Translocation was not mediated via opioid receptors. The potential of dynorphins to penetrate into cells correlates with their ability to induce non-opioid effects in animals. Translocation across the plasma membrane may represent a previously unknown mechanism by which dynorphins can signal information to the cell interior.

Single-molecule studies of diffusion by oligomer-bound dyes in organically modified sol-gel-derived silicate films. Martin-Brown, S.A., Fu, Y., Saroja, G., Collinson, M.M., and Higgins, D.A., Anal Chem, 2005. 77(2): pp. 486-94

Single-molecule fluorescence spectroscopy is used to study dye diffusion within organically modified silicate (ORMOSIL) films. ORMOSIL films are prepared from sols containing tetraethoxysilane and isobutyltrimethoxysilane in 2:1 and 1:9 molar ratios. Nile red and a new silanized form of nile red that can be covalently attached to the silicate matrix are used as fluorescent probe molecules. The number and rate of single molecules diffusing through these films increases dramatically with increasing film organic content. Autocorrelation of the fluorescence images yields a quantitative measure of the relative populations of fixed and diffusing species. Surprisingly, both "free" and silicate-bound nile red exhibit relatively facile translational motions. Single-molecule/single-point fluorescence correlation spectroscopy (FCS) is used to measure the dye diffusion coefficients in submicrometer-scale film regions. The most common diffusion coefficients for "free" and silicate-bound nile red molecules in the 1:9 films are 3.9 x 10(-10) and 1.6 x 10(-10) cm(2)/s, respectively. The unexpectedly rapid diffusion of silicate-bound nile red is attributed to the presence of liquidlike silicate oligomers in the films. A lower bound for the molecular weight of the oligomers is estimated at 2900. Bulk solution-phase FCS experiments performed on "free" and silicate-bound nile red species extracted into chloroform solutions provide valuable support for these conclusions. Comparison of the results derived from experimental and simulated time transients indicates film heterogeneity occurs on sub-100-nm-length scales and likely results from the presence of inorganic- and organic-rich domains.

New fluorescence correlation spectroscopy enabling direct observation of spatiotemporal dependence of diffusion constants as an evidence of anomalous transport in extracellular matrices. Masuda, A., Ushida, K., and Okamoto, T., Biophys J, 2005. 88(5): pp. 3584-91

The potential of fluorescence correlation spectroscopy (FCS) is extended to enable the direct observation of anomalous subdiffusion (ASD) in inhomogeneous media that are of great importance particularly in many biological systems, such as membranes, cytoplasm, and extracellular matrices (ECMs). Because ASD can be confirmed by monitoring the spatiotemporal dependence of observable diffusion coefficients (D(obs)), the size of the effective confocal volume (V(eff)) for FCS sampling (sampling volume) was continuously changed on a scale of 300-500 nm using a motorized variable beam expander through which an illuminating laser beam passes. This new method, namely, sampling-volume-controlled (SVC)-FCS, was applied to the analysis of hyaluronan (HA) aqueous solutions where the D(obs) of light-emitting solute (Alexa 488) markedly changed, corresponding to the change in V(eff) (220-340 nm in the half-axis), because the network structure of HA of 7-33 nm (nanostructure) interferes with the material transport within it. The results indicate that moderate ASD may occur even in the presence of a small amount ( approximately 0.1 wt %) of HA in ECM. Because the change in D(obs) along with the traveling distance (the mean-square displacement) can be identified even in systems with no deformation of the autocorrelation function, this technique has a great potential for general applications to many biological systems in which ASD shows complex time and space dependences.

An electrostatic engine model for autoinhibition and activation of the epidermal growth factor receptor (EGFR/ErbB) family. McLaughlin, S., Smith, S.O., Hayman, M.J., and Murray, D., J Gen Physiol, 2005. 126(1): pp. 41-53

We propose a new mechanism to explain autoinhibition of the epidermal growth factor receptor (EGFR/ErbB) family of receptor tyrosine kinases based on a structural model that postulates both their juxtamembrane and protein tyrosine kinase domains bind electrostatically to acidic lipids in the plasma membrane, restricting access of the kinase domain to substrate tyrosines. Ligand-induced dimerization promotes partial trans autophosphorylation of ErbB1, leading to a rapid rise in intracellular [Ca(2+)] that can activate calmodulin. We postulate the Ca(2+)/calmodulin complex binds rapidly to residues 645--660 of the juxtamembrane domain, reversing its net charge from +8 to -8 and repelling it from the negatively charged inner leaflet of the membrane. The repulsion has two consequences: it releases electrostatically sequestered phosphatidylinositol 4,5-bisphosphate (PIP(2)), and it disengages the kinase domain from the membrane, allowing it to become fully active and phosphorylate an adjacent ErbB molecule or other substrate. We tested various aspects of the model by measuring ErbB juxtamembrane peptide binding to phospholipid vesicles using both a centrifugation assay and fluorescence correlation spectroscopy; analyzing the kinetics of interactions between ErbB peptides, membranes, and Ca(2+)/calmodulin using fluorescence stop flow; assessing ErbB1 activation in Cos1 cells; measuring fluorescence resonance energy transfer between ErbB peptides and PIP(2); and making theoretical electrostatic calculations on atomic models of membranes and ErbB juxtamembrane and kinase domains.

Characterizing observation volumes and the role of excitation saturation in one-photon fluorescence fluctuation spectroscopy. Nagy, A., Wu, J., and Berland, K.M., J Biomed Opt, 2005. 10(4): p. 44015

Fluorescence correlation spectroscopy (FCS) and related distribution analysis techniques have become extremely important and widely used research tools for analyzing the dynamics, kinetics, interactions, and mobility of biomolecules. However, it is not widely recognized that photophysical dynamics can dramatically influence the calibration of fluctuation spectroscopy instrumentation. While the basic theories for fluctuation spectroscopy methods are well established, there have not been quantitative models to characterize the photophysical-induced variations observed in measured fluctuation spectroscopy data under varied excitation conditions. We introduce quantitative models to characterize how the fluorescence observation volumes in one-photon confocal microscopy are modified by excitation saturation as well as corresponding models for the effect of the volume changes in FCS. We introduce a simple curve fitting procedure to model the role of saturation in FCS measurements and demonstrate its accuracy in fitting measured correlation curves over a wide range of excitation conditions.

Observation volumes and {gamma}-factors in two-photon fluorescence fluctuation spectroscopy. Nagy, A., Wu, J., and Berland, K.M., Biophys J, 2005. 89(3): pp. 2077-90

Fluorescence fluctuation spectroscopy has become an important measurement tool for investigating molecular dynamics, molecular interactions, and chemical kinetics in biological systems. Although the basic theory of fluctuation spectroscopy is well established, it is not widely recognized that saturation of the fluorescence excitation can dramatically alter the size and profile of the fluorescence observation volume from which fluorescence fluctuations are measured, even at relatively modest excitation levels. A precise model for these changes is needed for accurate analysis and interpretation of fluctuation spectroscopy data. We here introduce a combined analytical and computational approach to characterize the observation volume under saturating conditions and demonstrate how the variation in the volume is important in two-photon fluorescence correlation spectroscopy. We introduce a simple approach for analysis of fluorescence correlation spectroscopy data that can fully account for the effects of saturation, and demonstrate its success for characterizing the observed changes in both the amplitude and relaxation timescale of measured correlation curves. We also discuss how a quantitative model for the observed phenomena may be of broader importance in fluorescence fluctuation spectroscopy.

A microscopic view of miniprotein folding: enhanced folding efficiency through formation of an intermediate. Neuweiler, H., Doose, S., and Sauer, M., Proc Natl Acad Sci U S A, 2005. 102(46): pp. 16650-5

The role of polypeptide collapse and formation of intermediates in protein folding is still under debate. Miniproteins, small globular peptide structures, serve as ideal model systems to study the basic principles that govern folding. Experimental investigations of folding dynamics of such small systems, however, turn out to be challenging, because requirements for high temporal and spatial resolution have to be met simultaneously. Here, we demonstrate how selective quenching of an extrinsic fluorescent label by the amino acid tryptophan (Trp) can be used to probe folding dynamics of Trp-cage (TC), the smallest protein known to date. Using fluorescence correlation spectroscopy, we monitor folding transitions as well as conformational flexibility in the denatured state of the 20-residue protein under thermodynamic equilibrium conditions with nanosecond time resolution. Besides microsecond folding kinetics, we reveal hierarchical folding of TC, hidden to previous experimental studies. We show that specific collapse of the peptide to a molten globule-like intermediate enhances folding efficiency considerably. A single point mutation destabilizes the intermediate, switching the protein to two-state folding behavior and slowing down the folding process. Our results underscore the importance of preformed structure in the denatured state for folding of even the smallest globular structures. A unique method emerges for monitoring conformational dynamics and ultrafast folding events of polypeptides at the nanometer scale.

Dead-time distortion in fluorescence correlation measurements. Nishimura, G. and Kinjo, M., Appl Opt, 2005. 44(17): pp. 3458-67

The dead time of the detector significantly distorts the fluorescence correlation function of fluorescent particles in solution. This distortion of the correlation function is similar to the saturation effect of the correlation function in a high-power excitation region. The correlation amplitude is significantly reduced by the dead time. The deviations in the number of molecules and the diffusion time are empirically given by the deviation of the fluorescence intensity linearity. The empirical curves of the deviations can be applied to the systematic error estimation of the parameters. The proportionality of the number of molecules to the concentration of fluorophores is no longer maintained with a large dead time, although almost all of the proportionality of the diffusion time to the inverse diffusion constant remains. This fact makes the dead-time effect different from the saturation effect, which is due to photokinetics. In practice, these distortions can be reduced by use of a smaller excitation power in which the proportionality of the fluorescence intensity is maintained.

Monitoring the interaction between DNA and a transcription factor (MEF2A) using fluorescence correlation spectroscopy. Octobre, G., Lemercier, C., Khochbin, S., Robert-Nicoud, M., and Souchier, C., C R Biol, 2005. 328(12): pp. 1033-40

Fluorescence correlation spectroscopy (FCS) is an analytical method that allows distinguishing different populations of fluorescent probes in solution and provides data on their concentrations and their diffusion coefficients. FCS was used to characterize the interaction of the transcription factor (MEF2A) with its DNA target sequence. The myocyte enhancer factor 2 (MEF2) belongs to the MADS-box family and activates transcription of numerous muscle genes during myogenesis. Measurements were made using TAMRA-labelled oligonucleotide duplexes derived from a wild type (WT) or a mutated MEF2 target gene. Binding of the protein to the WT DNA resulted in significant changes of the diffusion. Specificity of the interaction was confirmed using the mutated DNA. Bound to free probe ratios were determined at different MEF2A concentrations and the apparent equilibrium dissociation constant K(D) for the full-length MEF2A was estimated.

Hansenula polymorpha Pex20p is an oligomer that binds the peroxisomal targeting signal 2 (PTS2). Otzen, M., Wang, D., Lunenborg, M.G., and van der Klei, I.J., J Cell Sci, 2005. 118(Pt 15): pp. 3409-18

We have cloned and characterized the Hansenula polymorpha PEX20 gene. The HpPEX20 gene encodes a protein of 309 amino acids (HpPex20p) with a calculated molecular mass of approximately 35 kDa. In cells of an HpPEX20 disruption strain, PTS2 proteins were mislocalized to the cytosol, whereas PTS1 matrix protein import proceeded normally. Also, the PTS2 proteins amine oxidase and thiolase were normally assembled and active in these cells, suggesting HpPex20p is not involved in oligomerization/activation of these proteins. Localization studies revealed that HpPex20p is predominantly associated with peroxisomes. Using fluorescence correlation spectroscopy we determined the native molecular mass of purified HpPex20p and binding of a synthetic peptide containing a PTS2 sequence. The data revealed that purified HpPex20p forms oligomers, which specifically bind PTS2-containing peptides.

Calculation of photon count number distributions via Master equations. Palo, K., Mets, U., Loorits, V., and Kask, P., Biophys J, 2005

Fitting of photon count number histograms is a way of analysis of fluorescence intensity fluctuations, a successor to fluorescence correlation spectroscopy. First versions of the theory for calculating photon count number distributions have assumed constant emission intensity by a molecule during a counting time interval. For a long time a question has remained unanswered: to what extent is this assumption violated in experiments. Here we present a theory of photon count number distributions that takes account of intensity fluctuations during a counting time interval. Theoretical count number distributions are calculated via a numerical solution of Master equations (ME), which is a set of differential equations describing diffusion, singlet-triplet transitions and photon emission. After-pulsing and dead time corrections are also included. The ME-theory is tested by fitting a series of photon count number histograms corresponding to different lengths of the counting time interval. Compared to the first version of FIMDA theory introduced in 2000, the fit quality is significantly improved. It is discussed how a theory of photon count number distributions, which assumes constant emission intensity during a counting time interval, may also yield a good fit quality. We argue that the spatial brightness distribution used in calculations of the fit curve is not the "true" spatial brightness distribution. Instead, a number of dynamic processes, which cause fluorescence intensity fluctuations, are indirectly taken into account via the adjustment parameters.

Effect of bin time on the photon counting histogram for one-photon excitation. Perroud, T.D., Huang, B., and Zare, R.N., Chemphyschem, 2005. 6(5): pp. 905-12

We have demonstrated that our photon counting histogram (PCH) model with the correction for one-photon excitation is valid at multiple bin times. The fitted apparent brightness and concentration follow the three-dimensional diffusion model. More importantly, the semi-empirical parameter, F, introduced in the PCH model for one-photon excitation to correct for the non-Gaussian shape of the observation volume, shows small variations with different bin times. These variations are consistent with the physical interpretation of F, and they do not affect the resolving power of the PCH model for one-photon excitation. Based on these findings, we extend the time-independent PCH analysis to time-dependent photon counting multiple histograms (PCMH). This model considers the effect of bin time on the PCH parameters in a way that is similar to fluorescence intensity multiple distribution analysis (FIMDA). From the same set of data, PCMH extracts time-dependent parameters (diffusion time and triplet-state relaxation time) as well as time-independent parameters (true specific brightness and true average number of molecules). Given a three- to fourfold experimental difference in molecular brightness, we find that PCMH can resolve each species in a two-species sample and extract their respective diffusion times even when fluorescence correlation spectroscopy cannot.

The nanopore connection to cell membrane unitary permeability. Peters, R., Traffic, 2005. 6(3): pp. 199-204

Artificial nanopores have recently emerged as versatile tools for analyzing and sorting single molecules at high speed. However, the biological cell has already developed a large set of sophisticated protein nanopores that are able to selectively translocate all types of molecules through membranes. Therefore, hybrid devices combining artifical solid-state with biomimetic protein nanopores appear to us as a particularly promising approach to the creation of powerful diagnostic, preparative and therapeutic devices. Here, we discuss a technique, optical single-transporter recording (OSTR), in which arrays of artificial micropores and nanopores are employed to analyze protein nanopores of cellular membranes. After briefly summarizing some salient features of OSTR, the technique is compared with the electrical patch clamp method and the first results of our efforts to amalgamate optical and electrical recording are described. Finally, prospects for combining OSTR with 4Pi microscopy, single-molecule fluorescence spectroscopy and fluorescence correlation spectroscopy are discussed.

Imaging the secretory pathway: the past and future impact of live cell optical techniques. Presley, J.F., Biochim Biophys Acta, 2005. 1744(3): pp. 259-72

Classically, the secretory pathway has been studied using a combination of electron microscopic, biochemical and genetic approaches. In the last 20 years with the arrival of molecular biology and epitope tagging, fluorescence microscopy has become more important than previously. Moreover, with the common availability of Green Fluorescent Protein (GFP) and confocal microscopes in the last 10 years, live cell imaging has become a major experimental approach. This review highlights the impact of the recent introduction of single-cell quantitative time-lapse imaging and photobleach techniques on the study of the secretory pathway, and the potential impact of those optical techniques which may play a significant future role in the study of the Golgi apparatus and the secretory pathway. Particular attention is paid to techniques (Fluorescence Resonance Energy Transfer, Fluorescence Correlation Spectroscopy) which can monitor protein-protein interactions in living cells.

Fluorescence correlation spectroscopy studies of diffusion of a weak polyelectrolyte in aqueous solutions. Pristinski, D., Kozlovskaya, V., and Sukhishvili, S.A., J Chem Phys, 2005. 122(1): p. 14907

We apply fluorescent correlation spectroscopy (FCS) to investigate solution dynamics of a synthetic polyelectrolyte, i.e., a weak polycarboxylic acid in aqueous solutions. The technique brings single molecule sensitivity and molecular specificity to dynamic measurements of polyelectrolyte solutions. Translational diffusion of Alexa-labeled poly(methacrylic acid), PMAA*, chains was studied in very dilute, 10(-4) mg/ml, solutions as a function of solution pH and ionic strength. The observed changes in diffusion coefficients were consistent with about twofold expansion of PMAA* coils when pH was changed from 5 to 8, and with chain contraction for alkaline metal ion concentrations from 0.01 to 0.1 M. The dependence of the hydrodynamic size of PMAA* chains on the counterion type followed the sequence: Li(+)>Na(+) approximately equal to Cs(+)>K(+). The dependence of translational diffusion on polyacid concentration was weak at the low concentration limit, but chain motions were significantly slower at higher polymer concentrations when PMAA chains overlapped. Finally, measurements of dynamics of PMAA* chains in "salt-free" solutions showed that self-diffusion of PMAA* chains significantly slowed down when PMAA concentration was increased, probably reflecting the sensitivity of PMAA* translational motions to the onset of interchain domain formation. These results illustrate the utility of the FCS technique for studying hydrodynamic sizes of polyelectrolyte coils in response to variation in solution pH or concentration of salt and polyelectrolytes. They also suggest that FCS will be a promising technique for selective observation of the dynamics of polyelectrolyte components in complex polymer mixtures.

Alpha 1-antitrypsin polymerization: a fluorescence correlation spectroscopic study. Purkayastha, P., Klemke, J.W., Lavender, S., Oyola, R., Cooperman, B.S., and Gai, F., Biochemistry, 2005. 44(7): pp. 2642-9

Alpha(1)-antitrypsin (AT) is the most abundantly circulating human proteinase inhibitor in the serpin family. The polymerization of AT, leading to alpha(1)-antitrypsin deficiency, has been studied extensively in vitro by a variety of ensemble methods. Here we report the use of fluorescence correlation spectroscopy to gain further insight into this process. Measurements of the distributions of diffusion times of polymerizing AT, carried out at 45, 50, and 55 degrees C, clearly show the existence of a kinetic lag phase, during which short oligomers are formed, prior to the formation of heterogeneous mixtures of longer polymers, and suggest that long polymers, which appear to be metastable, are produced through the condensation of shorter oligomers.

FRET-FCS as a tool to evaluate the stability of oligonucleotide drugs after intracellular delivery. Remaut, K., Lucas, B., Braeckmans, K., Sanders, N.N., De Smedt, S.C., and Demeester, J., J Control Release, 2005. 103(1): pp. 259-71

The intracellular degradation of single-stranded, double-labeled oligonucleotides (ONs) was studied by following the disappearance of Fluorescence Resonance Energy Transfer (FRET) between the rhodamine green and Cy5 fluorophores attached to respectively the 3' and 5' end of the ONs. The green and red fluorescence intensities upon rhodamine green excitation were monitored using the ultra-sensitive detectors of a dual-color Fluorescence Correlation Spectroscopy (FCS) instrument. The ratio of the red to green fluorescence (R/G ratio) as obtained from such FRET-FCS measurements showed to give accurate information on the integrity of the ONs, without the need for additional auto- or cross-correlation analysis of the registered fluorescence intensity fluctuations. Intracellular measurements revealed that most of the 40mer phosphodiester ONs were degraded before they entered the nucleus. For the 20mer phosphodiester ONs, this degradation occurred more slowly, and both intact and degraded ONs entered the nucleus. For the 20mer phosphorothioate ONs, no intracellular degradation was observed during the measured time period. The sensitive detection of the intracellular fluorescence by the FCS setup will be particularly useful in situations where the expected fluorescence is too low to be detected by FRET-imaging as may occur after intracellular delivery of ONs by cationic carriers.

Protection of oligonucleotides against nucleases by pegylated and non-pegylated liposomes as studied by fluorescence correlation spectroscopy. Remaut, K., Lucas, B., Braeckmans, K., Sanders, N.N., Demeester, J., and De Smedt, S.C., J Control Release, 2005. 110(1): pp. 212-26

Antisense phosphodiester oligonucleotides (ONs), complexed to carriers such as cationic liposomes, inhibit the production of proteins. The biochemical and biophysical phenomena that govern the extent of this inhibition are still not fully understood. Major biological barriers limiting a pronounced antisense effect are the cellular entry and endosomal escape of the ONs containing liposomes, the release of the ONs from the liposomes and the extra- and intracellular degradation of the ONs. In this paper we focus on the latter barrier and evaluate, by fluorescence correlation spectroscopy (FCS), to what extent phosphodiester ONs complexed to DOTAP/DOPE liposomes, are protected against degradation by nucleases. Liposomes studied were either with or without a polyethyleneglycol (PEG) moiety at the surface. Using non-pegylated liposomes the phosphodiester ONs were initially adequately protected when exposed to DNase I. Indeed, in the mechanism for lipoplex formation as suggested by others, the ONs become trapped between lipid bilayers and are therefore shielded from the environment. However, after a few hours the phosphodiester ONs no longer stayed intact. This was explained by a gradual fusion of the lipoplexes in time thereby spontaneously releasing phosphodiester ONs. Using pegylated liposomes, a substantial fraction of the phosphodiester ONs degraded immediately after exposing the complexes to DNase I. Based on experimental evidence we suggest that the presence of the PEG-chains influences lipoplex formation so that the ONs are not trapped between lipid bilayers and therefore remain accessible by the DNase I enzyme.

Rapid, Diffusional Shuttling of Poly(A) RNA between Nuclear Speckles and the Nucleoplasm. Ritland Politz, J.C., Tuft, R.A., Prasanth, K.V., Baudendistel, N., Fogarty, K.E., Lifshitz, L.M., Langowski, J., Spector, D.L., and Pederson, T., Mol Biol Cell, 2005

Monitoring Editor: Greg Matera Speckles are nuclear bodies that contain pre-mRNA splicing factors and polyadenylated RNA. Because nuclear poly(A) RNA consists of both mRNA transcripts and nucleus-restricted RNAs, we tested whether poly(A) RNA in speckles is dynamic or rather an immobile, perhaps structural, component. Fluorescein-labeled oligo(dT) was introduced into HeLa cells stably expressing a red fluorescent protein chimera of the splicing factor SC35 and allowed to hybridize. Fluorescence correlation spectroscopy (FCS) showed that the mobility of the tagged poly(A) RNA was virtually identical in both speckles and at random nucleoplasmic sites. This same result was observed in photoactivation-tracking studies in which caged fluorescein-labeled oligo(dT) was used as hybridization probe and the rate of movement away from either a speckle or nucleoplasmic site was monitored using digital imaging microscopy after photoactivation. Furthermore, the tagged poly(A) RNA was observed to rapidly distribute throughout the entire nucleoplasm and other speckles, regardless whether the tracking observations were initiated in a speckle or the nucleoplasm. Finally, in both FCS and photoactivation-tracking studies, a temperature reduction from 37 degrees C to 22 degrees C had no discernable effect on the behavior of poly(A) RNA in either speckles or the nucleoplasm, strongly suggesting that its movement in and out of speckles does not require metabolic energy.

lambda-Repressor oligomerization kinetics at high concentrations using fluorescence correlation spectroscopy in zero-mode waveguides. Samiee, K.T., Foquet, M., Guo, L., Cox, E.C., and Craighead, H.G., Biophys J, 2005. 88(3): pp. 2145-53

Fluorescence correlation spectroscopy (FCS) has demonstrated its utility for measuring transport properties and kinetics at low fluorophore concentrations. In this article, we demonstrate that simple optical nanostructures, known as zero-mode waveguides, can be used to significantly reduce the FCS observation volume. This, in turn, allows FCS to be applied to solutions with significantly higher fluorophore concentrations. We derive an empirical FCS model accounting for one-dimensional diffusion in a finite tube with a simple exponential observation profile. This technique is used to measure the oligomerization of the bacteriophage lambda repressor protein at micromolar concentrations. The results agree with previous studies utilizing conventional techniques. Additionally, we demonstrate that the zero-mode waveguides can be used to assay biological activity by measuring changes in diffusion constant as a result of ligand binding.

Lipid--protein interactions revealed by two-photon microscopy and fluorescence correlation spectroscopy. Sanchez, S.A. and Gratton, E., Acc Chem Res, 2005. 38(6): pp. 469-77

Cellular processes involve a multitude of chemical reactions that must be kept in delicate equilibrium to maintain cellular homeostasis. Powerful biophysical techniques are needed to measure the localization and concentration of target molecules as well as to quantify complex molecular processes in model and in vivo systems. Two-photon microscopy and fluorescence correlation spectroscopy (FCS) can measure association and dynamics of appropriate molecules under equilibrium conditions. FCS provides information on motility (diffusion coefficients), concentration (number of particles), association (molecular brightness), and localization (image) of the target molecules. All of this information, in conjunction with computational modeling techniques, can help us to better understand the network of complex molecular interactions, which are at the basis of cellular processes. Fluorescence imaging techniques add the beauty of visualization to the scientific information. Photons emitted by a fluorescent dye are digitized, and the associated spatial information and intensity can be translated into different colors and shades providing the researcher not only with quantitative intensity information but also with spatial resolution and visual comprehension of two- or three-dimensional images. In this Account, we review the use of two-photon excitation microscopy and FCS in the study of lipid-protein interactions. We discuss these new methodologies and techniques, and we present examples of different complexity from qualitative to quantitative, from simple model systems to studies in living cells.

Metal-free catalysts for the hydrolysis of RNA derived from guanidines, 2-aminopyridines, and 2-aminobenzimidazoles. Scheffer, U., Strick, A., Ludwig, V., Peter, S., Kalden, E., and Gobel, M.W., J Am Chem Soc, 2005. 127(7): pp. 2211-7

2-aminopyridine and 2-aminobenzimidazole were chosen as structural analogues to substitute guanidinium groups in receptor molecules designed as phosphoryl transfer catalysts. Shifting the pKa of the guanidinium analogues toward 7 was expected to raise catalytic activities in aqueous buffer. Although the pKa's of both heterocycles are similar (6.2 and 7.0), only 2-aminobenzimidazole led to active RNA cleavers. All cleavage assays were run with fluorescently labeled substrates and a DNA sequencer. RNase contaminations would degrade RNA enantioselectively. In contrast, achiral catalysts such as 9b and 10b necessarily induce identical cleavage patterns in RNA and its mirror image. This principle allowed us to safely rule out contamination effects in this study. The most active catalysts, tris(2-aminobenzimidazoles) 9b and 10b, were shown by fluorescence correlation spectroscopy (FCS) to aggregate with oligonucleotides. However, at very low concentrations the compounds are still active in the nonaggregated state. Conjugates of 10b with antisense oligonucleotides or RNA binding peptides, therefore, will be promising candidates as site specific artificial ribonucleases.

The oxidation state of a protein observed molecule-by-molecule. Schmauder, R., Librizzi, F., Canters, G.W., Schmidt, T., and Aartsma, T.J., Chemphyschem, 2005. 6(7): pp. 1381-6

We report the observation of the redox state of the blue copper protein azurin on the single-molecule level. The fluorescence of a small fluorophore attached to the protein is modulated by the change in absorption of the copper center via fluorescence resonance energy transfer (FRET). In our model system, the fluorescence label Cy5 was coupled to azurin from Pseudomonas aeruginosa via cysteine K27C. The Cy5 fluorescence was partially quenched by the absorption of the copper center of azurin in its oxidized state. In the reduced state, absorption is negligible, and thus no quenching occurs. We report on single-molecule measurements, both in solution by using fluorescence correlation spectroscopy (FCS) combined with fluorescence intensity distribution analysis (FIDA), and on surfaces by using wide-field fluorescence microscopy.

Substrate binding stoichiometry and kinetics of the norepinephrine transporter. Schwartz, J.W., Novarino, G., Piston, D.W., and DeFelice, L.J., J Biol Chem, 2005. 280(19): pp. 19177-84

The human norepinephrine (NE) transporter (hNET) attenuates neuronal signaling by rapid NE clearance from the synaptic cleft, and NET is a target for cocaine and amphetamines as well as therapeutics for depression, obsessive-compulsive disorder, and post-traumatic stress disorder. In spite of its central importance in the nervous system, little is known about how NET substrates, such as NE, 1-methyl-4-tetrahydropyridinium (MPP+), or amphetamine, interact with NET at the molecular level. Nor do we understand the mechanisms behind the transport rate. Previously we introduced a fluorescent substrate similar to MPP+, which allowed separate and simultaneous binding and transport measurement (Schwartz, J. W., Blakely, R. D., and DeFelice, L. J. (2003) J. Biol. Chem. 278, 9768-9777). Here we use this substrate, 4-(4-(dimethylamino)styrl)-N-methyl-pyridinium (ASP+), in combination with green fluorescent protein-tagged hNETs to measure substrate-transporter stoichiometry and substrate binding kinetics. Calibrated confocal microscopy and fluorescence correlation spectroscopy reveal that hNETs, which are homomultimers, bind one substrate molecule per transporter subunit. Substrate residence at the transporter, obtained from rapid on-off kinetics revealed in fluorescence correlation spectroscopy, is 526 micros. Substrate residence obtained by infinite dilution is 1000 times slower. This novel examination of substrate-transporter kinetics indicates that a single ASP+ molecule binds and unbinds thousands of times before being transported or ultimately dissociated from hNET. Calibrated fluorescent images combined with mass spectroscopy give a transport rate of 0.06 ASP+/hNET-protein/s, thus 36,000 on-off binding events (and 36 actual departures) occur for one transport event. Therefore binding has a low probability of resulting in transport. We interpret these data to mean that inefficient binding could contribute to slow transport rates.

DNA length evaluation using cyanine dye and fluorescence correlation spectroscopy. Shimizu, M., Sasaki, S., and Tsuruoka, M., Biomacromolecules, 2005. 6(5): pp. 2703-7

To develop a high-performance method for measuring the length of double-stranded DNA (dsDNA) fragments, the capability of fluorescence correlation spectroscopy (FCS) was examined. To omit troublesome and time-consuming labeling operations such as PCR with fluorescently labeled mononucleotides or primers, intercalation of dimeric cyanine dye YOYO-1 iodide (YOYO) to dsDNA was utilized as a simple labeling method. Various lengths of dsDNA fragments were prepared and mixed with YOYO prior to FCS, and the dependence of the diffusion time of a dsDNA-YOYO complex on the length of dsDNA fragment and the dsDNA/YOYO ratio was investigated. It was successfully demonstrated that the dsDNA length can be measured using YOYO and FCS, and the calibration curve was developed taking into account the rewinding and expansion of the dsDNA fragment caused by YOYO intercalation.

Probing vesicle dynamics in single hippocampal synapses. Shtrahman, M., Yeung, C., Nauen, D.W., Bi, G.Q., and Wu, X.L., Biophys J, 2005. 89(5): pp. 3615-27

We use fluorescence correlation spectroscopy and fluorescence recovery after photobleaching to study vesicle dynamics inside the synapses of cultured hippocampal neurons labeled with the fluorescent vesicle marker FM 1-43. These studies show that when the cell is electrically at rest, only a small population of vesicles is mobile, taking seconds to traverse the synapse. Applying the phosphatase inhibitor okadaic acid causes vesicles to diffuse freely, moving 30 times faster than vesicles in control synapses. These results suggest that vesicles move sluggishly due to binding to elements of the synaptic cytomatrix and that this binding is altered by phosphorylation. Motivated by these results, a model is constructed consisting of diffusing vesicles that bind reversibly to the cytomatrix. This stick-and-diffuse model accounts for the fluorescence correlation spectroscopy and fluorescence recovery after photobleaching data, and also predicts the well-known exponential refilling of the readily releasable pool. Our measurements suggest that the movement of vesicles to the active zone is the rate-limiting step in this process.

Global analysis of fluorescence fluctuation data. Skakun, V.V., Hink, M.A., Digris, A.V., Engel, R., Novikov, E.G., Apanasovich, V.V., and Visser, A.J., Eur Biophys J, 2005. 34(4): pp. 323-34

Over the last decade the number of applications of fluorescence correlation spectroscopy (FCS) has grown rapidly. Here we describe the development and application of a software package, FCS Data Processor, to analyse the acquired correlation curves. The algorithms combine strong analytical power with flexibility in use. It is possible to generate initial guesses, link and constrain fit parameters to improve the accuracy and speed of analysis. A global analysis approach, which is most effective in analysing autocorrelation curves determined from fluorescence fluctuations of complex biophysical systems, can also be implemented. The software contains a library of frequently used models that can be easily extended to include user-defined models. The use of the software is illustrated by analysis of different experimental fluorescence fluctuation data sets obtained with Rhodamine Green in aqueous solution and enhanced green fluorescent protein in vitro and in vivo.

Position-sensitive scanning fluorescence correlation spectroscopy. Skinner, J.P., Chen, Y., and Muller, J.D., Biophys J, 2005. 89(2): pp. 1288-301

Fluorescence correlation spectroscopy (FCS) uses a stationary laser beam to illuminate a small sample volume and analyze the temporal behavior of the fluorescence fluctuations within the stationary observation volume. In contrast, scanning FCS (SFCS) collects the fluorescence signal from a moving observation volume by scanning the laser beam. The fluctuations now contain both temporal and spatial information about the sample. To access the spatial information we synchronize scanning and data acquisition. Synchronization allows us to evaluate correlations for every position along the scanned trajectory. We use a circular scan trajectory in this study. Because the scan radius is constant, the phase angle is sufficient to characterize the position of the beam. We introduce position-sensitive SFCS (PSFCS), where correlations are calculated as a function of lag time and phase. We present the theory of PSFCS and derive expressions for diffusion, diffusion in the presence of flow, and for immobilization. To test PSFCS we compare experimental data with theory. We determine the direction and speed of a flowing dye solution and the position of an immobilized particle. To demonstrate the feasibility of the technique for applications in living cells we present data of enhanced green fluorescent protein measured in the nucleus of COS cells.

Breakdown of axonal synaptic vesicle precursor transport by microglial nitric oxide. Stagi, M., Dittrich, P.S., Frank, N., Iliev, A.I., Schwille, P., and Neumann, H., J Neurosci, 2005. 25(2): pp. 352-62

The mechanism of axonal injury in inflammatory brain diseases is still unclear. Increased microglial production of nitric oxide (NO) is a common early sign in neuroinflammatory diseases. We found by fluorescence correlation spectroscopy that synaptophysin tagged with enhanced green fluorescence protein (synaptophysin-EGFP) moves anterogradely in axons of cultured neurons. Activated microglia focally inhibited the axonal movement of synaptophysin-EGFP in a NO synthase-dependent manner. Direct application of a NO donor to neurons resulted in inhibition of axonal transport of synaptophysin-EGFP and synaptotagmin I tagged with EGFP, mediated via phosphorylation of c-jun NH2-terminal kinase (JNK). Thus, overt production of reactive NO by activated microglia blocks the axonal transport of synaptic vesicle precursors via phosphorylation of JNK and could cause axonal and synaptic dysfunction.

Single molecule studies of quantum dot conjugates in a submicrometer fluidic channel. Stavis, S.M., Edel, J.B., Samiee, K.T., and Craighead, H.G., Lab Chip, 2005. 5(3): pp. 337-43

A microfluidic and optical system was created for the detection and analysis of single molecules in solution. Fluidic channels with submicrometer dimensions were used to isolate, detect and identify individual quantum dots conjugated with organic fluorophores. The channels were fabricated in fused silica with a 500 nm square cross section. The resulting focal volume of approximately 500 aL reduced fluorescent background and increased the signal to noise ratio of single molecule detection. The channels also enabled the rapid detection of 99% of quantum dots and organic fluorophores traversing the focal volume. Conjugates were driven through the channels electrokinetically at 2.3 kV cm(-1), excited with a single 476 nm wavelength laser and detected with a confocal microscope. Fluorescence emission was collected simultaneously from green (500-590 nm) and red (610-680 nm) regions of the spectrum. Signal rejection was minimized by the narrow and symmetric emission spectra of the quantum dots. To demonstrate efficient multicolor detection and characterization of single molecule binding, Qdot 655 Streptavidin Conjugates were bound to Alexa Fluor 488 molecules and individually detected. Photon counting histogram analysis was used to quantify coincident detection and degree of binding. Fluorescence correlation spectroscopy was used to measure the mobility of bound and unbound species. The union of fluidic channels with submicrometer dimensions and quantum dots as fluorescent labels resulted in efficient and rapid multiplexed single molecule detection and analysis.

Reversible aggregation of polystyrene-block-poly(2-vinylpyridine)-block-poly(ethylene oxide) block copolymer micelles in acidic aqueous Solutions. Stepanek, M., Matejicek, P., Humpolickova, J., and Prochazka, K., Langmuir, 2005. 21(23): pp. 10783-90

Micelles of polystyrene-block-poly(2-vinylpyridine)-block-poly(ethylene oxide) (PS-PVP-PEO) were studied in acidic aqueous solutions by static and dynamic light scattering, alkalimetric titration, fluorescence correlation spectroscopy, and after deposition on a mica surface by atomic force microscopy. The PS-PVP-PEO micelles prepared by dialysis in ternary 1,4-dioxane-methanol-acidic water mixtures have a very low association number and show a strong tendency to form aggregates. The aggregation, which is promoted at low pH, seems to be fully reversible. Possible mechanisms of the aggregation are discussed. Atomic force microscopy scans of PS-PVP-PEO micelles deposited on a mica surface reveal the formation of micellar aggregates and support the general concept of aggregation upon changes in conditions and deterioration of the stability of small micelles.

One-step analysis of protein complexes in microliters of cell lysate. Stoevesandt, O., Kohler, K., Fischer, R., Johnston, I.C., and Brock, R., Nat Methods, 2005. 2(11): pp. 833-5

We present 'mix and measure' procedures for the analysis of protein complexes in microliters of crude human and mouse cell lysates using fluorescence correlation and crosscorrelation spectroscopy. We labeled interacting endogenous proteins by indirect immunofluorescence with all primary and secondary reagents added in one step. Especially for the screening of compounds interfering with interactions that depend on signaling-induced posttranslational modifications, the approach represents a major advance over existing protocols.

Analysis of cellular functions by multipoint fluorescence correlation spectroscopy. Takahashi, Y., Sawada, R., Ishibashi, K., Mikuni, S., and Kinjo, M., Curr Pharm Biotechnol, 2005. 6(2): pp. 159-65

The biophysical investigation of living cells is currently possible by single molecular detection methods such as fluorescence correlation spectroscopy (FCS). FCS is applied for measuring the dynamic mobility of target molecules in living cells; however, the conventional FCS systems still lack quantitative analysis for many regions of interests (ROI) in real time. To improve this situation, we have developed a novel multipoint FCS system (M-FCS) that can measure multipoint correlation functions in the cell simultaneously. To evaluate its performance, we measured correlation functions for rhodamine 6G (Rh6G) in homogeneous conditions and for green fluorescence protein (GFP) in HeLa cells. We conclude that M-FCS possesses reliable performance. As a pharmacological application, glucocorticoid receptor protein fused GFP (GR-GFP) was transfected in HeLa cells and FCS measurements were carried out in the cytoplasm and the nucleus simultaneously. The translocation of GR-GFP from the cytoplasm to the nucleus by ligand stimulation was observed with laser scanning microscopy (LSM) and M-FCS. Particularly in the nucleus, the slower diffusion of GR-GFP suggested molecular interactions after the translocation. These data imply that M-FCS can be applied for quantitative analysis of kinetic processes in living cells.

Probing single DNA mobility with fluorescence correlation microscopy. Tatarkova, S.A. and Berk, D.A., Phys Rev E Stat Nonlin Soft Matter Phys, 2005. 71(4 Pt 1): p. 041913

Fluorescence correlation spectroscopy combined with microscopy (FCSM) is used to study the mobility of DNA fragments in aqueous solution and tissue models on the single molecule level. The effective hydrodynamic radius was measured for various lengths of ds-DNA chains and obeyed the theoretically inveterate [DNA length](0.5) relationship. Hindered diffusion of ds-DNA through the gel matrix of various densities is thought of as an extension of Kramer's problem for a flexible polymer chain. With increasing DNA length the average barrier crossing time rises as [DNA length](2) and this agrees with theory predictions for polymer molecules surmounting an entropic barrier.

Quantitative fluorescence microscopy of macromolecules in gel and biological tissue. Tatarkova, S.A., Kamra Verma, A., Berk, D.A., and Lloyd, C.J., Phys Med Biol, 2005. 50(23): pp. 5759-68

Quantitative fluorescence microscopy provides valuable insight into drug delivery and pharmacokinetics. The technique is based on analysis of statistical fluctuations in fluorescence that arises as fluorophores pass through a small volume illuminated by a focused laser beam, and has been applied to measure particle motion and binding interactions in solutions, on surfaces and inside the cells. We examined the use of fluorescence correlation spectroscopy combined with a microscope (FCSM) to assess the transport of fluorescent beads and macromolecules in aqueous solutions, gels and living biological tissue. Obstructed diffusion of fluorescent beads in gels of various densities was tested to get a sensible estimate of diffusion in the interstitial tissue matrix consistent with previous reports. Fluorescently labelled liposomes as an artificial drug or gene carrying vehicles were used for pharmacokinetic tests of drug delivery in living tissue. The results indicate that FCS is an accurate and valuable tool for measuring the physical properties of gene vectors in vitro and for characterizing interactions with tissue in vivo.

Highlighting protein kinase CK2 movement in living cells. Theis-Febvre, N., Martel, V., Laudet, B., Souchier, C., Grunwald, D., Cochet, C., and Filhol, O., Mol Cell Biochem, 2005. 274(1-2): pp. 15-22

Protein kinase CK2 has traditionally been described as a stable heterotetrameric complex (alpha2beta2) but new approaches that effectively capture the dynamic behavior of proteins, are bringing a new picture of this complex into focus. To track the spatio-temporal dynamics of CK2 in living cells, we fused its catalytic alpha and regulatory beta subunits with GFP and analog proteins. Beside the mostly nuclear localization of both subunits, and the identification of specific domains on each subunit that triggers their localization, the most significant finding was that the association of both CK2 subunits in a stable tetrameric holoenzyme eliminates their nuclear import (Mol Cell Biol 23: 975-987, 2003). Molecular movements of both subunits in the cytoplasm and in the nucleus were analyzed using different new and updated fluorescence imaging methods such as: fluorescence recovery after photo bleaching (FRAP), fluorescence loss in photo bleaching (FLIP), fluorescence correlation spectroscopy (FCS), and photoactivation using a biphoton microscope. These fluorescence-imaging techniques provide unprecedented ways to visualize and quantify the mobility of each individual CK2 subunit with high spatial and temporal resolution. Visualization of CK2 heterotetrameric complex formation could also be recorded using the fluorescence resonance energy transfer (FRET) technique. FRET imaging revealed that the assembling of this molecular complex can take place both in the cytoplasmic and nuclear compartments. The spatio-temporal organization of individual CK2 subunits and their dynamic behavior remain now to be correlated with the functioning of this kinase in the complex environment of the cell.

Cross talk free fluorescence cross correlation spectroscopy in live cells. Thews, E., Gerken, M., Eckert, R., Zapfel, J., Tietz, C., and Wrachtrup, J., Biophys J, 2005. 89(3): pp. 2069-76

Fluorescence correlation spectroscopy (FCS) is now a widely used technique to measure small ensembles of labeled biomolecules with single molecule detection sensitivity (e.g., low endogenous concentrations). Fluorescence cross correlation spectroscopy (FCCS) is a derivative of this technique that detects the synchronous movement of two biomolecules with different fluorescence labels. Both methods can be applied to live cells and, therefore, can be used to address a variety of unsolved questions in cell biology. Applications of FCCS with autofluorescent proteins (AFPs) have been hampered so far by cross talk between the detector channels due to the large spectral overlap of the fluorophores. Here we present a new method that combines advantages of these techniques to analyze binding behavior of proteins in live cells. To achieve this, we have used dual color excitation of a common pair of AFPs, ECFP and EYFP, being discriminated in excitation rather than in emission. This is made possible by pulsed excitation and detection on a shorter timescale compared to the average residence time of particles in the FCS volume element. By this technique we were able to eliminate cross talk in the detector channels and obtain an undisturbed cross correlation signal. The setup was tested with ECFP/EYFP lysates as well as chimeras as negative and positive controls and demonstrated to work in live HeLa cells coexpressing the two fusion proteins ECFP-connexin and EYFP-connexin.

Disassembly of structurally modified viral nanoparticles: characterization by fluorescence correlation spectroscopy. Toivola, J., Gilbert, L., Michel, P., White, D., Vuento, M., and Oker-Blom, C., C R Biol, 2005. 328(12): pp. 1052-6

Analysis of the breakdown products of engineered viral particles can give useful information on the particle structure. We used various methods to breakdown both a recombinant enveloped virus and virus-like particles (VLPs) from two non-enveloped viruses and analysed the resulting subunits by fluorescence correlation spectroscopy (FCS). Analysis of the enveloped baculovirus, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), displaying the green fluorescent protein (GFP) fused to its envelope protein gp64 was performed in the presence and absence of 5 mM SDS and 25 mM DTT. Without treatment, the viral particle showed a diffusion time of 3.3 ms. In the presence of SDS, fluorescent subunits with diffusion times of 0.2 ms were observed. Additional treatment with DTT caused a drop in the diffusion time to 0.1 ms. Changes in the amplitude of the autocorrelation function suggested a 3-fold increase in fluorescent particle number when viral particles were treated with SDS, and a further 1.5-fold increase with additional treatment with DTT. Thus, the data showed that an average of 4.5 molecules of gp64-GFP was incorporated in the membrane of the modified baculovirus. Further, this suggests that each fluorescent gp64 trimer carries on average 1.5 fluorescent units. Similar experiments were carried out with two non-enveloped fluorescent virus-like particles (fVLPs) that displayed enhanced green fluorescent protein (EGFP). These, fVLPs of canine and human B19 parvoviruses were treated with 6 M urea and 5 mM SDS, respectively. Correspondingly, the original hydrodynamic radii of 17 and 14 nm were reduced to 9 and 5 nm after treatment. Here, the change in the amplitude of the autocorrelation curve suggested a 10-fold increase in particle number when viral particles of CPV were treated with 6 M urea at 50 degrees C for 10 min. For EGFP-B19, there was a decrease in the amplitude, accompanied by a 9-fold increase in the number of fluorescent units with SDS treatment. The results showed that approximately 10 and 9 fluorescent units were associated with the corresponding CPV and B19 VLPs. In summary, we were able to estimate the number of fluorescent subunits in a baculovirus containing a GFP-fusion with its gp64 envelope protein and in two different parvo-VLPs containing EGFP-fused with their VP2 capsid proteins.

Study of molecular events in cells by fluorescence correlation spectroscopy. Vukojevic, V., Pramanik, A., Yakovleva, T., Rigler, R., Terenius, L., and Bakalkin, G., Cell Mol Life Sci, 2005. 62(5): pp. 535-50

To understand processes in a living cell, sophisticated and creative approaches are required that can be used for gathering quantitative information about large number of components interacting across temporal and spatial scales without major disruption of the integral network of processes. A physical method of analysis that can meet these requirements is fluorescence correlation spectroscopy (FCS), which is an ultrasensitive and non-invasive detection method capable of single-molecule and real-time resolution. Since its introduction about 3 decades ago, this until recently emerging technology has reached maturity. As commercially built equipment is now available, FCS is extensively applied for extracting biological information from living cells unattainable by other methods, and new biological concepts are formulated based on findings by FCS. In this review, we focus on examples in the field of molecular cellular biology. The versatility of the technique in this field is illustrated in studies of single-molecule dynamics and conformational flexibility of proteins, and the relevance of conformational flexibility for biological functions regarding the multispecificity of antibodies, modulation of activity of C5a receptors in clathrin-mediated endocytosis and multiplicity of functional responses mediated by the p53 tumor suppressor protein; quantitative characterization of physicochemical properties of the cellular interior; protein trafficking; and ligand-receptor interactions. FCS can also be used to study cell-to-cell communication, here exemplified by clustering of apoptotic cells via bystander killing by hydrogen peroxide.

Fluorescence correlation spectroscopy diffusion laws to probe the submicron cell membrane organization. Wawrezinieck, L., Rigneault, H., Marguet, D., and Lenne, P.F., Biophys J, 2005. 89(6): pp. 4029-42

To probe the complexity of the cell membrane organization and dynamics, it is important to obtain simple physical observables from experiments on live cells. Here we show that fluorescence correlation spectroscopy (FCS) measurements at different spatial scales enable distinguishing between different submicron confinement models. By plotting the diffusion time versus the transverse area of the confocal volume, we introduce the so-called FCS diffusion law, which is the key concept throughout this article. First, we report experimental FCS diffusion laws for two membrane constituents, which are respectively a putative raft marker and a cytoskeleton-hindered transmembrane protein. We find that these two constituents exhibit very distinct behaviors. To understand these results, we propose different models, which account for the diffusion of molecules either in a membrane comprising isolated microdomains or in a meshwork. By simulating FCS experiments for these two types of organization, we obtain FCS diffusion laws in agreement with our experimental observations. We also demonstrate that simple observables derived from these FCS diffusion laws are strongly related to confinement parameters such as the partition of molecules in microdomains and the average confinement time of molecules in a microdomain or a single mesh of a meshwork.

FCS cell surface measurements--photophysical limitations and consequences on molecular ensembles with heterogenic mobilities. Widengren, J. and Thyberg, P., Cytometry A, 2005. 68(2): pp. 101-12

BACKGROUND: Fluorescence Correlation Spectroscopy is a powerful method to analyze densities and diffusive behavior of molecules in membranes, but effects of photodegradation can easily be overlooked. METHOD: Based on experimental photophysical parameters, calculations were performed to analyze the consequences of photobleaching in fluorescence correlation spectroscopy (FCS) cell surface experiments, covering a range of standard measurement conditions. RESULTS: Cumulative effects of photobleaching can be prominent, although an absolute majority of the fluorescent molecules would pass the laser excitation beam without being photo-bleached. Given a distribution of molecules on a cell surface with different diffusive properties, the fraction of molecules that is actually analyzed depends strongly on the excitation intensities and measurement times, as well as on the size of the reservoir of freely diffusing molecules. Both the slower and the faster diffusing molecules can be disfavored. CONCLUSIONS: Apart from quantifying photobleaching effects, the calculations suggest that the effects can be used to extract additional information, for instance about the size of the reservoirs of free diffusion. By certain choices of measurement conditions, it may be possible to more specifically analyze certain species within a population, based on their different diffusive properties, different areas of free diffusion, or different kinetics of possible transient binding.

Consensus paper of the WFSBP Task Force on Biological Markers of Dementia: the role of CSF and blood analysis in the early and differential diagnosis of dementia. Wiltfang, J., Lewczuk, P., Riederer, P., Grunblatt, E., Hock, C., Scheltens, P., Hampel, H., Vanderstichele, H., Iqbal, K., Galasko, D., Lannfelt, L., Otto, M., Esselmann, H., Henkel, A.W., Kornhuber, J., and Blennow, K., World J Biol Psychiatry, 2005. 6(2): pp. 69-84

Aging of population, and increasing life expectancy result in an increasing number of patients with dementia. This symptom can be a part of a completely curable disease of the central nervous system (e.g, neuroinflammation), or a disease currently considered irreversible (e.g, Alzheimer's disease, AD). In the latter case, several potentially successful treatment approaches are being tested now, demanding reasonable standards of pre-mortem diagnosis. Cerebrospinal fluid and serum analysis (CSF/serum analysis), whereas routinely performed in neuroinflammatory diseases, still requires standardization to be used as an aid to the clinically based diagnosis of AD. Several AD-related CSF parameters (total tau, phosphorylated forms of tau, Abeta peptides, ApoE genotype, p97, etc.) tested separately or in a combination provide sensitivity and specificity in the range of 85%, the figure commonly expected from a good diagnostic tool. In this review, recently published reports regarding progress in neurochemical pre-mortem diagnosis of dementias are discussed with a focus on an early and differential diagnosis of AD. Novel perspectives offered by recently introduced technologies, e.g, fluorescence correlation spectroscopy (FCS) and surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) are briefly discussed.

Scanning fluorescence correlation spectroscopy: a tool for probing microsecond dynamics of surface-bound fluorescent species. Xiao, Y., Buschmann, V., and Weston, K.D., Anal Chem, 2005. 77(1): pp. 36-46

In this report, a combined imaging and fluorescence correlation spectroscopy (FCS) method is described and its ability to characterize microsecond fluctuations in the fluorescence emission of a sample is demonstrated. A sample scanning laser confocal microscope is operated in the customary way while recording the time that each photon is detected with a time resolution of 50 ns using a low-cost counting board. The serial data stream of photon detection times allows access to fluorescence signal fluctuations that can be used to characterize dynamics using correlation methods. The same data stream is used to generate images of the sample. Using the technique, we demonstrate that it is possible to characterize the kinetics of transitions to and from nonemitting or "dark" states of the fluorescent dyes DiIC(16) and ATTO 520. Results are similar to, but deviate slightly from, a model that has been frequently used for extracting singlet-triplet: conversion rates using conventional solution-based FCS. Like conventional FCS, the concentration, or in our case the areal density of coverage, of fluorescent species can also be obtained. Many single-molecule fluorescence experiments aim to extract kinetics from intensity trajectories; this method may be used as a rapid and convenient technique for characterization of surface-linked or thin-film samples prior to performing the more time and effort intensive single-molecule measurements. Besides the capacity to measure photophysical phenomena, the surface-sensitive FCS method could also be applied for measuring conformational changes or interaction kinetics for species immobilized on a surface. One possible scenario is measurements of the frequency and duration of association of ligand-receptor pairs where a fluorescently labeled component is freely diffusing and the other is surface immobilized. Given that microarrays of custom-designed, surface-immobilized peptides and nucleic acids are now readily available, the ability to sensitively measure association and dissociation rates of the surface-linked species with a freely diffusing species could be a useful extension to what has already become an extremely important tool for characterizing the interactions of biomolecules.

Blinking and nonradiant dark fraction of water-soluble quantum dots in aqueous solution. Yao, J., Larson, D.R., Vishwasrao, H.D., Zipfel, W.R., and Webb, W.W., Proc Natl Acad Sci U S A, 2005. 102(40): pp. 14284-9

Water-soluble quantum dots (qdots) are now being used in life sciences research to take advantage of their bright, easily excited fluorescence and high photostability. Although the frequent erratic blinking and substantial dark (never radiant) fractions that occur in all available qdots may interfere with many applications, these properties of individual particles in biological environments had not been fully evaluated. By labeling Qdot-streptavidin with organic dyes, we were able to distinguish individual dark and bright qdots and to observe blinking events as qdots freely diffused in aqueous solution. Bright fractions were measured by confocal fluorescence coincidence analysis (CFCA) and two-photon cross-correlation fluorescence correlation spectroscopy (FCS). The observed bright fractions of various preparations were proportional to the ensemble quantum yields (QYs), but the intrinsic brightness of individual qdots was found to be constant across samples with different QYs but the same emission wavelengths. Increasing qdots' illuminated dwell time by 10-fold during FCS did not change the fraction of apparently dark qdots but did increase the detected fraction of blinking qdots, suggesting that the dark population does not arise from millisecond blinking. Combining CFCA with wide-field imaging of arrays of qdots localized in dilute agarose gel, the blinking of qdots was measured across five orders of magnitude in time: approximately 0.001-100 s. This research characterizes photophysical pathologies of qdots in biologically relevant environments rather than adhered on dielectric surfaces and describes methods that are useful for studying various bioapplicable nanoparticles.

Electrically induced microflows probed by Fluorescence Correlation Spectroscopy. Ybert, C., Nadal, F., Salome, R., Argoul, F., and Bourdieu, L., Eur Phys J E Soft Matter, 2005

We report on the experimental characterisation of electrically induced flows at the micrometer scale through Fluorescence Correlation Spectroscopy (FCS) measurements. We stress the potential of FCS as a useful characterisation technique in microfluidics devices for transport properties cartography. The experimental results obtained in a model situation are in agreement with previous calculations (F. Nadal, F. Argoul, P. Kestener, B. Pouligny, C. Ybert, A. Ajdari, Eur. Phys. J. E 9, 387 (2002)) predicting the structure and electric-field dependency of the induced flow. Additionally, the present study evidences a complex behaviour of the probe nanobeads under electric field whose precise understanding might prove relevant for situations where nano-objects interact with an external electric field.

Investigation of a novel artificial antimicrobial peptide by fluorescence correlation spectroscopy: an amphipathic cationic pattern is sufficient for selective binding to bacterial type membranes and antimicrobial activity. Yu, L., Ding, J.L., Ho, B., and Wohland, T., Biochim Biophys Acta, 2005. 1716(1): pp. 29-39

Fluorescence Correlation Spectroscopy (FCS) is used to study the interaction of a recently designed antimicrobial peptide, called V4, with LPS and lipids of varying head and tail groups. V4 is designed based on a known amphipathic cationic pattern BHPHB (B: basic; H: hydrophobic; P: polar residue, respectively) and shows a good combination of high antimicrobial activity, low cytotoxic activity and low hemolytic activity. It is shown that V4 has high binding affinity for LPS, which is the major component of the outer membrane of Gram-negative bacteria, and shows selectivity for negatively charged lipids in contrast to zwitterionic lipids at a low peptide/lipid ratio. At high peptide/lipid ratio, V4 can permeabilize vesicles composed of negatively charged lipids and eventually cause vesicle fusion. The identification of the amphipathic cationic pattern as the mediator of selectivity and antimicrobial activity could be a first step in the rational design of better antimicrobial peptides.

Lipid diffusion compared in outer and inner leaflets of planar supported bilayers. Zhang, L. and Granick, S., J Chem Phys, 2005. 123(21): p. 211104

The translational diffusion coefficient (D) of lipids located in the outer and inner leaflets of planar supported DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine) bilayers in the fluid phase was measured using fluorescence correlation spectroscopy of dye-labeled lipids at the low concentration of 0.001% and using iodide quenching of dyes in the outer leaflet to distinguish diffusion in the inner leaflet from that in the outer leaflet. To confirm the generality of these findings, the bilayers were prepared not only by vesicle fusion but also by Langmuir-Blodgett deposition. We conclude that regardless of whether the bilayers were supported on quartz or on a polymer cushion, D in the inner and outer leaflets was the same within an experimental uncertainty of +/-10% but with a small systematic tendency to be slower (by <5%) within the inner leaflet.

Slaved diffusion in phospholipid bilayers. Zhang, L. and Granick, S., Proc Natl Acad Sci U S A, 2005. 102(26): pp. 9118-21

The translational diffusion of phospholipids in supported fluid bilayers splits into two populations when polyelectrolytes adsorb at incomplete surface coverage. Spatially resolved measurements using fluorescence correlation spectroscopy show that a slow mode, whose magnitude scales inversely with the degree of polymerization of the adsorbate, coexists with a fast mode characteristic of naked lipid diffusion. Inner and outer leaflets of the bilayer are affected nearly equally. Mobility may vary from spot to spot on the membrane surface, despite the lipid composition being the same. This work offers a mechanism to explain how nanosized domains with reduced mobility arise in lipid membranes.

Probing strong adsorption of solute onto C18-silica gel by fluorescence correlation imaging and single-molecule spectroscopy under RPLC conditions. Zhong, Z., Lowry, M., Wang, G., and Geng, L., Anal Chem, 2005. 77(8): pp. 2303-10

Understanding molecular adsorption at a chromatographic interface is of great interest for addressing the tailing problem in chemical separations. Single-molecule spectroscopy and confocal fluorescence correlation imaging are used to study the adsorption sites of C(18) silica beads under RPLC chromatographic conditions. The experiments show that cationic molecule rhodamine 6G laterally diffuses through the chromatographic interface of a C(18) hydrocarbon monolayer and acetonitrile with occasional reversible strong adsorptions. Fluorescence correlation imaging extracts the rare strong adsorption events from large data sets, revealing that the strong adsorption sites are randomly distributed throughout the silica beads. Virtually every imaging pixel of silica beads adsorbs molecules. Single-molecule spectroscopy of the 584 strong adsorption events observed indicates that the strong adsorptions persist on the time scales from several milliseconds to seconds, having an average desorption time of 61 ms. The strong adsorption events are rare, comprising 0.3% of the total observation time. The sizes of strong adsorption sites are within the optical resolution of confocal imaging.

Fluorescence quenching by TEMPO: a sub-30 A single-molecule ruler. Zhu, P., Clamme, J.P., and Deniz, A.A., Biophys J, 2005. 89(5): pp. L37-9

A series of DNA molecules labeled with 5-carboxytetramethylrhodamine (5-TAMRA) and the small nitroxide radical TEMPO were synthesized and tested to investigate whether the intramolecular quenching efficiency can be used to measure short intramolecular distances in small ensemble and single-molecule experiments. In combination with distance calculations using molecular mechanics modeling, the experimental results from steady-state ensemble fluorescence and fluorescence correlation spectroscopy measurements both show an exponential decrease in the quenching rate constant with the dye-quencher distance in the 10-30 A range. The results demonstrate that TEMPO-5-TAMRA fluorescence quenching is a promising method to measure short distance changes within single biomolecules.

2005 FCS abstracts