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Fluorescence Correlation Spectroscopy (fluorescence + correlation_spectroscopy)

Distribution by Scientific Domains

Chemistry	66%
Life Sciences	27%

Selected Abstracts

Performance of Fluorescence Correlation Spectroscopy for Measuring Diffusion and Concentration

CHEMPHYSCHEM, Issue 11 2005
Jörg Enderlein Dr.
Abstract 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. [source]

Fluorescence correlation spectroscopy for the detection and study of single molecules in biology

BIOESSAYS, Issue 8 2002
Miguel Ángel Medina
The recent development of single molecule detection techniques has opened new horizons for the study of individual macromolecules under physiological conditions. Conformational subpopulations, internal dynamics and activity of single biomolecules, parameters that have so far been hidden in large ensemble averages, are now being unveiled. Herein, we review a particular attractive solution-based single molecule technique, fluorescence correlation spectroscopy (FCS). This time-averaging fluctuation analysis which is usually performed in Confocal setups combines maximum sensitivity with high statistical confidence. FCS has proven to be a very versatile and powerful tool for detection and temporal investigation of biomolecules at ultralow concentrations on surfaces, in solution, and in living cells. The introduction of dual-color cross-correlation and two-photon excitation in FCS experiments is currently increasing the number of promising applications of FCS to biological research. BioEssays 24:758,764, 2002. © 2002 Wiley Periodicals, Inc. [source]

Performance of Fluorescence Correlation Spectroscopy for Measuring Diffusion and Concentration

Probing events with single molecule sensitivity in zebrafish and Drosophila embryos by fluorescence correlation spectroscopy

DEVELOPMENTAL DYNAMICS, Issue 12 2009
Xianke Shi
Abstract Zebrafish and Drosophila are animal models widely used in developmental biology. High-resolution microscopy and live imaging techniques have allowed the investigation of biological processes down to the cellular level in these models. Here, using fluorescence correlation spectroscopy (FCS), we show that even processes on a molecular level can be studied in these embryos. The two animal models provide different advantages and challenges. We first characterize their autofluorescence pattern and determine usable penetration depth for FCS especially in the case of zebrafish, where tissue thickness is an issue. Next, the applicability of FCS to study molecular processes is shown by the determination of blood flow velocities with high spatial resolution and the determination of diffusion coefficients of cytosolic and membrane-bound enhanced green fluorescent protein,labeled proteins in different cell types. This work provides an approach to study molecular processes in vivo and opens up the possibility to relate these molecular processes to developmental biology questions. Developmental Dynamics 238:3156,3167, 2009. © 2009 Wiley-Liss, Inc. [source]

Inhibition of human ether à go-go potassium channels by Ca2+/calmodulin binding to the cytosolic N- and C-termini

FEBS JOURNAL, Issue 5 2006
Ulrike Ziechner
Human ether à go-go potassium channels (hEAG1) open in response to membrane depolarization and they are inhibited by Ca2+/calmodulin (CaM), presumably binding to the C-terminal domain of the channel subunits. Deletion of the cytosolic N-terminal domain resulted in complete abolition of Ca2+/CaM sensitivity suggesting the existence of further CaM binding sites. A peptide array-based screen of the entire cytosolic protein of hEAG1 identified three putative CaM-binding domains, two in the C-terminus (BD-C1: 674,683, BD-C2: 711,721) and one in the N-terminus (BD-N: 151,165). Binding of GST-fusion proteins to Ca2+/CaM was assayed with fluorescence correlation spectroscopy, surface plasmon resonance spectroscopy and precipitation assays. In the presence of Ca2+, BD-N and BD-C2 provided dissociation constants in the nanomolar range, BD-C1 bound with lower affinity. Mutations in the binding domains reduced inhibition of the functional channels by Ca2+/CaM. Employment of CaM-EF-hand mutants showed that CaM binding to the N- and C-terminus are primarily dependent on EF-hand motifs 3 and 4. Hence, closure of EAG channels presumably requires the binding of multiple CaM molecules in a manner more complex than previously assumed. [source]

Bilayer localization of membrane-active peptides studied in biomimetic vesicles by visible and fluorescence spectroscopies

FEBS JOURNAL, Issue 22 2003
Tanya Sheynis
Depth of bilayer penetration and effects on lipid mobility conferred by the membrane-active peptides magainin, melittin, and a hydrophobic helical sequence KKA(LA)7KK (denoted KAL), were investigated by colorimetric and time-resolved fluorescence techniques in biomimetic phospholipid/poly(diacetylene) vesicles. The experiments demonstrated that the extent of bilayer permeation and peptide localization within the membrane was dependent upon the bilayer composition, and that distinct dynamic modifications were induced by each peptide within the head-group environment of the phospholipids. Solvent relaxation, fluorescence correlation spectroscopy and fluorescence quenching analyses, employing probes at different locations within the bilayer, showed that magainin and melittin inserted close to the glycerol residues in bilayers incorporating negatively charged phospholipids, but predominant association at the lipid,water interface occurred in bilayers containing zwitterionic phospholipids. The fluorescence and colorimetric analyses also exposed the different permeation properties and distinct dynamic influence of the peptides: magainin exhibited the most pronounced interfacial attachment onto the vesicles, melittin penetrated more into the bilayers, while the KAL peptide inserted deepest into the hydrophobic core of the lipid assemblies. The solvent relaxation results suggest that decreasing the lipid fluidity might be an important initial factor contributing to the membrane activity of antimicrobial peptides. [source]

Dynamics of yeast prion aggregates in single living cells

GENES TO CELLS, Issue 9 2006
Shigeko Kawai-Noma
Prions are propagating proteins that are ordered protein aggregates, in which the phenotypic trait is retained in the altered protein conformers. To understand the dynamics of the prion aggregates in living cells, we directly monitored the fate of the aggregates using an on-chip single-cell cultivation system as well as fluorescence correlation spectroscopy (FCS). Single-cell imaging revealed that the visible foci of yeast prion Sup35 fused with GFP are dispersed throughout the cytoplasm during cell growth, but retain the prion phenotype. FCS showed that [PSI+] cells, irrespective of the presence of foci, contain diffuse oligomers, which are transmitted to their daughter cells. Single-cell observations of the oligomer-based transmission provide a link between previous in vivo and in vitro analyses of the prion and shed light on the relationship between the protein conformation and the phenotype. [source]

Spatial fluorescence cross-correlation spectroscopy by means of a spatial light modulator

JOURNAL OF BIOPHOTONICS, Issue 5 2008
Yoann Blancquaert
Abstract Spatial fluorescence cross-correlation spectroscopy is a rarely investigated version of fluorescence correlation spectroscopy, in which the fluorescence signals from different observation volumes are cross-correlated. In the reported experiments, two observation volumes, typically shifted by a few ,m, are produced, with a spatial light modulator and two adjustable pinholes. We illustrated the feasibility and potentiality of this technique by: i) measuring molecular flows, in the range 0.2,1.5 ,m/ms, of solutions seeded with fluorescent nanobeads or rhodamine molecules (simulating active transport phenomenons); ii) investigating the permeability of the phospholipidic membrane of giant unilamellar vesicles versus hydrophilic or hydrophobic molecules (in that case the laser spots were set on both sides of the membrane). Theoretical descriptions are proposed together with a discussion about fluorescence-correlation-spectroscopy-based, alternative methods. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source]

The oligomeric state and stability of the mannitol transporter, EnzymeIImtl, from Escherichia coli: A fluorescence correlation spectroscopy study

PROTEIN SCIENCE, Issue 8 2006
Gertjan Veldhuis
Abstract Numerous membrane proteins function as oligomers both at the structural and functional levels. The mannitol transporter from Escherichia coli, EnzymeIImtl, is a member of the phosphoenolpyruvate-dependent phosphotransferase system. During the transport cycle, mannitol is phosphorylated and released into the cytoplasm as mannitol-1-phosphate. Several studies have shown that EIImtl functions as an oligomeric species. However, the oligomerization number and stability of the oligomeric complex during different steps of the catalytic cycle, e.g., substrate binding and/or phosphorylation of the carrier, is still under discussion. In this paper, we have addressed the oligomeric state and stability of EIImtl using fluorescence correlation spectroscopy. A functional double-cysteine mutant was site-specifically labeled with either Alexa Fluor 488 or Alexa Fluor 633. The subunit exchange of these two batches of proteins was followed in time during different steps of the catalytic cycle. The most important conclusions are that (1) in a detergent-solubilized state, EIImtl is functional as a very stable dimer; (2) the stability of the complex can be manipulated by changing the intermicellar attractive forces between PEG-based detergent micelles; (3) substrate binding destabilizes the complex whereas phosphorylation increases the stability; and (4) substrate binding to the phosphorylated species partly antagonizes the stabilizing effect. [source]

Fluorescence correlation spectroscopy for the detection and study of single molecules in biology

Review fluorescence correlation spectroscopy for probing the kinetics and mechanisms of DNA hairpin formation

BIOPOLYMERS, Issue 1 2008
Alan Van Orden
Abstract This article reviews the application of fluorescence correlation spectroscopy (FCS) and related techniques to the study of nucleic acid hairpin conformational fluctuations in free aqueous solutions. Complimentary results obtained using laser-induced temperature jump spectroscopy, single-molecule fluorescence spectroscopy, optical trapping, and biophysical theory are also discussed. The studies cited reveal that DNA and RNA hairpin folding occurs by way of a complicated reaction mechanism involving long- and short-lived reaction intermediates. Reactions occurring on the subnanoseconds to seconds time scale have been observed, pointing out the need for experimental techniques capable of probing a broad range of reaction times in the study of such complex, multistate reactions. © 2007 Wiley Periodicals, Inc. Biopolymers 89: 1,16, 2008. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source]

Red-Emitting Rhodamine Dyes for Fluorescence Microscopy and Nanoscopy

CHEMISTRY - A EUROPEAN JOURNAL, Issue 1 2010
Kirill Kolmakov Dr.
Abstract Fluorescent markers emitting in the red are extremely valuable in biological microscopy since they minimize cellular autofluorescence and increase flexibility in multicolor experiments. Novel rhodamine dyes excitable with 630,nm laser light and emitting at around 660,nm have been developed. The new rhodamines are very photostable and have high fluorescence quantum yields of up to 80,%, long excited state lifetimes of 3.4,ns, and comparatively low intersystem-crossing rates. They perform very well both in conventional and in subdiffraction-resolution microscopy such as STED (stimulated emission depletion) and GSDIM (ground-state depletion with individual molecular return), as well as in single-molecule-based experiments such as fluorescence correlation spectroscopy (FCS). Syntheses of lipophilic and hydrophilic derivatives starting from the same chromophore-containing scaffold are described. Introduction of two sulfo groups provides high solubility in water and a considerable rise in fluorescence quantum yield. The attachment of amino or thiol reactive groups allows the dyes to be used as fluorescent markers in biology. Dyes deuterated at certain positions have narrow and symmetrical molecular mass distribution patterns, and are proposed as new tags in MS or LC-MS for identification and quantification of various substance classes (e.g., amines and thiols) in complex mixtures. High-resolution GSDIM images and live-cell STED-FCS experiments on labeled microtubules and lipids prove the versatility of the novel probes for modern fluorescence microscopy and nanoscopy. [source]

Host,Guest Interaction of Chaperonin GroEL and Water-Soluble CdTe Quantum Dots and its Size-Selective Inclusion

CHEMPHYSCHEM, Issue 15 2008
Chaoqing Dong
Abstract Some nanoparticles, such as quantum dots (QDs), are widely used in the biological and biomedical fields due to their unique optical properties. However, little is currently known about the interaction between these nanoparticles and biomolecules. Herein, we systemically investigated the interaction between chaperonin GroEL and water-soluble CdTe QDs based on fluorescence correlation spectroscopy (FCS), capillary electrophoresis, and fluorescence spectrometry. We observed that some water-soluble CdTe QDs were able to enter the inner cavity of GroEL and formed an inclusion complex after the activation of chaperonin GroEL with ATP. The inclusion of GroEL was size-selective to QDs and only small QDs were able to enter the inner cavity. The inclusion could suppress the fluorescence quenching of the QDs. Meanwhile, we evaluated the association constant between chaperonin GroEL and CdTe QDs by FCS. Our results further demonstrated that FCS was a very useful tool for study of the interaction of QDs and biomolecules. [source]

Combined AFM and Two-Focus SFCS Study of Raft-Exhibiting Model Membranes,

CHEMPHYSCHEM, Issue 11 2006
Salvatore Chiantia
Abstract Dioleoylphosphatidylcholine/sphingomyelin/cholesterol (DOPC/SM/cholesterol) model membranes exhibit liquid,liquid phase separation and therefore provide a physical model for the putative liquid-ordered domains present in cells. Here we present a combination of atomic force microscopy (AFM) imaging, force measurements, confocal fluorescence imaging and two-focus scanning fluorescence correlation spectroscopy (two-focus SFCS) to obtain structural and dynamical information about this model membrane system. Partition coefficients and diffusion coefficients in the different phases were measured with two-focus SFCS for numerous fluorescent lipid analogues and proteins, while being directly related to the lateral organization of the membrane and its mechanical properties probed by AFM. Moreover we show how the combination of these different approaches is effective in reducing artifacts resulting from the use of a single technique. [source]

A Close Look at Fluorescence Quenching of Organic Dyes by Tryptophan

CHEMPHYSCHEM, Issue 11 2005
Sören Doose Dr.
Abstract 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 kdof 4.0×109s,1,M,1, 2) static quenching with a bimolecular association constant Ksof 61,M,1, and 3) a sphere-of-action contribution to static quenching described by an exponential factor with a quenching constant , of 22,M,1. The latter two are characterized as nonfluorescent complexes, formed with ,30,% efficiency upon encounter, that are stable for tens of nanoseconds. The measured binding energy of 20,30 kJmol,1is 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. [source]

Photophysical Aspects of Single-Molecule Detection by Two-Photon Excitation with Consideration of Sequential Pulsed Illumination

CHEMPHYSCHEM, Issue 5 2004
R. Niesner
Abstract An important goal in single molecule fluorescence correlation spectroscopy is the theoretical simulation of the fluorescence signal stemming from individual molecules and its autocorrelation function. The simulation approaches developed up to now are based exclusively on continuous-wave (cw) illumination and consequently on cw-excitation. However, this approximation is no longer valid in the case of two-photon excitation, for which pulsed illumination is usually employed. We present a novel theoretical model for the simulation of the fluorescence signal of single molecules and its autocorrelation function with consideration of the time dependence of the excitation flux and thus of all illumination-dependent photoprocesses: two-photon excitation, induced emission and photobleaching. Further important characteristics of our approach are the consideration of the dependence of the photobleaching rate on illumination and the low intersystem-crossing rates of the studied coumarins. Moreover, using our approach, we can predict quantitatively the effect of the laser pulse width on the fluorescence signal of a molecule, that is, the contributions of the photobleaching and saturation effects, and thus we can calculate the optimal laser pulse width. The theoretical autocorrelation functions were fitted to the experimental data, and we could ascertain a good agreement between the resulting and the expected parameters. The most important parameter is the photobleaching constant ,, the cross section of the transition Sn,S1, which characterises the photostability of the molecules independent of the experimental conditions. Its value is 1.7×10,23 cm2 for coumarin 153 and 5×10,23 cm2 for coumarin 314. [source]