Fluorescence Correlation Spectroscopy FCS

Under the same optical configuration, FCS (Fluorescence Correlation Spectroscopy) measurements (see Section 11.3) can be carried out on samples at the singlemolecule level under conditions where the average number of fluorescent molecules in the excitation volume is less than 1. It should be noted that at low fluorophore concentrations, the time required to obtain satisfactory statistics for the fluctuations may become problematic in practical applications (e.g. for a concentration of 1 fM, a fluorophore crosses a confocal excitation volume every 15 min). 

Abbreviations AOD, Acousto-optical deflection BCB, bisbenzyocyclobutadiene CCD, indirect contact conductivity detection CL, chemiluminescence ECD, electron capture detector FCS, fluorescence correlation spectroscopy FRET, fluorescence resonance energy transfer ICCD, integrated contact conductivity detection GMR, giant magnetoresistive LED-CFD, light emitting diode confocal fluorescence detector LIF, laser-induced fluorescence LOD, limit of detection MALDI, matrix-assisted laser desorption ionization PDMS, poly(dimethylsiloxane) PMMA, poly(methylmetha-crylate) SPR, surface plasmon resonance SVD, sinusoidal voltammetric detection TLS, thermal lens spectroscopy. 

FIGURE 4.1 Assays commonly used in GPCR research. SPA = scintillation proximity assay FP = fluorescence polarization TR-FRET = time-resolved fluorescence resonance energy transfer FCS = fluorescence correlation spectroscopy SeAP = secreted alkaline phosphate TF = transcription factor EFC = enzyme fragment complementation DMR = dynamic mass redistribution CDS = cellular dielectric spectroscopy. 

A = agar plate-based assay S = solution-based assay FRET = fluorescence resonance energy transfer FP = fluorescence polarization FCS = fluorescence correlation spectroscopy... 

Mets, U, in R Rigler and ES Elson (Eds), Antibunching and Rotational Difiusion in FCS. Fluorescence Correlation Spectroscopy TheoryandAppUcations.Spnn T, Berlin, 2001, pp.346 359. 

FRET fluorescence resonance energy transfer FCS fluorescence correlation spectroscopy TIRF total internal reflection fiuorescence PCFI photon counting histogram ICCD intensified charge coupled device EMCCD eiectron muitipiying charge coupled device CMOS complimentary metal oxide semiconductor AFM atomic force microscope. 

FCS fluorescence correlation spectroscopy PCH photon counting histogram TCSPC time correlated single photon counting MCS muiti-channei scaiar APDiavaianche photodiode PMT photo-mutipiiertube PCi peripherai component interconnect. 

FCS Fluorescence correlation spectroscopy FRET Fluorescence resonance energy transfer RFP Red fluorescent protein TPE Two-photon excitation... 

Concluding, FRAP has clearly shown its applicability in nuclear research and is expected to contribute largely to further unravelling and quantifying nuclear processes such as transcription, replication, RNA splicing and DNA repair. In addition, the combined application of FRAP with other quantitative techniques, like FRET (fluorescence resonance energy transfer) and FCS (fluorescence correlation spectroscopy) will be instrumental for future research of the functional organisation of the cell nucleus. 

Fluorescence intensity detected with a confocal microscope for the small area of diluted solution temporally fluctuates in sync with (i) motions of solute molecules going in/out of the confocal volume, (ii) intersystem crossing in the solute, and (hi) quenching by molecular interactions. The degree of fluctuation is also dependent on the number of dye molecules in the confocal area (concentration) with an increase in the concentration of the dye, the degree of fluctuation decreases. The autocorrelation function (ACF) of the time profile of the fluorescence fluctuation provides quantitative information on the dynamics of molecules. This method of measurement is well known as fluorescence correlation spectroscopy (FCS) [8, 9]. 

Fluorescence correlation spectroscopy (FCS) measures rates of diffusion, chemical reaction, and other dynamic processes of fluorescent molecules. These rates are deduced from measurements of fluorescence fluctuations that arise as molecules with specific fluorescence properties enter or leave an open sample volume by diffusion, by undergoing a chemical reaction, or by other transport or reaction processes. Studies of unfolded proteins benefit from the fact that FCS can provide information about rates of protein conformational change both by a direct readout from conformation-dependent fluorescence changes and by changes in diffusion coefficient. 

Czemey P, Lehmann F, Wenzel M, Buschmann V, Dietrich A, Mohr GJ (2001) Tailor-made dyes for fluorescence correlation spectroscopy (FCS). Biol Chem 382 495-498... 

In fluorescence correlation spectroscopy (FCS), the temporal fluctuations of the fluorescence intensity are recorded and analyzed in order to determine physical or chemical parameters such as translational diffusion coefficients, flow rates, chemical kinetic rate constants, rotational diffusion coefficients, molecular weights and aggregation. The principles of FCS for the determination of translational and rotational diffusion and chemical reactions were first described in the early 1970s. But it is only in the early 1990s that progress in instrumentation (confocal excitation, photon detection and correlation) generated renewed interest in FCS. 

TIRF Combined with Fluorescence Correlation Spectroscopy (FCS)... 

Gradl, G., Gunther, R., Sterrer, S. Fluorescence correlation spectroscopy (FCS) measuring biological interactions in microstructures. BioMethods 1999, 10, 331-351. 

Other fluorescence-based methods to investigate target-ligand interactions use fluorescence correlation spectroscopy (FCS) or fluorescence resonance energy transfer (FRET) [8, 12, 46]. 

The relatively new method of fluorescence correlation spectroscopy (FCS) is based on the fact that molecules with different molecular weights (usually) exhibit different diffusion times in solution. Thus, small molecules diffuse faster than larger ones. To determine K- values, one component must be labeled with a fluorescent dye. Due to the different molecular weights of the uncomplexed, labeled component and the complex, the diffusion times of the free and complexed molecule differ. This fact allows determining the distribution of free and complexed molecules in the solution. After measuring the distribution in different mixtures with varying ligand concentrations, the K- value can be calculated [44]. 

Dynamic processes at thermodynamic equilibrium that occur within a time range from sub-microseconds to seconds can be probed without the imposition of a transient disturbance by optical intensity fluctuation spectroscopy. As such, dynamic light scattering (DLS) [155] measures the fluctuation of quasielastic scattering intensity and fluorescence correlation spectroscopy (FCS) [156-158] measures concentration fluctuations of specific fluorescent molecules... 

Whether the components of the gene carriers actually remain associated during import into the nucleus or enter individually cannot be answered by optical methods as their resolution is limited. A possible technique to study the complexation of DNA within cells is fluorescence correlation spectroscopy (FCS). Clamme et al. studied the intracellular fate of PEI after transfection with polyplexes by two-photon fluorescence FCS [54]. They showed that PEI binds to the inner membrane of endosomes and lysosomes and shows free diffusion in the cytosol as well as the nucleus. However, they did not detect any PEI/DNA complexes inside the nucleus. 

Fluorescence Correlation Spectroscopy and Fluorescence Burst Analysis. Several nanoscopic chemical imaging approaches work very well for measurements of chemical kinetics, interactions, and mobility in solution. Fluorescence correlation spectroscopy (FCS) measures the temporal fluctuations of fluorescent markers as molecules diffuse or flow in solution through a femtoliter focal volume.54 Their average diffusive dwell times reveal their diffusion coefficients, and additional faster fluctuations can reveal chemical reactions and their kinetics if the reaction provides fluorescence modulation. Cross-correlation of the fluorescence of two distinguishable fluorophore types can very effectively reveal chemical binding kinetics and equilibria at nanomolar concentrations. 

FCS FT fMRI FTIR Fluorescence Correlation Spectroscopy Fourier Transform Functional Magnetic Resonance Imaging Fourier Transform Infrared Spectroscopy... 

The biophysical experiments of our cooperation partners focus mainly on two different techniques - a rough estimate of aggregation prevention is found by differential ultracentrifugation (UC), but more detailed information about the kinetics and aggregate size comes from fluorescence correlation spectroscopy (FCS) [24]. 

However, in an attempt to integrate the SFA and spectroscopic techniques, the use of silver for optical interferometry has been seen as a drawback due to the fact that it precluded sufficient excitation source intensity to illuminate the buried interface. In order to circumvent this problem Mukhopadhyay and co-workers in an experimental set-up where the SFA was combined with fluorescence correlation spectroscopy (FCS) used, instead of silver, multilayer dielectric coatings that allowed simultaneous interferometry and fluorescence measurements in different regions of the optical spectrum [75]. Using this set-up they succeeded in measuring diffusion in molecularly thin films with singlemolecule sensitivity. 

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