Fluorescence correlation spectroscopy fluctuations

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. 

Haupts U, Maiti S, Schwille P, Webb WW (1998) Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy. Proc Natl Acad Sci USA 95 13573-13578... 

This technique does not require the equilibrium of the chemical system to be perturbed to measure the host-guest binding dynamics. Fluorescence correlation spectroscopy is based on the measurement of changes in the fluorescence intensity of individual molecules, which lead to intensity fluctuations.58 63 For this reason, the measurements are made by detecting the emission from a small sample volume (femtoliters to microliters) containing a small number of fluorophores. 

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. 

Fig. 11.10. Schematic illustration of fluorescence correlation spectroscopy. The autocorrelation function characterises the fluctuations of the fluorescence intensity its decay time expresses the average duration of a...

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). 

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... 

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. 

Figure 17.4 Principle of fluorescence correlation spectroscopy the fluorescence intensity temporal fluctuations originating from a well-defined volume are recorded and correlated to estimate the average number of molecules observed and the characteristic fluctuation time. This data is used to compute the average detected fluorescence rate per molecule in the observation volume.

In recent years, FRAP for diffusion measurements in membranes has been superseded by fluorescence correlation spectroscopy (FCS). FCS is very similar to FRAP in both theoretical and experimental approaches to the observation of diffusion. The difference between these two closely related techniques is that FRAP measures relaxation from an initial nonequilib-rium state after photobleaching, whereas FCS detects stochastic fluctuations that occur even in a system remaining in equilibrium (46). 

Summary. This chapter summarizes a series of new single-molecule spectroscopy investigations in the life sciences at Cornell University that began with our invention of Fluorescence Correlation Spectroscopy (FCS) about 1970. Our invention of FCS became my first focus on the Molecular Dynamics of Life. It motivated my transition from research on quantum fluctuations and transport in condensed matter physics including superconductivity and in the molecular dynamics of coherent fluctuations and nano-transport in inanimate physical and chemical systems subject to the nonlinear dynamics of continuous phase transitions. These interdisciplinary transitions exemplify the productivity of such interdisciplinary interactions in science. 

D. Magde, E. Elson, W.W. Webb, Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy. Phys. Rev. Lett. 29(11), 705-708 (1972)... 

H. Chen, E. Rhoades, J.S. Butler, S.N. Loh, W.W. Webb, Dynamics of equilibrium folding fluctuations of apomyoglobin measured by fluorescence correlation spectroscopy. PNAS 104(25), 10459-10464 (2007)... 

In fluorescence correlation spectroscopy (FCS) a small volume element or a small area) of a sample is illuminated by a laser beam and the autocorrelation function of fluctuations in the fluorescence is determined by photon counting. From this autocorrelation function the mean number densities of the fluorophores and their diffusion coefficients can be extracted. Measurement and analysis of higher order correlation functions of the fluorescence has been shown to yield information concerning aggregation states of fluorophores ). 

Figure 33.6 Illustration of confocal volume in fluorescence correlation spectroscopy (FCS) describing the experimental principle for evaluation of diffusion coefficients from the fluctuation of photon signals, (a) Fluctuation due to large and less mobile molecules is slow and...

Fluorescence correlation spectroscopy analyses the temporal fluctuations of the fluorescence intensity by means of an autocorrelation function from which translational and rotational diffusion coefficients, flow rates and rate constants of chemical processes of single molecules can be determined. For example, the dynamics of complex formation between /3-cyclodextrin as a host for guest molecules was investigated with singlemolecule sensitivity, which revealed that the formation of an encounter complex is followed by a unimolecular inclusion reaction as the rate-limiting step.263... 

Picosecond photon correlation experiments have some similarities to fluorescence correlation spectroscopy (PCS). PCS investigates the fluctuations of the fluorescence intensity of a small number of molecules confined in a small sample volume (see Sect. 5.10, page 176). The intensity fluctuations are correlated on a time scale from microseconds to milliseconds. Therefore, PCS differs from picosecond correlation in the way the photons are correlated. Moreover, PCS effects are driven by diffusion, conformational changes, or other sample-internal effects, while antibunching is driven by the absorption of the photons of the excitation light. 

Fluorescence correlation spectroscopy (FCS) is based on exciting a small number of molecules in a femtoliter volume and correlating the fluctuations of the fluorescence intensity. The fluctuations are caused by diffusion, rotation, intersystem crossing, conformational changes, or other random effects. The technique dates back to a work of Magde, Elson and Webb published in 1972 [335]. Theory and applications of FCS are described in [51, 429, 430, 431, 456, 457, 497, 537, 556]. 

Abstract We discuss recent experiments and theories concerning protein collapse and folding. Experiments using multicomponent solutions have revealed much about the mechanism of folding. Simulation and theory have been used to interpret thermodynamic and fluorescence correlation spectroscopy experimental results. We consider the theoretical arguments using variations of the free energy with respect to fluctuations in number and composition to consider recent experiments. We find new measures of protein stability tendencies offer a different view than the often poorly defined hydrophobic effect. 

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