Fluorescence correlation analysis

Schwille, P, Oehlenschlager, F, and Walter, NG, Quantitative hybridization kinetics of DNA probes to RNA in solution followed by diffusional fluorescence correlation analysis. Biochemistry 35 (1996) 10182-10193. 

The methodical elaboration is included for estimation of random and systematic errors by using of single factor dispersion analysis. For this aim the set of reference samples is used. X-ray analyses of reference samples are performed with followed calculation of mass parts of components and comparison of results with real chemical compositions. Metrological characteristics of x-ray fluorescence silicate analysis are established both for a-correction method and simplified fundamental parameter method. It is established, that systematic error of simplified FPM is less than a-correction method, if the correction of zero approximation for simplified FPM is used by preliminary established correlation between theoretical and experimental set data. 

The formal approach of 2D correlation analysis to time-dependent spectral intensity fluctuations has been extended to UV, Raman [1010], and near-IR spectroscopy [1011-1014] 2D fluorescence is upcoming. 

Fluorescence correlation spectroscopy can be used to measure the binding dynamics of host-guest complexes when the fluorescence quantum yields for the free and bound hosts are different. Analysis of fluorescence correlation spectra depends on the profile for the excitation pulse, which impacts the shape of the emission profile and mechanistic assumptions are made with respect to the diffusion of the various species in solution.58 For each chemical system different assumptions are made. 

Single Bioparticle Analysis with Fluorescence Correlation Spectroscopy... 

Rudenko, M. I. Kuhn, S. Lunt, E. J. Deamer, D. W. Hawkins, A. R. Schmidt, H., Ultrasensitive Q > Phase Analysis Using Fluorescence Correlation Spectroscopy on an Opto fluidic Chip, Biosensors and Bioelectronics 2009, 24, 3258 3263... 

G(t) decays with correlation time because the fluctuation is more and more uncorrelated as the temporal separation increases. The rate and shape of the temporal decay of G(t) depend on the transport and/or kinetic processes that are responsible for fluctuations in fluorescence intensity. Analysis of G(z) thus yields information on translational diffusion, flow, rotational mobility and chemical kinetics. When translational diffusion is the cause of the fluctuations, the phenomenon depends on the excitation volume, which in turn depends on the objective magnification. The larger the volume, the longer the diffusion time, i.e. the residence time of the fluorophore in the excitation volume. On the contrary, the fluctuations are not volume-dependent in the case of chemical processes or rotational diffusion (Figure 11.10). Chemical reactions can be studied only when the involved fluorescent species have different fluorescence quantum yields. 

Similar experiments have also been done with optical sensors, which do not come into physical contact with the plume of fluorescent dye. In this case, the geometrical arrangement of the array and the optimum selection of the appropriate sensor pairs for correlation analysis have played an important role (Fig. 10.20). 

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 8.9 Time-resolved fluorescent lifetime analysis of Cy3 attached to double-stranded DNA (Iqbal et al., 2008b). Fluorescent decay curve for Cy3 attached to a 16 bp DNA duplex, showing the experimental data and the instrument response function (IRF), and the fit to three exponential functions (line). The decay curve was generated using time-correlated single-photon counting, after excitation by 200 fs pulses from a titanium sapphire laser at 4.7 MHz.

Fluorescence-based detection methods are the most commonly used readouts for HTS as these readouts are sensitive, usually homogeneous and can be readily miniaturised, even down to the single molecule level.7,8 Fluorescent signals can be detected by methods such as fluorescence intensity (FI), fluorescence polarisation (FP) or anisotropy (FA), fluorescence resonance energy transfer (FRET), time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence intensity life time (FLIM). Confocal single molecule techniques such as fluorescence correlation spectroscopy (FCS) and one- or two-dimensional fluorescence intensity distribution analysis (ID FID A, 2D FIDA) have been reported but are not commonly used. 

Comparison of four CPPs— penetratin (5), Tat (6), transportan (12), and model amphipathic peptide (MAP) (13)— revealed that a model peptide cargo was most efficiently delivered into Bowes melanoma cells by MAP and transportan peptides. As judged by energy transfer experiments (31), the intracellular concentration of a cargo peptide delivered into cells by penetratin or Tat remained three- to fourfold lower compared with transportan- and MAP-mediated delivery. On the other hand, transportan and MAP were more noxious to cells and increased the plasma membrane permeability at lower concentrations. Import of penetratin sequences by the melanoma-derived SKMel-37 cells was in turn three- to fourfold more efficient than uptake of MTS-sequences as measured by fluorescence correlation spectroscopy in living cells and by FACS analysis (32). 

In addition to its exquisite sensitivity, other key advantages of ZnO NR platforms include ease of array fabrication, mechanical and chemical robustness, no autofluoroescence, and direct correlation of observed signal to protein concentration. Unlike other commonly used biosupport materials, this unique proper of ZnO NRs exhibiting no spectral overlap with fluorophores can be conveniently used in fluorescence data analysis. Fluorescence signal in the ZnO NR-assisted assays... 

Pyenta PS, Holowka D, Baird B. Cross-correlation analysis of inner-leatlet-anchored green fluorescent protein co-redistributed with IgE receptors and outer leaflet lipid raft components. Biophys. J. 2001 80 2120-2132. 

Single-molecule fluorescence detection was subsequently demonstrated at room temperature, first by detecting the burst of light as a molecule passes through the focus of a laser beam [67,68], but each molecule could be detected only once in this way. Correlation analysis of many such bursts provides a window into a variety of dynamical effects ranging from diffusion to intersystem crossing to rotational correlation [69], and this area termed fluorescence correlation spectroscopy (FCS, ([70-72]) has been reviewed in [73]. 

One limitation, however, is that only a limited number of spots can be measured simultaneously. A compromise between the temporal analysis of FCS and fluorescence fluctuation analysis in the spatial domain [39] can be obtained by exploiting the time structure of sample/laser scanning confocal microscope images [40,41]. Thereby, spatial correlation analysis of the emitted fluorescence is combined with temporal characterization of the fluorescence emission from the serial data stream of subsequently scanned pixels. This... 

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 ). 

Mikuni, S., Tamura, M. and Kinjo, M. (2007) Analysis of intranuclear binding process of glucocorticoid receptor using fluorescence correlation spectroscopy. FEBS Lett., 581, 389-393. 

Nagao, I., Aoki, Y., Tanaka, M. and Kinjo, M. (2008) Analysis of the molecular dynamics of medaka nuage proteins by fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. FEBS J., 275, 341-349. 

The foundations for fluorescence correlation spectroscopy (ECS) were already laid in the early 1970s, but this technique did not become widely used tmtil singlemolecule 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 d5mamic range covering... 

Rudenko MI, Kiihn S, Lunt EJ, Deamer DW, Hawkins AR, Schmidt H (2009) Ultrasensitive QP Phage analysis using fluorescence correlation spectroscopy on an optofluidic chip. Biosensors and Bioelectronics 24 3258-3263... 

Fluorescence correlation spectroscopy in nucleic acid analysis 01MI147. 

Another point to consider is that the penetration depth for fluorescent X-rays depends on their energy. Thus, the map for Ca will represent material closer to the surface than that of Zn (Fig. 20). This effect can distort the results of correlation analysis because the maps for different elements will represent different volumes. Since the incident beam is usually not normal to the surface, features deep inside the sample will appear out of registry with those nearer the surface. The net effect is as if the sample is viewed from the direction from which the beam comes, rather than straight on, as is usually the case for optical microscopy. 

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