Fluorescence fluctuations

The dye-loaded dendrimer 110 can be detected by means of single-molecule fluorescence.48,57,281,282 This multichromophoric system was embedded into a thin polymeric film at very low concentrations so that individual dendrimer molecules could be studied by far-field fluorescence microscopy at room temperature. Temporal fluorescence intensity fluctuations, fluorescence spectra, and decay times were measured separately or simultaneously. Collective on/off jumps of the fluorescence intensity as well as different emissive levels were observed for single dendrimer molecules with eight perylenemonoimide chromo-... 

Figure 2.2 Schematic illustration of the conceptual stages in the development of a model to fit photon counting histograms, (a) The case of a non-fluctuating fluorescent particle fixed at the centre of a closed excitation/detection volume (I/q). (b) The case when fluctuations are created by diffusion of the fluorescent molecule around a closed volume with spatially varying excitation/detection efficiency. (c)The case of multiple diffusing molecules in the closed volume, (d) The case when molecules can enter and leave the volume, (e) The case when molecules with different molecular brightness can enter and leave the volume.

Now consider the time dependence of the fluctuating fluorescence illustrated in Eig. 5.17C. Although the fluctuations are stochastic, they contain information on the dynamics of transitions between the on and off states in the model. This information can be extracted by calculating the autocorrelation function of the fluctuations, which is a measure of the correlation between the amplitude of the... 

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]. 

The autocorrelation function, G(x), of the temporal fluctuation of the fluorescence intensity at the confocal volume is analytically represented by the following equation [8, 9] ... 

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 (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. 

A laser beam highly focused by a microscope into a solution of fluorescent molecules defines the open illuminated sample volume in a typical FCS experiment. The microscope collects the fluorescence emitted by the molecules in the small illuminated region and transmits it to a sensitive detector such as a photomultiplier or an avalanche photodiode. The detected intensity fluctuates as molecules diffuse into or out of the illuminated volume or as the molecules within the volume undergo chemical reactions that enhance or diminish their fluorescence (Fig. 1). The measured fluorescence at time t,F(t), is proportional to the number of molecules in the illuminated volume weighted by the... 

Fig. 1. Schematic of an FCS experiment. For simplicity we consider an FCS measurement on a chemical reaction system confined to a plane, e.g., a membrane. The reaction is a two-state isomerization A (circles) B (squares). In the region of the plane illuminated by a laser beam (dark gray), A and B molecules appear white and light gray, respectively. Fluorescence fluctuations arise from interconversion of A and B and by A and B molecules diffusing into or out of the illuminated region. Molecules outside the illuminated region (black) are not detected.

In principle, FCS can also measure very slow processes. In this limit the measurements are constrained by the stability of the system and the patience of the investigator. Because FCS requires the statistical analysis of many fluctuations to yield an accurate estimation of rate parameters, the slower the typical fluctuation, the longer the time required for the measurement. The fractional error of an FCS measurement, expressed as the root mean square of fluorescence fluctuations divided by the mean fluorescence, varies as 1V-1/2, where N is the number of fluctuations that are measured. If the characteristic lifetime of a fluctuation is r, the duration of a measurement to achieve a fractional error of E = N l,/- is T = Nr. Suppose, for example, that r = 1 s. If 1% accuracy is desired, N = 104 and so T = 104 s. 

The spectroscopic properties of the solute-solvent system accounting fluctuations of solution structures can be analyzed using a simple model that includes a fluorescent solute and its immediate surrounding contributing to full potential of... 

Fluctuations, Relaxation, Heterogeneity, and Nonexponential Fluorescence Decay... 

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... 

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