Single molecule fluctuation spectroscopy

Single molecule fluctuation spectroscopy as a high throughput screening tool... 

In this chapter, we developed a stochastic theory of single molecule fluorescence spectroscopy. Fluctuations described by Q are evaluated in terms of a three-time correlation function C iXi, X2, T3) related to the response function in nonlinear spectroscopy. This function depends on the characteristics of the spectral diffusion process. Important time-ordering properties of the three-time correlation function were investigated here in detail. Since the fluctuations (i.e., Q) depend on the three-time correlation function, necessarily they contain more information than the line shape that depends on the one-time correlation function Ci(ti) via the Wiener-Khintchine theorem. 

Some interesting behavior in single-molecule spectroscopy involves the stochastic migration of lines. Usual statistical quantum theory describes only mean values or dispersions of observables, but not the actual fluctuations in the dynamics of single quantum systems. In an individual formalism of quantum mechanics, such fluctuations are of great importance. 

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. 

FCS and the utilization of fluorescence intensity as the fluctuating quantity was originally introduced in the 1970s with a series of papers presenting the theory and the first experimental realization of the technique [8-11]. In the first FCS experiments, the average number of fluorescent molecules in the observation volume was about 10,000. However, it was still possible to extract the motion of individual molecules from the large uncorrelated bulk fluorescence. In this particular respect, FCS is perhaps the first realization of fluorescence-based single-molecule spectroscopy. 

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... 

In fact, single molecule spectroscopy (SMS) experiments have recently become a reality. The first experiments were performed on pentacene (the chromophore) in a p-terphenyl crystal [8-10]. I will focus here on the experiments of Ambrose, Basche, and Moemer [9, 10], which involved repeated fluorescence excitation spectrum scans of the same chromophore. For each chromophore molecule they found an identical (except for its center frequency) Lorentzian line shape whose line width is determined by fast phonon-induced fluctuations (and by the excited state lifetime), as discussed above. However, for each of a number of different chromophore molecules Moemer and coworkers found that the chromophore s center frequency changed from scan to scan, reflecting spectral dynamics on the time scale of many seconds The transition frequencies of each of the chromophores seemed to sample a nearly infinite number of possible values. Plotting the transition frequency as a function of time produces what has been called a spectral diffusion trajectory (although the frequency fluctuations are not necessarily diffusive ). These fascinating and totally... 

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