Fluorescence Correlation Spectroscopy as a Tool to Investigate Single Molecule Probe Dynamics in Thin Polymer Films

Abstract

Fluorescence correlation experiments were performed on rhodamine 6G in PDMS spin-coated films on glass surfaces. With polarised excitation, ensemble bleaching of the dye and single molecule intensity fluctuations were observed. From the statistics of single molecule intensity data taken at different positions in the film, correlation functions were calculated. Two modes of motion with exponential decay shapes and correlation times of tau(c) = 0.15 s and tau(c) = 0.7 s could be detected. Potential origins of intensity fluctuations are lateral diffusion, rotational diffusion or intramolecular fluctuations of dyes involving spectral diffusion or photoinduced processes. From the experimental results, lateral diffusion can be ruled out as a motional mode. Single molecule fluctuations are assigned to changes of the molecular configuration of the dyes, which are rigidly bound to the glass. To assess the environmental influence on such molecular motions, the bulk viscosity of the PDMS was varied over two orders of magnitude, leading to changes of tau(c) of the slow mode by a factor of four. This result proves the sensitivity of the single molecule fluctuations to the molecular scale dynamics of the surrounding polymer matrix and makes the correlation time a measure of the local environment of dye probes.

Probing Bio-Molecules across Polyelectrolyte Multilayers

“…To investigate out-of-plane transport in thin polyelectrolyte multilayers, other optical techniques such as the Fluorescence Resonance Energy Transfer (FRET) and Fluorescence Correlation Spectroscopy (FCS) are better alternatives [12][13][14][15][16]. For example, out-of-plane transport of chitosan (CH) has been observed in films of CH and heparin (HEP) by using FRET [12].…”

“…Fluorescence Correlation Spectroscopy is also among the optical technique, which provides valuable information on the diffusive polymeric exchange in solution surrounding PEM stack [14][15][16]. FCS is also known as Single Molecule Confocal Microscopy as it is very sensitive to very small molecular interactions within the PEM.…”

A New Dimension for the Development of Fluorescence-Based Assays in Solution: From Physical Principles of FCS Detection to Biological Applications

“…The value x is always a small integer, for example, x = 1 in Figure 3. Now, we use C to denote the (true) mean value of the population of specific fluorescent molecules (the average number) and summarize our approach of SPSM-FCS by [8] '""" -c l... -e P (X::s x;C) =--,-.…”

“…. For the first time, we have established this condensed, time-averaged Poisson-distributed relationship (Equation [8]). The analytical relationship of Equations [7] and [8] (see Table I in Ref.…”

“…For example, there are recent papers on the FCS calculations of Brownian motion and the factors that influence the observed results (7). In addition, effects of the local environment on dye probes were investigated (8). In such sensitive methods as FCS, the key issue is the signal-tonoise ratio.…”