Separation of the rotational contribution in fluorescence correlation experiments

Abstract

The theory of fluorescence correlation spectroscopy is reexamined with the aim of separating the contribution of rotational diffusion. Under constant excitation, fluorescence correlation experiments are characterized by three polarizations: one of the incident beam and two of the two photon detectors. A set of experiments of different polarizations is proposed for study. From the results of the experiments the isotropic factor of the fluorescence intensity correlation functions can be determined, which is independent of the rotational motion of the sample molecule. This function can be used to represent each fluorescence intensity correlation function as the product of the isotropic and the rotational factors. The theory is illustrated by an experiment in which rotational diffusion of porcine pancreatic lipase labeled with Texas Red was observed Texas Red is a label that allows precise fluorescence correlation experiments even in the nanosecond time range.

Rotational and Translational Diffusion of Peptide-Coated CdSe/CdS/ZnS Nanorods Studied by Fluorescence Correlation Spectroscopy

“…7,14-16 With typical translational diffusion times being on the order of milliseconds, the contributions are well separated in time for rotational diffusion times on the order of microseconds. According to Kask et al 16 and Widengren et al 31 the rotational diffusion term of the total auto-correlation function can be separated from the translational diffusion term and to first approximation be expressed as a single-exponential function: (5) This analytical expression of the correlation function has been derived assuming sphericalshape diffusors (having a linear dipole emission) and fluorescence lifetimes much shorter than the rotational correlation times. The coefficient R depends on the experimental geometry and the degree of polarization of the fluorophore.…”

“…2. We compared the pol-FCS results for all NRs with calculations based on analytical expressions for the correlation function as presented by Kask et al 16 The translational and rotational diffusion coefficients of samples derived from the experimental pol-FCS data were used to calculate and plot theoretical FCS curves (Fig. 3).…”

“…pol-FCS curves derived from analytical expressions given by Kask et al 16 taking into account fitted rotational and translational diffusion constants as measured for 5×25 nm NRs (Fig. 1).…”

Polarized fluorescence correlation spectroscopy of DNA‐DAPI complexes

“…Previously, both FCS, and time-resolved fluorescence anisotropy of long lifetime probes have been used to study the rotational diffusion of small molecules in solution (Kask et al, 1987(Kask et al, , 1988Mets 2001). Time-resolved fluorescence anisotropy studies are limited to the lifetime of the probe, often in the nanosecond range.…”

“…Time-resolved fluorescence anisotropy studies are limited to the lifetime of the probe, often in the nanosecond range. However, there have been only a few experiments in FCS exploiting the polarization of the excitation to study the rotational motions of macromolecules free in solution (Kask et al, 1987(Kask et al, , 1988Mets, 2001). Since FCS is not limited by the lifetime of the probe, then there should be no size limit to the types of molecular aggregates that could be studied by polarized FCS experiments.…”