An epi-illuminated microscope configuration for use in fluorescence correlation spectroscopy in bulk solutions has been analyzed. For determining the effective sample dimensions the spatial distribution of the molecule detection efficiency has been computed and conditions for achieving quasi-cylindrical sample shape have been derived. Model experiments on translational diffusion of rhodamine 6G have been carried out using strong focusing of the laser beam, small pinhole size and an avalanche photodiode in single photon counting mode as the detector. A considerable decrease in background light intensity and measurement time has been observed. The background light is 40 times weaker than the fluorescence signal from one molecule of Rh6G, and the correlation function with signal-to-noise ratio of 150 can be collected in 1 second. The effect of the shape of the sample volume on the autocorrelation function has been discussed.

Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion

http://www.biophysik.physik.uni-muenchen.de/download/biopheinf/seminar/Schwille_1997_JACS.pdf

Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution

In this paper, we outline a simple procedure for analyzing small angle neutron scattering (SANS) spectra from interacting colloids containing a continuous distribution of particle sizes. In particular, the effects of polydispersity on the apparent interparticle structure factor S′(Q) observed by SANS is investigated. We find that the oscillations in S′(Q) are significantly damped in comparison to those of the true structure factor S(Q). When our procedure is extended to the analysis of SANS spectra from nonspherical particles, we find that orientational averaging affects S′(Q) in a qualitatively similar way. The applicability of the procedure to the analysis of real data is demonstrated with spectra taken from water‐in‐oil microemulsions, ionic micelles, and interacting proteins.

Analysis of small angle neutron scattering spectra from polydisperse interacting colloids

XIAOTAO PAN,∗ CAILI AW,∗ YANAN DU,† HANRY YU†,‡ 7 and THORSTEN WOHLAND,∗,§ ∗Department of Chemistry, National University of Singapore, 9 3 Science Drive 3, Singapore 117543, Singapore †Institute of Bioengineering and Nanotechnology, A*STAR, 31 Biopolis Way, 11 The Nanos #04-01, Singapore 138669, Singapore ‡Department of Chemistry, National University of Singapore, 13 3 Science Drive 3, Singapore 117597, Singapore §chmwt@nus.edu.sg 15

Fluorescence correlation spectroscopy

Molecular Dynamics in Living Cells Observed by Fluorescence Correlation Spectroscopy with One- and Two-Photon Excitation

A general theory of fluorescence correlation spectroscopy (FCS), including the effects of translational and rotational motions and chemical reactions, in ideal solutions, is given. The development is carried out explicitly for symmetric rotors of parallel transition moments and generalized to arbitrary configurations of the moments and to asymmetric rotors in the appendices. In addition, the effects of experimental geometry on the measured FCS correlation function are discussed in detail and the optimum geometries, for experiments seeking to measure different quantities, are given. We conclude with a brief discussion of signal‐to‐noise ratios and prospects for applications.

Fluorescence correlation spectroscopy as a probe of molecular dynamics

Analytical expressions for the light scattering autocorrelation function of Schulz distributed Rayleigh–Debye particles are presented. The theory is shown to be valid for spheres, thin rods, disks, and Gaussian coils executing translational diffusion. The polydisperse form factors for these shapes are also given. Analysis of polydisperse data with the expressions given yields the diffusion coefficient of the number average species and the Schulz polydispersity parameter. The effects of polydispersity on the angular dependence of the decay time and the conditions under which the form factor can be neglected are discussed.

Theory of dynamic light scattering from polydisperse systems

Dynamique de polymere sans ecoulement, en solution diluee, a l'aide du modele de Kratky Porod

Dynamics of wormlike chains

The scattered electric field amplitude autocorrelation function of a dilute solution of large rigid anisotropic particles is computed in the Rayleigh–Debye approximation. The autocorrelation function for cylindrically symmetric particles is an infinite series of decaying exponentials with time constants τ−1l=q2D +l (l+1) Θ⊥ containing both translational and rotational diffusion coefficients. The dynamical terms are weighted by particle form factors and also by experimental geometry factors which are given in coordinate independent form. Explicit formulas for the form factors of spheres, rods, and thin disks are given. The general formulas reduce to the correct expressions for similar optically isotropic scattering systems.

S. R. Aragón

Papers

Fluorescence correlation spectroscopy as a probe of molecular dynamics

Theory of dynamic light scattering from polydisperse systems

Dynamics of wormlike chains

Theory of dynamic light scattering from large anisotropic particles

Forward depolarized dynamic light scattering from wormlike chains