Interpretation of fluorescence correlation spectra of biopolymer solutions

Abstract

Fluorescence correlation spectroscopy (FCS) is regularly used to study diffusion in non-dilute "crowded" biopolymer solutions, including the interior of living cells. For fluorophores in dilute solution, the relationship between the FCS spectrum G(t) and the diffusion coefficient D is well-established. However, the dilute-solution relationship between G(t) and D has sometimes been used to interpret FCS spectra of fluorophores in non-dilute solutions. Unfortunately, the relationship used to interpret FCS spectra in dilute solutions relies on an assumption that is not always correct in non-dilute solutions. This paper obtains the correct form for interpreting FCS spectra of non-dilute solutions, writing G(t) in terms of the statistical properties of the fluorophore motions. Approaches for applying this form are discussed.

Hydrodynamic size characterization of a self-emulsifying lipid pharmaceutical excipient by Taylor dispersion analysis with fluorescent detection

“…Another widely used technique, particularly in biological applications, is the fluorescence correlation spectroscopy (FCS). It is based on the measurement of the fluorescence fluctuations originating from a fluorescent probe diffusing in and out of a tiny observation volume (Phillies, 2016). The advantage of FCS is the possibility to work in dilute solutions due to the high sensitivity of the technique.…”

FCS and RICS Spectra of Probes in Complex Fluids

“…This paper is a continuation of our previous work on fluorescence correlation spectroscopy studies of probes in complex fluids 1 . The previous paper considered systems in which relaxations were moderately non-exponential, so that the time correlation function g (1) (q, t) = a q (0)a q (t) of a single spatial fourier component a q (t) of the fluorophore density at time t could effectively be described by the first few terms of its time cumulant expansion. While the cumulant expansion for g (1) (q, t) is always convergent, for severely non-exponential relaxations the cumulant expansion can become cumbersome.…”

“…The previous paper considered systems in which relaxations were moderately non-exponential, so that the time correlation function g (1) (q, t) = a q (0)a q (t) of a single spatial fourier component a q (t) of the fluorophore density at time t could effectively be described by the first few terms of its time cumulant expansion. While the cumulant expansion for g (1) (q, t) is always convergent, for severely non-exponential relaxations the cumulant expansion can become cumbersome. This paper considers an alternative approach to treating fluorescence correlation spectroscopy (FCS) spectra, beginning with the distribution function P (x, t) for a particle to diffuse a distance x during time t.…”

Mean squared displacement from fluorescence correlation spectroscopy

“…Very recently, a similar argument has been published by Phillies. 27 Only if H(r,t) is Gaussian, i.e. of the form…”

“…In the case of 0 o a o 1 or a 4 1, we attain subdiffusion or superdiffusion, respectively. In complex fluids, however, Gaussian approximation to the Van Hove correlation function is generally invalid 27,28 as soon as the underlying process is not normal diffusion. The form given in eqn (15) is just one of the infinite amount of possibilities, that lead to the subdiffusive scaling of MSD(t).…”