Fluorescence Correlation Spectroscopy in Small Cytosolic Compartments Depends Critically on the Diffusion Model Used

Abstract

Fluorescence correlation spectroscopy (FCS) is a powerful technique for measuring low concentrations of fluorescent molecules and their diffusion constants. In the standard case, fluorescence fluctuations are measured in an open detection volume defined by the confocal optics. However, if FCS measurements are carried out in cellular processes that confine the detection volume, the standard FCS model leads to erroneous results. In this paper, we derive a modified FCS model that takes into account the confinement of the detection volume. Using this model, we have carried out the first FCS measurements in dendrites of cultured neurons. We further derive, for the case of confined diffusion, the limits within which the standard two-and three-dimensional diffusion models give reliable results.

Near-field optical fluorescence correlation spectroscopy

“…If the particle is initially located at position z than the probability density p to find the particle after a time lag τ at the location z ′ is given by [65], [105] p(z, z…”

Simultaneous two‐photon fluorescence correlation spectroscopy and lifetime imaging of dye molecules in submicrometer fluidic structures

“…In a channel with the width similar to or several times larger than the horizontal width of the measurement volume, the effects of the channel walls on the autocorrelation cannot be neglected; the diffusion in the perpendicular direction (along the y axis) is confined by the channel walls. Gennerich and Schild (2000) derived a model taking into account the confinement by the walls, and calculated an approximation of this model useful for data analysis: In a situation where the excitation beam is moving uniformly along a line, the fluorescence autocorrelation is naturally affected by this movement. The description is equivalent to a uniform flow of molecules in addition to diffusion (Kuricheti et al, 2004).…”

“…The size reduction and better confinement of the measurement volume improves the signal-tonoise ratio and permits measurements at higher biochemically and physiologically relevant concentrations. However, impenetrable boundaries near the measurement volume affect the diffusion of the molecules and the theoretical description of the autocorrelation curve subsequently becomes more complicated (Gennerich and Schild, 2000).…”

Tracking-FCS: Fluorescence correlation spectroscopy of individual particles

“…In our experiment, microspheres are confined in the z direction by the boundaries of the sample volume, which we may take to be reflecting boundaries for the relevant case of low surface adhesion. Green's functions for free particle motion and corresponding fluorescence autocorrelation functions have been calculated as an infinite series for a Gaussian beam focused symmetrically between two reflecting planes [2]; the resulting series have been approximated efficiently in [27], but we are unaware of an extension of these results to reflecting planes in an asymmetric configuration. Instead of tackling this difficult analytical problem, we instead approximate the reflecting boundaries in a way that naturally fits the formalism developed here: we introduce a third Ornstein-Uhlenbeck particle trap in the z dimension with corresponding rate γ z .…”