Fluorescence Correlation Spectroscopy: Criteria for Analysis in Complex Systems

Abstract

We have evaluated the effect of varying three key parameters for Fluorescence Correlation Spectroscopy analysis, first in the context of a one species/one environment system, and then in a complex system composed of two species, or conversely, two environments. We establish experimentally appropriate settings for the (1) minimum lag time, (2) maximum lag time, and (3) averaging times over which an autocorrelation is carried out, as a function of expected diffusion decay time for a particular solute, and show that use of appropriate settings plays a critical role in recovering accurate and reliable decay times and resulting diffusion constants. Both experimental and simulated data were used to show that for a complex binary system, to extract accurate diffusion constants for both species, decay times must be bounded by adequate minimum and maximum lag times as dictated by the fast and slow diffusing species, respectively. We also demonstrate that even when constraints on experimental conditions do not permit achieving the necessary lag time limits for both of the species in a binary system, the accuracy of the recovered diffusion constant for the one species whose autocorrelation function is fully time-resolved is unaffected by uncertainty in fitting introduced by the presence of the second species.

Fluorescence correlation spectroscopy in vivo

“…With increasing averaging time the obtained results for τ D and N converge. Depending on the necessary accuracy, the minimum measurement time can be chosen from the standard deviation on τ D and N. As a rule of thumb, the measurement time should at least be 10000 times longer than the diffusion time [9].…”

Permeability of anti-fouling PEGylated surfaces probed by fluorescence correlation spectroscopy

“…Many of the details of the sample preparation, setup, and theory have been previously reported [38–41]. 100 nm orange fluorescent carboxylate-modified FluoSphere beads (max abs/em: 540/560 nm) beads (1:1000 dilution) were used to determine the focal volume for the FCS measurements.…”

“…We used confocal FCS and single event analysis to quantify the presence and extent of interactions between PEG and the cationic Rhodamine 6G (R6G) and anionic AlexaFluor 555 ® (Alexa) dyes as a function of distance from the surface. We have successfully used these techniques to study transport at charged and crowded interfaces and with heterogeneous mixtures [38–41]. Others have recently reported the successful use of FCS to understand probe–polymer brush interactions [42].…”

Microscopy of Model Membranes

“…They arise from proximity to the coverslip, astigmatism, and optical saturation [84]. Because diffusion in membranes is slower than in solution, one needs to have longer measuring times to get statistically accurate results [85]. This longer measurement times make the system susceptible to instability.…”