TY - JOUR
T1 - Fluorescence correlation spectroscopy
T2 - Criteria for analysis in complex systems
AU - Tcherniak, Alexei
AU - Reznik, Carmen
AU - Link, Stephan
AU - Landes, Christy F.
PY - 2009/1/15
Y1 - 2009/1/15
N2 - 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.
AB - 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.
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U2 - 10.1021/ac8013109
DO - 10.1021/ac8013109
M3 - Article
AN - SCOPUS:64349085439
SN - 0003-2700
VL - 81
SP - 746
EP - 754
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 2
ER -