Information Content in Fluorescence Correlation Spectroscopy: Binary Mixtures and Detection Volume Distortion

Abstract

When properly implemented, fluorescence correlation spectroscopy (FCS) reveals numerous static and dynamic properties of molecules in solution. However, complications arise whenever the measurement scenario is complex. Specific limitations occur when the detection region does not match the ideal Gaussian geometry ubiquitously assumed by FCS theory, or when properties of multiple fluorescent species are assessed simultaneously. A simple binary solution of diffusers, where both mole fraction and diffusion constants are sought, can face interpretive difficulty. In order to better understand the limits of FCS, this study systematically explores the relationship between detection-volume distortion, diffusion constants, species mole fraction, and fitting methodology in analyses that utilize a two-component autocorrelation model. FCS measurements from solution mixtures of dye-labeled protein and free dye are compared to simulations, which predict the performance of FCS under a variety of experimental circumstances. The results reveal a range of conditions necessary for performing accurate measurements and describe experimental scenarios that should be avoided. The findings also provide guidelines for obtaining meaningful measurements when grossly distorted detection volumes are utilized and generally assess the latent information contained in FCS datasets.

Proximal Capture Dynamics for a Single Biological Nanopore Sensor

“…Diffusion is simulated using Brownian dynamics software developed for modeling single-molecule translation in fluorescence correlation spectroscopy (FCS) over long time scales. , Routines from the single molecule diffusion simulator (SMDS) were adapted to implement diffusion with drift in the context of the geometric constraints typical of an αHL nanopore and the drift equations outlined above. The SMDS uses bulk physical parameters (such as the analyte diffusion constant, electrophoretic mobility, total transmembrane voltage, etc.)…”

“…The numerical vector map is then coupled with a Brownian dynamics computer simulation to track single-particle migration over large distances. 31,32 Previous computational studies have examined various dynamic aspects of translocation through αHL. 33−37 Most involve calculating many atom−atom interactions within a relatively small volume (∼10 3 nm 3 ) and are limited to short periods of time (10 −9 −10 −6 s).…”

“…This yields a vector map of analyte drift both inside and outside the nanopore. The numerical vector map is then coupled with a Brownian dynamics computer simulation to track single-particle migration over large distances. , …”

Analytical chemistry research at primarily undergraduate institutions: training tomorrow's investigators

“…For example, a recent publication applied kinetic models to ATR-UV-Vis and NIR measurements of small-batch slurry reactions to elucidate process chemistry, 17 and another group compared experimental results and simulations of uorescence correlation spectroscopy to provide insight on best practices. 18 In another study, a neural network was used to optimize conditions for a uorescence-based binding assay. 19 Other recent advances improve data analysis or lower costs.…”

Single-molecule assays for investigating protein misfolding and aggregation

“…[72][73][74] Extracting the distribution of species present is also non-trivial, even in systems with only two populations, where the accuracy of the results depends on the relative concentrations of the two components and their fluorescence quantum yield. 74,75 Although quantitative analysis of the aggregating species may be difficult, FCS has nevertheless proven to be a valuable tool for detecting early intermediates, their temporal progression, and at least their qualitative properties. The incorporation of new technical developments promises continuing improvements in the accuracy of FCS results.…”