![]() Here, the diffusion coefficient and molecular brightness of R6G have been held fixed at the correct value during fitting. Concentrations are considered local fit parameters while diffusion coefficients and molecular brightness are global. Shown here are experimental autocorrelation data of binary RhB and R6G mixtures, with individually calibrated parameters (a), subject to global analysis of FCS data only (no lifetime data) incorporating repeated titration data sets. 3, we show here that global analysis of FCS data alone does not return comparable accuracy, or even stable fitting results. To demonstrate that multi-method global analysis, here implemented as τFCS, is the key to the accuracy of the results shown in Fig. Data points and error bars indicate the average and standard deviation of three independent simulated data sets.įigure S4: Comparison of initial guesses in global analysis of experimental FCS data only. τFCS also returns accurate molecular brightnesses (c) and fluorescence lifetimes (d) across the majority of the titration range, and still distinguishes the diffusion coefficient where FCS analysis fails (e gold triangles). τFCS analysis (b gold data points) of the same titration data set, in which no assumptions or held parameters are enforced on the 2nd species, returns accurate results across the entire range, even in the case of a very small fraction of the less bright species (gold circles). This is corroborated by the transition of the returned diffusion coefficient, D b, from 0.1 to 0.3 µm 2ms −1 (e all blue data points). Beyond the C a/C b of approximately 3, FCS analysis fails to identify two species and transitions into a fit result that finds two identical species of equal concentration, that of half the total. Across concentration ratios C a/C b of 0.03 to 1, in which the amount of unknown species is sufficiently large, FCS analysis can distinguish the 2nd component, albeit with molecular brightness guess dependent errors. Three different analyses (b) using ψ b guesses below (ψ b = 8 kcpsm horizontal kites), the same as (ψ b = 10 kcpsm diamonds), and above (ψ b = 12.5 kcpsm horizontal kites) the correct molecular brightness value highlight the potential inaccuracies in two component FCS results. The covariant autocorrelation amplitudes require that the molecular brightnesses be held for both species using FCS analysis therefore, we have “guessed” ψ b in order to attain stable fits. Here, we have ‘calibrated’ species a and fixed the known values for D a, ψ a and τ a during all subsequent analyses. Comparison of simulated data sets depicting a binary system with a diffusion coefficient ratio of 3, molecular brightness and lifetime ratios of 2 (a), and titrated across a concentration ratio of three orders of magnitude (b). Figure S3: The effects of molecular brightness assumptions in two component FCS analyses compared to τFCS. ![]()
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