Maximum position of Shannon-information of FRET frequency distribution represented as a function of the α (or G) calibrating factor as calculated in the framework of dual-laser flow cytometric FRET method – also called 3-cube FRET – gives a fairly good approximation of the real α, using only a single FRET sample. The estimation is accurate for completely symmetric FRET distributions like Gaussian and uniform. For non-symmetric distributions, the committed error has been found to depend on the direction and degree of deviation from the complete symmetry. The maximum information is characterized also by zero skewness, and minima in kurtosis and in the χ-value, defined as the “absolute deviation from the Gaussian”, as other α- functions characterizing shape. As to practice, information maximum of FRET distribution can be used to determine α, whenever symmetry can be assumed. In other cases, the goodness of estimation can be inferred from the information content and shape parameters (skewness, kurtosis and “χ-value”) of the primarily measured FRET parameter A′, based on their inverse correlations with their counterparts computed with the target FRET distribution. Above a threshold in FRET efficiency or its coefficient of variation, resonant peaks (“Ω-curves”) in FRET information appear on the α-scale, with their positions stabilized around α values with reduced fluctuations dictated by symmetry of the target FRET distribution. The phenomenon is explained by a FRET-induced broadening of the A′ distribution reaching the domain containing A′ = −α.
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