Information theoretic FRET calibration on the cell surface

•In-situ, stand-alone calibration of 3 channel-ratiometric FRET using only the FRET sample to determine the α (G) factor.•Measurement theory of 3 channel-FRET has been extended with information theory of Shannon in a flow cytometer.•Information contents of flow cytometric distributions measured on the bit scale lead to balance equations in terms of α.•Information balances have been based on the data processing law, equipartition of information and Cramer-Rao bound.•The α factor is supplied by the solution of the informational balance equations via extremum finding or optimization.