Different fluorophores with distinct lifetimes can be discriminated because every species is associated with a precise phasor (the so-called “fingerprint”). Using the geometrical working principle at the basis of the phasor-plot, different lifetime values (a cloud of points) are distributed over a semicircle where the “longest” lifetime are on the left side, while the “shortest” ones on the right side of the plot.

Moreover, if the fingerprint cloud falls precisely on the semicircle, it means that the fluorescence signal can be described by a single exponential decay, while if the cloud falls within the semicircle area, the lifetime is a superposition of different values. Therefore, the FLIM-phasor approach is extremely straightforward for the interpretation of fluorescence lifetime fingerprints in the sample under investigation [2].