Stokes shift. A, upon excitation, 1, electrons in a fluorophore move from a resting state, S0 , to the excited electronic single state, S2. Some energy is released as heat, 2. The remaining energy is released as fluorescence, 3, as the electrons return to their ground state, S0. B, the difference between the excitation maxima,A , and the emissions maxima,C , of a fluorophore is called its Stokes shiftB .
Use of FMO controls to determine fluorescence spread and to set accurate gates. Dot plots showing the spread of fluorescence into the PE-Cy5 channel in the FMO-PE-Cy5 control compared to the unstained control allowing correct gating (black dotted line) for the fully stained sample.