Deciphering fluorescence signals by quantifying separately the excitation intensity from the number of emitters

Conventional fluorescence detection is sensitive to an intricate product of the number of fluorescent emitters times the local excitation intensity. Here, we describe a method to locally quantify the excitation intensity and the number of emitters separately, enabling a clear distinction between the phenomena responsible for a given fluorescence signal. Our technique is based on harmonic excitation modulation and higher-order fluorescence demodulation. It is compatible with a wide range of observations, opening new characterization routes for fluorescence detection or imaging on complex systems, such as plasmonic substrates or scattering media.