In this article the first principles of frequency domain (FD) fluorescence lifetime imaging microscopy (FLIM) are further explained through use of equations. Although these principles not necessary for the execution of basic lifetime measurements, a thorough understanding provides the groundwork that enables deeper insight into your results and into the possibilities of FD lifetime imaging.

Fluorescence Lifetime 

For an ensemble of fluorescent molecules the rate at which molecules decay from the excited state to the ground state (cf. Fig.1) is proportional to the number of excited state molecules N: