A FRET line is a projection of a parameterized model to a set of single-molecule FRET observables. Such FRET experiment typically jointly determined the fluorescence weighted average lifetime of the donor \(\langle \tau \rangle_f\) in the presence of an acceptor and the FRET efficiency, E, for every molecule. Next, a multidimensional frequency histogram of these parameters integrates the detected particles. Often, such histograms are called MFD histograms. The feature image shown to the top of this post displays a set of FRET lines of normally distributed donor-acceptor distances. The red and the blue line ranging from FRET efficiency zero to one corresponds to static FRET lines. Static FRET lines depend on the average donor and acceptor separation distance for a single state. The red line corresponds “broad” and the blue line to “narrow” donor-acceptor distance distributions. Below I will outline how to calculate a FRET line in ChiSurf.