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.
Time-resolved fluorescence spectroscopy experiments track the emission of fluorescence light from a sample. Time-resolved fluorescence intensities are also called fluorescence decays. Analysis thereof may resolve composite samples and report on dynamics (citations). Excited fluorescent molecules stay a particular time in their excited state before emitting light and delay the detection of fluorescence. This “delay” depends on molecular properties. Hence, time-resolved fluorescence informs on the studied molecules.
One might think the analysis of a fluorescence decay is easy. In the end, it’s just standard curve analysis. However, the art is to precisely describe the fluorescence decay down to the shot-noise of the experiment. When doing so, a number experimental aspects have to be considered for the analysis: the instrumental response function, IRF, the differential non-linearity of the device, DNL, pile-up effects, etc.