FCS stands for fluorescence correlations spectroscopy (FCS). FCS is a method which relies on fluctuations on the recorded signals to characterize molecular interaction such as binding and unbinding, chemical reaction kinetics, diffusion of fluorescent molecules. 1,2

Elson EL, Magde D. Fluorescence Correlation Spectroscopy. I. Conceptual Basis and Theory. Biopolymers. 1974;13:1-27.
Magde D, Elson EL, Webb WW. Thermodynamic fluctuations in a reacting system – measurement by fluorescence correlation spectroscopy. Physical Review Letters. 1972;29:705-708.


FRET positioning system, FPS, is an approach to determine structural models based on a set of FRET measurements. FPS explicitly considers the spatial distribution of the dyes. This way experimental observables, i.e., FRET efficiencies may be predicted precisely.

The FPS-toolkit is available from the web page of the Seidel group of the Heinrich Heine University.


Förster resonance energy transfer (FRET) is a spectroscopic process where energy between fluorophores is transferred through distance and orientation dependent dipolar coupling.

FRET efficiency

The FRET efficiency is the yield of a FRET process. A FRET process transfers energy from the excited state of a donor fluorophore to an acceptor fluorophore. The number of donor molecules in an excited state which transfers energy to an acceptor defines the yield of this energy transfer.

E = \frac{transfered}{excited}

Practically, mostly the donor and acceptor fluorescence intensities are used to recover this yield.

FRET line

A FRET line relates at least two independent FRET observables through the change of a parameter describing the system. Typically, a FRET line connects the average FRET efficiency and the fluorescence weighted average lifetime.

FRET rate constant

The FRET rate constant, k_{RET}, quantifies the FRET process by the number of quanta transferred from the donor’s excited state to the acceptor per time. It depends on the mutual dipole orientation of the donor and the acceptor fluorophore, the distance between the donor and acceptor, R_{DA}, the Förster radius, R_0 of the dye pair, and the corresponding fluorescence lifetime of the donor in the absence of FRET, \tau_{0}. The orientation factor \kappa^2 captures The mutual dipole orientation.

$$ k_{RET} = \frac{1}{\tau_0} \kappa^2 \left( \frac{R_0}{R_{DA}} \right)^{6} $$

Note, for the calculation of the FRET rate constant the fluorescence lifetime has to match the Förster radius. Meaning the fluorescence lifetime of the corresponding donor fluorescence quantum yield, \Phi_{F}^{D0} should be used.

FRET-induced donor decay

The FRET-induced donor decay is a time-resolved intensity independent measure of FRET similar to the time-resolved anisotropy defined by the ratio of the donor fluorescence decay in the presence and the absence of FRET.


The G-factor, G, corrects for differences in the detection efficiency for fluorescence light of the parallel and perpendicular detector for anisotropy and time-resolved anisotropy experiments.

The G-factor is measured using the integrated (steady-state) fluorescence intensities
Here, the HV and HH represent the parallel and perpendicular detected sample which is horizontally excited.

Alternatively, the G-factor can be determined by matching the tails of the time-resolved fluorescence decays of a fastly rotating/depolarizing dye for VH and VV detection.


IRF stands for instrument response function. In time-resolved fluorescence measurements the IRF is the temporal response of the fluorescence spectrometer to a delta-pulse. Suppose a initially sharp pulse defines the time of excitation / triggers the laser, then recorded response of the fluorescence spectrometer is broadened due to: (1) the temporal response of the exciting light source, (2) the temporal dispersion due to the optics of the instrument, (3) the delay of the light within the sample, and (4) the response of the detector. As the most intuitive contribution to the IRF is the excitation profile, the IRF is sometimes called ‘lamp function’. The IRF is typically recorded by minimising the contribution of (3), e.g., by measuring the response of the instrument using a scattering sample, or a short lived dye.


MFD stands for Multiparameter Fluorescence Detection. A MFD experimentis essentially a time-resolved fluorescence experiment which probes the absorption and fluorescence, the fluorescence quantum yield, the fluorescence lifetime, and the anisotropy of the studied chromophores simulatanously.1

Kühnemuth R, Seidel CAM. Principles of Single Molecule Multiparameter Fluorescence Spectroscopy. S. 2001;2(4):251-254. doi: 10.1002/1438-5171(200112)2:4<251::aid-simo251>;2-t [Source]