Analysis of FRET samples

Parameters

Donor in the absence of FRET

Parameter name	Abbreviation	Description	Type	Recommendation
Number of donor fluorescence lifetime components	Donor, add/del	By clicking on the add and the del button a donor species can be added.	NA	The number of donor fluorescence lifetime components should be determined using a separate sample without an acceptor.
Species fraction of species i	x(L,i)	The species fraction of a donor component that has a fluorescence lifetime tau(L,i) in the absence of FRET.	float	The fraction of a donor component should be determined using a separate donor sample.
Fluorescence lifetime of the species i	tau(L,i)	The fluorescence lifetime of the donor component i in the absence of FRET	float	The donor component should be determined using a separate donor sample.

Donor in the presence of FRET

Parameter name	Abbreviation	Description	Type	Recommendation
Förster radius	R0	The Förster radius that matches to the orientation factor kappa2 and the tau0	float
Orientation factor	kappa2	The orientation factor of the donor-acceptor pair. By default a dynamic averaging in assumed. Alternatively, a static averaging can be set up in the chisurf settings file.	float	For most cases (dynamic averaging) 2/3 should be used. For fluorescent proteins a static orientation factor distribution should be used by changing the chisurf setting files.
Fluorescence lifetime of the donor in the absence of FRET	tau0	The fluorescence lifetime used to calculate the Förster radius.	float	The numbers R0, kappa2, and tau0 are jointly used to calculate the FRET rate constants, kFRET.
Fraction of donor molecules lacking an acceptor molecule	x(D,0)	The species fraction of FRET inactive donor molecules. The faction is in the range of [0,1].	float	For large xD0 (xD0>0.7) consider analyzing additionally, the FRET sensitized acceptor decay.
Number of superimposed distributions, Gi	NA	By clicking on add and del distributions can be added to the total distance distribution	NA	The number of components depends on the data and the system. In a typical data set, depending on the separation of the components, usually 2-3 components can be resolved.
Parameter describing the position of a individual component.	R	Usually, the individual components are normal distributions. For normal distributions, R is the center of the distribution.	float	For short distances or very broad distributions, consider using a chi instead of a normal distribution.
Parameter describing the width of a component	sigma	For normal distributions, sigma is the standard deviation of the distribution. For a chi distribution, which is the distance distribution between two normal distributions, sigma is the width of the individual normal distributions.	float	For the organic dyes typically used in FRET experiments sigma is usually in the range of 5-12 Angström.

Homo FRET and FRET sensitized emission

Parameter name	Abbreviation	Description	Type	Recommendation
Prefactor of the amplitudes for the energy migration of the dye A to the dye B (A>B) and dye B to A (B>A)	A>B, B>A	This factor can be used to set if the model function is a homo FRET experiment or a hetero FRET experiment. In a homo FRET experiment A>B and B>A are both one. In a hetero FRET experiment either A>B or B>A is zero.	float	This value should be either 1 or 0 and be fixed depending on the type of experiment
The fraction incomplete molecules that contain only the dye A or dye B but not both dyes AB	pureA, pureB	In a hetero FRET experiment these numbers correspond to the fraction of a acceptor only and donor only molecules. Depending on the parameters either (see A>B and B>A parameter) pureA is either the donor or the acceptor fraction. For A>B=1 and B>A=0 pureA is the donor only fraction and pureB is the acceptor only fraction.	float	pureA can be linked in a global analysis to the donor only fraction of the donor decay in the presence of FRET.
Excitation probabilities of the dye	ExA, ExB	Factor scaling the fluorescence decays of A and B on the excitation side. If ExA=1 and ExB=0 only dye A is excited. If ExA=0 and ExB=1 only dye B is excited.	float	The excitation of A and B can be estimated based on the absoption spectra and the spectrum of the excitation source.
Emission probabilities of the dyes	EmA, EmB	Factor scaling the fluorescence decays of A and B on the detection side. This factor depends on the spectra of the dyes and the experimental setup.	float	This parameter can be estimated using the spectra of the indivudal components of the setup, the fluorescence spectra of the dyes, and the dyes fluorescence quantum yields.
Fluorescence lifetimes of the dyes in the absence of FRET	Lifetimes-A, Lifetimes-B	The fluorescence lifetimes of the dyes in the absence of FRET. Meaning, the fluorescence lifetimes of the donor without acceptor and the acceptor that is directly excited (for hetero FRET).	NA	The fluorescence lifetime of the donor in the ab