Table 1.

Terms used

Symbols	Description
fA	Ratio of FRET complexes over total acceptor, considering intact fluorophores only (Eq. 26).
fD	Ratio of FRET complexes over total donor, considering intact fluorophores only (Eq. 26).
fa	Fraction of acceptor-type molecules participating in complexes, irrespective of their labeling state (Eq. 34).
fd	Fraction of donor-type molecules participating in complexes, irrespective of their labeling state (Eq. 33).
[A]	Concentration of free acceptor fluorophores (Eq. 6).
[D]	Concentration of free donor fluorophores (Eq. 6).
[DA]	Concentration of complexes carrying both intact donor and acceptor fluorophore (Eq. 6).
[a], [d], [da]	Chemical concentrations of free acceptor, free donor, and complexes, irrespective of their labeling state (Eqs. 28–30).
[Aref]	Concentration of intact acceptor fluorophore in the calibration samples (Eq. 2).
[Dref]	Concentration of intact donor fluorophore in the calibration samples (Eq. 1).
[At]	Total concentration of labeled acceptors with intact fluorophore (Eq. 22).
[Dt]	Total concentration of labeled donor with intact fluorophore (Eq. 21).
[at],[ dt]	Total chemical concentrations of acceptor and donor (Eqs. 31 and 32).
Rt	Ratio of total abundances of labeled acceptor over total abundances of labeled donors (Eq. 27).
pa, pd	Probabilities, by which a given molecule of type a and d is labeled with an intact fluorophore (Eq. 28).
pa,tc, pd,tc	Labeling probabilities of donors and acceptors within the tandem construct (Eq. 35).
${p^{\prime }}_{\text{d}}$	Abbreviation for pdpa,tc/pd,tc (see above).
αi	Apparent relative acceptor concentrations* (Eq. 13).
δi	Apparent relative donor concentrations* (Eq. 12).
Fi(λ)	Measured spectrum (linear combination of $F_{\text{D}}^{\text{i,ref}}$ and $F_{\text{A}}^{\text{i,ref}}$) (Eq. 6; see also Eq. 9).
$F_{\text{A}}^{\text{i},\text{ref}}$	Reference fluorescence emission spectra of pure acceptor* (Eq. 2).
$F_{\text{D}}^{\text{i},\text{ref}}$	Reference fluorescence emission spectra of pure donor* (Eq. 1).
rex,i	Scaling factor reflecting the excitation ratios of two fluorophores at the given excitation wavelength (Eq. 3).
E	Characteristic FRET efficiency (Eq. 6).
ETC	FRET efficiency of the tandem construct.
K	Dissociation constant (Eq. 47).
${\epsilon }_{\text{A}}^{\text{i}},{\epsilon }_{\text{D}}^{\text{i}}$	Extinction coefficients of acceptor and donor* (Eqs. 1 and 2).
QA, QD	Quantum yields of acceptor and donor (Eqs. 1 and 2).
ɛA(λ), ɛD(λ)	Standard emission spectra of the two fluorophores normalized to unit area (Eq. 3).
Ii,ref	Excitation intensity* (Eq. 1).
ηi(λ)	Detection efficiencies of the instrument used* (Eq. 1).
B	Bleaching ratio (Eq. 53).
${\delta }^{1,\text{pre}},{\delta }^{1,\text{post}}$	Apparent relative donor concentration before and after acceptor bleaching (Eqs. 51 and 52).
$F_{\text{D}}^{\text{pre}},F_{\text{D}}^{\text{post}}$	Donor fluorescence intensities before and after photobleaching the acceptor (Eq. 60).
${\overline{e}}_{\text{A}}^{\text{k}},{\overline{e}}_{\text{D}}^{\text{k}}$	Averages over the emission range k of standard (literature) emission spectra of acceptor and donor (Eq. 61).
$F_{\text{A}}^{\text{i},\text{k},\text{ref}},F_{\text{D}}^{\text{i},\text{k},\text{ref}}$	Reference reading of the acceptor and donor sample* at emission channel k.

^*Note that index i refers to two excitation wavelengths λⁱ (i = 1,2).