ALEX Corrections

This tab allows specifying sample- and setup-related correction parameters affecting the calculation of FRET efficiency (E) and stoichiometry ratio (S).

• Use Other Corrections

Check this box if corrections need to be applied

• Correction Type

Depending on whether the data set was acquired using microsecond laser alternation (us ALEX) or nanosecond alternation (ns ALEX), a different set of parameters needs to be selected. Selecting an option shows the corresponding set of correction factors.

• us ALEX Correction Factors

As shown above, there are four us ALEX Correction Factors:

• Donor leakage (D-leakage)

• Direct Acceptor Excitation (Direct A Excitation)

• Gamma

• Beta

The donor leakage factor multiplied by F_D^D gives the amount of donor signal leaking into the acceptor channel (during D-excitation).

The direct acceptor excitation factor multiplied by either F_A^A or F_D^D (depending on the chosen definition, see below), gives the amount of acceptor signal during D-excitation, due to direct acceptor excitation (i.e. not due to Förster energy transfer).

Direct Acceptor Excitation can be expressed in terms of the acceptor excitation, acceptor emission signal (F_A^A), which is the default option and is used when the checkbox is not checked). The other option is to compute the direct acceptor excitation contribution as a function of the donor excitation. donor emission signal (F_D^D), which is selected by checking the checkbox. This choice is the only one possible in the absence of alternation (i.e. when a single, D-excitation laser is used).

The gamma and beta factors are defined as usual (see for instance (Lee et al., 2005)).

• ns ALEX Correction Factors