Another important parameter for defining the burst size is the \(\gamma\)-factor. As noted in section \ref{sec:burstsizeweights}, the \(\gamma\)-factor is used to compensate for different fluorescence quantum yields for the D and A fluorophores as well as different photon-detection efficiencies for the D and A channels. When \(\gamma\) is significantly different from 1, neglecting its effect on burst size leads to over-representing one FRET population versus the others.

When the \(\gamma\) factor is known (and \(\neq 1\)), a more unbiased selection of different FRET populations can be achieved passing the argument gamma to the selection function:

When not specified, \(\gamma=1\) is assumed. For more details on burst size selection, see the select_bursts.size documentation (link).

The method to compute \(\gamma\)-corrected burst sizes (with or without addition of naa ) is Data.burst_sizes (link).

In smFRET-ALEX experiments, donor-only (D-only) and acceptor-only (A-only) populations can be detected in addition to the FRET population(s). In most cases, the D-only and A-only populations are of no interest and need to be filtered out.

In principle, using the E-S representation, D-only and A-only bursts can be excluded by selecting bursts within a range of \(S\) values (e.g. S=0.2-0.8). This approach, however, simply truncates the burst distribution with arbitrary thresholds and is therefore not recommended for quantitative assessment of FRET populations.

An alternative approach consists in applying two selection filters sequentially. First, the A-only population is filtered out by applying a threshold on the number of photons during D excitation (Dex). Second, the D-only population is filtered out by applying a threshold on the number of A photons during A excitation (AexAem). The commands for these combined selections are:

Here, the variable ds2 contains the combined burst selection. Figure \ref{fig:alex_jointplot_fretsel} shows the resulting filtered FRET population obtained with the previous selection.