ALEX Settings

The ALEX Settings tab is used to control how the ALEX histogram (ALEX Histogram Bounds and ALEX Histogram Bins) and the corresponding E and S projection histograms are calculated (Project Outside Values). It also gives access to the definition of the Stoichiometry Boundaries used to define the stoichiometry- and brightness-compensated burst size (F_SBC).

The definitions of each parameter is given below.

• Alternation Mode

Allows selecting the proper way to interpret data acquired with a Periodic Acceptor Excitation (PAX) scheme (see ref. 11-12) rather than an Alternated Laser Excitation (ALEX) scheme. 

• Stoichiometry Ratio

Allows using a different definition of the stoichiometry ratio when analysing data acquired with a Periodic Acceptor Excitation (PAX) scheme (see ref. 11). The recommended approach is to use PAX when using a PAX alternation mode, and ALEX when using an ALEX alternation mode.

• ALEX Histogram Bounds

The 2-dimensional ALEX histogram has two axes: E and S. Uncorrected data (no background subtraction, no setup- and sample-specific corrections) are ratios of integer numbers limited to the interval [0, 1] (with the extra +Infinity value for S). However, corrected data (background-subtracted or corrected for D-leakage, direct A-excitation, etc) can cover the whole real number axis (-Inf, +Inf). The ALEX Histogram Bounds parameters allow limiting this range to a user-manageable region (and limit the memory footprint of the histograms).

In general, the default range of [-0.5, 1.5] is appropriate for most data sets.

• ALEX Histogram Bins

The bin size of the ALEX histogram can be adjusted independently for both axes.

• Project Outside Values

When checked, values falling outside the specified ALEX Histogram Bounds are artificially projected to the closest bound for histogramming purposes. The internally stored value for that burst is not modified.

For instance, if a A-only burst has a corrected F_D^A value of 0.34 and a corrected F_D^D value of -0.5, its calculated E will be 2.125. This falls outside of the default range [-0.5, 1.5], therefore this burst will be counted as belonging to the E bin [1.48, 1.5[.

Warning: when this option is not checked, the number of A-only bursts counted within a typical A-only region of interest [-0.5, 1.5] x [-0.5, 0.2] will be underestimating the actual number of A-only bursts in the data set.

• Weighted Histogram

This option allows to use a burst-specific weight when building the histograms. In the example shown below, a weight equal to (1 + (F_D^D)^2) is used.

This means that each burst falling within the specified histogram bounds is counted not as 1 but as (1 + (F_D^D)^2), giving more importance to bursts with large F_D^D signal than bursts with small F_D^D signal.

The resulting weighted histogram is normalized in such a way that the sum of all its bin contents is equal to the number of bursts within the histogram bounds. For instance, a constant weight will result in the exact same histogram as an unweighted histogram.

Any valid algebraic combination of burst statistics can be used as weight.

• Background Corrections

When checked, E and S values are computed using background-subtracted quantities.The background rates used in this calculation are those shown in the Background Rates tab of the Settings window and are usually identical to the result of the background rate analysis performed on the main Background Analysis page. However, the user is free to adjust these quantities in the Background Rates tab.

Note that other corrections are defined in the ALEX Corrections tab.

• Use Time-Dependent Background Rates

When time-dependent background rates have been determined in the Advanced Background Analysis page of ALiX, checking this box will use them when performing background subtraction.

If no time-dependent background rates have been defined, average background rates are used instead.

• Stoichiometry Boundaries

These boundaries define respectively:

- S_min^D: the minimum stoichiometry ratio which a burst needs to have in order to be identified as a D-only burst.

- S_max^A: the maximum stoichiometry ratio which a burst needs to have in order to be identified as a A-only burst.

These definitions, which are naturally associated with the ALEX histogram, where the appropriate boundaries are most easily determined, are used when computing the stoichiometry- and brightness-compensated burst size, F_SBC, a single burst statistics.