us Alternation Periods

This page is used to define the alternation period, and donor and acceptor excitation periods in μs ALEX experiments.

Three successive tasks need to be accomplished on this page:

1. Computation of the Macrotime Histogram

2. Definition of the acceptor and excitation periods

3. Validation of these definitions

1. Computation of the Macrotime Histogram

The alternation period's duration needs to be manually entered in the Alternation Period box if it has not been read from the file (currently, only photon-HDF5 files contain this information), or if the user chooses to ignore this information from the file by checking the Ignore us-ALEX Period Definitions from File checkbox. The value has a time unit which can be modified by the user. The default is us (microsecond).

The donor and acceptor excitation periods are defined using the Macrotime Histogram, computed by pressing the Macrotime Histo button. The histogram represents the distribution of macrotime stamp values modulo the alternation period.

A few parameters need to be defined before computing the histogram:

• Macrotime Bin: This is the bin size used to compute the histogram. The smallest sensible value is equal to the time stamp resolution. For files with a large number of photons, choosing the time stamp resolution for the macrotime bin value is fine, but for smaller files, this choice may result in a noisy histogram (low bin content). On the other hand, choosing too large a bin size will result in loss of precision in identifying the donor and acceptor excitation periods.

• Offset: This is the value subtracted from every time stamp before calculation of the remainder of the modulo operation. In general, this value needs to be determined once for a given setup, and represents the delay between the instant when the time stamp counter is started and the instant when the laser alternation task is started (typically a few microseconds). This value is also used to align time trace bins with the alternation period (see Time Trace Page for details).

For instance, in the Figure shown above, an offset value of 8.75 us has been chosen, which corresponds to the first midpoint between a donor and an acceptor excitation period.

Note that if successive data files are analyzed with the same experimental setup conditions, there is no need to compute the macrotime histogram for each new data file in order to define the alternation period. A simple click on the Define Periods button is enough (alternatively, using the Ctrl-P shortcut) in order to validate the previously define periods.

2. Definition of the acceptor and excitation periods

These periods can usually be easily identified: the Acceptor excitation period corresponds to the part of the histogram where the acceptor counts are largest and the donor counts the smallest. The Donor excitation period is obviously characterized by the opposite properties.

Ideally, the excitation intensity should follow a perfect square pattern, with the donor excitation period starting immediately after the acceptor excitation period, and vice versa. However, in practice, the opto-electronic component(s) used to generate the alternation pattern has(have) a finite response time and therefore, there exist short periods of time during which both lasers are exciting the sample at some intermediate intensity.

In order to eliminate these short intermediate periods from the analysis, the Macrotime Histogram provides 4 cursors which can be moved around to define the start and stop time for each period (donor or acceptor).

These cursors can be locked to one another, partially locked or completely free. This option can be selected using the pull-down list located to the right of the graph legend. There are 5 different modes:

• Free Boundaries: The Start and Stop locations of both periods can be located anywhere within the alternation period.

• D Stop = A Start: The donor excitation period stop time is identical to the acceptor start time; the donor start time and acceptor stop time can be located anywhere within the alternation period.

• A Stop = D Start: The acceptor excitation period stop time is identical to the donor start time; the acceptor start time and donor stop time can be located anywhere within the alternation period.

• D Stop = A Start, A Stop = D Start + Period: The donor excitation period start time is identical to the offset; the donor excitation period stop time is identical to the acceptor start time; the acceptor stop time is identical to the offset plus alternation period.

• A Stop = D Start, D Stop = A Start + Period: The acceptor excitation period start time is identical to the offset; the acceptor excitation period stop time is identical to the donor start time; the donor stop time is identical to the offset plus alternation period.

If the cursor location conflicts with the chosen mode, a "Conflicting Cursor Order!" message will be displayed in the message area (lower left of ALiX window), together with a red warning LED. An audible beep will also be emitted.

As an alternative, it is possible to use the period definitions included in the file itself, as is currently possible only with photon-HDF5 files. To ignore these file defintions, check the Ignore us-ALEX Period Definitions from File checkbox. If the checkbox is not checked, the file definitions will be used and the cursor moved to the corresponding locations. However, the periods will not be validated. This still requires user intervention (or a script action) as described in the next section.

Once a period definition has been validated, number statistics (us ALEX Photon Statistics) are computed and displayed under the Graph.

3. Validation of the acceptor and excitation period definitions

Once the donor and excitation periods have been defined with the corresponding cursors, clicking the Define Periods button will assign each photon to its designated period. Any photon/count not belonging to the donor or acceptor emission period will be assigned an "undefined" period.

The controls located below the graph provide information on:

• the respective percentage of photons within the D-excitation period,

• the percentage of photons outside both donor and acceptor excitation periods,

• the percentage of the alternation period rejected from the analysis.

Important Note: the 3 previous actions can be accomplished using a single Analysis menu item (Alternation Period Definition, shortcut: Ctrl-P)

Additional controls

The page comprises check boxes which allow:

• Moving on to the next analysis page: Switch to Background Analysis when Periods are Defined.

• Prevent moving on to another task without first defining excitation periods: Do not switch without defining periods.