Single-Spot Tutorial 1: us ALEX FRET Analysis

To execute this tutorial, the following files are needed:

• 004_dsDNA_17d_green100u_red40u.hdf5

• ALiX Settings & Controls 17d Single-Spot usALEX Analysis.txt

These files can be found on Figshare at:

• https://dx.doi.org/10.6084/m9.figshare.1098961

• https://dx.doi.org/10.6084/m9.figshare.3839427

Start ALiX if it is not running already. Remember that multiple copies of ALiX can be run in parallel (but be aware of memory limitations if you analyze large files). It is recommended to open the Notebook (Windows >> Notebook or Ctrl+N) at this stage, but this is not mandatory and can be done at any time during the analysis. Information regarding all steps are stored internally and sent to the Notebook in the order they were generated, the first time it is opened).

1. Loading the File

Switching to the Data File page, use the Browse button to the right of the File Path control to find the file mentioned above.

Note that if the Preview File Information checkbox (below the File Path control, to the left) is checked, the file will not be loaded in memory. Only basic information will be read from the file and displayed on the Data File page. Loading the file in memory will require pressing on the Load File button the the right of the Browse button.

Alternatively, use the File >> Open File (Ctrl+O) menu item. This will load the file in memory, skipping the preview step.

If this is the first time that you are opening a file, the following dialog window will appear:

This dialog will open each time a new file is loaded, if the option Reset all Graphs to Default when Loading a new File is selected (Settings window, General Settings page). If you do want to clear all graphs each time a new file is loaded but want to skip this warning dialog, check the Do not show this dialog again checkbox. It is possible to cancel this operation by clicking on the Cancel button.

ALiX will recognize that the file contains smFRET us-ALEX data (Measurement Type indicator above the File Path control) and that the File Type is .hdf5.

It will also indicate that the data has been successfully loaded by turning on the first status LED of the status bar at the bottom right.

Finally, it will indicate that two data channels have been found, one of which (Pixel ID 0) is the Donor channel, the other (Pixel ID 1) is the Acceptor channel:

2. Loading the Script

Important Note: this step is not indispensable, but is useful to specify a number of parameters and settings used in the analysis. These parameters and settings will be described below.

In order for this tutorial to be used with other files and different parameters, the steps which can be skipped if the script was loaded will be indicated in italics in the remainder of the tutorial.

In other words, if you have loaded the script (as described next), and plan to use parameters and settings as they are defined in this script, you do not need to execute the steps printed in italics.

• • Switch to the Scripting page (click on Scripting in the list of ALiX pages to the left of the main ALiX window, or select Pages >> Scripting from the menu).

  • Make sure the Mutispot Analysis panel is visible.

  • Check the Include All Control Values and Include All Settings checkboxes in the Script Archiving section (bottom right).

  • Load the script mentioned at the top of this page using the Load Multispot Script button (folder icon).

3. Defining the us-ALEX alternation period

• Switch to the us Alternation Periods page.

• Set the Offset to 8.75 us, the Alternation Period to 50 us and Macrotime Bin to 25 ns. These values may need to be adjusted if you are processing a different file.

• Press the Macrotime Histo button. The Macrotime Histogram should show two curves as illustrated below:

• Adjust the location of the 4 cursors (A Start, A Stop, D Start, D Stop) as indicated in the figure above.

• Press the Define Periods button: us ALEX Photon Statistics are updated and the Alternation Periods Defined LED lights up in the status bar.

4. Background Analysis

• Open the Settings window (Windows >> Settings or Ctrl+E) and switch to the Background Rates page.

• Set the MLE Algorithm to FRETBursts and the MLE Min Time Lag to 250 us.

• In the Background Analysis page of ALiX, set the Background Rate Used for Burst Search to MLE Rate.

• Press the Background button in the top left corner of the page. The program computes photon intervals and extracts the MLE values of the background rate computed for each stream. These values appear in the MLE Bkgd R array in the top right corner of the page, and the Background Rates Defined status LED lights up.

5. Advanced Background Analysis

• Switch to the Advanced Background Analysis page.

• Set the different analysis parameters as indicated in the figure below.

• • Press the Adv. Background button to compute the time-dependent background rates for each stream using a MLE algorithm and a 60 s time window.

  • The resulting time-dependent count rates are displayed in the top graph (see below) and the Time-Dependent Background Rates status LED lights up.

6. Burst Search

• • Switch to the Burst Search page.

• Set the burst search parameters as indicated in the figure below:

Note that the Background Rates array reproduce the result of step 4, but will not be used since the Use Time-Dependent Background Rates option has been selected.

• • Press the Burst Search button. The number of detected bursts (# Detected Bursts) and the duration covered by the whole time trace (Duration) are indicated, and the Burst Search Completed status LED lights up.

7. Burst Analysis

• • Switch to the Burst Analysis page.

• Make sure the following analysis options are chosen:

• Define the following burst statistics constraints:

These constraints set a minimum and maximum value for the corrected proximity ratio (this prevents very large positive or negative values of the proximity ratio to enforce a large number of histogram bins in the next step) and a minimum value of the photon counts during donor excitation (*).

• • Compute the burst statistics and apply the constraints by pressing the Burst Analysis button. The number of bursts satisfying these constraints is indicated (# Burst Statisfying Constraints) and the Burst Constraints Applied status LED lights up.

(*) The definition of the statistics aliases pr and XD can be looked up by opening the Statistics Aliases Definitions window (press the Statistics Aliases button at the bottom of the Burst Analysis page or Windows >> Statistics Aliases Definitions).

8. ALEX Analysis

• Switch to the ALEX Analysis page.

• Verify that the Use Burst Analysis Constraints checkbox is checked.

• Press the ALEX Histogram button. The 2D-ALEX Histogram is computed and plotted. Settings for this histograms can be modified in the ALEX Settings page of the Settings window.

• Define a region of interest (ROI) as indicated in the figure below:

• Make sure the E and S checkboxes below the SNA button are checked and both histogram bin sizes dE and dS are set to 0.03.

• Press the Histo button to the right of the E Histogram graph to compute the projections of the ROI-selected bursts ALEX Histogram along the E and S dimension. The corresponding histograms are computed and displayed in their respective graphs.

• Go to the Histogram Fits page of the Settings window and make sure the following settings are defined:

• Make sure that the fit models selected on top of the E and S Histogram graphs are set to Gaussian and press the Fit E and Fit S buttons (**).

This computes a Gaussian fit of the previous two histograms. To check the fitting parameters, right-click on the graphs and select Show Fit Parameters. To hide the parameters, repeat this action. Alternatively, open the Notebook, where the parameters as well as their uncertainty and other statistics have bee automatically appended.

• Go to the KDE Analysis page of the Settings window and define the following settings:

• • Make sure the KDE Bandwidth parameters below the E and S Histograms are set to 0.03 and press the KDE button (below the Histo button) and wait until the bootstrap analysis finishes and the KDE curves are computed. The KDE results are added to the Notebook.

(**) Pressing on the Fit E or Fit S buttons only fits the last computed histogram. To fit an older histogram (or fit the last histogram with a second model), right-click on the plot name in the graph legend and select a fit model among the different options under the Single Fit, Double Fit and Triple Fit submenus.

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Congratulations! You just completed the first ALiX tutorial.

For details about the different analysis steps and options, consult the respective manual pages. Alternatively, go to the Support page and post a question in the General Questions section.