Single-Spot Tutorial 2: us ALEX FCS Analysis

To execute this tutorial, the following files are needed:

• 001_4ChannelDarkCount.hdf5

• 006_dsDNA_7d_green100u_red40u.hdf5

and:

• Polimi donoror DC ACF (Multitau 12.5 ns, 1s, 8).dat

• Polimi Acceptor DC ACF (Multitau 12.5 ns, 1s, 8).dat

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.3817062

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).

The script comprises two steps:

A. Computation of the ACFs of the SPADs' afterpulsing using a SPAD DCR recording (the first file listed above).

This step can be skipped since the ACF curves are provided in the list of files shown above. However, it shows how to generate those curves in case the user is interested in analyzing data acquired with different detectors.

B. Computation of the data' ACFs with different corrections.

A. Computation of the SPAD DCR ACFs

1. Load File

• Load the 001_4ChannelDarkCount.hdf5 (for details on this step, check for instance step 1 in Single-Spot Tutorial 1).

• 4 channels are identified: Pixel ID 0 to Pixel ID 3. the first two corresponds to the SPADs we are interested in, the last two to two other SPADs which are not used in the measurements studied in step B. Select and define the first two pixels as indicated below:

While there is no "Donor" or "Acceptor" signal in this measurement, this choice allows treating both SPADs at once.

• The Measurement Type pull-down list should display smFRET (CW).

2. Compute Mean Count Rates

• Switch to the Background Analysis page.

• Press the PIH button (above the Photon Interval Histogram graph). This computes, among other things, the Average Rates (Hz), displayed in the top right corner of the page.

3. Compute the Autocorrelation Functions

• Switch to the Correlation page.

• In the ACF Photon Streams array to the left of the ACF Graph, select the Donor and Acceptor streams:

• Select the Multitau bin option and define the parameters as indicated below:

• Uncheck all options on the right of the ACF Graph as shown below:

• Compute the autocorrelation functions by pressing on the ACF button.

• Right-click on the Donor ACF plot legend and select Save as Afterpulsing ACF.

• Save the file as Polimi Donor DC ACF (Multitau 12.5 ns, 1s, 8).dat.

• Right-click on the Acceptor ACF plot legend and select Save as Afterpulsing ACF.

• Save the file as Polimi Acceptor DC ACF (Multitau 12.5 ns, 1s, 8).dat.

These files need to be specified as correction files as explained in the second part of the tutorial, in order to be available to correct any ACF of data acquired using these two SPADs.

Note: Remember which physical SPAD was associated with each saved ACF. In general, a setup will always be used in the same way and a detector used to record Donor emission will always be used for Donor emission. This is the reason why we have called these correction files "Donor" and "Acceptor". This makes it easy to remember which correction file to use to correct which ACF. However, what matters is not the nature of the signal ("Donor" or "Acceptor"), but which SPAD was used to record the signal. This means that if the two detectors are swapped, the "Acceptor" correction file above would have to be used to correct Donor channel ACFs.

B. Computation and correction of experimental ACFs

1. Load the ACF Correction Files

• • Open the Correlation Analysis panel of the Settings window.

  • If there are any definitions left, right-click on the scrollbar and select Empty Array.

  • Press the Load button and select the two files created in step A (or the last two files quoted at the beginning of the tutorial):

Note: This step needs to be done once per session (if the detectors remain the same and are affected to the same emission channel). Moreover, since there location and definitions are saved as part of ALiX settings, it is not necessary to repeat this step from one session to the next.

2. Load the Data File

• • Verify that the Measurement Type is smFRET us-ALEX

  • Verify that Pixel ID 0 is associated with the Donor channel and selected, and that Pixel ID 1 is associated with the Acceptor channel and selected:

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

• Switch to the Background Analysis page.

• 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.

Note: in this tutorial, we do not care too much about the actual method used to compute the background rates. The method currently selected in ALiX should work fine.

5. Correlation Analysis

• • Switch to the Correlation page.

  • Select the F_D^D and F_D^A Streams and the exact same bin definitions as in Step A3 (Multitau).

  • Uncheck all ACF options on the bottom right corner of the ACF Graph.

  • Press the ACF button.

  • In the ACF Graph, observe the oscillations due to us-ALEX:

• • Check the AP Correction and Normalize by Modulation Function ACF checkboxes:

• •  Press the ACF button and observe that oscillations and short time lag rises have now disappeared:

Note: Most of the noise at short time lag is due to noise in the afterpulsing ACFs. Because of this fact, it is important to compute afterpulsing ACF with large enough dark count files so that this noise is minimized. Taking data with a constant, but low, illumination is another alternative, as it allows acquiring better statistics in a shorter time. However, this approach is very sensitive to any instability in the illumination source.

• • Uncheck the AP Correction checkbox, leaving only the us-ALEX correction:

• • Press the ACF button and observe that oscillations are suppressed, but the short time lag rise due to afterpulsing is present:

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Congratulations! You have now completed Tutorial 2 and should now understand how to correct ACFs for afterpulsing and/or us ALEX alternation.

Note that the same us ALEX alternation correction is available for CCFs.