Analyze data

Follow this procedure to process your single molecule videos (SMVs) or trajectories and characterize the molecule dynamics in your sample.

Note: Skip step 1 if already in possession of intensity-time traces (ASCII files or in a mash project).

<< PREVIOUS: Create traces NEXT: Identify state network >>

Step 2: Find states in traces

In this step, single molecules are sorted, intensity-time traces are corrected from experimental bias and state sequences are inferred for individual traces.

  1. Import trajectories
  2. Correct for experimental bias
  3. Sort time traces
  4. Discretize time-traces
  5. Save and export
  6. Merge projects

Import trajectories

Intensity-time trace correction, sorting and discretization is done with module Trace processing. Traces can be read from a MASH project created in Step 1 or from a set of ASCII files.

In the first case, you can skip this step and go to step 2.

To import trajectories from ASCII files:

  1. Press New project in the project management area; a window pops up:

    Create a new project

  2. Select import trajectories to open the experiment settings window and fill in the description of your experiment setup and the structure of your trajectory files; please refer to Set experiment settings for help

  3. Set the default export destination by pressing ... in the project management area and selecting your root folder

  4. Save modifications to a .mash file by pressing Save project in the project management area.

  5. Select module Trace processing by pressing Trace processing in the main tool bar

Correct for experimental bias

Intensity-time traces must be corrected from experimental bias to obtain state trajectories that describe molecule dynamics the most accurately.

Experimental bias include shifts in molecule positions, background light and cross-talks between wavelength ranges used for emitter detection (bleedthrough) and excitation (direct excitation).

To adjust single molecule positions in panel Sub-images:

  1. Select the shifted molecule in the Molecule list of Sample management

  2. Select donor excitation in menu (a) of Single molecule images

  3. Press Recenter in Single molecule coordinates

  4. Go back to step 1 until all shifted molecules are recentered

To correct intensities from background light:

  1. Define Background correction settings for each trace selected in menu (a):

    default: method <N median values>
    default: option (h) activated and parameter (d) to 20 pixels

  2. Activate option Apply background correction for each trace selected in menu (a) of Background correction settings if not already done.

  3. Press all to apply the same settings to all molecules

To re-sample trajectories and increase SNR:

  1. Set the Trajectory sampling time to a larger value:

    default: Trajectory sampling time set to original video/trajectory sampling time

To correct intensities from cross-talks (if you are working with single detection channel and single laser illumination, ignore this step):

  1. Set for each emitter selected in the Emitter list:

    default: Bleedthrough coefficients set to 0
    default: Direct excitation coefficients set to 0

Sort time traces

Now that molecule positions and intensities are corrected, we can reliably exclude irrelevant intensity-time traces from the project, e. g incorrectly labelled species, and sort relevant traces into subgroups.

To remove incorrectly labelled species from the project, the molecule has first to be identified and tagged. Then, Tagged molecules can be deselected and cleared from the project.

To tag species missing emitter label [E]:

  1. Press Trace manager in Sample management to open the Trace manager

  2. Select tool Overview

  3. In Molecule selection, add the default tag no-[E] by typing the tag name in (b)

  4. Select tool Automatic sorting

  5. In panel Data, set menu (a) to total [E] (at [W]nm), the total intensity of emitter [E] with [W] the emitter’s specific excitation wavelength, menu (f) to none, and set parameters:

    default: option original time traces in menu (b)
    default: parameters (c) and (d) to minimum and maximum intensities respectively
    default: parameter (e) to 50

  6. Define a range including the histogram peak centered on zero by clicking on the Histogram plot

  7. In panel Range, set parameters:

    defaut: option at least in menu (e)
    defaut: parameter (f) to 90%
    defaut: option percentage of the trace in menu (h)

  8. Select the option total [E] (at [W]nm) in menu (a) of Concatenated trace plot and verify that selected traces are fluctuating around 0

  9. Press Save range in panel Range to save range settings

  10. Select menu (b) to no-[E] in Tags and press Tag to assign this label to the selected range

  11. Press TAG MOLECULES to tag with no-[E] all molecules missing emitter [E]

To clear species missing emitter [E] from the project:

  1. Select tool Overview

  2. In Molecule selection, select option remove no-[E] in menu (a) to deselect all molecules carrying this tag

  3. Press TO MASH in Overall plots to close Trace manager and export the current selection to Trace processing

  4. Press Clear selection in Sample management to delete deselected molecules from the project

Discretize time-traces

To obtain reliable state trajectories, photobleached or interrupted data must be ignored and ratio-time traces must be corrected prior applying the state finding algorithm. This is done by splitting trajectories manually, automatically detecting and truncating the trace at dye photobleaching and setting/calculating global gamma and beta factors.

To split trajectories at intensity interruptions:

  1. Define in Photobleaching:

    default: method Manual
    default: parameter Photobleaching cutoff to the ending position of the interruption
    default: option Cut deactivated

  2. Press Split to separate the left- and right-side of the splitting position to separate trajectories

To truncate trajectories at photobleaching:

  1. Define in Photobleaching:

    default: method Threshold
    default: data all intensities
    default: parameters in Automatic detection settings (b) to 0, (c) to 2 frames and (d) to 10 frames
    default: option Cut activated

  2. Press all to apply the same settings to all molecules

  3. Press UPDATE ALL in Sample management to process all molecules in the project and visualize truncated trajectories

To automatically calculate gamma and beta factors (if you are working without FRET calculations, ignore this step):

  1. For each FRET pair listed in the FRET pair list in Factor corrections, define:

    default: method ES linear regression (if you are working without stoichiometry calculations, use any other method)
    default: in ES linear regression, (b) to All molecules, (c) to -0.2, (d) to 50, (e) to 1.2, (f) to 1, (g) to 50, (h) to 3

    and press refresh calculations to calculate the ES histogram and perform linear regression, and Save and close to save settings

  2. Press all to apply the same settings to all molecules

  3. Press UPDATE ALL in the Control area to process all molecules in the project

To obtain state trajectories:

  1. Define in Find states:

    default: method STaSI
    default: apply to all
    default: Method parameters (b) to 5 for each trace selected in menu (a)
    default: no Post-processing methods with (b) an (c) set to 0

  2. Press all to apply the same settings to all molecules

  3. Press UPDATE ALL in the Control area to process all molecules in the project

Save and export

Project modifications must be saved to the .mash file in order to perform Step 3 and Step 4 with processed data.

Beside, various ASCII and image files containing processed data, data statistics and processing parameters can be exported for use in other software.

To save project modifications:

  1. Press Save project in the project management area and overwrite the current .mash file or create another project file to keep a backup of unprocessed data

To export processed data to ASCII and image files:

  1. Press Export... in the Control area to open and set export options; please refer to Set export options for help

  2. Press Next >> to start file export.

Merge projects

After applying proper corrections to intensities and sorting out molecules, samples from different projects with identical experiment settings can be merged into one big data set. This prevents to repeat the following data analysis procedure on multiple mash files and allows to constitute a more representative sample of molecules.

To merge projects:

  1. Select the multiple projects to merge in the Project list

  2. Right-click in the Project list, choose the option Merge projects and press Yes to start the merging process

    Note: The merging process induces a loss of single molecule videos that were used in individual projects. Make sure to perform all adjustments of molecule positions and background corrections prior merging.

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