Photobleaching

Photobleaching is the seventh panel of module Trace processing. Access the panel content by pressing Bottom arrow. The panel closes automatically after other panels open or after pressing Top arrow.

Photobleaching settings are specific to each molecule. Press all to apply current settings to all molecules. Corrections will be applied only after processing data by pressing UPDATE ALL; see Process all molecules data for more information.

Use this panel to detect dye photobleaching and suppress photobleached data.

Panel components

  1. Photobleaching detection method
  2. Photobleaching cutoff
  3. Truncate trajectories
  4. Split trajectories
  5. Automatic detection settings

Photobleaching detection method

Use this list to select the appropriate method for photobleaching detection.

Emitter photobleaching can either be detected visually or automatically, by respectively selecting Manual or Threshold in the list.

For Manual detection, the photobleaching cutoff must be set by hand in Photobleaching cutoff.

Threshold detection is performed by thresholding using the settings defined in Automatic detection settings.

Photobleaching cutoff

Shows the photobleaching position given in seconds or frame according to time-axis units defined in menu Units of the menu bar.

For method Threshold, the photobleaching cutoff detected with Automatic detection settings is shown here.

For method Manual, the photobleaching cutoff must be set here.

Truncate trajectories

Activate this option to truncate time traces at the Photobleaching cutoff, or deactivate this option to visualize a blue cursor at the cutoff position in Intensity-time traces and Ratio-time traces.

For more information about how photobleaching correction is used in smFRET data analysis, see Correct for photobleaching in Trace processing workflow.

Split trajectories

Intensity interruptions occurring in the middle of a trace (e. g., blinking) can be corrected by splitting intensity-time traces in two and truncating the end of the first trace (see Truncate trajectories) and the beginning of the second (see Time axis).

Press Split to split time traces in two at the Photobleaching cutoff. In this case, the right-side of the cutoff is saved as a separate molecule, which is added to the Molecule list. Both molecules will be given a “Split” tag in order to easily identify them in the list.

For more information about how blinking correction is used in smFRET data analysis, see Correct for photobleaching in Trace processing workflow.

Automatic detection settings

Us this interface to define the settings for automatic detection of photobleaching.

Photobleaching is detected when the time trace selected in menu (a) drops below a certain threshold defined in (b) and providing a minimum cutoff value set in (d).

Traces available for photobleaching detection are:

  • FRET [D]>[A] the FRET-time trace of the pair donor emitter [D] -acceptor emitter [A]
  • S [D]>[A] the Stoichiometry-time trace associated to the FRET pair donor emitter [D] -acceptor emitter [A]
  • [E] at [L]nm the single intensity-time trace of emitter [E] upon illumination with laser wavelength [L] (in nm)
  • all intensities the minimum values found in all intensity-time traces
  • summed intensities the sum of all intensity-time traces

In case of intensity-time traces, the threshold is given in counts or counts per second according to intensity units defined in menu Units of the menu bar.

Traces all intensities and summed intensities are calculated from intensities in absence of any acceptors, i. e. summed over all channels at emitter-specific illumination. This allows to exclude the zero-intensity signals collected at unspecific illuminations that are constantly “photobleached” and prevent the automatic photobleaching detection to function. Only emitters having a specific illumination defined in Channels are considered in these calculations.

To ensure detection at the very beginning of acceptor photobleaching, the detected cutoff position can be shifted downwards by a certain number of frames set in (c).

Parameters (b) and (c) are given in frame number or in second according to time units defined in menu Units of the menu bar.

The resulting photobleaching cutoff displayed in Photobleaching cutoff only after processing the current molecule, i.e., when pressing UPDATE; see Process current molecule data for more information.