Intensity integration

Intensity integration is the fourth panel of module Video processing. Access the panel content by pressing Bottom arrow. The panel closes automatically after other panels open or after pressing Top arrow.

Use this panel to create and export single molecule intensity-time traces.

Panel components

  1. Input coordinates
  2. Integration parameters

Input coordinates

Use this interface to import single molecule coordinates used to build intensity-time traces.

The calculation of intensity-time traces requires the single molecule coordinates transformed in all video channels. The availability of single moelcule coordinates are indicated by the icon located in (a) that displays ✔ if data is available and ✖ if data must still be calculated or imported.

In case single molecule coordinates are unavailable:

  • Start a spot finding procedure with Spotfinder and, for multi-channel video projects, transform spots coordinates in Coordinates transformation.
  • Or, import molecule coordinates from an external file by pressing Open and selecting the corresponding file; coordinates are read according to the Import options that can be accessed by pressing Import options.

Import options

Import options defined the way single molecule coordinates are read from file.

Single molecule coordinates are coordinates co-localized in each channel, with each channel corresponding to a specific x-range. The number of file header lines set in (b) is skipped before reading coordinates and channel-specific x- and y-coordinates are read from columns set in (c) and (d) respectively.

If all channel-specific data are organized in the same two columns, coordinates are sorted according to the specific x-range of each channel.

Save import settings by pressing Ok.

Integration parameters

Use this interface to define the settings for intensity calculation.

To obtain the single molecule intensity at one particular frame or time point, a square area of dimension (a) pixels around the molecule coordinates is defined. The positions of the (b) brightest pixels in the corresponding average sub-image are determined and used when summing up the (b) pixels in each frame.