Background in Single-Molecule FRET Microscopy

Background settings

By default the molecule background is a ring mask surrounding the molecule. This background is calculated as the average of the intensity within the ring mask multiplied by the number of pixels in the integration mask.

Starting from version 1.1 it is possible to additionally select the median or LSP background estimators. See the relevant reference for a description of these.

Open 'Settings->Background settings'.
In the settings menu select the background type. If you select the LSP background estimator make sure you have determined the optimal value of X. Determining the value of X is described under the documentation for the LSP background estimator.

Optimize background interactively

Suppose there is a close neighbouring molecule and the background ring includes the neighbouring molecule.
Activate the 'Select background pixels manually' button in the toolbar and move the cursor to the molecule image. Select the pixels that cover the other molecule. When clicking on pixels that are already in the background ring, they will be removed from the background ring. When finished, right-click within the molecule image.
After right-click, the new background trace has now been updated (black).