How to correct a fluorescent background

This guide shows you how to subtract the background bulk fluorescence from a fluorescence image.

Reference keys: preprocessing, background correction

Correction protocol

  1. Launch the software. Open a project.

  2. Expand the PROCESSING block. In the BACKGROUND CORRECTION section, click on the Fit tab.

  3. Set the fluorescence channel to correct.

  4. Click on the icon next to the threshold field to estimate this threshold value visually.

  5. Use the threshold slider to select as many cells as possible (in purple) while leaving most of the background regions unselected (not purple).

  6. Apply.

  7. Set the paraboloid model.

  8. Tick the subtract operation.

  9. Do not clip.

See celldetective.preprocessing.field_correction() to understand what the threshold represents. See celldetective.preprocessing.paraboloid() for a definition of the paraboloid model.

Note

Not clipping allows you to perform quality control on the resulting intensities. If the model is poor you should see many negative values.

Apply correction

  1. Press Add correction.

  2. Once all preprocessing protocols have been defined (background correction, other channel alignments), scroll down and press Submit. To apply the same protocol to all positions, accept the first popup.

  3. At the end of the preprocessing change the movie stack prefix in the experiment configuration to Corrected_.