Background subtraction

To obtain correct fluorescence and FRET values it is important to perform background correction. There are three types of background subtraction that can be performed. A temporal background subtraction that corrects variations in background over time (but not in x and y); a spatial background correction that corrects variations over the x and y (but not over time); and a general background correction, which is a single value for the entire movie. Any of these can be applied. If more than one are used, they have to be determined in the order temporal, spatial, general, as this is also the order in which they are applied. Determining the corrections in a different order, will delete the corrections that should have been determined later. The background corrections are stored in an _corrections.nc file for each movie.

Temporal background correction

If there are fluorophores in solutions, the background may change slightly over time due to e.g. bleaching. In this case a temporal background correction can be applied. The following line calculates a single background subtraction value per frame by taking the median.

file.movie.determine_temporal_background_correction(method='median')

Spatial background correction

To compensate for variations in background over the image, a spatial correction can be applied. For example by estimating the background using a median filter, which is then applied to all frames.

file.movie.determine_spatial_background_correction(method='median_filter', size=20)

General background correction

Because the median filter usually overestimates the signal, this can be compensated by applying a more accurate general background correction, a single value for each entire channel over all frames. The correction can be estimated for example by fitting the background peak in the intensity histogram.

file.movie.determine_general_background_correction(method='fit_background_peak')