Photobleach Correction Process Description

Photobleach Correction Process Description:

Photobleaching causes the fluorophores in each channel to lose intensity throughout the movie. The rate of photobleaching is usually different in the different channels and, therefore, can introduce artifacts in the ratio image if not corrected. This allows the photobleaching to be corrected by fitting a double-exponential to the average intensities in each channel. After the fitting, a figure will display the average ratio over time, and the fitted value along with confidence intervals. It is important to inspect this fit to ensure that the correction is valid and that the confidence intervals are not excessively large.

Parameter Descriptions:

Ratio Channels:

This channel should be set to the same as the numerator channel used in ratioing (usually FRET).

Photobleach Correction Type:

Ratio of Averages:

This method fits a double-exponential to the ratio of the average intensity in each of the channels used for ratioing and then uses this fit to correct the ratio images. This is generally the best method.

Average of Ratios:

This method fits a double-exponential to the average of the ratio image and then uses this fit to correct the ratio images. This will suppress any long-term changes in the ratio and should only be used if the other methods fail.

Ratio of Totals:

This method fits a double-exponential to the ratio of the total intensity in each of the channels used for ratioing and then uses this fit to correct the ratio images. This is generally only used when there is a drastic change in cell area throughout the movie (e.g., the cell rounds up). This sort of change will have a smaller effect on the total intensity than the average intensity, making the total a better measure in these cases.