Correction Factors in Quantitative Single-Molecule FRET Microscopy

Calculation of correction factors

See quick guide 2 for guide on how to determine correction factors using iSMS.

Specify correction factors

Correction factors are edited most quickly in the lower right corner of the FRET-pairs window. To use molecule-to-molecule dependent correction factors tick the 'Individualized' checkbox. Note how the histogram window updates immediately to the new settings.

Molecule-to-molecule individualized correction factors

To use individual correction factors for each molecule tick the checkbox in the lower right corner of the FRET-pair traces window (see figure above). When using individualized correction factors each molecule can be assigned an individual correction factor only applied to this particular molecule. For molecules where correction factors have been determined by iSMS the determined correction factors are applied by default. For molecules where correction factors have not been determined, the global value will be used.

The global value

Note that when using individualized correction factors changing the value of a correction factor in the FRET-pair trace window will only apply to the selected molecules. To change the global correction factor value open the 'Correction factors' menu located under the 'Settings'-menu.

A word of caution

In our experience using individualized correction factors is not a universally applicable fix since the correction factors of each molecule are determined with some uncertainty. For this reason, using individualized correction factors may distort FRET-histograms simply due to a few outliers with poorly determined correction factors.