LSP background estimator

Determining the value of X in the LSP background estimator

This protocol describes how to determine the value of X in the LSP background estimator using iSMS.

1. Identify a molecule that is isolated from any neighbors (by a radius > 12 pixels). Here we pick molecule #1 shown in the screen shot.
2. Open the FRET pair window from the toolbar of the main window. In the FRET pair window select the identified molecule. Here it is “1.1” corresponding to (file #1, molecule #1). Verify that the molecule is isolated in the images.
3. Go to Plot->Percentile plot. This will start the calculation of a chi-square surface over different X values (evaluated against the mean value).
4. Determine the value of X corresponding to the minimum on the plotted graph. Here X = 61%. The red line denotes the value of the median.