Specifying the time at which molecules blink or bleach is a critical step in the data analysis. The program uses this information to calculate backgrounds, correction factors, analyse transitional dynamics, group molecules and/or for visualization purposes.
Bleaching times are either detected automatically or set manually. It is recommended to use the bleach finder first and subsequently adjust the molecules that were missed by the bleach finder manually. You must optimize the settings of the bleach finder to match your setup in order for it to perform well.
Bleaching, blinking and time-intervals of interest are set in the FRET-pairs window. Activate the relevant crosshair-tool in the toolbar and point at the time-points inside the trace-axes of the selected molecule.
The time-interval of interest is used to specify a relevant sub-interval of the whole trace to be used for e.g. FRET-histogram plots or HMM analysis.