Bleaching Times, Blinking Intervals and Time-Intervals of Interest

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.

1. Set bleaching times manually

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.

Set donor bleaching time.
Set acceptor bleaching time.
The bleaching times are highlighted in the traces.

2. Time-intervals of interest

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.

Set time-interval of interest (two time-points within the same axes).
In this example the time-interval of interest is the interval after one of two acceptors has bleached.