Single-Molecule FRET Microscopy Software

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Automated Detection of Bleaching Times in Single-Molecule FRET

1. Set thresholds for the bleachfinder

In order for the bleach finder to perform well the threshold settings must be set properly. The bleach finder thresholds are set in 'Settings->Bleach-finder settings'.

The bleach finder settings are best explained by how the algorithm works:

The algorithms start at the end of the trace and move towards the start, one frame at a time. When an intensity above the chosen threshold level is encounted more than x consecutive frames this is defined as a bleaching event.

The optimal intensity threshold levels depend on your setup. For the detection of donor bleaching it is the sum of D and A intensities (on D excitation) that is used. For detection of acceptor bleaching it is the direct acceptor reference trace (AA) that is used in ALEX and the AD trace that is used in single-color mode. The traces are initially smoothed using a median filter.

Picture of bleachfinder settings

2. Run bleach finder

Instruction on how to find the tools button in Bleach Finder
Go to 'Tools->Bleach detection' in the FRET-pairs window. This will run the bleach finder. If some bleaching times have already been set, a dialog will be prompted asking which molecules to include in the scan.
Instruction on how to find the number of detected bleaching events
The number of detected bleaching events is shown in the lower left corner. To get an overview of the molecules showing bleaching you can create groups for these molecules by navigating to the menu 'Grouping->Create group for all with...'.
Revised 17.04.2023