FRET occurs between donor (D) and acceptor (A) dyes when these dyes are within certain proximity (< ca. 10 nm), resulting in a FRET signal (acceptor fluorescence upon donor excitation). There are many approaches to quantify FRET signal. In ZEN, the following methods are implemented:

• Sensitized Emission
• Acceptor Bleaching

Different acquisition set ups are necessary for each approach to enable the FRET view.

An analysis with Sensitized Emission requires a multichannel (multidimensional) data set (minimum 3 channels). The acquisition settings are determined by the used fluorophores. Refer to scientific papers for more information on sample preparation and necessary controls for sensitized emission experiments.

An analysis with Acceptor Bleaching requires a time series with a bleach event. The acquisition settings are determined by the used fluorophores. Refer to scientific papers for more information on sample preparation for acceptor bleaching.

Depending on the data set, the FRET view provides different parameter sets for image analysis.

To access the FRET view, the data set needs to be loaded or completely acquired. Online calculation of FRET data is not supported.