FRET
This module enables a mechanism which describes an energy transfer between two chromophores, typically fluorescent proteins. Upon excitation with light of suitable wavelength a donor chromophore, then in its electronic excited state, can transfer energy to an acceptor chromophore. The efficiency of this energy transfer depends on the distance between the two molecules. This makes FRET an indicator for even very small changes in the distance between two molecules.
When using confocal or super resolution techniques, FRET is typically used to determine whether two fluorophores are within a certain distance of each other and whether this distance changes due to external or internal influences.
Acquisiton Prerequisites
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.
Analysis Method for Acceptor Bleaching
Acceptor photobleaching analysis is available when a bleaching event is found in the image metadata. In this case the FRET efficiency can be calculated the following way:
E = (Dpost – Dpre)/Dpost
D is the donor signal and subscripts refer to pre- and post-bleach images.
In all cases the FRET values are calculated for every pixel that is considered “valid” and then the mean value is calculated and presented in the table. In case of acceptor photobleaching, the efficiency is also calculated from the pixel-averaged Dpre and Dpost signals.
Determining Donor Coefficient Values
- You have acquired or opened an image of a sample with a signal of the donor fluorophore only using the same imaging settings which are later used for imaging the FRET sample.
- You are in the FRET view.
- Go to the FRET view options tab and use the controls to define one or more analysis regions in the raw data image.
- The regions are displayed in a table on the FRET tab.
- Make sure that Object is activated for each region entry in the table.
- The regions are defined as objects that should be analyzed.
- On the Parameters tab, click Donor.
- The donor coefficient values are determined for the defined regions and displayed in the Parameters tab as well as the data table below the images.
Determining Acceptor Coefficient Values
- You have acquired or opened an image of a sample with a signal of the acceptor fluorophore only using the same imaging settings which are later used for imaging the FRET sample.
- You are in the FRET view.
- Go to the FRET view options tab and use the controls to define one or more analysis regions in the raw data image.
- The regions are displayed in a table on the FRET tab.
- Make sure that Object is activated for each region entry in the table.
- The regions are defined as objects that should be analyzed.
- On the Parameters tab, click Acceptor.
- The acceptor coefficient values are determined for the defined regions and displayed in the Parameters tab as well as the data table below the images.