Openlab Module - FRET

Technical Note: 320
Reads: 6268
Creation Date: 12/03/2003
Modification Date: 12/02/2004

FRET (Fluorescence Resonance Energy Transfer) is the non-radiative transfer of energy from an excited fluorophore (donor) to another fluorophore (acceptor). FRET only occurs when the molecules concerned are in very close proximity (1-10nm). This makes it a valuable technique for studies where distances and interactions between molecules are of interest.

The Openlab FRET module
The Openlab FRET module allows accurate FRET measurements and generation of net FRET (or ìcorrectedî FRET) and normalised FRET images for publication.

The analysis currently offered by the module is the 3-image FRET technique, as described by Gordon et al. (1998) and Xia and Liu (2001).

These references should be used to find further information on the technique and guidance on interpretation.

Data acquisition
Three images are collected for each data point, a Donor image, an Acceptor image, and a FRET image.

One typical donor/acceptor pair is CFP & YFP. Thus:

Donor image = Excite with CFP filter, CFP dichroic, emit with CFP filter

Acceptor image = Excite with YFP filter, YFP dichroic, emit with YFP filter

FRET image = Excite with CFP filter, CFP dichroic, emit with YFP filter

The Openlab Automator should be used to acquire these images using the appropriate filters. The order of image acquisition is important in analysis, see below.

The result is generated by the FRET module by combining the three input images on a pixel-by-pixel basis according to the chosen expression. The output is a floating - point value at each pixel.

Bleed-through
Reference to the expressions cited indicates that there are two constants required. To generate FRET images, the user requires two values (a and b) which represent the bleed-through or cross-talk of each dye into the FRET filter set. These values are calculated once and then used for all subsequent experiments.

ìAî, called Donor bleed-through in the interfac