Fret not, the new Openlab FRET module is here!

In a nutshell, FRET (Fluorescence Resonance Energy Transfer) is the transfer of energy from one excited fluorophore to another fluorophore. This only occurs when the molecules in question are really close together.

With the new module you can measure FRET using the 3-image technique (Gordon et al. 1998, Xia and Liu 2001). You can also produce net FRET and normalized FRET images for publication.

Before we head along into the intricacies of the module there are some additional points to think about. The Openlab FRET module is for 3-image FRET, so can only be used if you have a donor, an acceptor and a FRET image to use as input images. I will show you here how to use the Openlab FRET interface, however I cannot pass comment on sample preparation or interpretation of results. I would refer to those who have published on the subject.

In using the 3-image technique, a donor image, an acceptor image and a FRET image are required. Let's look at this using CFP and YFP as the donor/acceptor pair.

To acquire the donor image you would use a CFP excitation filter, a beam splitter and an emission filter.

For the acceptor image you would use a YFP excitation filter, a beam splitter and an emission filter.

For the FRET image use the CFP excitation filter, a CFP beam splitter and a YFP emission filter.

Remember to be mindful when considering the beam splitter as this will need to reflect the excitation wavelengths for CFP and YFP to the sample but also be able to transmit the emissions of both to the detector.

The order in which you acquire your images is also important - I'll get to this in a moment.

To generate your FRET result images you also need two constants, the donor and acceptor bleed-through constants. These need to be calculated before you use the FRET module.

1. The donor bleed-through- this is obtained by using a sample labeled only with the donor, in this case CFP. Two images are captured, a FRET image and a donor image.
The constant is generated as follows:

Donor bleed-through = Intensity of cell in FRET image/Intensity of cell in donor image

2. The acceptor bleed-through - this is obtained by using a sample labeled only with the acceptor, in this case YFP. Two images are captured, a FRET image and an acceptor image. The constant is generated as follows:


Acceptor bleed-through = Intensity of cell in FRET image/Intensity of cell in acceptor image

The constants will change if any alteration is made to the system, for example, if you invest in a nice new mercury or xenon lamp or feel the urge to change the objective.

Before using the FRET module your images must be background subtracted and if you need to align your images with the Openlab Registration module, now is the time to do it, before creating your FRET result images.

Images to be processed must be in sets of three and ordered in the Layers Manager in one of these two sequences:

Donor- FRET- Acceptor; Donor- FRET- Acceptor...
Or Donor- FRET- Acceptor; Acceptor- FRET- Donor; Donor- FRET ...
-this sequence is more efficient as fewer filter moves are required.

The Images must also be between 8 and 16 bit, all at the same depth and all general image layers.

OK, so you've done all that already, now go to the Special menu ... FRET.

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Lets start at the top of the dialog and move down.
Firstly pick the method of FRET calculation to be used.
Then select the order in which the images were acquired.
Enter the bleed-through constants from your control experiments.
Adjust the thresholds for each image, this improves contrast (Handy hint - use the HSI color spy before using the FRET module to give an idea of the background intensity of each image).
As for many other Openlab modules, there is a preview that shows the FRET result image that will be produced.
Notice that the Color LUT defaults to "Rainbow".
The Color LUT can be distributed Linearly or Logarithmically, over the desired output range, so adjust with sliders as desired.
The Auto button will scan all selected images finding the maximum range of FRET values and will use these as the output range.
Finally click on the OK button to apply the FRET calculation to all selected input images and create output images. These are added to the end of the Image Document.

Whoops - I forgot to mention Intensity Modulated Image.
This uses pseudocolor and intensity to display the output image.
Color represents the FRET value, Intensity represents the intensity of one of the input images.
You can pick from any of the source images or the brightest of the source images or the average of the source images.
This is particular good for spatial information since the intensity of an input image can be reflected in the result.