FRET (Foerster Resonance Energy Transfer) can happen between two fluors if they are close enough in distance and have the right spectral overlap. The donor fluor absorbs a photon within its excitation wavelength range, and can then transfer this energy, in a non-radiative manner, to the acceptor fluor, which can then emit light at its characteristic wavelength, which will be higher than the donor’s emission (you can think of it as the acceptor “stealing” energy from the donor and reducing its fluorescent signal). The two fluors must be very close (50 angstroms or less) for this to happen, and the orientations of the two molecules can also influence the efficiency of the energy transfer.
FRET is the gold standard for demonstrating that 2 molecules are indeed close enough to physically interact. There are also FRET sensors that allow one to detect in real time biological events such as substrate binding, conformational changes, vesicle fusion, or enzymatic activities through the gain or loss of FRET.
This application allows you to measure FRET efficiency by comparing the fluorescence of the donor and the acceptor before and after photobleaching of the acceptor (which will disrupt FRET and increase the signal from the donor). This is useful for measuring the average distance between the two fluors, as well as verifying FRET sensors.
Select the FRET-AB wizard from the TCS SP8 pull-down menu:
The first step is to set the lasers/detectors for the FRET donor. In this example I have used a linked CFP-YFP FRET sensor (with FRET as it default state), expressed in the mushroom body of an adult Drosophila brain. For CFP, 458nm is the best of our available laser wavelengths. Turn on the laser line (1), switch on PMT1 (2) and switch off PMT3. Use the pull down menus in PMT1 and PMT3 to call up the emission spectra for CFP and YFP, and set the detection window for PMT1 from 468nm to the left edge of the YFP curve (3). Live scan the specimen and adjust the gain/offset/laser% to get a clear image (4).
Now click “acceptor” under “Define the Acceptor setting” (1).
For YFP we use the 514nm laser. Turn the 458 to 0% and turn on the 514 (2). Activate PMT3, turn off PMT1, and set the detection window for YFP (3). Live scan again and adjust the controls until the image looks the way you want.
Now you need to set the bleach conditions. Click the “Bleach” tab on the top of the screen (1):
Turn the 514 laser to 100% (2). The “No. of frames” will determine the duration of the bleaching step (3). The optimal time will vary according to your type of specimen/ choice of FRET pairs, so you will need to experiment with various settings to find the one that works best for you. Finally, use the ROI tools (4) to select a portion of the specimen to photobleach. Click the “Run Experiment” tab at the bottom center of the panel when you are ready to begin the photobleaching protocol.
When the scan is finished the software will give you the intensities of the two fluors before and after photobleaching, as well as a calculation of FRET efficiency:
In this example, the loss of YFP and increase in CFP within the ROI is clearly visible in the images:
With the FRET Efficiency score, you can now calculate the distance (RDA) between the two fluors using this equation:
R0 is the distance required for ~50% efficiency of the maximum possible energy transfer from donor to acceptor. R0 values have been determined for many FRET pairs:
For this example the R0 for CFP-YFP is 4.7 angstroms (quite close!), and the measured efficiency was 0.4687.
This application is best used on fixed samples for calculating RDA. A live specimen (which was used in this example) will have much more variability (as the molecules will be more free to move), but it can be used to verify that a FRET sensor is functional.
(Coming next- how to set up the SP8 to use a FRET sensor for live imaging)