FRET - acceptor photobleaching

May 10, 2011
BALM Protocols – Ab FRET
FRET EXPERIMENTS DEMAND ADVANCED SKILLS IN USE OF MICROSCOPES AND IMAGE ANALYSIS and antibody staining
or transfections which are robust, clear and reproducible.
If you do not know what a FRET donor or a FRET acceptor is please go and read about FRET Theory. It is absolutely
essential you understand FRET theory before starting these experiments as you will not be able to analyse and interpret
the data otherwise. The BALM facility staff can technically support you with use of equipment but the design, and
analysis and interpretation of the experiments must be yours.
If you need more information please see the original reference for Ab FRET which is:
Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching
using confocal microscopy and a single laser. T. S. Karpova 1 and J. G. McNally 1,* Journal of Microscopy Volume 209,
Issue 1, pages 56–70, January 2003
Read about FRET theory here:
http://www.embl.de/eamnet/downloads/modules/FRET_teaching_module.pdf
http://www.olympusmicro.com/primer/techniques/fluorescence/fret/fretintro.html
See how to present the data by looking at these papers:
Tsang SM, Liu L, Teh M-T, Wheeler A, Grose R, et al. 2010 Desmoglein 3, via an Interaction with E-cadherin,
Is Associated with Activation of Src. PLoS ONE 5(12): e14211. doi:10.1371/journal.pone.0014211
Y. Gu, W. L. Di, D. P. Kelsell and D. Zicha. Quantitative fluorescence resonance energy transfer (FRET)
measurement with acceptor photobleaching and spectral unmixing Journal of Microscopy 215. Article first
published online: 16 JUL 2004 | DOI: 10.1111/j.0022-2720.2004.01365.x
An integrin-α4–14-3-3ζ–paxillin ternary complex mediates localised Cdc42 activity and accelerates cell
migration. Nicholas O. Deakin1,*‡, Mark D. Bass1,*, Stacey Warwood1, Julia Schoelermann2,3, Zohreh
Mostafavi-Pour1,§, David Knight1, Christoph Ballestrem1 and Martin J. Humphries1, J Cell Sci. 2009 May
15;122(Pt 10):1654-64. Epub 2009 Apr 28.
BASIC THEORY
Acceptor photobleaching works by eliminating the acceptor, so in a sample where FRET is occuring after Acceptor
photobleaching the energy which was being passed to the acceptor will be kept by the donor molecule. This leads to an
increase in the fluorescence of the donor molecule. The fluorescence increase can be measured quantitatively.
FRET Occurring
Acceptor
Photobleaching
Donor
Energy Acceptor
Donor FRET Acceptor
No energy transfer from
donor to acceptor

May 10, 2011
Recommended controls:
Positive and negative FRET controls – Donor alone, Acceptor alone,
Control to determine baseline of FRET A condition where FRET is not expected to happen. The condition where FRET is not
expected to happen can be unstimulated cells or where you have sent the donor fluorophore to a part of the cells where the
acceptor definitely won't bind to it. E.g. donor bound to nucleus, acceptor bound to plasma membrane. You can then
photobleach the acceptor and look to see if the donor fluorescence changes.
Control where you have a strong feeling FRET has worked or have identified an interaction by IP
When you start the experiment – Set up will take your first session.
Turn on microscope at least 4 hours prior to start of experiment to allow it to stabilise.
Find sample using microscope.
Check on camera that you can see enough cells / whatever you are measuring in the right amount of detail.
Set up acquisition read out channels to as follows:
1. Fluorescence of the donor (reading donor excitation, donor emission) – Data channel
2. Fluorescence of acceptor (reading acceptor excitation, acceptor emission) – Prooves photobleaching works
3. FRET Fluorescence (reading donor excitation, acceptor emission) – Just in case the reviewer asks….
Setting up detectors
The first sample to visualise under the microscope is the one where you suspect FRET has worked. Set up the microscope so
you can clearly record your image in all channels on the detector (i.e. not by eye). Ensure for all channels that the image is
in the middle of the intensity range of the detector. Set the detector so it is at the highest bit range, 16 bit for preference.
Now put the negative controls on the microscope. Alter the channels so that as little as possible is being recorded in the
FRET channel or. Make sure you take at least 3 pictures of both of the negative controls.
Go back to the positive control and check that the donor, acceptor and FRET channels can be recorded. Now save the
settings for the channels, write these settings down and do not change them during your experiment for a particular set of
donor and acceptor pairs.
Setting up bleach parameters
Using the positive control sample pick an area which doesn't contain FRET or isn't relevant to the analysis. Use this to set up
bleach conditions. This is done by selecting a small ROI and bleaching it with a given laser power and number of iterations.
The ideal bleach is one which reduces the acceptor intensity by between 25 – 50% but doesn't photodamage the cell. The
best bleach is one with the weakest laser power and smallest number of iterations to achieve 50% photobleaching. Check
photobleaching doesn't damage the donor channel.

May 10, 2011
Testing acceptor photobleaching
Using the positive control sample find a cell which potentially shows FRET. The FRET readout channel is useful here. To test
photobleaching set up regions as follows:
a. Regions which shows FRET – Termed data ROI, there can be as many of these as you like 3 is ideal
b. A region which doesn't show FRET – Termed internal control
c. A region which shows FRET but isn't bleached ‐ Termed Photodamage ROI
Set up an experiment which bleaches regions 'a' and 'b' by about 25 – 50% but not 'c'. Measure Fluorescent intensities in
donor channel in regions 'a', 'b' and 'c' before and after photobleaching.
Use the acceptor photobleaching equation to determine % FRET
% FRET for Data ROI= 100 x Intensity in donor channel post‐bleach + Photodamage in donor channel
Intensity of donor channel pre‐bleach
If % FRET is about 5% the acceptor photobleaching has definitely worked.
If for none of the data ROIS FRET is seen then:
I. The acceptor photobleach hasn't worked,
II. You have damaged the donor with your acceptor bleach so you need to check the bleaching parameters
III. You can’t detect the change in donor intensity
IV. There isn't any real FRET
If you see drift in the system between donor and acceptor imaging this will be caused by temperature changes. Ensure
the microscope is fully warmed up before you start.
PLEASE DISPOSE OF ALL YOUR CLINICAL WASTE IN THE YELLOW BAG/ LARGE SHARPS BIN PROVIDED