User manual | FRET with Leica TCS SP5 (LAS AF)

CONFOCAL APPLICATION LETTER
reSOLUTION
Red Channel
Emission
Green Channel
Wavelength
LAS AF APPLICATION WIZARD
FRET SENSITIZED EMISSION
Sep.
2006
No.20
FRET Sensitized Emission
FRET with Leica TCS SP5 (LAS AF)
FRET Sensitized Emission
Fluorescence Resonance Energy Transfer (FRET) is a technique,
Donor
Acceptor
S1
S1
which allows insight into the interactions between proteins or molecules in proximities beyond light microscopic resolution.
The Principle:
An excited fluorophore, called the donor, transfers its excited state
energy to a light absorbing molecule which is called the acceptor.
This transfer of energy is non-radiative.
Sensitized Emission is one established method for the evaluation of
FRET efficiencies. It can be applied to live cells as well as to fixed
samples.
The Method:
As this method is non-invasive, it is most frequently used for live cell
experiments. Because crosstalk is an important issue, controls and
complex correction calculations are inherent to the method.
Measurements are executed by detection of the fluorescent signals
of the donor and FRET as well as acceptor in a line by line sequential
scan acquisition.
Proper excitation conditions play a major role. In the first sequence,
the donor must be excited to generate donor fluorescence and sensitized emission of the acceptor. In the second sequence, the selective excitation of the acceptor will create acceptor fluorescence.
FRET sample preparations must therefore include references of
donor in the absence of the acceptor (donor only control) and acceptor in the absence of the donor (acceptor only control).
Ideally, all references are included in the same preparation. The
donor and acceptor references are used to obtain calibration coefficients to correct for excitation and emission cross talk.
It is important to be aware that throughout the entire experiment and
calibration routine, all measuring parameters such as gain, emission
detection window, excitation intensities, zoom, format, scan speed,
pinhole etc must remain constant.
2
Confocal Application Letter
FRET Sensitized Emission
FRET Wizards in Leica Application Suite
Advanced Fluorescence (LAS AF)
➀
Within the LAS AF ➀ menu bar you can find two wizards to perform
FRET experiments: FRET AB (Acceptor Photo-Bleaching) and FRET
SE (Sensitized Emission). This application note describes working
with the FRET SE Wizard ➁.
The wizard consists of 4 steps and in addition, an overview of the
experimental workflow.
•
•
•
➁
Step 1: is dedicated to the imaging set-up.
Step 2: executes control and FRET measurements.
Step 3: guides through the calibration routine and calculates the
calibration coefficients.
•
Step 4: is dedicated to the execution of time-lapse experiments,
evaluation of results and generation of experimental reports.
Confocal Application Letter
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Step A1: Setting of experimental conditions
B
5
3
6
2
C
1
4
A
C
B
Step 2: Acquire image sequences
of controls for calibration
2
3
Note:
It is vital to keep all measurements under
identical conditions. If your controls do
not match the actual imaging conditions
(saturation!) and if you have therefore to
readjust, all previous measurements under the changed conditions will have to be
repeated. All measurements performed
under differing conditions should be discarded!
4
Confocal Application Letter
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FRET Sensitized Emission
FRET Sensitized Emission – Step by Step
5. Slowly increase the excitation light of the acceptor (in our example 514 nm) until the signal of the acceptor (orange) is just below
Step 1: Setting of experimental conditions
saturation. Do not change the PMT gain or offset as this will
Begin the imaging set-up with the FRET sample with all fluorescence
also change the imaging conditions for the FRET (yellow) signal
information (donor, FRET and acceptor) in the same specimen. Define
detection!
imaging conditions for donor, FRET and acceptor fluorescence by following the workflow. You may start your experiment with previously
6. Define the number of averages for best imaging conditions. The
saved imaging conditions. Use the Load and Save options ➀. If you
desired acquisition mode may be chosen with the tools under
want to establish imaging conditions yourself you must start out in
Acquisition ➄.
the Beam Path Settings ➂.
7. Proceed to the next step Corr. Images to acquire control images of
1. Image acquisition of donor, FRET and acceptor will automatically
the specimen for calibration.
be done in a line by line sequential scan mode, after the definition
of donor, FRET and acceptor. Start defining the donor and the FRET
detection (Donor + FRET) ➁ via the Beam Path Settings window
➂. Begin by simultaneous excitation and detection of the donor
and acceptor (e.g.: Donor = CFP excitation 458nm; emission 462-
Step 2: Acquire image sequences of controls for calibration
510nm; Acceptor = YFP excitation 514nm; emission 518-580nm).
1. In step 2 you may first take an image set of your FRET sample
This enables you to assess the fluorescence intensities, PMT gain,
since it is already under the scope and nicely set-up. Acquire the
and laser dose for each label. Make all adjustments by using the
sequence via Capture Image ➀. The image set will be automati-
Live button ➃. Check for appropriate imaging resolution. You may
cally named ‘FRET’.
change the zoom factor via the control panel or with the tools
2. Continue with the measurements of the controls (Donor only and
under Acquisition ➄.
Acceptor only)
2. Reduce now the laser light of the acceptor down to 0% ➅. Re-ad-
➁. To keep track of the specimen choose the
correct radio button for the according specimen.
just the acceptor PMT to be slightly below saturation. You are now
exciting only selectively for the donor and have properly defined
3. If controls are on separate slides you will now change specimen.
For a better field of vision you may want to go back to zoom 1 to be
your conditions for the donor and FRET imaging.
able to find adequate cells to properly match the intensities and to
3. Continue by defining the acceptor set-up ➁ via the Beam Path
avoid saturated regions. Use the function button Search Speci-
Settings window ➂. Turn the donor excitation light down to 0%.
men ➂. You now have access to all the acquisition tools needed.
Choose a new LUT for the directly excited acceptor signal (e.g.:
When you exit Search Specimen you will automatically return to
light blue = donor; yellow = FRET; orange = acceptor) for better
the zoom factor and resolution conditions of all previous measure-
discrimination. Start Live Scan ➃.
ments in the experiment.
4. The images you see in the viewer are generated by means of a line
by line sequential scan. The first sequence
A
+
B
consists of
4. Proceed from Corr. Images to the next step Corr. Factors to generate calibration factors.
donor (light blue) and FRET (Yellow). The excitation wavelength is
selectively chosen for donor excitation. The second sequence
C
contains acceptor fluorescence (orange). The excitation light is
selective for acceptor excitation.
Confocal Application Letter
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2
Step 3: Calibration
1
3
Step 3: Calibration
➃
4
6
Confocal Application Letter
FRET Sensitized Emission
Step 3: Calibration
Calibration Factors:
1. Ensure you have the appropriate image set active in the experiment overview to match the indication marked by the radio button
1. Donor only reference generates correction factor
in the interface ➀.
Calibration Factors:
2. Draw a region of interest (ROI) in the image where only donor
b corrects for donor cross-talk: b = B/A
signal is found ➁. The mean intensities within the ROI are shown
1
in the workflow interface ➂. Press Accept to use values for cal-
CFP
0,9
culation of calibration coefficients.
0,8
images show background above 0. If this is the case, draw a ROI
in the image where only background signal is found and press
Intensity
0,7
3. Background subtraction is optional. It is only necessary if the
b = B/A
0,6
A = Donor Channel
B = FRET Channel
0,5
0,4
0,3
Accept to use these values.
0,2
A
B
0,1
4. Continue likewise for acceptor only sequence.
0
440
490
540
590
640
Emission spectra of CFP [nm]
For ease of interpretation intensity distribution check the table
showing the intensity distribution for measured specimen (e.g.
2. The Acceptor only reference generates correction
CFP-YFP):
factors a, g, d.
Channel 1 (A)
Channel 2 (B)
Channel 3 (C)
Specimen: FRET
Signal (Donor)
Signal (FRET)
Signal (Acceptor)
Specimen:
Signal (Donor)
Signal < Channel 1
No Signal
a corrects for acceptor cross-excitation cross-talk:
a = A/C
g corrects for acceptor cross-excitation:
g = B/C
(x-talk)
Donor only
Specimen:
Very little to no
Signal < Channel 3
Acceptor only
Signal (x-excited
(x-Excitation)
d corrects for FRET cross-talk:
Signal (Acceptor)
d = A/B
x-talk Acceptor)
1
YFP
0,9
5. Press Calculate Factors to generate correction factors ➃.
Acceptor excited by donor excitation
0,8
a = A/C
g = B/C
d = A/B
sequences (fixed sample analysis) or any time-lapse experiment to
follow. Precondition: all measurements remain under unchanged
imaging conditions.
Intensity
0,7
All calculated calibration factors will be applied to the existing FRET
C
0,6
0,5
A = Donor Channel
B = FRET Channel
C = Acceptor Channel
0,4
0,3
0,2
0,1
6. Continue to the next step Evaluation to perform live cell measurements after calibration and to retrieve FRET efficiencies.
Acceptor selectively excited
A
B
0
440
490
540
590
640
Emission spectra of YFP [nm]
Confocal Application Letter
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3
Step 4: Calculation of FRET efficiencies
and live cell measurements
1
2
4
5
8
Confocal Application Letter
FRET Sensitized Emission
Step 4: Calculation of FRET efficiencies and live cell measurements
Calculation of FRET efficiency:
Before running a time-lapse experiment you may choose the calcula-
EA is the apparent FRET efficiency. A, B, C correspond to the
tion method to calculate and represent the apparent FRET efficien-
intensities of the 3 signals (donor, FRET, acceptor) and a, b, g
cies ➀. You may also change the method after running the experi-
and d are the calibration factors generated by acceptor only
ment. Method 1 is automatically applied.
and donor only references.
Method 1:
If your FRET data shows a background above 0 you may run a background subtraction ➁. Draw a ROI in the image viewer where only
EA(i) =
B–Axb–Cxg
C
background signal is found ➂. Press Accept to include the background subtraction into calculations. You may also fill in a value by
yourself to match background variations more precisely.
Ref. Wouters et al., TRENDS in Cell Biology, Vol 11, No.5,
May 2001: 203-211
Method 2:
For time-lapse experiments choose imaging conditions under Acquisition in the Acquisition Mode ➃ and start your series with Run
Experiment ➄.
EA(i) =
B – A x b – C x (g – aÐ x b)
C x (1 – b x d)
Ref. Van Rheenen, J., M. Langeslag, K. Jalink: Correcting Confocal Acquisition
to Optimize Imaging of Fluorescence Resonance Energy Transfer by Sensitized Emission. Biophysical Journal, Vol. 86, April 2004: 1-13.
Method 3:
EA(i) = B
A
The Ratiometric Calculation is used in samples with a fixed
stochiometry (1:1) of donor and acceptor (e.g. Cameleons).
Confocal Application Letter
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Step 4: Calculation of FRET
efficiencies and live cell measurements
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Confocal Application Letter
FRET Sensitized Emission
To see FRET and intensity values displayed in a graph during the
experiment change to the tab sheet Graph. Draw one or multiple ROIs
in the image viewer to see intensities displayed in the graph.
For apparent FRET efficiencies change to tab sheet Statistics.
Single FRET images (e.g. fixed specimen) can be analyzed too, but
will show results only under Statistics and not in the graphical
display. Change to tab sheet Statistics and use ROI functionality
to choose the appropriate regions of interest in the image.
Suggested background reading:
• Gadella T.W.J., G.N.M. Van der Krogt, T. Bisseling. GFP-based
FRET Microscopy in Living Plant Cells. Trends Plant Sci. 4(7)
287-291 (1999)
• Gordon et al.: Quantitative Fluorescence Resonance Energy
Transfer Measurements Using Fluorescence Microscopy.
Biophysical Journal, Vol. 74 2702-2713 (1998)
• Lippincott-Schwartz, J., E. Snapp and A.K. Kenworthy. Studying protein dynamics in living cells. Molecular Cell Biology, 2
444-456 (2001)
• Van Rheenen, J., M. Langeslag, K. Jalink: Correcting Confocal
Acquisition to Optimize Imaging of Fluorescence Resonance
Energy Transfer by Sensitized Emission. Biophysical Journal,
Vol. 86 1-13 (2004)
• Wouters F.S., P.J. Verveer & P.I.H. Bastiaens. Imaging
biochemistry inside cells. Trends Cell Biol., Vol. 5: 203-211
(2001)
• Zal T., R.J. Gascoigne: Photobleaching-Corrected FRET
Efficiency Imaging of Live Cells. Biophysical Journal, Vol. 86
3923-3939 (2004)
• Zhang, J., R.E. Campbell, A.Y. Ting, R.Y. Tsien: Creating new
fluorescent probes for cell biology. Nature, Vol. 3 (2002)
Confocal Application Letter
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