Leica CONFOCAL APPLICATION LETTER User Manual
Sep.
2006
No.20
CONFOCAL APPLICATION LETTER
LAS AF APPLICATION WIZARD
FRET SENSITIZED EMISSION
reSOLUTION
Emission
Wavelength
Green Channel Red Channel
FRET Sensitized Emission
Donor
Acceptor
S1
S1
FRET with Leica TCS SP5 (LAS AF)
FRET Sensitized Emission
Fluorescence Resonance Energy Transfer (FRET) is a technique,
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.
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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.
FRET Sensitized Emission
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FRET Wizards in Leica Application Suite
Advanced Fluorescence (LAS AF)
Within the LAS AF ➀menu 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.
•
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.
➁
➀
Step 1: Setting of experimental conditions
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!
A
Step 2: Acquire image sequences
of controls for calibration
B
C
5
2
1
4
3
6
2
1
3
C
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