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IPC Friedrich-Schiller-Universität Jena1
7. Fluorescence microscopy
7.3 FRET microscopy - The different Dipoles
1. static electric Dipole:
Far away: E ~ r-3 along all directions
2. radiating (Hertz's) Dipole: (VERY DIFFERENT!)
far away:
in line: zero field (n x p = 0)orthogonal: E ~ r-1
r = = distance from middle to positionn = direction unit vectorp = dipole vector
IPC Friedrich-Schiller-Universität Jena2
7. Fluorescence microscopy
7.3 FRET microscopy - The different Dipoles
3. transition Dipole moment – an analogy :
-
+
"molecular"
conductor
E = electric Field
Induced Transition Dipole
Transition Dipole Moment : a vector along who's direction the dipole will be induced
IPC Friedrich-Schiller-Universität Jena3
Resolution of a light microscope is limited to several hundred nanometers
(< organelles) FRET allows detection of molecule-molecule interactions on a nanometer scale by
means of a light microscope
Decrease of donor-emission
Increase of acceptor emission
Reduction of donor fluorescence life-time
Energy transfer (FRET-efficiency) depends strongly on donor-acceptor distance
R0 = Förster-radius (distance for which energy transfer is half maximal)
7. Fluorescence microscopy
7.3 FRET microscopy
sensitizedemission
IPC Friedrich-Schiller-Universität Jena4
FRET ratio imaging = acceptor emission at donor excitation (sensitized emission SAkzeptor) divided by donor
emission at donor excitation (SDonor)
Advantages: Since both donor decrease as well as acceptor increase contribute to
the signal the signal-to-noise ratio is better than for solely recording the acceptor
fluorescence
SAkzeptorSDonor
7. Fluorescence microscopy
7.3 FRET microscopy
IPC Friedrich-Schiller-Universität Jena5
FRET ratio imaging – problems:
Correction for direct excitation of the acceptor when exciting donor (control measurement with YFP only) = correction factor rDE
Excitation wavelength
Correction for bleedthrough : Portion of CFP in yellow channel for blue excitation in absence of FRET (acceptor) = bleedthrough of CFP in YFP-channel (rBT,CY)
or bleedthrough of YFP in CFP-channel (rBT,YC)
FR
ET
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7. Fluorescence microscopy
7.3 FRET microscopy
IPC Friedrich-Schiller-Universität Jena6
FRET ratio imaging – 3-filter-set:
1. Donor excitation and emission(ICFP,430)
2. Acceptor excitation and emission(IYFP,514)
3. Donor excitation and acceptor emission(IYFP,430)
7. Fluorescence microscopy
7.3 FRET microscopy
IPC Friedrich-Schiller-Universität Jena7
FRET ratio imaging – 3-filter-set:
Model of FRET-detection of Src-
Csk protein interaction
(Src = protein tyrosine kinase
Csk = C-terminal Src kinase) Important signal transduction step
during blood coagulation
7. Fluorescence microscopy
7.3 FRET microscopy
No FRET
IPC Friedrich-Schiller-Universität Jena8
FRET ratio imaging – 3-filter-set:Visualization of Src-Csk-interaction during aIIbß3-induced fibrinogen adhesion in a
thrombocyte model cell line (A5-CHO) by means of FRET
7. Fluorescence microscopy
7.3 FRET microscopy
superposition
IPC Friedrich-Schiller-Universität Jena9
FRET ratio imaging – 3-filter-set:FRET for displaying Ca2+ in living cells via Yellow-Cameleon-2 (YC2) sensor
FRET-ratio image of HeLa-cells, expressing the YC2-sensor before and after adding ionomycin
FRET response of HEK/293 cells expressing YC2-seonsor after adding 1nM ionomycin and additional extracellular Ca21 (30 mM)
7. Fluorescence microscopy
7.3 FRET microscopy
Acceptor Bleaching
Free Donor +1 Donor (Pairs)
AcceptorRemoved
Free Donor +Donor (Pairs)
Donor excitation,Donor emission detection
1 can be calibrated
De-Quenching
Principle ofAcceptor-bleaching-FRET microscopy
Donor fluorescenceshould increase afteracceptor bleaching
PrincipleDonor fluorescence should
increase (dequenching) after“removal” of the acceptor
Acceptor depletion FRET
CFPExc 457Em 470-500
‘Sens-YFP’Exc 457Em 535-570
YFPExc 514Em 535-570
Combined
100 µm
Data of Dorus Gadella
Donor Emission channel
Acceptor Emission channel
CFPExc 457Em 470-500
‘Sens-YFP’Exc 457Em 535-570
YFPExc 514Em 535-570
Combined
100 µm
Data of Dorus Gadella
BUT is it:High concentration, low efficiency?
Low concentration, high efficiency?
Donor Emission channel
Acceptor Emission channel
IPC Friedrich-Schiller-Universität Jena14
FRET fluorescence life-time microscopy:
In case of FRET the donor fluorescence life-time is reduced. Determination of this
donor life time reduction yields a quantitative FRET measurement which is
independent of dye concentration or spectral contamination (crosstalk, bleedthrough).
Dimerization of C/EBP® – proteins in GHFT1-5 cell nuclei(donor/acceptor CFP/YFP-C/EBP®)
7. Fluorescence microscopy
7.3 FRET microscopy
IPC Friedrich-Schiller-Universität Jena15
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
7. Fluorescence microscopy
Laser pulse
Longer fluorescence life-time
Shorter fluorescence life-time
Methods of time-resolved fluorescence diagnostics
Time-resolved measurements
Sample is excited by a short laser pulse
Sample molecules relax individually according to the transition probability of the different relaxation pathways to the ground state
Fluorescence intensity exhibits mono-, multi or non-exponential decay depending on nature and number of fluorescence contributions.
IPC Friedrich-Schiller-Universität Jena16
Time-resolved measurements
Intensity integrating measurementsThe determination of the fluorescence decay time ¿ or times ¿i and relative amplitudes ®i in
case of multiple contributions is possible by recording the fluorescence signal for several measurement points after the excitation pulse. For a mono-exponential decay behavior or to determine the average decay time ¿ two sampling points are sufficient
For two times t1 and t2 after the excitation pulse the detector signal is integrated for a sampling window ¢ T. The ratio of the measurement signals D1 und D2 can be used to calculate the decay-time ¿ or the average decay-time ¿ :
Methods of time-resolved fluorescence diagnostics
7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
IPC Friedrich-Schiller-Universität Jena17
Methods of time-resolved fluorescence diagnostics
Time-resolved measurements Gated fluorescence detection
Gated optical image intensifiers (GOI) are capable of taking pictures with high (sub-nanosecond) time resolution i.e. camera with ultrafast shutter (gate < 100 ps) which can be opened and closed for different delay-times after the sample has been excited with an ultrashort laser-pulse. By collecting a series of time-scanned fluorescence intensity images for different delay-times after excitation the fluorescence decay profile for every pixel in the field of view can be accessed and displayed as false color plot = fluorescence life-time image
7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
IPC Friedrich-Schiller-Universität Jena18
Methods of time-resolved fluorescence diagnostics
Time-resolved measurements Gated fluorescence detection
Tissue section of a rat ear: (a) Brightfield microscopy image stained with orcein(b) Fluorescence intensity- and (c) FLIM images of an unstained parallel sample (tissue autofluorescence) (excitation 410 nm; FLIM false color plot from 200 ps (blue) to 1800 ps (red)
(a) (b) (c)
(Top) FLIM image of an unstained human pancreas section (tissue autofluorescence) with an endocrine tumor (below) Brightfield image of the same section after conventional histopathological staining
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
7. Fluorescence microscopy
IPC Friedrich-Schiller-Universität Jena19
Methods of time-resolved fluorescence diagnostics
Time-resolved measurements
TCSPC = time-correlated single photon countingIn case of intensive excitation light many electrons of the dye are getting excited for every laser pulse i.e. the average life-time can be deduced from the fluorescence decay-time after every pulse (multiple photon emission).
A common FLIM method is the measurement of the life-time for single fluorescence photons. In doing so the dye is excited by light pulses of extremely low intensity in a way that at most one electron per pulse gets excited. The individual life-time of every photon is measured and the average life-time is determined staistically.
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
7. Fluorescence microscopy
IPC Friedrich-Schiller-Universität Jena20
Methods of time-resolved fluorescence diagnostics
Time-resolved measurements
TCSPC = time-correlated single photon counting Detection of single photons of a periodic light
signal
Light intensity is so weak, that the probability to detect a photon within one period is very small.
Periods with more than one photon are extremely rare
For every detected photon its delay time with respect to the excitation pulse is determined
A delay distribution builds up over many pulse
Time resolution up to 25 ps
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
7. Fluorescence microscopy
IPC Friedrich-Schiller-Universität Jena21
Probe Stop watch = TAC: Time-to-Amplitude Converter
converts time between a start and a stop pulse by charging
a capacitor with constant current
Start can be reference (from laser) and photon is stop
-> Problem is loss of much time (due to reset time)
-> Reverse counting (start = photon, stop=next laser pulse)
Histogram of arrival times after excitation
-> fluorescence life-time.
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
Methods of time-resolved fluorescence diagnostics
Time-resolved measurements
TCSPC = time-correlated single photon counting
7. Fluorescence microscopy
IPC Friedrich-Schiller-Universität Jena22
Fluorescence intensity image of a vacuole which is labeled byfluorescent phospholipids
FLIM image and corresponding distribution of life-times. Long life-times (red) are found in the cell membrane while the cytoplasma exhibits shorter life-times pointing towards a less ordered environment.
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
Methods of time-resolved fluorescence diagnostics
Time-resolved measurements
TCSPC = time-correlated single photon counting
7. Fluorescence microscopy
IPC Friedrich-Schiller-Universität Jena23
Methods of time-resolved fluorescence diagnostics
Steady state measurements Phase modulation
Intensity of a continuous wave (CW) source is modulated at high frequency by
a standing wave acousto-optic modulator ( 50 MHz) which will
modulate the excitation intensity at double frequency.
Detected fluorescence is modulated at the same frequency.
The observed phase shift with respect to the excitation and
the modulation depth M (ratio of Ac signal to DC signal)
depends on the fluorescence life-time of the excited fluorophores.
Fluorescence lifetimes phase and phase can be calculated and should be
identical for single exponential decays.
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
7. Fluorescence microscopy
IPC Friedrich-Schiller-Universität Jena24
Methods of time-resolved fluorescence diagnostics
Steady state measurements Phase modulation
Measurement values:Demodulation (modulation depth) M Phase shift
Modulation of excitation light with , which is characterized by modulation depth ME = a/d and E :
leads to an accordingly modulated fluorescence signal F(t) with demodulation MF = A/D und phase F
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
Time
Inte
nsity Excitation
light
Fluorescence
7. Fluorescence microscopy
IPC Friedrich-Schiller-Universität Jena25
Methods of time-resolved fluorescence diagnostics
Steady state measurements Phase modulation
Rate equation of change of number of excited molecules
F(t) ~ N(t) Relationship between fluorescence life-time and
fluorescence emission behavior upon intensity modulated excitation light Relationship between measurement parameters:
M = MF / ME as well as = E - E and life-time :
Absorption rate:
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
7. Fluorescence microscopy
IPC Friedrich-Schiller-Universität Jena26
Methods of time-resolved fluorescence diagnostics
Steady state measurements Phase modulation
Continuous intensity modulated excitation of fluorescence transforms the
determination of fluorescence decay-times to measurements of phase shifts and
demodulation of the fluorescence signal
Demodulation M and phase shift of the fluorescence depend on the fluorescence life-time as well as on the modulation frequency = 2 of the excitation light. The simulation shows the dependency of M and for a decay time of = 4 ns and a frequency of = 40 MHz
Choosing frequency at 1
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
7. Fluorescence microscopy
IPC Friedrich-Schiller-Universität Jena27
Methods of time-resolved fluorescence diagnostics
Steady state measurements Phase modulation
Frozen section of portio biopsies in the spectral region of Em>500 nm (exc = 457 nm; = 40 MHz)Top right: Fluorescence intensity Bottom right: corresponding HE stain image.
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
7. Fluorescence microscopy