IPC Friedrich-Schiller-Universität Jena 1 7. Fluorescence microscopy 7.3 FRET microscopy - The different Dipoles 1. static electric Dipole: Far away: E

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



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


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)


7.3 FRET microscopy

sensitizedemission


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


7.3 FRET microscopy


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

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7.3 FRET microscopy


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)


7.3 FRET microscopy


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


7.3 FRET microscopy

No FRET


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


7.3 FRET microscopy

superposition


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)


7.3 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


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®)


7.3 FRET microscopy


7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)


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.



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 ¿ :






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





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






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.






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




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.




TCSPC = time-correlated single photon counting



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.




TCSPC = time-correlated single photon counting




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.






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


Time

Inte

nsity Excitation

light

Fluorescence





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:






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






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.



Documents

IPC Friedrich-Schiller-Universität Jena 1 7. Fluorescence microscopy 7.3 FRET microscopy - The different Dipoles 1. static electric Dipole: Far away: E