ipc friedrich-schiller-universität jena 1 6. fluorescence spectroscopy
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6. Fluorescence Spectroscopy
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Energy differences between vibrational states which determine vibronic band intensities are very often the same for ground and electronic excited state
Emission spectrum = mirror image of absorption spectrum
Emission bands are shifted bathochromically i.e. to higher wavelengths
= Stokes-Shift due to vibrational energy
relaxation within electronic excited state
6.1 Stokes-Shift
6. Basic concepts in fluorescence spectroscopy
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The following transitions will be considered:
S0
S1 T1
: rate constant for radiative S1S0 decay via fluorescence;
: rate constant for internal conversion (S1S0);
: rate constant for intersystem crossing;
: rate constant for radiative decay via phosphorescence (T1S0);
: rate constant for non-radiative decay (T1S0).
Non radiative transitions originating from S1 are combined in:
6.2 Fluorescence life-time
6. Basic concepts in fluorescence spectroscopy
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Dilute solution of fluorescent species A.
Short -laser pulse excites certain fraction of molecules A at t = 0.
Decay rate of excited molecules A*:
Integration:
together with: number of molecules A promoted in the excited state at t = 0
and life-time of excited state S1:
Fluorescence intensity is number of photons emitted per time and volume:
Fluorescence intensity IF at time t after excitation by a short light pulse:
Part of molecules can end up in triplet state.
Life-time of triplet state is defined as:
1A* 1A + Photon
6. Basic concepts in fluorescence spectroscopy
6.2 Fluorescence life-time
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6.3 Fluorescence quantum yield
Fluorescence quantum yield: Emitted Photons per Excitation events
It follows:
The quantum yields for ISC and phosphorescence can be expressed in analogy:
Integration over
complete decay
bzw.
6. Basic concepts in fluorescence spectroscopy
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Life-times & quantum yields
Attention:
Quantum yield is proportional to
life-time
but
other non-radiative decay
processes change lifetime
radiative rate depends on
refractive index of medium
6. Basic concepts in fluorescence spectroscopy
6.3 Fluorescence quantum yield
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It is advantageous to define the steady-state fluorescence per absorbed photon as
photon flux in dependence of wavelength
(Photon spectrum) :
Emission photon spectrum expresses the probability distribution of the
different transitions from the vibrational ground state of S1
down to the various vibrational states of S0.
The normalized steady-state fluorescence IF(F), recorded for the wavelength F is
proportional to as well as to the number of absorbed photons at the
excitation wavelength E.
Number of absorbed photons is given by:
irradiated transmittedIntensity
6. Basic concepts in fluorescence spectroscopy
6.4 Steady-State fluorescence emission
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Fluorescence intensity can be expressed as follows:
Considering the intensity of the transmitted light by Lambert-Beer‘s law yields:
Recording the intensity IF as function of the wavelength F for a fixed excitation
wavelength E yields fluorescence spectrum.
For low concentrations it follows:
Higher terms can be neglected for diluted solutions.
Thus it follows:
A(E) = absorbance at E
Proportionality between fluorescence intensity and concentration for diluted
solutions only
with k = proportionality constant dependent on numerous experimental values like e.g. collection angle, band width of monochromator, slid width, etc.
6. Basic concepts in fluorescence spectroscopy
6.4 Steady-State fluorescence emission
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6.4 Fluorescence excitation spectroscopy
Recording fluorescence intensity as function of excitation wavelength E for a fixed
observation wavelengthF yields fluorescence excitation spectrum.
According to: the fluorescence intensity
recorded as a function of the excitation wavelength reflects the product
In case the wavelength dependency of the incoming light can be compensated the
fluorescence excitation spectrum depends only on what corresponds to the
absorption spectrum.
As long as only one ground state species exists the corrected excitation spectrum
is identical to the absorption spectrum. Otherwise a comparison between
fluorescence excitation and absorption spectrum yields valuable information
about the sample species present.
6. Basic concepts in fluorescence spectroscopy
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Cinoxacin in H2O
NN
O
O
O COOH
CH3
6. Basic concepts in fluorescence spectroscopy
6.4 Fluorescence excitation spectroscopy
250 300 350 400 450 500 550 600
415
210
259
359.
5
350
267.
5
245
Fluorescence excitation (E = variabel,
F = 419 nm)
IF(
E=359 nm,
F = variable)
Wavelength / nm
Absorption-,
fluorescence- and fluorescence-
excitation spectra
Flu
ore
scenc
e in
tensi
ty, I
F(
E =
359
nm
, F)
Abs
orban
ce, A
(E)
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7.1 Fluorochromes
Fluorescence microscopy differentiates between two kinds of fluorochromes:
Primary fluorescence (autofluorescence)
Secondary fluorescence (fluorochromation)
Fluorescence dyes
Immunofluorescence (using Antibodies)
Molecular tags (SNAP Tag, ...)
Fluorescent Proteins
Applications of fluorochromes
Identification of otherwise visible structures
Localization and identification of otherwise invisible structures
Monitoring of physiological processes
Specific detection of a protein
Using Photophysical properties of dyes (e.g. switching) for superresolution
7. Fluorescence microscopy
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7.1 Primary fluorescence (autofluorescence) Most samples fluoresce when excited with short-wave light
Fluorescence very often occurs for systems containing many conjugated double bonds:
e.g. chlorophyll exhibits dark red fluorescence when excited by blue or red light
Moss reeds – green excitation
NNH
NH
N
Porphyrin ring – central unit in Chlorophyll
7. Fluorescence microscopy
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7. Fluorescence microscopy
http://en.wikipedia.org/wiki/File:Chlorophyll_ab_spectra2.PNG
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Further examples: Riboflavine (550nm) NAD(P)H (460nm, 400ps) Elastin und Collagen (305-450nm) Retinol (500nm) Cuticula (blue) Lignin (> 590nm) DNA (Ex @320nm, 390nm) Aminoacids:
Tryptophane (348nm, 2.6ns) Tyrosin (303nm, 3.6ns, weak) Phenylalanine (282nm weak)
Resins, Oils
Eucalyptus leaf section – UV excitation
Nematode living sample – UV excitation
7. Fluorescence microscopy
7.1 Primary fluorescence (autofluorescence)
http://en.wikipedia.org/wiki/Autofluorescence
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Staining (labeling) specific structures with fluorescent labels (dyes):
fluorochromation Small dye concentrations are sufficient due to high fluorescence contrast
fluorescence labels are superior than bright field dyes Single molecule sensitivity Fluorescence labels must selectively bind to structures
or selectively accumulate in specific compartments e.g. DAPI (= 4',6-diamidino-2-phenylindole) to label DNA (cell nuclei)
exc = 358 nmem = 461 nm
Fluorescence image of Endothelium cells. Microtubili are labeld in green, while actin filaments are labeled red. DNA within cell nuclei are stained with DAPI.
7.1 Secondary fluorescence (fluorochromation)
7. Fluorescence microscopy
DAPI:
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Radiationless excitation energy transfer requires interaction between donor and acceptor
Emission spectrum of donor must overlap with absorption spectrum of acceptor.
Several vibronic transitions within donor have the same energy than in the acceptor
Resonant coupling of the transitions
RET = resonance energy transfer
Resonant transitions
7.3 FRET microscopy
7. Fluorescence microscopy
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Assumption: 2 electrons one at the donor D and one at the acceptor A are involved in the transition:
Antisymmetric wavefunction (Fermions) for initially excited state i (D excited, but not A) and final state f (A excited, but not D):
Overall Hamiltonian:Interaction energy:
Coulomb term UC Exchange term Uex
Radiationless excitation energy transfer
7. Fluorescence microscopy
7.3 FRET microscopy
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Coulomb Interaction (CI)
Exchange Interaction
7. Fluorescence microscopy
7.3 FRET microscopyRadiationless excitation energy transfer
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Different interaction mechanism lead to excitation energy transfer:
Coulombinteraction
Inter molecular orbital overlap
Dipolar(Förster)
Multipolar
Electron exchange(Dexter)
Charge resonance interaction
Singlet energy transfer
TripletEnergy transfer
„LongRange“
„ShortRange“
7. Fluorescence microscopy
7.3 FRET microscopyRadiationless excitation energy transfer
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Förster Resonance Energy Transfer (very weak coupling):
D + h1 D* Absorption
D* + A A* + D Energy transfer
A* A + h2 Emission
The following conditions must hold:
D must be a fluorophore with sufficiently long life-time
Partial spectral overlap between emission spectrum of D and absorption spectrum of A
Transition dipole moments D and A must
be oriented properly to each other;
Distance between D and A shouldn‘t be too large
7. Fluorescence microscopy
7.3 FRET microscopy
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Coulomb interaction can be developed in a multipole series in which the dipole
term exhibits the term with the longest range Energy transfer via dipole-dipole transfer has been first calculated by Förster and
is therefore called Förster process Energy transfer rate from molecule D to molecule A at a distance r:
kD = radiative decay rate of donor
tD0 = donor life-time in absence of energy transfer
r-6-dependency as a result of dipole-dipole interaction
R0 = critical distance or Förster-radius (distance at which intensity
decrease caused by energy transfer and spontaneous decay are
equal ( = kD)).
7. Fluorescence microscopy
Förster Resonance Energy Transfer (very weak coupling):
7.3 FRET microscopy
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R0 can be determined via spectroscopic values:
For R0 in Å, in nm, A() in M-1 cm-1 (overlap integral in M-1 cm-1 nm4)
Typical values for Förster-radii R0, i.e. for distances, over which energy transfer is
important lie in the range of 15 -60 Å
2 = orientational factor0
D = quantum yield of donor in absence of energy transfer n = average refractive index for wavelength area of spectral overlapID() = normalized fluorescence spectrum of donor ( )A() = molar absorption coefficient of acceptor.
Overlap between fluorescence of donor and absorption of acceptor
7. Fluorescence microscopy
Förster Resonance Energy Transfer (very weak coupling):
7.3 FRET microscopy
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Transfer efficiency can be expressed by:
In combination with changed lifetime:
It follows:
D und D0 are excited state life-times of
donor in absence and presence of acceptor, respectively
7. Fluorescence microscopy
Förster Resonance Energy Transfer (very weak coupling):
7.3 FRET microscopy
distance dependency:ddDA
DD
k 0
11
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Besides the distance between the two chromophores also the relative orientation
of the transition dipole moments of the donor D and acceptor A plays a crucial
role for the energy transfer efficiency The orientation factor 2 is given by:
D
A
A: angle between D-A connecting line and
acceptor transition dipole moment
D: angle between D-A connecting line and
donor transition dipole moment
T: angle between donor and acceptor
transition dipole moment
7. Fluorescence microscopy
Förster Resonance Energy Transfer (very weak coupling):
7.3 FRET microscopy
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For systems where the orientation stays constant during the energy transfer (e.g. usage of highly viscose solvents or rigid coupling of chromophores to large and stiff molecules) can reach values between 0 (transition dipole moments are orthogonal) and 4 (collinear arrangement); = 1, for a parallel arrangement
If both acceptor and donor can rotate the orientational factor 2 must be replaced by an average value:
In case both chromophores undergo a fast isotropic rotation i.e. the rotation is considerably faster than the energy transfer rate the average orientation factor is given by = 2/3
In case donor and acceptor are freely movable but the rotation is significantly slower than the energy transfer the orientation factor results in: 2 = 0.476
7. Fluorescence microscopy
Förster Resonance Energy Transfer (very weak coupling):
7.3 FRET microscopy
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RET is utilized as „optical nano ruler“ (10 – 100 Å) in biochemistry and cell biology
Distance between donor and acceptor should be in the range of:
because R0 is a benchmark for donor-acceptor distances which can be determined
by FRET.
7. Fluorescence microscopy
Förster Resonance Energy Transfer (very weak coupling):
7.3 FRET microscopy
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RET as „optical nano ruler“ in biochemistry and cell biology
7. Fluorescence microscopy
Förster Resonance Energy Transfer (very weak coupling):
7.3 FRET microscopy
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7. Fluorescence microscopy
RET as „optical nano ruler“ in biochemistry and cell biologyFörster Resonance Energy Transfer (very weak coupling):
7.3 FRET microscopy
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RET as „optical nano ruler“ in biochemistry and cell biologyOne requires appropriate method to label specific intracellular proteins with suitable fluorophores (fluorescent proteins genetics):
Green Fluorescent Protein (GFP) first isolated from the jellyfish Aequorea victoria GFP can be combined with just about any other protein by attaching its gene to the gene of a target protein, thereby introducing it into a cell. Thus by recording the GFP fluorescence the spatial and temporal distribution of this target protein can be directly monitored in living cells, tissue and organism.
Several GFP mutants with altered fluorescence spectra exist. These mutants are named according to their color e.g. CFP (cyan) or YFP (yellow)
7. Fluorescence microscopy
Förster Resonance Energy Transfer (very weak coupling):
7.3 FRET microscopy
Excitation maxima at 395 und 475 nmEmission wavelength at 509 nm
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Agar plate of fluorescent bacteria
colonies
7. Fluorescence microscopy
7.3 FRET microscopy
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RET as „optical nano ruler“ in biochemistry and cell biology :GFP-mutants
no FRET
FRET
protein folding protein-protein interaction
R0 = 4.7 – 4.9 nm
7. Fluorescence microscopy
Förster Resonance Energy Transfer (very weak coupling):
7.3 FRET microscopy
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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
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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
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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
-de
tect
ion
ch
an
ne
l
7. Fluorescence microscopy
7.3 FRET microscopy
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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
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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
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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
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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
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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
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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.
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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)
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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)
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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