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6. Fluorescence Spectroscopy
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IPC Friedrich-Schiller-Universität Jena
6. Basic concepts in fluorescence spectroscopy
6.1 Stokes-Shift
= Stokes-Shift due to vibrational energy
relaxation within electronic excited state
 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
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6. Basic concepts in fluorescence spectroscopy
6.2 Fluorescence life-time
The following transitions will be considered:
S1
T1
S0
: rate constant for radiative S1S0 decay via fluorescence;
: rate constant for internal conversion (S1S0);
: rate constant for intersystem crossing;
: rate constant for radiative decay via phosphorescence (T1S0);
: rate constant for non-radiative decay (T1S0).
Non radiative transitions originating from S1 are combined in:
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6. Basic concepts in fluorescence spectroscopy
6.2 Fluorescence life-time
 Dilute solution of fluorescent species A.
 Short d-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:
1A*
1A
+ Photon
 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:
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IPC Friedrich-Schiller-Universität Jena
6. Basic concepts in fluorescence spectroscopy
6.3 Fluorescence quantum yield
 Fluorescence quantum yield: Emitted Photons per Excitation events
 It follows:
Integration over
complete decay
 The quantum yields for ISC and phosphorescence can be expressed in analogy:
bzw.
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IPC Friedrich-Schiller-Universität Jena
6. Basic concepts in fluorescence spectroscopy
6.3 Fluorescence quantum yield
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
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IPC Friedrich-Schiller-Universität Jena
6. Basic concepts in fluorescence spectroscopy
6.4 Steady-State fluorescence emission
 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(lF), recorded for the wavelength lF is
proportional to
as well as to the number of absorbed photons at the
excitation wavelength lE.
 Number of absorbed photons is given by:
Intensity
transmitted
irradiated
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IPC Friedrich-Schiller-Universität Jena
6. Basic concepts in fluorescence spectroscopy
6.4 Steady-State fluorescence emission
 Fluorescence intensity can be expressed as follows:
with k = proportionality constant dependent on numerous
experimental values like e.g. collection angle, band width of
monochromator, slid width, etc.
 Considering the intensity of the transmitted light by Lambert-Beer‘s law yields:
 Recording the intensity IF as function of the wavelength lF for a fixed excitation
wavelength lE yields fluorescence spectrum.
 For low concentrations it follows:
 Higher terms can be neglected for diluted solutions.
 Thus it follows:
A(lE) = absorbance at lE
 Proportionality between fluorescence intensity and concentration for diluted
solutions only
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IPC Friedrich-Schiller-Universität Jena
6. Basic concepts in fluorescence spectroscopy
6.4 Fluorescence excitation spectroscopy
 Recording fluorescence intensity as function of excitation wavelength lE for a fixed
observation wavelength lF 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.
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IPC Friedrich-Schiller-Universität Jena
6. Basic concepts in fluorescence spectroscopy
6.4 Fluorescence excitation spectroscopy
Fluorescence excitation (lE = variabel, lF = 419 nm)
Absorption-,
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CH3
N
O
fluorescence- and fluorescenceexcitation spectra
N
O
COOH
415
359.5
IF(lE=359 nm,lF = variable)
350
267.5
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Absorbance, A(lE)
O
250
300
350
400
450
500
550
Fluorescence intensity, IF(lE = 359 nm, lF)
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Cinoxacin in H2O
600
Wavelength / nm
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IPC Friedrich-Schiller-Universität Jena
7. Fluorescence microscopy
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
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7. Fluorescence microscopy
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
N
H
N
N
H
N
Porphyrin ring –
central unit in Chlorophyll
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Moss reeds – green excitation
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7. Fluorescence microscopy
http://en.wikipedia.org/wiki/File:Chlorophyll_ab_spectra2.PNG
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7. Fluorescence microscopy
7.1 Primary fluorescence (autofluorescence)
 Further examples:
 Riboflavine (550nm)
 NAD(P)H (460nm, 400ps)
 Elastin und Collagen (305-450nm)
 Retinol (500nm)
 Cuticula (blue)
 Lignin (> 590nm)
Eucalyptus leaf section – UV excitation
 DNA (Ex @320nm, 390nm)
 Aminoacids:
 Tryptophane (348nm, 2.6ns)
 Tyrosin (303nm, 3.6ns, weak)
 Phenylalanine (282nm weak)
 Resins, Oils
http://en.wikipedia.org/wiki/Autofluorescence
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Nematode living sample – UV excitation
IPC Friedrich-Schiller-Universität Jena
7. Fluorescence microscopy
7.1 Secondary fluorescence (fluorochromation)
 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)
DAPI:
lexc = 358 nm
lem = 461 nm
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Fluorescence image of
Endothelium cells.
Microtubili are labeld in
green,
while actin filaments are
labeled red.
DNA within cell nuclei
are stained with DAPI.
IPC Friedrich-Schiller-Universität Jena
7. Fluorescence microscopy
7.3 FRET microscopy
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
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IPC Friedrich-Schiller-Universität Jena
7. Fluorescence microscopy
7.3 FRET microscopy
Radiationless excitation energy transfer
 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
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
Exchange term Uex
IPC Friedrich-Schiller-Universität Jena
7. Fluorescence microscopy
7.3 FRET microscopy
Radiationless excitation energy transfer
Coulomb Interaction (CI)
Exchange Interaction
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7. Fluorescence microscopy
7.3 FRET microscopy
Radiationless excitation energy transfer
 Different interaction mechanism lead to excitation energy transfer:
Dipolar
(Förster)
„Long
Range“
Coulomb
interaction
Multipolar
Singlet
energy transfer
Inter molecular
orbital overlap
Triplet
Energy transfer
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Electron
exchange
(Dexter)
„Short
Range“
Charge
resonance
interaction
IPC Friedrich-Schiller-Universität Jena
7. Fluorescence microscopy
7.3 FRET microscopy
Förster Resonance Energy Transfer (very weak coupling):
D + hn1
D* + A
A*
 D*
Absorption
 A* + D
Energy transfer
 A + hn2
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
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IPC Friedrich-Schiller-Universität Jena
7. Fluorescence microscopy
7.3 FRET microscopy
Förster Resonance Energy Transfer (very weak coupling):
 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)).
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7. Fluorescence microscopy
7.3 FRET microscopy
Förster Resonance Energy Transfer (very weak coupling):
 R0 can be determined via spectroscopic values:
Overlap between fluorescence of donor and
absorption of acceptor
k2 = orientational factor
F0D = quantum yield of donor in absence of energy transfer
n = average refractive index for wavelength area of spectral overlap
ID(l) = normalized fluorescence spectrum of donor (
)
eA(l) = molar absorption coefficient of acceptor.
 For R0 in Å, l in nm, eA(l) 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
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7. Fluorescence microscopy
7.3 FRET microscopy
Förster Resonance Energy Transfer (very weak coupling):
 Transfer efficiency can be expressed by:
 In combination with changed lifetime:
1
D

1
 D0
 k Add D
distance dependency:
 It follows:
D und D0 are excited state life-times of
donor in absence and presence of
acceptor, respectively
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7. Fluorescence microscopy
7.3 FRET microscopy
Förster Resonance Energy Transfer (very weak coupling):
 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 k2 is given by:
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
D
A
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7. Fluorescence microscopy
7.3 FRET microscopy
Förster Resonance Energy Transfer (very weak coupling):
 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)
k2 can reach values between 0 (transition dipole moments
are orthogonal) and 4 (collinear arrangement); k2 = 1, for a
parallel arrangement
 If both acceptor and donor can rotate the orientational factor k2 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 k2 = 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: k2 = 0.476
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IPC Friedrich-Schiller-Universität Jena
7. Fluorescence microscopy
7.3 FRET microscopy
Förster Resonance Energy Transfer (very weak coupling):
 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.
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7. Fluorescence microscopy
7.3 FRET microscopy
Förster Resonance Energy Transfer (very weak coupling):
 RET as „optical nano ruler“ in biochemistry and cell biology
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IPC Friedrich-Schiller-Universität Jena
7. Fluorescence microscopy
7.3 FRET microscopy
Förster Resonance Energy Transfer (very weak coupling):
 RET as „optical nano ruler“ in biochemistry and cell biology
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IPC Friedrich-Schiller-Universität Jena
7. Fluorescence microscopy
7.3 FRET microscopy
Förster Resonance Energy Transfer (very weak coupling):
 RET as „optical nano ruler“ in biochemistry and cell biology
One 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)
Excitation maxima at 395 und 475 nm
Emission wavelength at 509 nm
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7. Fluorescence microscopy
7.3 FRET microscopy
Agar plate of
fluorescent bacteria
colonies
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IPC Friedrich-Schiller-Universität Jena
7. Fluorescence microscopy
7.3 FRET microscopy
Förster Resonance Energy Transfer (very weak coupling):
 RET as „optical nano ruler“ in biochemistry and cell biology :
GFP-mutants
FRET
R0 = 4.7 – 4.9 nm
no FRET
protein folding
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protein-protein interaction
IPC Friedrich-Schiller-Universität Jena
7. Fluorescence microscopy
7.3 FRET microscopy
 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 donoremission
 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)
sensitized
emission
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7. Fluorescence microscopy
7.3 FRET microscopy
FRET ratio imaging =
acceptor emission at donor excitation (sensitized emission SAkzeptor) divided by donor
emission at donor excitation (SDonor)
SDonor
SAkzeptor
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
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7. Fluorescence microscopy
7.3 FRET microscopy
FRET ratio imaging – problems:
Excitation
wavelength
FRET-detection
channel
 Correction for direct excitation of  Correction for bleedthrough :
the acceptor when exciting donor Portion of CFP in yellow channel for blue
excitation in absence of FRET (acceptor)
(control measurement with YFP
= bleedthrough of CFP in YFP-channel (rBT,CY)
only) = correction factor rDE
 or bleedthrough of YFP in CFP-channel (rBT,YC)
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IPC Friedrich-Schiller-Universität Jena
7. Fluorescence microscopy
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)

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IPC Friedrich-Schiller-Universität Jena
7. Fluorescence microscopy
7.3 FRET microscopy
FRET ratio imaging – 3-filter-set:
 Model of FRET-detection of SrcCsk protein interaction
(Src = protein tyrosine kinase
Csk = C-terminal Src kinase)
 Important signal transduction step
during blood coagulation
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No FRET
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7. Fluorescence microscopy
7.3 FRET microscopy
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
superposition
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IPC Friedrich-Schiller-Universität Jena
7. Fluorescence microscopy
7.3 FRET microscopy
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)
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IPC Friedrich-Schiller-Universität Jena
7. Fluorescence microscopy
7.3 FRET microscopy
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®)
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7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
Methods of time-resolved
fluorescence diagnostics
 Time-resolved measurements
 Sample is excited by a short
laser pulse
Laser pulse
Longer
fluorescence
life-time
Shorter
fluorescence
life-time
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 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 Jena
7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
Methods of time-resolved fluorescence diagnostics
 Time-resolved measurements
 Intensity integrating measurements
The 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
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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
¿ :
IPC Friedrich-Schiller-Universität Jena
7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
Methods of time-resolved fluorescence diagnostics
 Time-resolved measurements
 Gated fluorescence detection
Gated optical image intensifiers (GOI) are
capable of taking pictures with high (subnanosecond) 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
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7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
Methods of time-resolved fluorescence diagnostics
 Time-resolved measurements
 Gated fluorescence detection
(a)
(b)
(c)
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)
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(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
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7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
Methods of time-resolved fluorescence diagnostics
 Time-resolved measurements
 TCSPC = time-correlated single photon counting
In 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 lifetime of every photon is measured and the
average life-time is determined staistically.
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7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
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
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7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
Methods of time-resolved fluorescence diagnostics
 Time-resolved measurements
 TCSPC = time-correlated single photon counting
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.
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7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
Methods of time-resolved fluorescence diagnostics
 Time-resolved measurements
 TCSPC = time-correlated single photon counting
Fluorescence intensity image of a
vacuole which is labeled by
fluorescent phospholipids
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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.
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7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
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 (n 2 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.
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7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
Methods of time-resolved fluorescence diagnostics
Intensity
 Steady state measurements
 Phase modulation
Excitation
light
 Measurement values:
Demodulation (modulation depth) M
Phase shift DF
 Modulation of excitation light with n,
which is characterized by modulation
depth ME = a/d and FE :
Fluorescence
Time
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 leads to an accordingly modulated
fluorescence signal F(t) with
demodulation MF = A/D und phase FF
IPC Friedrich-Schiller-Universität Jena
7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
Methods of time-resolved fluorescence diagnostics
Steady state measurements
 Phase modulation
 Rate equation of change of number of excited molecules
Absorption rate:
 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 DF = FE - FE and life-time  :
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7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
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 DF of the
fluorescence depend on the fluorescence lifetime  as well as on the modulation frequency
w = 2p n of the excitation light. The simulation
shows the dependency of M and DF
for a decay time of  = 4 ns and
a frequency of n = 40 MHz
 Choosing frequency at w  1
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7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
Methods of time-resolved fluorescence diagnostics
 Steady state measurements
 Phase modulation
Frozen section of portio biopsies in the spectral region of
lEm>500 nm (lexc = 457 nm; n = 40 MHz)
Top right: Fluorescence intensity
Bottom right: corresponding HE stain image.
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