IPC Friedrich-Schiller-Universität Jena 1 6. Fluorescence Spectroscopy

IPC Friedrich-Schiller-Universität Jena1

6. Fluorescence Spectroscopy


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


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



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


6.2 Fluorescence life-time


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.



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


6.3 Fluorescence quantum yield


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


6.4 Steady-State fluorescence emission


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.


6.4 Steady-State fluorescence emission


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.



Cinoxacin in H2O

NN

O

O

O COOH

CH3


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)


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


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




http://en.wikipedia.org/wiki/File:Chlorophyll_ab_spectra2.PNG


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


7.1 Primary fluorescence (autofluorescence)

http://en.wikipedia.org/wiki/Autofluorescence


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)


DAPI:


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



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


7.3 FRET microscopy


Coulomb Interaction (CI)

Exchange Interaction


7.3 FRET microscopyRadiationless excitation energy transfer


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“


7.3 FRET microscopyRadiationless excitation energy transfer


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


7.3 FRET microscopy


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)).



7.3 FRET microscopy


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



7.3 FRET microscopy


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



7.3 FRET microscopy

distance dependency:ddDA

DD

k 0

11


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



7.3 FRET microscopy


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



7.3 FRET microscopy


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.



7.3 FRET microscopy


RET as „optical nano ruler“ in biochemistry and cell biology



7.3 FRET microscopy



RET as „optical nano ruler“ in biochemistry and cell biologyFörster Resonance Energy Transfer (very weak coupling):

7.3 FRET microscopy


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)



7.3 FRET microscopy

Excitation maxima at 395 und 475 nmEmission wavelength at 509 nm


Agar plate of fluorescent bacteria

colonies


7.3 FRET microscopy


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



7.3 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 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

ET

-de

tect

ion

ch

an

ne

l


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


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.



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IPC Friedrich-Schiller-Universität Jena 1 6. Fluorescence Spectroscopy