Johan Hofkens

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Theories and methods to study molecular
interactions fluorescence and its applications
K.U.LEUVEN
Johan Hofkens
Laboratory of Photochemistry and
Spectroscopy Katholieke Universiteit Leuven -
Belgium
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Principles of fluorescence and its applications
to study molecular interactions
30/11/2005 Basic principles of fluorescence -
absorption, emission, charateristics of a
probe - time resolved measurements - quenching,
anisotropy - energy transfer, electron
transfer - examples 02/12/2005 Fluorescence
microscopy (Dr J. Hotta) - definitions,
parameters - different types of
microscopy 07/12/2005 Single molecule
fluorescence microscopy - why single molecule
studies - different single molecule
approaches 09/12/2005 Applications of
fluorescence microscopy
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Fluorescence

• What is it?
• Where does it come from?
• Parameters, Advantages, Techniques
• Examples

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References additional reading
http//www.chem.kuleuven.ac.be/research/mds/bioinf
ormatics_courses.htm
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For light to be useful to us it must interact
with matter

• Types of interaction
• Reflection
• Refraction
• Absorption (followed by emission)

Fluorescence photons emitted by organic
molecules after interaction with light
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Dual Nature of light wave and particle

• Light as a wave

• c/l
• E hn hc/l

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Dual Nature of light wave and particle

• Light as a particle

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Visible light

• Why do we call this visible light

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Overview of electromagnetic radiation
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Overview of electromagnetic radiation
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Absorption electronic transition(s) in a
molecule
Orbitals, molecular orbitals
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Simplified Jablonski Diagram
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• Absorption of photon elevates chromophore to
  excited state.

• Return to ground state results in emission of
  radiation (fluorochrome).

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Absorption Franck Condon Principal, Vibrational
fine structure
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Absorption Franck Condon Principal, Vibrational
fine structure
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The Jablonski diagram
Characteristics of stationary molecular
fluorescence
- PES displaced, for molecules where the
excitation is delocalized. - Transition from S0
crosses S1 with highest probability in 1th
vibronic level. - The 0-0 transition is not the
most intense one anymore. - This results in a
more symmetric spectrum.
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The Jablonski diagram
Characteristics of stationary molecular
fluorescence
- Effect on emission is similar as for
absorption - For rigid molecules with little
displacement between PES mirror symmetry and
large overlap
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The Jablonski diagram
Characteristics of stationary molecular
fluorescence
- Effect on emission is similar as for
absorption - For rigid molecules with
displacement between PES mirror symmetry and
small overlap
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The Jablonski diagram
Characteristics of stationary molecular
fluorescence
- Effect on emission is similar as for
absorption - For rigid molecules with
displacement between PES mirror symmetry and
small overlap
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The Jablonski diagram
Characteristics of stationary molecular
fluorescence
- Repulsive S1 PES results in a broad
unstructured spectrum. - Maximum given by the AB
line. - Symmetric (Gaussian) absorption band.
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The Jablonski diagram
Characteristics of stationary molecular
fluorescence
- Repulsive ground state, emission will result in
a broad band - When stabilizing excited state
interaction is caused by two identical molecules
it is called excimer, when the interaction is
caused by two different molecules it is called
exciplex.
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Stokes shift

• is the energy difference between the lowest
  energy peak of absorbence and the highest energy
  of emission

Stokes Shift is 25 nm
Fluorescein molecule
520 nm
495 nm
Fluorescnece Intensity
Wavelength
result of vibrational relaxation solvent
reorganization
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Stokes shift
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Fluorophores/chromophores/probes

• Chromophores are compounds or molecules which
  absorb light
• They contain generally aromatic rings
• The longer the conjugated system, the longer
  wavelength of fluorescence.

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Fluorophores/chromophores/probes
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Allophycocyanin (APC)
Protein
632.5 nm (HeNe)
300 nm 400 nm 500 nm
600 nm 700 nm
Excitation
Emisson
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Excitation - Emission Peaks
Max Excitation at 488 568 647 nm
Fluorophore EXpeak EM peak
FITC 496 518 87 0 0 Bodipy 503 511 58 1 1 Tetra
-M-Rho 554 576 10 61 0 L-Rhodamine 572 590 5 92 0
Texas Red 592 610 3 45 1 CY5 649 666 1 11 98
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Probes for Proteins
Probe Excitation Emission

• FITC 488 525
• PE 488 575
• APC 630 650
• PerCP 488 680
• Cascade Blue 360 450
• Coumerin-phalloidin 350 450
• Texas Red 610 630
• Tetramethylrhodamine-amines 550 575
• CY3 (indotrimethinecyanines) 540 575
• CY5 (indopentamethinecyanines) 640 670

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Probes for Nucleic Acids

• Hoechst 33342 (AT rich) (uv) 346 460
• DAPI (uv) 359 461
• POPO-1 434 456
• YOYO-1 491 509
• Acridine Orange (RNA) 460 650
• Acridine Orange (DNA) 502 536
• Thiazole Orange (vis) 509 525
• TOTO-1 514 533
• Ethidium Bromide 526 604
• PI (uv/vis) 536 620
• 7-Aminoactinomycin D (7AAD) 555 655

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DNA Probes

• AO
• Metachromatic dye
• concentration dependent emission
• double stranded NA - Green
• single stranded NA - Red
• AT/GC binding dyes
• AT rich DAPI, Hoechst, quinacrine
• GC rich antibiotics bleomycin, chromamycin A3,
  mithramycin, olivomycin, rhodamine 800

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Probes for Ions

• INDO-1 Ex350 Em405/480
• QUIN-2 Ex350 Em490
• Fluo-3 Ex488 Em525
• Fura -2 Ex330/360 Em510

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pH Sensitive Indicators
Probe Excitation Emission

• SNARF-1 488 575
• BCECF 488 525/620
• 440/488 525

2,7-bis-(carboxyethyl)-5,6-carboxyfluorescein
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Probes for Oxidation States
Probe Oxidant Excitation Emission

• DCFH-DA (H2O2) 488 525
• HE (O2-) 488 590
• DHR 123 (H2O2) 488 525

DCFH-DA - dichlorofluorescin diacetate HE -
hydroethidine DHR-123 - dihydrorhodamine 123
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Specific Organelle Probes
Probe Site Excitation Emission

• BODIPY Golgi 505 511
• NBD Golgi 488 525
• DPH Lipid 350 420
• TMA-DPH Lipid 350 420
• Rhodamine 123 Mitochondria 488 525
• DiO Lipid 488 500
• diI-Cn-(5) Lipid 550 565
• diO-Cn-(3) Lipid 488 500

BODIPY - borate-dipyrromethene complexes NBD -
nitrobenzoxadiazole DPH - diphenylhexatriene TMA
- trimethylammonium
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Other Probes of Interest

• GFP - Green Fluorescent Protein
• GFP is from the chemiluminescent jellyfish
  Aequorea victoria
• excitation maxima at 395 and 470 nm (quantum
  efficiency is 0.8) Peak emission at 509 nm
• contains a p-hydroxybenzylidene-imidazolone
  chromophore generated by oxidation of the
  Ser-Tyr-Gly at positions 65-67 of the primary
  sequence
• Major application is as a reporter gene for assay
  of promoter activity
• requires no added substrates

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Other Probes of Interest
Excited State Dynamics of the Green Fluorescent
Proteins
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Other Probes of Interest
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Other Probes of Interest
Excited State Dynamics of the Green Fluorescent
Proteins
Applications

• monitoring proteins, organelles, cells in living
  tissue.
• protein-protein interaction using double labeling
  and FRET.
• membrane traffic studies.
• pH sensor.
• Ca2 sensor.
• .

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Fluorescent proteins
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DsRed a longer wavelength substitute for GFPs
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New trends in GFP-research

• Optical marking (following intracellular
  dynamics) or kindling

Patterson, G. H. Lippincott-Schwartz, J.
Science 2002, 297, 1873.
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Photo-Switchable Fluorescent Protein Dronpa

• Dronpa is a monomeric GFP-like fluorescent
  protein from coral Echinophyllia sp.

• Dronpa shows reversible photoswitching on
  irradiation with a 488 nm and 405 nm light.

On
Intensity
Off
405 nm
488 nm
Time
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Steady-State Spectra of Dronpa
pH 7.4
pH 5.0

• Deprotonated form (B form) fluorescent state,
  ffl488 0.85, tfl 3.6 ns

• Protonated form (A1 form) dim state, ffl390
  0.02, tfl 14 ps

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Photoswitching of Dronpa at the Ensemble Level
pH 7.4
488 nm
pH 7.4
405 nm
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Photoswitched Protonated (A2) Form
pH 5.0
488 nm
pH 5.0
405 nm
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Scheme of the Photoswitching
S1
f 0.37
14 ps
f 3.2 10-4
3.6 ns
S0
Protonated form
Photoswitched protonated form
Non-fluorescent intermediate
Fluorescent deprotonated form
On
Intensity
Off
405 nm
488 nm
Time
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New trends in GFP-research

• Diffraction-unlimited microscopy in far field

Hell, S. W. Curr. Opin. Neurobiol. 2004, 14, 599.
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New probes for fluorescence
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New probes for fluorescence
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Spectra
Emission versus excitation spectrum
- Emission spectrum or fluorescence spectrum one
excites at one wavelength and scan the emission-
monochromator. - Excitation spectrum one
fixes the emission monochromator at one
wavelength and scans the excitation
monochromator. - At low concentrations excitation
spectra and emission spectra should be the same.
Differences point to aggregation or other
processes (see energy tranfer).
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Excitation Sources

• Excitation Sources

Lamps Xenon Xenon/Mercury Lasers Argon Ion
(Ar) Krypton (Kr) Helium Neon (He-Ne) Helium
Cadmium (He-Cd) Krypton-Argon (Kr-Ar)
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Arc Lamp Excitation Spectra
Xe Lamp
???
???
Irradiance at 0.5 m (mW m-2 nm-1)
?
Hg Lamp
????
??? ????? ????????? ??????????????
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Common Laser Lines
PE-TR Conj.
Texas Red
PI
Ethidium
PE
FITC
cis-Parinaric acid
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Definitions
Definitions for fluorescence
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Characteristic times Absorption 10-15
s Vibrational relaxation 10-12 10-10 s Lifetime
of S1 10-10 10-7 s Intersystem crossing 10-10
10-8 s Internal conversion 10-11-10-9
s Lifetime T1 10-6 1 s
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Parameters

• Extinction Coefficient
• ? refers to a single wavelength (usually the
  absorption maximum)
• Quantum Yield
• Qf is a measure of the integrated photon
  emission over the fluorophore spectral band

Lifetime decay time
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Parameters

• Transition dipole moment direction of movement
  of electrons

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Photobleaching

• Defined as the irreversible destruction of an
  excited fluorophore (discussed in later lecture)
• Methods for countering photobleaching (see
  microscopy)
• Scan for shorter times
• Use high magnification, high NA objective
• Use wide emission filters
• Reduce excitation intensity
• Use antifade reagents (not compatible with
  viable cells)

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Definitions
Definitions for fluorescence
Principle of photoselection using polarized
excitation light mainly molecules excited that
have a transition dipole parallel to the
excitation light. As a result, the fluorescence
is also polarized, unless processes occur that
destroy the polarization Processes can be
rotation of the molecule, energy
transfer Relation between P and r In
ensemble measurements r is most frequently
used. In absence of depolarization processes the
fundamental of limiting anisotropy value r0 has a
value between 0.4 and -0.2 depending on the
angle between excitation and emission transition
dipole.
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Decay time of a fluorophore
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Time Correlated Single Photon Counting
Spectroscopy
Time resolved fluorecence
SAMPLE
Fluo. response function Ifl(t)? (1/?fl
)exp(-t/?fl )
Excitation (? pulse)
Time resolved fluorescence excitation of the
sample with a pulse that is shorter then the
decay time of the fluorophore, typically 5 ns.
d1M/dt - (kflkISCkICkQQ) 1M
Solving the differential equation
1M 1M0exp(-t/ ?fl )
?fl 1/(kflkIC kISCkQQ)
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Time Correlated Single Photon Counting
Spectroscopy
Basic principle of the TCSPC experiment
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Time Correlated Single Photon Counting
Spectroscopy
Basic principle of the TCSPC experiment
CFD
TAC
Excitation source - flash lamps
monochromatic filters (ns pulses up to 10 kHz
rep.) - mode-locked lasers (ps
pulses up to 82 MHz rep.) -
pulsed semiconductor diode lasers
- synchrotron radiation (UV excitation)
Optical components -
polarization accessories -
collection lens system -
monochromator
Detection system - PMT, MCP or
APD
Electronics - Delay, CFD, TAC,
Amplifier, MCA, PC.
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Time Correlated Single Photon Counting
Spectroscopy
Statistics
xi
i
start
ti
lt xigtltnigtq
? pulse
S
ltnigt
MCP
Large number of pulses for one event
Px(i) (1/x!)(ltxigt)x exp(-ltxigt) ? lt xigtltnigtq
Single Photon Counting !
Decay histogram
Yi (1/?fl ) ? SexcT ?t exp(-i ?t/?fl)
Fit functions for the decays iT(t) ? Ajexp(-t/
?j) exponential model iT(t) Aexp(-t/
?fl-2? (t/ ?fl)1/2) nonexponential model
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Time Correlated Single Photon Counting
Spectroscopy
TCSPC experiment at K.U.Leuven
polarizer
Measurements under magic angle in order to
avoid distortions by rotational diffusion (magic
angle is 54.7 degrees for vertical polarization).
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Time Correlated Single Photon Counting
Spectroscopy
Time-resolved emission spectroscopy (TRES)

• provide information on the evolution of kinetics
  in terms of intensity, time and spectral position
• solvent relaxation around fluorophores,
  short-lived species, molecules having two or more
  fluorescing configurations with different decay
  times are processes that can be studied using
  TRES.

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Time Correlated Single Photon Counting
Spectroscopy
Time-resolved fluorescence depolarization
measurements

• information about the molecular reorientational
  motion in solution.

r (t) (III(t) - I?(t))/ (III(t) 2I?(t)) IT(t)
III(t) 2I?(t) III(t)exp(- t/ ?fl)(12r0 exp(-
t/ ?)) I?(t)exp(- t/ ?fl)(1-r0 exp(- t/ ?)) r
r0exp(- t/ ?)
1. ?fllt ?r fluorescence decays before
anisotropy ?only r0 can be measured 2. ?flgt ?r
or ?fl ? ?r r0 and ?r can be measured.
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Time Correlated Single Photon Counting
Spectroscopy

• intra and intermolecular excited state processes
  taking place from picosecond to nanosecond time
  scale.
• determination of rates of competitive
  de-excitation pathways.
• reaction kinetics proton/electron and energy
  transfer, excimer or exciplex formation.
• environmental effects solvent relaxation,
  quenching of excited states, conformational
  dynamics in proteins.

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Energy Transfer
Radiative Non radiative -Dexter type - Forster
type
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Quenching processes in fluorescence
Energy transfer
- Energy transfer is iso-energetic, followed by
fast vibrational relaxation - Excited state of
acceptor should be lower than that of donor to
have driving force - Quantum yield of donor and
decay time of donor decrease. - Process can occur
between singulet as well as triplet excited
states. - Two mechanisms (except for trivial
mechanisms) Dexter and Förster transfer
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Quenching processes in fluorescence
Energy transfer
- Dexter transfer exchange mechanism, distances
between 0.5 and 1 nm, spin changes are allowed.
Overlap between donor fluorescence and acceptor
absorption required.
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Quenching processes in fluorescence
Energy transfer
- Förster transfer long distance, upto 10 nm,
dipole-dipole interaction, total spin maintained,
resonance energy transfer. Overlap between donor
fluorescence and acceptor absorption required.
Due to strong distance dependence also called
molecular ruler. - Förster transfer between
identical chromophores is called energy hopping
and can go in both directions.
E is called the efficiency of energy transfer
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Fluorescence

• Fluorescene (Forster) Resonance Energy Transfer
• FRET

Molecule 1
Molecule 2
Fluorescence
Fluorescence
ACCEPTOR
DONOR
Intensity
Absorbance
Absorbance
Wavelength
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Quenching processes in fluorescence
Energy transfer
E can be obtained from the fluorescence quantum
yield in the presence (QDA) and absence of the
acceptor (QD) (and in a similar way from decay
time in presence and absence of acceptor).
It can be shown that the rate constant for
transfer equals
?D is the decay time of the donor in absence of
the acceptor, R is the distance between donor and
acceptor and R0 is the Förster radius, the
distance at witch half of the excitation
energy undergoes transfer while half is
dissipated by all the other processes including
emission.
J is the so called overlap integral between
emission and absorption and ? is the orientation
factor (2/3 for random orientation).
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Quenching processes in fluorescence
Energy transfer
The overlap integral can be calculated as
The orientation factor can be written as
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Quenching processes in fluorescence
Energy transfer
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Forster type Energy Transfer(FRET)

• Effective between 10-100 Å only
• Emission and excitation spectrum must
  significantly overlap
• Donor transfers non-radiatively to the acceptor
• PE-Texas Red
• Carboxyfluorescein-Sulforhodamine B

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Quenching processes in fluorescence
Electron transfer
Intermolecular Electron transfer always occurs
via collision and requires diffusion (O2 will
diffuse 7 nm in 10 ns in aqueous
solution) maximum rate constant for bimolecular
reaction is in the order of 4x1010
Excited donor is a better donor, excited acceptor
is a better acceptor
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Markus theory for e-transfer theory that
describes how the rate constant of electron
transfer depends on parameters such as
orientation, ?G, solvent reorganization,
distance.
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Quenching processes in fluorescence
Kinetics of quenching
The case of bimolecular quenching (stationairy)
kd is the rate constant for deactivation without
quenching
Stern-Volmer equation
K is Stern-Volmer constant in l.mol-1
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Quenching processes in fluorescence
Kinetics of quenching
The case of bimolecular quenching (time resolved)
kd is the rate constant for deactivation without
quenching
with
Stern-Volmer equation
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Quenching processes in fluorescence
Kinetics of quenching
The case of intramolecular quenching
kd is the rate constant for deactivation without
quenching
Solving the equation leads to
or
Stern-Volmer equation
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Examples
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Examples
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Examples
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Examples
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Examples
Fluorescence polarization
Anisotropy to study micro-viscosity in membranes
and aggregation
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Examples
Kinetics of quenching
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Examples
Energy transfer
Distance determination form the extend of transfer
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Examples
Energy transfer
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Photosynthesis
Humans, animals, fungi, bacteria live by
degrading molecules provided by other organisms.
Life on earth obviously could not continue
indefinitely in this manner without an
independent mechanism for synthesizing complex
molecules from simple ones the energy provided
in this mechanism comes from the sun and is
captured in the process of photosynthesis. Plants
and other photosynthetic organisms fixe 1011
tons/year of carbon in organic compounds
(carbohydrate molecules, noted (CH2O)) from CO2.
But globally, the consumption is higher than the
synthesis. So, what will happen? CO2 H2O
light ? (CH2O) O2 ? Important to understand
the photosynthesis and how our activities affect
it! Note 1/3 of the fixed C is done by
microorganisms in the oceans. Some bacteria also
participate to the photosynthesis.
Equilibrium constant K 10-496 ? huge
thermodynamic gradient!
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Chlorophyll structure
Porphyrin ring
http//www.life.uiuc.edu/govindjee/papers/mileston
es.html
c.f. TZ 7.5
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The First Step absorption of light

• In addition to chlorophyll, plants contain
  several pigments that absorb light
• The accessory pigments have antioxidant functions
  as well

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Energy transfer after light absorption
e- acceptor
neighboring pigment molecule
excited pigment molecule
light heat
light
e- donor

-

-
e-
3. successive resonance energy transfers
2. resonance energy transfer
1. fluorescence
EXCITATION
3 POSSIBLE DECAY PATHWAYS
After Alberts Fig. 14-47
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Pigment molecules
Chlorophyll
Resonance transfer of light energy
Carotenoid or other pigment
Raven Fig 7-13 c.f. TZ 7.7
Electron acceptor
Special pair of chl a molecules
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Note for bacteria, the antenna systems are
called LH-I and LH-II. They have this
characteristic hollow cylinder shape. LH-I has a
reaction center (RC) within this cylinder. LH-II
has 9 bacteriochlorophylls outside the cylinder
(to take the light) and 18 within the cylinder
(to transfer the energy).
18 9 bacteriochlorophylls
32 bacteriochlorophylls
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Events at the PS II reaction center
c.f. TZ 7.24
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Photosynthesis and aerobic respiration complete a
cycle
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Examples
Energy hopping
Why investigate multichromophoric systems?
Energy transfer in multichromoporic systems
key-process in photosynthesis.
The energy transfer process influenced by -
extend of coupling between the chromophores. -
disorder (slow and fast fluctuations of the
surrounding proteins )...
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Examples
Energy hopping
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Examples
Energy hopping
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Examples
Energy hopping
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Examples
Energy hopping
Fluorescence decay analysis
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Examples
Energy hopping
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Examples
Energy hopping
khopp
khopp
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Examples
Energy hopping
khopp
khopp
4.6ns-1
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Examples
Energy transfer
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Examples
Energy transfer
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Examples
Energy transfer
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Examples
Energy transfer
Fluorescence decay analysis
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Cameleon protein YC3.1

• Fluorescent indicators for measuring Ca2
  concentration.

- Energy donor ECFP
- Energy acceptor EYFP
- Linker calmodulin (CaM)
calmodulin-binding peptide M13 (myosin light
chain kinase)
Binding of Ca2 makes calmodulin wrap around the
M13 domain, increasing the fluorescence resonance
energy transfer between the flanking GFPs.
4Ca2
-4Ca2
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Definitions

• FRET the excited donor transfers its energy to
  the acceptor via a dipole-dipole interaction.
• Requirements - emission spectrum of donor and
  acceptor must overlap. -
  transition dipole moments of donor and acceptor
  must be sufficiently aligned. -
  distance between donor and acceptor must be
  such that probability of transfer is high.
• FRET can be detected by - a decrease in donor
  decay time - a decrease in donor
  fluorescence intensity
  - an increase in acceptor fluorescence
  intensity - a change in
  fluorescence polarization - growing in
  component in acceptor decay

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Absorption and emission spectra of EYFP
- Absorption spectrum
400 nm protonated form.
514 nm deprotonated form.
- Emission spectrum
528 nm deprotonated form.
ff (400 nm excitaiton) 0.02
fESPT 0.03
ff (500 nm excitaiton) 0.61
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Excited-state photophysics of EYFP
a1
t1 (ns)
a2
t2 (ns)
excitation
detection
440 nm 0.9 0.006 0.1
0.06
400 nm
560 nm 1 3.4
560 nm 1 3.4
488 nm
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Excited-state photophysics of EYFP
- The A2 form having a conformation that allows
ESPT, will relax to the I state within 60 ps.
- The A1 form will decay radiatively to its
corresponding ground state, its fluorescence
being quenched down to 6 ps by a non-radiative
process.
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Photophysics of ECFP
a2
t2 (ns)
a3
t3 (ns)
a1
t1 (ns)
0.01 0.24 0.10 1.0
0.89 3.2
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ECFP and EYFP as an energy transfer pair
- The strong overlap of the emission spectrum of
ECFP with the absorption spectrum of EYFP.
- The relative high quantum yield of fluorescence
of ECFP (ff 0.4).
- The mono-exponential decaying of fluorescence
of EYFP when excited at the deprotonated band.
Although displaying complicated photophysics,
ECFP and EYFP still can be used to construct an
energy transfer pair.
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Emission spectra of YC3.1
ID the integrated fluorescence intensity of the
donor
IA the integrated fluorescence intensity of the
acceptor
fD the fluorescence quantum yield of the donor
fA the fluorescence quantum yield of the
acceptor
fDA the fluorescence quantum yield of the donor
in the presence of acceptor
Ca2-binding YC3.1 E 0.29
Ca2-free YC3.1 E 0.16
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The distance between ECFP and EYFP
R0 the critical transfer distance
R the distance between the donor and the
acceptor
kET the rate constant of energy transfer
kf the rate constant of donor in the absence of
acceptor
fDA the fluorescence quantum yield of the donor
in the presence of acceptor
k2 orientation factor
n the refractive index of the solvent
NA Avogadros number
f(n) the fluorescence spectrum of the donor
normalized on the wavenumber scale
e(n) the molar extinction coefficient of the
acceptor at that wavenomber
Ca2-binding YC3.1 R 57 Å
Ca2-free YC3.1 R 65 Å
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The distance between ECFP and EYFP

• Ca2-binding YC3.1 R 57 Å

• Ca2-free YC3.1 R 65 Å

gt120 Å
24 Å
- Even for assuming the perfectly oriented
transition dipole moment (k2 4), the efficiency
of the energy transfer is estimated to be E
0.027 if the protein adopt the most extended
conformation (R 120 Å).
47 32 30 Å
42 Å
The estimated R value is consistent with the
proposed structure.
Relatively compact conformation of the protein
construct, even in the Ca2-free condition.