BIOPHOTONICS 2 P. N. Prasad Introduction to Biophotonics, Wiley ed. - - PowerPoint PPT Presentation

• S. Haacke

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Ultrafast Biophotonics

1 – A general overview

CNRS-EWHA Winter School 9 & 10 Feb 2012

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BIOPHOTONICS

• P. N. Prasad „Introduction to Biophotonics“, Wiley ed.

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1.1 Making use of light

A) Light as a biomedical tool: Diagnostics

Louis Pasteur (1822-1895)

Microscope C. Zeiss 1891

Micro-organisms & infections Conservation by “heating”

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1.1 Making use of light

A) Light as a biomedical tool: Diagnostics

• (Fluorescence) Microscopy
• Optical coherence

tomography

A.M. Rollins et al., OPN, April 2002 2D images of human retinas (left) and with depth resolution obtained by OCT (right)

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1.1 Making use of light

A) Light as a biomedical tool: Diagnostics

• Non-linear effects:

2nd/3rd harmonic generation fsec laser sources

2 adhering lipid vesicles illuminated with a fs laser. Lipid/H2O boundaries generate SHG signal, while lipid/lipid do not (left). The latter is made visible when stained with a fluorophore (right). L. Moreaux et al. Opt.Lett. (2000)

New laser sources and

• bservation techniques

basics of molecules & light

Combined SHG & fluorescence imaging of artery walls and collagen. Courtesy H. Dorkenoo (IPCMS)

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1.1 Making use of light

B) Light as a biomedical tool: Therapy

• Skin cosmetics
• Eye surgery

LASIK: laser-assisted keratomileusis

• Tumour treatment

(ablation, photodynamic therapy,…)

Molecular mechanisms

• f light-matter interaction ?

Welding, ionisation, photochemistry…

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1.2 Light used by living organisms

VISION

Rod outer segment Disk membrane Rhodopsins Retina

Lens Fovea Iris

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Absorption spectra of human cone cells

From S.S. Deeb, Clin. Genet. (2005)

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Color vision of animals

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1.2 Light used by living organisms

Breakes down into a « light phase » Dissociation H2O, ATP and NADPH formation and a « dark phase » (Calvin cycle)

The basic photo-reaction

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1.2 Light used by living organisms

Chloroplasts as seen through a microscope

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1.2 Light used by living organisms

Light-induced e- transfer H2O dissociation H+ transport → ATP

Photosystem I and II Cytochrome ATPsynthase

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1.2 Light used by living organisms

• W. Zinth, « Festschrift » DPG 2005

Light-induced e- transfer H2O dissociation H+ transport → ATP

chlorophyll Rhodobacter sphaeroides: a bacterial model

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1.2 Light used by living organisms

Light-Harvesting Complexes and the Reaction Center

AFM image of the LH complexes

• f Rsp. photometricum

Ritz&Schulten, Phys. Journal (2001)

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1.2 Light used by living organisms

PHOTO-driven processes

Photomorphism: light-dependent plant growth Phototaxis: Light-driven movement of bacteria

A crowd of bacteria forms in the center of a yellow light spot (left), but they tend to avoid harmful blue/UV light.

How does that work ? Molecular mechanisms ?

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Can the events inside a living organism be explained (predicted) solely by physics and chemistry? Erwin Schrödinger: What is life ?

Conference series, Dublin 1943

Yes, on the molecular level

Schrödinger: « The obvious inability of present-day physics and chemistry to account for such events is no reason at all for doubting that they can be accounted for by those sciences. »

• S. Haacke

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Ultrafast Biophotonics

2 – Proteins and chromophores

CNRS-EWHA Winter School 9 & 10 Feb 2012

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2.1. Protein = chain of amino acids

Lysine Tyrosine Tryptophane Phenylalanine Arginine Aspartate Glutatmate …

Covalent peptide bond linking amino acids

Proteins are made of 20 « bricks », the amino acids. Differ by residues (side chains).

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2.2. Protein structure

Primary structure = sequence of amino acids Secondary structure: structural elements (building blocks) formed due to hydrogen bonding

• α helices
• β sheets
• loops

From L. Stryer « Biochemistry »

sequence of a nucleocapsid protein of HIV

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2.2 Protein structure

Structure of rod rhodopsin at 2.2 Å resolution From Okada et al., JMB 2004

Tertiary structure: x,y,z coordinates of atoms Protein “folding” Consequence of “hydrophobic interactions”: Unpolar side chains (residues) tend to maximize mutual contacts and minimize contact with polar water → hydrophobic core

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2.3. Absorption by amino acids

A) UV spectra

Strongest absorption in C=O and C=N bonds In a peptide, C=N dipoles couple and add their absorption (oscillator) strengths. « Excitonic coupling » Depends on relative orientation of dipoles Absorption spectrum sensitive to structure. See example Poly-Lysine

More in “Biophysical chemistry”, van Holde et al.

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2.3. Absorption by amino acids

A) UV spectra

“Aromatic” amino acids have ฀→π* transitions in conjugated C=C (C=N) bonds Tryptophan, Tyrosine, Phenylalanine π and π* orbitals

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2.3 Absorption by amino acids

Proteins do not absorb visible light This is done by covalently linked CHROMOPHORES

(retinal, chlorophyl, carotene, flavins,…)

Tetrapyrole found in phytochrome protein (photo- morphism)

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2.3.1 Vibronic transitions

The absorption of visible/UV light leads to “HOMO – LUMO” transitions A series of vibrational levels is found for each electronic state S0, S1,… if

PES is a bound potential.

LUMO (S1) HOMO (S0)

, n v ψ =

Light absorption promotes electrons from =0 in S0 to a manifold of vibrational levels ’ in S1.

n: electronic state : vibrational level

1 2 E

ν

ω ν   = +     

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Rhodopsin = “opsin” + retinal

Chromophore Retinal (-cation) ≈ Vitamine A

• G. Wald, R.Granit, H.K. Hartline

Nobel price Medicine 1967

Bovine rhodopsin at 2.2 Å resolution From Okada et al., JMB 2004

11-cis

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Absorption spectrum

• Opt. density of bacterio-rhodopsin

Wavelength (nm)

Amino Acids Retinal

OD λ

( )= − log I(d)

I0 = ε λ

( )cd

Optical density

I d

( )= I010

−ε λ

( )cd

Lambert-Beer’s law

ε: extinction coeff.

c: molar concentration d: sample thickness

2.4 Absorptions shifts in protein

S1 S0 A) Steric effects – chromophore under strain Energy

constrained in a protein unconstrained in a solvent

tilt angle

2.4 Absorptions shifts in protein

B) Electrostatic effects – protein has local charges S1 S0 Energy

ground state stabilized by Coulomb interaction

+ +

• ground state

destabilized

+ +

• change in amino acid composition
• r intermol. distances

δ+

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2.4. Band-shifts in protein environment

Absorption spectrum BChl a

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2.4. Band-shifts in protein environment

• J. Herek et al., Biophys. J. (2000)

Absorption spectrum LH2 LH2 contains 8 + 16 BChla’s and 7 carotenoïds BChl’s are in ~nm distance wavefunction delocalization (“exciton”) spectral red-shift

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2.5 Excitonic coupling

Light-harvesting complexes:

Absorption band red-shift upon aggregation of chromophore

UV spectra peptides: Coupling of C=N dipoles

1 2 12

H H H V = + +

V12 = 1 4pe

0R3

Ê Ë Á Á Á Á ˆ ¯ ˜ ˜ ˜ ˜ ˜ m u r

1g

m u r

2 -

3 m u r

1g

R u r

( )m

u r

2g

R u r

( )

R2 Ï Ì Ô Ô Ô Ô Ó Ô Ô Ô Ô ¸ ð Ô Ô Ô Ô ý Ô Ô Ô Ô

inter-acting dipoles Two 2-level systems Trial wavefunction solving H 1 1 2 2

, , , χ χ χ χ

+ +

1 2 1 1 2 2 1 2 3 1 2

C C C C χ χ χ χ χ χ χ χ

+ + + +

Ψ = + + +

Analogy. 2 QW’s separated by a thin barrier

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Solutions

( ) ( )

1 2 1 2 1 2 1 2 1 2 2 2 1 2

2 2 2 E E e V E e V E e χ χ χ χ χ χ χ χ χ χ χ χ

+ + + + + + − − + +

= Ψ = + = + Ψ = − = − Ψ = = Ψ =

1 1 1 2 2 2 2 1 12 2 1

e H H V V χ χ χ χ χ χ χ χ

+ + + +

= = =

with and assuming

χ2

0χ1 0 V12 χ2 0χ1 0 =

χ2

+χ1 + V12 χ2 +χ1 + = 0

E0 E- E+ E+=2e 2V if V>0

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Transition dipole moments

E0 E- E+ if V > 0 μ+ μ-

• ext. coeff

doubles

2

ε µ ∝

1 2 1 1 2

2 2 2 µ µ µ µ µ µ µ µ µ

+ + − −

+ = Ψ Ψ = − = Ψ Ψ = ฀ ฀

allowed forbidden blue-shift if V > 0 red-shift if V< 0 repulsive alignment

• trans. moment

attractive alignment E

∑

• V 0 +V

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2 - Summary

 Light acts on individual molecules:

amino acids or chromophores

 Experiments done in vitro on isolated, purified

molecules

 The structure and flexibility of proteins determine their

biological function

 Proteins absorb VIS light via chromophores  Absorption spectra are protein-specific

Photo-induced reactions ? Protein effects ?

• S. Haacke

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Ultrafast Biophotonics

3 – Basic concepts of ultrafast biomolecular spectroscopy

CNRS-EWHA Winter School 9 & 10 Feb 2012

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The ultrafast camera – w atching molecules move

Only short exposure times allow capturing fast motion Only lasers provide femto time resolution How to watch molecules ? SPECTROSCOPY

Electron / X-ray diffraction

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3.1 “Pump-probe” techniques

The lab equipment

A commercial Titane-Saphir laser 35 fs pulses, 800 nm, 0.4 mJ, 5 kHz Home-built « pump-probe » set-up Generation of 400 and 266 nm pulses & white-light continuum (280- 1100 nm)

Ti:Sapphire Amplified Laser System

λ = 800 nm Repetition rate = 5 kHZ Pulse duration = 40 fs P = 2.5 W E = 0.5 mJ/pulse

BBO CaF2

Delay line

Sample CCD camera Monochromator Continuum generation Frequency doubling Initial pulse train 280-950 nm 400 nm

Transient absorption “pump-probe”

Intense 400 nm pulses excite the sample “pump” Weak broadband pulses “probes” absorption changes

3.1.1. Non linear optics : Change laser wavelength

Second harmonic generation 1 + 2= 3 k1 + k2 = k3 k3 k1 k2

3 3 2 2 1 1

) ( ) ( ) ( λ λ λ λ λ λ n n n = +

Sum freq. generation

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Non linear optics

Example : White light (supercontinuum) generation Several non linear processes involved Self phase modulation (3rd order phenomenon): Instantaneous index modulation: n=n(t)=nlin+I(t)nNL

• Frequency modulation
• New spectral components

generated

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3.1 Ultrafast transient absorption “pump& probe”

400 nm pump pulse (40 fs) White-light continuum (290-950 nm) Delay line

Detection

Sample excitation probe

15 µm  100 fs

Other options: - Change probe wavelength (X-ray, mid-IR, THz,…)

• Transient reflectivity
• Transient Raman scattering (spectrally narrow probe)
• Multiple beam experiments (“pump-dump-probe”, 2D spectroscopy)

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The ultrafast camera – w atching molecules move

1999 Nobel Prize In Chemistry Ahmed Zewail

http://www.zewail.caltech.edu/index.html

Vibrational w avepacket induced by ultrafast laser excitation

42 Experimental measurements of vibrational coherence Precise information on molecular motion & vibrational modes activated

. . . .

) (

0 r

ψ ) (

1 r

ψ ) (

2 r

ψ 1 2 S1 S0 S1 Ultrashort laser pulse

3.2 What do w e measure ?

S1 Sn S0 Wavelength

Ground state bleach

ΔA < 0

GSB SE

ΔA < 0

• Stim. emission

ΔA > 0

ESA Excited state absorption

Spectral location of bleach and stim. emission can a priori be predicted from steady-state spectra. ESA revealed by pump-probe experiment Photo-reaction: appearance of a product band

S1 Sn S0 ΔA < 0

GSB

ΔA > 0

ESA SE

ΔA < 0 The signal measured

( ) ( )(

)

∑

= ∆

j i i i j i

c t c d t A

, pump wo , pump with , ,

• )

( , λ ε λ

    - ext. coeff. transition level i  j  - Sample thickness  - molar concentration in level i Ground state bleach (GSB)

( )

( )

0,with pump 0,wo pump

( )- , ( )

GSB

c t c A t c t λ < ⇒ ∆ ∝ ∆ <

Excited state absorption (ESA)

( )

( )

1,with pump 1,wo pump 1

( )- , ( )

ESA

c t c A t c t λ > ⇒ ∆ ∝ ∆ >

Excited state species

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Photoreaction kinetics – rate equations

S1 P S0

Excitation

ΔAp > 0

Photoproduct

( )

d r d

t t p r

C A t e e

τ τ

τ τ

− −

  ∆ = −   − r: rise time

S1  P relaxation d: decay time P life time Generic 3-level system here: Exc. state  photoproduct relaxation

• Ex. Photo-oxydation, -isomerisation, -addition, -

dissociation,…

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Limitations of pump-probe spectroscopy

S1 Sn S0 ΔA < 0

GSB

ΔA > 0

ESA SE

ΔA < 0

( ) ( )(

)

∑

= ∆

j i i i j i

c t c d t A

, pump wo , pump with , ,

• )

( , λ ε λ

   Spectral / temporal overlap of ∑ij’s:

Signals of different sign cancel

Spectral shifts due to molecular dynamics

( )

,

,

i j

f t ε λ =

Non-exponential transients UV/VIS pump-probe gives only indirect evidence for structural dynamics

( )

, A t λ ∆

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3.3 Photoreactions

electronic ground state struct. relax. Energy T1 S1 S2 Fluo (10-9…10-6 s) Phosphorescence

(10-6…1 s)

ISC IC

Jablonski diagram

IC IC: Internal conversion, transition between states of same spin 10-13…10-10 s ISC: Inter-system crossing between states of ≠ spin, 10-9…10-3 s

not represented photo-

• xydation

photo-dissociation

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3.4 Femtosecond fluorescence techniques

Electronic detection of fluorescence signals has limited time resolution (≈ 30 ps) Femtosecond processes require optical gating – fluorescence up-conversion

« Polychromatic up-conversion » S. Haacke et al. JOSA B (1998)

• G. Zgrablic, PhD thesis EPFL (2006)

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Femtosecond fluorescence up-conversion

Sum frequency signal = temporal convolution of fluorescence and gate pulse

( ) ( ) ( )

t I t S dt I

fluo gate SFG

∫

∞ ∞ −

− = τ τ

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☺Fluorescence = excited state

dynamics only  Signal easier to analyze and interpret

:-( NO Photoproduct signature

see also Kerr gating:

• B. Schmidt et al. Biochimica et Biophysica Acta 1706 (2005)

Femtosecond fluorescence

example all-trans retinal in MeOH

Broadband operation possible

• G. Zgrablic et al. JPC-B (2009)

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SUMMARY

Watching molecular motion requires a “femtosecond camera”:

Combination of impulsive excitation (pump), synchronizing the molecules AND spectroscopy at a precise delay (probe) time

Many experimental schemes

UV/VIS pump-probe (absorption, reflection) & femtosecond fluorescence

X-ray, mid-IR, THz absorption, Photon echo spectroscopy, multiple pulse configurations Raman scattering, electron X-ray diffraction

Spectroscopy  identify reaction species and their concentrations  reaction scheme & kinetics  advanced data analysis Text book examples – isomerisation, energy and charge transfer

• S. Haacke

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Ultrafast Biophotonics

4 – Photo-reactions energize proteins

CNRS-EWHA Winter School 9 & 10 Feb 2012

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4.1 Photo - isomerization

Cis – trans isomerization retinal

• G. Wald, Nobel price Medicine 1967

Hubbard&Kropf (1955-1960)

Bovine rhodopsin at 2.2 Å resolution Okada et al., JMB 2004

11-cis all-trans

Experimental proof ? How fast ? Effect on protein ?

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From light absorption to nerve signal

Light absorption decreases the Ca2+ and Na+ current across membrane channels

Rod cell from salamander and the effect of light on the ion flux

From Rieke & Baylor, Rev. Mod. Physics (1998)

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From light absorption to nerve signal

Light absorption decreases the Ca2+ and Na+ current across the membrane channels

Ion flux through channel

From Rieke & Baylor, Rev. Mod. Physics (1998)

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4.1 Photo-isomerization and vision

Isomerization time = 100 fs = 10-13 s !!!

Measure transient absorption

Δt

absorption cis retinal

ΔA

• +

Δt> reaction time signature of isomerization

λ

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4.1 Photo-isomerization and vision

ΔA

• +

λ Schoenlein et al, Science (1991) Pump-probe experiments with 40 fs resolution Bleach at 505 nm, induced absorption @ ≈550 nm

180 fs

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4.1. Isomerization is a fs process

Wang et al, Science (1994)

The molecular physics picture

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Other Photoreactions

electronic ground state struct. relax. Energy T1 S1 S2 Fluo (10-9…10-6 s) Phosphorescence

(10-6…1 s)

ISC IC

Jablonski diagram

IC Isomerization competes with ISC High speed  quantum efficiency

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3.4 Photoreactions

Conical intersection in polyatomic molecules

• cf. work by Olivucci & Robb
• W. Fuss et al, Chem. Phys. (1998)

Klessinger & Michl “Exc. states and photochemistry”, ed. VCH

Reaction speed limited by PES prior to CI (state R*) Barrier on the way to CI ?

CI’s spectroscopically “dark”

I J/K

Reaction-induced coherences

Torsional wavepacket launched by intramolecular C=C relaxation

Rhodopsin is unique:

Fastest reaction & displays coherences

Unidirectional speed matters: Molecules are bound to twist unisono

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Reaction-ind. coherence in NAIP switches

Briand et al. PCCP 2010

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Reaction-ind. coherence in NAIP switches

Briand et al. PCCP 2010 Kinetic traces best described by non-exponential models

ESA: Exc. state WP PA: Ground state WP

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Reaction-ind. coherences in NAIP switches

• Only observed when reaction fast enough ? Seems so…

Photoproduct transients perfectly exponential in « slow » switch 600 fs

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Is high speed sufficient ?

• NO: ZW-NAIP forward vs backward

Léonard et al., submitted

Same speed Same quant. eff. But no ground state coherences in E Z

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• Single trajectories

identify underlying motion

(M. Olivucci et al., unpublished)

What can quantum chemistry do ?

Challenge: Large enough no. of trajectories at high computational costs

Ex: Polli et al., Nature (2010)

Single trajectory calc for EZ

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4.2 Non-radiative energy transfer

FRET: Fluorescence resonance energy transfer

Process that brings the optical excitation energy from the light-harvesting antennas to the photo-synthetic reaction center

Sundström, Pullerits, van Grondelle, J Phys. Chem. B (1999)

Classical analogon: Pendulum transferring energy to a coupled, nearly resonant 2nd pendulum

transfer times at 77 K

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4.2 Non-radiative energy transfer

Resonance energy transfer quenches fluorescence

Donor S1 Acceptor Energy donor (substance A) and acceptor molecules (B) must have their emission/absorption spectra overlapp

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Light-harvesting complex 2

Top view of LH2 showing only the Bchla molecules in B800 (blue), B850 ring (green) and carotenoids (yellow). Side view of LH2 showing in addition the amino acids (C-grey, O-red, S-yellow, etc.)

B800 B850

Sundström, Pullerits, van Grondelle, J Phys. Chem. B (1999)

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B800 – B850 FRET

Absorption spectra of natural “wild-type” (WT) protein complex and of mutants LH2 LH1

Sundström, Pullerits, van Grondelle, J Phys. Chem. B (1999)

Emission decay in B800 band

0.7 ps 1.2 ps 1.5 ps

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B800 – B850 FRET

Absorption spectra of wild-type (WT) protein complex and of mutants LH2 LH1

Sundström, Pullerits, van Grondelle, J Phys. Chem. B (1999)

Absorption increase in B850 band

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4.3 Photo-induced electron transfer

A + B

hν

 →  A* + B

ET

 →  A+ + B−

ground state A excited ground state

Charge Transfer quenches fluorescence

Importance for biological function

• Photosynthetic reaction center
• Photoreceptor cryptochrome
• DNA repair enzyme photolyase
• ….

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4 - Summary

 Photo-active proteins are triggered by

ultrafast photochemistry:

isomerization, e- transfer, …

 (Indirect) information of molecular conformation

and charge state from UV/VIS spectroscopy

 Fs lasers monitor with <10 fs resolution  Large protein amount (>mg) needed

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Reading suggestions

There is no comprehensive book or article on « Ultrafast Biophotonics » today. But, in addition to the references given, we can suggest the following textbooks & review articles

J.C. Rullière: « Ultrafast Laser Pulses: Principles & Experiments », Springer Verlag, 2e edition (2005)

• M. Braun, P. Gilch. W. Zinth, « Ultrafast Laser Pulses in Biology and Medecine », Springer Verlag

2008.

• A. Zewail, «Femtochemistry: Atomic-Scale Dynamics of the Chemical Bond», J. Phys. Chem. A,

104, 5660 – 5694 (2001). G.D. Reid & K. Wynne, « Ultrafast Laser Technolgy and Spectroscopy», in Encyclopedia of Analytical Chemistry, R.A. Meyers (Ed.) pp. 13644–13670, John Wiley & Sons Ltd, 2000 Ultrafast Spectroscopy Retinal proteins:

• J. Briand, J. Léonard & S. Haacke, J. Optics, 12, 084004 (2010)

Photosynthetic systems & light - harvesting:

• T. Pullerits & V. Sundström, « From biological to synthetic light-harvesting materials - the elementary

steps », in Energy harvesting materials, ed. David I. Andrews, 143 – 186, World Scientific (2005).

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• 2. Structural dynamics prior to photo-

isomerisation

Sub-20 fs pulses excite vibrational wavepackets

Kobayashi et al., Nature (2001)

Bacteriorhodopsin: Pump-probe with 5 fs pulse Time-dependent frequency change observed 11-cis all-trans

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• 2. Structural dynamics prior to photo-

isomerisation

11-cis all-trans

7 fs pulses at 550 – 600 nm

• A. Kahan et al., JACS (2007)

Hydrogren out-of-plane motion in exc. state

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1.2 Light used by living organisms

Primitive PHOTOSYNTHESIS – ATP production in halobacteria