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

Ultrafast Biophotonics 1 – A general overview CNRS-EWHA Winter School S. Haacke 1 9 & 10 Feb 2012

BIOPHOTONICS 2 P. N. Prasad „Introduction to Biophotonics“, Wiley ed.

1.1 Making use of light A) Light as a biomedical tool: Diagnostics Louis Pasteur (1822-1895) Micro-organisms & infections Conservation by “heating” Microscope C. Zeiss 1891 3

1.1 Making use of light A) Light as a biomedical tool: Diagnostics A.M. Rollins et al., OPN, April 2002 • (Fluorescence) Microscopy • Optical coherence tomography 2D images of human retinas (left) and with depth 4 resolution obtained by OCT (right)

1.1 Making use of light A) Light as a biomedical tool: Diagnostics • Non-linear effects: 2 nd /3 rd harmonic generation fsec laser sources 2 adhering lipid vesicles illuminated with a fs laser. Lipid/H 2 O boundaries generate SHG signal, while lipid/lipid New laser sources and do not (left). The latter is made visible when stained with a fluorophore (right). L. Moreaux et al. Opt.Lett. (2000) observation techniques basics of molecules & light Combined SHG & fluorescence imaging of artery walls and collagen. Courtesy H. Dorkenoo (IPCMS) 5

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 of light-matter interaction ? Welding, ionisation, photochemistry… 6

1.2 Light used by living organisms VISION Rod outer segment Retina Disk membrane Iris Fovea Rhodopsins Lens 7

Absorption spectra of human cone cells From S.S. Deeb, Clin. Genet. (2005) 8

Color vision of animals 9

1.2 Light used by living organisms The basic photo-reaction Breakes down into a « light phase »  Dissociation H 2 O, ATP and NADPH formation and a « dark phase » (Calvin cycle) 10

1.2 Light used by living organisms Chloroplasts as seen through a microscope 11

1.2 Light used by living organisms Light-induced e - transfer H 2 O dissociation H + transport → ATP Photosystem I and II Cytochrome ATPsynthase 12

1.2 Light used by living organisms Light-induced e - transfer Rhodobacter sphaeroides: a bacterial model H 2 O dissociation H + transport → ATP W. Zinth, « Festschrift » DPG 2005 chlorophyll 13

1.2 Light used by living organisms Light-Harvesting Complexes and the Reaction Center AFM image of the LH complexes 14 Ritz&Schulten, Phys. Journal (2001) of Rsp. photometricum

1.2 Light used by living organisms PHOTO-driven processes 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 ? Photomorphism: light-dependent plant growth 15

Erwin Schrödinger: What is life ? Conference series, Dublin 1943 Can the events inside a living organism be explained (predicted) solely by physics and chemistry? 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. » 16

Ultrafast Biophotonics 2 – Proteins and chromophores CNRS-EWHA Winter School S. Haacke 17 9 & 10 Feb 2012

2.1. Protein = chain of amino acids Proteins are made of 20 « bricks », the amino acids. Differ by residues (side chains). Lysine Tyrosine Tryptophane Phenylalanine Arginine Aspartate Glutatmate … Covalent peptide bond linking amino acids 18

2.2. Protein structure sequence of a nucleocapsid protein of HIV 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 » 19

2.2 Protein structure 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 Structure of rod rhodopsin at 2.2 Å resolution From Okada et al., JMB 2004 20

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. 21

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 22

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) 23

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 S 0 , S 1 ,… if LUMO (S 1 ) PES is a bound potential . n: electronic state ψ = n v ,  : vibrational level   1 = ω ν +    E ν   2 HOMO (S 0 ) Light absorption promotes electrons from  =0 in S 0 to a manifold of vibrational levels  ’ in S 1. 24

Rhodopsin = “opsin” + retinal Chromophore Retinal (-cation) ≈ Vitamine A G. Wald, R.Granit, H.K. Hartline Nobel price Medicine 1967 11-cis Bovine rhodopsin at 2.2 Å resolution From Okada et al., JMB 2004 25

Absorption spectrum Lambert-Beer’s law ( ) = I 0 10 ( ) cd − ε λ I d Amino Acids Retinal Optical density ( ) = − log I ( d ) ( ) cd OD λ = ε λ I 0 ε : extinction coeff. c: molar concentration d: sample thickness Wavelength (nm) Opt. density of bacterio-rhodopsin 26

2.4 Absorptions shifts in protein A) Steric effects – chromophore under strain constrained unconstrained in a protein in a solvent S 1 S 0 Energy tilt angle

2.4 Absorptions shifts in protein B) Electrostatic effects – protein has local charges δ + + + + + ground state - ground state - destabilized stabilized by Coulomb interaction S 1 S 0 Energy change in amino acid composition or intermol. distances

2.4. Band-shifts in protein environment Absorption spectrum BChl a 29

2.4. Band-shifts in protein environment Absorption spectrum LH2 LH2 contains 8 + 16 BChla’s and 7 carotenoïds BChl’s are in ~nm distance wavefunction delocalization (“exciton”) spectral red-shift J. Herek et al., Biophys. J. (2000) 30

2.5 Excitonic coupling Light-harvesting complexes: Absorption band red-shift upon aggregation of chromophore UV spectra peptides: = + + H H H V Coupling of C=N dipoles 1 2 12 u r u r u r u r ( ) m ( ) Ï ¸ Ô Ô 3 m 1 g 2 g Ô u r u r Ô Ê ˆ R R Ô Ô inter-acting ˜ Á 1 Ô Ô ˜ Á V 12 = ˜ m 1 g m 2 - Ì ð ˜ Á ˜ dipoles Ô Ô Á 4 pe 0 R 3 R 2 Ë ¯ Ô Ô Ô Ô Ô Ô Ó ý + + χ χ χ χ 0 0 , , , Two 2-level systems 1 1 2 2 Trial wavefunction solving H Ψ = χ χ + χ χ + + χ χ + + χ χ + + 0 0 0 0 C C C C 0 1 2 1 1 2 2 1 2 3 1 2 Analogy. 2 QW’s separated by a thin barrier 31

with Solutions = χ + χ = χ + χ 0 0 e H H 1 1 1 2 2 2 = Ψ = χ χ = χ χ + χ χ + 0 0 0 0 V V E 0 2 1 12 2 1 0 0 1 2 ( ) χ χ + + χ χ + 0 0 E + = 2e = + Ψ = 1 2 1 2 E e V + + 2 ( ) χ χ + − χ χ + 0 0 = − Ψ = 1 2 1 2 E e V − − 2 + + = Ψ = χ χ E + E 2 e 2V 2 2 1 2 E - and assuming 0 = 0 V 12 χ 2 χ 2 0 χ 1 0 χ 1 + = 0 + V 12 χ 2 + χ 1 + χ 1 χ 2 E 0 if V>0 32

Transition dipole moments µ + µ µ = Ψ µ Ψ = µ if V > 0 allowed ฀ 1 2 2 + + 0 1 2 E + µ − µ µ = Ψ µ Ψ = ฀ 1 2 0 forbidden E - − − 0 2 μ + ε ∝ µ μ - 2 ext. coeff doubles E 0 ∑ repulsive alignment trans. moment blue-shift if V > 0 red-shift if V< 0 attractive alignment E -V 0 +V 33

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 ? 34

Ultrafast Biophotonics 3 – Basic concepts of ultrafast biomolecular spectroscopy CNRS-EWHA Winter School S. Haacke 35 9 & 10 Feb 2012

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 36