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

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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:

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

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

  3. 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

  4. 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)

  5. 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

  6. 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

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

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

  9. Color vision of animals 9

  10. 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

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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

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

  18. 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

  19. 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

  20. 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

  21. 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

  22. 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

  23. 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

  24. 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

  25. 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

  26. 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

  27. 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

  28. 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

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

  30. 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

  31. 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

  32. 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

  33. 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

  34. 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

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

  36. 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

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