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Jerker Widengren Exp. Biomol. Physics Dept. P hysics , KTH

Detection, identification and conformational dynamic characterization of single molecules by ultra-sensitive fluorescence spectroscopy techniques. Jerker Widengren Exp. Biomol. Physics Dept. P hysics , KTH. Topics of Discussion. F luorescence Correlation Spectroscopy (FCS) - Concept

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Jerker Widengren Exp. Biomol. Physics Dept. P hysics , KTH

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  1. Detection, identification and conformational dynamic characterization of single molecules by ultra-sensitive fluorescence spectroscopy techniques. Jerker Widengren Exp. Biomol. Physics Dept. Physics, KTH

  2. Topics of Discussion • Fluorescence Correlation Spectroscopy (FCS) - Concept - strategies to study molecular kinetics / applications • Single-molecule Multi-parameter fluorescence detection (smMFD) - Concept - single-molecule FRET studies

  3. Fluorescence Correlation Spectroscopy • Dynamic properties of molecules probed via their thermodynamic fluctuations • At equilibrium, no perturbation • Original concept Magde, Elson and Webb, 1972, Phys. Rev. Lett. 29, 705 Elson and Magde, 1974, Biopolymers, 13, 1 Magde, Elson and Webb, 1974, Biopolymers, 13, 29 Ehrenberg and Rigler, 1974, Chem. Phys., 4, 390

  4. FCS set-up • Higher spatial discrimination • Higher spectral discrimination • Enhanced detection efficiency • - increased fluor./(mol. x s) - reduced background • REF: • Rigler and Widengren, in Bioscience, • Klinge and Owman (Ed.), Lund University • Press, 180, 1990 • Rigler, Widengren and Mets, in Fluoresc. • Spectroscopy, Wolfbeis (Ed.), • Berlin:Springer, 13, 1992 • Rigler, Mets, Widengren and Kask, • Eur. Biophys. J. 22, 179, 1993

  5. Fluorescence fluctuations due to translational diffusion

  6. The Autocorrelation function: Translational diffusion for a 3D gaussianvolume element:

  7. The experimental FCS curve for translational diffusion:

  8. Change in diffusion properties

  9. Ligand-receptor interactions: A:nAChR in solution

  10. High sensitivity, ligand-receptor interactions at low conc. can be followed • low conc. of labelled ligands ---> facilitates displacements studies • No separation of bound from unbound • Low quantities of material needed • No radioactivity REF: Rauer, Neumann, Widengren, Rigler 1996, Biophys. Chem 58, 3-12

  11. Change in fluorescence upon chemical reaction

  12. Change in fluorescence upon chemical reaction

  13. Change in fluorescence upon chemical reaction

  14. Ion concentration monitoring:

  15. Buffer effects Widengren J, Terry B, Rigler R, Chem Phys. 249, 259-271, 1999

  16. Photophysics • - triplet state transitions • - electron transfer • - trans-cis isomerization

  17. Triplet state monitoring by FCS Fluctuations influorescence due to singlet-triplet transitions

  18. The fluorescence intensity correlation function:

  19. Environmental influence on the triplet state Effects of solvents and quenchers on the triplet state Triplet state properties of FITC

  20. Triplet state monitoring: • Distortion of FCS curves at high excitation intensities can to a large extent be attributed to triplet state build-up. • By FCS it is possible to measure intersystem crossing rates, triplet state lifetimes and excitation cross sections. • The environmental sensitivity of the triplet parameters suggests the use of FCS for micro-environmental probing. • Knowledge of triplet parameters important for optimization of fluorescence • REF: - Widengren, Rigler and Mets J. Fluoresc. 4(3), 255-258, 1994 • - Widengren, Mets and Rigler J. Phys. Chem. 99, 13368-13379, 1995 • - Mets, Widengren and Rigler Phys. Chem. 218, 191-198, 1997

  21. Dual colour FCS

  22. Photon counting histograms (PCH) / Fluorescence intensity distribution analysis (FIDA): • Fluorescence brightness • Concentration

  23. Figures of merit: Photophysical limitations: - Fluorescence saturation - Photodestruction

  24. Fluorescence saturation:

  25. Photobleaching Widengren J, Rigler R, Bioimaging, 4, 149-157, 1996 Eggeling C, Widengren J, Rigler R, Seidel, C, Anal Chem, 70, 2651-2659, 1998

  26. Photobleaching effects in a cell surface Exposure time: t Excitation power: Pexc Diffusion coeff: D Radius of cell area: Rcell Pexc Rcell Widengren J submitted to Biophys. J.

  27. How to maximize fluorescence information from single molecules: info n f n f

  28. Single-molecule Multi-parameter Fluorescence detection (smMFD)

  29. Model system Cy5 A488

  30. FRET and Coincidence Analysis

  31. Conformation-based identification

  32. Fit to a structural model of DNA :

  33. FRET studies with • smMFD: • High sensitivity, precision andaccuracy • resolution better than 1 nm • - identification based onconformational properties • (”conformational fingerprints”) • range: 10-100 Ångström • Detection and selective analysis of subpopulations

  34. Photodynamics of Cy5

  35. Trans-cis isomerization of Cy5 Widengren J. & Schwille P. J. Phys. Chem. 104(27), 6416-6428, 2000 Widengren J. & Seidel C. Phys. Chem. Chem. Phys. 2, 3435-3441, 2000

  36. FRET-mediated excitation:

  37. FRET-mediated excitation of Cy5

  38. FRET-mediated excitation of Cy5

  39. FRET-mediated excitation of Cy5

  40. FRET-mediated excitation of Cy5

  41. Determination of E via trans-cis isomerization of the acceptor dye • - Interference with other relaxation processes • * Photodynamic reaction to excitation monitored on acceptor side • + Independent read-out: • * donor-fluorescence cross-talk • * background • * labelling efficiencies • * absolute concentrations • * absolute fluorescence and detection Q.Y. • + Calibration on same sample possible • + wide range, good precision • *P: * lower than expectedWidengren, Schweinberger, Berger, and Seidel • * non-constantJ. Phys. Chem. A 105, 6851-6866, 2001

  42. Selective FCS:

  43. Traditional fluorescence parameters Fluctuation parameters four dimensions:- excitation and fluorescence spectra: lE, lF - quantum yield: FF - lifetime: t - anisotropy: r

  44. Acknowledgements: • Dept. Med. Biophysics, MBB, Karolinska • Insitutet, Stockholm: • Ylo Mets, Per Thyberg, Petra Schwille, Aladdin Pramanik, • Rudolf Rigler • MPI f. Biophys.Chem. Göttingen, Germany: • Enno Schweinberger, Christian Eggeling,Jörg Schaffer, Sylvia Berger, Matthew Antonik,Claus Seidel, Martin Margittai, Reinhard Jahn • Financial Support: • Swedish Foundation for Cooperation inHigher Educationand Research (STINT) • - BMBF-Biofuture Program • VW-Stiftung • The Swedish Research Council (Medicine) • - Magnus Bergwall Foundation • - The Swedish Society of Medicine • - Karolinska Intitutet Research Funds

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