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 n Fluorescence Correlation Spectroscopy (FCS) - Concept - strategies to study molecular kinetics / applications n Single-molecule Multi-parameter fluorescence detection (smMFD) - Concept - single-molecule FRET studies

3. Fluorescence Correlation Spectroscopy n Dynamic properties of molecules probed via their thermodynamic fluctuations n At equilibrium, no perturbation n 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 n Higher spatial discrimination n Higher spectral discrimination n Enhanced detection efficiency è - increased fluor./(mol. x s) - reduced background REF: - Rigler and Widengren, in Bioscience, Klinge and Owman (Ed.), Lund University Klinge and Owman (Ed.), Lund University Press, 180, 1990 Press, 180, 1990 - Rigler, Widengren and Mets, in Fluoresc. Spectroscopy, Wolfbeis (Ed.), Spectroscopy, Wolfbeis (Ed.), Berlin:Springer, 13, 1992 Berlin:Springer, 13, 1992 - Rigler, Mets, Widengren and Kask, Eur. Biophys. J. 22, 179, 1993 Eur. Biophys. J. 22, 179, 1993

5. Fluorescence fluctuations due to translational diffusion

6. The Autocorrelation function: Translational diffusion for a 3D gaussian volume 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 58, 3-12

11. 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 n - triplet state transitions n - electron transfer n - trans-cis isomerization

17. Triplet state monitoring by FCS Fluctuations in fluorescence 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 - Widengren, Mets and Rigler J. Phys. Chem. 99, 13368-13379, 1995 - Mets, Widengren and Rigler Phys. Chem. 218, 191-198, 1997 - 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. Photophysical limitations: - Fluorescence saturation - Photodestruction Figures of merit:

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 R cell P exc Exposure time: tExcitation power: P exc Diffusion coeff: DRadius of cell area: R cell Widengren J submitted to Biophys. J.

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

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

32. Cy5 A488 Model system

34. FRET and Coincidence Analysis

37. Conformation-based identification

38. : Fit to a structural model of DNA

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

40. Photodynamics of Cy5

41. 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

42. FRET-mediatedexcitation:

43. FRET-mediated excitation of Cy5

47. 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 * donor-fluorescence cross-talk * background * background * labelling efficiencies * labelling efficiencies * absolute concentrations * absolute concentrations * absolute fluorescence and detection Q.Y. * absolute fluorescence and detection Q.Y. + Calibration on same sample possible + wide range, good precision *P:* lower than expected Widengren, Schweinberger, Berger, and Seidel * non-constant J. Phys. Chem. A 105, 6851-6866, 2001

48. Selective FCS:

49. Traditional fluorescence parameters four dimensions: - excitation and fluorescence spectra: E, F - quantum yield:  F - lifetime:  - anisotropy: rFluctuationparameters

50. 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 in Higher Education and Research (STINT) - BMBF-Biofuture Program - VW-Stiftung - The Swedish Research Council (Medicine) - Magnus Bergwall Foundation - The Swedish Society of Medicine - Karolinska Intitutet Research Funds

51. Prospects for the future: -Basic research: Reveal structures and dynamics of molecules beyond ensemble averaging -Ultrasensitve diagnostics: Detection and identification of sparse amounts of disease-specific molecules on/inside cells or in body fluids. -Ultrasensitive characterization of disease specific molecules or target molecules for drug therapies -High-throughput-screening (small sample volumes, low concentrations, fast read-out)

52. The Experimental Biomolecular Physics group Senior researchers / post docs: Anders Hedqvist Per Thyberg vacant PhD students: Per-Åke Löfdahl vacantvacant