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

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

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

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, Rigler, Widengren and Mets, in Fluoresc. Spectroscopy, Wolfbeis (Ed.), Spectroscopy, Wolfbeis (Ed.), Berlin:Springer, 13, 1992 Berlin:Springer, 13, Rigler, Mets, Widengren and Kask, Eur. Biophys. J. 22, 179, 1993 Eur. Biophys. J. 22, 179, 1993

Fluorescence fluctuations due to translational diffusion

The Autocorrelation function: Translational diffusion for a 3D gaussian volume element:

The experimental FCS curve for translational diffusion:

Change in diffusion properties

Ligand-receptor interactions: A: nAChR in solution

: 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

Change in fluorescence upon chemical reaction

Ion concentration monitoring:

Buffer effects Widengren J, Terry B, Rigler R, Chem Phys. 249, , 1999

Photophysics n - triplet state transitions n - electron transfer n - trans-cis isomerization

Triplet state monitoring by FCS Fluctuations in fluorescence due to singlet-triplet transitions

The fluorescence intensity correlation function:

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

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), , Widengren, Mets and Rigler J. Phys. Chem. 99, , Widengren, Mets and Rigler J. Phys. Chem. 99, , Mets, Widengren and Rigler Phys. Chem. 218, , Mets, Widengren and Rigler Phys. Chem. 218, , 1997

Dual colour FCS

Photon counting histograms (PCH) / Fluorescence intensity distribution analysis (FIDA):  Fluorescence brightness  Concentration

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

Fluorescence saturation:

Photobleaching Widengren J, Rigler R, Bioimaging, 4, , 1996 Eggeling C, Widengren J, Rigler R, Seidel, C, Anal Chem, 70, , 1998

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.

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

Single-molecule Multi-parameter Fluorescence detection (smMFD)

Cy5 A488 Model system

FRET and Coincidence Analysis

Conformation-based identification

: Fit to a structural model of DNA

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

Photodynamics of Cy5

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

FRET-mediatedexcitation:

FRET-mediated excitation of Cy5

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, , 2001

Selective FCS:

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

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

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)

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