Download presentation
Presentation is loading. Please wait.
Published bySabina Young Modified over 9 years ago
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
Similar presentations
© 2025 SlidePlayer.com. Inc.
All rights reserved.