Detection, identification and conformational dynamic characterization of single molecules by ultrase

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Detection, identification and conformational
dynamic characterization of single molecules by
ultra-sensitive fluorescence spectroscopy
techniques.

• Jerker Widengren
• Exp. Biomol. Physics
• Dept. Physics, KTH

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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

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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

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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.),
• BerlinSpringer, 13, 1992
• Rigler, Mets, Widengren and Kask,
• Eur. Biophys. J. 22, 179, 1993

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Fluorescence fluctuations due to translational
diffusion
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The Autocorrelation function
Translational diffusion for a 3D gaussian volume
element
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The experimental FCS curve for translational
diffusion
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Change in diffusion properties
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Ligand-receptor interactions A nAChR in solution
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• High sensitivity, ligand-receptor interactions at
  low conc. can be followed
• low conc. of labelled ligands ---gt 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

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Change in fluorescence upon chemical reaction
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Change in fluorescence upon chemical reaction
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Change in fluorescence upon chemical reaction
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Ion concentration monitoring
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Buffer effects
Widengren J, Terry B, Rigler R, Chem Phys. 249,
259-271, 1999
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Photophysics

• - triplet state transitions
• - electron transfer
• - trans-cis isomerization

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Triplet state monitoring by FCS
Fluctuations in fluorescence due to
singlet-triplet transitions
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The fluorescence intensity correlation function
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Environmental influence on the triplet
state Effects of solvents and quenchers on the
triplet state
Triplet state properties of FITC
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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

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Dual colour FCS
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Photon counting histograms (PCH) / Fluorescence
intensity distribution analysis (FIDA)

• Fluorescence brightness
• Concentration

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

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Photobleaching
Widengren J, Rigler R, Bioimaging, 4, 149-157,
1996 Eggeling C, Widengren J, Rigler R, Seidel,
C, Anal Chem, 70, 2651-2659, 1998
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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.
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How to maximize fluorescence information from
single molecules
info
n
f
n
f
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Single-molecule Multi-parameter Fluorescence
detection (smMFD)
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Model system
Cy5
A488
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FRET and Coincidence Analysis
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Conformation-based identification
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Fit to a structural model of DNA

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

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Photodynamics of Cy5
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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
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FRET-mediated excitation
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FRET-mediated excitation of Cy5
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FRET-mediated excitation of Cy5
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FRET-mediated excitation of Cy5
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FRET-mediated excitation of Cy5
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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
expected Widengren, Schweinberger, Berger, and
Seidel non-constant J. Phys. Chem. A 105,
6851-6866, 2001
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Selective FCS
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Traditional fluorescence parameters
Fluctuation parameters
four dimensions- excitation and
fluorescence spectra lE, lF - quantum yield
FF - lifetime t - anisotropy r
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• 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

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• 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)

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