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Single Molecule Optics

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leah-randall
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Single Molecule Optics. Michel Orrit Mo lecular N ano- O ptics and S pins Leiden University Winterschool: Spectroscopy and Theory 15-18 December 2008, Han-sur-Lesse, Belgium. 1. Introduction. Optical detection of single molecules by fluorescence

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Single Molecule Optics
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  1. Single Molecule Optics Michel Orrit Molecular Nano-Optics and Spins Leiden University Winterschool: Spectroscopy and Theory 15-18 December 2008, Han-sur-Lesse, Belgium

  2. 1. Introduction • Optical detection of single molecules by fluorescence • High signal/background ratio thanks to resonance • Made possible by advances in sources, detectors, optics

  3. Molecular Photophysics • Electronic levels are split in a series of harmonic oscillator levels • Transitions between levels are related to overlaps between oscillator wavefunctions

  4. Mirror Image Absorption and fluorescence spectra are related by a mirror symmetry around the 0-0 transition

  5. Absorption & Emission of Cy3 wavenumber (cm-1) S1S0 S1S0 vibronic vibronic S2S0

  6. m12 F2 F1 Transition dipole moment characterizes the strength of the optical transition

  7. Kasha’s Rule • Radiative and non-radiative relaxation between electronic levels • Fluorescence can only arise from the lowest excited singlet state S1; • higher excited states relax to S1 faster than they can emit; • triplet states emit weak phosphorescence.

  8. Fluorescence quantum yield S1 knr kr S0

  9. Jablonski diagram: relaxation between electronic states

  10. tryptophane l = 280 nm TMR l = 514 nm TDI l = 630 nm Cy5 l = 630 nm DAPI l = 355 nm eGFP l = 490 nm Typical fluorophores +

  11. 1 nm green-fluorescent protein (GFP) K. Brejc et.al., PNAS 94 (1997) 2306

  12. 2. Optical Microscopy intermediate image object objective lens eye-piece Working principle of an optical microscope Diffraction-limited resolution

  13. Both Collection Efficiency and Point Spread Function dependon the Numerical Aperture NA

  14. Immersion favors collection efficiency

  15. Aberrations must be corrected Spherical aberration, coma, pincushion/barrel deformation

  16. Polarization structure in focal plane

  17. Confocal Scanning Microscope sample scanning beam scanning

  18. Total Internal Reflection

  19. Other focusing and collecting elements used in single-molecule experiments

  20. Near-Field Optics

  21. 3. Excitation and Detection of Fluorescence • Sources: Lasers cw (ion), pulsed (Nd-YAG, Ti-sapphire, diodes) • Photon Detectors:PhotoMultiplier Tube, Avalanche PhotoDiode, Charge Coupling Device • Spectral Filters: colored glass, notch holographic, multidielectric

  22. Photon Counting Analyses • Field and Intensity Correlation Functions Field Intensity

  23. Thermal or Chaotic Light (Lamp)

  24. Coherent Light (cw Laser) Poisson statistics: - independent photons, - correspondence with classical light wave for large numbers

  25. Correlation function versus Start-Stop The two functions are identical for short times, but differ for long times

  26. Histogram of delays between fluorescence photon and laser pulse Full time information: macroscopic arrival time of photon, and delay with respect to laser pulse Time-Correlated Single Photon Counting

  27. 4. Single Molecules in Fluid SolutionsMolecules diffuse, bursts of fluorescence, photon-by-photon studies . • Burst analysis (intensity) • Multiparameter analysis, statistical correlations • Fluorescence Correlation Spectroscopy

  28. Translational Diffusion D diffusion coefficient, t and a transverse and axial beam waists N average number of molecules in the excitation volume.

  29. Rotational Diffusion For isotropic diffusion on a sphere, the correlation function decays exponentially, on some nanoseconds for small fluorophores in water, more slowly for bigger molecules or more viscous fluids.

  30. Blinking due to a Dark State (Triplet) Single-exponential decay of the correlation function with the sum of the two rates.

  31. 5. Immobilized Molecules Signal/Background ratio must be large enough or

  32. Sample preparation Spin-coating Langmuir-Blodgett films

  33. Microscopy images • Counting, stoichiometry, colocalization • Orientation Comparison of polarization modulation for epi-illumination and total internal reflection.

  34. I t I t Photophysics, Blinking flickering blinking Triplet state Electron transfer Other photochemical reactions Bleaching

  35. Donor Acceptor 6. Fluorescence Resonance Energy Transfer (FRET) Dipole-dipole interaction (near-field)

  36. Förster transfer rate -6 • Decreases as (distance) • Spectral Overlap between Donor Fluorescence and Acceptor Absorption • Angular dependence 2 isotropic: <2> = 2/3

  37. Transfer Efficiency • Fraction of excitations transferred to acceptor • R0 Förster radius, maximum 10 nm for large overlap

  38. FRET Histograms and Dynamics