1 / 38

Single Molecule Optics

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

leah-randall
Download Presentation

Single Molecule Optics

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related