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Introduction t o Fluorescence Correlation Spectroscopy Oleg Krichevsky

Introduction t o Fluorescence Correlation Spectroscopy Oleg Krichevsky Ben-Gurion University in t he Negev. Outline. Tutorial on FCS The basic idea of the technique Instrumentation Standard applications: - measurements of concentrations - diffusion kinetics - binding assay

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Introduction t o Fluorescence Correlation Spectroscopy Oleg Krichevsky

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  1. Introduction to Fluorescence Correlation Spectroscopy Oleg Krichevsky Ben-Gurion University in the Negev

  2. Outline • Tutorial on FCS • The basic idea of the technique • Instrumentation • Standard applications: - measurements of concentrations - diffusion kinetics - binding assay • DNA dynamics

  3. t (ms) Fluorescence Correlation Spectroscopy (FCS) Magde, Elson & Webb (1972); Rigler et al (1993)

  4. What is correlation function?

  5. What is correlation function (of fluorescence fluctuations)? logarithmic scale in time

  6. General Properties of FCS Correlation Function

  7. Rh6G t (ms) Correlation function for simple diffusion:

  8. Principles of confocal setup Sampling volume 0.5 fl (Ø 0.45 x 2 mm) Incident light power 10 - 50mW 0.1-300 molecules per sampling volume on average

  9. Enhancements and variations of the standard setup: • Two-color FCS (Schwille et al) • Two-photon FCS (Berland et al) • Scanning FCS(Petersen et al) References and technical details in G. Bonnet and O.K., Reports on Progress in Physics, 65(2002), 251-297

  10. Standard applications: • Amplitude of G(t)→ concentration of moving molecules • Decay → diffusion kinetics (in vitro and in vivo) • Binding assay

  11. Fast Diffusion DNA + Few mm Slow Diffusion FCS as a Binding Assay Protein Few nm

  12. In general, for two-component diffusion: Methyltransferase + Lambda-DNA (methyltransferase – courtesy of Albert Jeltsch and Vikas Handa)

  13. DNA hairpin • opening-closing kinetics to (k-) tc (k+) with Grégore Altan-Bonnet Albert Libchaber Rockefeller University

  14. DNA hairpin fluctuations: Molecular beacon design Tyagi&Kramer (1996) to (k-) tc (k+) 5’ - Rh6G – CCCAA – (Xn) – TTGGG – [DABCYL] – 3’ (n=12-30) Signal/background:Io/ Ic ~ 50-100 I (kHz) T (oC)

  15. FCS on Molecular beacons: two processes – two characteristic time scales

  16. HOPE!!! Correlation function of a molecular beacon: G t (ms) structural fluctuations diffusion

  17. to (k-) tc (k+) to (k-) tc (k+) Control: Beacon:

  18. t (ms) Correlation functions of beacon & control Ratio of the correlation functions: pure conformational kinetics

  19. Conformational kinetics at different temperatures: Gconf t (ms)

  20. The experimental procedure: 1) Melting curves: I(T) I T 2) FCS on beacons: 3) FCS on controls:

  21. Characteristic time scales of opening and closing of T21 loop hairpin:

  22. Different lengths of T-loops:

  23. The loops of equal length but different sequence: T21 vs. A21

  24. Stacking interaction between bases

  25. Closing enthalpy (kcal/mol) vs. loop length (poly-A) 0.55 kcal/mol/stacked base Opening and closing times of different poly-A loops

  26. Placing a defect in a poly-A loop no defect

  27. In some simple situations we have some understanding of the sequence-dependence of hairpin closing kinetics • In a number of other situations we have no undersanding • poly-C loops • short poly-T loops (below 7 bases(

  28. 2) DNA “breathing” The experimental construct:

  29. Conformational dynamics of polymers in good solvents: on the model of dsDNA and ssDNA molecules

  30. G(t) lag (ms) lag (ms) Diffusion of dsDNA 6700bp

  31. Polymer Statistics Freely Jointed Chain model: Random Walks inSpace b Ree Ree

  32. center of mass polymer end • The kinetics of monomer random motion: • double-stranded DNA (dsDNA) • single-stranded DNA (ssDNA) Polymer conformational dynamics: Rouse (1953) Zimm (1956)

  33. Theory: b2 t

  34. Basic length scale: b Basic timescale: Polymer size: N Rouse theory of Polymer Dynamics: b g

  35. Mean-square displacement of an end-monomer: Center-of-mass internal Rouse modes: n 0 N

  36. r Exact: Rouse model: connectivity + friction of polymer segments

  37. 1) Experimental measurements of polymer coil diffusion (dynamic light scattering) 2) Hydrodynamic interactions between polymer segments cannot be neglected Rouse model is nice but wrong:

  38. Zimm model: Rouse model + hydrodynamic interactions Diverge with N => cannot be neglected even for distant monomers

  39. Hydrodynamic shell: r Exact Zimm model: Rouse model + hydrodynamic interactions

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