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FCS Club Sept 8 th 2006

FCS Club Sept 8 th 2006. Schwille, P., F. Meyer-Almes and R. Rigler (1997). "Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution." Biophys. J. 72(4): 1878-1886. The Point.

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FCS Club Sept 8 th 2006

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  1. FCS ClubSept 8th 2006 Schwille, P., F. Meyer-Almes and R. Rigler (1997). "Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution." Biophys. J. 72(4): 1878-1886

  2. The Point Establishes FCCS is a variable method when using two laser lines and two independent dyes. Requires a well characterized system Provides the means to calibrate the system Measures annealing reactions

  3. Nomenclature for the paper • G = Green Dye • R = Red Dye • GR = Double Labeled (G + R) • D = Detector • DG = Detector Green • DR = Detector Red (or Doctor)

  4. FCCS requires spectrally separated dyes Rhodamine Green Cy5

  5. The setup was custom built

  6. The telescope system was a key element to generate equal volumes “Beam diameter 1/e2 values at the objecitve back aperature are 3.5 mm (488) and 5 mm (647, giving a Gaussian focal sport diameter r0 of 0.7 µ for both wavelengths.”

  7. Fluorescence fluctuations are attributed to particle concentration and collection efficiency Detector Properties Beam Geometry Collection Efficiency Function (Qian and Elson, 1991; Riggler et al., 1993) Particle Concentration Equation 1

  8. The “emission characteristics” provides the molecule detection efficiency function Photons released for each molecule per second The combination of detector, volume, and CEF Rigler et al. 1993 Equation 2

  9. Basic definitions: Equation 3 In text Three dimensional diffusion

  10. Single component three dimensional diffusion model including a triplet state Triplet State Average Particle Number 3D diffusion Equation 9 (Modified)

  11. In an ideal world, the cross correlation would have no cross talk

  12. Cross talk between channels is accounted for by examining the photons per molecule ηGGG Dye Laser Channel

  13. Cross talk is established by measuring the amount of red dye in the green channel ηGGG, ηRGG: Green and red emission of green dye excited by green laser ηGRG, ηRRG: Green and red emission of red dye excited by green laser ηGRR,ηRRR: Green and red emission of red dye excited by red laser ηGGR and ηRGR can be set to zero, because the green dye is proved not to be excitable by the used red laser. Although present in our system, the green anti-Stokes fluorescence of the Cy-5 is very weak; therefore we may also approximate ηGRG≈ 0 and ηGRR ≈ 0

  14. Color corrections in the objective influence matching focal volumes Good Objective Bad Objective

  15. A cy5 only sample establishes overlapping confocal volumes 488 Excitation 647 Excitation

  16. The cross correlated GR complex moves slowers Green Dye Cy5 Cross correlation

  17. FCCS measurements monitor the production of the complex

  18. The irreversible reaction allows for a affinity measurement

  19. What about reversible reactions??

  20. Assumes diffusion limited association: 106 M-1 sec-1

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