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):

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

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)

FCCS requires spectrally separated dyes Rhodamine Green Cy5

The setup was custom built

The telescope system was a key element to generate equal volumes “Beam diameter 1/e 2 values at the objecitve back aperature are 3.5 mm (488) and 5 mm (647, giving a Gaussian focal sport diameter r 0 of 0.7 µ for both wavelengths.”

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

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

Basic definitions: Equation 3 In text Three dimensional diffusion

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

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

Cross talk between channels is accounted for by examining the photons per molecule η GGG Dye Channel Laser

Cross talk is established by measuring the amount of red dye in the green channel η GGG, η RGG : Green and red emission of green dye excited by green laser η GRG, η RRG : Green and red emission of red dye excited by green laser η GRR, η RRR : Green and red emission of red dye excited by red laser η GGR and η RGR 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 η GRG ≈ 0 and η GRR ≈ 0

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

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

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

FCCS measurements monitor the production of the complex

The irreversible reaction allows for a affinity measurement

What about reversible reactions??

Assumes diffusion limited association: 10 6 M -1 sec -1