Fluorescence Cross-correlation Spectroscopy (FCCS) Single wavelength FCCS (SW-FCCS) SW-FCCS with 2 and 3 colors Dimerization of the Epidermal Growth Factor Receptor (EGFR) Kd measurements in zebrafish embryos (Cdc42, IQGAP1) How fluorescence are fluorescent proteins? Imaging Total Internal Reflection FCS
SW-FCCS Single Wavelength Excitation Fluorescence Cross-correlation Spectroscopy
2.2 2.0
G(τ)
1.8 1.6 1.4 1.2 1.0 10
-6
10
-5
10
10
-6
10
-4
10 τ[s]
-3
10
-2
10
-5
10
-1
10
-4
10 τ[s]
-3
10
-2
10
0
-1
10
2.2 2.0
G(τ)
1.8 1.6 1.4 1.2 1.0 0
Ricka and Binkert, Phys Rev A, 39(5) :2646-52 (1989) Hwang and Wohland, ChemPhysChem 5, 549-551 (2004)
Confocal FCS setup FCS/FCCS measurements can be performed with self-built addition to commercial confocal microscope
Pan et.al. Rev. Sci. Instr. (78): 053711 (2007)
4 Hwang and Wohland, JCP, 122, 114708 (2005)
The Stokes’ shift problem Quantum Dots
Tandem Dyes
Measurements can be performed with one- or two-photon excitation.
Quantum dots and tandem dyes both have very large Stokes’ shifts allowing the simultaneous excitation with fluorescein at 488 nm but separate detection.
FCCS taken on a normal FV300 confocal microscope
FCS microscope performance Diffusion time profile
DC-FCCS: Biotin-Alexa488 (Ex: 488, Em 520) Streptavidin-Cy5 (Ex: 633, Em 670)
