Fluorescence Cross-correlation Spectroscopy (FCCS) is a powerful tool to measure molecular interactions. It is based on the observation of fluorescence from a small observation volume at two or more distinct wavelength ranges. By cross-correlating the different signals one can determine whether the different labeled molecules/particles interact. Most commonly FCCS is performed in a dual-color mode in which two different labels are excited by two different laser lines. However, the necessary alignment of the two lasers to the same focal spot can be difficult. In addition, any changes of the alignment during the measurement (e.g. by thermal drift) will lead to changes in the overlap of the excitation volumes and makes quantitation of measurements very difficult. Another problem arises in measurements in tissues or organisms in which the chromatic aberrations induced by the tissue can lead to changes in overlap of excitation volumes and problems in quantification.
Another solution is the one-photon excitation of a fluorophore pair with similar excitation but distinct emission spectra, i.e. with widely different Stokes shifts, by a single laser line (for a review of FCCS an related methods see ref . First measurements of cross-correlations using a single laser wavelength were performed by Ricka and Binkert in 1989 in which a scattering particle and a fluorophore were used . However, the use of scatterers is problematic since they have to be large and the separation of scattered laser light and laser can be very difficult. In 2004 we therefore used tandem dyes (a covalently coupled energy transfer pair) and quantum dots, both of which can exhibit very large Stokes shifts, in combination with normal organic dyes to demonstrate that FCCS can be performed using a single laser wavelength for excitation . We called the techniques single wavelength FCCS or SW-FCCS. We then proceeded to show that the technique can be used with pairs of organic dyes and that stoichiometry and affinity of interactions can be determined , and that even multi-color approaches are feasible with the technique [5-7].
Below is a schematic diagram which shows the typical configuration for SW-FCCS.
A transition to cells, however, needed either efficient labelling techniques or the use of genetically encoded labels, e.g. fluorescent proteins . We therefore used an enhanced green fluorescent protein (EGFP) and a monomeric red fluorescent protein (mRFP) to measure the dimerization fraction of a transmembrane protein, the epidermal growth factor receptor (EGFR) [9,10]. The final step was the transition of the technique into tissues or organisms which can pose a problem for techniques using two lasers. For this purpose we measured the interaction of a small Rho-GTPase (cdc42-mRFP) with a scaffolding protein (IQGAP-EGFP) in zebrafish embryos. Using SW-FCCS we were able to determine the dissociation constant of this pair within living zebrafish.In 2012, Foo Yong Hwee of my group in a collaboration with Don Lamb (LMU, Germany) demonstrated how FCCS data in live cells can be corrected for artifacts including non-fluorescent proteins, FRET, and endogenous proteins to provide more quantitative affinity constants . This study was performed with EGFP and mRFP or mCherry, as the green and red labels, respectively, making quantitative SW-FCCS analysis widely accessible for many biological problems. Overall, we went from the inception of SW-FCCS to the testing of a wide range of fluorophores to the quantitative measurement of biomolecular interactions (dimerization and dissociation/binding) in cells and organisms. In the future we aim to apply SW-FCCS to the quantification of biomolecular interactions in signal transduction pathways for the elucidation of biological problems in cells and organisms.
Here we show auto (red and green) and cross correlation (blue) curves obtained from CHO cells expressing different chimeric fluorescent proteins. (A) PMTGFP/-mRFP (negative control 2). No cross correlation, hence no interaction, between the two FPs was observed. (B) Cell expressing mRFP-EGFR-GFP (positive control), in which mRFP and GFP were fused in tandem to N- and C-termini of EGFR, respectively. (C and D) Cell coexpressing EGFR-GFP/mRFP-EGFR or EGFR-GFP/EGFR-mRFP. Similar auto- and cross-correlation curves were observed in both the cases. (Ref. fig. 3 in )
PTB-EGFP/mRFP-EGFR proteins were co expressed in cells. (C) shows the auto (red) and cross-correlation (blue) after a short exposure of EGF (3 min). (E) shows the auto anc cross-correlation after a long exposure of EGF. No cross-correlation in seen in C. In the case of E, binding of PTB to EGFR leads to an increase in the cross-correlation. (Ref. )
Interactions of IQGAP1 with Cdc42G12V and Cdc42T17N in zebrafish embryos. (a) SW-FCCS result of the protein pair of mRFP-Cdc42G12V and EGFP-IQGAP1. The insets are schematic drawing and confocal images of the muscle fiber cell that shows both green and red channels. (b and c) KD determination results using scattering plot and log normal distribution histogram. (d–f) Corresponding results for the protein pair of mRFP-Cdc42T17N and EGFP-IQGAP1. SD ln , standard deviation factor of log-normal distribution. Scale bars, 20 μm. (Ref. fig 2 in )
 Hwang, L.C. and Wohland, T., Recent advances in fluorescence cross-correlation spectroscopy. Cell Biochemistry and Biophysics, 2007. 49(1): 1-13.
 Ricka, J. and Binkert, T., Direct measurement of a distinct correlation function using fluorescence cross correlation. Phys. Rev. A. 1989, 39: 2646-2652.
 Hwang, L.C. and Wohland, T., Dual-color fluorescence cross-correlation spectroscopy using single laser wavelength excitation. Chemphyschem, 2004. 5(4): 549-551.
 Hwang, L.C. and Wohland, T., Single wavelength excitation fluorescence cross-correlation spectroscopy with spectrally similar fluorophores: Resolution for binding studies. Journal of Chemical Physics, 2005. 122(11).
 Hwang, L.C., Gosch, M., Lasser, T. and Wohland, T., Simultaneous multicolor fluorescence cross-correlation spectroscopy to detect higher order molecular interactions using single wavelength laser excitation. Biophysical Journal, 2006. 91(2): 715-727.
 Hwang, L.C., Leutenegger, M., Gosch, M., Lasser, T., Rigler, P., Meier, W. and Wohland, T., Prism-based multicolor fluorescence correlation spectrometer. Optics Letters, 2006. 31(9): 1310-1312.
 Hogan, H., One laser does the work of three, June 2006, Biophotonics, June 2006, p. 21-23.
 Pan, X.T., Foo, W., Lim, W., Fok, M.H.Y., Liu, P., Yu, H., Maruyama, I. and Wohland, T., Multifunctional fluorescence correlation microscope for intracellular and microfluidic measurements. Review of Scientific Instruments, 2007. 78(5).
 Liu, P., Sudhaharan, T., Koh, R.M.L., Hwang, L.C., Ahmed, S., Maruyama, I.N. and Wohland, T., Investigation of the dimerization of proteins from the epidermal growth factor receptor family by single wavelength fluorescence cross-correlation spectroscopy. Biophysical Journal, 2007. 93(2): 684-698.
 Ma, X., Ahmed, S., Wohland, T., EGFR activation monitored by SW-FCCS in live cells, FrontBiosci (Elite Ed), 3(2011), 22-23.
 Foo, Y.H., Naredi-Rainer, N., Lamb, D.C., Ahmed, S., Wohland, T., Factors affecting the quantification of biomolecular interactions by fluorescence cross-correlation spectroscopy, Biophys J, 102 (2012) 1174-1183.
 Shi, X.; Foo, Y.H.; Sudhaharan, T.; Chong, S.W.; Korzh, V.; Ahmed, S.; Wohland, T. Determination of dissociation constants in living zebrafish embryos with single wavelength fluorescence cross-correlation spectroscopy, Biophys J, 97 (2009) 678-686.