Dual-color cross-correlation

Unfortunately, because of space, we have not covered important new developments in light scattering for the study of nontransparent systems such as diffusion wave spectroscopy (DWS), fiber-optic quasi-elastic light scattering (FOQELS), dual-color cross-correlation, 3D crosscorrelation DLS, and improved techniques followed from these techniques [1-4]. 

In a dual-color cross-correlation fluorescence spectroscopy (DCCFS) experiment [46], a sample containing two fluorophores with different emissions in each molecule was irradiated with two lasers (or with one laser) to perform simultaneous excitation of the fluorophores. The DCCFS in combination with the confocal laser microscopy allows the separation of microscopic volume with two different fluorophores from volume with only one of them and, therefore, the monitoring of dissociation of the dual-labeled molecules or association of two single-labeled molecules. Optical setup as realized in an inverted microscope to perform simultaneous excitation of the fluorophores (Figure 11.14). 

Kogure, T., Karasawa, S., Araki, T., Saito, K., Kinjo, M., and Miyawaki, A. 2006. A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color singlelaser fluorescence cross-correlation spectroscopy. Nature Biotechnol. 24 577-81. 

Bacia, K. and Schwille, P. (2007) Practical guidelines for dual-color fluorescence cross-correlation spectroscopy. Nature Protocols, 2, 2842-2856. 

There are, however, limits in the resolution of heterogeneous populations. In the particular case of multiple diffusing fluorescent species, for example in binding studies, the diffusion times must differ by a factor of 1.6-2.0 to be resolved, corresponding to a 4-8-fold increase in molecular weight. This limitation is alleviated in dual-color fluorescence cross-correlation spectroscopy (DC-FCCS) [116], in which the fluorescence fluctuation is simultaneously recorded in the same observation volume for two different species labeled with distinguishable fluorophores. The fraction of double-labeled and hence associated species is derived from the cross-correlation function between the two fluorescences (fig. 3.4B). 

As in most other experimental techniques, there is also a constant development and improvement involved in FCS which broadens its range, applicability, and accuracy. These developments resulted in advances such as dual-focus FCS, total internal reflection FCS, and STED-FCS which will be discussed in this section. Apart from this progress, a multitude of other variations have been reported which cannot be covered within this book chapter. These include FCS with two-photon excitation [57-61], spatial fluorescence cross-correlation spectroscopy (FCCS) which can be used to investigate microflows [62], dual-color FCCS to correlate... 

Schwille P, Meyer-Almes FJ, Rigler R (1997) Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution. Biophys J 72 (4) 1878-1886... 

Jahnz, M., Schwille, P. An ultrasensitive site-specific DNA recombination assay based on dual-color fluorescence cross-correlation spectroscopy. Nucleic Acids Res. 33, e60 (2005)... 

Rigler, R. Mets, U. Widengren, J. et al. Fluorescence Correlation Spectroscopy with high Count Rate and low Background - Analysis of Translational Diffusion. Eur. Biophys. J. Biophys. Lett. 1993, 22, 169-175. Bacia, K. Majoul, I. V. Schwille, P. Probing the endocytic pathway in live cells using dual-color fluorescence cross-correlation analysis. Biophys. J. 2002, , 1184-1193. 

Leutenegger, M. Blom, H. Widengren, J. Eggeling, C. GOsch, M. Leitgeb, R. A. Lasser T. Dual-color Total Internal Reflection Fluorescence cross-Correlation Spectroscopy. J. Biomed. Opt. 2006, II, accepted for publication. 

DC-FCCS Dual-color fluorescence cross-correlation spectroscopy DLS Dynamic light scattering... 

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