Fluorescence cross-correlation spectroscopy

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. 

Dittrich, P.S., Schwille, R, Spatial two-photon fluorescence cross-correlation spectroscopy for controlling molecular transport in microfluidic structures. Anal. Chem. 2002, 74(17), 4472-4479. 

Reviews listed in Further Reading provide excellent introductions to PCS. Related techniques have been developed to detect other molecular properties. These properties include fluorescence cross-correlation spectroscopy (FCCS) (28) to detect codiffusing fluorophores and photon-counting histograms (PCH) (29), or fluorescence intensity distribution analysis (FIDA) (29) to distinguish fluorescent species according to their brightness. 

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). 

Foldes-Papp, Z and Rigler, R, Quantitative two-color fluorescence cross-correlation spectroscopy in the analysis of polymerase chain reaction. Biological Chemistry3S2 (2001) 473-478. 

LeCaptain, DJ and Van Orden, A, Two-beam fluorescence cross-correlation spectroscopy in an electrophoretic mobflity shift assay. Analytical Chemistry 74 (2002) 1171 1176. 

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... 

Yordanov S, Best A, Butt H-J, Koynov K (2009) Direct studies of liquid flows near solid surfaces by total internal reflection fluorescence cross-correlation spectroscopy. Opt Express 17(23) 21149-21158... 

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)... 

Weidemann, T. Wachsmuth, M. Tewes, M. Rippe, K. Langowski, J. Analysis of Ligand Binding by Two-Colour Fluorescence Cross-Correlation Spectroscopy. Single Mol. 2002, 3, 49-61. 

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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