Dual color FCCS

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... 

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). 

This study demonstrated dual-color single molecule FCCS measurements based on epi-illumination TIR. This TIR-FCCS concept offers distinct advantages to confocal FCCS for coincidence assays at solid/liquid surfaces, in particular by virtue of the much higher fluorescence collection efficiency and the high confinement of the excitation field at the surface. 

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

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