Two- and Three-color fluorescence cross-correlation spectroscopy with single laser wavelength excitation
Hwang L.C., Gösch M., Lasser T., Wohland T.
Two-color fluorescence cross-correlation spectroscopy had been recently performed with a single laser wavelength excitation (SW-FcCS) [1]. Using only one laser source simplifies the FcCS setup as there is no need of aligning two laser beams to the same confocal volume. However, this method was limited to the use of long Stokes-shift dyes such as tandem dyes and quantum dots to reduce cross-talk between the two detection channels.
Here, we show that SW-FcCS can be performed even with spectrally similar organic dyes such as fluorescein and tetramethylrhodamine. To investigate the applicability of SW-FcCS for binding studies, limits for the measurement of dissociation constants in dependence on sample concentration, sample purity, quenching and spectral cross-talk between the different detection channels were determined. This method is further extended to three-color SW-FcCS binding measurements with biotin-streptavidin using a combination of dyes, fluorescein, R-phycoerythrin and Alexa Fluor 647-R-phycoerythrin. This extends the potential application of SW-FcCS to detection of biomolecular systems of multiple binding sites or partners.
[1] Hwang L.C., Wohland T., Chem Phys Chem 5: 549-551 (2004)