|
Fluorescence cross correlation spectroscopy (FCCS) is now widely used as a powerful technique to analyze molecular
interactions in vitro and also in vivo. This method basically requires two laser excitations for two target molecules
distinctly labeled with fluorophores. Coincidence of the two molecules can be analyzed at detectors, corresponding to
each emission wavelength. However, due to spectral overlap of the two fluorophores, a cross-talk signal causes critical
difficulties of data assessments by false positive cross correlations. To overcome this problem, we have developed a new
cross-talk free FCCS system; the excitation laser pulses are switched alternatively by modulating of a conventional
Acousto-optic tunable filter (AOTF) in the excitation laser unit. Alternatively, the specimen is illuminated with two
different wavelengths, and cross correlation can be correctly calculated by this switching state, and therefore, we can
eliminate spectra cross-talk. In this report, we show system outlines and demonstrate the feasibility of the switching
FCCS for enzymatic cleavage of proteins in living cells. A fusion protein of two fluorophores (EGFP and mRFP)
inserted with cleavage site of caspase3 was expressed in HeLa cells, and the cleaving process during apoptotic cell death
was monitored. We measured a relative cross correlation amplitude obtained by FCCS with and without switching
system. More significant changing of relative cross correlation amplitude by protein cleavage was detected with
switching than that without switching. This result indicates that the switching FCCS measurement can exclude cross-talk
signal. Hence, the switching FCCS enables a reliable analysis of molecular interaction in living cells with higher
sensitivity than FCCS without switching.
|
