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Proceedings Paper

Cross-talk-free fluorescence cross-correlation spectroscopy by the switching method
Author(s): Junichi Nishimura; Yasuo Takahashi; Masataka Kinjo; Atsushi Miyawaki
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Paper Abstract

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.

Paper Details

Date Published: 14 February 2007
PDF: 9 pages
Proc. SPIE 6444, Ultrasensitive and Single-Molecule Detection Technologies II, 64440I (14 February 2007); doi: 10.1117/12.699992
Show Author Affiliations
Junichi Nishimura, Olympus Corp. (Japan)
Yasuo Takahashi, Olympus Corp. (Japan)
Masataka Kinjo, Hokkaido Univ. (Japan)
Atsushi Miyawaki, RIKEN (Japan)

Published in SPIE Proceedings Vol. 6444:
Ultrasensitive and Single-Molecule Detection Technologies II
Jorg Enderlein; Zygmunt K. Gryczynski, Editor(s)

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