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          Institute: MPI für biophysikalische Chemie     Collection: Abteilungsunabhängige Arbeitsgruppen     Display Documents

ID: 16648.0, MPI für biophysikalische Chemie / Abteilungsunabhängige Arbeitsgruppen
Two-photon fluorescence coincidence analysis: Rapid measurements of enzyme kinetics
Authors:Heinze, K. G.; Rarbach, M.; Jahnz, M.; Schwille, P.
Date of Publication (YYYY-MM-DD):2002-09
Title of Journal:Biophysical Journal
Issue / Number:3
Start Page:1671
End Page:1681
Review Status:Peer-review
Audience:Not Specified
Abstract / Description:Dual-color fluorescence cross-correlation analysis is a powerful tool for probing interactions of different fluorescently labeled molecules in aqueous solution. The concept is the selective observation of coordinated spontaneous fluctuations in two separate detection channels that unambiguously reflect the existence of physical or chemical linkages among the different fluorescent species. It has previously been shown that the evaluation of cross-correlation amplitudes, i.e., coincidence factors, is sufficient to extract essential information about the kinetics of formation or cleavage of chemical or physical bonds. Confocal fluorescence coincidence analysis (CFCA) (Winkler et al., Proc. Natl. Acad. Sci. U.S.A. 96:13751378, 1999) emphasizes short analysis times and simplified data evaluation and is thus particularly useful for screening applications or measurements on live cells where small illumination doses need to be applied. The recent use of two-photon fluorescence excitation has simplified dual- or multicolor measurements by enabling the simultaneous excitation of largely different dye molecules by a single infra-red laser line (Heinze et al., Proc. Natl. Acad. Sci. U.S.A. 97:10377- 10382, 2000). It is demonstrated here that a combination of CFCA with two-photon excitation allows for minimization of analysis times for multicomponent systems down to some hundreds of milliseconds, while preserving all known advantages of two- photon excitation. By introducing crucial measurement parameters, experimental limits for the reduction of sampling times are discussed for the special case of distinguishing positive from negative samples in an endonucleolytic cleavage assay.
Comment of the Author/Creator:Date: 2002, SEP
External Publication Status:published
Document Type:Article
Communicated by:N. N.
Affiliations:MPI für biophysikalische Chemie/AG Petra Schwille
External Affiliations:DIREVO Biotech AG, D-50829 Cologne, Germany
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