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  Two-photon fluorescence coincidence analysis: Rapid measurements of enzyme kinetics

Heinze, K. G., Rarbach, M., Jahnz, M., & Schwille, P. (2002). Two-photon fluorescence coincidence analysis: Rapid measurements of enzyme kinetics. Biophysical Journal, 83(3), 1671-1681. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1302263/pdf/12202390.pdf.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0012-F315-2 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0028-EC7F-8
Genre: Journal Article

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Heinze, K. G.1, Author              
Rarbach, M.1, Author              
Jahnz, M.1, Author              
Schwille, P.2, Author
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1Research Group of Experimental Biophysics, MPI for biophysical chemistry, Max Planck Society, ou_578554              
2Max Planck Society, ou_persistent13              

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 Abstract: 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.

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Language(s): eng - English
 Dates: 2002-09
 Publication Status: Published in print
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 Rev. Method: Peer
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Title: Biophysical Journal
Source Genre: Journal
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Pages: - Volume / Issue: 83 (3) Sequence Number: - Start / End Page: 1671 - 1681 Identifier: -