High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence

Jin Chen; Ravindra V. Dalal; Alexey N. Petrov; Albert Tsai; Seán E. O'Leary; Karen Chapin; Janice Cheng; Mark Ewan; Pei Lin Hsiung; Paul Lundquist; Stephen W. Turner; David R. Hsu; Joseph D. Puglisi

doi:10.1073/pnas.1315735111

High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence

Jin Chen, Ravindra V. Dalal, Alexey N. Petrov, Albert Tsai, Seán E. O'Leary, Karen Chapin, Janice Cheng, Mark Ewan, Pei Lin Hsiung, Paul Lundquist, Stephen W. Turner, David R. Hsu, Joseph D. Puglisi

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98 Scopus citations

Abstract

Zero-mode waveguides provide a powerful technology for studying single-molecule real-time dynamics of biological systems at physiological ligand concentrations. We customized a commercial zero-mode waveguide-based DNA sequencer for use as a versatile instrument for single-molecule fluorescence detection and showed that the system provides long fluorophore lifetimes with good signal to noise and low spectral cross-talk. We then used a ribosomal translation assay to show real-time fluidic delivery during data acquisition, showing it is possible to follow the conformation and composition of thousands of single biomolecules simultaneously through four spectral channels. This instrument allows high-throughput multiplexed dynamics of single-molecule biological processes over long timescales. The instrumentation presented here has broad applications to single-molecule studies of biological systems and is easily accessible to the biophysical community.

Original language	English (US)
Pages (from-to)	664-669
Number of pages	6
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	111
Issue number	2
DOIs	https://doi.org/10.1073/pnas.1315735111
State	Published - 2014
Externally published	Yes

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Chen, J., Dalal, R. V., Petrov, A. N., Tsai, A., O'Leary, S. E., Chapin, K., Cheng, J., Ewan, M., Hsiung, P. L., Lundquist, P., Turner, S. W., Hsu, D. R., & Puglisi, J. D. (2014). High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence. Proceedings of the National Academy of Sciences of the United States of America, 111(2), 664-669. https://doi.org/10.1073/pnas.1315735111

Chen, J, Dalal, RV, Petrov, AN, Tsai, A, O'Leary, SE, Chapin, K, Cheng, J, Ewan, M, Hsiung, PL, Lundquist, P, Turner, SW, Hsu, DR & Puglisi, JD 2014, 'High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence', Proceedings of the National Academy of Sciences of the United States of America, vol. 111, no. 2, pp. 664-669. https://doi.org/10.1073/pnas.1315735111

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title = "High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence",

abstract = "Zero-mode waveguides provide a powerful technology for studying single-molecule real-time dynamics of biological systems at physiological ligand concentrations. We customized a commercial zero-mode waveguide-based DNA sequencer for use as a versatile instrument for single-molecule fluorescence detection and showed that the system provides long fluorophore lifetimes with good signal to noise and low spectral cross-talk. We then used a ribosomal translation assay to show real-time fluidic delivery during data acquisition, showing it is possible to follow the conformation and composition of thousands of single biomolecules simultaneously through four spectral channels. This instrument allows high-throughput multiplexed dynamics of single-molecule biological processes over long timescales. The instrumentation presented here has broad applications to single-molecule studies of biological systems and is easily accessible to the biophysical community.",

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year = "2014",

doi = "10.1073/pnas.1315735111",

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AU - Chen, Jin

AU - Dalal, Ravindra V.

AU - Petrov, Alexey N.

AU - Tsai, Albert

AU - O'Leary, Seán E.

AU - Chapin, Karen

AU - Cheng, Janice

AU - Ewan, Mark

AU - Hsiung, Pei Lin

AU - Lundquist, Paul

AU - Turner, Stephen W.

AU - Hsu, David R.

AU - Puglisi, Joseph D.

PY - 2014

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AB - Zero-mode waveguides provide a powerful technology for studying single-molecule real-time dynamics of biological systems at physiological ligand concentrations. We customized a commercial zero-mode waveguide-based DNA sequencer for use as a versatile instrument for single-molecule fluorescence detection and showed that the system provides long fluorophore lifetimes with good signal to noise and low spectral cross-talk. We then used a ribosomal translation assay to show real-time fluidic delivery during data acquisition, showing it is possible to follow the conformation and composition of thousands of single biomolecules simultaneously through four spectral channels. This instrument allows high-throughput multiplexed dynamics of single-molecule biological processes over long timescales. The instrumentation presented here has broad applications to single-molecule studies of biological systems and is easily accessible to the biophysical community.

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High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence

Abstract

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