@ARTICLE{Horrocks2012-ct,
  title     = "Single molecule fluorescence under conditions of fast flow",
  author    = "Horrocks, Mathew H and Li, Haitao and Shim, Jung-Uk and
               Ranasinghe, Rohan T and Clarke, Richard W and Huck, Wilhelm T S
               and Abell, Chris and Klenerman, David",
  abstract  = "We have experimentally determined the optimal flow velocities to
               characterize or count single molecules by using a simple
               microfluidic device to perform two-color coincidence detection
               (TCCD) and single pair F{\"o}rster resonance energy transfer
               (spFRET) using confocal fluorescence spectroscopy on molecules
               traveling at speeds of up to 10 cm s(-1). We show that flowing
               single fluorophores at $\geq$0.5 cm s(-1) reduces the
               photophysical processes competing with fluorescence, enabling
               the use of high excitation irradiances to partially compensate
               for the short residence time within the confocal volume (10-200
               $\mu$s). Under these conditions, the data acquisition rate can
               be increased by a maximum of 38-fold using TCCD at 5 cm s(-1) or
               18-fold using spFRET at 2 cm s(-1), when compared with
               diffusion. While structural characterization requires more
               photons to be collected per event and so necessitates the use of
               slower speeds (2 cm s(-1) for TCCD and 1 cm s(-1) for spFRET), a
               considerable enhancement in the event rate could still be
               obtained (33-fold for TCCD and 16-fold for spFRET). Using flow
               under optimized conditions, analytes could be rapidly quantified
               over a dynamic range of up to 4 orders of magnitude by direct
               molecule counting; a 50 fM dual-labeled model sample can be
               detected with 99.5\% statistical confidence in around 8 s using
               TCCD and a flow velocity of 5 cm s(-1).",
  journal   = "Anal. Chem.",
  publisher = "American Chemical Society (ACS)",
  volume    =  84,
  number    =  1,
  pages     = "179--185",
  month     =  jan,
  year      =  2012,
  language  = "en"
}

{"_id":"vhdMjbRqLCGuaDmec","bibbaseid":"horrocks-li-shim-ranasinghe-clarke-huck-abell-klenerman-singlemoleculefluorescenceunderconditionsoffastflow-2012","author_short":["Horrocks, M. H","Li, H.","Shim, J.","Ranasinghe, R. T","Clarke, R. W","Huck, W. T S","Abell, C.","Klenerman, D."],"bibdata":{"bibtype":"article","type":"article","title":"Single molecule fluorescence under conditions of fast flow","author":[{"propositions":[],"lastnames":["Horrocks"],"firstnames":["Mathew","H"],"suffixes":[]},{"propositions":[],"lastnames":["Li"],"firstnames":["Haitao"],"suffixes":[]},{"propositions":[],"lastnames":["Shim"],"firstnames":["Jung-Uk"],"suffixes":[]},{"propositions":[],"lastnames":["Ranasinghe"],"firstnames":["Rohan","T"],"suffixes":[]},{"propositions":[],"lastnames":["Clarke"],"firstnames":["Richard","W"],"suffixes":[]},{"propositions":[],"lastnames":["Huck"],"firstnames":["Wilhelm","T","S"],"suffixes":[]},{"propositions":[],"lastnames":["Abell"],"firstnames":["Chris"],"suffixes":[]},{"propositions":[],"lastnames":["Klenerman"],"firstnames":["David"],"suffixes":[]}],"abstract":"We have experimentally determined the optimal flow velocities to characterize or count single molecules by using a simple microfluidic device to perform two-color coincidence detection (TCCD) and single pair Förster resonance energy transfer (spFRET) using confocal fluorescence spectroscopy on molecules traveling at speeds of up to 10 cm s(-1). We show that flowing single fluorophores at $≥$0.5 cm s(-1) reduces the photophysical processes competing with fluorescence, enabling the use of high excitation irradiances to partially compensate for the short residence time within the confocal volume (10-200 $μ$s). Under these conditions, the data acquisition rate can be increased by a maximum of 38-fold using TCCD at 5 cm s(-1) or 18-fold using spFRET at 2 cm s(-1), when compared with diffusion. While structural characterization requires more photons to be collected per event and so necessitates the use of slower speeds (2 cm s(-1) for TCCD and 1 cm s(-1) for spFRET), a considerable enhancement in the event rate could still be obtained (33-fold for TCCD and 16-fold for spFRET). Using flow under optimized conditions, analytes could be rapidly quantified over a dynamic range of up to 4 orders of magnitude by direct molecule counting; a 50 fM dual-labeled model sample can be detected with 99.5% statistical confidence in around 8 s using TCCD and a flow velocity of 5 cm s(-1).","journal":"Anal. Chem.","publisher":"American Chemical Society (ACS)","volume":"84","number":"1","pages":"179–185","month":"January","year":"2012","language":"en","bibtex":"@ARTICLE{Horrocks2012-ct,\n title = \"Single molecule fluorescence under conditions of fast flow\",\n author = \"Horrocks, Mathew H and Li, Haitao and Shim, Jung-Uk and\n Ranasinghe, Rohan T and Clarke, Richard W and Huck, Wilhelm T S\n and Abell, Chris and Klenerman, David\",\n abstract = \"We have experimentally determined the optimal flow velocities to\n characterize or count single molecules by using a simple\n microfluidic device to perform two-color coincidence detection\n (TCCD) and single pair F{\\\"o}rster resonance energy transfer\n (spFRET) using confocal fluorescence spectroscopy on molecules\n traveling at speeds of up to 10 cm s(-1). We show that flowing\n single fluorophores at $\\geq$0.5 cm s(-1) reduces the\n photophysical processes competing with fluorescence, enabling\n the use of high excitation irradiances to partially compensate\n for the short residence time within the confocal volume (10-200\n $\\mu$s). Under these conditions, the data acquisition rate can\n be increased by a maximum of 38-fold using TCCD at 5 cm s(-1) or\n 18-fold using spFRET at 2 cm s(-1), when compared with\n diffusion. While structural characterization requires more\n photons to be collected per event and so necessitates the use of\n slower speeds (2 cm s(-1) for TCCD and 1 cm s(-1) for spFRET), a\n considerable enhancement in the event rate could still be\n obtained (33-fold for TCCD and 16-fold for spFRET). Using flow\n under optimized conditions, analytes could be rapidly quantified\n over a dynamic range of up to 4 orders of magnitude by direct\n molecule counting; a 50 fM dual-labeled model sample can be\n detected with 99.5\\% statistical confidence in around 8 s using\n TCCD and a flow velocity of 5 cm s(-1).\",\n journal = \"Anal. Chem.\",\n publisher = \"American Chemical Society (ACS)\",\n volume = 84,\n number = 1,\n pages = \"179--185\",\n month = jan,\n year = 2012,\n language = \"en\"\n}\n\n","author_short":["Horrocks, M. H","Li, H.","Shim, J.","Ranasinghe, R. T","Clarke, R. W","Huck, W. T S","Abell, C.","Klenerman, D."],"bibbaseid":"horrocks-li-shim-ranasinghe-clarke-huck-abell-klenerman-singlemoleculefluorescenceunderconditionsoffastflow-2012","role":"author","urls":{},"metadata":{"authorlinks":{}}},"bibtype":"article","biburl":"https://bibbase.org/f/SnzpCnctps9apFAde/MyRefs.bib","dataSources":["P4DL9yCkBYWMTxTMC","6RizfyANYFwZbeYCF","hos8GKTiSWJrJ8p57","jYJBHEqqdSqSw3E7J","DrBTLzMk52FTGMmNJ"],"keywords":[],"search_terms":["single","molecule","fluorescence","under","conditions","fast","flow","horrocks","li","shim","ranasinghe","clarke","huck","abell","klenerman"],"title":"Single molecule fluorescence under conditions of fast flow","year":2012}