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Standardization and Quality Assurance in Fluorescence Measurements II pp 199–212Cite as

Single Molecule Spectroscopy: Instrumentation and Multiparameter Detection

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Part of the book series: Springer Series on Fluorescence ((SS FLUOR,volume 6))

Abstract

Single molecule spectroscopy has become a standard tool in analytics. It is possible not only to detect single molecule fluorescence, but to characterize individual fluorophores by their excitation and emission spectra, quantum yield, anisotropy, and fluorescence lifetime. Dedicated setups based on pulsed excitation, confocal detection, and intelligent data processing allow to simultaneously record and analyze several properties of a single dye fluorescence. A substantial advantage of single molecule measurements is the detection and characterization of subpopulations even in heterogeneous mixtures. A popular example is the analysis of macromolecules labeled with fluorescent donor and acceptor molecules capable of Förster resonance energy transfer (FRET). As an example, we show a multiparameter analysis of single molecule measurements of recombinant bovine liver rhodanese labeled with Alexa 488 and Alexa 594. Native and unfolded states can be clearly resolved and exhibit distinct FRET efficiencies, donor lifetimes, and anisotropies. In FRET measurements, pulsed interleaved excitation can be applied to distinguish molecules with and without fluorescent acceptor, as illustrated on a peptide labeled with Alexa 555 and Alexa 647.

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Authors and Affiliations

  1. PicoQuant GmbH, Rudower Chaussee 29, 12489, Berlin, Germany

    Volker Buschmann & Felix Koberling

  2. Universität Zürich, Biochemisches Institut, Winterthurerstrasse 190, 8057, Zürich, Switzerland

    Benjamin Schuler, Frank Hillger & Daniel Nettels

Authors
  1. Volker Buschmann
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  2. Felix Koberling
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  3. Benjamin Schuler
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  4. Frank Hillger
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  5. Daniel Nettels
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Corresponding authors

Correspondence to Volker Buschmann or Benjamin Schuler .

Editor information

Ute Resch-Genger

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© 2008 Springer-Verlag Berlin Heidelberg

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Buschmann, V., Koberling, F., Schuler, B., Hillger, F., Nettels, D. (2008). Single Molecule Spectroscopy: Instrumentation and Multiparameter Detection. In: Resch-Genger, U. (eds) Standardization and Quality Assurance in Fluorescence Measurements II. Springer Series on Fluorescence, vol 6. Springer, Berlin, Heidelberg. https://doi.org/10.1007/4243_2008_051

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