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MFD-PIE and PIE-FI: Ways to Extract More Information with TCSPC

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Advanced Photon Counting

Part of the book series: Springer Series on Fluorescence ((SS FLUOR,volume 15))

Abstract

Pulsed interleaved excitation (PIE) is the method of fast alternation of pulsed lasers for quasi-simultaneous observation of fluorophores with different spectral properties. PIE was originally introduced to enable artifact-free fluorescence cross-correlation measurements, while first experiments with alternating laser excitation (ALEX) used the dual excitation of donor and acceptor for single-pair Förster resonance energy transfer (spFRET). In this article, we will review the benefit of PIE for spFRET experiments with multiparameter fluorescence detection (MFD). The direct probing of the acceptor fluorophore in PIE increases the robustness of the quantitative MFD analysis and extends it to even more parameters.

Recently, PIE has been combined with commonly used fluorescence fluctuation imaging techniques such as raster image correlation spectroscopy (RICS) and the number and brightness analysis (N&B). We highlight how PIE improves these methods, and how artifacts in the analysis can be avoided. Similar to PIE-FCS, quantitative cross-correlation raster image correlation spectroscopy (ccRICS) is greatly simplified. Additionally, the lifetime information can be used to further increase the contrast and sensitivity of the method with raster lifetime image correlation spectroscopy (RLICS).

*These authors contributed equally to this work.

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Acknowledgments

We thank Dr. Jelle Hendrix for the data on PIE-FI and Carolina Sanchéz Rico, Dr. Lisa Warner, and Prof. Dr. Michael Sattler for providing the U2AF65 protein. We gratefully acknowledge the financial support of the Deutsche Forschungsgemeinschaft through the Excellence Cluster Nanosystems Initiative Munich (NIM) and the Collaborate Research Center (SFB1035) and the Ludwig-Maximilians-University Munich (LMUinnovativ BioImaging Network).

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    Authors and Affiliations

    1. Department of Chemistry and Center for Nano Science, Ludwig-Maximilians-Universität München, Butenandtstr. 11, 81377, Munich, Germany

      Anders Barth, Lena Voith von Voithenberg & Don C. Lamb

    2. Center for Integrated Protein Science, Munich, Germany

      Anders Barth, Lena Voith von Voithenberg & Don C. Lamb

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    1. Anders Barth
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    2. Lena Voith von Voithenberg
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    3. Don C. Lamb
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    Correspondence to Don C. Lamb .

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    Editors and Affiliations

    1. J. Heyrovsky Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, Prague 8, Czech Republic

      Peter Kapusta

    2. PicoQuant GmbH, Berlin, Germany

      Michael Wahl

    3. PicoQuant GmbH, Berlin, Germany

      Rainer Erdmann

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    Barth, A., von Voithenberg, L.V., Lamb, D.C. (2014). MFD-PIE and PIE-FI: Ways to Extract More Information with TCSPC. In: Kapusta, P., Wahl, M., Erdmann, R. (eds) Advanced Photon Counting. Springer Series on Fluorescence, vol 15. Springer, Cham. https://doi.org/10.1007/4243_2014_66

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