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Implementation and Application of Pulsed Interleaved Excitation for Dual-Color FCS and RICS

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Fluorescence Spectroscopy and Microscopy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1076))

Abstract

Pulsed interleaved excitation (PIE) employs pulsed laser sources that are interleaved such that differentially colored fluorophores can be measured or imaged quasi-simultaneously in the absence of spectral crosstalk. PIE improves the robustness and reduces data analysis complexity of many fluorescence techniques, such as fluorescence cross-correlation spectroscopy (FCCS) and raster image cross-correlation spectroscopy (ccRICS), two methods used for quantitative investigation of molecular interactions in vitro and in living cells. However, as PIE is most often used for fluorescence fluctuation spectroscopy and burst analysis experiments and utilizes time-correlated single-photon counting detection and advanced optoelectronics, it has remained a technique that is mostly used by specialized single-molecule research groups. This protocols chapter provides an accessible overview of PIE for anyone considering implementing the method on a homebuilt or commercial microscope. We give details on the instrumentation, data collection and analysis software, on how to properly set up and align a PIE microscope, and finally, on how to perform proper dual-color FCS and RICS experiments.

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Acknowledgements

J.H. wishes to acknowledge the Research Foundation Flanders for a postdoctoral fellowship and travel grant. D.C.L. gratefully acknowledges the financial support of the Deutsche Forschungsgemeinschaft through the SFB1032 and the Nanoinitiative Munich (NIM) and of the LMU via the Center for NanoScience (CeNS) and the LMUinnovativ BioImaging Network. We thank Dr. Matthias Höller, Dr. Volodymyr Kudryavtsev, Dipl. Chem. Waldemar Schrimpf and Dr. Martin Sikor for helping develop the data acquisition and analysis software. Dr. Nick Smisdom and Prof. Marcel Ameloot (Hasselt University, Belgium) are thanked for fruitful discussions.

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Hendrix, J., Lamb, D.C. (2014). Implementation and Application of Pulsed Interleaved Excitation for Dual-Color FCS and RICS. In: Engelborghs, Y., Visser, A. (eds) Fluorescence Spectroscopy and Microscopy. Methods in Molecular Biology, vol 1076. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-649-8_30

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  • DOI: https://doi.org/10.1007/978-1-62703-649-8_30

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-648-1

  • Online ISBN: 978-1-62703-649-8

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