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Imaging Lifetimes

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Perspectives on Fluorescence

Part of the book series: Springer Series on Fluorescence ((SS FLUOR,volume 17))

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Abstract

This chapter discusses the critical contributions of Gregorio Weber to the development of techniques to measure fluorescence lifetimes. The fluorescence lifetime is the average time required for a population of fluorophores in the excited state to decay to the ground state. Events in a fluorophore’s environment that influence the excited state can alter the lifetime, and this is measured using fluorescence lifetime imaging microscopy (FLIM). This chapter describes the application of FLIM to quantify Förster resonance energy transfer (FRET) between labeled proteins inside living cells. FRET is a non-radiative pathway through which a donor fluorophore in the excited state transfers energy to nearby acceptor molecules. The transfer of energy reduces the donor’s fluorescence lifetime, and this can be quantified by FLIM. Since energy transfer occurs through near-field electromagnetic interactions, it can only occur over a distance of 80 angstroms or less. Thus, FRET microscopy has become a valuable tool for investigating biochemical networks inside living cells. In this regard, Gregorio Weber recognized the importance of measuring the biological and physical properties of proteins as integrated systems. Here, proteins labeled with the genetically encoded fluorescent proteins (FPs) are used to demonstrate how FRET-FLIM enables robust and sensitive measurements of protein interactions inside living cells.

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Acknowledgements

This chapter is dedicated in memory of Dr. Robert (Bob) M. Clegg, a colleague of Gregorio Weber at the University of Illinois at Urbana-Champaign. In 2011, Bob presented a lecture entitled “History of trials, blunders, tribulations and finally success in the dark ages of fluorescence lifetime measurements” that contained many historical insights referenced here. The author thanks Drs. Yuansheng Sun and Shih-Chu (Jeff) Liao (ISS Inc., Champaign, IL) for their advice and technical help with FLIM, Michael Davidson (FSU) for providing plasmids encoding the FPs, and Dr. Jing Qi for excellent laboratory support.

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Authors and Affiliations

  1. Department of Cellular and Integrative Physiology, Indiana University School of Medicine, 635 Barnhill Dr., MS 333, Indianapolis, IN, 46202, USA

    Richard N. Day

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  1. Richard N. Day
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Correspondence to Richard N. Day .

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  1. John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii, USA

    David M. Jameson

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Day, R.N. (2016). Imaging Lifetimes. In: Jameson, D. (eds) Perspectives on Fluorescence. Springer Series on Fluorescence, vol 17. Springer, Cham. https://doi.org/10.1007/4243_2016_1

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