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Time-resolved fluorescence microscopy

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Abstract

In fluorescence microscopy, the fluorescence emission can be characterised not only by intensity and position, but also by lifetime, polarization and wavelength. Fluorescence lifetime imaging (FLIM) can report on photophysical events that are difficult or impossible to observe by fluorescence intensity imaging, and time-resolved fluorescence anisotropy imaging (TR-FAIM) can measure the rotational mobility of a fluorophore in its environment. We compare different FLIM methods: a chief advantage of wide-field time-gating and phase modulation methods is the speed of acquisition whereas for time-correlated single photon counting (TCSPC) based confocal scanning it is accuracy in the fluorescence decay. FLIM has been used to image interactions between proteins such as receptor oligomerisation and to reveal protein phosphorylation by detecting fluorescence resonance energy transfer (FRET). In addition, FLIM can also probe the local environment of fluorophores, reporting, for example, on the local pH, refractive index, ion or oxygen concentration without the need for ratiometric measurements.

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Authors and Affiliations

  1. Department of Physics, King’s College London, Strand, London, UK, WC2R 2LS

    Klaus Suhling

  2. Photonics Group, Department of Physics, Imperial College London, South Kensington Campus, London, UK, SW7 2AZ

    Paul M. W. French

  3. Department of Chemistry, Imperial College London, South Kensington Campus, London, UK, SW7 2AZ

    David Phillips

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  1. Klaus Suhling
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  2. Paul M. W. French
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Correspondence to Klaus Suhling.

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Dedicated to Professor Hiroshi Masuhara on the occasion of his 60th birthday

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Suhling, K., French, P.M.W. & Phillips, D. Time-resolved fluorescence microscopy. Photochem Photobiol Sci 4, 13–22 (2005). https://doi.org/10.1039/b412924p

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