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Wide-Field Fluorescence Lifetime Imaging with Multi-anode Detectors

  • Roland Hartig
  • Yury Prokazov
  • Evgeny Turbin
  • Werner Zuschratter
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1076)

Abstract

Fluorescence lifetime imaging microscopy (FLIM) has become a powerful and widely used tool to monitor inter- and intramolecular dynamics of fluorophore-labeled proteins inside living cells.

Here, we present recent achievements in the construction of a positional sensitive wide-field single-photon counting detector system to measure fluorescence lifetimes in the time domain and demonstrate its usage in FRET applications.

The setup is based on a conventional fluorescence microscope equipped with synchronized short-pulse lasers that illuminate the entire field of view at minimal invasive intensities, thereby enabling long-term experiments of living cells. The system is capable to acquire single-photon counting images and measures directly the transfer rate of fast photophysical processes as, for instance, FRET, in which it can resolve complex fluorescence decay kinetics.

Key words

Time-correlated single-photon counting (TCSPC) Fluorescence lifetime imaging (FLIM) Förster resonance energy transfer (FRET) Positional sensitive photon multiplier Multichannel plate 

Notes

Acknowledgments

This work was supported by the following grants: DFG FOR 521 HA3498/1-3; BMBF “Quantum” VDI 13N10077 and DFG SFB 854 TPZ (W.Z.). The authors thank Thomas Kuner, Institute of Anatomy and Cell Biology, University of Heidelberg, Germany, Fernando Picazo and Juan Llopis, University of Castilla-La Mancha, Albacete, Spain for providing FRET constructs.

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Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Roland Hartig
    • 1
  • Yury Prokazov
    • 2
  • Evgeny Turbin
    • 2
  • Werner Zuschratter
    • 2
  1. 1.Multidimensional Microscopy and Cellular Diagnostics, Medical FacultyOtto-von-Guericke-UniversityMagdeburgGermany
  2. 2.Special Laboratory Electron- and Laserscanning MicroscopyLeibniz Institute for Neurobiology (LIN)MagdeburgGermany

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