Wide-field fluorescence lifetime imaging with multi-anode detectors

Methods Mol Biol. 2014:1076:457-80. doi: 10.1007/978-1-62703-649-8_20.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Electrodes
  • Fluorescence Resonance Energy Transfer*
  • Fluorescence*
  • Kinetics
  • Microscopy, Fluorescence / methods*
  • Optical Imaging*
  • Photons
  • Proteins / analysis
  • Proteins / chemistry

Substances

  • Proteins