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

Wide-field fluorescence lifetime imaging with multi-anode detectors.

Author information

1
Multidimensional Microscopy and Cellular Diagnostics, Medical Faculty, Otto-von-Guericke-University, Magdeburg, Germany.

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.

PMID:
24108639
DOI:
10.1007/978-1-62703-649-8_20
[Indexed for MEDLINE]

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