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PLoS One. 2011 Feb 2;6(2):e15820. doi: 10.1371/journal.pone.0015820.

Wide-Field Multi-Parameter FLIM: long-term minimal invasive observation of proteins in living cells.

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  • 1Leibniz Institute for Neurobiology, Magdeburg, Germany.

Abstract

Time-domain Fluorescence Lifetime Imaging Microscopy (FLIM) is a remarkable tool to monitor the dynamics of fluorophore-tagged protein domains inside living cells. We propose a Wide-Field Multi-Parameter FLIM method (WFMP-FLIM) aimed to monitor continuously living cells under minimum light intensity at a given illumination energy dose. A powerful data analysis technique applied to the WFMP-FLIM data sets allows to optimize the estimation accuracy of physical parameters at very low fluorescence signal levels approaching the lower bound theoretical limit. We demonstrate the efficiency of WFMP-FLIM by presenting two independent and relevant long-term experiments in cell biology: 1) FRET analysis of simultaneously recorded donor and acceptor fluorescence in living HeLa cells and 2) tracking of mitochondrial transport combined with fluorescence lifetime analysis in neuronal processes.

PMID:
21311595
[PubMed - indexed for MEDLINE]
PMCID:
PMC3032730
Free PMC Article
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