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.
Original language | English |
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Article number | e15820 |
Number of pages | 12 |
Journal | PLoS ONE |
Volume | 6 |
Issue number | 2 |
DOIs | |
Publication status | Published - 2011 |
Keywords
- lifetime imaging microscopy
- green fluorescent protein
- resonance energy-transfer
- single molecules
- fret microscopy
- global analysis
- excitation
- resolution
- transport
- oxygen