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

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

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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms
  • Biological Transport
  • Cells / metabolism
  • Cells / ultrastructure*
  • Efficiency
  • Fluorescence Resonance Energy Transfer / methods*
  • Green Fluorescent Proteins / metabolism
  • HeLa Cells
  • Humans
  • Microscopy, Fluorescence / methods
  • Mitochondria / metabolism
  • Mitochondria / physiology
  • Observation / methods
  • Photobleaching
  • Proteins / analysis
  • Proteins / metabolism*
  • Single-Cell Analysis / methods
  • Time Factors
  • Time-Lapse Imaging / methods

Substances

  • Proteins
  • Green Fluorescent Proteins