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Improving FRET accuracy with FLIM & environmental control

Mary-Ann Mycek and colleagues from the University of Michigan have used fluorescence lifetime imaging microscopy (FLIM) combined with environmental controls to increase the accuracy of detecting molecular interactions in living cells with FRET. They set out to examine the effect of controlling physiological parameters during FLIM detection of FRET when examining Ras Homology Protein C (RhoC) inactive-form interactions in living cells. This protein has been identified as a specific oncogene marker of aggressive breast cancers.

Using FLIM to detect FRET (rather than intensity-based methods) let the researchers remove intensity-based artifacts, improving fluorescence measurement seven-fold. And using a FLIM system that allowed temperature control and CO2 stabilization eliminated nonspecific FRET in the live cells they studied. The researchers report that the technique, called physiological FLIM, clearly and unambiguously showed the presence of specific molecular interactions in the cells they studied and that it should be generally applicable to various live cell systems.

See other FRET posts including "White light laser allows orange fluorescent protein to be useful acceptor FRET" here.

Research paper:
Ching-Wei Chang, Mei Wu, Sofia D. Merajver, and Mary-Ann Mycek. Physiological fluorescence lifetime imaging microscopy improves Förster resonance energy transfer detection in living cells. J. Biomed. Opt. Vol. 14, 060502 (Nov. 5, 2009)



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