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Journal of Biomedical Optics • Open Access

Physiological fluorescence lifetime imaging microscopy improves Förster resonance energy transfer detection in living cells
Author(s): Ching-Wei Chang; Mei Wu; Sofia D. Merajver; Mary-Ann Mycek

Paper Abstract

Accurate, unambiguous detection of molecular interactions in living cells via measurements of Förster (or fluorescence) resonance energy transfer (FRET) events is experimentally challenging. We develop and apply a physiological fluorescence lifetime imaging microscopy (physiological FLIM) system to significantly improve FRET detection in living cells. Multiple positive and negative cellular controls are implemented to validate the experimental method developed. FLIM measurement techniques were found to remove fluorescence intensity-based artifacts, resulting in a seven-fold improvement in fluorescence measurement precision. The addition of cellular environmental controls, including both temperature and CO2 stabilization, for physiological FLIM eliminates nonspecific FRET in the live-cell system studied. Overall, only physiological FLIM results in statistically significant results that clearly indicated the presence of specific molecular interactions in the live-cell system. This approach can be applied generally to improve the accuracy and precision of FRET measurements in living cells.

Paper Details

Date Published: 1 November 2009
PDF: 3 pages
J. Biomed. Opt. 14(6) 060502 doi: 10.1117/1.3257254
Published in: Journal of Biomedical Optics Volume 14, Issue 6
Show Author Affiliations
Ching-Wei Chang, Univ. of Michigan (United States)
Mei Wu, Univ. of Michigan (United States)
Sofia D. Merajver, Univ. of Michigan (United States)
Mary-Ann Mycek, Univ. of Michigan (United States)

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