Physiological fluorescence lifetime imaging microscopy improves Förster resonance energy transfer detection in living cells

Ching-Wei Chang; Mei Wu; Sofia D. Merajver M.D.; Mary-Ann Mycek

doi:10.1117/1.3257254

1 November 2009 Physiological fluorescence lifetime imaging microscopy improves Förster resonance energy transfer detection in living cells

Ching-Wei Chang, Mei Wu, Sofia D. Merajver M.D., Mary-Ann Mycek

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Journal of Biomedical Optics, Vol. 14, Issue 6, 060502 (November 2009). https://doi.org/10.1117/1.3257254

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.

©(2009) Society of Photo-Optical Instrumentation Engineers (SPIE)

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Ching-Wei Chang, Mei Wu, Sofia D. Merajver M.D., and Mary-Ann Mycek "Physiological fluorescence lifetime imaging microscopy improves Förster resonance energy transfer detection in living cells," Journal of Biomedical Optics 14(6), 060502 (1 November 2009). https://doi.org/10.1117/1.3257254

Published: 1 November 2009

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