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Monitoring Biosensor Activity in Living Cells with Fluorescence Lifetime Imaging Microscopy
Department of Cellular and Integrative Physiology, Indiana University School of Medicine, 635 Barnhill Dr., MS 333, Indianapolis, IN 46202, USA
* Author to whom correspondence should be addressed.
Received: 29 September 2012; in revised form: 27 October 2012 / Accepted: 1 November 2012 / Published: 7 November 2012
Abstract: Live-cell microscopy is now routinely used to monitor the activities of the genetically encoded biosensor proteins that are designed to directly measure specific cell signaling events inside cells, tissues, or organisms. Most fluorescent biosensor proteins rely on Förster resonance energy transfer (FRET) to report conformational changes in the protein that occur in response to signaling events, and this is commonly measured with intensity-based ratiometric imaging methods. An alternative method for monitoring the activities of the FRET-based biosensor proteins is fluorescence lifetime imaging microscopy (FLIM). FLIM measurements are made in the time domain, and are not affected by factors that commonly limit intensity measurements. In this review, we describe the use of the digital frequency domain (FD) FLIM method for the analysis of FRET signals. We illustrate the methods necessary for the calibration of the FD FLIM system, and demonstrate the analysis of data obtained from cells expressing “FRET standard” fusion proteins. We then use the FLIM-FRET approach to monitor the changes in activities of two different biosensor proteins in specific regions of single living cells. Importantly, the factors required for the accurate determination and reproducibility of lifetime measurements are described in detail.
Keywords: fluorescent protein; biosensor probe; fluorescence resonance energy transfer (FRET); fluorescence lifetime imaging microscopy (FLIM); cell signaling
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MDPI and ACS Style
Hum, J.M.; Siegel, A.P.; Pavalko, F.M.; Day, R.N. Monitoring Biosensor Activity in Living Cells with Fluorescence Lifetime Imaging Microscopy. Int. J. Mol. Sci. 2012, 13, 14385-14400.
Hum JM, Siegel AP, Pavalko FM, Day RN. Monitoring Biosensor Activity in Living Cells with Fluorescence Lifetime Imaging Microscopy. International Journal of Molecular Sciences. 2012; 13(11):14385-14400.
Hum, Julia M.; Siegel, Amanda P.; Pavalko, Fredrick M.; Day, Richard N. 2012. "Monitoring Biosensor Activity in Living Cells with Fluorescence Lifetime Imaging Microscopy." Int. J. Mol. Sci. 13, no. 11: 14385-14400.