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.
Highlights
The use of microscopy to monitor fluorescent reporters that “sense” specific subcellular events inside single living cells are providing an unprecedented view of the spatial and temporal regulation of cell signaling events
Many different design strategies have been used to develop the genetically encoded biosensor proteins, but most rely on Förster resonance energy transfer (FRET) microscopy to report the changes in protein conformation that accompany the targeted signaling event [2,3,4,5,6]
Since the distance over which efficient energy transfer can occur is limited to less than 80 Å, FRET can be used monitor dynamic conformational changes in biosensor proteins that occur in response to signaling events inside living cells, tissues, and organisms [5,6,7,8,9]
Summary
The use of microscopy to monitor fluorescent reporters that “sense” specific subcellular events inside single living cells are providing an unprecedented view of the spatial and temporal regulation of cell signaling events. These genetically encoded fluorescent reporters, known as biosensor proteins, combine sensing units to detect specific cell signaling events with reporter modules that incorporate the fluorescent proteins (FPs). The evolution of the newer generations of FPs with optimized characteristics has improved the detection of signals from the biosensor proteins expressed inside living cells [1] This has given cell biologists access to an impressive collection of molecular tools to investigate the spatial and temporal regulation of cell signaling events. The frequency domain (FD) FLIM method is used to characterize the activity of a Src-biosensor inside living bone cells in response to a mechanical stimulus applied by dynamic fluid flow over the cell surface, and to evaluate cAMP activity within pituitary cells in response to an agonist
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