Abstract
FRET biosensors have proven very useful tools for studying the activation of specific signalling pathways in living cells. Most biosensors designed to date have been predicated on fluorescent protein pairs that were identified by, and for use in, intensity based measurements, however fluorescence lifetime provides a more reliable measurement of FRET. Both the technology and fluorescent proteins available for FRET have moved on dramatically in the last decade. Lifetime imaging systems have become increasingly accessible and user-friendly, and there is an entire field of biology dedicated to refining and adapting different characteristics of existing and novel fluorescent proteins. This growing pool of fluorescent proteins includes the long-lifetime green and cyan fluorescent proteins Clover and mTurquoise2, the red variant mRuby2, and the dark acceptor sREACh. Here, we have tested these donors and acceptors in appropriate combinations against the standard or recommended norms (EGFP and mTFP as donors, mCherry and either Ypet or Venus as acceptors) to determine if they could provide more reliable, reproducible and quantifiable FLIM-FRET data to improve on the dynamic range compared to other donors and breadth of application of biosensor technologies. These tests were performed for comparison on both a wide-field, frequency domain system and a multiphoton, TCSPC time domain FLIM system. Clover proved to be an excellent donor with extended dynamic range in combination with mCherry on both platforms, while mRuby2 showed a high degree of variability and poor FRET efficiencies in all cases. mTFP-Venus was the most consistent cyan-yellow pair between the two FLIM methodologies, but mTurquoise2 has better dynamic range and transfers energy consistently over time to the dark acceptor sRCh. Combination of mTFP-sRCh with Clover-mCherry would allow the simultaneous use of two FLIM-FRET biosensors within one sample by eliminating the crosstalk between the yellow acceptor and green donor emissions.
Highlights
Fluorescent labels have been used to help solve biological questions for over 5 decades [1, 2]
Fluorescence lifetime measurements and Forster Resonance Energy Transfer (FRET) efficiencies of fluorophore pairs In order to compare the fluorescence lifetimes of our selected donors and the relative FRET efficiencies of different donor-acceptor pairings in the context of living cells, fluorescent protein constructs were expressed in HEK 293 cells grown in standard serum-supplemented conditions
Fluorescence lifetime readings were taken on the frequency and multiphoton time domain FLIM-systems from separate samples of donor-expressing and donor-acceptor fusion-expressing cells, on three separate occasions per fluorophore pair
Summary
Fluorescent labels have been used to help solve biological questions for over 5 decades [1, 2]. Detection of photo-physical effects in, and interactions between, such labels has elevated the technique from a purely qualitative imaging process to a potentially quantitative measurement system. Methods such as FRAP and FRET can provide information on molecular dynamics and interactions that were previously unavailable. FRET-based probes have given unique insights into the biochemical activities of a number of critical signalling molecules, such as the dissociation of G-Proteins from GPCRs upon receptor stimulation [4] and the highly localised production of cAMP in cardiac myocytes [5]. Fluorescent protein biology and DNA manipulation technology have provided researchers with an exciting, but often bewildering, array of options for such experiments
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
