Quantitative real-time imaging of intracellular FRET biosensor dynamics using rapid multi-beam confocal FLIM

James A. Levitt, Simon P. Poland, Nikola Krstajic, Karin Pfisterer, Ahmet Erdogan, Paul R. Barber, Maddy Parsons, Robert K. Henderson, Simon M. Ameer-Beg (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)
9 Downloads (Pure)

Abstract

Fluorescence lifetime imaging (FLIM) is a quantitative, intensity-independent microscopical method for measurement of diverse biochemical and physical properties in cell biology. It is a highly effective method for measurements of Förster resonance energy transfer (FRET), and for quantification of protein-protein interactions in cells. Time-domain FLIM-FRET measurements of these dynamic interactions are particularly challenging, since the technique requires excellent photon statistics to derive experimental parameters from the complex decay kinetics often observed from fluorophores in living cells. Here we present a new time-domain multi-confocal FLIM instrument with an array of 64 visible beamlets to achieve parallelised excitation and detection with average excitation powers of ~ 1–2 μW per beamlet. We exemplify this instrument with up to 0.5 frames per second time-lapse FLIM measurements of cAMP levels using an Epac-based fluorescent biosensor in live HeLa cells with nanometer spatial and picosecond temporal resolution. We demonstrate the use of time-dependent phasor plots to determine parameterisation for multi-exponential decay fitting to monitor the fractional contribution of the activated conformation of the biosensor. Our parallelised confocal approach avoids having to compromise on speed, noise, accuracy in lifetime measurements and provides powerful means to quantify biochemical dynamics in living cells.

Original languageEnglish
Article number5146
JournalScientific Reports
Volume10
Issue number1
DOIs
Publication statusPublished - 20 Mar 2020

Fingerprint Dive into the research topics of 'Quantitative real-time imaging of intracellular FRET biosensor dynamics using rapid multi-beam confocal FLIM'. Together they form a unique fingerprint.

Cite this