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.

Details

Title
Quantitative real-time imaging of intracellular FRET biosensor dynamics using rapid multi-beam confocal FLIM
Author
Levitt, James A 1 ; Poland, Simon P 2 ; Krstajic Nikola 3   VIAFID ORCID Logo  ; Pfisterer, Karin 4 ; Erdogan Ahmet 3   VIAFID ORCID Logo  ; Barber, Paul R 5 ; Parsons Maddy 4   VIAFID ORCID Logo  ; Henderson, Robert K 3 ; Ameer-Beg, Simon M 2   VIAFID ORCID Logo 

 Microscopy Innovation Centre, Guy’s Campus, Kings College, London, UK (GRID:grid.13097.3c) (ISNI:0000 0001 2322 6764); Richard Dimbleby Laboratories, School of Cancer and Pharmaceutical Sciences, Guy’s Campus, Kings College London, London, UK (GRID:grid.13097.3c) (ISNI:0000 0001 2322 6764) 
 Richard Dimbleby Laboratories, School of Cancer and Pharmaceutical Sciences, Guy’s Campus, Kings College London, London, UK (GRID:grid.13097.3c) (ISNI:0000 0001 2322 6764) 
 College of Science and Engineering, University of Edinburgh, Institute for Microelectronics and Nanosystems, School of Engineering, Edinburgh, UK (GRID:grid.4305.2) (ISNI:0000 0004 1936 7988) 
 Randall Centre for Cell and Molecular Biophysics, Guy’s Campus, Kings College, London, UK (GRID:grid.13097.3c) (ISNI:0000 0001 2322 6764) 
 UCL Cancer Institute, Paul O’Gorman Building, University College London, London, UK (GRID:grid.83440.3b) (ISNI:0000000121901201) 
Publication year
2020
Publication date
2020
Publisher
Nature Publishing Group
e-ISSN
20452322
Source type
Scholarly Journal
Language of publication
English
ProQuest document ID
2380033489
Copyright
This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.