Multiplexed Time Lapse Fluorescence Lifetime Readouts in an Optically Sectioning Time-Gated Imaging Microscope

Abstract

The aim of our study is to follow the temporal and spatial interactions of proteins within different signalling networks in live cells. To map protein-protein interactions, we apply Forster resonant energy transfer (FRET) readout using fluorescence lifetime imaging (FLIM) and we present an optically sectioned FLIM microscope capable of time-lapse readouts of multiplexed FRET pairs. We note that FLIM FRET provides robust measurements, permits the use of dark acceptors and helps minimise the impact of spectral cross-talk.Wide-field FLIM is implemented using a gated optical intensifier synchronized with the excitation pulses from a fibre-laser pumped supercontinuum source or a frequency doubled Ti:Sapphire laser and enables FLIM FRET in live cells with acquisition times below ∼10 seconds. Optical sectioning is implemented using a Nipkow disk unit and improves quantitation compared to wide-field imaging, e.g. permitting separation of signals from the plasma membrane and the cytosol.To demonstrate the capabilities of this instrument, we have mapped the intracellular changes of calcium levels using interleaved time lapse FLIM acquisitions of live cells labelled with two calcium probes following stimulation with ionomycin and calcium solutions. HEK293 cells were labelled with the genetically expressed biosensor, Troponin TN-XL, for which changes in calcium levels are read out by FRET between the CFP and YFP fluorophores, and with a calcium sensing dye, GFP-Certified™ FluoForte™. The emission profile of the latter is spectrally separate from CFP and YFP, making it useful for multiplexing with the many available CFP/YFP FRET biosensors.We aim to apply this instrument to follow the spatial and temporal changes of simultaneous activation of signalling pathways during fibroblast migration in a concentration gradient of a chemo-attractant (PDGF), particularly using specifically designed FRET biosensors for small GTPases (Rac), phosphoinositide pathway (IP3) and calcium.