Spectrally resolved fluorescence lifetime imaging microscopy: Förster resonant energy transfer global analysis with a one- and two-exponential donor model

Abstract

In many fields of life science, visualization of spatial proximity, as an indicator of protein interactions in living cells, is of outstanding interest. A method to accomplish this is the measurement of Förster resonant energy transfer (FRET) by means of spectrally resolved fluorescence lifetime imaging microscopy. The fluorescence lifetime is calculated using a multiple-wavelength fitting routine. The donor profile is assumed first to have a monoexponential time-dependent behavior, and the acceptor decay profile is solved analytically. Later, the donor profile is assumed to have a two-exponential time-dependent behavior and the acceptor decay profile is derived analytically. We develop and apply a multispectral fluorescence lifetime imaging microscopy analysis system for FRET global analysis with time-resolved and spectrally resolved techniques, including information from donor and acceptor channels in contrast to using just a limited spectral data set from one detector only and a model accounting only for the donor signal. This analysis is used to demonstrate close vicinity of β-secretase (BACE) and GGA1, two proteins involved in Alzheimer's disease pathology. We attempt to verify if an improvement in calculating the donor lifetimes could be achieved when time-resolved and spectrally resolved techniques are simultaneously incorporated.

Multi-target immunofluorescence by separation of antibody cross-labelling via spectral-FLIM-FRET

“…In total, the inherent ability of the sFLIM method to resolve fluorophores with spectroscopically similar fluorescent signatures or even FRET of cross-labelling ABs, as shown here, potentially enhances the possible number of combinations of fluorophores which could be multiplexed, imaged and visualized simultaneously. This is supported by comparable studies which likewise demonstrate that sFLIM successfully enables FRET quantitation with high accuracy [23][24][25] .…”

A Method to Quantify FRET Stoichiometry with Phasor Plot Analysis and Acceptor Lifetime Ingrowth

“…All of these are of interest, but they cannot be quantified without knowledge of the bound acceptor fraction (fraction of acceptors that are in complex with their binding partners), which is not traditionally available when only donor lifetimes are measured. Spectrally resolved FLIM has been applied for FRET measurements to improve both the separation of multiple lifetime components and the accuracy of recovered FRET efficiencies ( 6,15–17 ), but they have not been extended to the recovery of the acceptor stoichiometry. The lifetime ingrowth of acceptors has been exploited for the analysis of FRET stoichiometry ( 18,19 ); however, these methods are impractical when fluorescence bleedthrough from donor fluorophores contaminates the FRET signal, a problem for most FRET pairs, because then the bound acceptor fraction becomes difficult to retrieve.…”

Auto-Phase-Locked Time-Resolved Luminescence Detection: Principles, Applications, and Prospects

“…In addition to the excitation sources and detectors, precise circuit systems with small timing jitter are usually used to synchronize the camera or shutter with the pulse excitation in many time-resolved detection systems (Krishnan et al, 2003;Urayama et al, 2003;Biskup et al, 2004;Connally et al, 2006;Qu et al, 2006;Sun et al, 2009;Gahlaut and Miller, 2010;Cicchi and Pavone, 2011;Strat et al, 2011;Becker, 2012;Hirvonen et al, 2014;Bergmann et al, 2016;Pominova et al, 2016;Chen T. et al, 2017;Luo et al, 2017;Liu et al, 2019Liu et al, , 2020. For Damayanti et al, 2013;Lu et al, 2014;Wang et al, 2017 Streak camera Scanning <1 ps 0.26 ns−1 ms Krishnan et al, 2003;Biskup et al, 2004;Qu et al, 2006;Bergmann et al, 2016;Pominova et al, 2016 Intensified camera Wide-field 0.2 ns 0.6 ns−1 ms Urayama et al, 2003;Connally et al, 2006;Sun et al, 2009;Gahlaut and Miller, 2010;Hirvonen et al, 2014;Chen T. et al, 2017;Liu et al, 2020 Gated detecting the delayed luminescence within nanoseconds, the related instruments are always composed of elements, such as ultrafast lasers, high-speed cameras and precise control circuits, which are very complicated and expensive to be popularized.…”

“…Time-resolved techniques have been widely applied to the study of ultrafast photophysical processes (Wirth, 1990 ; Walker et al, 2013 ), the research of temporal behavior of chemical systems (Piatkowski et al, 2014 ), biological detection and imaging (Connally and Piper, 2008 ; Berezin and Achilefu, 2010 ; Bünzli, 2010 ; Cicchi and Pavone, 2011 ; Strat et al, 2011 ; Becker, 2012 ; Yang et al, 2013 ; Grichine et al, 2014 ; Lemmetyinen et al, 2014 ; Lu et al, 2014 ; Bui et al, 2017 ; Luo et al, 2017 ; Zhang et al, 2018 ; Zhu et al, 2018b ; Liu et al, 2019 ). Since these techniques can detect the luminescence change in time domain, they play a more and more important role as many long-lived luminescence materials and probes were developed (Connally and Piper, 2008 ; Bünzli, 2010 ; Yang et al, 2013 ; Zhang et al, 2018 ).…”