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

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.

The biochemical resolving power of fluorescence lifetime imaging: untangling the roles of the instrument response function and photon-statistics

“…We have made significant progress in ameliorating pulse pile-up effects, for example, by utilizing multi-hit time-to-digital [7,8] or multiplexed time-to-analog converters [9], ultra-fast digitizers [10], fast detectors (e.g. hybrid photomultipliers [11]), and detector arrays [12][13][14][15]. Such innovations permit contemporary FLIM systems to acquire images with fluorescence lifetime contrast at spatial resolutions typical of confocal microscopy without significantly compromising imaging speed.…”

Investigations on Average Fluorescence Lifetimes for Visualizing Multi-Exponential Decays

“…Fluorescence lifetime imaging (FLIM) is a crucial technique for assessing microenvironments of fluorescent molecules [1,2], such as pH [3], Ca 2+ [4,5], O 2 [6], viscosity [7], or temperature [8]. Combining with Förster Resonance Energy Transfer (FRET) techniques, FLIM can be a powerful "quantum ruler" to measure protein conformations and interactions [9][10][11][12]. Compared with fluorescence intensity imaging, FLIM is independent of the signal intensity and fluorophore concentrations, making FLIM a powerful quantitative imaging technique for applications in life sciences [13], medical diagnosis [14][15][16], drug developments [17][18][19], and flow diagnosis [20][21][22].…”

“…FLIM techniques can build on time-correlated single-photon counting (TCSPC) [23][24][25], time-gating [26][27][28], or streak cameras [29]; they record time-resolved fluorescence intensity profiles to extract lifetimes with a lifetime determination algorithm (LDA) [1]. There is a rapid growth of real-time applications that fast analysis is sought after [12,30]. Traditional LDAs usually use the least square method (LSM) or maximum likelihood estimation (MLE) [31] to analyze decay models chosen by users, and model-fitting analysis follows a reduced chi-squared criterion [1].…”

A Guide to Fluorescence Lifetime Microscopy and Förster's Resonance Energy Transfer in Neuroscience

“…The higher depth resolution is achieved by focusing a one‐photon laser onto a defined spot at a specific depth in the sample, while a pinhole inside the optical path is used to cut off fluorescence generated from out‐of‐focus fluorophores. Confocal microscopy can be combined with FLIM (Levitt et al., 2020) for neuroscience applications. For example, activation of AMPA receptor was measured by confocal FLIM using CFP/YFP‐tagged GluA2 subunit constructs, and electrophysiological properties of the cultured cells were simultaneously measured with a patch clamp technique (Zachariassen et al., 2016).…”