Abstract
Intensity- and amplitude-weighted average lifetimes, denoted as τI and τA hereafter, are useful indicators for revealing Förster resonance energy transfer (FRET) or fluorescence quenching behaviors. In this work, we discussed the differences between τI and τA and presented several model-free lifetime determination algorithms (LDA), including the center-of-mass, phasor, and integral equation methods for fast τI and τA estimations. For model-based LDAs, we discussed the model-mismatch problems, and the results suggest that a bi-exponential model can well approximate a signal following a multi-exponential model. Depending on the application requirements, suggestions about the LDAs to be used are given. The instrument responses of the imaging systems were included in the analysis. We explained why only using the τI model for FRET analysis can be misleading; both τI and τA models should be considered. We also proposed using τA/τI as a new indicator on two-photon fluorescence lifetime images, and the results show that τA/τI is an intuitive tool for visualizing multi-exponential decays.
Highlights
Fluorescence lifetime imaging (FLIM) is a crucial technique for assessing microenvironments of fluorescent molecules [1, 2], such as pH [3], Ca2+ [4, 5], O2 [6], viscosity [7], or temperature [8]
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]
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]
Summary
Fluorescence lifetime imaging (FLIM) is a crucial technique for assessing microenvironments of fluorescent molecules [1, 2], such as pH [3], Ca2+ [4, 5], O2 [6], viscosity [7], or temperature [8]. The question about which average lifetime we should use according to the applications has been investigated in [38] They can be directly obtained with model-free LDAs, such as hardware-friendly center-of-mass methods (CMM) [41,42,43,44], the phasor method (Phasor) [45,46,47], the rapid lifetime determination method (RLD) [30, 48,49,50,51], or the integral extraction method (IEM) [52, 53], without assuming any decay model. We theoretically investigated two types of average lifetimes evaluated by model-free LDAs, examined the performances of τA and τI estimations using different LDAs, and suggested the choices of LDAs in terms of accuracy, precision, and estimation speeds according to the applications. Experimental results demonstrate the performance of τA/τI in comparison with Phasor
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