Intensity-and amplitude-weighted average lifetimes, denoted as τ I and τ A hereafter, are useful indicators for revealing Förster resonance energy transfer (FRET) or fluorescence quenching behaviors. In this work, we discussed the differences between τ I and τ A and presented several model-free lifetime determination algorithms (LDA), including the center-of-mass, phasor, and integral equation methods for fast τ I and τ A 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 τ I model for FRET analysis can be misleading; both τ I and τ A models should be considered. We also proposed using τ A /τ I as a new indicator on two-photon fluorescence lifetime images, and the results show that τ A /τ I is an intuitive tool for visualizing multi-exponential decays.
Measuring fluorescence lifetimes of fast-moving cells or particles have broad applications in biomedical sciences. This paper presents a dynamic fluorescence lifetime sensing (DFLS) system based on the time-correlated single-photon counting (TCSPC) principle. It integrates a CMOS 192 × 128 single-photon avalanche diode (SPAD) array, offering an enormous photon-counting throughput without pile-up effects. We also proposed a quantized convolutional neural network (QCNN) algorithm and designed a field-programmable gate array embedded processor for fluorescence lifetime determinations. The processor uses a simple architecture, showing unparallel advantages in accuracy, analysis speed, and power consumption. It can resolve fluorescence lifetimes against disturbing noise. We evaluated the DFLS system using fluorescence dyes and fluorophore-tagged microspheres. The system can effectively measure fluorescence lifetimes within a single exposure period of the SPAD sensor, paving the way for portable time-resolved devices and shows potential in various applications.
Facultative intracellular pathogens are able to live inside and outside host cells. It is highly desirable to differentiate their cellular locations for the purposes of fundamental research and clinical applications. In this work, we developed a novel analysis platform that allows users to choose two analysis models: amplitude weighted lifetime (τ A) and intensity weighted lifetime (τ I) for fluorescence lifetime imaging microscopy (FLIM). We applied these two models to analyse FLIM images of mouse Raw macrophage cells that were infected with bacteria Shigella Sonnei, adherent and invasive E. coli (AIEC) and Lactobacillus. The results show that the fluorescence lifetimes of bacteria depend on their cellular locations. The τ A model is superior in visually differentiating bacteria that are in extra- and intra-cellular and membrane-bounded locations, whereas the τ I model show excellent precision. Both models show speedy performances that analysis can be performed within 0.3 s. We also compared the proposed models with a widely used commercial software tool (τ C, SPC Image, Becker & Hickl GmbH), showing similar τ I and τ C results. The platform also allows users to perform phasor analysis with great flexibility to pinpoint the regions of interest from lifetime images as well as phasor plots. This platform holds the disruptive potential of replacing z-stack imaging for identifying intracellular bacteria.
Time-correlated single-photon counting (TCSPC) has been the gold standard for fluorescence lifetime imaging (FLIM) techniques due to its high signal-to-noize ratio and high temporal resolution. The sensor system's temporal instrument response function (IRF) should be considered in the deconvolution procedure to extract the real fluorescence decay to compensate for the distortion on measured decays contributed by the system imperfections. However, to measure the instrument response function is not trivial, and the measurement setup is different from measuring the real fluorescence. On the other hand, automatic synthetic IRFs can be directly derived from the recorded decay profiles and provide appropriate accuracy. This paper proposed and examined a synthetic IRF strategy. Compared with traditional automatic synthetic IRFs, the new proposed automatic synthetic IRF shows a broader dynamic range and better accuracy. To evaluate its performance, we examined simulated data using nonlinear least square deconvolution based on both the Levenberg-Marquardt algorithm and the Laguerre expansion method for bi-exponential fluorescence decays. Furthermore, experimental FLIM data of cells were also analyzed using the proposed synthetic IRF. The results from both the simulated data and experimental FLIM data show that the proposed synthetic IRF has a better performance compared to traditional synthetic IRFs. Our work provides a faster and precise method to obtain IRF, which may find various FLIM-based applications. We also reported in which conditions a measured or a synthesized IRF can be applied.
We propose a histogram clustering (HC) method to accelerate fluorescence lifetime imaging (FLIM) analysis in pixel-wise and global fitting modes. The proposed method’s principle was demonstrated, and the combinations of HC with traditional FLIM analysis were explained. We assessed HC methods with both simulated and experimental datasets. The results reveal that HC not only increases analysis speed (up to 106 times) but also enhances lifetime estimation accuracy. Fast lifetime analysis strategies were suggested with execution times around or below 30 μ s per histograms on MATLAB R2016a, 64-bit with the Intel Celeron CPU (2950M @ 2GHz).
Fluorescence lifetime imaging (FLIM) is powerful for monitoring cellular microenvironments, protein conformational changes, and protein interactions. It can facilitate metabolism research, drug screening, DNA sequencing, and cancer diagnosis. Lifetime determination algorithms (LDAs) adopted in FLIM analysis can influence biological interpretations and clinical diagnoses. Herein, we discuss the commonly used and advanced time-domain LDAs classified in fitting and non-fitting categories. The concept and explicit mathematical forms of LDAs are reviewed. The output lifetime parameter types are discussed, including lifetime components, average lifetimes, and graphic representation. We compare their performances, identify trends, and provide suggestions for end users in terms of multi-exponential decay unmixing ability, lifetime estimation precision, and processing speed.
We present a deep learning approach to obtain high-resolution (HR) fluorescence lifetime images from low-resolution (LR) images acquired from fluorescence lifetime imaging (FLIM) systems. We first proposed a theoretical method for training neural networks to generate massive semi-synthetic FLIM data with various cellular morphologies, a sizeable dynamic lifetime range, and complex decay components. We then developed a degrading model to obtain LR-HR pairs and created a hybrid neural network, the spatial resolution improved FLIM net (SRI-FLIMnet) to simultaneously estimate fluorescence lifetimes and realize the nonlinear transformation from LR to HR images. The evaluative results demonstrate SRI-FLIMnet’s superior performance in reconstructing spatial information from limited pixel resolution. We also verified SRI-FLIMnet using experimental images of bacterial infected mouse raw macrophage cells. Results show that the proposed data generation method and SRI-FLIMnet efficiently achieve superior spatial resolution for FLIM applications. Our study provides a solution for fast obtaining HR FLIM images.
We present a deep learning (DL) framework, termed few-photon fluorescence lifetime imaging (FPFLI), for fast analysis of fluorescence lifetime imaging (FLIM) data under highly low-light conditions with only a few photons per pixel. FPFLI breaks the conventional pixel-wise lifetime analysis paradigm and fully exploits the spatial correlation and intensity information of fluorescence lifetime images to estimate lifetime images, pushing the photon budget to an unprecedented low level. The DL framework can be trained by synthetic FLIM data and easily adapted to various FLIM systems. FPFLI can effectively and robustly estimate FLIM images within seconds using synthetic and experimental data. The fast analysis of low-light FLIM images made possible by FPFLI promises a broad range of potential applications.
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