Time-domain Fluorescence Lifetime Imaging Research Articles

Cellular Imaging and Time-Domain FLIM Studies of Meso-Tetraphenylporphine Disulfonate as a Photosensitising Agent in 2D and 3D Models.

Fluorescence lifetime imaging (FLIM) and confocal fluorescence studies of a porphyrin-based photosensitiser (meso-tetraphenylporphine disulfonate: TPPS2a) were evaluated in 2D monolayer cultures and 3D compressed collagen constructs of a human ovarian cancer cell line (HEY). TPPS2a is known to be an effective model photosensitiser for both Photodynamic Therapy (PDT) and Photochemical Internalisation (PCI). This microspectrofluorimetric study aimed firstly to investigate the uptake and subcellular localisation of TPPS2a, and evaluate the photo-oxidative mechanism using reactive oxygen species (ROS) and lipid peroxidation probes combined with appropriate ROS scavengers. Light-induced intracellular redistribution of TPPS2a was observed, consistent with rupture of endolysosomes where the porphyrin localises. Using the same range of light doses, time-lapse confocal imaging permitted observation of PDT-induced generation of ROS in both 2D and 3D cancer models using fluorescence-based ROS together with specific ROS inhibitors. In addition, the use of red light excitation of the photosensitiser to minimise auto-oxidation of the probes was investigated. In the second part of the study, the photophysical properties of TPPS2a in cells were studied using a time-domain FLIM system with time-correlated single photon counting detection. Owing to the high sensitivity and spatial resolution of this system, we acquired FLIM images that enabled the fluorescence lifetime determination of the porphyrin within the endolysosomal vesicles. Changes in the lifetime dynamics upon prolonged illumination were revealed as the vesicles degraded within the cells.

Label-Free Identification of Carbonaceous Particles Using Nonlinear Optical Microscopy.

The adverse health effects of ambient carbonaceous particles (CPs) such as carbon black (CB), black carbon (BC), and brown carbon (BrC) are becoming more evident and depend on their composition and emission source. Therefore, identifying and quantifying these particles in biological samples are important to better understand their toxicity. Here, we report the development of a nonlinear optical approach for the identification of CPs such as CB and BrC using imaging conditions compatible with biomedical samples. The unique visible light fingerprint of CB and BrC nanoparticles (NPs) upon illumination with a femtosecond (fs) pulsed laser at 1300 nm excitation wavelength is an effective approach for their identification in their biological context. The emission from spectral features of these CPs was investigated with time-domain fluorescence lifetime imaging (FLIM) to further support their identification. This study is performed for different types of CPs embedded in agarose gel as well as in in vitro mammalian cells. The unique nonlinear emissive behavior of CP NPs used for their label-free identification is further complementary with fluorophores typically used for specific staining of biological samples thus providing the relevant bio-context.

Compact and robust deep learning architecture for fluorescence lifetime imaging and FPGA implementation

This paper reports a bespoke adder-based deep learning network for time-domain fluorescence lifetime imaging (FLIM). By leveraging the -norm extraction method, we propose a 1D Fluorescence Lifetime AdderNet (FLAN) without multiplication-based convolutions to reduce the computational complexity. Further, we compressed fluorescence decays in temporal dimension using a log-scale merging technique to discard redundant temporal information derived as log-scaling FLAN (FLAN+LS). FLAN+LS achieves 0.11 and 0.23 compression ratios compared with FLAN and a conventional 1D convolutional neural network (1D CNN) while maintaining high accuracy in retrieving lifetimes. We extensively evaluated FLAN and FLAN+LS using synthetic and real data. A traditional fitting method and other non-fitting, high-accuracy algorithms were compared with our networks for synthetic data. Our networks attained a minor reconstruction error in different photon-count scenarios. For real data, we used fluorescent beads’ data acquired by a confocal microscope to validate the effectiveness of real fluorophores, and our networks can differentiate beads with different lifetimes. Additionally, we implemented the network architecture on a field-programmable gate array (FPGA) with a post-quantization technique to shorten the bit-width, thereby improving computing efficiency. FLAN+LS on hardware achieves the highest computing efficiency compared to 1D CNN and FLAN. We also discussed the applicability of our network and hardware architecture for other time-resolved biomedical applications using photon-efficient, time-resolved sensors.

Effect of UV light illumination in humid air on the optical and electronic properties of the orthorhombic α-MoO3 and monoclinic β-MoO3

We present reflectivity measurements on monoclinic MoO2, orthorhombic α-MoO3, and monoclinic β-MoO3 in a wide frequency range of 190–2500 nm. The extracted optical conductivity [σ(ω)] showed that MoO2 has a metallic character while α-MoO3 and β-MoO3 have an insulating behavior. In addition, the photochromic properties of both α-MoO3 and β-MoO3 have been studied. The σ(ω) spectra for both samples showed a different spectral weight of the optical transition due to the formation of color center bands, which formed as a result of UV exposure. The spectral weight of optical transition from the bulk sixfold cations Mob5+ to Mo6+ cations is higher in case of the illuminated β-MoO3 sample than the illuminated α-MoO3 sample. The XRD results showed that both α-MoO3 and β-MoO3 were transformed to monoclinic molybdenum oxide dihydrate (H4MoO5) after exposure to UV irradiation in humid air. The σ(ω) spectra revealed that photoinjection of hydrogen into the β-MoO3 film is higher than in the case of the α-MoO3 film. In addition, the time domain fluorescence lifetime imaging microscopy data showed that the lifetime due to the optical transition from surface fourfold cations Mos5+ to Mo6+ cations in the case of illuminated β-MoO3 is higher than that for the illuminated α-MoO3 for the same optical transition. Meaning that, in the case of illuminated β-MoO3, the surface Mos5+ cations disperse and penetrate into the bulk, lowering the spectral weight of the [Mos5+ Mos5+] dimers and enhancing the spectral weight of the bulk centers.

Time-Domain Fluorescence Lifetime Imaging of cAMP Levels with EPAC-Based FRET Sensors.

Second messenger molecules in eukaryotic cells relay the signals from activated cell surface receptors to intracellular effector proteins. FRET-based sensors are ideal to visualize and measure the often rapid changes of second messenger concentrations in time and place. Fluorescence Lifetime Imaging (FLIM) is an intrinsically quantitative technique for measuring FRET. Given the recent development of commercially available, sensitive and photon-efficient FLIM instrumentation, it is becoming the method of choice for FRET detection in signaling studies. Here, we describe a detailed protocol for time domain FLIM, using the EPAC-based FRET sensor to measure changes in cellular cAMP levels with high spatiotemporal resolution as an example.

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

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.

Selective plane illumination microscopy (SPIM) with time-domain fluorescence lifetime imaging microscopy (FLIM) for volumetric measurement of cleared mouse brain samples.

We have developed an imaging technique which combines selective plane illumination microscopy with time-domain fluorescence lifetime imaging microscopy (SPIM-FLIM) for three-dimensional volumetric imaging of cleared mouse brains with micro- to mesoscopic resolution. The main features of the microscope include a wavelength-adjustable pulsed laser source (Ti:sapphire) (near-infrared) laser, a BiBO frequency-doubling photonic crystal, a liquid chamber, an electrically focus-tunable lens, a cuvette based sample holder, and an air (dry) objective lens. The performance of the system was evaluated with a lifetime reference dye and micro-bead phantom measurements. Intensity and lifetime maps of three-dimensional human embryonic kidney (HEK) cell culture samples and cleared mouse brain samples expressing green fluorescent protein (GFP) (donor only) and green and red fluorescent protein [positive Förster (fluorescence) resonance energy transfer] were acquired. The results show that the SPIM-FLIM system can be used for sample sizes ranging from single cells to whole mouse organs and can serve as a powerful tool for medical and biological research.

FRET Image Correlation Spectroscopy Reveals RNAPII-Independent P-TEFb Recruitment on Chromatin

Biochemical studies have revealed that the RNA Polymerase II (RNAPII) pause release is triggered by phosphorylation of the transcription machinery by the positive transcription elongation factor b (P-TEFb). However, there are no direct report that P-TEFb and RNA polymerase II interact in single living cells and the biophysical mechanisms mediating this association are still unclear. Förster resonance energy transfer (FRET) detects molecular interactions at the subcellular level. Time domain fluorescence lifetime imaging provides an accurate quantification of FRET efficiency, EFRET, because it is fluorochrome concentration-independent and insensitive to fluorescence bleed-through. However, the way FRET signal is usually analyzed does not provide information about the areas where protein-protein interactions take place. In this work, we developed a method, dubbed FRET image correlation spectroscopy (FICS), which relied on FRET fluorescence lifetime imaging image acquisition and image correlation spectroscopy of EFRET clusters to quantify the spatial distribution of interaction clusters in the nucleus. The combination of high content FRET microscopy with batch image analysis allowed a robust statistical analysis. By applying FICS, we characterized the area and density of interaction clusters between P-TEFb and RNAPII or histone H2A in single living cells. The FICS method applied to cells expressing genetically engineered mutated proteins confirmed that the histidine-rich domain of P-TEFb is required for its interaction with RNAPII. Furthermore, it demonstrated that P-TEFb was also located in close vicinity to histone H2A, independently of its interactions with RNAPII. These results support the hypothesis that P-TEFb dynamics on chromatin regulate its recruitment on RNAPII.

Time-domain fluorescence lifetime imaging by nonlinear fluorescence microscopy constructed of a pump-probe setup with two-wavelength laser pulses.

We propose a time-domain approach for fluorescence lifetime measurements using nonlinear fluorescence microscopy constructed of a pump-probe setup with two-wavelength laser pulses. Nonlinear fluorescence signals generated by fluorescence reduction due to stimulated emission were detectable through a lock-in technique. Changing the time delay between the two-wavelength pulses enables acquisition of a time-resolved nonlinear fluorescence signal, which directly reflects the fluorescence lifetime of the sample and is thus applicable to fluorescence lifetime imaging. We also quantitatively demonstrate that nonlinear fluorescence microscopy possesses better optical resolution than conventional laser-scanning fluorescence microscopy. Experimental trials indicate that straightforward fluorescence lifetime imaging with high optical resolution is readily available.

Estimating fluorescence lifetimes using extended Kalman filter

The extended Kalman filter (EKF) has been widely used in communication, signal processing and navigation control. In this Letter, the authors applied EKF, for the first time to their knowledge, to simultaneously estimate fluorescence lifetimes and instrument response functions (IRF) for time-domain fluorescence lifetime imaging microscopy (FLIM) systems (we focus on gating and time-correlated single-photon counting techniques in this work). Monte-Carlo simulations were performed to test its performances in comparison with previously reported methods. Simulation results show that the proposed algorithm can achieve comparable or better results than the others. More importantly, with EKF there is no need to measure the IRF of the FLIM system.

Noise-Corrected Principal Component Analysis of fluorescence lifetime imaging data.

Fluorescence Lifetime Imaging (FLIM) is an attractive microscopy method in the life sciences, yielding information on the sample otherwise unavailable through intensity-based techniques. A novel Noise-Corrected Principal Component Analysis (NC-PCA) method for time-domain FLIM data is presented here. The presence and distribution of distinct microenvironments are identified at lower photon counts than previously reported, without requiring prior knowledge of their number or of the dye's decay kinetics. A noise correction based on the Poisson statistics inherent to Time-Correlated Single Photon Counting is incorporated. The approach is validated using simulated data, and further applied to experimental FLIM data of HeLa cells stained with membrane dye di-4-ANEPPDHQ. Two distinct lipid phases were resolved in the cell membranes, and the modification of the order parameters of the plasma membrane during cholesterol depletion was also detected. Noise-corrected Principal Component Analysis of FLIM data resolves distinct microenvironments in cell membranes of live HeLa cells.

Towards unsupervised fluorescence lifetime imaging using low dimensional variable projection.

Analyzing large fluorescence lifetime imaging (FLIM) data is becoming overwhelming; the latest FLIM systems easily produce massive amounts of data, making an efficient analysis more challenging than ever. In this paper we propose the combination of a custom-fit variable projection method, with a Laguerre expansion based deconvolution, to analyze bi-exponential data obtained from time-domain FLIM systems. Unlike nonlinear least squares methods, which require a suitable initial guess from an experienced researcher, the new method is free from manual interventions and hence can support automated analysis. Monte Carlo simulations are carried out on synthesized FLIM data to demonstrate the performance compared to other approaches. The performance is also illustrated on real-life FLIM data obtained from the study of autofluorescence of daisy pollen and the endocytosis of gold nanorods (GNRs) in living cells. In the latter, the fluorescence lifetimes of the GNRs are much shorter than the full width at half maximum of the instrument response function. Overall, our proposed method contains simple steps and shows great promise in realising automated FLIM analysis of large data sets.

GPU acceleration of time-domain fluorescence lifetime imaging.

Fluorescence lifetime imaging microscopy (FLIM) plays a significant role in biological sciences, chemistry, and medical research. We propose a Graphic Processing Units (GPUs) based FLIM analysis tool suitable for high-speed and flexible time-domain FLIM applications. With a large number of parallel processors, GPUs can significantly speed up lifetime calculations compared to CPU - OpenMP (parallel computing with multiple CPU cores) based analysis. We demonstrate how to implement and optimize FLIM algorith ms on GPUs for both iterative and non-iterative FLIM analysis algorithms. The implemented algorithms have been tested on both synthesized and experimental FLIM data. The results show that at the same precision the GPU analysis can be up to 24-fold faster than its CPU - OpenMP counterpart. This means that even for high precision but time-consuming iterative FLIM algorithms, GPUs enable fast or even real-time analysis.

Time-Domain Fluorescence Lifetime Imaging Microscopy: A Quantitative Method to Follow Transient Protein–Protein Interactions in Living Cells

Quantitative analysis in Förster resonance energy transfer (FRET) imaging studies of protein-protein interactions within live cells is still a challenging issue. Many cellular biology applications aim at the determination of the space and time variations of the relative amount of interacting fluorescently tagged proteins occurring in cells. This relevant quantitative parameter can be, at least partially, obtained at a pixel-level resolution by using fluorescence lifetime imaging microscopy (FLIM). Indeed, fluorescence decay analysis of a two-component system (FRET and no FRET donor species), leads to the intrinsic FRET efficiency value (E) and the fraction of the donor-tagged protein that undergoes FRET (fD). To simultaneously obtain fD and E values from a two-exponential fit, data must be acquired with a high number of photons, so that the statistics are robust enough to reduce fitting ambiguities. This is a time-consuming procedure. However, when fast-FLIM acquisitions are used to monitor dynamic changes in protein-protein interactions at high spatial and temporal resolutions in living cells, photon statistics and time resolution are limited. In this case, fitting procedures are unreliable, even for single lifetime donors. We introduce the concept of a minimal fraction of donor molecules involved in FRET (mfD), obtained from the mathematical minimization of fD. Here, we discuss different FLIM techniques and the compromises that must be made between precision and time invested in acquiring FLIM measurements. We show that mfD constitutes an interesting quantitative parameter for fast FLIM because it gives quantitative information about transient interactions in live cells.

New Functional Handle for Use as a Self-Reporting Contrast and Delivery Agent in Nanomedicine

The synthesis and photophysical characterization of a chromophore-bridged block copolymer system is presented. This system is based on a dithiomaleimide (DTM) functional group as a highly emissive functionality which can readily be incorporated into polymeric scaffolds. A key advantage of this new reporter group is its versatile chemistry, ease of further functionalization, and notably small size, which allows for ready incorporation without affecting or disrupting the self-assembly process critical to the formation of core-shell polymeric contrast and drug delivery agents. We demonstrate the potential of this functionality with a diblock system which has been shown to be appropriate for micellization and, when in the micellar state, does not self-quench. The block copolymer is shown to be significantly more emissive than the lone dye, with a concentration-independent emission and anisotropy profile from 1.5 mM to 0.15 μM. An emission lifetime and anisotropy decay comparison of the block copolymer to its micelle displays that time-domain fluorescence lifetime imaging (FLIM) is able to rapidly resolve differences in the supramolecular state of this block-dye-block polymer system. Furthermore, the ability to resolve these differences in the supramolecular state means that the DTM micelles are capable of self-reporting when disassembly occurs, simply by monitoring with FLIM. We demonstrate the great potential for in vitro applications that this system provides by using FLIM to observe micelle disassembly in different vascular components of rat hippocampal tissue. In total this system represents a new class of in-chain emitter which is appropriate for application in quantitative imaging and the tracking of particle degradation/disassembly events in biological environments.

A Time-Resolved CMOS Image Sensor With Draining-Only Modulation Pixels for Fluorescence Lifetime Imaging

This paper presents a time-resolved CMOS image sensor with draining-only modulation (DOM) pixels, for time-domain fluorescence lifetime imaging. In the DOM pixels using a pinned photodiode (PPD) technology, a time-windowed signal charge transfer from a PPD to a pinned storage diode (PSD) is controlled by a draining gate only, without a transfer gate between the two diodes. This structure allows a potential barrierless and trapless charge transfer from the PPD to the PSD. A 256 × 256 pixel time-resolved CMOS imager with 7.5 × 7.5 μm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> DOM pixels has been implemented using 0.18-μm CMOS image sensor process technology with PPD option. The prototype demonstrates high sensitivity for weak signal of less than one electron per light pulse and accurate measurement of fluorescence decay process with subnanosecond time resolution.

Generalization of the polar representation in time domain fluorescence lifetime imaging microscopy for biological applications: practical implementation

The polar representation or phasor, which provides a fast and visual indication on the number of exponentials present in the intensity decay of the fluorescence lifetime images is increasingly used in time domain fluorescence lifetime imaging microscopy experiments. The calculations of the polar coordinates in time domain fluorescence lifetime imaging microscopy experiments involve several experimental parameters (e.g. instrumental response function, background, angular frequency, number of temporal channels) whose role has not been exhaustively investigated. Here, we study theoretically, computationally and experimentally the influence of each parameter on the polar calculations and suggest parameter optimization for minimizing errors. We identify several sources of mistakes that may occur in the calculations of the polar coordinates and propose adapted corrections to compensate for them. For instance, we demonstrate that the numerical integration method employed for integrals calculations may induce errors when the number of temporal channels is low. We report theoretical generalized expressions to compensate for these deviations and conserve the semicircle integrity, facilitating the comparison between fluorescence lifetime imaging microscopy images acquired with distinct channels number. These theoretical generalized expressions were finally corroborated with both Monte Carlo simulations and experiments.

Fluorescence lifetime imaging – techniques and applications

Fluorescence lifetime imaging (FLIM) uses the fact that the fluorescence lifetime of a fluorophore depends on its molecular environment but not on its concentration. Molecular effects in a sample can therefore be investigated independently of the variable, and usually unknown concentration of the fluorophore. There is a variety of technical solutions of lifetime imaging in microscopy. The technical part of this paper focuses on time-domain FLIM by multidimensional time-correlated single photon counting, time-domain FLIM by gated image intensifiers, frequency-domain FLIM by gain-modulated image intensifiers, and frequency-domain FLIM by gain-modulated photomultipliers. The application part describes the most frequent FLIM applications: Measurement of molecular environment parameters, protein-interaction measurements by Förster resonance energy transfer (FRET), and measurements of the metabolic state of cells and tissue via their autofluorescence. Measurements of local environment parameters are based on lifetime changes induced by fluorescence quenching or conformation changes of the fluorophores. The advantage over intensity-based measurements is that no special ratiometric fluorophores are needed. Therefore, a much wider selection of fluorescence markers can be used, and a wider range of cell parameters is accessible. FLIM-FRET measures the change in the decay function of the FRET donor on interaction with an acceptor. FLIM-based FRET measurement does not have to cope with problems like donor bleedthrough or directly excited acceptor fluorescence. This relaxes the requirements to the absorption and emission spectra of the donors and acceptors used. Moreover, FLIM-FRET measurements are able to distinguish interacting and noninteracting fractions of the donor, and thus obtain independent information about distances and interacting and noninteracting protein fractions. This is information not accessible by steady-state FRET techniques. Autofluorescence FLIM exploits changes in the decay parameters of endogenous fluorophores with the metabolic state of the cells or the tissue. By resolving changes in the binding, conformation, and composition of biologically relevant compounds FLIM delivers information not accessible by steady-state fluorescence techniques.

Total variation versus wavelet‐based methods for image denoising in fluorescence lifetime imaging microscopy

We report the first application of wavelet-based denoising (noise removal) methods to time-domain box-car fluorescence lifetime imaging microscopy (FLIM) images and compare the results to novel total variation (TV) denoising methods. Methods were tested first on artificial images and then applied to low-light live-cell images. Relative to undenoised images, TV methods could improve lifetime precision up to 10-fold in artificial images, while preserving the overall accuracy of lifetime and amplitude values of a single-exponential decay model and improving local lifetime fitting in live-cell images. Wavelet-based methods were at least 4-fold faster than TV methods, but could introduce significant inaccuracies in recovered lifetime values. The denoising methods discussed can potentially enhance a variety of FLIM applications, including live-cell, in vivo animal, or endoscopic imaging studies, especially under challenging imaging conditions such as low-light or fast video-rate imaging.

Quantitative comparison of polar approach versus fitting method in time domain FLIM image analysis

We calculate here analytically the performance of the polar approach (or phasor) in terms of signal-to-noise ratio and F values when performing time-domain Fluorescence Lifetime Imaging Microscopy (FLIM) to determine the minimal number of photons necessary for FLIM measurements (which is directly related to the F value), and compare them to those obtained from a well-known fitting strategy using the Least Square Method (LSM). The importance of the fluorescence background on the lifetime measurement precision is also investigated. We demonstrate here that the LSM does not provide the best estimator of the lifetime parameter for fluorophores exhibiting mono-exponential intensity decays as soon as fluorescence background is superior to 5%. The polar approach enables indeed to determine more precisely the lifetime values for a limited range corresponding to usually encountered fluorescence lifetime values. These theoretical results are corroborated with Monte Carlo simulations. We finally demonstrate experimentally that the polar approach allows distinguishing in living cells two fluorophores undetectable with usual time-domain LSM fitting software.