Fluorescence Lifetime Imaging Microscopy System Research Articles

Metabolic imaging of human embryos is predictive of ploidy status but is not associated with clinical pregnancy outcomes: a pilot trial.

Does fluorescence lifetime imaging microscopy (FLIM)-based metabolic imaging assessment of human blastocysts prior to frozen transfer correlate with pregnancy outcomes? FLIM failed to distinguish consistent patterns in mitochondrial metabolism between blastocysts leading to pregnancy compared to those that did not. FLIM measurements provide quantitative information on NAD(P)H and flavin adenine dinucleotide (FAD+) concentrations. The metabolism of embryos has long been linked to their viability, suggesting the potential utility of metabolic measurements to aid in selection. This was a pilot trial enrolling 121 IVF couples who consented to have their frozen blastocyst measured using non-invasive metabolic imaging. After being warmed, 105 couples' good-quality blastocysts underwent a 6-min scan in a controlled temperature and gas environment. FLIM-assessed blastocysts were then transferred without any intervention in management. Eight metabolic parameters were obtained from each blastocyst (4 for NAD(P)H and 4 for FAD): short and long fluorescence lifetime, fluorescence intensity, and fraction of the molecule engaged with enzyme. The redox ratio (intensity of NAD(P)H)/(intensity of FAD) was also calculated. FLIM data were combined with known metadata and analyzed to quantify the ability of metabolic imaging to differentiate embryos that resulted in pregnancy from embryos that did not. De-identified discarded aneuploid human embryos (n = 158) were also measured to quantify correlations with ploidy status and other factors. Statistical comparisons were performed using logistic regression and receiver operating characteristic (ROC) curves with 5-fold cross-validation averaged over 100 repeats with random sampling. AUC values were used to quantify the ability to distinguish between classes. No metabolic imaging parameters showed significant differences between good-quality blastocysts resulting in pregnancy versus those that did not. A logistic regression using metabolic data and metadata produced an ROC AUC of 0.58. In contrast, robust AUCs were obtained when classifying other factors such as comparison of Day 5 (n = 64) versus Day 6 (n = 41) blastocysts (AUC = 0.78), inner cell mass versus trophectoderm (n = 105: AUC = 0.88) and aneuploid (n = 158) versus euploid and positive pregnancy embryos (n = 108) (AUC = 0.82). The study protocol did not select which embryo to transfer and the cohort of 105 included blastocysts were all high quality. The study was also limited in number of participants and study sites. Increased power and performing the trial in more sites may have provided a stronger conclusion regarding the merits of the use of FLIM clinically. FLIM failed to distinguish consistent patterns in mitochondrial metabolism between good-quality blastocysts leading to pregnancy compared to those that did not. Blastocyst ploidy status was, however, highly distinguishable. In addition, embryo regions and embryo day were consistently revealed by FLIM. While metabolic imaging detects mitochondrial metabolic features in human blastocysts, this pilot trial indicates it does not have the potential to serve as an effective embryo viability detection tool. This may be because mitochondrial metabolism plays an alternative role post-implantation. This study was sponsored by Optiva Fertility, Inc. Boston IVF contributed to the clinical site and services. Becker Hickl, GmbH, provided the FLIM system on loan. T.S. was the founder and held stock in Optiva Fertility, Inc., and D.S. and E.S. had options with Optiva Fertility, Inc., during this study. The study was approved by WCG Connexus IRB (Study Number 1298156).

Light-sheet autofluorescence lifetime imaging with a single-photon avalanche diode array.

Fluorescence lifetime imaging microscopy (FLIM) of the metabolic co-enzyme nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] is a popular method to monitor single-cell metabolism within unperturbed, living 3D systems. However, FLIM of NAD(P)H has not been performed in a light-sheet geometry, which is advantageous for rapid imaging of cells within live 3D samples. We aim to design, validate, and demonstrate a proof-of-concept light-sheet system for NAD(P)H FLIM. A single-photon avalanche diode camera was integrated into a light-sheet microscope to achieve optical sectioning and limit out-of-focus contributions for NAD(P)H FLIM of single cells. An NAD(P)H light-sheet FLIM system was built and validated with fluorescence lifetime standards and with time-course imaging of metabolic perturbations in pancreas cancer cells with 10s integration times. NAD(P)H light-sheet FLIM in vivo was demonstrated with live neutrophil imaging in a larval zebrafish tail wound also with 10s integration times. Finally, the theoretical and practical imaging speeds for NAD(P)H FLIM were compared across laser scanning and light-sheet geometries, indicating a to acquisition speed advantage for the light sheet compared to the laser scanning geometry. FLIM of NAD(P)H is feasible in a light-sheet geometry and is attractive for 3D live cell imaging applications, such as monitoring immune cell metabolism and migration within an organism.

Computational Photon Counting Using Multithreshold Peak Detection for Fast Fluorescence Lifetime Imaging Microscopy.

Time-resolved photon counting methods have a finite bandwidth that restricts the acquisition speed of techniques like fluorescence lifetime imaging microscopy (FLIM). To enable faster imaging, computational methods can be employed to count photons when the output of a detector is directly digitized at a high sampling rate. Here, we present computational photon counting using a hybrid photodetector in conjunction with multithreshold peak detection to count instances where one or more photons arrive at the detector within the detector response time. This method can be used to distinguish up to five photon counts per digitized point, whereas previous demonstrations of computational photon counting on data acquired with photomultiplier tubes have only counted one photon at a time. We demonstrate in both freely moving C. elegans and a human breast cancer cell line undergoing apoptosis that this novel multithreshold peak detection method can accurately characterize the intensity and fluorescence lifetime of samples producing photon rates up to 223%, higher than previously demonstrated photon counting FLIM systems.

Use of nanosecond excitation pulses in fluorescence lifetime measurement via phasor analysis.

We investigated the possibility of using long excitation pulses in fluorescence lifetime imaging microscopy (FLIM) using phasor analysis. It has long been believed that the pulse width of an excitation laser must be shorter than the lifetime of a fluorophore in a time-domain FLIM system. Even though phasor analysis can effectively minimize the pulse effect by using deconvolution, the precision of a measured lifetime can be degraded seriously. Here, we provide a fundamental theory on pulse-width-dependent measurement precisions in lifetime measurement in the phasor plane. Our theory predicts that high-precision lifetimes can be obtained even with a laser whose pulse width is four times larger than the lifetime of a fluorophore. We have experimentally demonstrated this by measuring the lifetimes of fluorescence probes with 2.57 ns and 3.75 ns lifetimes by using various pulse widths (0.52-38 ns) and modulation frequencies (10-200 MHz). We believe our results open a new possibility of using long pulse-width lasers for high-precision FLIM.

Phasor S-FLIM for the physiological profiling of living cells

Fluorescence Lifetime Imaging Microscopy (FLIM) and spectral imaging are two broadly applied methods for increasing dimensionality in microscopy. However, their combination is typically inefficient and slow in terms of both acquisition and processing. By integrating technological and computational advances, we developed a robust and unbiased Spectral FLIM (S-FLIM) system capable of extracting detailed and precise information about the photophysics of fluorescent specimens at optical resolution. The data are processed with the phasor approach to provide unbiased and fast analysis of S-FLIM data.

Simple phasor-based deep neural network for fluorescence lifetime imaging microscopy

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to probe the molecular environment of fluorophores. The analysis of FLIM images is usually performed with time consuming fitting methods. For accelerating this analysis, sophisticated deep learning architectures based on convolutional neural networks have been developed for restrained lifetime ranges but they require long training time. In this work, we present a simple neural network formed only with fully connected layers able to analyze fluorescence lifetime images. It is based on the reduction of high dimensional fluorescence intensity temporal decays into four parameters which are the phasor coordinates, the mean and amplitude-weighted lifetimes. This network called Phasor-Net has been applied for a time domain FLIM system excited with an 80 MHz laser repetition frequency, with negligible jitter and afterpulsing. Due to the restricted time interval of 12.5 ns, the training range of the lifetimes was limited between 0.2 and 3.0 ns; and the total photon number was lower than 106, as encountered in live cell imaging. From simulated biexponential decays, we demonstrate that Phasor-Net is more precise and less biased than standard fitting methods. We demonstrate also that this simple architecture gives almost comparable performance than those obtained from more sophisticated networks but with a faster training process (15 min instead of 30 min). We finally apply successfully our method to determine biexponential decays parameters for FLIM experiments in living cells expressing EGFP linked to mCherry and fused to a plasma membrane protein.

Real-time pixelwise phasor analysis for video-rate two-photon fluorescence lifetime imaging microscopy.

Two-photon fluorescence lifetime imaging microscopy (FLIM) is a widely used technique in biomedical optical imaging. Presently, many two-photon time-domain FLIM setups are limited by long acquisition and postprocessing times that decrease data throughput and inhibit the ability to image fast sub-second processes. Here, we present a versatile two-photon FLIM setup capable of video-rate (up to 25 fps) imaging with graphics processing unit (GPU)-accelerated pixelwise phasor analysis displayed and saved simultaneously with acquisition. The system uses an analog output photomultiplier tube in conjunction with 12-bit digitization at 3.2 GHz to overcome the limited maximum acceptable photon rate associated with the photon counting electronics in many FLIM systems. This allows for higher throughput FLIM acquisition and analysis, and additionally enables the user to assess sample fluorescence lifetime in real-time. We further explore the capabilities of the system to examine the kinetics of Rhodamine B uptake by human breast cancer cells and characterize the effect of pixel dwell time on the reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) autofluorescence lifetime estimation accuracy.

Focus on analytical equipment, spectroscopy, and spectrometry

Focus on analytical equipment, spectroscopy, and spectrometry

Phasor S-FLIM: a new paradigm for fast and robust spectral fluorescence lifetime imaging.

Fluorescence lifetime imaging microscopy (FLIM) and spectral imaging are two broadly applied methods for increasing dimensionality in microscopy. However, their combination is typically inefficient and slow in terms of acquisition and processing. By integrating technological and computational advances, we developed a robust and unbiased spectral FLIM (S-FLIM) system. Our method, Phasor S-FLIM, combines true parallel multichannel digital frequency domain electronics with a multidimensional phasor approach to extract detailed and precise information about the photophysics of fluorescent specimens at optical resolution. To show the flexibility of the Phasor S-FLIM technology and its applications to the biological and biomedical field, we address four common, yet challenging, problems: the blind unmixing of spectral and lifetime signatures from multiple unknown species, the unbiased bleedthrough- and background-free Förster resonance energy transfer analysis of biosensors, the photophysical characterization of environment-sensitive probes in living cells and parallel four-color FLIM imaging in tumor spheroids.

Label-free characterization of single extracellular vesicles using two-photon fluorescence lifetime imaging microscopy of NAD(P)H

The heterogeneous nature of extracellular vesicles (EVs) creates the need for single EV characterization techniques. However, many common biochemical and functional EV analysis techniques lack single EV resolution. Two-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to functionally characterize the reduced form of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate (NAD(P)H) in cells and tissues. Here, we demonstrate that FLIM can also be used to image and characterize NAD(P)H in single isolated EVs. EVs were isolated using standard differential ultracentrifugation techniques from multiple cell lines and imaged using a custom two-photon FLIM system. The presented data show that the NAD(P)H fluorescence lifetimes in isolated cell-derived EVs follow a wide Gaussian distribution, indicating the presence of a range of different protein-bound and free NAD(P)H species. EV NAD(P)H fluorescence lifetime distribution has a larger standard deviation than that of cells and a significantly different fluorescence lifetime distribution than the nuclei, mitochondria, and cytosol of cells. Additionally, changes in the metabolic conditions of cells were reflected in changes in the mean fluorescence lifetime of NAD(P)H in the produced EVs. These data suggest that FLIM of NAD(P)H could be a valuable tool for EV research.

Visualization of Lipid Droplets in Living Cells and Fatty Livers of Mice Based on the Fluorescence of π-Extended Coumarin Using Fluorescence Lifetime Imaging Microscopy.

Lipid droplets (LDs) are closely related to lipid metabolism in living cells and are highly associated with diverse diseases such as fatty liver, diabetes, and cancer. Herein we describe a π-extended fluorescent coumarin (PC6S) for visualizing LDs in living cells and in the tissues of living mice using confocal fluorescence lifetime imaging microscopy (FLIM). PC6S showed a large positive solvatochromic shift and high fluorescence quantum yield (>0.80) in both nonpolar and polar solvents. Additionally, the fluorescence lifetimes of PC6S were largely dependent on solvent polarity. The excellent spectral and photophysical properties of PC6S allowed its selective staining of LDs in living and fixed cells, and multicolor imaging. Fluorescence lifetime measurements of PC6S allowed estimation of the apparent polarity of LDs. The high photostability and long intracellular retention of PC6S supported in situ visualization of the formation processes of LDs resulting from the accumulation of fatty acid. Furthermore, intravenous administration of PC6S and use of the FLIM system allowed the imaging of LDs in hepatocytes in living normal mice and the growth of LDs resulting from the excess accumulation of lipids in high-fat-diet-fed mice (fatty liver model mice). Taking advantage of the high selectivity and sensitivity of PC6S for LDs in liver, we could visualize the adipocytes of lipid-rich tissues and LDs in kidney peritubular cells by PC6S fluorescence. These results demonstrated that PC6S combined with a FLIM system can be useful for monitoring and tracking the formation of LDs in both cultured cells and specific tissues and organs.

Live-cell FLIM-FRET using a commercially available system.

Förster resonance energy transfer (FRET)-based sensors have been powerful tools in cell biologists' toolkit for decades. Informed by fundamental understanding of fluorescent proteins, protein-protein interactions, and the structural biology of reporter components, researchers have been able to employ creative design approaches to build sensors that are uniquely capable of probing a wide range of phenomena in living cells including visualization of localized calcium signaling, sub-cellular activity gradients, and tension generation to name but a few. While FRET sensors have significantly impacted many fields, one must also be cognizant of the limitations to conventional, intensity-based FRET measurements stemming from variation in probe concentration, sensitivity to photobleaching, and bleed-through between the FRET fluorophores. Fluorescence lifetime imaging microscopy (FLIM) largely overcomes the limitations of intensity-based FRET measurements. In general terms, FLIM measures the time, which for the reporters described in this chapter is nanoseconds (ns), between photon absorption and emission by a fluorophore. When FLIM is applied to FRET sensors (FLIM-FRET), measurement of the donor fluorophore lifetime provides valuable information such as FRET efficiency and the percentage of reporters engaged in FRET. This chapter introduces fundamental principles of FLIM-FRET toward informing the practical application of the technique and, using two established FRET reporters as proofs of concept, outlines how to use a commercially available FLIM system.

Fast single-cell biochemistry: theory, open source microscopy and applications

Fluorescence lifetime sensing enables researchers to probe the physicochemical environment of a fluorophore providing a window through which we can observe the complex molecular make-up of the cell. Fluorescence lifetime imaging microscopy (FLIM) quantifies and maps cell biochemistry, a complex ensemble of dynamic processes. Unfortunately, typical high-resolution FLIM systems exhibit rather limited acquisition speeds, often insufficient to capture the time evolution of biochemical processes in living cells. Here, we describe the theoretical background that justifies the developments of high-speed single photon counting systems. We show that systems with low dead-times not only result in faster acquisition throughputs but also improved dynamic range and spatial resolution. We also share the implementation of hardware and software as an open platform, show applications of fast FLIM biochemical imaging on living cells and discuss strategies to balance precision and accuracy in FLIM. The recent innovations and commercialisation of fast time-domain FLIM systems are likely to popularise FLIM within the biomedical community, to impact biomedical research positively and to foster the adoption of other FLIM techniques as well. While supporting and indeed pursuing these developments, with this work we also aim to warn the community about the possible shortcomings of fast single photon counting techniques and to highlight strategies to acquire data of high quality.

Automatic segmentation of intravital fluorescence microscopy images by K-means clustering of FLIM phasors

Fluorescence lifetime imaging microscopy (FLIM) provides additional contrast for fluorophores with overlapping emission spectra. The phasor approach to FLIM greatly reduces the complexity of FLIM analysis and enables a useful image segmentation technique by selecting adjacent phasor points and labeling their corresponding pixels with different colors. This phasor labeling process, however, is empirical and could lead to biased results. In this Letter, we present a novel and unbiased approach to automate the phasor labeling process using an unsupervised machine learning technique, i.e., K-means clustering. In addition, we provide an open-source, user-friendly program that enables users to easily employ the proposed approach. We demonstrate successful image segmentation on 2D and 3D FLIM images of fixed cells and living animals acquired with two different FLIM systems. Finally, we evaluate how different parameters affect the segmentation result and provide a guideline for users to achieve optimal performance.

Generalized stepwise optical saturation enables super-resolution fluorescence lifetime imaging microscopy.

We present a novel super-resolution fluorescence lifetime microscopy technique called generalized stepwise optical saturation (GSOS) that generalizes and extends the concept of the recently demonstrated stepwise optical saturation (SOS) super-resolution microscopy [Biomed. Opt. Express 9, 1613 (2018)29675306]. The theoretical basis of GSOS is developed based on exploring the dynamics of a two-level fluorophore model and using perturbation theory. We show that although both SOS and GSOS utilize the linear combination of M raw images to increase the imaging resolution by a factor of , SOS is a special and the simplest case of GSOS. The super-resolution capability is demonstrated with theoretical analysis and numerical simulations for GSOS with sinusoidal and pulse-train modulations. Using GSOS with pulse-train modulation, super-resolution and fluorescence lifetime imaging microscopy (FLIM) images can be obtained simultaneously. The super-resolution FLIM capability is experimentally demonstrated with a cell sample on a custom-built two-photon frequency-domain (FD) FLIM system based on radio frequency analog signal processing. To our knowledge, this is the first implementation of super-resolution imaging in FD-FLIM.

Parallelized fluorescence lifetime imaging microscopy (FLIM) based on photon reassignment

We propose a novel parallelized fluorescence lifetime imaging microscopy (FLIM) method based on photon reassignment with a detector array and time-correlated single photon counting (TCSPC) array. The detector array composed of seven avalanche photon diodes (APDs) substitutes for original pinhole and detector to constitute an imaging scanning microscopy (ISM) system. The introduction of parallel detection can solve the pile-up effect caused by the dead-time in TCSPC measurement, which can enhance the effective photon detection efficiency and notably increase the fluorescence lifetime imaging speed. More importantly, the resolution and signal–noise ratio (SNR) can be enhanced based on photon reassignment. The simulation results embody the superiority of our FLIM system, and the experiments with different samples validate the improvement of the resolution and speed of fluorescence lifetime imaging. The experiment results show that the resolution of our method is enhanced by a factor of 1.5 and the imaging speed can be at least increased by a factor of 2.5 compared with conventional confocal TCSPC FLIM system. We envision that the proposed FLIM method based on parallel detection will allow for super-resolution fluorescence lifetime real-time imaging of live cell.

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.

Single pulse two photon fluorescence lifetime imaging (SP-FLIM) with MHz pixel rate.

Two-photon-excited fluorescence lifetime imaging microscopy (FLIM) is a chemically specific 3-D sensing modality providing valuable information about the microstructure, composition and function of a sample. However, a more widespread application of this technique is hindered by the need for a sophisticated ultra-short pulse laser source and by speed limitations of current FLIM detection systems. To overcome these limitations, we combined a robust sub-nanosecond fiber laser as the excitation source with high analog bandwidth detection. Due to the long pulse length in our configuration, more fluorescence photons are generated per pulse, which allows us to derive the lifetime with a single excitation pulse only. In this paper, we show high quality FLIM images acquired at a pixel rate of 1 MHz. This approach is a promising candidate for an easy-to-use and benchtop FLIM system to make this technique available to a wider research community.

Automated High-Throughput Fluorescence Lifetime Imaging Microscopy to Detect Protein-Protein Interactions.

Fluorescence resonance energy transfer (FRET) is widely used to study conformational changes of macromolecules and protein-protein, protein-nucleic acid, and protein-small molecule interactions. FRET biosensors can serve as valuable secondary assays in drug discovery and for target validation in mammalian cells. Fluorescence lifetime imaging microscopy (FLIM) allows precise quantification of the FRET efficiency in intact cells, as FLIM is independent of fluorophore concentration, detection efficiency, and fluorescence intensity. We have developed an automated FLIM system using a commercial frequency domain FLIM attachment (Lambert Instruments) for wide-field imaging. Our automated FLIM system is capable of imaging and analyzing up to 50 different positions of a slide in less than 4 min, or the inner 60 wells of a 96-well plate in less than 20 min. Automation is achieved using a motorized stage and controller (Prior Scientific) coupled with a Zeiss Axio Observer body and full integration into the Lambert Instruments FLIM acquisition software. As an application example, we analyze the interaction of the oncoprotein Ras and its effector Raf after drug treatment. In conclusion, our automated FLIM imaging system requires only commercial components and may therefore allow for a broader use of this technique in chemogenomics projects.

High-speed multifocal array scanning using refractive window tilting.

Confocal microscopy has several advantages over wide-field microscopy, such as out-of-focus light suppression, 3D sectioning, and compatibility with specialized detectors. While wide-field microscopy is a faster approach, multiplexed confocal schemes can be used to make confocal microscopy more suitable for high-throughput applications, such as high-content screening (HCS) commonly used in drug discovery. An increasingly powerful modality in HCS is fluorescence lifetime imaging microscopy (FLIM), which can be used to measure protein-protein interactions through Förster resonant energy transfer (FRET). FLIM-FRET for HCS combines the requirements of high throughput, high resolution and specialized time-resolving detectors, making it difficult to implement using wide-field and spinning disk confocal approaches. We developed a novel foci array scan method that can achieve uniform multiplex confocal acquisition using stationary lenslet arrays for high resolution and high throughput FLIM. Unlike traditional mirror galvanometers, which work in Fourier space between scan lenses, this scan method uses optical flats to steer a 2-dimension foci array through refraction. After integrating this scanning scheme in a multiplexing confocal FLIM system, we demonstrate it offers clear benefits over traditional mirror galvanometer scanners in scan linearity, uniformity, cost and complexity.