Real-time phasor analysis of fast FLIM for visualizing metabolic dynamics

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.

Fluorescence lifetime imaging microscopy (FLIM) provides valuable quantitative insights into fluorophores' chemical microenvironment. Due to long computation times and the lack of accessible, open-source real-time analysis toolkits, traditional analysis of FLIM data, particularly with the widely used time-correlated single-photon counting (TCSPC) approach, typically occurs after acquisition. As a result, uncertainties about the quality of FLIM data persist even after collection, frequently necessitating the extension of imaging sessions. Unfortunately, prolonged sessions not only risk missing important biological events but also cause photobleaching and photodamage. We present the first open-source program designed for real-time FLIM analysis during specimen scanning to address these challenges. Our approach combines acquisition with real-time computational and visualization capabilities, allowing us to assess FLIM data quality on the fly. Our open-source real-time FLIM viewer, integrated as a Napari plugin, displays phasor analysis and rapid lifetime determination (RLD) results computed from real-time data transmitted by acquisition software such as the open-source Micro-Manager-based OpenScan package. Our method facilitates early identification of FLIM signatures and data quality assessment by providing preliminary analysis during acquisition. This not only speeds up the imaging process, but it is especially useful when imaging sensitive live biological samples.

Probing of local molecular environment in cells is of significant value in creating a fundamental understanding of cellular processes and molecular profiles of diseases, as well as studying drug cell interactions. In order to investigate the dynamically changing in subcellular environment during RNA synthesis, we applied two-photon excited fluorescence lifetime imaging microscopy (FLIM) method to monitor the green fluorescent protein (GFP) fused nuclear protein ASF/SF2. The fluorescence lifetime of fluorophore is known to be in inverse correlation with a local refractive index, and thus fluorescence lifetimes of GFP fusions provide real-time information of the molecular environment of ASF/SF2- GFP. The FLIM results showed continuous and significant fluctuations of fluorescence lifetimes of the fluorescent protein fusions in live HeLa cells under physiological conditions. The fluctuations of fluorescence lifetime values indicated the variations of activities of RNA polymerases. Moreover, treatment with pharmacological drugs inhibiting RNA polymerase activities led to irreversible decreases of fluorescence lifetime values. In summary, our study of FLIM imaging of GFP fusion proteins has provided a sensitive and real-time method to investigate RNA synthesis in live cell nuclei.

Artificial intelligence (AI)/machine-learning (ML) algorithms have been heavily used in data processing in various biological and clinical applications. Sensitive biological signaling can be monitored using two-photon fluorescence lifetime imaging microscopy (FLIM). Lifetime fitting, processing, and analyzing FLIM data of biological specimens can be a challenging and time-consuming affair. The recently developed Fluorescence Lifetime Redox Ratio (FLIRR) focuses on tracking metabolic changes ‘before-and-after-treatment’ in live cells using only two lifetime parameters. FLIM data produces many data parameters which are all associated with drug response in living cells. To predict drug cellular responses, we have chosen the Becker & Hickl SPCImage software to fit the lifetime images and the resultant data was used in ML analysis. With the objective of achieving even more robust statistical power predicting earliest drug effects, we developed Python software and autoencoder (AE) models to analyze the multiple biophysical FLIM parameters acquired in 2p-FLIM images of drug response in cervical cancer cells. The use of systematic hyperparameter (HP) tuning shows the variation in performance across the different models, enabling the selection of the highest performing model and HPs for repetition. Our results show that our optimized multi-parameter trained AE models can statistically outperform single FLIRR time-course analysis in discriminating earliest metabolic changes following drug treatment.

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.

.Two-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to capture autofluorescence signals from cellular components to investigate dynamic physiological changes in live cells and tissues. Among these intrinsic fluorophores, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD)—essential coenzymes in cellular respiration—have been used as intrinsic fluorescent biomarkers for metabolic states in cancer and other pathologies. Traditional FLIM imaging for NAD(P)H, FAD, and in particular fluorescence lifetime redox ratio (FLIRR) requires a sequential multiwavelength excitation to avoid spectral bleed-through (SBT). This sequential imaging complicates image acquisition, may introduce motion artifacts, and reduce temporal resolution. Testing several two-photon excitation wavelengths in combination with optimized emission filters, we have proved a FLIM imaging protocol, allowing simultaneous image acquisition with a single 800-nm wavelength excitation for NADH and FAD with negligible SBT. As a first step, standard NADH and FAD single and mixed solutions were tested that mimic biological sample conditions. After these optimization steps, the assay was applied to two prostate cancer live cell lines: African-American (AA) and Caucasian-American (LNCaP), used in our previous publications. FLIRR result shows that, in cells, the 800-nm two-photon excitation wavelength is suitable for NADH and FAD FLIM imaging with negligible SBT. While NAD(P)H signals are decreased, sufficient photons are present for accurate lifetime fitting and FAD signals are measurably increased at lower laser power, compared with the common 890-nm excitation conditions. This single wavelength excitation allows a simplification of NADH and FAD FLIM imaging data analysis, decreasing the total imaging time. It also avoids motion artifacts and increases temporal resolution. This simplified assay will also make it more suitable to be applied in a clinical setting.

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.

Metabolism is a complex phenomenon in the biological systems, falling into one of two general categories: catabolic and anabolic reactions, or the processes of breaking down and building up, respectively. Changes in cell or tissue metabolic states respond to changes in physiological demands or the onset of pathological conditions. Auto-fluorescent endogenous biomolecules associated with metabolism such as coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are key players in the production of adenosine triphosphate (ATP)—the energy currency of life. Thus, changes in the abundance of NADH and FAD can serve as noninvasive biomarkers in cells and tissues. There are other auto-fluorescent endogenous molecules, but none as thoroughly investigated as NADH and FAD by advancements in technology such as intensity and lifetime microscopy. The importance of the coenzymes NADH and FAD in metabolism was fully recognized to the scientific world by the great innovator, the late Prof. Britton Chance (1913–2010). He demonstrated cellular NADH auto-fluorescence in 1958 by fluorescence spectrophotometry 1. He developed a fiber optics based fluorimeter to monitor by NADH, the mitochondrial metabolic state of awake rat brains during 1962 2. He has published over 1,500 research articles. Prof. Chance's passion was in translational research and mentoring diverse student populations—regardless of their gender, ethnicity, nationality, religion, or social status—to influence research communities all over the world. Prof. Chance students regarded him as a role model, great mentor, and dear friend. Prof. Chance has tirelessly promoted scientific cooperation and academic exchanges between East and West 3. The ability to make these visible the endogenous biomarkers introduced by Prof. Chance led to their characterization and pathways. During glycolysis, two molecules of ATP are produced in the cytoplasm. Cytoplasmic NADH is transported to mitochondria by malate–aspartate shuttle 4 where, NADH is oxidized to NAD+ producing malate; malate is transported to mitochondrial matrix, mitochondrial NAD+ is reduced to NADH which is in turn is oxidized to NAD+ by the electron transport chain (ETC) in the respiratory chain in the mitochondrial matrix 4, 5. Mitochondrial NADH is produced by citric acid cycle or Kreb's cycle based on several substrates. The energy stored in mitochondrial NADH is transferred to an electrochemical proton gradient that is essential to drive phosphorylation of ADP to ATP by F0F1 ATP synthase, to produce approximately 30 units of ATP, the energy currency of the cell (see Fig. 1 reproduced from the review by Schaefer et al. [in this issue, 34]). The coenzymes FMN/FAD are derived from riboflavin (vitamin B 2) and are covalently bound to flavoproteins in enzymatic reactions of the Kreb's cycle and ETC which takes place in the mitochondria 4. The ETC complex II of succinate dehydrogenase contains bound FAD cofactor which is reduced to FADH2. FADH2 is then oxidized to FAD by Coenzyme Q 4, 5. Only the oxidized form of the flavins, FpOx (FAD or FMN) are fluorescent whereas, the reduced forms Fp (FADH2 or FMNH2), are nonfluorescent. On the other hand, NAD(P)H is fluorescent whereas, the oxidized form NAD(P)+ is nonfluorescent 5, 6. Three out of the 20 amino acids, Tyrosine (Try or Y), Phenylalanine (Phe or F), and Tryptophan (Trp or W), all aromatic, are autofluorescent absorbing and emitting in the UV region. Tryptophan (Trp), an essential amino acid is of particular interest in the biomedical sciences, their fluorescence intensity and lifetime providing information on protein abundance and is dysregulated in cancer 7, 8. Trp is moderately fluorescent with a quantum yield of 0.1–0.5 and has the highest absorption coefficient of the 3 amino acids, and therefore the highest brightness and the longest emission wavelength of 350 nm 8. Alterations in the tryptophan metabolism are suggested to be a common pathological pathway for clinically distinct neurodegenerative disorders, like Parkinson's, Huntington's, and Alzheimer's diseases 9. Modern technologies such as fluorescence lifetime imaging (FLIM) produce endogenous fluorophore data as readouts of mitochondrial function in their natural environment for investigators in the biological and clinical sciences. The unique characteristics of lifetime are that it is sensitive to environmental changes and insensitive to change in fluorophore concentration or excitation intensity. Lifetime helps investigators to fingerprint the molecular functions under various conditions 10. Lifetime techniques have been used for measuring NADH decays since 1967 by spectrofluorometry 11. FLIM provides both spatial and temporal information of molecules and discriminates the bound from free NADH (ref) and NADH from NAD(P)H 12. In this special Cytometry A issue on Metabolism, 10 well-established authors used multiple imaging techniques to explore metabolism in various directions, by: different cell types in 2D and 3D, single cell analyses and their intra- and inter-cell heterogeneities, response to treatment. While imaging the auto-fluorescent NADH and FAD molecules have come a long way to provide insights into the metabolic states of cells and tissue, Schaefer et al. (in this issue, 34) in their very thorough review discuss the “confounding factors” and potential pitfalls—and how to mitigate them. Lukina et al. (in this issue, 47) and Cao et al. (in this issue, 100) discuss additional correlative biomarkers to NADH in second harmonic generation (SHG) of collagen and the putative FRET interaction of Tryptophan (Trp) and NADH, respectively, both in tumors. Lukina et al. (in this issue, 47) investigated the cellular metabolism collagen in colorectal cancer model. Their results demonstrates that the changes in cellular metabolism and fibrotic stroma organization precede morphological alterations and tumor size reduction, and that indicates that NAD(P)H and collagen can be considered as intrinsic indicators of the response to colorectal cancer treatment. Cao et al. (in this issue, 110) presents data in HeLa cervical cancer cells upon treatment with doxorubicin to show the correlations of Trp-NADH energy transfer (E%) of putative Trp residues on enzymes interacting with NADH during NADH to NAD+ transition to a novel, entirely FLIM-based redox ratio – FLIRR 13. In addition, the NADPH/NADH ratio as per Blacker 12 is established suggesting the changing balance between catabolism and anabolism/ROS defense. Cong et al. (in this issue, 80) applied 2-photon FLIM to characterize the critical factors such as physical and chemical microenvironmental signaling in in vitro 3-dimensional tumor models. Ramanujan (in this issue, 101) used a flow cytometry assay to monitor NAD(p)H oxidation kinetics and metabolic status in human breast cancer cells. He also investigated tumor cell heterogeneity in primary tumor epithelial cells isolated from preclinical animal models. Ma et al. (in this issue, 93) sorted out the leukemia cell lines based on their metabolic state differences between proliferation and quiescent cells using the droplet microfluidic technology and phasor FLIM. This approach may be useful to upstream phenotypic platform to correlate with genomic statistics. Chorvatova et al. (in this issue, 13) provided a detailed analysis of endogenous flavins fluorescence photobleaching arising in living cardiac cells during spectrally resolved confocal imaging. This investigation led to significant modifications of the cell responsiveness to metabolic modulators and its contractility, suggesting functional metabolic alterations in the recorded cells. FLIM based AFI imaging is a sensitive way to monitor the cellular metabolism but there are many issues involved in the complex biological system and that may influence the variability in the lifetime values. Chacko and Eliceiri (in this issue, 56) described in this article that it is important to consider the cellular microenvironment such as confluency, pH, inter-/intra-cellular heterogeneity, and choice of cell line for accurate quantitative FLIM based AFI measurement of cellular metabolism. Altarkistany et al. (in this issue, 70) monitored the shift in the fluorescence decay of NAD(P)H and correlate this shift to changes in metabolism that cells undergo during apoptosis using cytometry based FLIM acquisition with phasor analysis. They have demonstrated free to bound ratios as the ratio of “short-to-long” (S/L) fluorescence lifetime, where S/L was found to consistently decrease with an increase in apoptosis. This special issue on metabolism highlights the challenges involved in the investigation using biomarkers but also provides great opportunities to advance research by using the advanced technologies such as FLIM and data analysis at single cells for tissue/in vivo metabolism mapping using biomarkers such as FLIRR, Tryptophan and SHG for changes in collagen. We would like to thank Dr. Attila Tarnok, Editor-in-Chief and Steven Perez, Associate Managing Editor for their valuable guidance and help during this administration of this special issue.

We intend to develop an efficient method of measuring respiratory function of the cornea. With this purpose, we resorted to fluorescence lifetime imaging microscopy (FLIM) to monitor the metabolic co-factor flavin adenine dinucleotide (FAD). FAD and nicotinamide adenine dinucleotide (NADH) are co-factors of the electron transport chain. Therefore alterations in amount of these molecules reflect alterations in the metabolism. For assessing the potential of FLIM for metabolic imaging of the cornea, we performed a series of experiments using a time-correlated single photon counting (TCSPC) fluorescence lifetime microscope (Picoquant to MicroTime 100 coupled to an Olympus BX51 Microscope). In this technique, the acquired signal is the convolution between the instrument response function (IRF) and the fluorescence signal from the sample. IRF was acquired using an Erythrosin B solution. In this work we show that it is possible to acquire fluorescence lifetime images of rat and bovine corneas using FAD autofluorescence.

Raman fingerprint spectroscopy and fluorescence lifetime imaging are emerging tools for studying metabolic profiles of biological specimens. While Raman fingerprint spectroscopy detects intrinsic molecular vibrations that reflect the molecular composition and chemical environment of a sample, fluorescence lifetime imaging measures changes in the excited-state lifetime of fluorophores that are sensitive to their microenvironments. Here, we present a multimodal imaging platform combining broadband coherent anti-Stokes Raman scattering (BCARS) and two-photon fluorescence lifetime imaging (2p-FLIM) microscopy that can acquire biologically relevant Raman fingerprint spectra and fluorescence lifetime signals in vivo and simultaneously. The tremendous chemical information obtained from spatially co-registered BCARS and 2p-FLIM images allows us to characterize the subtle differences between sub-cellular compartments and verify the potential false-positive results generated by fluorescence imaging alone. This is demonstrated by directly comparing the BCARS, 2p-FLIM, and two-photon excitation fluorescence (TPEF) signals simultaneously obtained from the same dye-stained organelle in the live, intact C. elegans expressing a green fluorescent protein (GFP) marker. In this work, we introduce the BCARS/2p-FLIM/TPEF setup scheme, the image acquisition steps, data processing, and representative results showing that the cross-modality imaging method enables rigorous characterization and in vivo detection at sub-cellular resolution. This protocol provides a framework for simultaneous chemical and fluorescence lifetime imaging to improve the accuracy of biological interpretation in complex living systems.

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

Cellular metabolic state can serve as a biomarker to indicate the differentiation potential of stem cells into other specialized cell lineages. In this study, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was applied to determine the fluorescence lifetime and the amounts of the auto-fluorescent metabolic co-factor reduced nicotinamide adenine dinucleotide (NADH) to elucidate the cellular metabolism of human mesenchymal stem cells (hMSCs) in osteogenic and adipogenic differentiation processes. 2P-FLIM provides the free to protein-bound NADH ratio which can serve as the indicator of cellular metabolic state. We measured NADH fluorescence lifetime at 0, 7, and 14 days after hMSCs were induced for either osteogenesis or adipogenesis. In both cases, the average fluorescence lifetime increased significantly at day 14 (P < 0.001), while the ratio of free to protein-bound NADH ratio decreased significantly in 7- days (P < 0.001) and 14-days (P < 0.001). Thus, our results indicated a higher metabolic rate in both osteogenic and adipogenic differentiation processes when compared with undifferentiated hMSCs. This approach may be further utilized to study proliferation efficiency and differentiation potential of stem cells into other specialized cell lineages.

Metabolic imaging with the use of fluorescence lifetime imaging microscopy (FLIM) accurately detects mitochondrial dysfunction in mouse oocytes

IntroductionMetabolic reprogramming of cancer and immune cells occurs during tumorigenesis and has a significant impact on cancer progression. Unfortunately, current techniques to measure tumor and immune cell metabolism require sample destruction and/or cell isolations that remove the spatial context. Two-photon fluorescence lifetime imaging microscopy (FLIM) of the autofluorescent metabolic coenzymes nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) provides in vivo images of cell metabolism at a single cell level.MethodsHere, we report an immunocompetent mCherry reporter mouse model for immune cells that express CD4 either during differentiation or CD4 and/or CD8 in their mature state and perform in vivo imaging of immune and cancer cells within a syngeneic B78 melanoma model. We also report an algorithm for single cell segmentation of mCherry-expressing immune cells within in vivo images.ResultsWe found that immune cells within B78 tumors exhibited decreased FAD mean lifetime and an increased proportion of bound FAD compared to immune cells within spleens. Tumor infiltrating immune cell size also increased compared to immune cells from spleens. These changes are consistent with a shift towards increased activation and proliferation in tumor infiltrating immune cells compared to immune cells from spleens. Tumor infiltrating immune cells exhibited increased FAD mean lifetime and increased protein-bound FAD lifetime compared to B78 tumor cells within the same tumor. Single cell metabolic heterogeneity was observed in both immune and tumor cells in vivo.DiscussionThis approach can be used to monitor single cell metabolic heterogeneity in tumor cells and immune cells to study promising treatments for cancer in the native in vivo context.

Pregnancy-associated breast cancer (PABC) is a rare disease, which is frequently diagnosed at an advanced stage due to limitations in current diagnostic methods. In this study, fluorescence lifetime imaging microscopy (FLIM) was used to study the metabolic changes by measuring maternal blood and umbilical cord blood via the autofluorescence of coenzymes, reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), and flavin adenine dinucleotide (FAD). The NAD(P)H data showed that a PABC case had significant differences compared with normal cases, which may indicate increased glycolysis. The FAD data showed that both maternal and cord blood of PABC had shorter mean lifetimes and higher bound-FAD ratios. The significant differences suggested that FLIM testing of blood samples may be a potential method to assist in PABC non-radiative screening.