Fluorescence Lifetime Imaging Research Articles

UNET-FLIM: A Deep Learning-Based Lifetime Determination Method Facilitating Real-Time Monitoring of Rapid Lysosomal pH Variations in Living Cells.

Lifetime determination plays a crucial role in fluorescence lifetime imaging microscopy (FLIM). We introduce UNET-FLIM, a deep learning architecture based on a one-dimensional U-net, specifically designed for lifetime determination. UNET-FLIM focuses on handling low photon count data with high background noise levels, which are commonly encountered in fast FLIM applications. The proposed network can be effectively trained using simulated decay curves, making it adaptable to various time-domain FLIM systems. Our evaluations of simulated data demonstrate that UNET-FLIM robustly estimates lifetimes and proportions, even when the signal photon count is extremely low and background noise levels are high. Remarkably, UNET-FLIM's insensitivity to noise and minimal photon count requirements facilitate fast FLIM imaging and real-time lifetime analysis. We demonstrate its potential by applying it to monitor rapid lysosomal pH variations in living cells during in situ acetic acid treatment, all without necessitating any modifications to existing FLIM systems.

Accelerating biopharmaceutical cell line selection with label-free multimodal nonlinear optical microscopy and machine learning

The selection of high-performing cell lines is crucial for biopharmaceutical production but is often time-consuming and labor-intensive. We investigated label-free multimodal nonlinear optical microscopy for non-perturbative profiling of biopharmaceutical cell lines based on their intrinsic molecular contrast. Employing simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy with fluorescence lifetime imaging microscopy (FLIM), we characterized Chinese hamster ovary (CHO) cell lines at early passages (0–2). A machine learning (ML)-assisted analysis pipeline leveraged high-dimensional information to classify single cells into their respective lines. Remarkably, the monoclonal cell line classifiers achieved balanced accuracies exceeding 96.8% as early as passage 2. Correlation features and FLIM modality played pivotal roles in early classification. This integrated optical bioimaging and machine learning approach presents a promising solution to expedite cell line selection process while ensuring identification of high-performing biopharmaceutical cell lines. The techniques have potential for broader single-cell characterization applications in stem cell research, immunology, cancer biology and beyond.

Styryl dyes for viscosity measurement and detection of pathological processes in mitochondria of living cells using fluorescence lifetime imaging microscopy, a critical study

In the present work, two molecular rotors based on styryl dyes were synthesized and investigated. Both dyes showed significant sensitivity to the viscosity of the medium. Due to their significant positive charge, they efficiently accumulated in mitochondria of living cells. Binding to mitochondria resulted in increased fluorescence intensity and fluorescence lifetime of the dyes, allowing them to be visualized without washing off the unbound dye. We have also shown that the fluorescence lifetime of the studied molecular rotors is determined not only by the viscosity of the medium, but also by interactions with cell components such as proteins and nucleic acids. The present work clearly shows that these interactions do not allow a reliable estimation of viscosity in cell organelles using the synthesized dyes. This compromises the results of previous viscosity studies using molecular rotors at least of the styryl type. Nevertheless, induction of cell apoptosis by benzylviologen led to an increase in brightness and fluorescence lifetime of the dyes, which can be caused by both changes in viscosity and changes in the expression profile of proteins localized in mitochondria and interacting with the dyes. Thus, the obtained styryl dyes, like previously published homologous compounds, cannot be used for viscosity measurements, but can be used for identification of pathological states of the cell.

A correlation of polymorphic G-quadruplex formation in vitro and in the lysosomes of live cancer cells.

Guanine-rich oligonucleotides (GROs) can fold into G-quadruplex (G4) structures. The diverse roles of G4 structures, particularly as targets for drug design, anticancer agents, and drug delivery systems, highlight their critical significance in cancer research. However, the formation of G4 structures is highly dependent on the specific nucleotide sequences and the number of G-tracts within each GRO. In vitro studies using circular dichroism (CD), nuclear magnetic resonance (NMR), and polyacrylamide gel electrophoresis (PAGE) demonstrated that GROs with fewer than four G-tracts can form intermolecular G4 structures in K+ solution at 37°C. In fluorescence lifetime imaging microscopy study, intermolecular parallel G4 structures, formed by single-stranded GROs containing three G-tracts with three guanines each, were observed to be detectable in the lysosomes of live CL1-0 cancer cells. In contrast, a mutated sequence with only two G-tracts was rarely detected in the lysosomes of CL1-0 cancer cells, highlighting its incapability of forming intermolecular parallel G4s. Furthermore, polymorphic G4 formation in vitro and in-cell studies revealed a potential correlation. Our findings demonstrate that exogenous GROs can be introduced to explore the structural dynamics of G4 formation in live cancer cells, as well as their potential as anticancer agents and drug delivery carriers targeting lysosomes.

Advanced fluorescence lifetime-enhanced multiplexed nanoscopy of cells

Abstract In this review paper, we summarize the significant advancements in the field of fluorescence lifetime imaging microscopy (FLIM), particularly wide-field FLIM with single-molecule sensitivity, achieved using the time-correlated single-photon counting-based position-sensitive LINCam system. Fluorescence lifetime adds valuable information beyond conventional intensity-based imaging, enabling diverse applications across research fields. Here, we focus on three primary bioimaging applications: (I) single-molecule FLIM in the far-red spectral region, (II) fast and multiplexed super-resolution imaging of cells, and (III) three-dimensional super-resolution imaging with high axial localization precision. Recent advances in position-sensitive detector technologies offer exciting opportunities for high-throughput super-resolution imaging with enhanced localization precision.

From principles to practice: a comprehensive guide to FRET-FLIM in plants

Abstract Förster Resonance Energy Transfer combined with Fluorescence Lifetime Imaging Microscopy (FRET-FLIM) is revolutionizing plant biology, by enabling the study of protein–protein interactions (PPIs) within live cells. This manuscript describes the principles of FRET and the practical application of FRET acceptor photobleaching (FRET-APB) in exploring PPIs in vivo. It mainly focuses on the superior characteristics of FRET-FLIM and details the materials and methods for implementing this technique in plants. It provides a profound overview about the required instruments, protocols for sample preparation, methods for calibration and acquisition, and pipelines for data analyses including novel analyses for binding and FRET efficiencies. Furthermore, it discusses the potential pitfalls and challenges related to the sample autofluorescence, protein expression heterogeneity, and acquisition photodamage or bleaching. This works aims to highlight the great prospects of FRET-FLIM in advancing our understanding of PPIs in living plant cells.

Quantitative Profiling pH Heterogeneity of Acidic Endolysosomal Compartments using Fluorescence Lifetime Imaging Microscopy.

The endo-lysosomal system plays a crucial role in maintaining cellular homeostasis and promoting organism fitness. The pH of its acidic compartments is a crucial parameter for proper function, and it is dynamically influenced by both intracellular and environmental factors. Here, we present a method based on fluorescence lifetime imaging microscopy (FLIM) for quantitatively analyzing the pH profiles of acidic endolysosomal compartments in diverse types of primary mammalian cells and in live organism Caenorhabditis elegans. This FLIM-based method exhibits high sensitivity in resolving subtle pH differences, thereby revealing heterogeneity within a cell and across cell types. This method enables rapid measurement of pH changes in the acidic endolysosomal system in response to various environmental stimuli. Furthermore, the fast FLIM measurement of pH-sensitive dyes circumvents the need for transgenic reporters and mitigates potential confounding factors associated with varying dye concentrations or excitation light intensity. This FLIM approach offers absolute pH quantification and highlights the significance of pH heterogeneity and dynamics, offering a valuable tool for investigating lysosomal functions and their regulation in various physiological and pathological contexts.

Smart Applications of Lanthanide Chelates-based Luminescent Probes in Bio-Imaging.

Luminescent Lanthanide (III) (Ln(III)) bioprobes (LLBs) have been extensively used in the last two decades as intracellular molecular probes in bio-imaging for the efficient revelation of analytes, to signal intracellular events (enzymes/protein activity, antigen-antibody interaction), target specific organelles, and determine parameters of particular biophysical interest, to gain important insights on pathologies or diseases. The choice of using a luminescent Ln(III) coordination compound with respect to a common organic fluorophore is intimately connected to how their photophysical sensitization (antenna effect) can be finely tuned and especially triggered to respond (even quantitatively) to a certain biophysical event, condition or analyte. While there are other reviews focused on how to design chromophoric ligands for an efficient sensitization of Ln(III) ions, both in the visible and NIR region, this review is application-driven: it is a small collection of particularly interesting examples where the LLB's emissive information is acquired by imaging the emission intensity and/or the fluorescence lifetime (fluorescence lifetime imaging microscopy, FLIM).

Subcellular Compartmentalization of Glucose Mediated Insulin Secretion

Regulation of blood glucose levels depends on the property of beta cells to couple glucose sensing with insulin secretion. This is accomplished by the concentration-dependent flux of glucose through glycolysis and oxidative phosphorylation, generating ATP. The resulting rise in cytosolic ATP/ADP inhibits KATP channels, inducing membrane depolarization and Ca2+ influx, which prompts insulin secretion. Evidence suggests that this coupling of glucose sensing with insulin secretion may be compartmentalized in the submembrane regions of the beta cell. We investigated the subcellular responses of key components involved in this coupling and found mitochondria in the submembrane zone, some tethered to the cytoskeleton near capillaries. Using Fluorescent Lifetime Imaging Microscopy (FLIM), we observed that submembrane mitochondria were the fastest to respond to glucose. In the most glucose-responsive beta cells, glucose triggers rapid, localized submembrane increases in ATP and Ca2+ as synchronized ~4-min oscillations, consistent with pulsatile insulin release after meals. These findings are consistent with the hypothesis that glucose sensing is coupled with insulin secretion in the submembrane zone of beta cells. This zonal adaptation would enhance both the speed and energy efficiency of beta cell responses to glucose, as only a subset of the most accessible mitochondria would be required to trigger insulin secretion.

Could metabolic imaging and artificial intelligence provide a novel path to non-invasive aneuploidy assessments? A certain clinical need.

Pre-implantation genetic testing for aneuploidy (PGT-A) via embryo biopsy helps in embryo selection by assessing embryo ploidy. However, clinical practice needs to consider the invasive nature of embryo biopsy, potential mosaicism, and inaccurate representation of the entire embryo. This creates a significant clinical need for improved diagnostic practices that do not harm embryos or raise treatment costs. Consequently, there has been an increasing focus on developing non-invasive technologies to enhance embryo selection. Such innovations include non-invasive PGT-A, artificial intelligence (AI) algorithms, and non-invasive metabolic imaging. The latter measures cellular metabolism through autofluorescence of metabolic cofactors. Notably, hyperspectral microscopy and fluorescence lifetime imaging microscopy (FLIM) have revealed unique metabolic activity signatures in aneuploid embryos and human fibroblasts. These methods have demonstrated high accuracy in distinguishing between euploid and aneuploid embryos. Thus, this review discusses the clinical challenges associated with PGT-A and emphasizes the need for novel solutions such as metabolic imaging. Additionally, it explores how aneuploidy affects cell behaviour and metabolism, offering an opinion perspective on future research directions in this field of research.

Perspective: fluorescence lifetime imaging and single-molecule spectroscopy for studying biological condensates

Abstract Biological condensates, often formed via liquid-liquid phase separation (LLPS), are membraneless compartments organizing biochemical reactions. Recent advances have shifted the focus from identifying condensates to elucidating their dynamic biological functions, such as buffering concentrations, mediating reactions, and regulating signaling. These are critical for cellular processes and implicated in diseases like cancer and neurodegeneration. Advanced microscopy techniques, including fluorescence lifetime imaging microscopy (FLIM), FLIM-FRET, and fluorescence correlation spectroscopy (FCS), enable quantitative, real-time investigations of condensate composition, dynamics, material properties, and their responses to environmental stimuli in live cells. This perspective highlights the utility of time-resolved fluorescence and single-molecule spectroscopy techniques for shedding light on condensate functions, properties, and interactions with membranes, offering insights into cellular physiology and pathology.

Multispectral laser-scanning pulse-sampling fluorescence lifetime system for large-scale tissue imaging.

We report a multispectral laser-scanning pulse-sampling fluorescence lifetime imaging (LSPS-FLIm) system designed for rapid, high-resolution imaging of large tissue specimens. This system provides a substantial imaging field of view (FoV) of 6 × 15 cm2 with a high spatial resolution of ∼17.5 µm. The LSPS-FLIm system has been tested on a range of fluorescent dyes, endogenous tissue fluorophores, and tissue specimens with varied sizes and properties. These tests demonstrate the system's versatility in resolving morphological and molecular features, enabling centimeter-scale FoV imaging, and distinguishing complex microstructures in tissue specimens. With the capability to maintain high imaging quality and acquisition speed while minimizing tissue damage, LSPS-FLIm represents a promising advancement in the field of fluorescence lifetime imaging for biological and clinical applications.

Fluorescence lifetime imaging in drug delivery research.

Once an exotic add-on to fluorescence microscopy for life science research, fluorescence lifetime imaging (FLIm) has become a powerful and increasingly utilised technique owing to its self-calibration nature, which affords superior quantification over conventional steady-state fluorescence imaging. This review focuses on the state-of-the-art implementation of FLIm related to the formulation, release, dosage, and mechanism of action of drugs aimed for innovative diagnostics and therapy. Quantitative measurements using FLIm have appeared instrumental for encapsulated drug delivery design, pharmacokinetics and pharmacodynamics, pathological investigations, early disease diagnosis, and evaluation of therapeutic efficacy. Attention is paid to the latest advances in lifetime-engineered nanomaterials and practical instrumentation, which begin to show preclinical and clinical translation potential beyond in vitro samples of cells and tissues. Finally, major challenges that need to be overcome in order to facilitate future perspectives are discussed.

A Single Early Life Acetaminophen Exposure Causes Persistent Abnormalities in the Murine Lung.

Whether early life acetaminophen (APAP) exposures injure the developing lung is controversial. We sought to correlate murine pulmonary developmental expression profiles of Cyp2e1 to susceptibility to APAP exposure. P14 C57BL/6 mice were exposed to APAP (140 mg/kg x 1, IP) and assessed for evidence of a histologic, metabolic, functional, and/or transcriptional pulmonary response. Similar experiments were performed in P14 IL6-/- mice given the controversial role of IL6 in APAP-induced tissue injury. No evidence of hepatic injury was noted in APAP exposed P14 mice. In contrast, within 6 hours of exposure pulmonary tissue demonstrated histologic and functional evidence of injury and increased mitochondrial load by fluorescent lifetime imaging microscopy (FLIM). The pulmonary transcriptional response was marked by increased expression of Cyp2e1, Nrf2 targets, and pro-inflammatory genes. Specifically, APAP exposure increased pulmonary IL6 mRNA, protein and associated STAT3 signaling. In contrast, IL6-/- demonstrated attenuated STAT3 signaling and injury at 6 hours of exposure. At P28, functional and stereologic assessment of both WT and IL6-/- mice exposed to a single dose of APAP at P14 revealed persistent abnormalities consistent with lung enlargement and alveolar simplification. Developmentally regulated surges in pulmonary Cyp2e1 expression correlate with sensitivity to APAP exposures that do not cause recognizable hepatic injury. A single exposure during this developmental window is enough to cause persistent functional and stereological abnormalities. These results highlight the need to further study the relationship between developmentally-regulated pulmonary Cyp2e1 expression, APAP exposures and long-term pulmonary dysfunction.

In vivo two-photon FLIM resolves photosynthetic properties of maize bundle sheath cells.

Maize (Zea mays L.) performs highly efficient C4 photosynthesis by dividing photosynthetic metabolism between mesophyll and bundle sheath cells. In vivo physiological measurements are indispensable for C4 photosynthesis research as photosynthetic activities are easily interrupted by leaf section or cell isolation. Yet, direct in vivo observation regarding bundle sheath cells in the delicate anatomy of the C4 leaf is still challenging. In the current work, we used two-photon fluorescence-lifetime imaging microscopy (two-photon-FLIM) to access the photosynthetic properties of bundle sheath cells on intact maize leaves. The results provide spectroscopic evidence for the diminished total PSII activity in bundle sheath cells at its physiological level and show that the single PSIIs could undergo charge separation as usual. We also report an acetic acid-induced chlorophyll fluorescence quenching on intact maize leaves, which might be a physiological state related to the nonphotochemical quenching mechanism.

Label-free Detection and Characterization of Metabolism in Fresh and Cryopreserved Macrophages.

Cryopreservation is a widely used technique to preserve biological samples for extended periods of time at low temperatures. Even though it is known to have significant effects on cell viability, its effect on their metabolism remains unexplored. Studying how cryopreservation influences the metabolism of cells is important to guarantee the reliability of samples transported between sites for analysis. Optical metabolic imaging allows for the study of cellular metabolism in a label-free manner by using the autofluorescence properties of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), two metabolic coenzymes. The goal of this research is to study the metabolic changes in macrophages after cryopreservation and compare these results with freshly isolated macrophage samples to evaluate if the metabolic data is retained after cryopreservation. The metabolism of macrophages was analyzed with fluorescence lifetime imaging microscopy (FLIM) using a multiphoton microscope. Monocytes were isolated from human whole blood and separated into two groups. In Group 1, freshly isolated monocytes were differentiated into macrophages using macrophage-colony stimulating factor (M-CSF) over 8 days. In Group 2, isolated monocytes were cryopreserved after separation from whole blood and then thawed and stimulated with M-CSF for 8 days. Single-cell analysis showed there are significant changes in FLIM parameters between the groups suggesting that the metabolic data of macrophages is altered after a cryopreservation and cell thawing cycle. Our research bridges the current gap by studying the metabolic changes of cells after cryopreservation using a non-invasive and label-free imaging technique.

Nanoscale Viscometry Reveals an Inherent Mucus Defect in Cystic Fibrosis.

The abnormally viscous and thick mucus is a hallmark of cystic fibrosis (CF). How the mutated CF gene causes abnormal mucus remains an unanswered question of paramount interest. Mucus is produced by the hydration of gel-forming mucin macromolecules that are stored in intracellular granules prior to release. Current understanding of mucin/mucus structure before and after secretion remains limited, and contradictory models exist. Here, we used a molecular viscometer and fluorescence lifetime imaging of human bronchoepithelial cells (Normal and CF) to measure nanometer-scale viscosity. We found significantly elevated intraluminal nanoviscosity in a population of CF mucin granules, indicating an intrinsic, presecretory mucin defect. Nanoviscosity influences protein conformational dynamics and function. Its elevation along the protein secretory pathway could arise from molecular overcrowding, impacting mucin's post-translational processing, hydration, and mucus rheology after release. The nanoviscosity of secreted CF mucus was elevated compared to that of non-CF. Interestingly, it was higher after release than in granules. Validation experiments indicate that reduced mobility of water hydrating mucin macromolecules may contribute to the high nanoviscosity in mucus and mucin granules. This suggests that mucins have a weakly ordered state in granules but adopt a highly ordered, nematic crystalline structure when secreted. This challenges the traditional view of mucus as a porous agarose-like gel and suggests an alternative model for mucin organization before and after secretion. Our study also indicates that endoplasmic reticulum stress due to molecular overcrowding could contribute to mucus pathogenesis in CF cells. It encourages the development of therapeutics that target presecretory mechanisms in CF and other muco-obstructive lung diseases.

Fluorescence Lifetime Endoscopy with a Nanosecond Time-Gated CAPS Camera with IRF-Free Deep Learning Method.

Fluorescence imaging has been widely used in fields like (pre)clinical imaging and other domains. With advancements in imaging technology and new fluorescent labels, fluorescence lifetime imaging is gradually gaining recognition. Our research department is developing the tauCAMTM, based on the Current-Assisted Photonic Sampler, to achieve real-time fluorescence lifetime imaging in the NIR (700-900 nm) region. Incorporating fluorescence lifetime into endoscopy could further improve the differentiation of malignant and benign cells based on their distinct lifetimes. In this work, the capabilities of an endoscopic lifetime imaging system are demonstrated using a rigid endoscope involving various phantoms and an IRF-free deep learning-based method with only 6-time points. The results show that this application's fluorescence lifetime image has better lifetime uniformity and precision with 6-time points than the conventional methods.

Unveiling Microscopic Variations during Photodynamic Therapy via Polarity-Responsive Fluorescence Lifetime Imaging.

Photodynamic therapy is a highly promising method for cancer adjuvant treatment. However, the current research on the microscopic changes during the photodynamic therapy process is still quite limited, which seriously impedes the deep understanding of the procedure. For this purpose, a novel polarity-responsive probe, MI-PPF, with excellent mitochondrial targeting and anchoring capabilities has been rationally designed and synthesized. Notably, MI-PPF has successfully realized the in situ detection of mitochondrial morphology and polarity alterations during the photodynamic therapy process in cancer cells through fluorescence lifetime imaging. The results showed that a series of phenomena such as deformation, shrinkage, vacuolation, and aggregation occurred in the mitochondrial morphology during photodynamic therapy. Concurrently, a decline in mitochondrial polarity is also noted, which may be closely linked to the mitochondrial oxidative stress response during this process. Furthermore, MI-PPF can be used for photodynamic therapy on tumor mouse models and has successfully achieved fluorescence lifetime imaging of tumor sections before and after photodynamic therapy, uncovering multifaceted changes in cell morphology, polarity, and polarity distribution within the mouse tumor model during the process. It is anticipated that this study will offer valuable insights and guidance to the research of mitochondrial-related fields and will boost the advancement of diagnostic and therapeutic areas for associated diseases.

Rapid Acquisition of High-Pixel Fluorescence Lifetime Images of Living Cells via Image Reconstruction Based on Edge-Preserving Interpolation.

Fluorescence lifetime imaging (FLIM) has established itself as a pivotal tool for investigating biological processes within living cells. However, the extensive imaging duration necessary to accumulate sufficient photons for accurate fluorescence lifetime calculations poses a significant obstacle to achieving high-resolution monitoring of cellular dynamics. In this study, we introduce an image reconstruction method based on the edge-preserving interpolation method (EPIM), which transforms rapidly acquired low-resolution FLIM data into high-pixel images, thereby eliminating the need for extended acquisition times. Specifically, we decouple the grayscale image and the fluorescence lifetime matrix and perform an individual interpolation on each. Following the interpolation of the intensity image, we apply wavelet transformation and adjust the wavelet coefficients according to the image gradients. After the inverse transformation, the original image is obtained and subjected to noise reduction to complete the image reconstruction process. Subsequently, each pixel is pseudo-color-coded based on its intensity and lifetime, preserving both structural and temporal information. We evaluated the performance of the bicubic interpolation method and our image reconstruction approach on fluorescence microspheres and fixed-cell samples, demonstrating their effectiveness in enhancing the quality of lifetime images. By applying these techniques to live-cell imaging, we can successfully obtain high-pixel FLIM images at shortened intervals, facilitating the capture of rapid cellular events.