Fluorescence Lifetime Imaging Microscopy Analysis Research Articles

Deciphering the Influence of Zwitterionic Surfactants on Pluronic Co-assemblies: A Synergistic Odyssey through Spectroscopic, Microscopic, and Scattering Techniques.

Fundamental investigations into the photophysical properties and microenvironmental features of pluronic-zwitterionic surfactant mixed assemblies are essential for advancing our understanding of molecular interactions at the nanoscale, setting the stage for innovative solutions in drug delivery, diagnostics, and other applications of pluronic-zwitterionic surfactant assemblies. This investigation explores the intricate photophysics of pluronic-zwitterionic surfactant mixed assemblies, utilizing the twisted intramolecular charge transfer state forming styryl dye trans-2-[(4-dimethylamino) styryl] benzothiazole as a probe. By comparing the behaviors of two distinct poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) block copolymers with block composition of (PEO)132-(PPO)50-(PEO)132 [F108] and (PEO)100-(PPO)65-(PEO)100 [F127] at concentrations of 5 and 10 wt %, this study systematically examines the impact of the addition of zwitterionic surfactants. Through a comprehensive set of analytical techniques, including UV-visible absorption, steady-state emission, and time-resolved fluorescence emission studies, significant alterations in the emission spectra and quantum yields were observed upon the addition of zwitterionic surfactants. These modifications suggest a dynamic equilibrium for the transitioning of dye molecules between various microenvironments within the assemblies. Additionally, dynamic light scattering, zeta potential (ζ), nuclear Overhauser effect spectroscopy, small-angle neutron scattering, cryogenic transmission electron microscopy, field emission scanning electron microscopy, and fluorescence lifetime imaging microscopy analyses have shed light on the substantial impact of surfactant incorporation on the structural properties of assemblies.

Net O2 exchange rates under dark and light conditions across different stem compartments.

Woody plants display some photosynthetic activity in stems, but the biological role of stem photosynthesis and the specific contributions of bark and wood to carbon uptake and oxygen evolution remain poorly understood. We aimed to elucidate the functional characteristics of chloroplasts in stems of different ages in Fraxinus ornus. Our investigation employed diverse experimental approaches, including microsensor technology to assess oxygen production rates in whole stem, bark, and wood separately. Additionally, we utilized fluorescence lifetime imaging microscopy (FLIM) to characterize the relative abundance of photosystems I and II (PSI : PSII chlorophyll ratio) in bark and wood. Our findings revealed light-induced increases in O2 production in whole stem, bark, and wood. We present the radial profile of O2 production in F. ornus stems, demonstrating the capability of stem chloroplasts to perform light-dependent electron transport. Younger stems exhibited higher light-induced O2 production and dark respiration rates than older ones. While bark emerged as the primary contributor to net O2 production under light conditions, our data underscored that wood chloroplasts are also photosynthetically active. The FLIM analysis unveiled a lower PSI abundance in wood than in bark, suggesting stem chloroplasts are not only active but also acclimate to the spectral composition of light reaching inner compartments.

Delignification of wood fibers using a eutectic carvacrol–methanesulfonic acid mixture analyses of the structure and fractional distribution of lignin, cellulose, and hemicellulose

AbstractDelignification and fractional pretreatments are essential for valorization of wood biomass in various bioproducts. Herein, lignocellulose wood fibers were exposed to a eutectic mixture (EM) of carvacrol and methanesulfonic acid for different times. The resulting structural and chemical alterations in biomass were explored in terms of the fiber morphology and fractional chemical composition through fiber image analysis, field-emission scanning electron microscopy, high-performance liquid chromatography, and a novel approach based on fluorescence-lifetime imaging microscopy (FLIM). The autofluorescence of the lignocellulose fibers, which was primarily due to lignin with contributions from cellulose and hemicellulose, enabled application of FLIM in lignocellulose compositional analysis in micro-scale. FLIM analysis revealed that EM treatment efficiently removed lignin from the outer fiber layers. Furthermore, the effective EM treatment time was 3 h (with a residual lignin content of ~ 7 wt%), after which defects were observed on the fibers and the cellulose chains started breaking. This degradation was also indicated by a shift of the lifetime spectra toward the fluorescence lifetime of cellulose with increasing treatment time. Overall, our findings provide valuable insights to the response of lignocellulose fibers to EM treatment, contributing to the important goal of wood biomass application in bioproducts.

Altered metabolism and DAM-signatures in female brains and microglia with aging

Despite Alzheimer’s disease (AD) disproportionately affecting women, the mechanisms remain elusive. In AD, microglia undergo ‘metabolic reprogramming’, which contributes to microglial dysfunction and AD pathology. However, how sex and age contribute to metabolic reprogramming in microglia is understudied. Here, we use metabolic imaging, transcriptomics, and metabolic assays to probe age- and sex-associated changes in brain and microglial metabolism. Glycolytic and oxidative metabolism in the whole brain was determined using Fluorescence Lifetime Imaging Microscopy (FLIM). Young female brains appeared less glycolytic than male brains, but with aging, the female brain became ‘male-like.’ Transcriptomic analysis revealed increased expression of disease-associated microglia (DAM) genes (e.g., ApoE, Trem2, LPL), and genes involved in glycolysis and oxidative metabolism in microglia from aged females compared to males. To determine whether estrogen can alter the expression of these genes, BV-2 microglia-like cell lines, which abundantly express DAM genes, were supplemented with 17β-estradiol (E2). E2 supplementation resulted in reduced expression of DAM genes, reduced lipid and cholesterol transport, and substrate-dependent changes in glycolysis and oxidative metabolism. Consistent with the notion that E2 may suppress DAM-associated factors, LPL activity was elevated in the brains of aged female mice. Similarly, DAM gene and protein expression was higher in monocyte-derived microglia-like (MDMi) cells derived from middle-aged females compared to age-matched males and was responsive to E2 supplementation. FLIM analysis of MDMi from young and middle-aged females revealed reduced oxidative metabolism and FAD+ with age. Overall, our findings show that altered metabolism defines age-associated changes in female microglia and suggest that estrogen may inhibit the expression and activity of DAM-associated factors, which may contribute to increased AD risk, especially in post-menopausal women.

Real-time open-source FLIM analysis.

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.

Phasor identifier: A cloud-based analysis of phasor-FLIM data on Python notebooks

This paper introduces an innovative approach utilizing Google Colaboratory for the versatile analysis of phasor fluorescence lifetime imaging microscopy (FLIM) data collected from various samples (e.g., cuvette, cells, tissues) and in various input file formats. In fact, phasor-FLIM widespread adoption has been hampered by complex instrumentation and data analysis requirements. We mean to make advanced FLIM analysis more accessible to researchers through a cloud-based solution that 1) harnesses robust computational resources, 2) eliminates hardware limitations, and 3) supports both CPU and GPU processing. We envision a paradigm shift in FLIM data accessibility and potential, aligning with the evolving field of artificial intelligence-driven FLIM analysis. This approach simplifies FLIM data handling and opens doors for diverse applications, from studying cellular metabolism to investigating drug encapsulation, benefiting researchers across multiple domains. The comparative analysis of freely distributed FLIM tools highlights the unique advantages of this approach in terms of adaptability, scalability, and open-source nature.

Fluorescence Lifetime Nanoscopy of Liposomal Irinotecan Onivyde: From Manufacturing to Intracellular Processing.

Onivyde was approved by the Food and Drug Administration (FDA) in 2015 for the treatment of solid tumors, including metastatic pancreatic cancer. It is designed to encapsulate irinotecan at high concentration, increase its blood-circulation lifetime, and deliver it to cells where it is enzymatically converted into SN-38, a metabolite with 100- to 1000-fold higher anticancer activity. Despite a rewarding clinical path, little is known about the physical state of encapsulated irinotecan within Onivyde and how this synthetic identity changes throughout the process from manufacturing to intracellular processing. Herein, we exploit irinotecan intrinsic fluorescence and fluorescence lifetime imaging microscopy (FLIM) to selectively probe the supramolecular organization of the drug. FLIM analysis on the manufacturer's formulation reveals the presence of two coexisting physical states within Onivyde liposomes: (i) gelated/precipitated irinotecan and (ii) liposome-membrane-associated irinotecan, the presence of which is not inferable from the manufacturer's indications. FLIM in combination with high-performance liquid chromatography (HPLC) and a membrane-impermeable dynamic quencher of irinotecan reveals rapid (within minutes) and complete chemical dissolution of the gelated/precipitated phase upon Onivyde dilution in standard cell-culturing medium with extensive leakage of the prodrug from liposomes. Indeed, confocal imaging and cell-proliferation assays show that encapsulated and nonencapsulated irinotecan formulations are similar in terms of cell-uptake mechanism and cell-division inhibition. Finally, 2-channel FLIM analysis discriminates the signature of irinotecan from that of its red-shifted SN-38 metabolite, demonstrating the appearance of the latter as a result of Onivyde intracellular processing. The findings presented in this study offer fresh insights into the synthetic identity of Onivyde and its transformation from production to in vitro administration. Moreover, these results serve as another validation of the effectiveness of FLIM analysis in elucidating the supramolecular organization of encapsulated fluorescent drugs. This research underscores the importance of leveraging advanced imaging techniques to deepen our understanding of drug formulations and optimize their performance in delivery applications.

O-205 Poor mitochondrial metabolism impairs meiosis and contributes to reduced oocyte maturation rates in patients with advanced maternal age

Abstract Study question Is there a relationship between poor mitochondrial metabolism and impaired meiotic progression of germinal vesicle (GVs) oocytes in women of advanced maternal age (AMA)? Summary answer Poor mitochondrial metabolism impairs meiotic progression in human oocytes, contributing to a lower oocyte maturation capacity in women of AMA. What is known already High aneuploidy rates and impaired metabolism are characteristic features of AMA oocytes. Studies in mouse suggest that successful meiotic maturation and metabolic competency may be functionally linked, however this relationship has not been clearly established in human. Metabolic dynamics can be visualized by indirect measurements through mitochondrial staining and quantified more directly using Fluorescence Lifetime Imaging Microscopy (FLIM). This live-imaging approach can generate metabolic timelapse profiles of oocytes throughout meiosis. We performed an extensive characterization of oocyte metabolism in maturing GVs, obtained from both young and AMA patients, to establish the importance of mitochondrial metabolism during meiosis. Study design, size, duration A total of 401 GVs from young (≤30 years, n = 264) and AMA (>37 years, n = 137) women were included in the study. GVs were matured in vitro in G2-plus medium for 30 hours. Maturation was determined by the presence of an extruded polar body. Oocytes were used for FLIM (n = 175) and immunofluorescence (IF) analysis (n = 226). Further, mitochondrial metabolism loss of function studies (n = 64) were performed by treating young GVs with 1μM Trifluoromethoxy-carbonylcyanide-phenylhydrazone (FCCP) for 30 minutes. Participants/materials, setting, methods The proteins Dihydrolipoamide-S-Acetyltransferase (D-LAT) and Translocase-of-outer mitochondrial-membrane (TOMM20) were analysed in young and AMA oocytes by IF (Arbitrary-Unit, AU) to assess mitochondrial activity and localization, respectively. Fluorescence mean intensities were quantified with ImageJ and compared by t-test; maturation rates were compared by Chi-squared test. FLIM comprehensive metabolism (NADH; FAD+) were taken at GV stage. Different FLIM parameters (fluorescence intensity, fraction bound, short/long lifetime) were evaluated individually and combined into the Redox ratio (NADH intensity/FAD+ intensity). Main results and the role of chance Mitochondrial staining showed a common pattern in young and AMA GV oocytes, with a uniform localization of mitochondria in the ooplasm (TOMM20) and a subcortical localization of active organelles (D-LAT). These patterns were confirmed by in-live FLIM analysis. The total mitochondria abundance was comparable between AMA and young GV oocytes (intensity of 61674±24322 AU in young, 32186±33414 AU in AMA, p = 0.195), however, active mitochondria were diminished in AMA compared to young GVs both by IF and FLIM (intensity of 78614±58534 AU in young, 12517±10187 AU in AMA, p = 0.003; Redox ratio in young 2e+00±0.15, in AMA 1e+00 ± 0.16, p = 2.969e-05). Notably, young oocytes matured at a significantly higher rate (86.3%; 63/73) than AMA oocytes (62.3%; 38/61; p = 0.002). Moreover, FLIM imaging revealed that GVs with a higher metabolism were more likely to complete meiosis to MII (Redox ratio 2e+00±0.17 in GVs matured to MII, 1e+00±0.18 in non-matured, p = 1.054e-06). Following treatment with FCCP, young GVs showed a similar phenotype to AMA oocytes, with a significant decrease in mitochondrial activity (intensity of 78614±58534 IU in untreated and 11554±16131 IU in treated GVs, p = 0.019), and an associated drop-in maturation rate. Ultimately, only 39.5% (17/43) of the young treated GVs were able to accomplish meiotic maturation. Limitations, reasons for caution Maturation rates were assessed by the presence of an extruded PB and variations in spindle assembly timings may have been overlooked. The quantification of mitochondrial activity in loss of function studies was assessed only by IF staining. Wider implications of the findings Our findings demonstrate the presence of a functional link between oocyte metabolism and impaired meiosis, which may contribute to the decline of oocyte quality with age. Given that live-metabolic imaging is able to discern GVs that progress through meiosis, this approach may represent a valuable non-invasive tool in oocyte selection. Trial registration number not applicable

FLUTE: A Python GUI for interactive phasor analysis of FLIM data.

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique used to probe the local environment of fluorophores. The fit-free phasor approach to FLIM data is increasingly being used due to its ease of interpretation. To date, no open-source graphical user interface (GUI) for phasor analysis of FLIM data is available in Python, thus limiting the widespread use of phasor analysis in biomedical research. Here, we present Fluorescence Lifetime Ultimate Explorer (FLUTE), a Python GUI that is designed to fill this gap. FLUTE simplifies and automates many aspects of the analysis of FLIM data acquired in the time domain, such as calibrating the FLIM data, performing interactive exploration of the phasor plot, displaying phasor plots and FLIM images with different lifetime contrasts simultaneously, and calculating the distance from known molecular species. After applying desired filters and thresholds, the final edited datasets can be exported for further user-specific analysis. FLUTE has been tested using several FLIM datasets including autofluorescence of zebrafish embryos and in vitro cells. In summary, our user-friendly GUI extends the advantages of phasor plotting by making the data visualization and analysis easy and interactive, allows for analysis of large FLIM datasets, and accelerates FLIM analysis for non-specialized labs.

SCFOsFBK1 E3 ligase mediates jasmonic acid-induced turnover of OsATL53 and OsCCR14 to regulate lignification of rice anthers and roots.

The rice F-box protein OsFBK1, which mediates the turnover of a cinnamoyl CoA-reductase, OsCCR14, has previously been shown to regulate anther and root lignification. Here, we identify OsATL53, a member of the ATL family of RING-H2 proteins that interacts with OsCCR14 in the cytoplasm. OsATL53 was identified in the same yeast two-hybrid library screening as reported previously for OsCCR14, and we show it to have cytoplasmic localization and E3 ligase ubiquitination properties. SCFOsFBK1 mediates turnover of OsATL53 in the cytoplasm and the nucleus, and that of OsCCR14 only in the nucleus, as shown by cell-free degradation assays. Confocal fluorescence lifetime imaging microscopy analyses demonstrate that in presence of jasmonic acid (JA), which plays a role in anther dehiscence, OsATL53-OsCCR14 undergoes conformational changes that trigger the complex to accumulate around the nuclear periphery and signals OsFBK1 to initiate degradation of the proteins in the respective cellular compartments. OsATL53 decreases the enzymatic activity of OsCCR14 and sequesters it in the cytoplasm, thereby regulating the lignification process. Transgenic rice with knockdown of OsATL53 display increased lignin deposition in the anthers and roots compared to the wild type, whilst knockdown of OsCCR14 results in decreased lignin content. Our results show that OsATL53 affects the activity of OsCCR14, and that their JA-induced degradation by SCFOsFBK1 regulates lignification of rice anthers and roots.

Glutathione-capped gold nanoclusters as near-infrared-emitting efficient contrast agents for confocal fluorescence imaging of tissue-mimicking phantoms.

An innovative research has been conducted focused on demonstrating the ability of novel dual-emissive glutathione-stabilized gold nanoclusters (GSH-AuNCs) to perform bright near-infrared (NIR)-emitting contrast agents inside tissue-mimicking agarose-phantoms via two complementary confocal fluorescence imaging techniques. First, using a new and fast microwave-assisted approach, we synthesized photostable dual-emitting GSH-AuNCs with an average size of 3.2 ± 0.4 nm and NIR emission quantum yield of 9.9%. Steady-state fluorescence measurements coupled with fluorescence lifetime imaging microscopy (FLIM) assays performed on lyophilized GSH-AuNCs revealed that the obtained GSH-AuNCs exhibit PL emissions at 610nm (red PL) and, respectively, 800nm (NIR PL) in both solution and powder solid-state. Time-resolved fluorescence measurements showed that the two PL components are characterized by average lifetimes of 407ns (red PL) and 1821ns (NIR PL), respectively. Additionally, due to a partial overlap between the red PL and the absorption of the NIR PL, an energy transfer between the two coexisting emissive centers was discovered and confirmed via steady-state and time-resolved fluorescence measurements. Furthermore, the FLIM analysis performed on powder GSH-AuNCs under 640nm, an excitation more suitable for bioimaging applications, revealed a homogeneous and photostable NIR PL signal from GSH-AuNCs. Finally, the ability of GSH-AuNCs to operate as reliable NIR-emitting contrast agents inside tissue-mimicking agarose-phantoms was demonstrated here for the first time via complementary FLIM and re-scan confocal fluorescence imaging techniques. In consequence, GSH-AuNCs show great promise for future in vivo imaging applications via confocal fluorescence microscopy.

Metabolic state oscillations in cerebral nuclei detected using two-photon fluorescence lifetime imaging microscopy

The fluorescence lifetime of nicotinamide adenine dinucleotide (NADH), a key endogenous coenzyme and metabolic biomarker, can reflect the metabolic state of cells. To implement metabolic imaging of brain tissue at high resolution, we assembled a two-photon fluorescence lifetime imaging microscopy (FLIM) platform and verified the feasibility and stability of NADH-based two-photon FLIM in paraformaldehyde-fixed mouse cerebral slices. Furthermore, NADH based metabolic state oscillation was observed in cerebral nuclei suprachiasmatic nucleus (SCN). The free NADH fraction displayed a relatively lower level in the daytime than at the onset of night, and an ultradian oscillation at night was observed. Through the combination of high-resolution imaging and immunostaining data, the metabolic tendency of different cell types was detected after the first two hours of the day and at night. Thus, two-photon FLIM analysis of NADH in paraformaldehyde-fixed cerebral slices provides a high-resolution and label-free method to explore the metabolic state of deep brain regions.

Development and characterization of phasor-based analysis for FLIM to evaluate the metabolic and epigenetic impact of HER2 inhibition on squamous cell carcinoma cultures.

.Significance: Deranged metabolism and dysregulated growth factor signaling are closely associated with abnormal levels of proliferation, a recognized hallmark in tumorigenesis. Fluorescence lifetime imaging microscopy (FLIM) of endogenous nicotinamide adenine dinucleotide (NADH), a key metabolic coenzyme, offers a non-invasive, diagnostic indicator of disease progression, and treatment response. The model-independent phasor analysis approach leverages FLIM to rapidly evaluate cancer metabolism in response to targeted therapy.Aim: We combined lifetime and phasor FLIM analysis to evaluate the influence of human epidermal growth factor receptor 2 (HER2) inhibition, a prevalent cancer biomarker, on both nuclear and cytoplasmic NAD(P)H of two squamous cell carcinoma (SCC) cultures. While better established, the standard lifetime analysis approach is relatively slow and potentially subject to intrinsic fitting errors and model assumptions. Phasor FLIM analysis offers a rapid, model-independent alternative, but the sensitivity of the bound NAD(P)H fraction to growth factor signaling must also be firmly established.Approach: Two SCC cultures with low- and high-HER2 expression, were imaged using multiphoton-excited NAD(P)H FLIM, with and without treatment of the HER2 inhibitor AG825. Cells were challenged with mitochondrial inhibition and uncoupling to investigate AG825’s impact on the overall metabolic capacity. Phasor FLIM and lifetime fitting analyses were compared within nuclear and cytoplasmic compartments to investigate epigenetic and metabolic impacts of HER2 inhibition.Results: NAD(P)H fluorescence lifetime and bound fraction consistently decreased following HER2 inhibition in both cell lines. High-HER2 SCC74B cells displayed a more significant response than low-HER2 SCC74A in both techniques. HER2 inhibition induced greater changes in nuclear than cytoplasmic compartments, leading to an increase in NAD(P)H intensity and concentration.Conclusions: The use of both, complementary FLIM analysis techniques together with quantitative fluorescence intensity revealed consistent, quantitative changes in NAD(P)H metabolism associated with inhibition of growth factor signaling in SCC cell lines. HER2 inhibition promoted increased reliance on oxidative phosphorylation in both cell lines.

Recent innovations in fluorescence lifetime imaging microscopy for biology and medicine.

.Significance: Fluorescence lifetime imaging microscopy (FLIM) measures the decay rate of fluorophores, thus providing insights into molecular interactions. FLIM is a powerful molecular imaging technique that is widely used in biology and medicine.Aim: This perspective highlights some of the major advances in FLIM instrumentation, analysis, and biological and clinical applications that we have found impactful over the last year.Approach: Innovations in FLIM instrumentation resulted in faster acquisition speeds, rapid imaging over large fields of view, and integration with complementary modalities such as single-molecule microscopy or light-sheet microscopy. There were significant developments in FLIM analysis with machine learning approaches to enhance processing speeds, fit-free techniques to analyze images without a priori knowledge, and open-source analysis resources. The advantages and limitations of these recent instrumentation and analysis techniques are summarized. Finally, applications of FLIM in the last year include label-free imaging in biology, ophthalmology, and intraoperative imaging, FLIM of new fluorescent probes, and lifetime-based Förster resonance energy transfer measurements.Conclusions: A large number of high-quality publications over the last year signifies the growing interest in FLIM and ensures continued technological improvements and expanding applications in biomedical research.

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.

Fluorescently Labeled Cellulose Nanofibers for Environmental Health and Safety Studies.

An optimal methodology for locating and tracking cellulose nanofibers (CNFs) in vitro and in vivo is crucial to evaluate the environmental health and safety properties of these nanomaterials. Here, we report the use of a new boron-dipyrromethene (BODIPY) reactive fluorescent probe, meso-DichlorotriazineEthyl BODIPY (mDTEB), tailor-made for labeling CNFs used in simulated or in vivo ingestion exposure studies. Time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) was used to confirm covalent attachment and purity of mDTEB-labeled CNFs. The photoluminescence properties of mDTEB-labeled CNFs, characterized using fluorescence spectroscopy, include excellent stability over a wide pH range (pH2 to pH10) and high quantum yield, which provides detection at low (μM) concentrations. FLIM analysis also showed that lignin-like impurities present on the CNF reduce the fluorescence of the mDTEB-labeled CNF, via quenching. Therefore, the chemical composition and the methods of CNF production affect subsequent studies. An in vitro triculture, small intestinal, epithelial model was used to assess the toxicity of ingested mDTEB-labeled CNFs. Zebrafish (Danio rerio) were used to assess in vivo environmental toxicity studies. No cytotoxicity was observed for CNFs, or mDTEB-labeled CNFs, either in the triculture cells or in the zebrafish embryos.

Label-free identification and differentiation of different microplastics using phasor analysis of fluorescence lifetime imaging microscopy (FLIM)-generated data

As plastic pollution is becoming an increasing worldwide problem, a variety of different techniques for the detection and in-depth characterization of plastics, including spectroscopy and chromatography methods, were introduced to the public. Recently we presented fluorescence lifetime imaging microscopy (FLIM) a new approach for the identification and characterization of microplastics using their fluorescence lifetime (τ) for differentiation. A very powerful extension of the recently established FLIM could be phasor analysis, which allows data representation in an interactive 2D graphical phasor plot thereby enabling a global view of the fluorescence decay in each pixel of the measured image. Microplastic particles generated from six different types of plastics were subjected to excitation wavelengths of 440 nm, upon which specific fluorescence lifetimes as well as the photon yield were determined using FLIM and phasor analysis. We could show that phasor analysis for FLIM with a laser pulse repetition frequency of 40 MHz was able to generate specific locations in the phasor plot for the plastics for fast differentiation, e.g. resulting in well-defined phasor plot positions for ABS at 3.019 ns, PPE at 6.239 ns, PET bottle from Germany at 2.703 ns and PET bottle from USA at 2.711 ns. Phasor analysis for FLIM proves to be a fast, label-free, and sensitive method for the identification and differentiation of plastics also with the aid of visualization variation enabling techniques such as heat treatment of plastics.

Fluorescence Lifetime Imaging Microscopy Maps Fluorophore Concentrations, a Gaussian Process Approach

Fluorescence microscopy techniques have made many contributions to unravel rules of biological processes in the past few decades. These procedures make use of different properties, such as life-time, emission spectrum and intensity of fluorophores to learn about sub-cellular environments. In particular, fluorescence lifetime imaging microscopy (FLIM) makes use of lifetimes of fluorescent dyes to map sub-cellular environment variations, such as temperature, pH, molecule concentrations, and protein-protein interactions. In FLIM, the number of photons collected by the detector depends on the lifetimes and concentrations of the fluorescent dyes present in the sample. Therefore, FLIM equips us with a powerful tool to learn the fluorophore concentrations with high spatial resolution. FLIM analysis can be performed in either time or frequency domain. In time domain, photon arrival times are fitted to a mixture of decay profiles, which are convolutions of exponential components and the instrument response function. The current time domain approaches group the input data into multiple bins which leads to loss of information. Moreover, these techniques report sub-cellular heterogeneity with a resolution limited to the pixel size. We developed a time domain FLIM data analysis technique in a Bayesian paradigm that uses Gaussian processes to map the fluorophore concentrations with sub-pixel resolution. This method efficiently uses all the available photon arrival time information and, as such, requires a minimum number of photons to infer accurate fluorophore concentrations. The approach was validated using both empirical and simulated data.

Developing Analysis Protocols for Monitoring Intracellular Oxygenation Using Fluorescence Lifetime Imaging of Myoglobin-mCherry.

Oxygen (O2) is a critical metabolite for cellular function as it fuels aerobic cellular metabolism; further, it is a known regulator of gene expression. Monitoring oxygenation within cells and organelles can provide valuable insights into how O2, or lack thereof, both influences and responds to cell processes. In recent years, fluorescence lifetime imaging microscopy (FLIM) has been used to track several probe concentration independent intracellular phenomena, such as pH, viscosity, and, in conjunction with Förster resonance energy transfer (FRET), protein-protein interactions. Here, we describe methods for synthesizing and expressing the novel FLIM-FRET intracellular O2 probe Myoglobin-mCherry (Myo-mCherry) in cultured cell lines, as well as acquiring FLIM images using a laser scanning confocal microscope configured for two-photon excitation and a time-correlated single photon counting (TCSPC) module. Finally, we provide step-by-step protocols for FLIM analysis of Myo-mCherry using the commercial software SPCImage and conversion of fluorescence lifetime values in each pixel to apparent intracellular oxygen partial pressures (pO2).

FLIMJ: An open-source ImageJ toolkit for fluorescence lifetime image data analysis.

In the field of fluorescence microscopy, there is continued demand for dynamic technologies that can exploit the complete information from every pixel of an image. One imaging technique with proven ability for yielding additional information from fluorescence imaging is Fluorescence Lifetime Imaging Microscopy (FLIM). FLIM allows for the measurement of how long a fluorophore stays in an excited energy state, and this measurement is affected by changes in its chemical microenvironment, such as proximity to other fluorophores, pH, and hydrophobic regions. This ability to provide information about the microenvironment has made FLIM a powerful tool for cellular imaging studies ranging from metabolic measurement to measuring distances between proteins. The increased use of FLIM has necessitated the development of computational tools for integrating FLIM analysis with image and data processing. To address this need, we have created FLIMJ, an ImageJ plugin and toolkit that allows for easy use and development of extensible image analysis workflows with FLIM data. Built on the FLIMLib decay curve fitting library and the ImageJ Ops framework, FLIMJ offers FLIM fitting routines with seamless integration with many other ImageJ components, and the ability to be extended to create complex FLIM analysis workflows. Building on ImageJ Ops also enables FLIMJ's routines to be used with Jupyter notebooks and integrate naturally with science-friendly programming in, e.g., Python and Groovy. We show the extensibility of FLIMJ in two analysis scenarios: lifetime-based image segmentation and image colocalization. We also validate the fitting routines by comparing them against industry FLIM analysis standards.