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We present high-resolution, high-speed fluorescence lifetime imaging microscopy (FLIM) of live cells based on a compressed sensing scheme. By leveraging the compressibility of biological scenes in a specific domain, we simultaneously record the time-lapse fluorescence decay upon pulsed laser excitation within a large field of view. The resultant system, referred to as compressed FLIM, can acquire a widefield fluorescence lifetime image within a single camera exposure, eliminating the motion artifact and minimizing the photobleaching and phototoxicity. The imaging speed, limited only by the readout speed of the camera, is up to 100 Hz. We demonstrated the utility of compressed FLIM in imaging various transient dynamics at the microscopic scale., Competing Interests: The authors declare no competing interest.

Plants contain abundant autofluorescent molecules that can be used for biochemical, physiological, or imaging studies. The two most studied molecules are chlorophyll (orange/red fluorescence) and lignin (blue/green fluorescence). Chlorophyll fluorescence is used to measure the physiological state of plants using handheld devices that can measure photosynthesis, linear electron flux, and CO 2 assimilation by directly scanning leaves, or by using reconnaissance imaging from a drone, an aircraft or a satellite. Lignin fluorescence can be used in imaging studies of wood for phenotyping of genetic variants in order to evaluate reaction wood formation, assess chemical modification of wood, and study fundamental cell wall properties using Förster Resonant Energy Transfer (FRET) and other methods. Many other fluorescent molecules have been characterized both within the protoplast and as components of cell walls. Such molecules have fluorescence emissions across the visible spectrum and can potentially be differentiated by spectral imaging or by evaluating their response to change in pH (ferulates) or chemicals such as Naturstoff reagent (flavonoids). Induced autofluorescence using glutaraldehyde fixation has been used to enable imaging of proteins/organelles in the cell protoplast and to allow fluorescence imaging of fungal mycelium.

α-Synuclein (aS) aggregation has been amply investigated for its involvement in Parkinson's disease because its amyloid fibrils are the main constituent of Lewy bodies, one of the hallmarks of the disease. aS aggregation was studied here in vitro and in cellular models to correlate aggregation products with toxicity mechanisms. Independent results published elsewhere suggested that aS overexpression and/or aggregation may impair cellular metabolism and cause mitochondrial damage. In this context, we report the characterization of changes in NADH fluorescence properties in vitro and in human embryonic kidney 293 cells upon aS aggregation. The application of the phasor approach to study NADH fluorescence lifetime and emission allowed us to identify changes that correlate with aS aggregation. In particular, the fraction of bound NADH, characterized by longer lifetimes in comparison to free NADH, is increased, and the maximum of the NADH emission is shifted toward shorter wavelengths in the presence of aggregating aS both in vitro and in cells. These data suggest that NADH binds to aggregated aS. NMR experiments in vitro substantiate such binding, which occurs during aggregation. NADH fluorescence is thus useful to detect aS aggregation and by extension the associated oxidative stress., (© FASEB.)

Nonmuscle myosin II (NMII) generates cytoskeletal forces that drive cell division, embryogenesis, muscle contraction and many other cellular functions. However, at present there is no method that can directly measure the forces generated by myosins in living cells. Here, we describe a Förster resonance energy transfer (FRET)-based tension sensor that can detect myosin-associated force along the filamentous actin network. Fluorescence lifetime imaging microscopy (FLIM)-FRET measurements indicate that the forces generated by NMII isoform B (NMIIB) exhibit significant spatial and temporal heterogeneity as a function of donor lifetime and fluorophore energy exchange. These measurements provide a proxy for inferred forces that vary widely along the actin cytoskeleton. This initial report highlights the potential utility of myosin-based tension sensors in elucidating the roles of cytoskeletal contractility in a wide variety of contexts.

In this work, we examine the use of environment-sensitive fluorescent dyes in fluorescence lifetime imaging microscopy (FLIM) biosensors. We screened merocyanine dyes to find an optimal combination of environment-induced lifetime changes, photostability, and brightness at wavelengths suitable for live-cell imaging. FLIM was used to monitor a biosensor reporting conformational changes of endogenous Cdc42 in living cells. The ability to quantify activity using phasor analysis of a single fluorophore (e.g., rather than ratio imaging) eliminated potential artifacts. We leveraged these properties to determine specific concentrations of activated Cdc42 across the cell.

The relative amount of AMPA receptors expressed at the surface of neurons can be measured using superecliptic pHluorin (SEP) labeling at their N-terminus. However, the high signal variability resulting from protein overexpression in neurons and the low signal observed in intracellular vesicles make quantitative characterization of receptor trafficking difficult. Here, we establish a real-time live-cell assay of AMPAR trafficking based on fluorescence lifetime imaging (FLIM), which allows for simultaneous visualization of both surface and intracellular receptors. Using this assay, we found that elevating amyloid-beta (Ab) levels leads to a strong increase in intracellular GluA1 and GluA2-containing receptors, indicating that Aβ triggers the endocytosis of these AMPARs. In APP/PS1 Alzheimer's disease model mouse neurons, FLIM revealed strikingly different AMPAR trafficking properties for GluA1- and GluA3- containing receptors, suggesting that chronic Aβ exposure triggered the loss of both surface and intracellular GluA3-containing receptors. Interestingly, overexpression of protein phosphatase 1 (PP1) also resulted inGluA1 endocytosis as well as depressed synaptic transmission, confirming the important role of phosphorylation in regulating AMPAR trafficking. This new approach allows for the quantitative measurement of extracellular pH, small changes in receptor trafficking, as well as simultaneous measurement of surface and internalized AMPARs in living neurons, and could therefore be applied to several different studies in the future. [ABSTRACT FROM AUTHOR]

The ongoing expansion of fifth-generation (5G) and future sixth-generation (6G) mobile communications is expected to result in widespread human exposure to millimeter-wave (mmWave) radiation globally. Given the short penetration depth of mmWaves and their high absorption by the skin, it is imperative to investigate the potential effects of 5G radiation not only in terms of temperature increase but also at the cellular level. To understand the biological mechanisms of mmWave effects, accurate methods for assessing mmWave absorption in the skin are crucial. In this study, we use fluorescence lifetime imaging microscopy (FLIM) to explore these effects. Employing a mmWave exposure system operating at 26 gigahertz (GHz), porcine skin is irradiated for varying durations (5, 10, 20, and 30 min). We investigate changes in tissue temperature and the autofluorescence of flavin adenine dinucleotide (FAD). Our findings suggest that operating our mmWave exposure systems at the configured power level of 26 GHz is unlikely to cause damage to FADs, even after a 30 min exposure duration. [ABSTRACT FROM AUTHOR]

The successful trajectory of liposome-encapsulated doxorubicin (e.g., Doxil, which has been approved by the U.S. Food and Drug Administration) as an anticancer nanodrug in clinical applications is contradicted by in vitro cell viability data that highlight its reduced efficacy in promoting cell death compared with non-encapsulated doxorubicin. No reports to date have provided a mechanistic explanation for this apparently discordant evidence. Taking advantage of doxorubicin intrinsic fluorescence and time-resolved optical microscopy, we analyze the uptake and intracellular processing of liposome-encapsulated doxorubicin (L-DOX) in several in vitro cellular models. Cell entry of L-DOX was found to lead to a rapid (seconds to minutes), energy- and temperature-independent release of crystallized doxorubicin nanorods into the cell cytoplasm, which then disassemble into a pool of fibril-shaped derivatives capable of crossing the cellular membrane while simultaneously releasing active drug monomers. Thus, a steady state is rapidly established in which the continuous supply of crystal nanorods from incoming liposomes is counteracted by a concentration-guided efflux in the extracellular medium of fibril-shaped derivatives and active drug monomers. These results demonstrate that liposome-mediated delivery is constitutively less efficient than isolated drug in establishing favorable conditions for drug retention in the cell. In addition to explaining previous contradictory evidence, present results impose careful rethinking of the synthetic identity of encapsulated anticancer drugs.

This article discusses the development of a novel pH probe called Mem-C0C18 for quantitative monitoring of cell surface pH using fluorescence lifetime imaging. The surface pH of tumor cells is lower than that of normal cells due to the production of acidic metabolites. The probe targets the plasma membrane and accurately measures pH changes using fluorescence lifetime imaging. The article provides details on the molecular design of the probe, its spectral properties, establishment of standard curves, and its application in evaluating the effects of extracellular fluid acidity and glycolysis on cell surface pH. The probe shows promise as a tool for studying pH fluctuations in the cell membrane. [Extracted from the article]

Genetically encoded fluorescence lifetime biosensors have emerged as powerful tools for quantitative imaging, enabling precise measurement of cellular metabolites, molecular interactions, and dynamic cellular processes. This review provides an overview of the principles, applications, and advancements in quantitative imaging with genetically encoded fluorescence lifetime biosensors using fluorescence lifetime imaging microscopy (go-FLIM). We highlighted the distinct advantages of fluorescence lifetime-based measurements, including independence from expression levels, excitation power, and focus drift, resulting in robust and reliable measurements compared to intensity-based approaches. Specifically, we focus on two types of go-FLIM, namely Förster resonance energy transfer (FRET)–FLIM and single-fluorescent protein (FP)-based FLIM biosensors, and discuss their unique characteristics and benefits. This review serves as a valuable resource for researchers interested in leveraging fluorescence lifetime imaging to study molecular interactions and cellular metabolism with high precision and accuracy. [ABSTRACT FROM AUTHOR]

Fluorescence lifetime imaging (FLIm) is an optical spectroscopic imaging technique capable of real-time assessments of tissue properties in clinical settings. Label-free FLIm is sensitive to changes in tissue structure and biochemistry resulting from pathological conditions, thus providing optical contrast to identify and monitor the progression of disease. Technical and methodological advances over the last two decades have enabled the development of FLIm instrumentation for real-time, in situ, mesoscopic imaging compatible with standard clinical workflows. Herein, we review the fundamental working principles of mesoscopic FLIm, discuss the technical characteristics of current clinical FLIm instrumentation, highlight the most commonly used analytical methods to interpret fluorescence lifetime data and discuss the recent applications of FLIm in surgical oncology and cardiovascular diagnostics. Finally, we conclude with an outlook on the future directions of clinical FLIm.

Fluorescence lifetime imaging microscopy (FLIM) has emerged as a promising tool for all scientific studies in recent years. However, the utilization of FLIM data requires complex data modeling techniques, such as curve-fitting procedures. These conventional curve-fitting procedures are not only computationally intensive but also time-consuming. To address this limitation, machine learning (ML), particularly deep learning (DL), can be employed. This review aims to focus on the ML and DL methods for FLIM data analysis. Subsequently, ML and DL strategies for evaluating FLIM data are discussed, consisting of preprocessing, data modeling, and inverse modeling. Additionally, the advantages of the reviewed methods are deliberated alongside future implications. Furthermore, several freely available software packages for analyzing the FLIM data are highlighted. [ABSTRACT FROM AUTHOR]

Deposition of calcium-containing minerals such as hydroxyapatite and whitlockite in the subretinal pigment epithelial (sub-RPE) space of the retina is linked to the development of and progression to the end-stage of age-related macular degeneration (AMD). AMD is the most common eye disease causing blindness amongst the elderly in developed countries; early diagnosis is desirable, particularly to begin treatment where available. Calcification in the sub-RPE space is also directly linked to other diseases such as Pseudoxanthoma elasticum (PXE). We found that these mineral deposits could be imaged by fluorescence using tetracycline antibiotics as specific stains. Binding of tetracyclines to the minerals was accompanied by increases in fluorescence intensity and fluorescence lifetime. The lifetimes for tetracyclines differed substantially from the known background lifetime of the existing natural retinal fluorophores, suggesting that calcification could be visualized by lifetime imaging. However, the excitation wavelengths used to excite these lifetime changes were generally shorter than those approved for retinal imaging. Here, we show that tetracycline-stained drusen in post mortem human retinas may be imaged by fluorescence lifetime contrast using multiphoton (infrared) excitation. For this pilot study, ten eyes from six anonymous deceased donors (3 female, 3 male, mean age 83.7 years, range 79–97 years) were obtained with informed consent from the Maryland State Anatomy Board with ethical oversight and approval by the Institutional Review Board. [ABSTRACT FROM AUTHOR]

Fluorescence thermometry is a microscopy technique in which a fluorescent temperature sensor records temperature changes as alterations of fluorescence signals. Fluorescence lifetime imaging (FLIM) is a promising method for quantitative analysis of intracellular temperature. Recently, we developed small-molecule thermometers, termed Organelle Thermo Greens, that target various organelles and achieved quantitative temperature mapping using FLIM. Despite its highly quantitative nature, FLIM-based thermometry cannot be used widely due to expensive instrumentation. Here, we investigated the applicability and limitations of fluorescence intensity (FI)-based analysis, which is more commonly used than FLIM-based thermometry. Temperature gradients generated by artificial heat sources and physiological heat produced by brown adipocytes were visualized using FI- and FLIM-based thermometry. By comparing the two thermometry techniques, we examined how the shapes of organelles and cells affect the accuracy of the temperature measurements. Based on the results, we concluded that FI-based thermometry could be used for "qualitative", rather than quantitative, thermometry under the limited condition that the shape change and the dye leakage from the target organelle were not critical. [ABSTRACT FROM AUTHOR]

We describe the performance of a new wide area time-gated single-photon avalanche diode (SPAD) array for phasor-FLIM, exploring the effect of gate length, gate number and signal intensity on the measured lifetime accuracy and precision. We conclude that the detector functions essentially as an ideal shot noise limited sensor and is capable of video rate FLIM measurement. The phasor approach used in this work appears ideally suited to handle the large amount of data generated by this type of very large sensor (512 × 512 pixels), even in the case of small number of gates and limited photon budget.

The availability of energy has significant impact on cell physiology. However, the role of cellular metabolism in bacterial pathogenesis is not understood. We investigated the dynamics of central metabolism during virulence induction by surface sensing and quorum sensing in early-stage biofilms of the multidrug-resistant bacterium Pseudomonas aeruginosa We established a metabolic profile for P. aeruginosa using fluorescence lifetime imaging microscopy (FLIM), which reports the activity of NADH in live cells. We identified a critical growth transition period during which virulence is activated. We performed FLIM measurements and direct measurements of NADH and NAD+ concentrations during this period. Here, planktonic (low-virulence) and surface-attached (virulence-activated) populations diverged into distinct metabolic states, with the surface-attached population exhibiting FLIM lifetimes that were associated with lower levels of enzyme-bound NADH and decreasing total NAD(H) production. We inhibited virulence by perturbing central metabolism using citrate and pyruvate, which further decreased the enzyme-bound NADH fraction and total NAD(H) production and suggested the involvement of the glyoxylate pathway in virulence activation in surface-attached populations. In addition, we induced virulence at an earlier time using the electron transport chain oxidase inhibitor antimycin A. Our results demonstrate the use of FLIM to noninvasively measure NADH dynamics in biofilms and suggest a model in which a metabolic rearrangement accompanies the virulence activation period.IMPORTANCE The rise of antibiotic resistance requires the development of new strategies to combat bacterial infection and pathogenesis. A major direction has been the development of drugs that broadly target virulence. However, few targets have been identified due to the species-specific nature of many virulence regulators. The lack of a virulence regulator that is conserved across species has presented a further challenge to the development of therapeutics. Here, we identify that NADH activity has an important role in the induction of virulence in the pathogen P. aeruginosa This finding, coupled with the ubiquity of NADH in bacterial pathogens, opens up the possibility of targeting enzymes that process NADH as a potential broad antivirulence approach.

Single‐walled carbon nanotubes (SWCNTs) are versatile near infrared (NIR) fluorescent building blocks for biosensors. Their surface is chemically tailored to respond to analytes by a change in fluorescence. However, intensity‐based signals are easily affected by external factors such as sample movements. Here, we demonstrate fluorescence lifetime imaging microscopy (FLIM) of SWCNT‐based sensors in the NIR. We tailor a confocal laser scanning microscope (CLSM) for NIR signals (>800 nm) and employ time correlated single photon counting of (GT)10‐DNA functionalized SWCNTs. They act as sensors for the important neurotransmitter dopamine. Their fluorescence lifetime (>900 nm) decays biexponentially and the longer lifetime component (370 ps) increases by up to 25 % with dopamine concentration. These sensors serve as paint to cover cells and report extracellular dopamine in 3D via FLIM. Therefore, we demonstrate the potential of fluorescence lifetime as a readout of SWCNT‐based NIR sensors. [ABSTRACT FROM AUTHOR]

Einwandige Kohlenstoffnanoröhren (single‐walled carbon nanotubes, SWCNTs) sind vielseitig einsetzbare Bausteine für Biosensoren, die im nahen Infrarot (NIR) fluoreszieren. Ihre Oberfläche kann chemisch so modifiziert werden, dass sie auf Analyten mit einer Veränderung ihrer Fluoreszenz reagieren. Intensitätsbasierte Signale werden jedoch leicht durch äußere Faktoren wie Bewegungen der Probe beeinflusst. Hier zeigen wir Fluoreszenz‐Lebensdauer Mikroskopie (fluorescence lifetime imaging microscopy, FLIM) von SWCNT‐basierten Sensoren im NIR. Dafür wurde ein konfokales Laser‐Scanning‐Mikroskop (CLSM) für NIR‐Signale (>800 nm) angepasst und zeitkorrelierte Einzelphotonenzählung von (GT)10‐DNA‐funktionalisierten SWCNTs verwendet. (GT)10‐SWCNTs fungieren als Sensoren für den wichtigen Neurotransmitter Dopamin. Ihre Fluoreszenzlebensdauer (>900 nm) fällt biexponentiell ab, wobei die längere Lebensdauerkomponente (370 ps) mit steigender Dopaminkonzentration um bis zu 25 % ansteigt. Mit diesen Sensoren können Zellen beschichtet werden, um extrazelluläres Dopamin in 3D mittels FLIM zu messen. Wir demonstrieren damit das Potenzial der Fluoreszenzlebensdauer als Messgröße für SWCNT‐basierte NIR‐Sensoren. [ABSTRACT FROM AUTHOR]

Fluorescence lifetime imaging is an important tool in bioimaging that allows one to detect subtle changes in cell dynamics and their environment. Most time-domain approaches currently involve scanning a single illumination point across the sample, which can make imaging dynamic scenes challenging, while single-shot “rapid lifetime determination” can suffer from large uncertainties when the lifetime is not appropriately sampled. Here, we propose a time-folded fluorescence lifetime imaging microscopy (TFFLIM) approach, whereby a time-folding cavity provides multiple spatially sheared replicas of the lifetime, each shifted temporally with respect to a fixed time gate. This provides a robust, single-shot FLIM approach that we experimentally validate across a broad lifetime range on fluorescent beads and Convallaria samples. [ABSTRACT FROM AUTHOR]

We describe an advanced DIVER (Deep Imaging Via Emission Recovery) detection system for two-photon fluorescence microscopy that allows imaging in multiple scattering media, including biological tissues, up to a depth of a few mm with micron resolution. This detection system is more sensitive to low level light signals than conventional epi-detection used in two-photon fluorescence microscopes. The DIVER detector efficiently collects scattered emission photons from a wide area of turbid samples at almost any entrance angle in a 2π spherical angle. Using an epi-detection scheme only photons coming from a relatively small area of a sample and at narrow acceptance angle can be detected. The transmission geometry of the DIVER imaging system makes it exceptionally suitable for Second and Third Harmonic Generation (SHG, THG) signal detection. It also has in-depth fluorescence lifetime imaging (FLIM) capability. Using special optical filters with sin-cos spectral response, hyperspectral analysis of images acquired in-depth in scattering media can be performed. The system was successfully employed in imaging of various biological tissues. The DIVER detector can be plugged into a standard microscope stage and used as an external detector with upright commercial two-photon microscopes.

Nicotinamide adenine dinucleotide (reduced form: NADH) serves as a vital redox-energy currency for reduction-oxidation homeostasis and fulfilling energetic demands. While NADH exists as free and bound forms, only free NADH is utilized for complex I to power oxidative phosphorylation, especially important in neurons. Here, we studied how much free NADH remains available for energy production in mitochondria of old living neurons. We hypothesize that free NADH in neurons from old mice is lower than the levels in young mice and even lower in neurons from the 3xTg-AD Alzheimer's disease (AD) mouse model. To assess free NADH, we used lifetime imaging of NADH autofluorescence with 2-photon excitation to be able to resolve the pool of NADH in mitochondria, cytoplasm, and nuclei. Primary neurons from old mice were characterized by a lower free/bound NADH ratio than young neurons from both non-transgenic (NTg) and more so in 3xTg-AD mice. Mitochondrial compartments maintained 26 to 41% more reducing NADH redox state than cytoplasm for each age, genotype, and sex. Aging diminished the mitochondrial free NADH concentration in NTg neurons by 43% and in 3xTg-AD by 50%. The lower free NADH with age suggests a decline in capacity to regenerate free NADH for energetic supply to power oxidative phosphorylation which further worsens in AD. Applying this non-invasive approach, we showed the most explicit measures yet of bioenergetic deficits in free NADH with aging at the subcellular level in live neurons from in-bred mice and an AD model.

The growing demand for tissue engineered vascular grafts (TEVG) motivates the development of optimized fabrication and monitoring procedures. Bioreactors which provide physiologically-relevant conditions are important for improving holistic TEVG properties and performance. Herein we describe a fiber-based intraluminal imaging system that allows for in situ assessment of vascular materials and re-cellularization processes inside a bioreactor by simultaneous and co-registered measurements of endogenous fluorescence lifetime and exogenous marker fluorescence intensity. The lumen of 6 vascular grafts (∼4 mm diameter) were scanned by reciprocally rotating a 41° angle polished multimode optical fiber inside a protective glass tube with outer diameter of 3 mm. Tubular bovine pericardium constructs were recellularized using enhanced Green Fluorescent Protein (eGFP) transfected cells in a custom bioreactor. The imaging system has resolved consistently the cellular autofluorescence from that of tissue matrix in situ based on the lifetime fluorescence properties of endogenous molecular species. The location of the re-cellularized area was validated by the eGFP emission. Current results demonstrate the potential of this system as a valuable tool in tissue engineering for in situ studies of cell-tissue interactions in cylindrical or other 3-dimensional structures.

Easily accessible blue‐emitting materials are in the focus of ongoing research, as they still lack the efficiency and lifetime of their red and green counterparts. The new multidentate phosphine oxide ligands and two respective ZnCl2 complexes presented here combine a straightforward synthesis with high yields and show interesting luminescent properties. The free ligand exhibits blue luminescence in the crystalline state, but not in amorphous films or diluted solution. In contrast, the Zn(II) complexes shows intense blue luminescence in the crystalline state as well as in amorphous thin films and in solution. Fluorescence lifetime imaging microscopy measurements show luminescence lifetimes of 3–6 ns indicative of fluorescence. By combining the experimental data with quantum chemical calculations, we propose a model where the conformation of the molecule is restricted, either via the crystal environment, aggregation, or the steric fixation by the coordinating central atom, blocking the nonradiative relaxation from the excited into the ground electronic state. However, this nonradiative relaxation is still possible in the gas phase via elongation of a PC bond. These results may provide a general mechanism to explain the luminescence properties in a whole class of organic phosphine oxides. [ABSTRACT FROM AUTHOR]

Mammalian spermatozoa are highly energized cells in which most of the proteins and activated signaling cascades are involved in the metabolic pathways. Flavin adenine dinucleotide (FAD) has one of the most important roles in the correct functional activity of spermatozoa since it acts as a cofactor for flavoenzymes, critical for proper metabolism and predominantly located in mitochondria. Non-invasive, vital and non-traumatic examination of sperm FAD level and microenvironment could be performed by fluorescence lifetime imaging microscopy (FLIM). In this study, we assessed the metabolic status of spermatozoa from healthy donors and found that FLIM could be used to segregate and separate the male germ cells according to the type of metabolic activity which corresponds with spermatozoa motility measured in standard spermogram tests. [Display omitted] • FLIM parameters reveal the heterogeneity of the sperm sample. • The Type of sperm motility are associated with FLIM parameters. • FLIM technology could be used in ART centers due to the non-invasive procedure and gentle effect on the cell. [ABSTRACT FROM AUTHOR]

Soil microbial communities are involved in most biogeochemical processes creating hotspots for nutrient cycling. The spatial visualization of such soil hotspots via microscopic techniques is still challenging caused by the intrinsic fluorescence and opacity of the soil. One way to differentiate microbial cells from the heterogeneous soil matrix is a fluorescence lifetime-based technique (FLIM) with subsequent phasor plot separation; it separates and visualizes the distinctly different photon arrival times of all photons per pixel. FLIM delivers additional independent information behind intensity-based image processing and image analysis which is often hampered by, e.g., autofluorescence, resolution issues, and photobleaching artifacts caused by the prevailing minerals and organic substances. We determined characteristic fluorescence lifetime profiles of BacLight™ Green for Rhodotorula mucilaginosa and Bacillus subtilits in phosphate-buffered saline (PBS) solution and water as well as in natural, autoclaved, glucose-activated, and soil mineral particles by FLIM measurements via confocal laser scanning fluorescence microscopy. Rhodotorula mucilaginosa and Bacillus subtilits from pure cultures measured in water and PBS accounted for 1.20 (± 0.2) ns and 1.3 (± 0.1) ns respectively. The lifetime profile within the cells was rather homogeneous for both microbial species tested. This suggests stable photon arrival times for microbial strains with minor effects of matrix components as tested in PBS and water. We identified a clear difference in fluorescence lifetime profiles between microorganisms (around 1 ns) and the surrounding soil matrix (0.2 to 0.7 ns, > 3.6 ns) via phasor plot separation. The results presented raise the feasibility to extend the applicability of FLIM to other soils and their accompanying microbiota. [ABSTRACT FROM AUTHOR]

Quantitative single-molecule and time-resolved fluorescence techniques offer new insights into many samples in life and materials sciences. So far, their adoption has been slow because expert knowledge is required for correct data acquisition and analysis. Now, PicoQuant has developed a single-photon counting confocal microscope, called Luminosa. It combines state-of-the-art hardware with cutting-edge software to deliver high-quality data while simplifying daily operation. The system software includes context-based workflows for each technique, which improve accuracy, reproducibility, and quality of the data. This article showcases how this is achieved for fluorescence lifetime imaging (FLIM). [ABSTRACT FROM AUTHOR]

The phasor approach to FLIM (Fluorescence Lifetime Imaging Microscopy) is becoming popular due to the powerful fit free analysis and the visualization of the decay at each point in images of cells and tissues. However, although several implementation of the method are offered by manufactures of FLIM accessories for microscopes, the details of the conversion of the decay to phasors at each point in an image requires some consideration. Here, we show that if the decay is not properly acquired, the apparently simple phasor transformation can provide incorrect phasor plots and the results may be misinterpreted. In particular, we show the disagreement in experimental data acquired on the same samples using the two cards (FLIMbox, frequency domain and Becker & Hickl BH 830, time domain) and the effect produced by using the BH 830 card with different settings. This difference in data acquisition translates to the assignment of phasor components calculated using different acquisition parameters. This effect is already present in the original data that are not acquired with the proper parameters for the phasor conversion. We also show that the difference in the resolution of components already exists in the data acquired in the time domain when used with settings that do not allow acquisition of the fluorescence decay on a sufficient large time scale. RESEARCH HIGHLIGHTS: This paper is intended to made researchers aware of some simple requirements for the conversion of time-domain data (typically TCSPC) to phasors. The use of phasors for FLIM analysis has seen a surge of popularity. Since the phasor approach is a fit free method and has a powerful visualization of the data, it appears very simple to use. This paper shows that when the original data in the time domain is not acquired with the proper time range to cover the lifetimes in a sample, the conversion to phasors can produce very erroneous results. These results are appearing more frequently in the literature since many of the manufacturers of FLIM accessories for microscopes are now offering the phasor analysis in their software. Here, we show that the phasor transformation per se cannot correct for the problems with data acquisition and that one is misled to think that the "phasor approach" is a universal fix for the lack of the proper time range for data acquisition.

Having access to fluorescence lifetime, researchers can reveal in-depth details about the microenvironment as well as the physico-chemical state of the molecule under investigation. However, the high number of influencing factors might be an explanation for the strongly deviating values of fluorescent lifetimes for the same fluorophore reported in the literature. This could be the reason for the impression that inconsistent results are obtained depending on which detection and excitation scheme is used. To clarify this controversy, the two most common techniques for measuring fluorescence lifetimes in the time-domain and in the frequency-domain were implemented in one single microscopy setup and applied to a variety of fluorophores under different environmental conditions such as pH-value, temperature, solvent polarity, etc., along with distinct state forms that depend, for example, on the concentration. From a vast amount of measurement results, both setup- and sample-dependent parameters were extracted and represented using a single display form, the phasor-plot. The measurements showed consistent results between the two techniques and revealed which of the tested parameters has the strongest influence on the fluorescence lifetime. In addition, quantitative guidance as to which technique is most suitable for which research task and how to perform the experiment properly to obtain consistent fluorescence lifetimes is discussed. [ABSTRACT FROM AUTHOR]

The real-time monitoring of the intracellular pH in live cells with high precision represents an important methodological challenge. Although genetically encoded fluorescent indicators can be considered as a probe of choice for such measurements, they are hindered mostly by the inability to determine an absolute pH value and/or a narrow dynamic range of the signal, making them inefficient for recording the small pH changes that typically occur within cellular organelles. Here, we study the pH sensitivity of a green-fluorescence-protein (GFP)-based emitter (EGFP-Y145L/S205V) with the alkaline-shifted chromophore's pKa and demonstrate that, in the pH range of 7.5–9.0, its fluorescence lifetime changes by a factor of ~3.5 in a quasi-linear manner in mammalian cells. Considering the relatively strong lifetime response in a narrow pH range, we proposed the mitochondria, which are known to have a weakly alkaline milieu, as a target for live-cell pH measurements. Using fluorescence lifetime imaging microscopy (FLIM) to visualize the HEK293T cells expressing mitochondrially targeted EGFP-Y145L/S205V, we succeeded in determining the absolute pH value of the mitochondria and recorded the ETC-uncoupler-stimulated pH shift with a precision of 0.1 unit. We thus show that a single GFP with alkaline-shifted pKa can act as a high-precision indicator that can be used in a specific pH range. [ABSTRACT FROM AUTHOR]

We report an investigation of the in vivo accumulation of Radachlorin photosensitizer in a murine model in several types of normal and tumor tissues based on an FLIM-assisted analysis of fluorescence intensity images, time-resolved fluorescence signals, and phasor plots. Experiments were performed on ex vivo histological samples of normal and tumor tissues. It was shown that the investigation of fluorescence intensity distributions combined with that of time-resolved fluorescence images can be used for qualitative and—under some limitations—quantitative analyses of the relative uptake of this photosensitizer in tissues. The phasor plot representations of time-resolved fluorescence signals were shown to be suitable for identification of the accumulation of predominant photosensitizers in tissues. [ABSTRACT FROM AUTHOR]

Fluorescence thermometry is a microscopy technique in which a fluorescent temperature sensor records temperature changes as alterations of fluorescence signals. Fluorescence lifetime imaging (FLIM) is a promising method for quantitative analysis of intracellular temperature. Recently, we developed small-molecule thermometers, termed Organelle Thermo Greens, that target various organelles and achieved quantitative temperature mapping using FLIM. Despite its highly quantitative nature, FLIM-based thermometry cannot be used widely due to expensive instrumentation. Here, we investigated the applicability and limitations of fluorescence intensity (FI)-based analysis, which is more commonly used than FLIM-based thermometry. Temperature gradients generated by artificial heat sources and physiological heat produced by brown adipocytes were visualized using FI- and FLIM-based thermometry. By comparing the two thermometry techniques, we examined how the shapes of organelles and cells affect the accuracy of the temperature measurements. Based on the results, we concluded that FI-based thermometry could be used for “qualitative”, rather than quantitative, thermometry under the limited condition that the shape change and the dye leakage from the target organelle were not critical.

Fluorescence lifetime imaging (FLIM) allows the quantification of sub‐cellular processes in situ, in living cells. A number of approaches have been developed to extract the lifetime from time‐domain FLIM data, but they are often limited in terms of speed, photon efficiency, precision or the dynamic range of lifetimes they can measure. Here, we focus on one of the best performing methods in the field, the centre‐of‐mass method (CMM), that conveys advantages in terms of speed and photon efficiency over others. In this paper, however, we identify a loss of photon efficiency of CMM for short lifetimes when background noise is present. We subsequently present a new development and generalization of CMM that provides for the rapid and accurate extraction of fluorescence lifetime over a large lifetime dynamic range. We provide software tools to simulate, validate and analyse FLIM data sets and compare the performance of our approach against the standard CMM and the commonly employed least‐square minimization (LSM) methods. Our method features a better photon efficiency than standard CMM and LSM and is robust in the presence of background noise. The algorithm is applicable to any time‐domain FLIM data set. [ABSTRACT FROM AUTHOR]

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 610 nm (red PL) and, respectively, 800 nm (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 407 ns (red PL) and 1821 ns (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 640 nm, 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. [ABSTRACT FROM AUTHOR]

Fluorescence lifetime imaging microscopy (FLIM) evaluates the metabolic state of tissue based on reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD). Fluorescence lifetime imaging ophthalmoscopy (FLIO) can image the fundus of the eyes, but cannot detect NAD(P)H. We used multiphoton FLIM to study the metabolic state of the retina in fixed eyes of wild-type mice C57BL6/J. We sectioned the eye using a polyacrylamide gel-embedding technique and estimated the percentage of bound NAD(P)H. We found that oxidative phosphorylation was the predominant metabolic state, particularly in the inner retina, when a fixed retina was used. We also demonstrated the feasibility of FAD imaging of the retina. In addition, we demonstrated that autofluorescence and various FLIM channels, such as hemoglobin, melanin and collagen, can be used to evaluate the structure of the retina and other parts of the eye without any special staining. [ABSTRACT FROM AUTHOR]

The effects of metal‐enhanced fluorescence (MEF) of rhodamine dye by gold sub‐micro hexagonal plates were studied. A multilayered avidin–biotin complex was designed and used as a spacer to connect the fluorophore and the sub‐microplate. Fluorescence lifetime image microscopy (FLIM) combined with time‐correlated single‐photon counting was used to obtain lifetime images and intensities of dye on single particles. Gold hexagon plates ~900 nm were synthesized, and the avidin–biotin complexes were placed layer‐by‐layer for up to 4 layers onto the sub‐microplates. All emission curves of fluorophore displayed biexponential decay with short lifetimes of 18–23 ps and long lifetimes of 249, 271, 304, and 348 ps for 1–4 layers on gold sub‐microplates, respectively. Using the kinetic model of energy transfer between fluorophore and nanoparticles to explain the coupling mechanism, we found that the excited fluorophore transferred energy mostly to the high‐order modes for the 1‐layered avidin–biotin complexes enclosing gold nanospheres, consequently resulting in a non‐emissive pathway. The 4‐layered samples had the highest emission intensity because, at those distances, the enhancement by the gold sub‐microplates during plasmonic excitation remained high and became comparable to the energy quenching rate. Using the composed complexes, the MEF effect achieved the maximal enhancement in this system. [ABSTRACT FROM AUTHOR]

Previous work characterizing ovarian bioenergetics has defined follicular metabolism by measuring metabolic by-products in culture media. However, culture conditions perturb the native state of the follicle, and these methods do not distinguish between metabolism occurring within oocytes or granulosa cells. We applied the phasor approach to fluorescence lifetime imaging microscopy (phasor FLIM) at 740-nm two-photon excitation to examine the spatial distribution of free and protein-bound nicotinamide adenine dinucleotide hydride (NADH) during primordial through preovulatory stages of follicular development in fresh ex vivo murine neonatal and gonadotropin stimulated prepubertal ovaries. We obtained subcellular resolution phasor FLIM images of primordial through primary follicles and quantified the free/bound NADH ratio (relative NADH/NAD+) separately for oocyte nucleus and oocyte cytoplasm. We found that dynamic changes in oocyte nucleus free/bound NADH paralleled the developmental maturation of primordial to primary follicles. Immunohistochemistry of NAD+-dependent deacetylase SIRTUIN 1 (SIRT1) in neonatal ovary revealed that increasing SIRT1 expression in oocyte nuclei was inversely related to decreasing free/bound NADH during the primordial to primary follicle transition. We characterized oocyte metabolism at these early stages to be NADH producing (glycolysis/Krebs). We extended the results of prior studies to show that cumulus and mural granulosa cell metabolism in secondary through preovulatory follicles is mainly NADH producing (glycolysis/Krebs cycle), while oocyte metabolism is mainly NADH consuming (oxidative phosphorylation). Taken together, our data characterize dynamic changes in free/bound NADH and SIRT1 expression during early follicular development and confirm results from previous studies defining antral and preovulatory follicle metabolism in culture.

All forms of progressive renal diseases develop a final pathway of tubulointerstitial fibrosis and glomerulosclerosis. Renal fibrosis is usually quantified using histological staining, a process that is time-consuming and pathologist dependent. Here we develop a fast and operator-independent method to measure fibrosis utilizing the murine unilateral ureteral obstruction model which manifests a time-dependent fibrotic increase in obstructed kidneys while the contralateral kidneys are used as controls. After ureteral obstruction, kidneys were analyzed at 7, 14, and 21 days. Fibrosis was quantified using fluorescence lifetime imaging (FLIM) and second harmonic generation (SHG) in a Deep Imaging via Enhanced photon Recovery deep tissue imaging microscope. This microscope was developed for deep tissue along with second and third harmonic generation imaging and has extraordinary sensitivity toward harmonic generation. SHG data suggest the presence of more fibrillar collagen in the obstructed kidneys. The combination of short-wavelength FLIM and SHG analysis results in a robust assessment procedure independent of observer interpretation and let us create criteria to quantify the extent of fibrosis directly from the image. Thus, the FLIM-SHG technique shows remarkable improvement in quantification of renal fibrosis compared to standard histological techniques.

Study Question: Can non-invasive imaging with fluorescence lifetime imaging microscopy (FLIM) detect metabolic differences in euploid versus aneuploid human blastocysts?Summary Answer: FLIM has identified significant metabolic differences between euploid and aneuploid blastocysts.What Is Known Already: Prior studies have demonstrated that FLIM can detect metabolic differences in mouse oocytes and embryos and in discarded human blastocysts.Study Design, Size, Duration: This was a prospective observational study from August 2019 to February 2020. Embryo metabolic state was assessed using FLIM to measure the autofluorescence metabolic factors nicotinamide adenine dinucleotide dehydrogenase together with nicotinamide adenine phosphate dinucleotide dehydrogenase (NAD(P)H) and flavin adenine dinucleotide (FAD). Eight metabolic FLIM parameters were obtained from each blastocyst (four for NAD(P)H and four for FAD): short (T1) and long (T2) fluorescence lifetime, fluorescence intensity (I) and fraction of the molecules engaged with enzymes (F). The redox ratio (NAD(P)H-I)/(FAD-I) was also calculated for each image.Participants/materials, Setting, Methods: This study was performed at a single academically affiliated centre where there were 156 discarded frozen blastocysts (n = 17 euploids; 139 aneuploids) included. Ploidy status was determined by pre-implantation genetic testing for aneuploidy (PGT-A). Discarded human blastocysts were compared using single FLIM parameters. Additionally, inner cell mass (ICM) and trophectoderm (TE) were also evaluated. Multilevel models were used for analysis. A post-hoc correction used Benjamini-Hochberg's false discovery rate, at a q-value of 0.05.Main Results and the Role Of Chance: Comparing euploid (n = 17) versus aneuploid (n = 139) embryos, a significant difference was seen in NAD(P)H-F (P < 0.04), FAD-I (P < 0.04) and redox ratio (P < 0.05). Euploid ICM (n = 15) versus aneuploid ICM (n = 119) also demonstrated significantly different signatures in NAD(P)H-F (P < 0.009), FAD-I (P < 0.03) and redox ratio (P < 0.03). Similarly, euploid TE (n = 15) versus aneuploid TE (n = 119) had significant differences in NAD(P)H-F (P < 0.0001) and FAD-I (P < 0.04).Limitations, Reasons For Caution: This study utilized discarded human blastocysts, and these embryos may differ metabolically from non-discarded human embryos. The blastocysts analysed were vitrified after PGT-A biopsy and it is unclear how the vitrification process may affect the metabolic profile of blastocysts. Our study was also limited by the small number of rare donated euploid embryos available for analysis. Euploid embryos are very rarely discarded due to their value to patients trying to conceive, which limits their use for research purposes. However, we controlled for the imbalance with the bootstrap resampling analysis.Wider Implications Of the Findings: These findings provide preliminary evidence that FLIM may be a useful non-invasive clinical tool to assist in identifying the ploidy status of embryos.Study Funding/competing Interest(s): The study was supported by the Blavatnik Biomedical Accelerator Grant at Harvard University. Becker and Hickl GmbH and Boston Electronics sponsored research with the loaning of equipment for FLIM. D.J.N. is an inventor on patent US20170039415A1. There are no other conflicts of interest to declare.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Fluorescence imaging is an indispensable method for analysis of diverse cellular and molecular processes, enabling, for example, detection of ions, second messengers, or metabolites. Intensity-based approaches, however, are prone to artifacts introduced by changes in fluorophore concentrations. This drawback can be overcome by fluorescence lifetime imaging (FLIM) based on time-correlated single-photon counting. FLIM often necessitates long photon collection times, resulting in strong temporal binning of dynamic processes. Recently, rapidFLIM was introduced, exploiting ultra-low dead-time photodetectors together with rapid electronics. Here, we demonstrate the applicability of rapidFLIM, combined with new and improved correction schemes, for spatiotemporal fluorescence lifetime imaging of low-emission fluorophores in a biological system. Using tissue slices of hippocampi of mice of either sex, loaded with the Na+ indicator ING2, we show that improved rapidFLIM enables quantitative, dynamic imaging of neuronal Na+ signals at a full-frame temporal resolution of 0.5Hz. Induction of transient chemical ischemia resulted in unexpectedly large Na+ influx, accompanied by considerable cell swelling. Both Na+ loading and cell swelling were dampened on inhibition of TRPV4 channels. Together, rapidFLIM enabled the spatiotemporal visualization and quantification of neuronal Na+ transients at unprecedented speed and independent from changes in cell volume. Moreover, our experiments identified TRPV4 channels as hitherto unappreciated contributors to neuronal Na+ loading on metabolic failure, suggesting this pathway as a possible target to ameliorate excitotoxic damage. Finally, rapidFLIM will allow faster and more sensitive detection of a wide range of dynamic signals with other FLIM probes, most notably those with intrinsic low-photon emission. [ABSTRACT FROM AUTHOR]

Widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) is a fast and accurate method to measure the fluorescence lifetime of entire images. However, the complexity and high costs involved in construction of such a system limit the extensive use of this technique. PCO AG recently released the first luminescence lifetime imaging camera based on a high frequency modulated CMOS image sensor, QMFLIM2. Here we tested and provide operational procedures to calibrate the camera and to improve the accuracy using corrections necessary for image analysis. With its flexible input/output options, we are able to use a modulated laser diode or a 20 MHz pulsed white supercontinuum laser as the light source. The output of the camera consists of a stack of modulated images that can be analyzed by the SimFCS software using the phasor approach. The nonuniform system response across the image sensor must be calibrated at the pixel level. This pixel calibration is crucial and needed for every camera settings, e.g. modulation frequency and exposure time. A significant dependency of the modulation signal on the intensity was also observed and hence an additional calibration is needed for each pixel depending on the pixel intensity level. These corrections are important not only for the fundamental frequency, but also for the higher harmonics when using the pulsed supercontinuum laser. With these post data acquisition corrections, the PCO CMOS-FLIM camera can be used for various biomedical applications requiring a large frame and high speed acquisition.

We present the first realisation of simultaneous multi‐spectral fluorescence imaging using a single‐photon avalanche diode (SPAD) array, where the spectral unmixing is facilitated by a plasmonic metasurface mosaic colour filter array (CFA). A 64 × 64 pixel format silicon SPAD array is used to record widefield fluorescence and brightfield data from four biological samples. A plasmonic metasurface composed of an arrangement of circular and elliptical nanoholes etched into an aluminium thin film deposited on a glass substrate provides the high transmission efficiency CFA, enabling a bespoke spectral unmixing algorithm to reconstruct high fidelity, full colour images from as few as ∼3 photons per pixel. This approach points the way toward real‐time, single‐photon sensitive multi‐spectral fluorescence imaging. Furthermore, this is possible without additional bulky components such as a filter wheel, prism or diffraction grating, nor the need for multiple sample exposures or multiple detectors. [ABSTRACT FROM AUTHOR]

We describe a method based on fluorescence-lifetime imaging microscopy (FLIM) to assess the fluidity of various membranes in neuronal cells at different stages of development [day 12 (E12) and day 16 (E16) of gestation]. For the FLIM measurements, we use the Laurdan probe which is commonly used to assess membrane water penetration in model and in biological membranes using spectral information. Using the FLIM approach, we build a fluidity scale based on calibration with model systems of different lipid compositions. In neuronal cells, we found a marked difference in fluidity between the internal membranes and the plasma membrane, being the plasma membrane the less fluid. However, we found no significant differences between the two cell groups, E12 and E16. Comparison with NIH3T3 cells shows that the plasma membranes of E12 and E16 cells are significantly more fluid than the plasma membrane of the cancer cells.

We have developed a two-photon fluorescence microscope capable of imaging up to 4mm in turbid media with micron resolution. The key feature of this instrument is the innovative detector, capable of collecting emission photons from a wider surface area of the sample than detectors in traditional two-photon microscopes. This detection scheme is extremely efficient in the collection of emitted photons scattered by turbid media which allows eight fold increase in the imaging depth when compared with conventional two-photon microscopes. Furthermore, this system also has in-depth fluorescence lifetime imaging microscopy (FLIM) imaging capability which increases image contrast. The detection scheme captures emission light in a transmission configuration, making it extremely efficient for the detection of second harmonic generation (SHG) signals, which is generally forward propagating. Here we present imaging experiments of tissue phantoms and in vivo and ex vivo biological tissue performed with this microscope.

Mechanical force modulates myriad cellular functions including migration, alignment, proliferation, and gene transcription. Mechanotransduction, the transmission of mechanical forces and its translation into biochemical signals, may be mediated by force induced protein conformation changes, subsequently modulating protein signaling. For the paxillin and focal adhesion kinase interaction, we demonstrate that force-induced changes in protein complex conformation, dissociation constant, and binding Gibbs free energy can be quantified by lifetime-resolved fluorescence energy transfer microscopy combined with intensity imaging calibrated by fluorescence correlation spectroscopy. Comparison with in vitro data shows that this interaction is allosteric in vivo. Further, spatially resolved imaging and inhibitor assays show that this protein interaction and its mechano-sensitivity are equal in the cytosol and in the focal adhesions complexes indicating that the mechano-sensitivity of this interaction must be mediated by soluble factors but not based on protein tyrosine phosphorylation.

Advanced stage glioma is the most aggressive form of malignant brain tumors with a short survival time. Real-time pathology assisted, or image guided surgical procedures that eliminate tumors promise to improve the clinical outcome and prolong the lives of patients. Our work is focused on the development of a rapid and sensitive assay for intraoperative diagnostics of glioma and identification of optical markers essential for differentiation between tumors and healthy brain tissues. We utilized fluorescence lifetime imaging (FLIM) of endogenous fluorophores related to metabolism of the glioma from freshly excised brains tissues. Macroscopic time-resolved fluorescence images of three intracranial animal glioma models and surgical samples of patients' glioblastoma together with the white matter have been collected. Several established and new algorithms were applied to identify the imaging markers of the tumors. We found that fluorescence lifetime parameters characteristic of the glioma provided background for differentiation between the tumors and intact brain tissues. All three rat tumor models demonstrated substantial differences between the malignant and normal tissue. Similarly, tumors from patients demonstrated statistically significant differences from the peritumoral white matter without infiltration. While the data and the analysis presented in this paper are preliminary and further investigation with a larger number of samples is required, the proposed approach based on the macroscopic FLIM has a high potential for diagnostics of glioma and evaluation of the surgical margins of gliomas. [ABSTRACT FROM AUTHOR]

When OLED materials are depositing on the back‐plane in the evaporation process, they can be mixed in pixels by the FMM shadow. As a result, the luminance is decreased and abnormal display panels are made. Previously, we measured the level of shadow using AFM (atomic force microscopy) technique, but this technique cannot be applied to actual display panel. So we have introduced the FLIM (fluorescence lifetime imaging microscopy) technique to identify the cause of the problem. We can analyze the mixed OLED materials using the lifetime of organic materials at a specific wavelength. Finally, we can define the level of shadow and distinguish the normal and abnormal pixels while the general PL (photoluminescence) method cannot measure the shadow. Therefore, this technique is very useful to confirm the shadow level with non‐destructive and high‐resolution imaging. [ABSTRACT FROM AUTHOR]

flimview is a bio-imaging Python software package to read, explore, manage and visualize Fluorescence-Lifetime Imaging Microscopy (FLIM) images. It can open the standard FLIM data file conventions (e.g., sdt and ptu) and processes them from the raw format to a more readable and manageable binned and fitted format. It allows customized kernels for binning the data as well as user defined masking operations for pre-processing the images. It also allows customized fluorescence decay fitting functions and preserves all of the metadata generated for provenance and reproducibility. Outcomes from the analysis are lossless compressed and stored in an efficient way providing the necessary open-source tools to access and explore the data. flimview is open source and includes example data, example Jupyter notebooks and tutorial documentation. The package, test data and documentation are available on Github.