Lifetime-based Method Research Articles

A fluorescence lifetime-based novel method for accurate lipid quantification of BODIPY vital-stained C. elegans

Lipid droplets (LDs) are organelles associated with lipid storage and energy metabolism; thus, their morphology and quantity are of significant research interest. While commercially available BODIPY dye effectively labels LDs in various cell types, it also labels lysosome-related organelles (LROs) in C. elegans, leading to non-specific LD quantification. Here, we report that the fluorescent signals of BODIPY exhibit distinct fluorescence lifetime patterns for LROs and LDs, which can be captured, visualized, and filtered by fluorescence lifetime imaging microscopy. Furthermore, we proposed and validated a method based on fluorescence lifetime that can improve the accuracy of fat storage quantification in BODIPY vital-staining worms, which holds broad applications, including rapid and accurate LD quantification in forward genetic screening. Additionally, our method enables observing dynamic LD-LRO interactions in living worms, a unique capability of BODIPY vital staining. Our findings highlight distinct BODIPY fluorescence lifetime characteristics of LDs and LROs, providing a valuable tool for future research on LDs, LROs, or their interactions.

Pixel Screening in Lifetime-Based Temperature Mapping Using β-NaYF4:Nd3+,Yb3+ by Time-Gated Near-Infrared Fluorescence Imaging on Deep Tissue in Live Mice.

Near-infrared fluorescence (NIRF) thermometry is an emerging method for the noncontact measurement of in vivo deep temperatures. Fluorescence-lifetime-based methods are effective because they are unaffected by optical loss due to excitation or detection paths. Moreover, the physiological changes in body temperature in deep tissues and their pharmacological effects are yet to be fully explored. In this study, we investigated the potential application of the NIRF lifetime-based method for temperature measurement of in vivo deep tissues in the abdomen using rare-earth-based particle materials. β-NaYF4 particles codoped with Nd3+ and Yb3+ (excitation: 808 nm, emission: 980 nm) were used as NIRF thermometers, and their fluorescence decay curves were exponential. Slope linearity analysis (SLA), a screening method, was proposed to extract pixels with valid data. This method involves performing a linearity evaluation of the semilogarithmic plot of the decay curve collected at three delay times after cutting off the pulsed laser irradiation. After intragastric administration of the thermometer, the stomach temperature was monitored by using an NIRF time-gated imaging setup. Concurrently, a heater was attached to the lower abdomens of the mice under anesthesia. A decrease in the stomach temperature under anesthesia and its recovery via the heater indicated changes in the fluorescence lifetime of the thermometer placed inside the body. Thus, NaYF4:Nd3+/Yb3+ functions as a fluorescence thermometer that can measure in vivo temperature based on the temperature dependence of the fluorescence lifetime at 980 nm under 808 nm excitation. This study demonstrated the ability of a rare-earth-based NIRF thermometer to measure deep tissues in live mice, with the proposed SLA method for excluding the noisy deviations from the analysis for measuring temperature using the NIRF lifetime of a rare-earth-based thermometer.

Phosphor thermometry evaluation of heat transfer enhancement on a hot plate achieved by a vortex-based fluidic oscillator

Recently, a vortex-based feedback fluidic oscillator has been proposed as an alternative to conventional feedback fluidic oscillators. This study aimed to compare the heat transfer performance of a newly designed fluidic oscillator with that of conventional feedback fluidic oscillators with and without an output diffuser using phosphor thermometry. To obtain the instantaneous temperature field in a wide range of high temperatures, a lifetime-based method was employed in two-dimensional phosphor thermography, with Mg4FGeO6: Mn serving as the sensor material and being excited by a UV-LED. A high-speed camera was used to capture images of the hot plate at 4,000 fps. The calibration test was conducted in the temperature range of 25–325 ˚C, and the results were compared with data from thermocouples. The study investigated the impact of the mass flow rate and throat-to-plate distance ratio of three fluidic oscillators to obtain the optimum Nusselt number. The results demonstrated that the newly designed fluidic oscillator exhibited significantly superior cooling effects in comparison to other types of fluidic oscillators. At the lowest ratio of the throat-to-wall distance to the outlet throat height (Z/D = 4.5) and a flow rate of 100 Lpm, the vortex-based fluidic oscillator demonstrated 78 % and 45 % higher heat transfer rates compared to the conventional oscillator with and without an external diffuser, respectively. Furthermore, the newly designed oscillator was more sensitive to the mass flow rate and throat-to-plate distance ratio than the other oscillators.

Lifetime based pressure and temperature sensitive paint measurement on a civil aircraft wing under conditions near the onset of shock-induced separation

Lifetime-based pressure- and temperature-sensitive paint (PSP and TSP) measurements were conducted in a large-scale industrial transonic wind tunnel to obtain high-quality pressure data for validation of computational fluid dynamics (CFD) simulations. A wind tunnel test was performed using the NASA common research model in the JAXA 2 m × 2 m transonic wind tunnel. The freestream Mach number was varied in the range of 0.70–0.89, and aerodynamic forces and moments were acquired in order to obtain the pitch break condition related to the onset of shock-induced separation. Polymer-based PSP was coated on a main wing of the model, and TSP was used in tandem with PSP to correct the temperature dependence of PSP. The two-gate lifetime-based method was applied to obtain the PSP and TSP emissions. An a-priori/in-situ hybrid calibration was conducted to convert ratio-of-ratios to pressure, and measured pressure distributions were mapped onto a three-dimensional (3D) model grid. The root-mean-square error for the pressure measurements was evaluated by pressure tap data and was approximated to be 0.8 kPa for all Mach numbers tested. The obtained pressure distributions exhibited a significantly high signal-to-noise ratio and were used for comparison with CFD results on a 3D grid. The high-spatial-resolution PSP measurements helped to accurately localize the differences from the CFD simulation results and showed that the prediction of the shock location along the main wing is still a relevant challenge.

A Prototype Wearable Device for Noninvasive Monitoring of Transcutaneous Oxygen.

Transcutaneous oxygen monitoring is a noninvasive method for measuring the partial pressure of oxygen diffusing through the skin, which strongly correlates with changes in dissolved oxygen in the arteries. Luminescent oxygen sensing is one of the techniques for assessing transcutaneous oxygen. Intensity- and lifetime-based measurements are two well-known methods used in this technique. The latter is more immune to optical path changes and reflections, making the measurements less vulnerable to motion artifacts and skin color changes. Although the lifetime-based method is promising, the acquisition of high-resolution lifetime data is crucial for accurate transcutaneous oxygen measurements from the human body when skin is not heated. We have built a compact prototype along with its custom firmware for the lifetime estimation of transcutaneous oxygen with a provision of a wearable device. Furthermore, we performed a small experiment study on three healthy human volunteers to prove the concept of measuring oxygen diffusing from the skin without heating. Lastly, the prototype successfully detected changes in lifetime values driven by the changes in transcutaneous oxygen partial pressure due to pressure-induced arterial occlusion and hypoxic gas delivery. The prototype resolved a minimum change of 1.34ns in a lifetime that corresponds to ∼0.031mmHg in response to slow changes in the oxygen pressure in the volunteer's body caused by hypoxic gas delivery. The prototype is believed to be the first in the literature to successfully conduct measurements in human subjects using the lifetime-based technique.

Optimal gate selection method for simultaneous lifetime-based measurement of PSP and TSP

In this study, a new method that optimizes a measurement condition in a lifetime-based simultaneous measurement of a pressure-sensitive paint (PSP) and a temperature-sensitive paint (TSP) is proposed for the improvement of the accuracy of the pressure measurement. An optimal gate is selected based on a pressure measurement error when calculating the pressure and the temperature simultaneously from measurement values of a PSP and a TSP. A shot noise of a PSP, a temperature error, and a fluctuation in an emission intensity ratio due to blurring were considered error factors of the PSP measurement. The pressure measurement error propagated from each error source was considered as an evaluation index in an optimization of a measurement condition. We evaluated 17 types of TSP characteristics and selected an optimal TSP and a measurement condition for the PSP measurement. Further, the optimized measurement condition was evaluated in a PSP/TSP simultaneous measurement using a coupon-based test. The optimal measurement condition obtained based on the proposed method and an empirical selection method were compared by a PSP/TSP simultaneous measurement using a coupon-based test. A small-pressure measurement error, i.e. high pressure-measurement accuracy, was realized by the proposed method in the simultaneous lifetime-based method of a PSP and a TSP. In addition to the analyses above, the blurring effects were found to be minor and briefly summarized in appendices.

Characterization and Improvement of Heat Resistance of a Polymer-Ceramic Pressure-Sensitive Paint at High Temperatures.

Degradation of fast response pressure-sensitive paints (PSP) above room temperature is a serious problem for PSP measurements in high-temperature environments. A standard polymer-ceramic PSP (PC-PSP) composed of platinum(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorphenyl)-porphyrin (PtTFPP), titania particles and poly(isobutyl methacrylate) (polyIBM) was characterized to elucidate the degradation mechanism. Applying a two-gate lifetime-based method, the PC-PSP has sufficient pressure and temperature sensitivities even at 100 °C, while the luminescence intensity significantly decreases during the test. Subsequent measurements on thermal and photostability as well as luminescence spectra reveal that the main cause of the degradation is the photodegradation of PtTFPP due to direct exposure of the dye molecules to the atmosphere. In order to suppress such degradation, a small amount of urethane resin is added to the dye solution as a simple additional step in the preparation of PC-PSP. The addition of the urethane resin significantly reduces the degradation of the PSP, although its time response is slightly slower than that of the standard PC-PSP.

Quantitative Mapping of Liver Hypoxia in Living Mice Using Time-Resolved Wide-Field Phosphorescence Lifetime Imaging.

Hypoxia has been identified to contribute the pathogenesis of a wide range of liver diseases, and therefore, quantitative mapping of liver hypoxia is important for providing critical information in the diagnosis and treatment of hepatic diseases. However, the existing imaging methods are unsuitable to quantitatively assess liver hypoxia due to the need of liver‐specific contrast agents and be easily affected by other imaging factors. Here, a time‐resolved lifetime‐based imaging method is established for quantitative mapping of the distribution of hypoxia in the livers of mice by combining a wide‐field luminescence lifetime imaging system with an oxygen‐sensitive nanoprobe. It is shown that the method is suitable for real‐time quantification of the change of oxygen pressure in the process of hepatic ischemia‐reperfusion of the mouse. Moreover, the developed lifetime imaging methodology is used to quantitatively map liver hypoxia regions in the mouse model of orthotopic liver tumor, where the average oxygen pressure in tumorous liver is far below the normal liver.

Simultaneous measurement method of pressure and temperature using dual-layer PSP/TSP with lifetime-based method

Recently, the application field of the pressure measurement technique using pressure sensitive paint (PSP) has been expanding. However, the temperature dependence of PSP is a serious obstacle to accurate pressure measurement in low gauge pressure conditions because PSP is an absolute pressure sensor. Therefore, the temperature information of PSP is indispensably required for accurate pressure measurement, especially in low gauge pressure conditions. As a result, both temperature information and pressure information with a function of temperature are required simultaneously to use the PSP in low gauge pressure conditions. Dual-layer PSP/temperature sensitive paint (TSP) (DL-PTSP) is one solution, but simultaneous measurement of pressure and temperature requires two optical filters to separate the luminescence of PSP and TSP, resulting in complex equipment and additional error caused by parallax. To solve the problem, we introduce a lifetime-based method for DL-PTSP that allows simultaneous measurement of pressure and temperature. It is a method which separates each component of luminescence with different lifetimes. Firstly, the DL-PTSP developed in this study was optimized by reducing the luminescence intensity of the PSP with a longer lifetime while maintaining the luminescence intensity of the TSP with a shorter lifetime, considering the lifetime measurement method. Secondly, we have clarified the optimum time conditions of the monolayer TSP and the monolayer PSP by analysing their luminescence decay process, by controlling the irradiation delay of the light source relative to the camera shutter. Thirdly, the sensitivity of the DL-PTSP was examined using conditions optimized for mono-layer TSP and PSP. The luminescence decay of the TSP component shows sufficient temperature dependence, while the pressure dependence is very small. On the other hand, the dependence of the luminescence decay of the PSP component on the temperature and pressure is almost equal to that of the mono-layer PSP. The results confirm the feasibility of the simultaneous measurement of pressure and temperature using DL-PTSP.

A novel lifetime-based phosphor thermography using three-gate scheme and a low frame-rate camera

Lifetime-based method using multiple-frame technique and sophisticated high-speed cameras has been developed for two-dimensional phosphor thermography in recent years, which is however limited due to high cost and long time in data processing. In this study, a novel lifetime-based method using three-gate scheme and a low frame-rate camera was proposed, which is able to significantly reduce equipment cost, simplify data processing, and remarkably increase spatial resolution. Here, Mg4FGeO6:Mn was used as sensor material, which was excited by a pulsed UV LED. A low frame-rate CCD camera at 4Hz was sufficient to conduct phosphor thermometry using a three-gate scheme: one in fully excited region, one in decay region and one for background subtraction. Calibration was performed with a temperature range from 200°C to 550°C, and the results agreed well with data obtained by a high-speed camera sampling at 40kHz. The effect of exposure time was investigated to yield an optimum value of 2ms in this study. Finally, this new method was demonstrated in temperature measurements of the steady and impulsively started impingement jet heat transfer.

Studying Kinetochores In Vivo Using FLIM-FRET.

Kinetochores play essential roles in coordinating mitosis, as a mechanical connector between chromosome and microtubule and as a source of numerous biochemical signals. These mechanical and biochemical behaviors of kinetochores change dynamically in cells during mitosis. Therefore, understanding kinetochore function requires an imaging tool that quantifies the protein-protein interactions or biochemical changes with high spatiotemporal resolution. FRET has previously been used in combination with biosensors to probe protein-protein interactions and biochemical activity. In this chapter, we introduce FLIM-FRET, a lifetime-based method that quantifies FRET, and describe the use of FLIM-FRET as a method for studying dynamic kinetochore behavior in cells with high spatiotemporal resolution.

A 3D aligning method for stimulated emission depletion microscopy using fluorescence lifetime distribution.

Optimal resolution by stimulated emission depletion (STED) microscopy requires precise alignment of the donut-shaped depletion focus to the excitation focus. In this article, we demonstrate that fluorescence lifetime distribution can be implemented to align the STED system. Different from the traditional aligning methods in which a scattering imaging module is often equipped, the lifetime-based method is free from probable mismatches between the scattering mode and the fluorescent mode, drift errors caused by separate imaging and complex fitting methods. Based on this method, a spatial resolution of 38 nm by time-gated detection has been achieved.

Room temperature phosphorescence study on the structural flexibility of single tryptophan containing proteins

In this study, we have undertaken efforts to find correlation between phosphorescence lifetimes of single tryptophan containing proteins and some structural indicators of protein flexibility/rigidity, such as the degree of tryptophan burial or its exposure to solvent, protein secondary and tertiary structure of the region of localization of tryptophan as well as B factors for tryptophan residue and its immediate surroundings. Bearing in mind that, apart from effective local viscosity of the protein/solvent matrix, the other factor that concur in determining room temperature tryptophan phosphorescence (RTTP) lifetime in proteins is the extent of intramolecular quenching by His, Cys, Tyr and Trp side chains, the crystallographic structures derived from the Brookhaven Protein Data Bank were also analyzed concentrating on the presence of potentially quenching amino acid side chains in the close proximity of the indole chromophore. The obtained results indicated that, in most cases, the phosphorescence lifetimes of tryptophan containing proteins studied tend to correlate with the above mentioned structural indicators of protein rigidity/flexibility. This correlation is expected to provide guidelines for the future development of phosphorescence lifetime-based method for the prediction of structural flexibility of proteins, which is directly linked to their biological function.

Lifetime-based measurement of stress using mechanoluminescence of SrAl_2O_4:Eu^2+

The stress and loading rate on disks of SrAl2O4:Eu mixed epoxy resin were measured by a lifetime-based method. The concentration of the mechanoluminescence (ML) phosphor and the excitation power did not affect the measured lifetime. The lifetime was proportional to the stress and was inversely proportional to the loading rate. The concentration, the excitation power, the stress, and the loading rate affected the peak intensity of ML, while previous work estimated the stress using the absolute intensity under well-controlled excitation and concentration conditions. The method used here can shorten the measurement time and enhance the practicality of a ML phosphor.

Measurement of the inclusive and dijet cross-sections of b-jets in pp collisions at $\sqrt{s}=7\mbox{~TeV}$ with the ATLAS detector

The inclusive and dijet production cross-sections have been measured for jets containing b-hadrons (b-jets) in proton-proton collisions at a centre-of-mass energy of sqrt(s) = 7 TeV, using the ATLAS detector at the LHC. The measurements use data corresponding to an integrated luminosity of 34 pb^-1. The b-jets are identified using either a lifetime-based method, where secondary decay vertices of b-hadrons in jets are reconstructed using information from the tracking detectors, or a muon-based method where the presence of a muon is used to identify semileptonic decays of b-hadrons inside jets. The inclusive b-jet cross-section is measured as a function of transverse momentum in the range 20 < pT < 400 GeV and rapidity in the range |y| < 2.1. The bbbar-dijet cross-section is measured as a function of the dijet invariant mass in the range 110 < m_jj < 760 GeV, the azimuthal angle difference between the two jets and the angular variable chi in two dijet mass regions. The results are compared with next-to-leading-order QCD predictions. Good agreement is observed between the measured cross-sections and the predictions obtained using POWHEG + Pythia. MC@NLO + Herwig shows good agreement with the measured bbbar-dijet cross-section. However, it does not reproduce the measured inclusive cross-section well, particularly for central b-jets with large transverse momenta.

Multiplex bacterial growth monitoring in 24‐well microplates using a dual optical sensor for dissolved oxygen and pH

Non-invasive, simultaneous optical monitoring of oxygen and pH during bacterial cultivation in 24-well microplates is presented using an integrated dual sensor for dissolved oxygen and pH values. The dual sensor is based on oxygen-sensitive organosilica microparticles and pH-sensitive microbeads from a polymethacrylate derivative embedded into a polyurethane hydrogel. The readout is based on a phase-domain fluorescence lifetime-based method referred to as modified frequency domain dual lifetime referencing using a commercially available detector system for 24-well microplates. The sensor was used for monitoring the growth of Pseudomonas putida bacterial cultures. The method is suitable for parallelized, miniaturized bioprocessing, and cell-based high-throughput screening applications.

An empirical study of a reverse engineering method for the aggregation relationship based on operation propagation

One of the major obstacles in reverse engineering legacy object-oriented systems is the identification of aggregation relationships. An aggregation relationship, also called whole---part relationship, is a form of association relationship where an object is considered as a part of another object. This characteristic is mostly of semantic nature; therefore, it is difficult to distinguish aggregation from association relationships by implementation mechanism. Most reverse engineering methods for aggregation relationships are based on the lifetime dependence of an object on another object since many implementations of aggregation relationships result in such dependence. However, research literature shows that lifetime dependence is not really a primary property of the aggregation relationships. A reverse engineering approach is proposed on the basis of a primary characteristic for aggregation relationship--propagation of operations. To compare the propagation-based method with the lifetime-based method, we apply both methods to ten class libraries, collect their output, and perform statistical analysis to determine the effectiveness of the two methods. The analysis results show that the propagation-based method performs significantly better than the lifetime-based method and by combining both methods simultaneously the complete aggregation relationships can be uncovered for the class libraries in our experiment.

A102 寿命法による感圧塗料計測の低速流への適用と評価

The lifetime-based method has been applied to Pressure-Sensitive Paint (PSP) measurement in low-speed flow. Based on theoretical considerations and sample tests, a method to calculate pressure using a-priori method was proposed. In this method, pressure is related to the ratio of the luminescent intensity ratios obtained at two different gates between the wind-on and wind-off conditions, multiplied by a correction factor calculated from the single-point measurement of model substrate temperature. In order to evaluate the effectiveness of this method, a simplified car model was tested in a low-speed wind tunnel. It was shown that the effects of non-uniform distributions of lifetime and temperature could be cancelled out by the present method. The result obtained by the lifetime method was in good agreement with those of pressure taps and the intensity method.

B311 低温風洞へのLifetime Imaging法の適用可能性

The intensity-based method is a most common technique by which Pressure-Sensitive Paint images can be converted to the surface pressure. However, this method cannot be applied to a cryogenic wind tunnel because the thermal equilibrium condition cannot be achieved when stopping the tunnel to take the wind-off reference image. In the present experiment, we applied the lifetime-based method to a cryogenic wind tunnel to calculate surface pressure from two images obtained during the wind-on condition. In this experiment, the circular-arc bump model sprayed with PtTFPP/PTMSP paint was tested at the JAXA 0.1m Cryogenic Transonic Wind Tunnel using a UV-LED pulse illuminator and a multi-gated CCD camera. As a result, we could succeed in visualizing surface pressure distribution induced by a shock wave on the model by using lifetime PSP imaging method. This result has demonstrated a feasibility of using lifetime imaging method to a cryogenic wind tunnel.

Discrimination of damaged/dead cells by propidium iodide uptake in immunofluorescently labeled populations analyzed by phase-sensitive flow cytometry

We report a flow cytometric fluorescence lifetime-based method to discriminate damaged/dead from viable cells in immunofluorescently labeled populations using propidium iodide as a dye-exclusion viability probe. Fluorescence signals from propidium iodide and the anti-thymus cell-surface immunofluorescence marker fluorochromes, phycoerythrin and phycoerythrin/Texas Red (tandem conjugate), which have overlapping emission spectra with propidium iodide, are resolved based on differences in their fluorescence emission lifetimes using phase-sensitive detection. Mouse thymus cell samples were first labeled separately with anti-Thy 1.2 antibody directly conjugated to phycoerythrin and to phycoerythrin/Texas Red and propidium iodide. Labeled cells were then analyzed to determine the lifetimes of the immunofluorescence markers and propidium iodide. Based on these results, rat and mouse thymocytes labeled with anti-Thy 1.1 conjugated to phycoerythrin and anti-Thy 1.2 conjugated to phycoerythrin/Texas Red, respectively, were suspended in phosphate buffered saline containing propidium iodide, and were analyzed as they passed through a flow chamber and crossed a high-frequency, intensity-modulated (sinusoidal) laser excitation beam. The resulting immunofluorescence and propidium iodide signals were resolved based on differences in fluorescence lifetimes expressed as phase shifts using phase-sensitive detection and displayed as frequency distribution histograms and bivariate contour diagrams. This technology provides a new method to resolve immunofluorescence and propidium iodide signals from overlapping fluorescence emission spectra and a flow cytometric lifetime-based technique to quantify damaged/dead cells in immunofluorescence studies.