Fluorescence lifetime is not only associated with the molecular structure f fuorophores,but alsostrongly depends on the environment around them,which llows fuorescence lifetime imagingmicroscopy(FLIM)to be used as a t...Fluorescence lifetime is not only associated with the molecular structure f fuorophores,but alsostrongly depends on the environment around them,which llows fuorescence lifetime imagingmicroscopy(FLIM)to be used as a tool for precise measurement of the cell or tisue microenvironment,This review introduces the basic principle of fuorescence lifetime imagingtechnology and its application in clinical medicine,including research and diagnosis of diseases inskin,brain,eyes,mouth,bone,blood vessels and cavity organs,and drug evaluation.As anoninvasive,nontoxic and nonionizing radiation technique,FLIM demonstrates excellent per-formance with high sensitivity and specificity,which allows to determine precise position of thelesion and,thus,has good potential for application in biomedical research and clinical diagnosis.展开更多
Fluorescence litime imaging(FLIM)is an effective noninvasive bioanalytical tol based onmeasuring fuorescent lifetime of fuorophores.A growing number of FLIM studies utilizes ge-netically engineered fluorescent protein...Fluorescence litime imaging(FLIM)is an effective noninvasive bioanalytical tol based onmeasuring fuorescent lifetime of fuorophores.A growing number of FLIM studies utilizes ge-netically engineered fluorescent proteins targeted to specific subcellular structures to probe localmolecular environment,which opens new directions in cell science.This paper highlights theunconventional applications of FLIM for studies of molecular processes in diverse organelles oflive cultured cells.展开更多
Fluorescence lifetime imaging microscopy(FLIM)is increasingly used in biomedicine,material science,chemistry,and other related research fields,because of its advantages of high specificity and sensitivity in monitorin...Fluorescence lifetime imaging microscopy(FLIM)is increasingly used in biomedicine,material science,chemistry,and other related research fields,because of its advantages of high specificity and sensitivity in monitoring cellular microenvironments,studying interaction between proteins,metabolic state,screening drugs and analyzing their efficacy,characterizing novel materials,and diagnosing early cancers.Understandably,there is a large interest in obtaining FLIM data within an acquisition time as short as possible.Consequently,there is currently a technology that advances towards faster and faster FLIM recording.However,the maximum speed of a recording technique is only part of the problerm.The acquisition time of a FLIM image is a complex function of many factors.These include the photon rate that can be obtained from the sample,the amount of information a technique extracts from the decay functions,the fficiency at which it determines fluorescence decay parameters from the recorded photons,the demands for the accuracy of these parameters,the number of pixels,and the lateral and axial resolutions that are obtained in biological materials.Starting from a discussion of the parameters which determine the acquisition time,this review will describe existing and emerging FLIM techniques and data analysis algo-rithms,and analyze their performance and recording speed in biological and biomedical applications.展开更多
Apoptosis is very important for the maintenance of cellular homeostasis and is closely related to the occurrence and treatment of many diseases.Mitochondria in cells play a crucial role in programmed cell death and re...Apoptosis is very important for the maintenance of cellular homeostasis and is closely related to the occurrence and treatment of many diseases.Mitochondria in cells play a crucial role in programmed cell death and redox processes.Nicotinamide adenine dinucleotide(NAD(P)H)is the primary producer of energy in mitochondria,changing NAD(P)H can directly reflect the physiological state of mitochondria.Therefore,NAD(P)H can be used to evaluate metabolic response.In this paper,we propose a noninvasive detection method that uses two-photon fluorescence lifetime imaging microscopy(TP-FLIM)to characterize apoptosis by observing the binding kinetics of cellular endogenous NAD(P)H.The result shows that the average fluorescence lifetime of NAD(P)H and the fluorescence lifetime of protein-bound NAD(P)H will be affected by the changing pH,serum content,and oxygen concentration in the cell culture environment,and by the treatment with reagents such as H2O2 and paclitaxel.Taxol(PTX).This noninvasive detection method realized the dynamic detection of cellular endogenous substances and the assessment of apoptosis.展开更多
Fluorescence lifetime(FLT)of fluorophores is sensitive to the changes in their surrounding microenvironment,and hence it can quantitatively reveal the physiological characterization of the tissue under investigation.F...Fluorescence lifetime(FLT)of fluorophores is sensitive to the changes in their surrounding microenvironment,and hence it can quantitatively reveal the physiological characterization of the tissue under investigation.Fluorescence lifetime imaging microscopy(FLIM)provides not only morphological but also functional information of the tisse by producing spatially resolved image of fuorophore lifetime,which can be used as a signature of disorder and/or malignancy in diseased tissues.In this paper,we begin by introducing the basic principle and common detection methods of FLIM.Then the recent advances in the FLIM-based diagnosis of three different skin cancers,including basal cell carcinoma(BCC),squamous cell carcinoma(SCC)and malignant melanoma(MM)are reviewed.Furthermore,the potential advantages of FLIM in skin cancer diagnosis and the challenges that may be faced in the future are prospected.展开更多
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 re...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 paraformaldehydefixed 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.展开更多
Fluorescence lifetime and anisotropy has become a prevalent tool to detect the structure change and motility property of proteins. YgaP is the only membrane-integrated rhodanese in E. coli. The sulfur transfer process...Fluorescence lifetime and anisotropy has become a prevalent tool to detect the structure change and motility property of proteins. YgaP is the only membrane-integrated rhodanese in E. coli. The sulfur transfer process has been characterized by various studies. However, the mechanism of the outward transportation of SCN-remains unclear. In this work, we examined the fluorescence lifetime and anisotropy of site-specific incorporated unnatural amino acid 7-HC to study the conformational change of YgaP upon SCN-binding. We also compared the fluorescence changes between detergent-wrapped environment in DPC and intact native membrane environment in SMA. Our results suggested the presence of at least two different conformations in YgaP protein. Both the residues in the middle of TMH2 and the residues near extracellular side play important roles in the binding and/or output of SCN-. SMA is a good material to reflect the in situ conformation changes of protein than micelles.展开更多
Fluorescence microscopy, as a sensitive method to detect microenvironment of molecules, is widely used in protein conformation and dynamic studies in live cells. Fluorescence lifetime imaging microscopy（FLIM）, which...Fluorescence microscopy, as a sensitive method to detect microenvironment of molecules, is widely used in protein conformation and dynamic studies in live cells. Fluorescence lifetime imaging microscopy（FLIM）, which is independent of fluorophore concentrations, scattering and bleaching, is a suitable tool to analyze membrane proteins in a single cell. Ferroportin（FPN）, a multi-ion exporter in vertebrates, was modulated by metal ions with unknown mechanism. Herein, we fused green fluorescence protein on Cterminal of FPN（FPN-eGFP） and applied fluorescence lifetime to monitor conformation changes of FPN in a live cell. The fluorescence lifetime distribution showed a shift to shorter lifetime upon Mn^（2＋） treatment,suggesting a preference conformation of FPN in Mn^（2＋） exposure. It is also observed that the lifetime（rather than intensity） measurement was not strongly influenced by laser power. The observed fluorescence lifetime changes of FPN-eGFP upon Mn^（2＋） treatments indicated that extracellular metal ions can modulate FPN through conformation exchanges between several different states.展开更多
Objective：To study the mitochondrial redox state in experimental animals to sensitively detect early signs of mitochondrial function in pathophysiologieal conditions, such as isehemia. Methods： Fluorescence of nieot...Objective：To study the mitochondrial redox state in experimental animals to sensitively detect early signs of mitochondrial function in pathophysiologieal conditions, such as isehemia. Methods： Fluorescence of nieotinamide adenine dinucleotide （phosphate） , or NAD（P）H, the principal electron donor in mitochondrial respiration responsible for vital ATP supply of cardiomyocytes, is studied for non-invasive fluorescent probing of the mitochondrial function. Examination of NAD （P）H fluorescence in living cardiomyocytes following excitation by UV-pulsed laser diode and detection by spectrally-resolved time-correlated single photon counting （TCSPC） , is based on the simultaneous measurement of the fluorescence spectra and lifetime. Results ： The dynamic characteristics of NAD （P） H fluorescence decay in living rat cardiomyocytes show that at least a 3-exponential decay model, with 0.4 - 0.7 ns, 1.2 - 1.9 ns and 8.0 - 13.0 ns lifetimes, is necessary to describe cardiomyocyte autofluorescenee （AF）. Decay-associated spectra （DSA） revealed the presence of 4 spectrally-distinct populations of NADH molecules in eardiomyocytes with spectral maximum at 470 nm for short-lifetime pool for the first time, and emission peaks at 450 nm, 470 nm and 490 nm for intermediate and long-lifetime pools. Increased mitochondrial NADH content ratio by ketone bodies enhanced the AF intensity, without the significant change in fluorescent lifetimes. Rotenone, the inhibitor of Complex I of the mitochondrial respiratory chain, increased AF and shortened the average fluorescence lifetime. Dinitrophenol （DNP）, an uncoupling agent of the mitochondrial oxidative phosphorylation, lowered AF,broadened the spectral shoulder at 520 nm and increased the average lifetime. These effects, comparable to the changes in the concentration and in the rate of dehydrogenation of NADH in vitro, were also examined under ischemia-mimetic conditions. Conclusion： Our findings anticipate a contribution of both conformational NADH changes and energy transfer from NADH to lipoamide dehydrogenase （LipDH）-bound flavins, to explain observed fluorescence kinetics. Presented spectrally resolved fluorescence lifetime approach provides promising new tool for analysis of mitochondrial NAD （P） H in living cardiomyocytes, and hence for investigation of energy metabolism and mitoehondrial dysfunction at a cellular level.展开更多
Octadecylamine was derivatized with dansyl chloride (5-dimethylaminonaphthalene-1-sulfonyl chloride) In order to simplify and understand the LB films of fluorescent probe labeling proteins. its monolayer and multilaye...Octadecylamine was derivatized with dansyl chloride (5-dimethylaminonaphthalene-1-sulfonyl chloride) In order to simplify and understand the LB films of fluorescent probe labeling proteins. its monolayer and multilayers in the absence and presence of stearic acid were deposited by LB technique. Fluorescence spectra and lifetimes of the fluorescent products were studied to elucidate the microenvironment of molecules in the LB films.展开更多
Fluorescence lifetime imaging microscopy(FLIM)is a powerful tool to discriminate fluorescent molecules or probe their nanoscale environment.Traditionally,FLIM uses time-correlated single-photon counting(TCSPC),which i...Fluorescence lifetime imaging microscopy(FLIM)is a powerful tool to discriminate fluorescent molecules or probe their nanoscale environment.Traditionally,FLIM uses time-correlated single-photon counting(TCSPC),which is precise but intrinsically low-throughput due to its dependence on point detectors.Although time-gated cameras have demonstrated the potential for high-throughput FLIM in bright samples with dense labeling,their use in single-molecule microscopy has not been explored extensively.Here,we report fast and accurate single-molecule FLIM with a commercial time-gated single-photon camera.Our optimized acquisition scheme achieves single-molecule lifetime measurements with a precision only about three times less than TCSPC,while imaging with a large number of pixels(512×512)allowing for the spatial multiplexing of over 3000 molecules.With this approach,we demonstrate parallelized lifetime measurements of large numbers of labeled pore-forming proteins on supported lipid bilayers,and temporal single-molecule Förster resonance energy transfer measurements at 5-25 Hz.This method holds considerable promise for the advancement of multi-target single-molecule localization microscopy and biopolymer sequencing.展开更多
Bacterial resistance,primarily stemming from misdiagnosis,misuse,and overuse of antibacterial medications in humans and animals,is a pressing issue.To address this,we focused on developing a fluorescent probe for the ...Bacterial resistance,primarily stemming from misdiagnosis,misuse,and overuse of antibacterial medications in humans and animals,is a pressing issue.To address this,we focused on developing a fluorescent probe for the detection of bacteria,with a unique feature--an exceptionally long fluorescence lifetime,to overcome autofluorescence limitations in biological samples.The polymyxin-based probe(ADOTA-PMX)selectively targets Gram-negative bacteria and used the red-emitting fluorophore azadioxatriangulenium(with a reported fluorescence lifetime of 19.5 ns).Evaluation of ADOTA-PMX’s bacterial labeling efficacy revealed strong specificity for Gram-negative bacteria,and full spectral fluorescence lifetime imaging microscopy demonstrated the potential of ADOTA-PMX for bacterial imaging applications.The probe exhibited a lifetime of 4.5 ns when bound to bacteria,possibly indicating interactions with the bacterial outer membrane.Furthermore,the fluorescence lifetime measurements of ADOTA-PMX labeled bacteria could be performed using a benchtop fluorimeter without the need of sophisticated microscopes.This study represents the first targeted probe for fluorescence lifetime imaging,offering sensitivity for detecting Gram-negative bacteria and enabling multiplexing via fluorescence lifetime imaging.展开更多
Fluorescence lifetime imaging can reveal the high-resolution structure of various biophysical and chemical parameters in a microenvironment quantitatively.However,the depth of imaging is generally limited to hundreds ...Fluorescence lifetime imaging can reveal the high-resolution structure of various biophysical and chemical parameters in a microenvironment quantitatively.However,the depth of imaging is generally limited to hundreds of micrometers due to aberration and light scattering in biological tissues.This paper introduces an iterative multi-photon adaptive compensation technique(IMPACT)into a two-photon fluorescence lifetime microscopy system to successfully overcome aberrations and multiple scattering problems in deep tissues.It shows that 400 correction modes can be achieved within 5 min,which was mainly limited by the frame rate of a spatial light modulator.This system was used for high-resolution imaging of mice brain tissue and live zebrafish,further verifying its superior performance in imaging quality and photon accumulation speed.展开更多
Time-resolved flow cytometry(TRFC)was used to measure metabolic differences in estrogen receptor-positive breast cancer cells.This specialty cytometry technique measures fluorescence lifetimes as a single-cell paramet...Time-resolved flow cytometry(TRFC)was used to measure metabolic differences in estrogen receptor-positive breast cancer cells.This specialty cytometry technique measures fluorescence lifetimes as a single-cell parameter thereby providing a unique approach for high-throughput cell counting and screening.Differences in fluorescence lifetime were detected and this was associated with sensitivity to the commonly prescribed therapeutic tamoxifen.Differences in fluorescence lifetime are attributed to the binding states of the autofluorescent metabolite NAD(P)H.The function of NAD(P)H is well described and in general involves cycling from a reduced to oxidized state to facilitate electron transport for the conversion of pyruvate to lactate.NAD(P)H fluorescence lifetimes depend on the bound or unbound state of the metabolite,which also relates to metabolic transitions between oxidative phosphorylation and glycolysis.To determine if fundamental metabolic profiles differ for cells that are sensitive to tamoxifen compared to those that are resistant,large populations of MCF-7 breast cancer cells were screened and fluorescence lifetimes were quantified.Additionally,metabolic differences associated with tamoxifen sensitivity were measured with a Seahorse HS mini metabolic analyzer(Agilent Technologies Inc.Santa Clara,CA)and confocal imaging.Results show that tamoxifen-resistant breast cancer cells have increased utilization of glycolysis for energy production compared to tamoxifen-sensitive breast cancer cells.This work is impacting because it establishes an early step toward developing a reliable screening technology in which large cell censuses can be differentiated for drug sensitivity in a label-free fashion.展开更多
Two-dimensional(2D)transition-metal dichalcogenide(TMD)materials have aroused noticeable interest due to their distinguished electronic and optical properties.However,little is known about their complex exciton proper...Two-dimensional(2D)transition-metal dichalcogenide(TMD)materials have aroused noticeable interest due to their distinguished electronic and optical properties.However,little is known about their complex exciton properties together with the exciton dynamics process which have been expected to influence the performance of optoelectronic devices.The process of fluorescence can well reveal the process of exciton transition after excitation.In this work,the room-temperature layer-dependent exciton dynamics properties in layered WSe2 are investigated by the fluorescence lifetime imaging microscopy(FLIM)for the first time.This paper focuses on two mainly kinds of excitons including the direct transition neutral excitons and trions.Compared with the lifetime of neutral excitons(<0.3 ns within four-layer),trions possess a longer lifetime(~6.6 ns within four-layer)which increases with the number of layers.We attribute the longer-lived lifetime to the increasing number of trions as well as the varieties of trion configurations in thicker WSe2.Besides,the whole average lifetime increases over 10%when WSe2 flakes added up from monolayer to four-layer.This paper provides a novel tuneable layer-dependent method to control the exciton dynamics process and finds a relatively longer transition lifetime of trions at room temperature,enabling to investigate in the charge transport in TMD-based optoelectronics devices in the future.展开更多
By using fluorescence lifetime image microscope （FLIM） and time-correlated single photon counting （TCSPC） technique, we measured fluorescence lifetime of crude oils with density of 0.9521-0.7606 g/cm3 and multiple...By using fluorescence lifetime image microscope （FLIM） and time-correlated single photon counting （TCSPC） technique, we measured fluorescence lifetime of crude oils with density of 0.9521-0.7606 g/cm3 and multiple petroleum inclusions from Tazhong uplift of Tarim Basin. As indicated by the test results, crude oil density is closely correlated with average fluorescence lifetime following the regression equation Y=-0.0319X＋0.9411, which can thus be used to calculate density of oil inclusions in relation to fluorescence lifetime and density of corresponding surface crude. For type A oil inclusions showing brown-yellow fluorescence from Tazhong 1 well in Tarim Basin, their average fluorescence lifetime was found to be 2.144-2.765 ns, so the density of surface crude corresponding to crude trapping these oil inclusions is 0.852-0.873 g/cm3, indicating that they are matured oil inclusions trapped at earlier stage ofoil formation. For type B oil inclusions with light yellow-white fluorescence, their average fluorescence lifetime was found to be 4.0294.919 ns, so the density of surface crude corresponding to crude trapping these oil inclusions is 0.784-0.812 g/cm3, indicating that they are higher matured oil inclusions trapped at the second stage of oil formation. For type C oil inclusions showing light blue-green fluorescence, their average fluorescence lifetime was found to be 5.0634.168 ns, so the density of surface crude corresponding to crude trapping these oil inclusions is 0.743-0.779 g/cm3, indicating that they are highly-matured light oil inclusions trapped at the third stage of oil formation.展开更多
Phagocytosis is a biological process that plays a key role in host defense and tissue homeostasis.Efficient approaches for real-time imaging of phagocytosis are highly desired but limited.Herein,an AIE-active near-inf...Phagocytosis is a biological process that plays a key role in host defense and tissue homeostasis.Efficient approaches for real-time imaging of phagocytosis are highly desired but limited.Herein,an AIE-active near-infrared fluorescent probe,named TBTCP,was developed for fluorescence lifetime imaging of phagocytosis.TBTCP could selectively label the cell plasma membrane with fast staining,wash-free process,high signal-to-background ratio,and excellent photostability.Cellular membrane statuses under different osmolarities as well as macrophage phagocytosis of bacteria or large silica particles in early stages could be reported by the fluorescence lifetime changes of TBTCP.Compared with current fluorescence imaging methods,which target the bioenvironmental changes in the late phagocytosis stage,this approach detects the changes in the cell membrane,thus giving a faster response to phagocytosis.This article provides a functional tool to report the phagocytic dynamics of macrophages which may greatly contribute to the studies of phagocytic function-related diseases.展开更多
Intracellular pH plays a critical role in biological functions,and abnormal pH values are related to various diseases.Here,we report on an intracellular pH sensor AgInS_(2)(AIS)/ZnS quantum dots(QDs)that show long flu...Intracellular pH plays a critical role in biological functions,and abnormal pH values are related to various diseases.Here,we report on an intracellular pH sensor AgInS_(2)(AIS)/ZnS quantum dots(QDs)that show long fluorescence lifetimes of hundreds of nanoseconds and low toxicity.Fluorescence lifetime imaging microscopy(FLIM)combined with AIS/ZnS QDs is used for the imaging of live cells in different pH buffers and different cell lines.The FLIM images of AIS/ZnS QDs in live cells demonstrate different intracellular pH values in different regions,such as in lysosomes or cytoplasm.This method can also distinguish cancer cells from normal cells,and the fluorescence lifetime difference of the AIS/ZnS QDs between the two types of cells is 100±7 ns.Most importantly,the exfoliated cervical cells from 20 patients are investigated using FLIM combined with AIS/ZnS QDs.The lifetime difference value between the normal and cervical cancer(CC)groups is 115±9 ns,and the difference between the normal and the precancerous lesion group is 64±9 ns.For the first time,the noninvasive method has been used for cervical cancer screening,and it has shown great improvement in sensitivity compared with a clinical conventional cytology examination.展开更多
Fluorescence lifetime measurement in the time domain requires excitation from a well separated single bunch using synchrotron light sources. In the colliding mode of the Beijing Electron Positron Collider Ⅱ （BEPCⅡ...Fluorescence lifetime measurement in the time domain requires excitation from a well separated single bunch using synchrotron light sources. In the colliding mode of the Beijing Electron Positron Collider Ⅱ （BEPCⅡ）, a hybrid filling pattern was realized such that a single bunch was placed in the middle of a large gap between two multi-bunch groups. Detection of fluorescence lifetime, based on the excitation of the light pulse from this designated single-bunch, was established at Beamline 4B8 of the Beijing Synchrotron Radiation Facility （BSRF）. The timing signal of the BEPCII was utilized as a trigger to gate this fluorescence event. L-Tryptophan amino acid, a known lifetime standard, was selected to assess the lifetime measurement performance. The measured lifetime was consistent in both colliding and single-bunch mode with the time resolution down to 450 ps. Moreover, both the bunch purity and the fine structure of the hybrid filling pattern were characterized.展开更多
Fluorescence correlation spectroscopy （FCS） is a widely used method for measuring molecular diffusion and chemical kinetics. However, when a mixture of fluorescent species is taken into account, the conven- tional F...Fluorescence correlation spectroscopy （FCS） is a widely used method for measuring molecular diffusion and chemical kinetics. However, when a mixture of fluorescent species is taken into account, the conven- tional FCS method has limitations in extracting autocorrelations for different species and cross correla- tions between different species. Recently developed fluorescence lifetime correlation spectroscopy （FLCS） based on time-tagged time-resolved （TITR） photon recording, which can record the global and micro arrival time for each individual photon, has been used to discriminate different species according to fluorescence lifetime. Here, based on two-dimensional lifetime decay maps constructed from TITR photon stream, we have developed a quantitative lifetime-deconvolution FCS model （LDFCS） to extract precise chemical rates for chemical conversions in multi-species systems. The key point of LDFCS model is separation of different species according to the global distribution of fluorescence lifetime and then deconvolution of autocorrelations and cross-correlations from the two-dimensional lifetime decay maps constructed bv the micro arrival times of photon pairs at each delay time.展开更多
基金funded by the Science and Technology Planning Fundamental Research Project of Shenzhen(No.JCYJ20150324140036853)National Natural Science Foundation of China(No.61378091)+1 种基金Ningbo Natural Science Foundation Project(No.2016A610032)the Central University Basic Scientic Research Business Expenses Project(No.NSIY051405).
文摘Fluorescence lifetime is not only associated with the molecular structure f fuorophores,but alsostrongly depends on the environment around them,which llows fuorescence lifetime imagingmicroscopy(FLIM)to be used as a tool for precise measurement of the cell or tisue microenvironment,This review introduces the basic principle of fuorescence lifetime imagingtechnology and its application in clinical medicine,including research and diagnosis of diseases inskin,brain,eyes,mouth,bone,blood vessels and cavity organs,and drug evaluation.As anoninvasive,nontoxic and nonionizing radiation technique,FLIM demonstrates excellent per-formance with high sensitivity and specificity,which allows to determine precise position of thelesion and,thus,has good potential for application in biomedical research and clinical diagnosis.
基金supported by the National Basic Research Program of China(2015CB352005)the National Natural Science Foundation of China(61525503/61378091/61620106016)+2 种基金Guangdong Natural Science Foundation Innovation Team(2014A030312008)Hong Kong,Macao and Taiwan cooperation innovation platform and major projects of international cooperation in Colleges and Universities in Guangdong Province(2015KGJHZ002)Shenzhen Basic Research Project(JCYJ20150930104948169/JCYJ20160328144746940/GJHZ 20160226202139185).
文摘Fluorescence litime imaging(FLIM)is an effective noninvasive bioanalytical tol based onmeasuring fuorescent lifetime of fuorophores.A growing number of FLIM studies utilizes ge-netically engineered fluorescent proteins targeted to specific subcellular structures to probe localmolecular environment,which opens new directions in cell science.This paper highlights theunconventional applications of FLIM for studies of molecular processes in diverse organelles oflive cultured cells.
基金support from the National Key R&D Program of China(2017YFA0700500)National Natural Science Foundation of China(61775144/61525503/61620106016/61835009/81727804)+2 种基金(Key)Project of Department of Education of Guangdong Province(2015KGJHZ002/2016KCXTD007)Guangdong Natural Science Foundation(2014A030312008,2017A030310132,2018A030313362)Shenzhen Basic Research Project(JCYJ20170818144012025/JCYJ20170818141701667/JCYJ20170412105003520/JCYJ20150930104948169).
文摘Fluorescence lifetime imaging microscopy(FLIM)is increasingly used in biomedicine,material science,chemistry,and other related research fields,because of its advantages of high specificity and sensitivity in monitoring cellular microenvironments,studying interaction between proteins,metabolic state,screening drugs and analyzing their efficacy,characterizing novel materials,and diagnosing early cancers.Understandably,there is a large interest in obtaining FLIM data within an acquisition time as short as possible.Consequently,there is currently a technology that advances towards faster and faster FLIM recording.However,the maximum speed of a recording technique is only part of the problerm.The acquisition time of a FLIM image is a complex function of many factors.These include the photon rate that can be obtained from the sample,the amount of information a technique extracts from the decay functions,the fficiency at which it determines fluorescence decay parameters from the recorded photons,the demands for the accuracy of these parameters,the number of pixels,and the lateral and axial resolutions that are obtained in biological materials.Starting from a discussion of the parameters which determine the acquisition time,this review will describe existing and emerging FLIM techniques and data analysis algo-rithms,and analyze their performance and recording speed in biological and biomedical applications.
基金supported in part by the National Key R&D Program of China(2017YFA0700402)National Natural Science Foundation of China(61961136005/61935012/62175163/61835009)+1 种基金Shenzhen Key projects(JCYJ20200109105404067)Shenzhen International Cooperation Project(GJHZ 20190822095420249).
文摘Apoptosis is very important for the maintenance of cellular homeostasis and is closely related to the occurrence and treatment of many diseases.Mitochondria in cells play a crucial role in programmed cell death and redox processes.Nicotinamide adenine dinucleotide(NAD(P)H)is the primary producer of energy in mitochondria,changing NAD(P)H can directly reflect the physiological state of mitochondria.Therefore,NAD(P)H can be used to evaluate metabolic response.In this paper,we propose a noninvasive detection method that uses two-photon fluorescence lifetime imaging microscopy(TP-FLIM)to characterize apoptosis by observing the binding kinetics of cellular endogenous NAD(P)H.The result shows that the average fluorescence lifetime of NAD(P)H and the fluorescence lifetime of protein-bound NAD(P)H will be affected by the changing pH,serum content,and oxygen concentration in the cell culture environment,and by the treatment with reagents such as H2O2 and paclitaxel.Taxol(PTX).This noninvasive detection method realized the dynamic detection of cellular endogenous substances and the assessment of apoptosis.
基金supported by The 111 Project(B17035)Open Research Fund Program of the State Key Laboratory of Low Dimensional Quantum Physics(KF201713)+1 种基金State Key Laboratory of Transient Optics and Photonics,Chinese Academy of Sciences(SKLST201804)the Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province(GD201711).
文摘Fluorescence lifetime(FLT)of fluorophores is sensitive to the changes in their surrounding microenvironment,and hence it can quantitatively reveal the physiological characterization of the tissue under investigation.Fluorescence lifetime imaging microscopy(FLIM)provides not only morphological but also functional information of the tisse by producing spatially resolved image of fuorophore lifetime,which can be used as a signature of disorder and/or malignancy in diseased tissues.In this paper,we begin by introducing the basic principle and common detection methods of FLIM.Then the recent advances in the FLIM-based diagnosis of three different skin cancers,including basal cell carcinoma(BCC),squamous cell carcinoma(SCC)and malignant melanoma(MM)are reviewed.Furthermore,the potential advantages of FLIM in skin cancer diagnosis and the challenges that may be faced in the future are prospected.
基金supported by the National Key R&D Program of China(Nos.2016YFA0400900 and 2017YFA0505301)National Natural Science Foundation of China(No.U1832181)。
文摘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 paraformaldehydefixed 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.
基金supported by the National Key R&D Program of China (Nos. 2016YFA0400900, 2017YFA0505300)the Instrument Developing Project of the Chinese Academy of Sciences (No. YZ201564)
文摘Fluorescence lifetime and anisotropy has become a prevalent tool to detect the structure change and motility property of proteins. YgaP is the only membrane-integrated rhodanese in E. coli. The sulfur transfer process has been characterized by various studies. However, the mechanism of the outward transportation of SCN-remains unclear. In this work, we examined the fluorescence lifetime and anisotropy of site-specific incorporated unnatural amino acid 7-HC to study the conformational change of YgaP upon SCN-binding. We also compared the fluorescence changes between detergent-wrapped environment in DPC and intact native membrane environment in SMA. Our results suggested the presence of at least two different conformations in YgaP protein. Both the residues in the middle of TMH2 and the residues near extracellular side play important roles in the binding and/or output of SCN-. SMA is a good material to reflect the in situ conformation changes of protein than micelles.
基金supported by the National Key R&D Program of China (Nos. 2016YFA0400900, 2017YFA0505300)the Instrument Developing Project of the Chinese Academy of Sciences (No. YZ201564)
文摘Fluorescence microscopy, as a sensitive method to detect microenvironment of molecules, is widely used in protein conformation and dynamic studies in live cells. Fluorescence lifetime imaging microscopy（FLIM）, which is independent of fluorophore concentrations, scattering and bleaching, is a suitable tool to analyze membrane proteins in a single cell. Ferroportin（FPN）, a multi-ion exporter in vertebrates, was modulated by metal ions with unknown mechanism. Herein, we fused green fluorescence protein on Cterminal of FPN（FPN-eGFP） and applied fluorescence lifetime to monitor conformation changes of FPN in a live cell. The fluorescence lifetime distribution showed a shift to shorter lifetime upon Mn^（2＋） treatment,suggesting a preference conformation of FPN in Mn^（2＋） exposure. It is also observed that the lifetime（rather than intensity） measurement was not strongly influenced by laser power. The observed fluorescence lifetime changes of FPN-eGFP upon Mn^（2＋） treatments indicated that extracellular metal ions can modulate FPN through conformation exchanges between several different states.
基金Canadian Institute for Health Researchgrant number:MOP74600+3 种基金Canadian Foundation for Innovationgrant number:N°9b84Groupe de Recherche Universitaire sur Mdicament grant to AC,FRSQ-NSFCgrant number:N°5540
文摘Objective：To study the mitochondrial redox state in experimental animals to sensitively detect early signs of mitochondrial function in pathophysiologieal conditions, such as isehemia. Methods： Fluorescence of nieotinamide adenine dinucleotide （phosphate） , or NAD（P）H, the principal electron donor in mitochondrial respiration responsible for vital ATP supply of cardiomyocytes, is studied for non-invasive fluorescent probing of the mitochondrial function. Examination of NAD （P）H fluorescence in living cardiomyocytes following excitation by UV-pulsed laser diode and detection by spectrally-resolved time-correlated single photon counting （TCSPC） , is based on the simultaneous measurement of the fluorescence spectra and lifetime. Results ： The dynamic characteristics of NAD （P） H fluorescence decay in living rat cardiomyocytes show that at least a 3-exponential decay model, with 0.4 - 0.7 ns, 1.2 - 1.9 ns and 8.0 - 13.0 ns lifetimes, is necessary to describe cardiomyocyte autofluorescenee （AF）. Decay-associated spectra （DSA） revealed the presence of 4 spectrally-distinct populations of NADH molecules in eardiomyocytes with spectral maximum at 470 nm for short-lifetime pool for the first time, and emission peaks at 450 nm, 470 nm and 490 nm for intermediate and long-lifetime pools. Increased mitochondrial NADH content ratio by ketone bodies enhanced the AF intensity, without the significant change in fluorescent lifetimes. Rotenone, the inhibitor of Complex I of the mitochondrial respiratory chain, increased AF and shortened the average fluorescence lifetime. Dinitrophenol （DNP）, an uncoupling agent of the mitochondrial oxidative phosphorylation, lowered AF,broadened the spectral shoulder at 520 nm and increased the average lifetime. These effects, comparable to the changes in the concentration and in the rate of dehydrogenation of NADH in vitro, were also examined under ischemia-mimetic conditions. Conclusion： Our findings anticipate a contribution of both conformational NADH changes and energy transfer from NADH to lipoamide dehydrogenase （LipDH）-bound flavins, to explain observed fluorescence kinetics. Presented spectrally resolved fluorescence lifetime approach provides promising new tool for analysis of mitochondrial NAD （P） H in living cardiomyocytes, and hence for investigation of energy metabolism and mitoehondrial dysfunction at a cellular level.
基金the National Natural Science Foundation of China (296333010) and The President Science Foundation of the Chinese Academy of Scie
文摘Octadecylamine was derivatized with dansyl chloride (5-dimethylaminonaphthalene-1-sulfonyl chloride) In order to simplify and understand the LB films of fluorescent probe labeling proteins. its monolayer and multilayers in the absence and presence of stearic acid were deposited by LB technique. Fluorescence spectra and lifetimes of the fluorescent products were studied to elucidate the microenvironment of molecules in the LB films.
基金support from the EPFL Center for Imaging(A.R.,N.R.,E.C.and C.B.)European Research Council(grant 101020445 to A.R.)+2 种基金the Swiss National Science Foundation(grant 200021-184687 to G.P.A.,grant 200021L-212128 to M.D.P.and grant IZSEZ0-224299 to R.R.)the National Center of Competence in Research Bio-Inspired Materials(NCCR 51NF40-182881 to G.P.A.and A.R.)the European Union Program HORIZON-Pathfinder-Open(grant 101099125 to G.P.A.).
文摘Fluorescence lifetime imaging microscopy(FLIM)is a powerful tool to discriminate fluorescent molecules or probe their nanoscale environment.Traditionally,FLIM uses time-correlated single-photon counting(TCSPC),which is precise but intrinsically low-throughput due to its dependence on point detectors.Although time-gated cameras have demonstrated the potential for high-throughput FLIM in bright samples with dense labeling,their use in single-molecule microscopy has not been explored extensively.Here,we report fast and accurate single-molecule FLIM with a commercial time-gated single-photon camera.Our optimized acquisition scheme achieves single-molecule lifetime measurements with a precision only about three times less than TCSPC,while imaging with a large number of pixels(512×512)allowing for the spatial multiplexing of over 3000 molecules.With this approach,we demonstrate parallelized lifetime measurements of large numbers of labeled pore-forming proteins on supported lipid bilayers,and temporal single-molecule Förster resonance energy transfer measurements at 5-25 Hz.This method holds considerable promise for the advancement of multi-target single-molecule localization microscopy and biopolymer sequencing.
基金funded by Engineering and Physical Sciences Research Council(EPSRC,United Kingdom)Interdisciplinary Research Collaboration,Grant Nos.EP/K03197X/1 and EP/R005257/1funded by a Royal Society,Grant No.RGS\R1\201163+1 种基金funded via the BBSRC EastBIO doctoral training program,grant BB/M010996/1funded by a Newton-Bhabha International Fellowship from The Royal Society,UK and The Science and Engineering Research Board,India,Grant No.NIF/R1/192688.
文摘Bacterial resistance,primarily stemming from misdiagnosis,misuse,and overuse of antibacterial medications in humans and animals,is a pressing issue.To address this,we focused on developing a fluorescent probe for the detection of bacteria,with a unique feature--an exceptionally long fluorescence lifetime,to overcome autofluorescence limitations in biological samples.The polymyxin-based probe(ADOTA-PMX)selectively targets Gram-negative bacteria and used the red-emitting fluorophore azadioxatriangulenium(with a reported fluorescence lifetime of 19.5 ns).Evaluation of ADOTA-PMX’s bacterial labeling efficacy revealed strong specificity for Gram-negative bacteria,and full spectral fluorescence lifetime imaging microscopy demonstrated the potential of ADOTA-PMX for bacterial imaging applications.The probe exhibited a lifetime of 4.5 ns when bound to bacteria,possibly indicating interactions with the bacterial outer membrane.Furthermore,the fluorescence lifetime measurements of ADOTA-PMX labeled bacteria could be performed using a benchtop fluorimeter without the need of sophisticated microscopes.This study represents the first targeted probe for fluorescence lifetime imaging,offering sensitivity for detecting Gram-negative bacteria and enabling multiplexing via fluorescence lifetime imaging.
基金supported by the National Key Research and Development Program of China(No.2021YFF0502900)the National Natural Science Foundation of China(Nos.62175163,62225505,61935012,61835009,62127819,and 62205220)+2 种基金the Shenzhen Key Projects(No.JCYJ20200109105404067)the Shenzhen Talent Innovation Project(No.RCJC20210706091949022)the Shenzhen Science and Technology Planning Project(No.ZDSYS20210623092006020)。
文摘Fluorescence lifetime imaging can reveal the high-resolution structure of various biophysical and chemical parameters in a microenvironment quantitatively.However,the depth of imaging is generally limited to hundreds of micrometers due to aberration and light scattering in biological tissues.This paper introduces an iterative multi-photon adaptive compensation technique(IMPACT)into a two-photon fluorescence lifetime microscopy system to successfully overcome aberrations and multiple scattering problems in deep tissues.It shows that 400 correction modes can be achieved within 5 min,which was mainly limited by the frame rate of a spatial light modulator.This system was used for high-resolution imaging of mice brain tissue and live zebrafish,further verifying its superior performance in imaging quality and photon accumulation speed.
基金the National Institute of Health for supporting this research under grants NIH R35GM152076,NIH 1SC1GM127175-01,NIH T32GM148394.
文摘Time-resolved flow cytometry(TRFC)was used to measure metabolic differences in estrogen receptor-positive breast cancer cells.This specialty cytometry technique measures fluorescence lifetimes as a single-cell parameter thereby providing a unique approach for high-throughput cell counting and screening.Differences in fluorescence lifetime were detected and this was associated with sensitivity to the commonly prescribed therapeutic tamoxifen.Differences in fluorescence lifetime are attributed to the binding states of the autofluorescent metabolite NAD(P)H.The function of NAD(P)H is well described and in general involves cycling from a reduced to oxidized state to facilitate electron transport for the conversion of pyruvate to lactate.NAD(P)H fluorescence lifetimes depend on the bound or unbound state of the metabolite,which also relates to metabolic transitions between oxidative phosphorylation and glycolysis.To determine if fundamental metabolic profiles differ for cells that are sensitive to tamoxifen compared to those that are resistant,large populations of MCF-7 breast cancer cells were screened and fluorescence lifetimes were quantified.Additionally,metabolic differences associated with tamoxifen sensitivity were measured with a Seahorse HS mini metabolic analyzer(Agilent Technologies Inc.Santa Clara,CA)and confocal imaging.Results show that tamoxifen-resistant breast cancer cells have increased utilization of glycolysis for energy production compared to tamoxifen-sensitive breast cancer cells.This work is impacting because it establishes an early step toward developing a reliable screening technology in which large cell censuses can be differentiated for drug sensitivity in a label-free fashion.
基金This work is supported by the National Natural Science Foundation of China(Nos.51527901,51575298,51705285,and 11890672)And we are grateful to Tsinghua-Nikon Imaging Core Facility for providing technical support and to Yanli Zhang for assistance with confocal microscopy and image processing.
文摘Two-dimensional(2D)transition-metal dichalcogenide(TMD)materials have aroused noticeable interest due to their distinguished electronic and optical properties.However,little is known about their complex exciton properties together with the exciton dynamics process which have been expected to influence the performance of optoelectronic devices.The process of fluorescence can well reveal the process of exciton transition after excitation.In this work,the room-temperature layer-dependent exciton dynamics properties in layered WSe2 are investigated by the fluorescence lifetime imaging microscopy(FLIM)for the first time.This paper focuses on two mainly kinds of excitons including the direct transition neutral excitons and trions.Compared with the lifetime of neutral excitons(<0.3 ns within four-layer),trions possess a longer lifetime(~6.6 ns within four-layer)which increases with the number of layers.We attribute the longer-lived lifetime to the increasing number of trions as well as the varieties of trion configurations in thicker WSe2.Besides,the whole average lifetime increases over 10%when WSe2 flakes added up from monolayer to four-layer.This paper provides a novel tuneable layer-dependent method to control the exciton dynamics process and finds a relatively longer transition lifetime of trions at room temperature,enabling to investigate in the charge transport in TMD-based optoelectronics devices in the future.
基金supported by the National Natural Science Foundation of China(Grant Nos.41321002,41402116)
文摘By using fluorescence lifetime image microscope （FLIM） and time-correlated single photon counting （TCSPC） technique, we measured fluorescence lifetime of crude oils with density of 0.9521-0.7606 g/cm3 and multiple petroleum inclusions from Tazhong uplift of Tarim Basin. As indicated by the test results, crude oil density is closely correlated with average fluorescence lifetime following the regression equation Y=-0.0319X＋0.9411, which can thus be used to calculate density of oil inclusions in relation to fluorescence lifetime and density of corresponding surface crude. For type A oil inclusions showing brown-yellow fluorescence from Tazhong 1 well in Tarim Basin, their average fluorescence lifetime was found to be 2.144-2.765 ns, so the density of surface crude corresponding to crude trapping these oil inclusions is 0.852-0.873 g/cm3, indicating that they are matured oil inclusions trapped at earlier stage ofoil formation. For type B oil inclusions with light yellow-white fluorescence, their average fluorescence lifetime was found to be 4.0294.919 ns, so the density of surface crude corresponding to crude trapping these oil inclusions is 0.784-0.812 g/cm3, indicating that they are higher matured oil inclusions trapped at the second stage of oil formation. For type C oil inclusions showing light blue-green fluorescence, their average fluorescence lifetime was found to be 5.0634.168 ns, so the density of surface crude corresponding to crude trapping these oil inclusions is 0.743-0.779 g/cm3, indicating that they are highly-matured light oil inclusions trapped at the third stage of oil formation.
基金the Start-up Funding from Ming Wai Lau Centre for Reparative Medicine, Karolinska Institutetthe National Natural Science Foundation of China (22177094, 21708030)+1 种基金the Applied Basic Research of Sichuan Province (2021YJ0397)the Fundamental Research Funds for the Central University (2682021ZTPY039)。
文摘Phagocytosis is a biological process that plays a key role in host defense and tissue homeostasis.Efficient approaches for real-time imaging of phagocytosis are highly desired but limited.Herein,an AIE-active near-infrared fluorescent probe,named TBTCP,was developed for fluorescence lifetime imaging of phagocytosis.TBTCP could selectively label the cell plasma membrane with fast staining,wash-free process,high signal-to-background ratio,and excellent photostability.Cellular membrane statuses under different osmolarities as well as macrophage phagocytosis of bacteria or large silica particles in early stages could be reported by the fluorescence lifetime changes of TBTCP.Compared with current fluorescence imaging methods,which target the bioenvironmental changes in the late phagocytosis stage,this approach detects the changes in the cell membrane,thus giving a faster response to phagocytosis.This article provides a functional tool to report the phagocytic dynamics of macrophages which may greatly contribute to the studies of phagocytic function-related diseases.
基金supported by the National Natural Science Foundation of China(NSFC,Nos.62074044,61904036,and 11804350)the Medical Engineering Fund of Fudan University(No.yg2021-022)+7 种基金Zhongshan-Fudan Joint Innovation Center and Jihua Laboratory Projects of Guangdong Province(No.X190111UZ190)Fudan University-CIOMP Joint Fund(No.FC2018-001)Pioneering Project of Academy for Engineering and Technology of Fudan University(Nos.gyy2018-001 and gyy2018-002)Shanghai Natural Science Foundation(Nos.20ZR1405100 and 20ZR1403700)Science and Technology Research Program of Shanghai(No.19DZ2282100)Shanghai key discipline construction plan(2020-2022)(No.GWV-10.1-XK01)Shanghai Hong Kong,Macao,and Taiwan Cooperation Project(No.19490760900)Shanghai Engineering Technology Research Center of Hair Medicine(No.19DZ2250500).
文摘Intracellular pH plays a critical role in biological functions,and abnormal pH values are related to various diseases.Here,we report on an intracellular pH sensor AgInS_(2)(AIS)/ZnS quantum dots(QDs)that show long fluorescence lifetimes of hundreds of nanoseconds and low toxicity.Fluorescence lifetime imaging microscopy(FLIM)combined with AIS/ZnS QDs is used for the imaging of live cells in different pH buffers and different cell lines.The FLIM images of AIS/ZnS QDs in live cells demonstrate different intracellular pH values in different regions,such as in lysosomes or cytoplasm.This method can also distinguish cancer cells from normal cells,and the fluorescence lifetime difference of the AIS/ZnS QDs between the two types of cells is 100±7 ns.Most importantly,the exfoliated cervical cells from 20 patients are investigated using FLIM combined with AIS/ZnS QDs.The lifetime difference value between the normal and cervical cancer(CC)groups is 115±9 ns,and the difference between the normal and the precancerous lesion group is 64±9 ns.For the first time,the noninvasive method has been used for cervical cancer screening,and it has shown great improvement in sensitivity compared with a clinical conventional cytology examination.
基金Supported by National Natural Science Foundation of China (10635060,20871116)Innovation Fund of Institute of HighEnergy Physics (IHEP)
文摘Fluorescence lifetime measurement in the time domain requires excitation from a well separated single bunch using synchrotron light sources. In the colliding mode of the Beijing Electron Positron Collider Ⅱ （BEPCⅡ）, a hybrid filling pattern was realized such that a single bunch was placed in the middle of a large gap between two multi-bunch groups. Detection of fluorescence lifetime, based on the excitation of the light pulse from this designated single-bunch, was established at Beamline 4B8 of the Beijing Synchrotron Radiation Facility （BSRF）. The timing signal of the BEPCII was utilized as a trigger to gate this fluorescence event. L-Tryptophan amino acid, a known lifetime standard, was selected to assess the lifetime measurement performance. The measured lifetime was consistent in both colliding and single-bunch mode with the time resolution down to 450 ps. Moreover, both the bunch purity and the fine structure of the hybrid filling pattern were characterized.
基金supported by ‘‘Strategic Priority Research Program” of Chinese Academy of Sciences (XDA09040300)Beijing Science and Technology Project (Z151100003915077)+1 种基金Beijing Nova Programme (Z151100000315081)Beijing Talents Fund (2015000021223ZK17)
文摘Fluorescence correlation spectroscopy （FCS） is a widely used method for measuring molecular diffusion and chemical kinetics. However, when a mixture of fluorescent species is taken into account, the conven- tional FCS method has limitations in extracting autocorrelations for different species and cross correla- tions between different species. Recently developed fluorescence lifetime correlation spectroscopy （FLCS） based on time-tagged time-resolved （TITR） photon recording, which can record the global and micro arrival time for each individual photon, has been used to discriminate different species according to fluorescence lifetime. Here, based on two-dimensional lifetime decay maps constructed from TITR photon stream, we have developed a quantitative lifetime-deconvolution FCS model （LDFCS） to extract precise chemical rates for chemical conversions in multi-species systems. The key point of LDFCS model is separation of different species according to the global distribution of fluorescence lifetime and then deconvolution of autocorrelations and cross-correlations from the two-dimensional lifetime decay maps constructed bv the micro arrival times of photon pairs at each delay time.
