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
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
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
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
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 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
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
Fluorescence lifetime imaging microscopy(FLIM)has emerged as a transformative imaging technique in cancer research,offering quantitative insights into cellular metabolism,tumor microenvironments,and therapeutic respon...Fluorescence lifetime imaging microscopy(FLIM)has emerged as a transformative imaging technique in cancer research,offering quantitative insights into cellular metabolism,tumor microenvironments,and therapeutic responses.By measuring the fluorescence lifetimes of metabolic cofactors such as NADH and FAD,FLIM facilitates the analysis of cancer-specific metabolic reprogramming and heterogeneity.Integration with deep learning further enhances FLIM’s diagnostic and therapeutic potential,enabling high-resolution imaging,automated data analysis,and biomarker identification.This review provides a comprehensive overview of the principles and technological advancements of FLIM,highlighting its applications in cancer diagnostics,drug delivery,and therapy,as well as its integration with deep learning to increase imaging precision and data interpretation.Challenges such as high costs,high computational complexity,and the need for standardized imaging protocols are also addressed.By bridging FLIM with cutting-edge computational techniques,this review highlights its potential to revolutionize cancer research,paving the way for early diagnosis,personalized therapies,and deeper insights into tumor biology.展开更多
Increased micro-and nanoplastic(MNP)pollution poses significant health risks,yet the mechanisms of their accumulation and effects on absorptive tissues remain poorly understood.Addressing this knowledge gap requires t...Increased micro-and nanoplastic(MNP)pollution poses significant health risks,yet the mechanisms of their accumulation and effects on absorptive tissues remain poorly understood.Addressing this knowledge gap requires tractable models coupled to dynamic live cell imaging methods,enabling multi-parameter single cell analysis.We report a new method combining adult stem cell-derived small intestinal organoid cultures with live fluorescence lifetime imaging microscopy(FLIM)to study MNP interactions with gut epithelium.To facilitate this,we optimized live imaging of porcine and mouse small intestinal organoids with an‘apical-out’topology.Subsequently,we produced a set of pristine MNPs based on PMMA and PS(<200 nm,doped with deep-red fluorescent dye)and evaluated their interaction with organoids displaying controlled epithelial polarity.We found that nanoparticles interacted differently with apical and basal membranes of the organoids and showed a species-specific pattern of cellular uptake.Using a phasor analysis approach,we demonstrate improved sensitivity of FLIM over conventional intensity-based microscopy.The resulting‘fluorescence lifetime barcoding’enabled distinguishing of different types of MNP and their interaction sites within organoids.Finally,we studied short(1 day)-and long(3 day)-term exposure effects of PMMA and PS-based MNPs on mitochondrial function,total cell energy budget and epithelial inflammation.We found that even pristine MNPs could disrupt chemokine production and mitochondrial membrane potential in intestinal epithelial cells.The presented FLIM approach will advance the study of MNP toxicity,their biological impacts on gastrointestinal tissue and enable the tracing of other fluorescent nanoparticles in live organoid and 3D ex vivo systems.展开更多
To address the current limitations of time-gated Raman spectroscopy,specifically its narrow spectral range and low spectral resolution,and simultaneously acquire Raman and fluorescence life-time images,we have develop...To address the current limitations of time-gated Raman spectroscopy,specifically its narrow spectral range and low spectral resolution,and simultaneously acquire Raman and fluorescence life-time images,we have developed a Fourier-transform photon counting spectroscopy platform.A Mach-Zehnder interferometer employing a high accuracy linear motor stage was combined with photon-counting avalanche diodes and time-tagged acquisition,allowing to sort photons into a matrix of stage positions determined using their coarse arrival time with 50 ns steps of the excitation laser repetition period,and a fine arrival time of 80 ps resolution relative to the excitation pulse of 100 ps duration.The instrument achieves a time resolution of 547 ps,a wide spectral range of−1000 to 10,000 cm−1 Raman shift from the excitation at 532 nm wavelength,and a high spectral resolution of 0.05 cm−1.For experimental validation,we used fluorescently coated silicon wafers and fluorescent plastic microspheres.Raman signal was observed during the laser excitation pulse within the time-resolution,while fluorescence signals dominate afterwards.The results confirm that the instrument can effectively separate Raman and fluorescence signals.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
Rheumatoid arthritis,being a chronic autoimmune malady,may culminate in joint malformation and incapacitation in severe instances.Nevertheless,monitoring the heterogeneity of the viscosity microenvironment in local jo...Rheumatoid arthritis,being a chronic autoimmune malady,may culminate in joint malformation and incapacitation in severe instances.Nevertheless,monitoring the heterogeneity of the viscosity microenvironment in local joint areas remains challenging.Hence,we have developed a near-infrared(NIR)-emitting fluorescence lifetime probe WY-V for dual fluorescence lifetime imaging microscopy(FLIM)/NIR optical imaging,featuring precise targeting capabilities to the endoplasmic reticulum(ER)and lipid droplets(LD).This probe modulates distinct fluorescence lifetimes in the varying viscosity environments of these organelles,allowing for the quantification of their respective viscosities.Using FLIM/NIR optical imaging of joint tissues from arthritic mice,the probe accurately discerned the viscosity of inflamed cells at diverse sites,reveals the viscosity heterogeneity present within the arthritic tissues.Therefore,this research offers a potent biological instrument for clinical diagnosis and pathological examination of rheumatoid arthritis.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
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
A novel fluorescence lifetime imaging microscopy(FLIM) working with deep UV 240–280 nm wavelength excitations has been developed. UV-FLIM is used for measurement of defect-related fluorescence and its changes upon an...A novel fluorescence lifetime imaging microscopy(FLIM) working with deep UV 240–280 nm wavelength excitations has been developed. UV-FLIM is used for measurement of defect-related fluorescence and its changes upon annealing from femtosecond laser-induced modifications in fused silica. This FLIM technique can be used with microfluidic and biosamples to characterize temporal characteristics of fluorescence upon UV excitation, a capability easily added to a standard microscope-based FLIM. UV-FLIM was tested to show annealing of the defects induced by silica structuring with ultrashort laser pulses. Frequency-domain fluorescence measurements were converted into the time domain to extract long fluorescence lifetimes from defects in silica.展开更多
Fluorescence liftime imaging （FLIM） of modified hydrophobic bodipy dyes that act as fluorescent molecular rotors shows that the fluorescence lifetime of these probes is a function of the microviscosity of their envi...Fluorescence liftime imaging （FLIM） of modified hydrophobic bodipy dyes that act as fluorescent molecular rotors shows that the fluorescence lifetime of these probes is a function of the microviscosity of their environment. Incubating cells with these dyes, we find a punctate and continuous distribution of the dye in cells. The viscosity value obtained in what appears to be endocytotic vesicles in living cells is around 100 times higher than that of water and of cellular cytoplasm.Time-resolved fluorescence anisotropy measurements also yield rotational correlation times consistent with large microviscosity values. In this way, we successfully develop a practical and versatile approach to map the microviscosity in cells based on imaging fluorescent molecular rotors.展开更多
Compared with visible light,near infrared(NIR)light has deeper penetration in biological tisues.Three-photon fuorescence microscopy(3PFM)can effectively utilize the NIR excitation to obtain high-contrast images in the...Compared with visible light,near infrared(NIR)light has deeper penetration in biological tisues.Three-photon fuorescence microscopy(3PFM)can effectively utilize the NIR excitation to obtain high-contrast images in the deep tisue.However,the weak three photon fluorescence signals may be not well presented in the traditional fuorescence intensity imaging mode.Fluorescence lifetime of certain probes is insensitive to the intensity of the excitation laser.Moreover,fluorescence lifetimne imaging microscopy(FLIM)can detect weak signals by utilizing time correlated single photon counting(TCSPC)technique.Thus,it would be an improved strategy to combine the 3PFM imaging with the FLIM together.Herein,DCDPP-2TPA,a novel agegation-induced emission luminogen(AIEgen),was adopted as the fluorescent probes.The three-photon absorption cros-section of the AlEgen,which has a deep-red fluorescence emission,was proved to be large.DCDPP-2TPA nanoparticles were synthesized,and the three photon fluorescence lifetime of which was measured in water.Moreover,in vrivo thre-photon fuorescence lifetime microscopic imaging of a craniotomy mouse was conducted via a home made optical system.High contrast cerebrovascular images of different vertical depths were obtained and the maximun depth was about 600 pumn.Even reaching the depth of 600 pum,tiny capillary vessels as small as 1.9 pum could still be distinguished.The three photon fuorescence lifetimes of the capillaries in some representative images were in accord with that of DCDPP-2TPA nanoparticles in water.A vivid 3D reconstruction was further organized to present a wealth of lifetime information.In the future,the combination strategy of 3PFM and FLIM could be further applied in the brain functional imaging.展开更多
Fluorescence imaging in the second near-infrared window(NIR-II,900–1880 nm)with less scattering background in biological tissues has been combined with the confocal microscopic system for achieving deep in vivo imagi...Fluorescence imaging in the second near-infrared window(NIR-II,900–1880 nm)with less scattering background in biological tissues has been combined with the confocal microscopic system for achieving deep in vivo imaging with high spatial resolution.However,the traditional NIR-IIfluorescence confocal microscope with separate excitation focus and detection pinhole makes it possess low confocal e±ciency,as well as di±cultly to adjust.Two types of upgraded NIR-IIfluorescence confocal microscopes,sharing the same pinhole by excitation and emission focus,leading to higher confocal e±ciency,are built in this work.One type is-ber-pinhole-based confocal microscope applicable to CW laser excitation.It is constructed forfluorescence intensity imaging with large depth,high stabilization and low cost,which could replace multiphotonfluorescence microscopy in some applications(e.g.,cerebrovascular and hepatocellular imaging).The other type is air-pinhole-based confocal microscope applicable to femtosecond(fs)laser excitation.It can be employed not only for NIR-IIfluorescence intensity imaging,but also for multi-channelfluorescence lifetime imaging to recognize different structures with similarfluorescence spectrum.Moreover,it can be facilely combined with multiphotonfluorescence microscopy.A single fs pulsed laser is utilized to achieve up-conversion(visible multiphotonfluorescence)and down-conversion(NIR-II one-photonfluorescence)excitation simultaneously,extending imaging spectral channels,and thus facilitates multi-structure and multi-functional observation.展开更多
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 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 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 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Indian Council of Medical Research(ITR/Ad-hoc/43/2020-21,ID No.2020-3286)Department of Science and Technology,(GITA/DST/TWN/P-95/2021)Government of India,India for financial support.
文摘Fluorescence lifetime imaging microscopy(FLIM)has emerged as a transformative imaging technique in cancer research,offering quantitative insights into cellular metabolism,tumor microenvironments,and therapeutic responses.By measuring the fluorescence lifetimes of metabolic cofactors such as NADH and FAD,FLIM facilitates the analysis of cancer-specific metabolic reprogramming and heterogeneity.Integration with deep learning further enhances FLIM’s diagnostic and therapeutic potential,enabling high-resolution imaging,automated data analysis,and biomarker identification.This review provides a comprehensive overview of the principles and technological advancements of FLIM,highlighting its applications in cancer diagnostics,drug delivery,and therapy,as well as its integration with deep learning to increase imaging precision and data interpretation.Challenges such as high costs,high computational complexity,and the need for standardized imaging protocols are also addressed.By bridging FLIM with cutting-edge computational techniques,this review highlights its potential to revolutionize cancer research,paving the way for early diagnosis,personalized therapies,and deeper insights into tumor biology.
基金supported by the Special Research Fund(BOF)grants(BOF/STA/202009/003,BOF/BAF/1 y/25/1/004)Research Foundation Flanders(FWO,I001922N,I004124N)the European Union,fliMAGIN3D-DN Horizon Europe-MSCA-DN No.101073507 grants.
文摘Increased micro-and nanoplastic(MNP)pollution poses significant health risks,yet the mechanisms of their accumulation and effects on absorptive tissues remain poorly understood.Addressing this knowledge gap requires tractable models coupled to dynamic live cell imaging methods,enabling multi-parameter single cell analysis.We report a new method combining adult stem cell-derived small intestinal organoid cultures with live fluorescence lifetime imaging microscopy(FLIM)to study MNP interactions with gut epithelium.To facilitate this,we optimized live imaging of porcine and mouse small intestinal organoids with an‘apical-out’topology.Subsequently,we produced a set of pristine MNPs based on PMMA and PS(<200 nm,doped with deep-red fluorescent dye)and evaluated their interaction with organoids displaying controlled epithelial polarity.We found that nanoparticles interacted differently with apical and basal membranes of the organoids and showed a species-specific pattern of cellular uptake.Using a phasor analysis approach,we demonstrate improved sensitivity of FLIM over conventional intensity-based microscopy.The resulting‘fluorescence lifetime barcoding’enabled distinguishing of different types of MNP and their interaction sites within organoids.Finally,we studied short(1 day)-and long(3 day)-term exposure effects of PMMA and PS-based MNPs on mitochondrial function,total cell energy budget and epithelial inflammation.We found that even pristine MNPs could disrupt chemokine production and mitochondrial membrane potential in intestinal epithelial cells.The presented FLIM approach will advance the study of MNP toxicity,their biological impacts on gastrointestinal tissue and enable the tracing of other fluorescent nanoparticles in live organoid and 3D ex vivo systems.
基金Funded by Chinese Academy of Sciences President’s International Fellowship Initiative.Grant No.2022VBA0028.
文摘To address the current limitations of time-gated Raman spectroscopy,specifically its narrow spectral range and low spectral resolution,and simultaneously acquire Raman and fluorescence life-time images,we have developed a Fourier-transform photon counting spectroscopy platform.A Mach-Zehnder interferometer employing a high accuracy linear motor stage was combined with photon-counting avalanche diodes and time-tagged acquisition,allowing to sort photons into a matrix of stage positions determined using their coarse arrival time with 50 ns steps of the excitation laser repetition period,and a fine arrival time of 80 ps resolution relative to the excitation pulse of 100 ps duration.The instrument achieves a time resolution of 547 ps,a wide spectral range of−1000 to 10,000 cm−1 Raman shift from the excitation at 532 nm wavelength,and a high spectral resolution of 0.05 cm−1.For experimental validation,we used fluorescently coated silicon wafers and fluorescent plastic microspheres.Raman signal was observed during the laser excitation pulse within the time-resolution,while fluorescence signals dominate afterwards.The results confirm that the instrument can effectively separate Raman and fluorescence signals.
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financially supported by Guangxi Natural Science Foundation(Nos.AD21220061,2021GXNSFDA075003)National Natural Science Foundation of China(Nos.22277014,22077048)the startup fund of Guangxi University(No.A3040051003)。
文摘Rheumatoid arthritis,being a chronic autoimmune malady,may culminate in joint malformation and incapacitation in severe instances.Nevertheless,monitoring the heterogeneity of the viscosity microenvironment in local joint areas remains challenging.Hence,we have developed a near-infrared(NIR)-emitting fluorescence lifetime probe WY-V for dual fluorescence lifetime imaging microscopy(FLIM)/NIR optical imaging,featuring precise targeting capabilities to the endoplasmic reticulum(ER)and lipid droplets(LD).This probe modulates distinct fluorescence lifetimes in the varying viscosity environments of these organelles,allowing for the quantification of their respective viscosities.Using FLIM/NIR optical imaging of joint tissues from arthritic mice,the probe accurately discerned the viscosity of inflamed cells at diverse sites,reveals the viscosity heterogeneity present within the arthritic tissues.Therefore,this research offers a potent biological instrument for clinical diagnosis and pathological examination of rheumatoid arthritis.
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 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support via the Australian Research Council Discovery DP130101205 and DP120102980 grantsproject with Altechna Ltd
文摘A novel fluorescence lifetime imaging microscopy(FLIM) working with deep UV 240–280 nm wavelength excitations has been developed. UV-FLIM is used for measurement of defect-related fluorescence and its changes upon annealing from femtosecond laser-induced modifications in fused silica. This FLIM technique can be used with microfluidic and biosamples to characterize temporal characteristics of fluorescence upon UV excitation, a capability easily added to a standard microscope-based FLIM. UV-FLIM was tested to show annealing of the defects induced by silica structuring with ultrashort laser pulses. Frequency-domain fluorescence measurements were converted into the time domain to extract long fluorescence lifetimes from defects in silica.
文摘Fluorescence liftime imaging （FLIM） of modified hydrophobic bodipy dyes that act as fluorescent molecular rotors shows that the fluorescence lifetime of these probes is a function of the microviscosity of their environment. Incubating cells with these dyes, we find a punctate and continuous distribution of the dye in cells. The viscosity value obtained in what appears to be endocytotic vesicles in living cells is around 100 times higher than that of water and of cellular cytoplasm.Time-resolved fluorescence anisotropy measurements also yield rotational correlation times consistent with large microviscosity values. In this way, we successfully develop a practical and versatile approach to map the microviscosity in cells based on imaging fluorescent molecular rotors.
基金supported by National Natural Science Foundation of China(61735016)Zhejiang Provincial Natural Science Foundation of China(LR17F050001).
文摘Compared with visible light,near infrared(NIR)light has deeper penetration in biological tisues.Three-photon fuorescence microscopy(3PFM)can effectively utilize the NIR excitation to obtain high-contrast images in the deep tisue.However,the weak three photon fluorescence signals may be not well presented in the traditional fuorescence intensity imaging mode.Fluorescence lifetime of certain probes is insensitive to the intensity of the excitation laser.Moreover,fluorescence lifetimne imaging microscopy(FLIM)can detect weak signals by utilizing time correlated single photon counting(TCSPC)technique.Thus,it would be an improved strategy to combine the 3PFM imaging with the FLIM together.Herein,DCDPP-2TPA,a novel agegation-induced emission luminogen(AIEgen),was adopted as the fluorescent probes.The three-photon absorption cros-section of the AlEgen,which has a deep-red fluorescence emission,was proved to be large.DCDPP-2TPA nanoparticles were synthesized,and the three photon fluorescence lifetime of which was measured in water.Moreover,in vrivo thre-photon fuorescence lifetime microscopic imaging of a craniotomy mouse was conducted via a home made optical system.High contrast cerebrovascular images of different vertical depths were obtained and the maximun depth was about 600 pumn.Even reaching the depth of 600 pum,tiny capillary vessels as small as 1.9 pum could still be distinguished.The three photon fuorescence lifetimes of the capillaries in some representative images were in accord with that of DCDPP-2TPA nanoparticles in water.A vivid 3D reconstruction was further organized to present a wealth of lifetime information.In the future,the combination strategy of 3PFM and FLIM could be further applied in the brain functional imaging.
基金supported by National Natural Science Foundation of China(61975172,82001874 and 61735016).
文摘Fluorescence imaging in the second near-infrared window(NIR-II,900–1880 nm)with less scattering background in biological tissues has been combined with the confocal microscopic system for achieving deep in vivo imaging with high spatial resolution.However,the traditional NIR-IIfluorescence confocal microscope with separate excitation focus and detection pinhole makes it possess low confocal e±ciency,as well as di±cultly to adjust.Two types of upgraded NIR-IIfluorescence confocal microscopes,sharing the same pinhole by excitation and emission focus,leading to higher confocal e±ciency,are built in this work.One type is-ber-pinhole-based confocal microscope applicable to CW laser excitation.It is constructed forfluorescence intensity imaging with large depth,high stabilization and low cost,which could replace multiphotonfluorescence microscopy in some applications(e.g.,cerebrovascular and hepatocellular imaging).The other type is air-pinhole-based confocal microscope applicable to femtosecond(fs)laser excitation.It can be employed not only for NIR-IIfluorescence intensity imaging,but also for multi-channelfluorescence lifetime imaging to recognize different structures with similarfluorescence spectrum.Moreover,it can be facilely combined with multiphotonfluorescence microscopy.A single fs pulsed laser is utilized to achieve up-conversion(visible multiphotonfluorescence)and down-conversion(NIR-II one-photonfluorescence)excitation simultaneously,extending imaging spectral channels,and thus facilitates multi-structure and multi-functional observation.
