Human error(HE) is the most important factor influencing on structural safety because its effect often exceeds the random deviation.Large numbers of facts have shown that structural failures may be caused by the gross...Human error(HE) is the most important factor influencing on structural safety because its effect often exceeds the random deviation.Large numbers of facts have shown that structural failures may be caused by the gross error due to HE.So it is essential to analyze HE in construction.The crucial work of human error analysis(HEA) is the estimation of human error probability(HEP) in construction.The method for estimating HEP,analytic hierarchy process and failure likelihood index method(AHP-FLIM),is introduced in this paper.The method also uses the process of expert judgment within the failure likelihood index method(FLIM).A numerical example shows the effectiveness of the methods proposed.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
Owing to the anaerobic metabolism in the tumor,abundant acidic metabolites are produced and accumulated in the cells.Therefore,the cells in different tumor layers are directly linked to the pH micro-environment.Nevert...Owing to the anaerobic metabolism in the tumor,abundant acidic metabolites are produced and accumulated in the cells.Therefore,the cells in different tumor layers are directly linked to the pH micro-environment.Nevertheless,due to the lack of robust tools,the high-efficient evaluation of the acidic micro-environment of tumor stratification faces the challenge of accurate diagnosis.We designed a new pH sensitive fluorescent lifetime probe target to lysosomes.As we expected,the fluorescence lifetime of PLN possesses a good linear fit to the pH value,which could detect the pH change at a single lysosome level in real time,and then evaluate the different acidity of tumor stratification.The probe PLN is successfully used to evaluate the tumor stratification by fluorescence lifetime imaging microscopy(FLIM)for the first time,which is of great significance in the preoperative diagnosis of clinical tumor treatment or evaluation of drug delivery effect.展开更多
Recently,photothermal therapy(PTT)has been proved to have great potential in tumor therapy.In the last several years,MoS_(2),as one novel member of nanomaterials,has been applied into PTT due to its excellent photothe...Recently,photothermal therapy(PTT)has been proved to have great potential in tumor therapy.In the last several years,MoS_(2),as one novel member of nanomaterials,has been applied into PTT due to its excellent photothermal conversion efficacy.In this work,we applied fuorescence lifetime imaging microscopy(FLIM)techniques into monitoring the PPT-triggered cell death under MoS_(2) nanosheet treatment.Two types of MoS_(2) nanosheets(single layer nanosheets and few layer nanosheets)were obtained,both of which exhibited presentable photothermal conversion fficacy,leading to high cell death rates of 4T1 cells(mouse breast cancer cells)under PTT.Next,live cell images of 4T1 cells were obtained via directly labeling the mitochondria with Rodamine123,which were then continuously observed with FLIM technique.FLIM data showed that the fuorescence lifetimes of mitochondria targeting dye in cells treated with each type of MoS_(2) nanosheets significantly increased during PTT treatment.By contrast,the fuorescence lifetime of the same dye in control cells(without nanomaterials)remained constant after laser irradiation.These findings suggest that FLIM can be of great value in monitoring cell death process during PTT of cancer cells,which could provide dynamic data of the cellular microenvironment at single cell level in multiple biomedical applications.展开更多
To test the hypothesis that the microviscosity changes of Endoplasmic Reticulum(ER)can be a useful indicator of ferroptosis promoted by ER Stresses(ERS),a new ER targeting viscosity rotor,L-Vis-1 was developed and app...To test the hypothesis that the microviscosity changes of Endoplasmic Reticulum(ER)can be a useful indicator of ferroptosis promoted by ER Stresses(ERS),a new ER targeting viscosity rotor,L-Vis-1 was developed and applied in the quantitation of viscosity by FLIM imaging in live cells.The FLIM imag-ing exhibited an excellent resolution almost as good as the corresponding confocal imaging,more sig-nificantly,during ferroptosis processes promoted by different types of ERS,the viscosity increases were clearly monitored by FLIM of L-Vis-1 within ER,which has not been demonstrated before.展开更多
Fluorescence lifetime imaging(FLIM)is increasingly used to read out cellular autofluorescenceoriginating from the coenzyme NADH in the context of investigating cell metabolic state.Wepresent here an automated multiwel...Fluorescence lifetime imaging(FLIM)is increasingly used to read out cellular autofluorescenceoriginating from the coenzyme NADH in the context of investigating cell metabolic state.Wepresent here an automated multiwell plate reading FLIM microscope optimized for UV illumi-nation with the goal of extending high content fluorescence lifetime asays to readouts ofmetabolism,We demonstrate its application to automated cellular autofluorescence lifetime imaging and discuss the key practical issues associated with its implementation.In particular,weillustrate its capability to read out the NADH-lifetime response of cells to metabolic modulators,thereby illustrating the potential of the instrument for cytotoxicity studies,assays for drugdiscovery and stratified medicine.展开更多
We have developed a two-photon fluorescence microscope capable of imaging up to 4mm inturbid media with micron resolution.The key feature of this instrument is the innovative de-tector,capable of collecting emission p...We have developed a two-photon fluorescence microscope capable of imaging up to 4mm inturbid media with micron resolution.The key feature of this instrument is the innovative de-tector,capable of collecting emission photons from a wider surface area of the sample thandetectors in traditional two-photon microscopes.This detection scheme is extremely efficient inthe collection of emitted photons scattered by turbid media which allows eight fold increase in theimaging depth when compared with conventional two-photon microscopes.Furthermore,thissystem also has in-depth fluorescence lifetime imaging microscopy(FLiM)imaging capabilitywhich increases image contrast.The detection scheme captures emission light in a transmissionconfiguration,making it extremely efficient for the detection of second harmonic generation(SHG)signals,which is generally forward propagating.Here we present imaging experiments oftissue phantoms and in vivo and ea vivo biological tissue performed with this microscope.展开更多
Inorganic quantum dots(QDs)have excellent optical properties,such as high°uorescence intensity,excellent photostability and tunable emission wavelength,etc.,facilitating them to be used as labels and probes for b...Inorganic quantum dots(QDs)have excellent optical properties,such as high°uorescence intensity,excellent photostability and tunable emission wavelength,etc.,facilitating them to be used as labels and probes for bioimaging.In this study,CdSe@ZnS QDs are used as probes for Fluorescence lifetime imaging microscope(FLIM)and stimulated emission depletion(STED)nanoscopy imaging.The emission peak of CdSe@ZnS QDs centered at 526 nm with a narrow width of 19 nm and the photoluminescence quantum yield(PLQY)was 64%.The QDs presented excellent anti-photobleaching property which can be irradiated for 400 min by STED laser with 39.8 mW.The lateral resolution of 42.0 nm is demonstrated for single QDs under STED laser(27.5 mW)irradiation.Furthermore,the CdSe@ZnS QDs were for the first time used to successfully label the lysosomes of living HeLa cells and 81.5 nm lateral resolution is obtained indicating the available super-resolution applications in living cells for inorganic QD probes.Meanwhile,Eca-109 cells labeled with the CdSe@ZnS QDs was observed with FLIM,and their fluorescence lifetime was around 3.1 ns,consistent with the in vitro value,suggesting that the QDs could act as a satisfactory probe in further FLIM-STED experiments.展开更多
Mitochondria are crucial sites for protein quality control within cells.When mitochondrial stress is triggered by protein misfolding,it can accelerate abnormal protein aggregation,potentially inducing various diseases...Mitochondria are crucial sites for protein quality control within cells.When mitochondrial stress is triggered by protein misfolding,it can accelerate abnormal protein aggregation,potentially inducing various diseases.This study developed a cascade-responsive sensor,named AggHX,to monitor the microenvironment of protein aggregation induced by zinc(II)ions and the accompanying mitochondrial dysfunction.The AggHX consists of two key components:(1)A Zn^(2+) þrecognition group for triggering a fluorescent enhance response,and(2)a near-infrared BODIPY scaffold that detects viscosity changes in cell aggregation via HaloTag.This sensor's mechanism of action is elucidated through photochemical and biochemical characterizations.To further investigate the relationship between protein aggregation and mitochondrial homeostasis,we employ fluorescence lifetime imaging microscopy to assess viscosity changes in protein aggregates under intracellular Zn2þstress.This research provides insights into the dynamic behavior and spatial distribution of protein aggregates and mitochondria,contributing to a deeper understanding of their physiological roles in cellular processes and potential implications in disease pathology.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
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
Significantly reduced tissue scattering of fluorescence signals in the second near-infrared(NIR-Ⅱ,1,000–1,700 nm)spectral region offers opportunities for large-depth in vivo bioimaging.Nowadays,most reported works c...Significantly reduced tissue scattering of fluorescence signals in the second near-infrared(NIR-Ⅱ,1,000–1,700 nm)spectral region offers opportunities for large-depth in vivo bioimaging.Nowadays,most reported works concerning NIR-II fluorescence in vivo bioimaging are realized by wide-field illumination and 2D-arrayed detection(e.g.,via InGaAs camera),which has high temporal resolution but limited spatial resolution due to out-of-focus signals.Combining NIR-II fluorescence imaging with confocal microscopy is a good approach to achieve high-spatial resolution visualization of biosamples even at deep tissues.In this presented work,a NIR-II fluorescence confocal microscopic system was setup.By using a kind of aggregation-induced emission(AIE)dots as NIR-II fluorescent probes,800 lm-deep 3D in vivo cerebrovascular imaging of a mouse was obtained,and the spatial resolution at 700 lm depth could reach 8.78 lm.Moreover,the time-correlated single photon counting(TCSPC)technique and femtosecond laser excitation were introduced into NIR-II fluorescence confocal microscopy,and in vivo confocal NIR-II fluorescence lifetime microscopic imaging(FLIM)of mouse cerebral vasculature was successfully realized.展开更多
Spectro-microscopy, a combination of fluorescence microscopy with spatially resolved spectroscopic techni- ques, provides new and exciting tools for functional cell biology in living organisms. This review focuses on ...Spectro-microscopy, a combination of fluorescence microscopy with spatially resolved spectroscopic techni- ques, provides new and exciting tools for functional cell biology in living organisms. This review focuses on recent devel- opments in spectro-microscopic applications for the investigation of living plant cells in their native tissue context. The application of spectro-microscopic methods led to the recent discovery of a fast signal response pathway for the brassi- nosteroide receptor BRI1 in the plasma membrane of living plant cells. Moreover, the competence of different plant cell types to respond to environmental or endogenous stimuli was determined in vivo by correlation analysis of different optical and spectroscopic readouts such as fluorescence lifetime （FLT）. Furthermore, a new spectro-microscopic technique, fluorescence intensity decay shape analysis microscopy （FIDSAM）, has been developed. FIDSAM is capable of imaging low- expressed fluorophore-tagged proteins at high spatial resolution and precludes the misinterpretation of autofluorescence artifacts. In addition, FIDSAM provides a very effective and sensitive tool on the basis of F6rster resonance energy transfer （FRET） for the qualitative and quantitative determination of protein-protein interaction. Finally, we report on the quan- titative analysis of the photosystem I and II （PSI/PSII） ratio in the chloroplasts of living Arabidopsis plants at room tem- perature, using high-resolution, spatially resolved fluorescence spectroscopy. With this technique, it was not only possible to measure PSI/PSII ratios, but also to demonstrate the differential competence of wild-type and carbohydrate-deficient plants to adapt the PSI/PSII ratio to different light conditions. In summary, the information content of standard microscopic images is extended by several dimensions by the use of spectro-microscopic approaches. Therefore, novel cell physiolog- ical and molecular topics can be addressed and valuable insights into molecular and subcellular processes can be obtained in living plants.展开更多
The aim of this study is to develop a novel technique for improving the intraoperative margin assessment of glioblastoma by examining the total extrinsic extracellular matrix（ECM） with eosin staining using fluoresce...The aim of this study is to develop a novel technique for improving the intraoperative margin assessment of glioblastoma by examining the total extrinsic extracellular matrix（ECM） with eosin staining using fluorescence lifetime imaging microscopy（FLIM） and scale-invariant feature transform（SIFT） descriptor analysis. Pseudocolor FLIM images obviously exhibit ECM distributions, changes in sequential sections, and different regions of interest. Meanwhile, SIFT descriptors are first utilized for the discrimination of glioblastoma margins by matching similar ECM regions and extracting keypoint orientations from FLIM images obtained from a series of continuous slices. The findings indicate that FLIM imaging with SIFT analysis of the total ECM is a promising method for improving intraoperative diagnosis of frozen and surgically excised brain specimen sections.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
文摘Human error(HE) is the most important factor influencing on structural safety because its effect often exceeds the random deviation.Large numbers of facts have shown that structural failures may be caused by the gross error due to HE.So it is essential to analyze HE in construction.The crucial work of human error analysis(HEA) is the estimation of human error probability(HEP) in construction.The method for estimating HEP,analytic hierarchy process and failure likelihood index method(AHP-FLIM),is introduced in this paper.The method also uses the process of expert judgment within the failure likelihood index method(FLIM).A numerical example shows the effectiveness of the methods proposed.
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financially supported by the National Natural Science Foundation of China(Nos.21877048,22077048 and 22277014)Guangxi Natural Science Foundation(Nos.2021GXNSFDA075003,AD21220061)the Startup Fund of Guangxi University(No.A3040051003)。
文摘Owing to the anaerobic metabolism in the tumor,abundant acidic metabolites are produced and accumulated in the cells.Therefore,the cells in different tumor layers are directly linked to the pH micro-environment.Nevertheless,due to the lack of robust tools,the high-efficient evaluation of the acidic micro-environment of tumor stratification faces the challenge of accurate diagnosis.We designed a new pH sensitive fluorescent lifetime probe target to lysosomes.As we expected,the fluorescence lifetime of PLN possesses a good linear fit to the pH value,which could detect the pH change at a single lysosome level in real time,and then evaluate the different acidity of tumor stratification.The probe PLN is successfully used to evaluate the tumor stratification by fluorescence lifetime imaging microscopy(FLIM)for the first time,which is of great significance in the preoperative diagnosis of clinical tumor treatment or evaluation of drug delivery effect.
基金supported by the National Key R&D Program of China(2018YFC0910602)the National Natural Science Foundation of China(Grant Nos.31771584/61775145/61605121,61620106016/61525503/61835009/81727804)+2 种基金Guangdong Natural Science Foundation Innovation Team(2014A030312008)Shenzhen Basic Research Project(JCYJ20170818100153423/JCYJ20170412110212234/JCYJ20160328144746940/JCYJ20170412105003520/JCYJ20170302142902581)Science Foundation of SZU(Grant No.000193).
文摘Recently,photothermal therapy(PTT)has been proved to have great potential in tumor therapy.In the last several years,MoS_(2),as one novel member of nanomaterials,has been applied into PTT due to its excellent photothermal conversion efficacy.In this work,we applied fuorescence lifetime imaging microscopy(FLIM)techniques into monitoring the PPT-triggered cell death under MoS_(2) nanosheet treatment.Two types of MoS_(2) nanosheets(single layer nanosheets and few layer nanosheets)were obtained,both of which exhibited presentable photothermal conversion fficacy,leading to high cell death rates of 4T1 cells(mouse breast cancer cells)under PTT.Next,live cell images of 4T1 cells were obtained via directly labeling the mitochondria with Rodamine123,which were then continuously observed with FLIM technique.FLIM data showed that the fuorescence lifetimes of mitochondria targeting dye in cells treated with each type of MoS_(2) nanosheets significantly increased during PTT treatment.By contrast,the fuorescence lifetime of the same dye in control cells(without nanomaterials)remained constant after laser irradiation.These findings suggest that FLIM can be of great value in monitoring cell death process during PTT of cancer cells,which could provide dynamic data of the cellular microenvironment at single cell level in multiple biomedical applications.
基金supported by the National Natural Science Foundation of China(Nos.21776037,22174009)Dalian Sci-ence and Technology Innovation Fund(No.2020JJ25CY014)Quanzhou Science and Technology Plan Project(No.2019C033R).
文摘To test the hypothesis that the microviscosity changes of Endoplasmic Reticulum(ER)can be a useful indicator of ferroptosis promoted by ER Stresses(ERS),a new ER targeting viscosity rotor,L-Vis-1 was developed and applied in the quantitation of viscosity by FLIM imaging in live cells.The FLIM imag-ing exhibited an excellent resolution almost as good as the corresponding confocal imaging,more sig-nificantly,during ferroptosis processes promoted by different types of ERS,the viscosity increases were clearly monitored by FLIM of L-Vis-1 within ER,which has not been demonstrated before.
基金funding from the UK Biotechnology and Biological Sciences Research Council(BBSRCBB/H00713X/1)the UK Engineering and PhysicalSciences Research Council(EPSRC EP/IO2770X/1)the UK Technology Strategy Board Technology Award(CHBT/007/00030,EP/C54269X,inpartnership with Astra Zeneca,GE Healthcare,GSK,Kentech Instruments Ltd).
文摘Fluorescence lifetime imaging(FLIM)is increasingly used to read out cellular autofluorescenceoriginating from the coenzyme NADH in the context of investigating cell metabolic state.Wepresent here an automated multiwell plate reading FLIM microscope optimized for UV illumi-nation with the goal of extending high content fluorescence lifetime asays to readouts ofmetabolism,We demonstrate its application to automated cellular autofluorescence lifetime imaging and discuss the key practical issues associated with its implementation.In particular,weillustrate its capability to read out the NADH-lifetime response of cells to metabolic modulators,thereby illustrating the potential of the instrument for cytotoxicity studies,assays for drugdiscovery and stratified medicine.
基金Funding was provided by National Institutes of Health P50 GM076516by the National Center for Research Resources(5P41RR003155-27)the National Instit ute of General Medical Sciences(8 P41GM103540-27).
文摘We have developed a two-photon fluorescence microscope capable of imaging up to 4mm inturbid media with micron resolution.The key feature of this instrument is the innovative de-tector,capable of collecting emission photons from a wider surface area of the sample thandetectors in traditional two-photon microscopes.This detection scheme is extremely efficient inthe collection of emitted photons scattered by turbid media which allows eight fold increase in theimaging depth when compared with conventional two-photon microscopes.Furthermore,thissystem also has in-depth fluorescence lifetime imaging microscopy(FLiM)imaging capabilitywhich increases image contrast.The detection scheme captures emission light in a transmissionconfiguration,making it extremely efficient for the detection of second harmonic generation(SHG)signals,which is generally forward propagating.Here we present imaging experiments oftissue phantoms and in vivo and ea vivo biological tissue performed with this microscope.
基金supported by the National Key R&D Program of China(2018YFC0910600)the National Natural Science Foundation of China(61605124/41603059/61525503/61620106016/61835009/81727804)+3 种基金Project of Department of Education of Guangdong Province(2015KGJHZ002/2016KCXTD007)Guangdong Natural Science Foundation Innovation Team(2014A030312008)Shenzhen Basic Research Project(JCYJ20150930104948169/JCYJ20160328144746940/JCYJ20170412105003520)the Natural Science Foundation of Shenzhen University(2019108).
文摘Inorganic quantum dots(QDs)have excellent optical properties,such as high°uorescence intensity,excellent photostability and tunable emission wavelength,etc.,facilitating them to be used as labels and probes for bioimaging.In this study,CdSe@ZnS QDs are used as probes for Fluorescence lifetime imaging microscope(FLIM)and stimulated emission depletion(STED)nanoscopy imaging.The emission peak of CdSe@ZnS QDs centered at 526 nm with a narrow width of 19 nm and the photoluminescence quantum yield(PLQY)was 64%.The QDs presented excellent anti-photobleaching property which can be irradiated for 400 min by STED laser with 39.8 mW.The lateral resolution of 42.0 nm is demonstrated for single QDs under STED laser(27.5 mW)irradiation.Furthermore,the CdSe@ZnS QDs were for the first time used to successfully label the lysosomes of living HeLa cells and 81.5 nm lateral resolution is obtained indicating the available super-resolution applications in living cells for inorganic QD probes.Meanwhile,Eca-109 cells labeled with the CdSe@ZnS QDs was observed with FLIM,and their fluorescence lifetime was around 3.1 ns,consistent with the in vitro value,suggesting that the QDs could act as a satisfactory probe in further FLIM-STED experiments.
基金support from the Qinglan Project of Jiangsu Province of China,National Natural Science Foundation of China(Grant No.22007048)the Natural Science Foundation of Jiangsu Basic Research Program(BK20221324).
文摘Mitochondria are crucial sites for protein quality control within cells.When mitochondrial stress is triggered by protein misfolding,it can accelerate abnormal protein aggregation,potentially inducing various diseases.This study developed a cascade-responsive sensor,named AggHX,to monitor the microenvironment of protein aggregation induced by zinc(II)ions and the accompanying mitochondrial dysfunction.The AggHX consists of two key components:(1)A Zn^(2+) þrecognition group for triggering a fluorescent enhance response,and(2)a near-infrared BODIPY scaffold that detects viscosity changes in cell aggregation via HaloTag.This sensor's mechanism of action is elucidated through photochemical and biochemical characterizations.To further investigate the relationship between protein aggregation and mitochondrial homeostasis,we employ fluorescence lifetime imaging microscopy to assess viscosity changes in protein aggregates under intracellular Zn2þstress.This research provides insights into the dynamic behavior and spatial distribution of protein aggregates and mitochondria,contributing to a deeper understanding of their physiological roles in cellular processes and potential implications in disease pathology.
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supported by the National Natural Science Foundation of China(61735016)Zhejiang Provincial Natural Science Foundation of China(LR17F050001)
文摘Significantly reduced tissue scattering of fluorescence signals in the second near-infrared(NIR-Ⅱ,1,000–1,700 nm)spectral region offers opportunities for large-depth in vivo bioimaging.Nowadays,most reported works concerning NIR-II fluorescence in vivo bioimaging are realized by wide-field illumination and 2D-arrayed detection(e.g.,via InGaAs camera),which has high temporal resolution but limited spatial resolution due to out-of-focus signals.Combining NIR-II fluorescence imaging with confocal microscopy is a good approach to achieve high-spatial resolution visualization of biosamples even at deep tissues.In this presented work,a NIR-II fluorescence confocal microscopic system was setup.By using a kind of aggregation-induced emission(AIE)dots as NIR-II fluorescent probes,800 lm-deep 3D in vivo cerebrovascular imaging of a mouse was obtained,and the spatial resolution at 700 lm depth could reach 8.78 lm.Moreover,the time-correlated single photon counting(TCSPC)technique and femtosecond laser excitation were introduced into NIR-II fluorescence confocal microscopy,and in vivo confocal NIR-II fluorescence lifetime microscopic imaging(FLIM)of mouse cerebral vasculature was successfully realized.
文摘Spectro-microscopy, a combination of fluorescence microscopy with spatially resolved spectroscopic techni- ques, provides new and exciting tools for functional cell biology in living organisms. This review focuses on recent devel- opments in spectro-microscopic applications for the investigation of living plant cells in their native tissue context. The application of spectro-microscopic methods led to the recent discovery of a fast signal response pathway for the brassi- nosteroide receptor BRI1 in the plasma membrane of living plant cells. Moreover, the competence of different plant cell types to respond to environmental or endogenous stimuli was determined in vivo by correlation analysis of different optical and spectroscopic readouts such as fluorescence lifetime （FLT）. Furthermore, a new spectro-microscopic technique, fluorescence intensity decay shape analysis microscopy （FIDSAM）, has been developed. FIDSAM is capable of imaging low- expressed fluorophore-tagged proteins at high spatial resolution and precludes the misinterpretation of autofluorescence artifacts. In addition, FIDSAM provides a very effective and sensitive tool on the basis of F6rster resonance energy transfer （FRET） for the qualitative and quantitative determination of protein-protein interaction. Finally, we report on the quan- titative analysis of the photosystem I and II （PSI/PSII） ratio in the chloroplasts of living Arabidopsis plants at room tem- perature, using high-resolution, spatially resolved fluorescence spectroscopy. With this technique, it was not only possible to measure PSI/PSII ratios, but also to demonstrate the differential competence of wild-type and carbohydrate-deficient plants to adapt the PSI/PSII ratio to different light conditions. In summary, the information content of standard microscopic images is extended by several dimensions by the use of spectro-microscopic approaches. Therefore, novel cell physiolog- ical and molecular topics can be addressed and valuable insights into molecular and subcellular processes can be obtained in living plants.
基金supported by the National Basic Research Program of China(No.2015CB352005)the National Natural Science Foundation of China(Nos.61525503,61378091,and 61620106016)+2 种基金the Guangdong Natural Science Foundation Innovation Team(No.2014A030312008)the Hong Kong,Macao and Taiwan cooperation innovation platform&major projects of international cooperation in Colleges and the Universities in Guangdong Province(No.2015KGJHZ002)the Shenzhen Basic Research Project(Nos.JCYJ20150930104948169,JCYJ2016032814 4746940,and GJHZ20160226202139185)
文摘The aim of this study is to develop a novel technique for improving the intraoperative margin assessment of glioblastoma by examining the total extrinsic extracellular matrix（ECM） with eosin staining using fluorescence lifetime imaging microscopy（FLIM） and scale-invariant feature transform（SIFT） descriptor analysis. Pseudocolor FLIM images obviously exhibit ECM distributions, changes in sequential sections, and different regions of interest. Meanwhile, SIFT descriptors are first utilized for the discrimination of glioblastoma margins by matching similar ECM regions and extracting keypoint orientations from FLIM images obtained from a series of continuous slices. The findings indicate that FLIM imaging with SIFT analysis of the total ECM is a promising method for improving intraoperative diagnosis of frozen and surgically excised brain specimen sections.
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.
