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
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
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
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
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 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.
