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(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
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
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
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
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.
