Measurement of bloodflow velocity is key to understanding physiology and pathology in vivo.While most measurements are performed at the middle of the blood vessel,little research has been done on characterizing the in...Measurement of bloodflow velocity is key to understanding physiology and pathology in vivo.While most measurements are performed at the middle of the blood vessel,little research has been done on characterizing the instantaneous bloodflow velocity distribution.This is mainly due to the lack of measurement technology with high spatial and temporal resolution.Here,we tackle this problem with our recently developed dual-wavelength line-scan third-harmonic generation(THG)imaging technology.Simultaneous acquisition of dual-wavelength THG line-scanning signals enables measurement of bloodflow velocities at two radially symmetric positions in both venules and arterioles in mouse brain in vivo.Our results clearly show that the instantaneous bloodflow velocity is not symmetric under general conditions.展开更多
Myelin sheaths wrapping axons are key structures that help maintain the propagation speed of action potentials in both central and peripheral nervous systems(CNS and PNS).However,noninvasive,deep imaging technologies ...Myelin sheaths wrapping axons are key structures that help maintain the propagation speed of action potentials in both central and peripheral nervous systems(CNS and PNS).However,noninvasive,deep imaging technologies visualizing myelin sheaths in the digital skin in vivo are lacking in animal models.3-photon°uorescence(3PF)imaging excited at the 1700-nm window enables deep imaging of myelin sheaths,but necessitates labeling by exogenous°uorescent dyes.Since myelin sheaths are lipid-rich structures which generate strong third-harmonic signals,in this paper,we perform a detailed comparative experimental study of both third-harmonic generation(THG)and 3PF imaging in the mouse digital skin in vivo.Our results show that THG imaging also enables visualization of myelin sheaths deep in the mouse digital skin,which shows colocalization with 3PF signals from labeled myelin sheaths.Besides its superior label-free advantage,THG does not su®er from photobleaching due to its 3PF property.展开更多
Multiphoton microscopy(MPM)is a powerful imaging technology for brain research.The imaging depth in MPM is partly determined by emission wavelength of fluorescent labels.It has been demonstrated that a longer emission...Multiphoton microscopy(MPM)is a powerful imaging technology for brain research.The imaging depth in MPM is partly determined by emission wavelength of fluorescent labels.It has been demonstrated that a longer emission wavelength is favorable for signal detection as imaging depth increases.However,there has been no comparison with near-infrared(NIR)emission.In order to quantitatively analyze the effect of emission wavelength on 3-photon imaging of mouse brains in vivo,we utilize the same excitation wavelength to excite a single fluorescent dye and simultaneously collect NIR and orange-red emission fluorescence at 828 nm and 620 nm,respectively.Both experimental and simulation results show that as the imaging depth increases,NIR emission decays less than orange-red fluorescent emission.These results show that it is preferable to shift the emission wavelength to NIR to enable more e±cient signal collection deep in the brain.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
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
The algorithm used for reconstruction or resolution enhancement is one of the factors affectingthe quality of super-resolution images obtained by fluorescence microscopy.Deep-learning-basedalgorithms have achieved sta...The algorithm used for reconstruction or resolution enhancement is one of the factors affectingthe quality of super-resolution images obtained by fluorescence microscopy.Deep-learning-basedalgorithms have achieved stateof-the-art performance in super-resolution fluorescence micros-copy and are becoming increasingly attractive.We firstly introduce commonly-used deep learningmodels,and then review the latest applications in terms of the net work architectures,the trainingdata and the loss functions.Additionally,we discuss the challenges and limits when using deeplearning to analyze the fluorescence microscopic data,and suggest ways to improve the reliability and robustness of deep learning applications.展开更多
The skin is heterogeneous and exerts strong scattering and aberration onto excitation light in multiphoton microscopy(MPM).Shifting to longer excitation wavelengths may help reduce skin scattering and aberration,poten...The skin is heterogeneous and exerts strong scattering and aberration onto excitation light in multiphoton microscopy(MPM).Shifting to longer excitation wavelengths may help reduce skin scattering and aberration,potentially enabling larger imaging depths.However,previous demonstrations of skin MPM employ excitation wavelengths only up to the 1700 nm window,leaving an open question as to whether longer excitation wavelengths are suitable for deep-skin MPM.Here,in order to explore the longer-wavelength territory,first,we demonstrate characterization of the broadband transmittance of excised mouse skin,revealing a high transmittance window at 2200nm.Then,we demonstrate third-harmonic generation(THG)imaging in mouse skin in vivo excited at this window.With 9mW optical power on the skin surface operating at 1MHz repetition rate,we can get THG signals of 250m below the skin surface.Comparative THG imaging excited at the 1700nm window shows that as imaging depth increases,THG signals decay even faster than those excited at 2200 nm.Our results thus uncover the 2200 nm window as a new,promising excitation window potential for deep-skin MPM.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
基金funded by the National Natural Science Foundation of China(Grant/Award Numbers 62075135 and 61975126)the Science and Technology Innovation Commission of Shenzhen(Grant/Award Numbers JCYJ20190808174819083 and JCYJ20190808175201640)Shenzhen Science and Technology Planning Project(ZDSYS 20210623092006020).
文摘Measurement of bloodflow velocity is key to understanding physiology and pathology in vivo.While most measurements are performed at the middle of the blood vessel,little research has been done on characterizing the instantaneous bloodflow velocity distribution.This is mainly due to the lack of measurement technology with high spatial and temporal resolution.Here,we tackle this problem with our recently developed dual-wavelength line-scan third-harmonic generation(THG)imaging technology.Simultaneous acquisition of dual-wavelength THG line-scanning signals enables measurement of bloodflow velocities at two radially symmetric positions in both venules and arterioles in mouse brain in vivo.Our results clearly show that the instantaneous bloodflow velocity is not symmetric under general conditions.
基金National Natural Science Foundation of China(NSFC)(61775143,61975126)the Science and Technology Innovation Commission of Shenzhen under(No.JCYJ20190808174819083,JCYJ20190808175201640,KQTD20150710165601017).
文摘Myelin sheaths wrapping axons are key structures that help maintain the propagation speed of action potentials in both central and peripheral nervous systems(CNS and PNS).However,noninvasive,deep imaging technologies visualizing myelin sheaths in the digital skin in vivo are lacking in animal models.3-photon°uorescence(3PF)imaging excited at the 1700-nm window enables deep imaging of myelin sheaths,but necessitates labeling by exogenous°uorescent dyes.Since myelin sheaths are lipid-rich structures which generate strong third-harmonic signals,in this paper,we perform a detailed comparative experimental study of both third-harmonic generation(THG)and 3PF imaging in the mouse digital skin in vivo.Our results show that THG imaging also enables visualization of myelin sheaths deep in the mouse digital skin,which shows colocalization with 3PF signals from labeled myelin sheaths.Besides its superior label-free advantage,THG does not su®er from photobleaching due to its 3PF property.
基金work is funded by the National Natural Sci-ence Foundation of China(Grant/Award Numbers 62075135 and 61975126)Shenzhen Science and Technology Planning Project(ZDSYS2021-0623092006020)+2 种基金Key R&D Program of Shandong Province(Grant Number 2021CXGC010202)the Science and Technology Innovation Commission of Shenzhen(Grant/Award Numbers JCYJ201908-08174819083 and JCYJ20190808175201640)and Natural Science Foundation of Shandong Province(Grant Number ZR2022MA046)Major Innovation Projects for Integrating Science,Education&Industry of Qilu University of Technology(Shan-dong Academy of Sciences,Grant Number 2022JBZ01-04).
文摘Multiphoton microscopy(MPM)is a powerful imaging technology for brain research.The imaging depth in MPM is partly determined by emission wavelength of fluorescent labels.It has been demonstrated that a longer emission wavelength is favorable for signal detection as imaging depth increases.However,there has been no comparison with near-infrared(NIR)emission.In order to quantitatively analyze the effect of emission wavelength on 3-photon imaging of mouse brains in vivo,we utilize the same excitation wavelength to excite a single fluorescent dye and simultaneously collect NIR and orange-red emission fluorescence at 828 nm and 620 nm,respectively.Both experimental and simulation results show that as the imaging depth increases,NIR emission decays less than orange-red fluorescent emission.These results show that it is preferable to shift the emission wavelength to NIR to enable more e±cient signal collection deep in the brain.
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 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 National Key R&D Program of China(2021YFF0502900)the National Natural Science Foundation of China(61835009/62127819).
文摘The algorithm used for reconstruction or resolution enhancement is one of the factors affectingthe quality of super-resolution images obtained by fluorescence microscopy.Deep-learning-basedalgorithms have achieved stateof-the-art performance in super-resolution fluorescence micros-copy and are becoming increasingly attractive.We firstly introduce commonly-used deep learningmodels,and then review the latest applications in terms of the net work architectures,the trainingdata and the loss functions.Additionally,we discuss the challenges and limits when using deeplearning to analyze the fluorescence microscopic data,and suggest ways to improve the reliability and robustness of deep learning applications.
基金supported by National Natural Science Foundation of China (NSFC) (Nos.61775143,61975126 and 62075135)the Science and Technology Innovation Commission of Shenzhen under Nos.JCYJ20190808174819083,JCYJ20190808175201640 and KQTD20150710165601017China Postdoctoral Science Foundation (No.2021M702241).
文摘The skin is heterogeneous and exerts strong scattering and aberration onto excitation light in multiphoton microscopy(MPM).Shifting to longer excitation wavelengths may help reduce skin scattering and aberration,potentially enabling larger imaging depths.However,previous demonstrations of skin MPM employ excitation wavelengths only up to the 1700 nm window,leaving an open question as to whether longer excitation wavelengths are suitable for deep-skin MPM.Here,in order to explore the longer-wavelength territory,first,we demonstrate characterization of the broadband transmittance of excised mouse skin,revealing a high transmittance window at 2200nm.Then,we demonstrate third-harmonic generation(THG)imaging in mouse skin in vivo excited at this window.With 9mW optical power on the skin surface operating at 1MHz repetition rate,we can get THG signals of 250m below the skin surface.Comparative THG imaging excited at the 1700nm window shows that as imaging depth increases,THG signals decay even faster than those excited at 2200 nm.Our results thus uncover the 2200 nm window as a new,promising excitation window potential for deep-skin MPM.
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.