The high-content image-based assay is commonly leveraged for identifying the phenotypic impact of genetic perturbations in biology field.However,a persistent issue remains unsolved during experiments:the interferentia...The high-content image-based assay is commonly leveraged for identifying the phenotypic impact of genetic perturbations in biology field.However,a persistent issue remains unsolved during experiments:the interferential technical noises caused by systematic errors(e.g.,temperature,reagent concentration,and well location)are always mixed up with the real biological signals,leading to misinterpretation of any conclusion drawn.Here,we reported a mean teacher-based deep learning model(Deep Noise)that can disentangle biological signals from the experimental noises.Specifically,we aimed to classify the phenotypic impact of 1108 different genetic perturbations screened from 125,510 fluorescent microscopy images,which were totally unrecognizable by the human eye.We validated our model by participating in the Recursion Cellular Image Classification Challenge,and Deep Noise achieved an extremely high classification score(accuracy:99.596%),ranking the 2nd place among 866 participating groups.This promising result indicates the successful separation of biological and technical factors,which might help decrease the cost of treatment development and expedite the drug discovery process.The source code of Deep Noise is available at https://github.com/Scu-sen/Recursion-Cellular-Image-Classification-Challenge.展开更多
Traumatic penumbra(TP)is a region with recoverable potential around the primary lesion of brain injury.Rapid and accurate imaging for identifying TP is essential for treating traumatic brain injury(TBI).In this study,...Traumatic penumbra(TP)is a region with recoverable potential around the primary lesion of brain injury.Rapid and accurate imaging for identifying TP is essential for treating traumatic brain injury(TBI).In this study,we first established traumatic brain injuries(TBIs)in rats using a modified Feeney method,followed by label-free imaging of brain tissue sections with multiphoton fluorescence microscopy.The results showed that the technique effectively imaged normal and traumatic brain tissues,and revealed pathological features such as extracellular matrix changes,vascular cell proliferation,and intracellular edema in the traumatic penumbra.Compared with normal brain tissue,the extracellular matrix in the TP was sparse,cells were disorganized,and hyperplastic vascular cells emitted higher two-photon excited fluorescence(TPEF)signals.Our research demonstrates the potential of multiphoton fluorescence technology in the rapid diagnosis and therapeutic evaluation of TBI.展开更多
INSULIN secretion was traditionally measured with biochemical and immunological methods such as enzyme linked immunosorbant assay and radioimmunoassay. However, these methods can only tell the amount of insulin secret...INSULIN secretion was traditionally measured with biochemical and immunological methods such as enzyme linked immunosorbant assay and radioimmunoassay. However, these methods can only tell the amount of insulin secreted; they give no information about the secretion process or mechanism of exocytosis. In recent years, an imaging technique known as total internal reflection fluorescence （TIRF） microscopy has been employed to study insulin secretion.展开更多
An ultimate goal of neuroscience is to decipher the principles underlying neuronal information processing at the molecular,cellular,circuit,and system levels.The advent of miniature fluorescence microscopy has further...An ultimate goal of neuroscience is to decipher the principles underlying neuronal information processing at the molecular,cellular,circuit,and system levels.The advent of miniature fluorescence microscopy has furthered the quest by visualizing brain activities and structural dynamics in animals engaged in self-determined behaviors.In this brief review,we summarize recent advances in miniature fluorescence microscopy for neuroscience,focusing mostly on two mainstream solutions-miniature single-photon microscopy,and miniature two-photon microscopy.We discuss their technical advantages and limitations as well as unmet challenges for future improvement.Examples of preliminary applications are also presented to reflect on a new trend of brain imaging in experimental paradigms involving body movements,long and complex protocols,and even disease progression and aging.展开更多
Fluorescence polarization is related to the dipole orientation of chromophores,making fuores-cence polarization microscopy possible to_reveal structures and functions of tagged cellularorganelles and biological macrom...Fluorescence polarization is related to the dipole orientation of chromophores,making fuores-cence polarization microscopy possible to_reveal structures and functions of tagged cellularorganelles and biological macromolecules.Several recent super resolution techniques have beenapplied to fluorescence polarization microscopy,achieving dipole measurement at nanoscale.In this review,we summarize both difraction limited and super resolution fluorescence polari-zation microscopy techniques,as well as their applications in biological imaging.展开更多
Ischemic stroke is one of the most common causes of mortality and disability worldwide.However,treatment efficacy and the progress of research remain unsatisfactory.As the critical support system and essential compone...Ischemic stroke is one of the most common causes of mortality and disability worldwide.However,treatment efficacy and the progress of research remain unsatisfactory.As the critical support system and essential components in neurovascular units,glial cells and blood vessels(including the bloodbrain barrier)together maintain an optimal microenvironment for neuronal function.They provide nutrients,regulate neuronal excitability,and prevent harmful substances from entering brain tissue.The highly dynamic networks of this support system play an essential role in ischemic stroke through processes including brain homeostasis,supporting neuronal function,and reacting to injuries.However,most studies have focused on postmortem animals,which inevitably lack critical information about the dynamic changes that occur after ischemic stroke.Therefore,a high-precision technique for research in living animals is urgently needed.Two-photon fluorescence laser-scanning microscopy is a powerful imaging technique that can facilitate live imaging at high spatiotemporal resolutions.Twophoton fluorescence laser-scanning microscopy can provide images of the whole-cortex vascular 3D structure,information on multicellular component interactions,and provide images of structure and function in the cranial window.This technique shifts the existing research paradigm from static to dynamic,from flat to stereoscopic,and from single-cell function to multicellular intercommunication,thus providing direct and reliable evidence to identify the pathophysiological mechanisms following ischemic stroke in an intact brain.In this review,we discuss exciting findings from research on the support system after ischemic stroke using two-photon fluorescence laser-scanning microscopy,highlighting the importance of dynamic observations of cellular behavior and interactions in the networks of the brain’s support systems.We show the excellent application prospects and advantages of two-photon fluorescence laser-scanning microscopy and predict future research developments and directions in the study of ischemic stroke.展开更多
Digestive tract tumors acount for 15%and 19.3%of the cancer incidence and deaths,respec-tively.Early detection of digestive tract tumors is crucial to the reduction of global cancer burden.Two-photon excitation fuores...Digestive tract tumors acount for 15%and 19.3%of the cancer incidence and deaths,respec-tively.Early detection of digestive tract tumors is crucial to the reduction of global cancer burden.Two-photon excitation fuorescence lifetime imaging microscopy(TP-FLIM)allows non-invasive,label free,three-dimensional,high-resolution imaging of living tisues with not only histological but also biochemical characterization ability in both qualitative and quantitative way.Benefiting from these advantages,this technology is protmising for clinical diagnosis of digestive tract tumors.In recent years,many efforts have'been made in this field and some remarkable progress has been achieved.In this paper,we overview the recent progress of TP-FLIM-based researches on digestive tract tumor detection.Among them,our latest results on the gastric cancer and esophageal cancer are elaborately depicted.Finally,we outlook and discuss the potential advantages and challenges of TP-FLIM in future clinical applications.展开更多
AIM: To assess potential contributions of biliary IgA for crystal agglomeration into gallstones, we visualized cholesterol crystal binding of biliary IgA. METHODS: Crystal binding biliary proteins were extracted from ...AIM: To assess potential contributions of biliary IgA for crystal agglomeration into gallstones, we visualized cholesterol crystal binding of biliary IgA. METHODS: Crystal binding biliary proteins were extracted from human gallbladder bile using lectin affinity chromatography.Biliary IgA was isolated from the bound protein fraction by immunoaffinity chromatography. Pure cholesterol monohydrate crystals were incubated with biliary IgA and fluoresceine isothiocyanate (FITC)conjugated anti IgA at 37 degree. Samples were examined under polarizing and fluorescence light microscopy with digital image processing. RESULTS: Binding of biliary IgA to cholesterol monohydrate crystals could be visualized with FITC conjugated anti IgA antibodies.Peak fluorescence occurred at crystal edges and dislocations. Controls without biliary IgA or with biliary IgG showed no significant fluorescence. CONCLUSION: Fluorescence light microscopy provided evidence for cholesterol crystal binding of biliary IgA. Cholesterol crystal binding proteins like IgA might be important mediators of crystal agglomeration and growth of cholesterol gallstones by modifying the evolving crystal structures in vivo.展开更多
Optical microscopy is an essential tool for exploring the structures and activities of cells and tissues.To break the limit of resolution caused by diffraction,researchers have made continuous advances and innovations...Optical microscopy is an essential tool for exploring the structures and activities of cells and tissues.To break the limit of resolution caused by diffraction,researchers have made continuous advances and innovations to improve the resolution of optical microscopy since the 1990s.These contributions,however,still make sub-10nm imaging an obstacle.Here,we name a series of technologies as modulated illumination localization microscopy(MILM),which makes ultra-high-resolution imaging practical.Besides,we review the recent progress since 2017 when MINFLUX was proposed and became the inspiration and foundation for the follow-up devel-opment of MILM.This review divides MILM into two types:point-scanning and wide-field.The schematics,principles and future research directions of MILM are discussed elaborately.展开更多
Apoptosis is very important for the maintenance of cellular homeostasis and is closely related to the occurrence and treatment of many diseases.Mitochondria in cells play a crucial role in programmed cell death and re...Apoptosis is very important for the maintenance of cellular homeostasis and is closely related to the occurrence and treatment of many diseases.Mitochondria in cells play a crucial role in programmed cell death and redox processes.Nicotinamide adenine dinucleotide(NAD(P)H)is the primary producer of energy in mitochondria,changing NAD(P)H can directly reflect the physiological state of mitochondria.Therefore,NAD(P)H can be used to evaluate metabolic response.In this paper,we propose a noninvasive detection method that uses two-photon fluorescence lifetime imaging microscopy(TP-FLIM)to characterize apoptosis by observing the binding kinetics of cellular endogenous NAD(P)H.The result shows that the average fluorescence lifetime of NAD(P)H and the fluorescence lifetime of protein-bound NAD(P)H will be affected by the changing pH,serum content,and oxygen concentration in the cell culture environment,and by the treatment with reagents such as H2O2 and paclitaxel.Taxol(PTX).This noninvasive detection method realized the dynamic detection of cellular endogenous substances and the assessment of apoptosis.展开更多
The fluorescence lifetime of nicotinamide adenine dinucleotide(NADH),a key endogenous coenzyme and metabolic biomarker,can reflect the metabolic state of cells.To implement metabolic imaging of brain tissue at high re...The fluorescence lifetime of nicotinamide adenine dinucleotide(NADH),a key endogenous coenzyme and metabolic biomarker,can reflect the metabolic state of cells.To implement metabolic imaging of brain tissue at high resolution,we assembled a two-photon fluorescence lifetime imaging microscopy(FLIM)platform and verified the feasibility and stability of NADH-based two-photon FLIM in paraformaldehydefixed mouse cerebral slices.Furthermore,NADH based metabolic state oscillation was observed in cerebral nuclei suprachiasmatic nucleus(SCN).The free NADH fraction displayed a relatively lower level in the daytime than at the onset of night,and an ultradian oscillation at night was observed.Through the combination of high-resolution imaging and immunostaining data,the metabolic tendency of different cell types was detected after the first two hours of the day and at night.Thus,two-photon FLIM analysis of NADH in paraformaldehyde-fixed cerebral slices provides a high-resolution and label-free method to explore the metabolic state of deep brain regions.展开更多
A new two-photon fluorescent probe, ADNO, for nitric oxide （NO） based on intramolecular photoinduced electron transfer （PET） mechanism d/splays a rapid response to NO with a remarkable fluorescent enhancement in P...A new two-photon fluorescent probe, ADNO, for nitric oxide （NO） based on intramolecular photoinduced electron transfer （PET） mechanism d/splays a rapid response to NO with a remarkable fluorescent enhancement in PBS buffer. The excellent chemoselectivity of ADNO for NO over other ROS/RNS （reactive oxygen species or nitrogen species） and common metal ions was observed. Moreover, ADNO has been successfully applied in fluorescence imaging of NO of living cells using both one-photon microscopy （OPM） and two-~hoton microscopy （TPM）,展开更多
Fluorescence microscopy, as a sensitive method to detect microenvironment of molecules, is widely used in protein conformation and dynamic studies in live cells. Fluorescence lifetime imaging microscopy（FLIM）, which...Fluorescence microscopy, as a sensitive method to detect microenvironment of molecules, is widely used in protein conformation and dynamic studies in live cells. Fluorescence lifetime imaging microscopy（FLIM）, which is independent of fluorophore concentrations, scattering and bleaching, is a suitable tool to analyze membrane proteins in a single cell. Ferroportin（FPN）, a multi-ion exporter in vertebrates, was modulated by metal ions with unknown mechanism. Herein, we fused green fluorescence protein on Cterminal of FPN（FPN-eGFP） and applied fluorescence lifetime to monitor conformation changes of FPN in a live cell. The fluorescence lifetime distribution showed a shift to shorter lifetime upon Mn^（2＋） treatment,suggesting a preference conformation of FPN in Mn^（2＋） exposure. It is also observed that the lifetime（rather than intensity） measurement was not strongly influenced by laser power. The observed fluorescence lifetime changes of FPN-eGFP upon Mn^（2＋） treatments indicated that extracellular metal ions can modulate FPN through conformation exchanges between several different states.展开更多
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
In light-sheet fluorescence microscopy,the axial resolution and field of view are mutually constrained.Axially swept light-sheet microscopy(ASLM)can decouple the trade-off,but the confocal detection scheme using a rol...In light-sheet fluorescence microscopy,the axial resolution and field of view are mutually constrained.Axially swept light-sheet microscopy(ASLM)can decouple the trade-off,but the confocal detection scheme using a rolling shutter also rejects fluorescence signals from the specimen in the field of interest,which sacrifices the photon efficiency.Here,we report a laterally swept light-sheet microscopy(LSLM)scheme in which the focused beam is first scanned along the axial direction and subsequently laterally swept with the rolling shutter.We show that LSLM can obtain a higher photon efficiency when similar axial resolution and field of view can be achieved.Moreover,based on the principle of image scanning microscopy,applying the pixel reassignment to the LSLM images,hereby named iLSLM,improves the optical sectioning.Both simulation and experimental results demonstrate the higher photon efficiency with similar axial resolution and optical sectioning.Our proposed scheme is suitable for volumetric imaging of specimens that are susceptible to photobleaching or phototoxicity.展开更多
Fluorescence microscopy has become an essential tool for biologists,to visualize the dynamics of intracellular structures with specific labeling.Quantitatively measuring the dynamics of moving objects inside the cell ...Fluorescence microscopy has become an essential tool for biologists,to visualize the dynamics of intracellular structures with specific labeling.Quantitatively measuring the dynamics of moving objects inside the cell is pivotal for understanding of the underlying regulatory mechanism.Protein-containing vesicles are involved in various biological processes such as material transportation,organelle interaction,and hormonal regulation,whose dynamic characteristics are signi¯cant to disease diagnosis and drug screening.Although some algorithms have been developed for vesicle tracking,most of them have limited performance when dealing with images with low resolution,poor signal-to-noise ratio(SNR)and complicated motion.Here,we proposed a novel deep learning-based method for intracellular vesicle tracking.We trained the U-Net for vesicle localization and motion classification,with demonstrates great performance in both simulated datasets and real biological samples.By combination with fan-shaped tracker(FsT)we have previously developed,this hybrid new algorithm significantly improved the performance of particle tracking with the function of subsequently automated vesicle motion classification.Furthermore,its performance was further demonstrated in analyzing with vesicle dynamics in different temperature,which achieved reasonable outcomes.Thus,we anticipate that this novel method would have vast applications in analyzing the vesicle dynamics in living cells.展开更多
Single-particle microbeam as a powerful tool can open a research field to find answers to many enigmas in radiobiology. A single-particle microbeam facility has been constructed at the Key Laboratory of Ion Beam Bioen...Single-particle microbeam as a powerful tool can open a research field to find answers to many enigmas in radiobiology. A single-particle microbeam facility has been constructed at the Key Laboratory of Ion Beam Bioengineering （LIBB）, Chinese Academy of Sciences （CAS）, China. However there has been less research activities in this field concerning the original process of the interaction between low-energy ions and complicated organisms. To address this challenge, an in situ multi-dimensional quantitative fluorescence microscopy system combined with the CAS-LIBB single-particle microbeam II endstation is proposed. In this article, the rationale, logistics and development of many aspects of the proposed system are discussed.展开更多
Lipid droplets(LDs)participate in many physiological processes,the abnormality of which will cause chronic diseases and pathologies such as diabetes and obesity.It is crucial to monitor the distribution of LDs at high...Lipid droplets(LDs)participate in many physiological processes,the abnormality of which will cause chronic diseases and pathologies such as diabetes and obesity.It is crucial to monitor the distribution of LDs at high spatial resolution and large depth.Herein,we carried three-photon imaging of LDs in fat liver.Owing to the large three-photon absorption cross-section of the luminogen named NAP-CF_(3)(1:67×10^(-79) cm^(6) s^(2)),three-photon fluorescence fat liver imaging reached the largest depth of 80μm.Fat liver diagnosis was successfully carried out with excellent performance,providing great potential for LDs-associated pathologies research.展开更多
In this paper,we propose a new fluorescence emission difference microscopy(FED)technique based on polarization modulation.An electro-optical modulator(EOM)is used to switch the excitation beam between the horizontal a...In this paper,we propose a new fluorescence emission difference microscopy(FED)technique based on polarization modulation.An electro-optical modulator(EOM)is used to switch the excitation beam between the horizontal and vertical polarization states at a high frequency,which leads to solid-and donut-shaped beams after spatial light modulation.Experiment on the fluorescent nanoparticles demonstrates that the proposed method can achieve~λ=4 spatial resolution.Using the proposed system,the dynamic imaging of subcellular structures in living cells over time is achieved.展开更多
TIRF microscopy has provided a means to view mobile granules within 100 nm in size in two dimensions.However quantitative analysis of the position and motion of those granules requires an appropriate tracking method.I...TIRF microscopy has provided a means to view mobile granules within 100 nm in size in two dimensions.However quantitative analysis of the position and motion of those granules requires an appropriate tracking method.In this paper,we present a new tracking algorithm combined with the unique features of TIRF.Firstly a fluorescence correction procedure was processed to solve the problem of fluorescence bleaching over time.Mobile granules were then segmented from a time-lapse image stack by an adaptive background subtraction method.Kalman filter was introduced to estimate and track the granules that allowed reducing searching range and hence greater reliability in tracking process.After the tracked granules were located in x-y plane,the z-position was indirectly inferred from the changes in their intensities.In the experiments the algorithm was applied in tracking GLUT4 vesicles in living adipose cells.The results indicate that the algorithm has achieved robust estimation and tracking of the vesicles in three dimensions.展开更多
文摘The high-content image-based assay is commonly leveraged for identifying the phenotypic impact of genetic perturbations in biology field.However,a persistent issue remains unsolved during experiments:the interferential technical noises caused by systematic errors(e.g.,temperature,reagent concentration,and well location)are always mixed up with the real biological signals,leading to misinterpretation of any conclusion drawn.Here,we reported a mean teacher-based deep learning model(Deep Noise)that can disentangle biological signals from the experimental noises.Specifically,we aimed to classify the phenotypic impact of 1108 different genetic perturbations screened from 125,510 fluorescent microscopy images,which were totally unrecognizable by the human eye.We validated our model by participating in the Recursion Cellular Image Classification Challenge,and Deep Noise achieved an extremely high classification score(accuracy:99.596%),ranking the 2nd place among 866 participating groups.This promising result indicates the successful separation of biological and technical factors,which might help decrease the cost of treatment development and expedite the drug discovery process.The source code of Deep Noise is available at https://github.com/Scu-sen/Recursion-Cellular-Image-Classification-Challenge.
基金funded by the Science and Technology Research Program of Chongqing Municipal Education Commission(KJZD-K202301105,KJQN202201107)the Scienti¯c and Technological Transformative Program of Chongqing Banan District(KY202208161124020).
文摘Traumatic penumbra(TP)is a region with recoverable potential around the primary lesion of brain injury.Rapid and accurate imaging for identifying TP is essential for treating traumatic brain injury(TBI).In this study,we first established traumatic brain injuries(TBIs)in rats using a modified Feeney method,followed by label-free imaging of brain tissue sections with multiphoton fluorescence microscopy.The results showed that the technique effectively imaged normal and traumatic brain tissues,and revealed pathological features such as extracellular matrix changes,vascular cell proliferation,and intracellular edema in the traumatic penumbra.Compared with normal brain tissue,the extracellular matrix in the TP was sparse,cells were disorganized,and hyperplastic vascular cells emitted higher two-photon excited fluorescence(TPEF)signals.Our research demonstrates the potential of multiphoton fluorescence technology in the rapid diagnosis and therapeutic evaluation of TBI.
文摘INSULIN secretion was traditionally measured with biochemical and immunological methods such as enzyme linked immunosorbant assay and radioimmunoassay. However, these methods can only tell the amount of insulin secreted; they give no information about the secretion process or mechanism of exocytosis. In recent years, an imaging technique known as total internal reflection fluorescence （TIRF） microscopy has been employed to study insulin secretion.
基金We thank Dr.Zhe Zhao and Dr.Haitao Wu for helping with the experiments for Fig.2D,and Dr.Weijian Zong for discussion.This work was supported by grants from the National Natural Science Foundation of China(31327901,31570839,61975002,31830036,31821091,and 8182780030)the Major State Basic Research Program of China(2016 YFA0500400 and 2016YFA0500403)and the National Postdoctoral Program for Innovative Talents of China(BX20190011).
文摘An ultimate goal of neuroscience is to decipher the principles underlying neuronal information processing at the molecular,cellular,circuit,and system levels.The advent of miniature fluorescence microscopy has furthered the quest by visualizing brain activities and structural dynamics in animals engaged in self-determined behaviors.In this brief review,we summarize recent advances in miniature fluorescence microscopy for neuroscience,focusing mostly on two mainstream solutions-miniature single-photon microscopy,and miniature two-photon microscopy.We discuss their technical advantages and limitations as well as unmet challenges for future improvement.Examples of preliminary applications are also presented to reflect on a new trend of brain imaging in experimental paradigms involving body movements,long and complex protocols,and even disease progression and aging.
基金supported by the National Instrument Development Special Program(2013YQ03065102)the Natural Science Foundation of China(614-75010,61428501)Science and Technology Commission of Shanghai Municipality(16DZ-1100300).
文摘Fluorescence polarization is related to the dipole orientation of chromophores,making fuores-cence polarization microscopy possible to_reveal structures and functions of tagged cellularorganelles and biological macromolecules.Several recent super resolution techniques have beenapplied to fluorescence polarization microscopy,achieving dipole measurement at nanoscale.In this review,we summarize both difraction limited and super resolution fluorescence polari-zation microscopy techniques,as well as their applications in biological imaging.
基金supported by grants from the National Natural Science Foundation of China,Nos.92148206,82071330(to ZPT)82201745(to HN)the Natural Science Foundation of Hubei Province,China,Nos.2021BCA109(to ZPT)and 2021CFB067(to HN)。
文摘Ischemic stroke is one of the most common causes of mortality and disability worldwide.However,treatment efficacy and the progress of research remain unsatisfactory.As the critical support system and essential components in neurovascular units,glial cells and blood vessels(including the bloodbrain barrier)together maintain an optimal microenvironment for neuronal function.They provide nutrients,regulate neuronal excitability,and prevent harmful substances from entering brain tissue.The highly dynamic networks of this support system play an essential role in ischemic stroke through processes including brain homeostasis,supporting neuronal function,and reacting to injuries.However,most studies have focused on postmortem animals,which inevitably lack critical information about the dynamic changes that occur after ischemic stroke.Therefore,a high-precision technique for research in living animals is urgently needed.Two-photon fluorescence laser-scanning microscopy is a powerful imaging technique that can facilitate live imaging at high spatiotemporal resolutions.Twophoton fluorescence laser-scanning microscopy can provide images of the whole-cortex vascular 3D structure,information on multicellular component interactions,and provide images of structure and function in the cranial window.This technique shifts the existing research paradigm from static to dynamic,from flat to stereoscopic,and from single-cell function to multicellular intercommunication,thus providing direct and reliable evidence to identify the pathophysiological mechanisms following ischemic stroke in an intact brain.In this review,we discuss exciting findings from research on the support system after ischemic stroke using two-photon fluorescence laser-scanning microscopy,highlighting the importance of dynamic observations of cellular behavior and interactions in the networks of the brain’s support systems.We show the excellent application prospects and advantages of two-photon fluorescence laser-scanning microscopy and predict future research developments and directions in the study of ischemic stroke.
基金supports from the National Key Research and Development Program of China(2017YFC0110200)Program 973(2015CB755502)+4 种基金the National Natural Science Foundation of China(NSFC)(81571724,81701744,81822023)the Natural Science Foundation of Guangdong Province(2014A030312006,2017A 030310308)the Scientific Instrument Innovation Team of Chinese Academy of Sciences(GJJSTD 20180002)the Shenzhen Science and Technology Program(JCYJ20170818164343304,JCYJ20170818155006471,JCYJ20160608214524052,JCYJ20180507182432303)the SIAT Innovation Program for Excellent Young Researchers(201821).
文摘Digestive tract tumors acount for 15%and 19.3%of the cancer incidence and deaths,respec-tively.Early detection of digestive tract tumors is crucial to the reduction of global cancer burden.Two-photon excitation fuorescence lifetime imaging microscopy(TP-FLIM)allows non-invasive,label free,three-dimensional,high-resolution imaging of living tisues with not only histological but also biochemical characterization ability in both qualitative and quantitative way.Benefiting from these advantages,this technology is protmising for clinical diagnosis of digestive tract tumors.In recent years,many efforts have'been made in this field and some remarkable progress has been achieved.In this paper,we overview the recent progress of TP-FLIM-based researches on digestive tract tumor detection.Among them,our latest results on the gastric cancer and esophageal cancer are elaborately depicted.Finally,we outlook and discuss the potential advantages and challenges of TP-FLIM in future clinical applications.
文摘AIM: To assess potential contributions of biliary IgA for crystal agglomeration into gallstones, we visualized cholesterol crystal binding of biliary IgA. METHODS: Crystal binding biliary proteins were extracted from human gallbladder bile using lectin affinity chromatography.Biliary IgA was isolated from the bound protein fraction by immunoaffinity chromatography. Pure cholesterol monohydrate crystals were incubated with biliary IgA and fluoresceine isothiocyanate (FITC)conjugated anti IgA at 37 degree. Samples were examined under polarizing and fluorescence light microscopy with digital image processing. RESULTS: Binding of biliary IgA to cholesterol monohydrate crystals could be visualized with FITC conjugated anti IgA antibodies.Peak fluorescence occurred at crystal edges and dislocations. Controls without biliary IgA or with biliary IgG showed no significant fluorescence. CONCLUSION: Fluorescence light microscopy provided evidence for cholesterol crystal binding of biliary IgA. Cholesterol crystal binding proteins like IgA might be important mediators of crystal agglomeration and growth of cholesterol gallstones by modifying the evolving crystal structures in vivo.
基金This work was financially sponsored by National Natural Science Foundation of China(61735017,61827825)Major Program of the Natural Science Foundation of Zhejiang Province(LD21F050002)+1 种基金Key Research and Development Program of Zhejiang Province(2020C01116)Fundamental Research Funds for the Central Universities(K20200132),Zhejiang Lab(2020MC0AE01)and Zhejiang Provincial Ten Thousand Plan for Young Top Talents(2020R52001).Y.S.and L.Y.contributed equally to this work.
文摘Optical microscopy is an essential tool for exploring the structures and activities of cells and tissues.To break the limit of resolution caused by diffraction,researchers have made continuous advances and innovations to improve the resolution of optical microscopy since the 1990s.These contributions,however,still make sub-10nm imaging an obstacle.Here,we name a series of technologies as modulated illumination localization microscopy(MILM),which makes ultra-high-resolution imaging practical.Besides,we review the recent progress since 2017 when MINFLUX was proposed and became the inspiration and foundation for the follow-up devel-opment of MILM.This review divides MILM into two types:point-scanning and wide-field.The schematics,principles and future research directions of MILM are discussed elaborately.
基金supported in part by the National Key R&D Program of China(2017YFA0700402)National Natural Science Foundation of China(61961136005/61935012/62175163/61835009)+1 种基金Shenzhen Key projects(JCYJ20200109105404067)Shenzhen International Cooperation Project(GJHZ 20190822095420249).
文摘Apoptosis is very important for the maintenance of cellular homeostasis and is closely related to the occurrence and treatment of many diseases.Mitochondria in cells play a crucial role in programmed cell death and redox processes.Nicotinamide adenine dinucleotide(NAD(P)H)is the primary producer of energy in mitochondria,changing NAD(P)H can directly reflect the physiological state of mitochondria.Therefore,NAD(P)H can be used to evaluate metabolic response.In this paper,we propose a noninvasive detection method that uses two-photon fluorescence lifetime imaging microscopy(TP-FLIM)to characterize apoptosis by observing the binding kinetics of cellular endogenous NAD(P)H.The result shows that the average fluorescence lifetime of NAD(P)H and the fluorescence lifetime of protein-bound NAD(P)H will be affected by the changing pH,serum content,and oxygen concentration in the cell culture environment,and by the treatment with reagents such as H2O2 and paclitaxel.Taxol(PTX).This noninvasive detection method realized the dynamic detection of cellular endogenous substances and the assessment of apoptosis.
基金supported by the National Key R&D Program of China(Nos.2016YFA0400900 and 2017YFA0505301)National Natural Science Foundation of China(No.U1832181)。
文摘The fluorescence lifetime of nicotinamide adenine dinucleotide(NADH),a key endogenous coenzyme and metabolic biomarker,can reflect the metabolic state of cells.To implement metabolic imaging of brain tissue at high resolution,we assembled a two-photon fluorescence lifetime imaging microscopy(FLIM)platform and verified the feasibility and stability of NADH-based two-photon FLIM in paraformaldehydefixed mouse cerebral slices.Furthermore,NADH based metabolic state oscillation was observed in cerebral nuclei suprachiasmatic nucleus(SCN).The free NADH fraction displayed a relatively lower level in the daytime than at the onset of night,and an ultradian oscillation at night was observed.Through the combination of high-resolution imaging and immunostaining data,the metabolic tendency of different cell types was detected after the first two hours of the day and at night.Thus,two-photon FLIM analysis of NADH in paraformaldehyde-fixed cerebral slices provides a high-resolution and label-free method to explore the metabolic state of deep brain regions.
基金the National Natural Science Foundation of China(Nos.21102148 and 21125205)National Basic Research Program of China(No.2011CB935800)the State Key Laboratory of Fine Chemicals,Department of Chemical Engineering,Dalian University of Technology for financial supports
文摘A new two-photon fluorescent probe, ADNO, for nitric oxide （NO） based on intramolecular photoinduced electron transfer （PET） mechanism d/splays a rapid response to NO with a remarkable fluorescent enhancement in PBS buffer. The excellent chemoselectivity of ADNO for NO over other ROS/RNS （reactive oxygen species or nitrogen species） and common metal ions was observed. Moreover, ADNO has been successfully applied in fluorescence imaging of NO of living cells using both one-photon microscopy （OPM） and two-~hoton microscopy （TPM）,
基金supported by the National Key R&D Program of China (Nos. 2016YFA0400900, 2017YFA0505300)the Instrument Developing Project of the Chinese Academy of Sciences (No. YZ201564)
文摘Fluorescence microscopy, as a sensitive method to detect microenvironment of molecules, is widely used in protein conformation and dynamic studies in live cells. Fluorescence lifetime imaging microscopy（FLIM）, which is independent of fluorophore concentrations, scattering and bleaching, is a suitable tool to analyze membrane proteins in a single cell. Ferroportin（FPN）, a multi-ion exporter in vertebrates, was modulated by metal ions with unknown mechanism. Herein, we fused green fluorescence protein on Cterminal of FPN（FPN-eGFP） and applied fluorescence lifetime to monitor conformation changes of FPN in a live cell. The fluorescence lifetime distribution showed a shift to shorter lifetime upon Mn^（2＋） treatment,suggesting a preference conformation of FPN in Mn^（2＋） exposure. It is also observed that the lifetime（rather than intensity） measurement was not strongly influenced by laser power. The observed fluorescence lifetime changes of FPN-eGFP upon Mn^（2＋） treatments indicated that extracellular metal ions can modulate FPN through conformation exchanges between several different states.
基金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(62005116 and 51720105015)the Science and Technology Innovation Commission of Shenzhen(KQTD20170810110913065 and 20200925174735005).
文摘In light-sheet fluorescence microscopy,the axial resolution and field of view are mutually constrained.Axially swept light-sheet microscopy(ASLM)can decouple the trade-off,but the confocal detection scheme using a rolling shutter also rejects fluorescence signals from the specimen in the field of interest,which sacrifices the photon efficiency.Here,we report a laterally swept light-sheet microscopy(LSLM)scheme in which the focused beam is first scanned along the axial direction and subsequently laterally swept with the rolling shutter.We show that LSLM can obtain a higher photon efficiency when similar axial resolution and field of view can be achieved.Moreover,based on the principle of image scanning microscopy,applying the pixel reassignment to the LSLM images,hereby named iLSLM,improves the optical sectioning.Both simulation and experimental results demonstrate the higher photon efficiency with similar axial resolution and optical sectioning.Our proposed scheme is suitable for volumetric imaging of specimens that are susceptible to photobleaching or phototoxicity.
基金supported by the National Key Research and Development Program of China(2021YFF0700305 and 2018YFE0119000)the National Natural Science Foundation of China(22104129 and 62105288)+1 种基金Zhejiang Province Science and Technology Research Plan(2022C03014)the Fundamental Research Funds for the Central Universities(2021XZZX022)and Alibaba Cloud.
文摘Fluorescence microscopy has become an essential tool for biologists,to visualize the dynamics of intracellular structures with specific labeling.Quantitatively measuring the dynamics of moving objects inside the cell is pivotal for understanding of the underlying regulatory mechanism.Protein-containing vesicles are involved in various biological processes such as material transportation,organelle interaction,and hormonal regulation,whose dynamic characteristics are signi¯cant to disease diagnosis and drug screening.Although some algorithms have been developed for vesicle tracking,most of them have limited performance when dealing with images with low resolution,poor signal-to-noise ratio(SNR)and complicated motion.Here,we proposed a novel deep learning-based method for intracellular vesicle tracking.We trained the U-Net for vesicle localization and motion classification,with demonstrates great performance in both simulated datasets and real biological samples.By combination with fan-shaped tracker(FsT)we have previously developed,this hybrid new algorithm significantly improved the performance of particle tracking with the function of subsequently automated vesicle motion classification.Furthermore,its performance was further demonstrated in analyzing with vesicle dynamics in different temperature,which achieved reasonable outcomes.Thus,we anticipate that this novel method would have vast applications in analyzing the vesicle dynamics in living cells.
文摘Single-particle microbeam as a powerful tool can open a research field to find answers to many enigmas in radiobiology. A single-particle microbeam facility has been constructed at the Key Laboratory of Ion Beam Bioengineering （LIBB）, Chinese Academy of Sciences （CAS）, China. However there has been less research activities in this field concerning the original process of the interaction between low-energy ions and complicated organisms. To address this challenge, an in situ multi-dimensional quantitative fluorescence microscopy system combined with the CAS-LIBB single-particle microbeam II endstation is proposed. In this article, the rationale, logistics and development of many aspects of the proposed system are discussed.
基金supported by National Natural Science Foundation of China (61975172,82001874,62105184)the Guangdong Basic and Applied Basic Research Foundation (2020A1515110578).
文摘Lipid droplets(LDs)participate in many physiological processes,the abnormality of which will cause chronic diseases and pathologies such as diabetes and obesity.It is crucial to monitor the distribution of LDs at high spatial resolution and large depth.Herein,we carried three-photon imaging of LDs in fat liver.Owing to the large three-photon absorption cross-section of the luminogen named NAP-CF_(3)(1:67×10^(-79) cm^(6) s^(2)),three-photon fluorescence fat liver imaging reached the largest depth of 80μm.Fat liver diagnosis was successfully carried out with excellent performance,providing great potential for LDs-associated pathologies research.
基金supported in part by the National Natural Science Foundation of China(61827825,62125504,and 61735017)Major Program of the Natural Science Foundation of Zhejiang Province(LD21F050002)+2 种基金Key Research and Development Program of Zhejiang Province(2020C01116)Zhejiang Lab(2020MC0AE01)China Postdoctoral Science Foundation(BX2021272).
文摘In this paper,we propose a new fluorescence emission difference microscopy(FED)technique based on polarization modulation.An electro-optical modulator(EOM)is used to switch the excitation beam between the horizontal and vertical polarization states at a high frequency,which leads to solid-and donut-shaped beams after spatial light modulation.Experiment on the fluorescent nanoparticles demonstrates that the proposed method can achieve~λ=4 spatial resolution.Using the proposed system,the dynamic imaging of subcellular structures in living cells over time is achieved.
基金Project supported by the National Natural Science Foundation ofChina (No. 30770596)the Key Laboratory for Biomedical En-gineering of Ministry of Education of China
文摘TIRF microscopy has provided a means to view mobile granules within 100 nm in size in two dimensions.However quantitative analysis of the position and motion of those granules requires an appropriate tracking method.In this paper,we present a new tracking algorithm combined with the unique features of TIRF.Firstly a fluorescence correction procedure was processed to solve the problem of fluorescence bleaching over time.Mobile granules were then segmented from a time-lapse image stack by an adaptive background subtraction method.Kalman filter was introduced to estimate and track the granules that allowed reducing searching range and hence greater reliability in tracking process.After the tracked granules were located in x-y plane,the z-position was indirectly inferred from the changes in their intensities.In the experiments the algorithm was applied in tracking GLUT4 vesicles in living adipose cells.The results indicate that the algorithm has achieved robust estimation and tracking of the vesicles in three dimensions.
