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
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
Deciphering the neuronal response to injury in the spinal cord is essential for exploring treatment strategies for spinal cord injury(SCI).However,this subject has been neglected in part because appropriate tools are ...Deciphering the neuronal response to injury in the spinal cord is essential for exploring treatment strategies for spinal cord injury(SCI).However,this subject has been neglected in part because appropriate tools are lacking.Emerging in vivo imaging and labeling methods offer great potential for observing dynamic neural processes in the central nervous system in conditions of health and disease.This review first discusses in vivo imaging of the mouse spinal cord with a focus on the latest imaging techniques,and then analyzes the dynamic biological response of spinal cord sensory and motor neurons to SCI.We then summarize and compare the techniques behind these studies and clarify the advantages of in vivo imaging compared with traditional neuroscience examinations.Finally,we identify the challenges and possible solutions for spinal cord neuron 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
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
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
Fluorescence lifetime is not only associated with the molecular structure f fuorophores,but alsostrongly depends on the environment around them,which llows fuorescence lifetime imagingmicroscopy(FLIM)to be used as a t...Fluorescence lifetime is not only associated with the molecular structure f fuorophores,but alsostrongly depends on the environment around them,which llows fuorescence lifetime imagingmicroscopy(FLIM)to be used as a tool for precise measurement of the cell or tisue microenvironment,This review introduces the basic principle of fuorescence lifetime imagingtechnology and its application in clinical medicine,including research and diagnosis of diseases inskin,brain,eyes,mouth,bone,blood vessels and cavity organs,and drug evaluation.As anoninvasive,nontoxic and nonionizing radiation technique,FLIM demonstrates excellent per-formance with high sensitivity and specificity,which allows to determine precise position of thelesion and,thus,has good potential for application in biomedical research and clinical diagnosis.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
Background and Aims: Accurate endoscopic detection of premalignant lesions and earlycancers in the colon is essential for cure, since prognosis is closely related to lesion size andstage. Although it has great clinica...Background and Aims: Accurate endoscopic detection of premalignant lesions and earlycancers in the colon is essential for cure, since prognosis is closely related to lesion size andstage. Although it has great clinical potential, autofluorescence endoscopy has limited tumorto-normal tissue image contrast for detecting small preneoplastic lesions. We have developed amolecularly specific, near-infrared fluorescent monoclonal antibody (CC49) bioconjugate whichtargets tumor-associated glycoprotein 72 (TAG72), as a contrast agent to improve fluorescencebased endoscopy of colon cancer. Methods: The fluorescent anti-TAG72 conjugate was evaluated in vitro and in vivo in athymic nude mice bearing human colon adenocarcinoma (LS174T)subcutaneous tumors. Autofluorescence, a fluorescent but irrelevant antibody and the free fluorescent dye served as controls. Fluorescent agents were injected intravenously, and in vivowhole body fluorescence imaging was performed at various time points to determine pharmacokinetics, followed by ex vivo tissue analysis by confocal fluorescence microscopy and histology Results: Fluorescence microscopy and histology confirmed specific LS174T cell membrane targeting of labeled CC49 in vitro and ex vivo. In vivo fluorescence imaging demonstrated significant tumor-to-normal tissue contrast enhancement with labeled-CC49 at three hours postinjection, with maximum contrast after 48 h. Accumulation of tumor fluorescence demonstratedthat modification of CC49 antibodies did not alter their specific tumor-localizing properties, andwas antibody-dependent since controls did not produce detectable tumor fluorescence. Conclusions: These results show proof-of-principle that our near-infrared fluorescent-antibody probetargeting a tumor-associated mucin detects colonic tumors at the molecular level in real time,and offer a basis for future improvement of image contrast during clinical fluorescence endoscopy.展开更多
Fluorescence lifetime imaging can reveal the high-resolution structure of various biophysical and chemical parameters in a microenvironment quantitatively.However,the depth of imaging is generally limited to hundreds ...Fluorescence lifetime imaging can reveal the high-resolution structure of various biophysical and chemical parameters in a microenvironment quantitatively.However,the depth of imaging is generally limited to hundreds of micrometers due to aberration and light scattering in biological tissues.This paper introduces an iterative multi-photon adaptive compensation technique(IMPACT)into a two-photon fluorescence lifetime microscopy system to successfully overcome aberrations and multiple scattering problems in deep tissues.It shows that 400 correction modes can be achieved within 5 min,which was mainly limited by the frame rate of a spatial light modulator.This system was used for high-resolution imaging of mice brain tissue and live zebrafish,further verifying its superior performance in imaging quality and photon accumulation speed.展开更多
Fluorescence imaging can be employed in fields of medical treatment,astronomical exploration,and national defense security.Traditional fluorescence imaging often takes the single-photon techniques,which is vulnerable ...Fluorescence imaging can be employed in fields of medical treatment,astronomical exploration,and national defense security.Traditional fluorescence imaging often takes the single-photon techniques,which is vulnerable to background interference and photobleaching.Remedially,two-photon fluorescence imaging can achieve much higher-resolution fluorescence imaging for reducing scattering and deeper depth.Hence,by assembling the tetraphenylethylene backbones with nontoxic and non-noble K^(+)ions,compound 1([(Hdma)K(H_(2)ettc)]_(n),H_(4)ettc=4',4''',4''''',4'''''''-(ethene-1,1,2,2-tetrayl)tetrakis(([1,1'-biphenyl]-4-carboxylic acid)))with the crystallization-induced emissions exhibited charming fluorescence imaging under two-photon excitation microscopy(TPEM).Besides,luminescent powders based on compound 1 can achieve high-resolution fingerprint recognition,providing secure access control and identification for a novel authentication method.Compared with the commercial fluorescent dyes coumarin-6,the as-synthesized compound 1 showed great solvent stability,indicating its durability against harsh environment.Moreover,compound 1 shows mechanoluminescent properties for the perturbation of weak supramolecular interactions within ordered arrangements of the H_(2)ettc^(2−)ligands.This novel compound has provided an important insight to the development of twophoton fluorescence imaging and advanced external-stimuli responsive materials.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
AIM: To evaluate a newly developed hand-held confocal probe for in vivo microscopic imaging of the complete gastrointestinal tract in rodents. METHODS: A novel rigid confocal probe (diameter 7 mm) was designed wit...AIM: To evaluate a newly developed hand-held confocal probe for in vivo microscopic imaging of the complete gastrointestinal tract in rodents. METHODS: A novel rigid confocal probe (diameter 7 mm) was designed with optical features similar to the flexible endomicroscopy system for use in humans using a 488 nm single line laser for fluorophore excitation, Light emission was detected at 505 to 750 nm. The field of view was 475 μm × 475 μm. Optical slice thickness was 7 μm with a lateral resolution of 0.7 μm. Subsurface serial images at different depths (surface to 250 μm) were generated in real time at 1024 × 1024 pixels (0.8 frames/s) by placing the probe onto the tissue in gentle, stable contact. Tissue specimens were sampled for histopathological correlation.RESULTS: The esophagus, stomach, small and large intestine and meso, liver, pancreas and gall bladder were visualised in vivo at high resolution in n = 48 mice. Real time microscopic imaging with the confocal minimicroscopy probe was easy to achieve. The different staining protocols (fluorescein, acriflavine, FITC-labelled dextran and L. esculentum lectin) each highlighted specific aspects of the tissue, and in vivo imaging correlated excellently with conventional histology. In vivo blood flow monitoring added a functional quality to morphologic imaging.CONCLUSION: Confocal microscopy is feasible in vivo allowing the visualisation of the complete GI tract at high resolution even of subsurface tissue structures. The new confocal probe design evaluated in this study is compatible with laparoscopy and significantly expands the field of possible applications to intra-abdominal organs. It allows immediate testing of new in vivo staining and application options and therefore permits rapid transfer from animal studies to clinical use in patients.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
Gastrointestinal stromal tumors(GISTs)are the most common mesenchymal tumors arising in the digest tract.It brings a challenge to diagnosis because it is asymptomatic clinically.It is well known that tumor development...Gastrointestinal stromal tumors(GISTs)are the most common mesenchymal tumors arising in the digest tract.It brings a challenge to diagnosis because it is asymptomatic clinically.It is well known that tumor development is often accompanied by the changes in the morphology of collagen fibers.Nowadays,an emerging optical imaging technique,second-harmonic generation(SHG),can directly identify collagen fibers without staining due to its noncentrosymmetric properties.Therefore,in this study,we attempt to assess the feasibility of SHG imaging for detecting GISTs by monitoring the morphological changes of collagen fibers in tumor microenvironment.We found that collagen alterations occurred obviously in the GISTs by comparing with normal tissues,and furthermore,two morphological features from SHG images were extracted to quantitatively assess the morphological difference of collagen fibers between normal muscular layer and GISTs by means of automated image analysis.Quantitative analyses show a significant difference in the two collagen features.This study demonstrates the potential of SHG imaging as an adjunctive diagnostic tool for label-free identification of GISTs.展开更多
Compared with visible light,near infrared(NIR)light has deeper penetration in biological tisues.Three-photon fuorescence microscopy(3PFM)can effectively utilize the NIR excitation to obtain high-contrast images in the...Compared with visible light,near infrared(NIR)light has deeper penetration in biological tisues.Three-photon fuorescence microscopy(3PFM)can effectively utilize the NIR excitation to obtain high-contrast images in the deep tisue.However,the weak three photon fluorescence signals may be not well presented in the traditional fuorescence intensity imaging mode.Fluorescence lifetime of certain probes is insensitive to the intensity of the excitation laser.Moreover,fluorescence lifetimne imaging microscopy(FLIM)can detect weak signals by utilizing time correlated single photon counting(TCSPC)technique.Thus,it would be an improved strategy to combine the 3PFM imaging with the FLIM together.Herein,DCDPP-2TPA,a novel agegation-induced emission luminogen(AIEgen),was adopted as the fluorescent probes.The three-photon absorption cros-section of the AlEgen,which has a deep-red fluorescence emission,was proved to be large.DCDPP-2TPA nanoparticles were synthesized,and the three photon fluorescence lifetime of which was measured in water.Moreover,in vrivo thre-photon fuorescence lifetime microscopic imaging of a craniotomy mouse was conducted via a home made optical system.High contrast cerebrovascular images of different vertical depths were obtained and the maximun depth was about 600 pumn.Even reaching the depth of 600 pum,tiny capillary vessels as small as 1.9 pum could still be distinguished.The three photon fuorescence lifetimes of the capillaries in some representative images were in accord with that of DCDPP-2TPA nanoparticles in water.A vivid 3D reconstruction was further organized to present a wealth of lifetime information.In the future,the combination strategy of 3PFM and FLIM could be further applied in the brain functional imaging.展开更多
We report three-dimensional fluorescence emission difference(3D-FED)microscopy using a spatial light modulator(SLM).Zero phase,0–2vortex phase and binary 0-pi phase are loaded on the SLM to generate the correspondin...We report three-dimensional fluorescence emission difference(3D-FED)microscopy using a spatial light modulator(SLM).Zero phase,0–2vortex phase and binary 0-pi phase are loaded on the SLM to generate the corresponding solid,doughnut and z-axis hollow excitation spot,respectively.Our technique achieves super-resolved image by subtracting three di®erently acquired images with proper subtractive factors.Detailed theoretical analysis and simulation tests are proceeded to testify the performance of 3D-FED.Also,the improvement of lateral and axial resolution is demonstrated by imaging 100 nm°uorescent beads.The experiment yields lateral resolution of 140 nm and axial resolution of approximate 380 nm.展开更多
In this paper, we describe an algorithm that performs automatic detection and tracking of astral microtubules in fluorescence confocal images. This sub-population of microtubules only exists during and immediately bef...In this paper, we describe an algorithm that performs automatic detection and tracking of astral microtubules in fluorescence confocal images. This sub-population of microtubules only exists during and immediately before mitosis and aids in the spindle orientation by connecting it to the cell cortex. Anomalies in their dynamic behaviour play a causal role in many diseases, such as development disorders and cancer. The main novelty of the proposed algorithm lies in the fact it provides a fully automated estimation of parameters related to microtubule dynamic instability (growth velocity, track length and track lifetime), and helps in understanding the effects of intermediate drug concentrations. Its performance has been objectively assessed using publicly available synthetic data and largely employed metrics. Moreover, we present experiments addressing cell cultures doped with different concentrations of taxol and nocodazole. Such drugs are known to suppress the microtubule dynamic instability, but their effects at intermediate concentrations are not completely assessed. The algorithm has been compared with other state-of-the-art approaches, tested on consistent real datasets. The results are encouraging in terms of performance, robustness and simplicity of use, and the algorithm is now routinely employed in our Department of Molecular Biotechnology.展开更多
A novel carbazole quaternary ammonium compound(abbreviated as T_2) had been synthesized and characterized by ~1H NMR, ^(13)C NMR and Mass spectrometry. The single-crystal structure has been determined by X-ray sin...A novel carbazole quaternary ammonium compound(abbreviated as T_2) had been synthesized and characterized by ~1H NMR, ^(13)C NMR and Mass spectrometry. The single-crystal structure has been determined by X-ray single-crystal diffraction. The electrochemical and two-photon absorption properties of T_2 were systematically studied by cyclic voltammetry and Z-scan determination methods, respectively. The results suggested that T_2 had a good oxidation-reduction and excellent nonlinear optical property. The two-photon absorption(TPA) value has a maximum corresponding to cross section σ = 7963.3 GM(Goeppert-Mayer units) at 700 nm, indicating potential applications in nonlinear optical materials. Furthermore, attributing to the excellent water solubility and low cytotoxicity, the compound was explored on its primary application in biological imaging.展开更多
Imaging three-dimensional,subcellular structures with high axial resolution has always been the core purpose of fluorescence microscopy.However,trade-offs exist between axial resolution and other important technical i...Imaging three-dimensional,subcellular structures with high axial resolution has always been the core purpose of fluorescence microscopy.However,trade-offs exist between axial resolution and other important technical indicators,such as temporal resolution,optical power density,and imaging process complexity.We report a new imaging modality,fluorescence interference structured illumination microscopy(FI-SIM),which is based on three-dimensional structured illumination microscopy for wide-field lateral imaging and fluorescence interference for axial reconstruction.FI-SIM can acquire images quickly within the order of hundreds of milliseconds and exhibit even 30 nm axial resolution in half the wavelength depth range without z-axis scanning.Moreover,the relatively low laser power density relaxes the requirements for dyes and enables a wide range of applications for observing fixed and live subcellular structures.展开更多
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(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,No.82272478(to PT)。
文摘Deciphering the neuronal response to injury in the spinal cord is essential for exploring treatment strategies for spinal cord injury(SCI).However,this subject has been neglected in part because appropriate tools are lacking.Emerging in vivo imaging and labeling methods offer great potential for observing dynamic neural processes in the central nervous system in conditions of health and disease.This review first discusses in vivo imaging of the mouse spinal cord with a focus on the latest imaging techniques,and then analyzes the dynamic biological response of spinal cord sensory and motor neurons to SCI.We then summarize and compare the techniques behind these studies and clarify the advantages of in vivo imaging compared with traditional neuroscience examinations.Finally,we identify the challenges and possible solutions for spinal cord neuron 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.
基金funded by the Science and Technology Planning Fundamental Research Project of Shenzhen(No.JCYJ20150324140036853)National Natural Science Foundation of China(No.61378091)+1 种基金Ningbo Natural Science Foundation Project(No.2016A610032)the Central University Basic Scientic Research Business Expenses Project(No.NSIY051405).
文摘Fluorescence lifetime is not only associated with the molecular structure f fuorophores,but alsostrongly depends on the environment around them,which llows fuorescence lifetime imagingmicroscopy(FLIM)to be used as a tool for precise measurement of the cell or tisue microenvironment,This review introduces the basic principle of fuorescence lifetime imagingtechnology and its application in clinical medicine,including research and diagnosis of diseases inskin,brain,eyes,mouth,bone,blood vessels and cavity organs,and drug evaluation.As anoninvasive,nontoxic and nonionizing radiation technique,FLIM demonstrates excellent per-formance with high sensitivity and specificity,which allows to determine precise position of thelesion and,thus,has good potential for application in biomedical research and clinical diagnosis.
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.
文摘Background and Aims: Accurate endoscopic detection of premalignant lesions and earlycancers in the colon is essential for cure, since prognosis is closely related to lesion size andstage. Although it has great clinical potential, autofluorescence endoscopy has limited tumorto-normal tissue image contrast for detecting small preneoplastic lesions. We have developed amolecularly specific, near-infrared fluorescent monoclonal antibody (CC49) bioconjugate whichtargets tumor-associated glycoprotein 72 (TAG72), as a contrast agent to improve fluorescencebased endoscopy of colon cancer. Methods: The fluorescent anti-TAG72 conjugate was evaluated in vitro and in vivo in athymic nude mice bearing human colon adenocarcinoma (LS174T)subcutaneous tumors. Autofluorescence, a fluorescent but irrelevant antibody and the free fluorescent dye served as controls. Fluorescent agents were injected intravenously, and in vivowhole body fluorescence imaging was performed at various time points to determine pharmacokinetics, followed by ex vivo tissue analysis by confocal fluorescence microscopy and histology Results: Fluorescence microscopy and histology confirmed specific LS174T cell membrane targeting of labeled CC49 in vitro and ex vivo. In vivo fluorescence imaging demonstrated significant tumor-to-normal tissue contrast enhancement with labeled-CC49 at three hours postinjection, with maximum contrast after 48 h. Accumulation of tumor fluorescence demonstratedthat modification of CC49 antibodies did not alter their specific tumor-localizing properties, andwas antibody-dependent since controls did not produce detectable tumor fluorescence. Conclusions: These results show proof-of-principle that our near-infrared fluorescent-antibody probetargeting a tumor-associated mucin detects colonic tumors at the molecular level in real time,and offer a basis for future improvement of image contrast during clinical fluorescence endoscopy.
基金supported by the National Key Research and Development Program of China(No.2021YFF0502900)the National Natural Science Foundation of China(Nos.62175163,62225505,61935012,61835009,62127819,and 62205220)+2 种基金the Shenzhen Key Projects(No.JCYJ20200109105404067)the Shenzhen Talent Innovation Project(No.RCJC20210706091949022)the Shenzhen Science and Technology Planning Project(No.ZDSYS20210623092006020)。
文摘Fluorescence lifetime imaging can reveal the high-resolution structure of various biophysical and chemical parameters in a microenvironment quantitatively.However,the depth of imaging is generally limited to hundreds of micrometers due to aberration and light scattering in biological tissues.This paper introduces an iterative multi-photon adaptive compensation technique(IMPACT)into a two-photon fluorescence lifetime microscopy system to successfully overcome aberrations and multiple scattering problems in deep tissues.It shows that 400 correction modes can be achieved within 5 min,which was mainly limited by the frame rate of a spatial light modulator.This system was used for high-resolution imaging of mice brain tissue and live zebrafish,further verifying its superior performance in imaging quality and photon accumulation speed.
基金supported by the National Natural Science Foundation of China(Nos.22205237,22271283,21971240,and 21827813)the National Key Research and Development Program of China(No.2017YFA0206802)the Scientific Instrument Developing Project of the Chinese Academy of Sciences(No.YJKYYQ20210039).
文摘Fluorescence imaging can be employed in fields of medical treatment,astronomical exploration,and national defense security.Traditional fluorescence imaging often takes the single-photon techniques,which is vulnerable to background interference and photobleaching.Remedially,two-photon fluorescence imaging can achieve much higher-resolution fluorescence imaging for reducing scattering and deeper depth.Hence,by assembling the tetraphenylethylene backbones with nontoxic and non-noble K^(+)ions,compound 1([(Hdma)K(H_(2)ettc)]_(n),H_(4)ettc=4',4''',4''''',4'''''''-(ethene-1,1,2,2-tetrayl)tetrakis(([1,1'-biphenyl]-4-carboxylic acid)))with the crystallization-induced emissions exhibited charming fluorescence imaging under two-photon excitation microscopy(TPEM).Besides,luminescent powders based on compound 1 can achieve high-resolution fingerprint recognition,providing secure access control and identification for a novel authentication method.Compared with the commercial fluorescent dyes coumarin-6,the as-synthesized compound 1 showed great solvent stability,indicating its durability against harsh environment.Moreover,compound 1 shows mechanoluminescent properties for the perturbation of weak supramolecular interactions within ordered arrangements of the H_(2)ettc^(2−)ligands.This novel compound has provided an important insight to the development of twophoton fluorescence imaging and advanced external-stimuli responsive materials.
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Supported by Group for Minimal-invasive Chirurgie, Johannes Gutenberg-Universitat, Mainz, Germany
文摘AIM: To evaluate a newly developed hand-held confocal probe for in vivo microscopic imaging of the complete gastrointestinal tract in rodents. METHODS: A novel rigid confocal probe (diameter 7 mm) was designed with optical features similar to the flexible endomicroscopy system for use in humans using a 488 nm single line laser for fluorophore excitation, Light emission was detected at 505 to 750 nm. The field of view was 475 μm × 475 μm. Optical slice thickness was 7 μm with a lateral resolution of 0.7 μm. Subsurface serial images at different depths (surface to 250 μm) were generated in real time at 1024 × 1024 pixels (0.8 frames/s) by placing the probe onto the tissue in gentle, stable contact. Tissue specimens were sampled for histopathological correlation.RESULTS: The esophagus, stomach, small and large intestine and meso, liver, pancreas and gall bladder were visualised in vivo at high resolution in n = 48 mice. Real time microscopic imaging with the confocal minimicroscopy probe was easy to achieve. The different staining protocols (fluorescein, acriflavine, FITC-labelled dextran and L. esculentum lectin) each highlighted specific aspects of the tissue, and in vivo imaging correlated excellently with conventional histology. In vivo blood flow monitoring added a functional quality to morphologic imaging.CONCLUSION: Confocal microscopy is feasible in vivo allowing the visualisation of the complete GI tract at high resolution even of subsurface tissue structures. The new confocal probe design evaluated in this study is compatible with laparoscopy and significantly expands the field of possible applications to intra-abdominal organs. It allows immediate testing of new in vivo staining and application options and therefore permits rapid transfer from animal studies to clinical use in patients.
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supported by the National Natural Science Foundation of China(Grant Nos.82171991 and 82172800)Joint Funds for the Innovation of Science and Technology of Fujian Province(Grant No.2019Y9101)+1 种基金Fujian Major Scientific and Technological Special Project for"Social Development"(No.2020YZ016002)Special Funds of the Central Government Guiding Local Science and Technology Development(No.2020L3008).
文摘Gastrointestinal stromal tumors(GISTs)are the most common mesenchymal tumors arising in the digest tract.It brings a challenge to diagnosis because it is asymptomatic clinically.It is well known that tumor development is often accompanied by the changes in the morphology of collagen fibers.Nowadays,an emerging optical imaging technique,second-harmonic generation(SHG),can directly identify collagen fibers without staining due to its noncentrosymmetric properties.Therefore,in this study,we attempt to assess the feasibility of SHG imaging for detecting GISTs by monitoring the morphological changes of collagen fibers in tumor microenvironment.We found that collagen alterations occurred obviously in the GISTs by comparing with normal tissues,and furthermore,two morphological features from SHG images were extracted to quantitatively assess the morphological difference of collagen fibers between normal muscular layer and GISTs by means of automated image analysis.Quantitative analyses show a significant difference in the two collagen features.This study demonstrates the potential of SHG imaging as an adjunctive diagnostic tool for label-free identification of GISTs.
基金supported by National Natural Science Foundation of China(61735016)Zhejiang Provincial Natural Science Foundation of China(LR17F050001).
文摘Compared with visible light,near infrared(NIR)light has deeper penetration in biological tisues.Three-photon fuorescence microscopy(3PFM)can effectively utilize the NIR excitation to obtain high-contrast images in the deep tisue.However,the weak three photon fluorescence signals may be not well presented in the traditional fuorescence intensity imaging mode.Fluorescence lifetime of certain probes is insensitive to the intensity of the excitation laser.Moreover,fluorescence lifetimne imaging microscopy(FLIM)can detect weak signals by utilizing time correlated single photon counting(TCSPC)technique.Thus,it would be an improved strategy to combine the 3PFM imaging with the FLIM together.Herein,DCDPP-2TPA,a novel agegation-induced emission luminogen(AIEgen),was adopted as the fluorescent probes.The three-photon absorption cros-section of the AlEgen,which has a deep-red fluorescence emission,was proved to be large.DCDPP-2TPA nanoparticles were synthesized,and the three photon fluorescence lifetime of which was measured in water.Moreover,in vrivo thre-photon fuorescence lifetime microscopic imaging of a craniotomy mouse was conducted via a home made optical system.High contrast cerebrovascular images of different vertical depths were obtained and the maximun depth was about 600 pumn.Even reaching the depth of 600 pum,tiny capillary vessels as small as 1.9 pum could still be distinguished.The three photon fuorescence lifetimes of the capillaries in some representative images were in accord with that of DCDPP-2TPA nanoparticles in water.A vivid 3D reconstruction was further organized to present a wealth of lifetime information.In the future,the combination strategy of 3PFM and FLIM could be further applied in the brain functional imaging.
基金This work was financially supported by grants from the National Basic Research Program of China (973 Program)(No.2015CB352003)the National Natural Science Foundation of China (Nos.61377013,61335003,61378051,and 61427818)+1 种基金NSFC of Zhejiang province LR16F050001,Innovation Joint Research Center for iCPS (2015XZZX005-01)Open Foundation of the State Key Laboratory of Modern Optical Instrumentation.
文摘We report three-dimensional fluorescence emission difference(3D-FED)microscopy using a spatial light modulator(SLM).Zero phase,0–2vortex phase and binary 0-pi phase are loaded on the SLM to generate the corresponding solid,doughnut and z-axis hollow excitation spot,respectively.Our technique achieves super-resolved image by subtracting three di®erently acquired images with proper subtractive factors.Detailed theoretical analysis and simulation tests are proceeded to testify the performance of 3D-FED.Also,the improvement of lateral and axial resolution is demonstrated by imaging 100 nm°uorescent beads.The experiment yields lateral resolution of 140 nm and axial resolution of approximate 380 nm.
文摘In this paper, we describe an algorithm that performs automatic detection and tracking of astral microtubules in fluorescence confocal images. This sub-population of microtubules only exists during and immediately before mitosis and aids in the spindle orientation by connecting it to the cell cortex. Anomalies in their dynamic behaviour play a causal role in many diseases, such as development disorders and cancer. The main novelty of the proposed algorithm lies in the fact it provides a fully automated estimation of parameters related to microtubule dynamic instability (growth velocity, track length and track lifetime), and helps in understanding the effects of intermediate drug concentrations. Its performance has been objectively assessed using publicly available synthetic data and largely employed metrics. Moreover, we present experiments addressing cell cultures doped with different concentrations of taxol and nocodazole. Such drugs are known to suppress the microtubule dynamic instability, but their effects at intermediate concentrations are not completely assessed. The algorithm has been compared with other state-of-the-art approaches, tested on consistent real datasets. The results are encouraging in terms of performance, robustness and simplicity of use, and the algorithm is now routinely employed in our Department of Molecular Biotechnology.
基金Supported by the National Natural Science Foundation of China(21271004,51372003,21271003,51432001,21101001)the Natural Science Foundation of Anhui Province(1308085MB24)Scientific Innovation Team Foundation of Educational Commission of Anhui Province(KJ2012A025,2006KJ007TD)
文摘A novel carbazole quaternary ammonium compound(abbreviated as T_2) had been synthesized and characterized by ~1H NMR, ^(13)C NMR and Mass spectrometry. The single-crystal structure has been determined by X-ray single-crystal diffraction. The electrochemical and two-photon absorption properties of T_2 were systematically studied by cyclic voltammetry and Z-scan determination methods, respectively. The results suggested that T_2 had a good oxidation-reduction and excellent nonlinear optical property. The two-photon absorption(TPA) value has a maximum corresponding to cross section σ = 7963.3 GM(Goeppert-Mayer units) at 700 nm, indicating potential applications in nonlinear optical materials. Furthermore, attributing to the excellent water solubility and low cytotoxicity, the compound was explored on its primary application in biological imaging.
基金sponsored by the National Natural Science Foundation of China(Grant Nos.62125504,61827825,and 31901059)STI 2030—Major Projects(Grant No.2021ZD0200401)+3 种基金Major Program of the Natural Science Foundation of Zhejiang Province(Grant No.LD21F050002)Zhejiang Provincial Ten Thousand Plan for Young Top Talents(Grant No.2020R52001)Croucher Foundation(Grant No.CM/CT/CF/CIA/0688/19ay)Hong Kong Innovation and Technology Fund(ITS/178/20FP and ITS/148/20).
文摘Imaging three-dimensional,subcellular structures with high axial resolution has always been the core purpose of fluorescence microscopy.However,trade-offs exist between axial resolution and other important technical indicators,such as temporal resolution,optical power density,and imaging process complexity.We report a new imaging modality,fluorescence interference structured illumination microscopy(FI-SIM),which is based on three-dimensional structured illumination microscopy for wide-field lateral imaging and fluorescence interference for axial reconstruction.FI-SIM can acquire images quickly within the order of hundreds of milliseconds and exhibit even 30 nm axial resolution in half the wavelength depth range without z-axis scanning.Moreover,the relatively low laser power density relaxes the requirements for dyes and enables a wide range of applications for observing fixed and live subcellular structures.