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
Three-dimensional (3D) visualization of dynamic biological processes in deep tissue remains challenging due to the trade-off between temporal resolution and imaging depth. Here, we present a novel near-infrared-II (NI...Three-dimensional (3D) visualization of dynamic biological processes in deep tissue remains challenging due to the trade-off between temporal resolution and imaging depth. Here, we present a novel near-infrared-II (NIR-II, 900–1880nm) fluorescence volumetric microscopic imaging method that combines an electrically tunable lens (ETL) with deep learning approaches for rapid 3D imaging. The technology achieves volumetric imaging at 4.2 frames per second (fps) across a 200 μm depth range in live mouse brain vasculature. Two specialized neural networks are utilized: a scale-recurrent network (SRN) for image enhancement and a cerebral vessel interpolation (CVI) network that enables 16-fold axial upsampling. The SRN, trained on two-photon fluorescence microscopic data, improves both lateral and axial resolution of NIR-II fluorescence wide-field microscopic images. The CVI network, adapted from video interpolation techniques, generates intermediate frames between acquired axial planes, resulting in smooth and continuous 3D vessel reconstructions. Using this integrated system, we visualize and quantify blood flow dynamics in individual vessels and are capable of measuring blood velocity at different depths. This approach maintains high lateral resolution while achieving rapid volumetric imaging, and is particularly suitable for studying dynamic vascular processes in deep tissue. Our method demonstrates the potential of combining optical engineering with artificial intelligence to advance biological imaging capabilities.展开更多
A new scheme of super-resolution optical fluctuation imaging(SOFI)is proposed to broaden its application in the high-order cumulant reconstruction by optimizing blinking characteristics,eliminating noise in raw data a...A new scheme of super-resolution optical fluctuation imaging(SOFI)is proposed to broaden its application in the high-order cumulant reconstruction by optimizing blinking characteristics,eliminating noise in raw data and applying multi-resolution analysis in cumulant reconstruction.A motor-driven rotating mask optical modulation system is designed to adjust the excitation lightfield and allows for fast deployment.Active-modulated fluorescence fluctuation superresolution microscopy with multi-resolution analysis(AMF-MRA-SOFI)demonstrates enhanced resolution ability and reconstruction quality in experiments performed on sample of conventional dyes,achieving a resolution of 100 nm in the fourth order compared to conventional SOFI reconstruction.Furthermore,our approach combining expansion super-resolution achieved a resolution at-57 nm.展开更多
White matter,a densely packed collection of myelinated axons,plays an essential part in neural networks.With high spatial resolution and deep penetration,multi-photon microscopy(MPM)is promising for white matter imagi...White matter,a densely packed collection of myelinated axons,plays an essential part in neural networks.With high spatial resolution and deep penetration,multi-photon microscopy(MPM)is promising for white matter imaging in animal models in vivo.The third harmonic generation(THG)signal can be generated from white matter,but the bottom part of the white matter layer generates weak THG due to its high scattering.Here,we demonstrate an in vivo labeling and imaging technology,capable of visualizing the white matter layer in the mouse brain,combining°uorescence labeling with MitoTracker Red and three-photon°uorescence(3PF)microscopy excited at the 1700 nm window.3PF signals are several times higher than THG signals,resulting in deeper imaging of the white matter layer with the former.Our results indicate that 3PF microscopy is a promising technology for white matter imaging in the deep brain in vivo.展开更多
Fluorescence imaging in the second near-infrared window(NIR-II,900–1880 nm)with less scattering background in biological tissues has been combined with the confocal microscopic system for achieving deep in vivo imagi...Fluorescence imaging in the second near-infrared window(NIR-II,900–1880 nm)with less scattering background in biological tissues has been combined with the confocal microscopic system for achieving deep in vivo imaging with high spatial resolution.However,the traditional NIR-IIfluorescence confocal microscope with separate excitation focus and detection pinhole makes it possess low confocal e±ciency,as well as di±cultly to adjust.Two types of upgraded NIR-IIfluorescence confocal microscopes,sharing the same pinhole by excitation and emission focus,leading to higher confocal e±ciency,are built in this work.One type is-ber-pinhole-based confocal microscope applicable to CW laser excitation.It is constructed forfluorescence intensity imaging with large depth,high stabilization and low cost,which could replace multiphotonfluorescence microscopy in some applications(e.g.,cerebrovascular and hepatocellular imaging).The other type is air-pinhole-based confocal microscope applicable to femtosecond(fs)laser excitation.It can be employed not only for NIR-IIfluorescence intensity imaging,but also for multi-channelfluorescence lifetime imaging to recognize different structures with similarfluorescence spectrum.Moreover,it can be facilely combined with multiphotonfluorescence microscopy.A single fs pulsed laser is utilized to achieve up-conversion(visible multiphotonfluorescence)and down-conversion(NIR-II one-photonfluorescence)excitation simultaneously,extending imaging spectral channels,and thus facilitates multi-structure and multi-functional observation.展开更多
Hematologic malignancies are one of the most common malignant tumors caused by the clonal proliferation and differentiation of hematopoietic and lymphoid stem cells.The examination of bone marrow cells combined with i...Hematologic malignancies are one of the most common malignant tumors caused by the clonal proliferation and differentiation of hematopoietic and lymphoid stem cells.The examination of bone marrow cells combined with immunodeficiency typing is of great significance to the diagnostic type,treatment and prognosis of hematologic malignancies.Super-resolution fluorescence microscopy(SRM)is a special kind of optical microscopy technology,which breaks the resolution limit and was awarded the Nobel Prize in Chemistry in 2014.With the development of SRM,many related technologies have been applied to the diagnosis and treatment of clinical diseases.It was reported that a major type of SRM technique,single molecule localization microscopy(SMLM),is more sensitive than flow cytometry(FC)in detecting cell membrane antigens'expression,thus enabling better chances in detecting antigens on hematopoietic cells than traditional analytic tools.Furthermore,SRM may be applied to clinical pathology and may guide precision medicine and personalized medicine for clone hematopoietic cell diseases.In this paper,we mainly discuss the application of SRM in clone hematological malignancies.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
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
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
The interphase nuclei of parenchyma cells and epidermal cells of garlic ( Allium sativum L.) clove were labelled with rabbit anti_actin antibody and FITC_conjugated goat anti_rabbit IgG antibody. The authors observ...The interphase nuclei of parenchyma cells and epidermal cells of garlic ( Allium sativum L.) clove were labelled with rabbit anti_actin antibody and FITC_conjugated goat anti_rabbit IgG antibody. The authors observed results with fluorescence microscopy and confocal laser scanning microscopy. The nuclei showed prominent green_yellow fluorescence, indicating the presence of actin in the nuclei. Fluorescence examination with TRITC_phalloidin showed distinctive red fluorescence in the nuclei, indicating that F_actin is present in the nuclei. Confocal laser scanning microscopy indicated the presence of F_actin containing network structures in the nuclei, but the network structures were absent and the nuclei still showed red fluorescence when the cells were treated with cytochalasin D before fixation; the red fluorescence in the nuclei was hard to be observed when the cells were treated with unlabelled phalloidin before the cells were stained with TRITC_phalloidin. These results indicate that F_actin is in the nuclei and forms network structures in the nuclei of garlic cells.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
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
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
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
Time-resolved volumetric fluorescence imaging over an extended duration with high spatial/temporal resolution is a key driving force in biomedical research for investigating spatial-temporal dynamics at organism-level...Time-resolved volumetric fluorescence imaging over an extended duration with high spatial/temporal resolution is a key driving force in biomedical research for investigating spatial-temporal dynamics at organism-level systems,yet it remains a major challenge due to the trade-off among imaging speed,light exposure,illumination power,and image quality.Here,we present a deep-learning enhanced light sheet fluorescence microscopy(LSFM)approach that addresses the restoration of rapid volumetric time-lapse imaging with less than 0.03%light exposure and 3.3%acquisition time compared to a typical standard acquisition.We demonstrate that the convolutional neural network(CNN)-transformer network developed here,namely U-net integrated transformer(UI-Trans),successfully achieves the mitigation of complex noise-scattering-coupled degradation and outperforms state-of-the-art deep learning networks,due to its capability of faithfully learning fine details while comprehending complex global features.With the fast generation of appropriate training data via flexible switching between confocal line-scanning LSFM(LS-LSFM)and conventional LSFM,this method achieves a three-to five-fold signal-to-noise ratio(SNR)improvement and~1.8 times contrast improvement in ex vivo zebrafish heart imaging and long-term in vivo 4D(3D morphology+time)imaging of heartbeat dynamics at different developmental stages with ultra-economical acquisitions in terms of light dosage and acquisition time.展开更多
Background: Confirmatory diagnosis of Trypanosoma brucei gambiense human African trypanosomiasis (HAT) is based on demonstration of parasites by microscopy. However, the sensitivity of routine microscopy methods is ve...Background: Confirmatory diagnosis of Trypanosoma brucei gambiense human African trypanosomiasis (HAT) is based on demonstration of parasites by microscopy. However, the sensitivity of routine microscopy methods is very low, and many cases are missed and left untreated. A clinical study was conducted in the Democratic Republic of the Congo to evaluate the accuracy of improved microscopy methods in diagnosis of HAT. These included examination by fluorescence microscopy (FM) of acridine orange (AO) stained smears of whole blood and smears made following a new procedure for concentrating trypanosomes by selective lysis of red blood cells (RBC). Methodology/Principal Findings: Venous blood was collected from 213 HAT cases, 101 HAT suspects and 95 controls and used to determine the accuracy of four microscopy methods: bright field microscopy of Giemsa-stained thick blood smears, FM of AO-stained thick blood smears, FM of AO-stained thick blood smears prepared after RBC lysis and concentration, and FM of AO-stained thin blood smears prepared after RBC lysis and concentration. The sensitivity of FM using thick blood smears stained with AO was 3 times higher than bright field microscopy using Giemsa-stained thick blood smears [19.7% (95% CI: 14.9% - 25.6%) versus 6.1% (95% CI: 3.6% - 10.2%)]. When the RBC lysis and concentration procedure was included, sensitivity of the test was further enhanced to 23.0% (95% CI: 17.9% - 29.1%) with thick blood smears and 34.3% (95% CI: 28.2% - 40.9%) with thin blood smears. Specificity of all four microscopy methods was 100% (95% CI: 96.1% - 100.0%). However, the miniature anion exchange chromatography technique (mAECT) and capillary tube centrifugation (CTC) method remained more sensitive. Conclusions: These new methods have practical advantages, including shorter staining time, ease of demonstration of parasites, and the possibility of archiving slides. They could, therefore, be alternative methods to improve case detection where concentration procedures such as mAECT or CTC are not performed.展开更多
Diagnosis of Trypanosoma brucei rhodesiense human African trypanosomiasis requires demonstration of parasites in body fluids by microscopy. The microscopy methods that are routinely used are difficult to deploy in res...Diagnosis of Trypanosoma brucei rhodesiense human African trypanosomiasis requires demonstration of parasites in body fluids by microscopy. The microscopy methods that are routinely used are difficult to deploy in resource-limited settings due to practical challenges, including lengthy and tedious procedures, and the need for specific equipment to centrifuge samples in glass capillary tubes. We report here on a study that was conducted in a rural region of eastern Uganda to evaluate new methods that take advantage of a field-deployable LED fluorescence microscope. Examination of acridine orange-stained blood smears by LED fluorescence microscopy resulted in a diagnostic accuracy that was similar to that of routine methods, while the time needed to identify parasites was shortened significantly. These findings make these new microscopy methods attractive alternatives to procedures that are currently used for diagnosis of T. b. rhodesiense human African trypanosomiasis.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
Recently,photothermal therapy(PTT)has been proved to have great potential in tumor therapy.In the last several years,MoS_(2),as one novel member of nanomaterials,has been applied into PTT due to its excellent photothe...Recently,photothermal therapy(PTT)has been proved to have great potential in tumor therapy.In the last several years,MoS_(2),as one novel member of nanomaterials,has been applied into PTT due to its excellent photothermal conversion efficacy.In this work,we applied fuorescence lifetime imaging microscopy(FLIM)techniques into monitoring the PPT-triggered cell death under MoS_(2) nanosheet treatment.Two types of MoS_(2) nanosheets(single layer nanosheets and few layer nanosheets)were obtained,both of which exhibited presentable photothermal conversion fficacy,leading to high cell death rates of 4T1 cells(mouse breast cancer cells)under PTT.Next,live cell images of 4T1 cells were obtained via directly labeling the mitochondria with Rodamine123,which were then continuously observed with FLIM technique.FLIM data showed that the fuorescence lifetimes of mitochondria targeting dye in cells treated with each type of MoS_(2) nanosheets significantly increased during PTT treatment.By contrast,the fuorescence lifetime of the same dye in control cells(without nanomaterials)remained constant after laser irradiation.These findings suggest that FLIM can be of great value in monitoring cell death process during PTT of cancer cells,which could provide dynamic data of the cellular microenvironment at single cell level in multiple biomedical applications.展开更多
基金funded by the 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.
基金supported by the National Key R&D Program of China (No. 2024YFF1206700)the National Natural Science Foundation of China (No. U23A20487)the Hangzhou Chengxi Sci-tech Innovation Corridor Management Committee.
文摘Three-dimensional (3D) visualization of dynamic biological processes in deep tissue remains challenging due to the trade-off between temporal resolution and imaging depth. Here, we present a novel near-infrared-II (NIR-II, 900–1880nm) fluorescence volumetric microscopic imaging method that combines an electrically tunable lens (ETL) with deep learning approaches for rapid 3D imaging. The technology achieves volumetric imaging at 4.2 frames per second (fps) across a 200 μm depth range in live mouse brain vasculature. Two specialized neural networks are utilized: a scale-recurrent network (SRN) for image enhancement and a cerebral vessel interpolation (CVI) network that enables 16-fold axial upsampling. The SRN, trained on two-photon fluorescence microscopic data, improves both lateral and axial resolution of NIR-II fluorescence wide-field microscopic images. The CVI network, adapted from video interpolation techniques, generates intermediate frames between acquired axial planes, resulting in smooth and continuous 3D vessel reconstructions. Using this integrated system, we visualize and quantify blood flow dynamics in individual vessels and are capable of measuring blood velocity at different depths. This approach maintains high lateral resolution while achieving rapid volumetric imaging, and is particularly suitable for studying dynamic vascular processes in deep tissue. Our method demonstrates the potential of combining optical engineering with artificial intelligence to advance biological imaging capabilities.
基金supported by the National Natural Science Foundation of China(62175034,62175036,32271510)the National Key R&D Program of China(2021YFF0502900)+2 种基金the Science and Technology Research Program of Shanghai(Grant No.19DZ2282100)the Shanghai Key Laboratory of Metasurfaces for Light Manipulation(23dz2260100)the Shanghai Engineering Technology Research Center of Hair Medicine(19DZ2250500).
文摘A new scheme of super-resolution optical fluctuation imaging(SOFI)is proposed to broaden its application in the high-order cumulant reconstruction by optimizing blinking characteristics,eliminating noise in raw data and applying multi-resolution analysis in cumulant reconstruction.A motor-driven rotating mask optical modulation system is designed to adjust the excitation lightfield and allows for fast deployment.Active-modulated fluorescence fluctuation superresolution microscopy with multi-resolution analysis(AMF-MRA-SOFI)demonstrates enhanced resolution ability and reconstruction quality in experiments performed on sample of conventional dyes,achieving a resolution of 100 nm in the fourth order compared to conventional SOFI reconstruction.Furthermore,our approach combining expansion super-resolution achieved a resolution at-57 nm.
基金funded by the National Natural Science Foundation of China(62075135,61975126)Shenzhen Key Laboratory of Photonics and Biophotonics(ZDSYS20210623092006020).
文摘White matter,a densely packed collection of myelinated axons,plays an essential part in neural networks.With high spatial resolution and deep penetration,multi-photon microscopy(MPM)is promising for white matter imaging in animal models in vivo.The third harmonic generation(THG)signal can be generated from white matter,but the bottom part of the white matter layer generates weak THG due to its high scattering.Here,we demonstrate an in vivo labeling and imaging technology,capable of visualizing the white matter layer in the mouse brain,combining°uorescence labeling with MitoTracker Red and three-photon°uorescence(3PF)microscopy excited at the 1700 nm window.3PF signals are several times higher than THG signals,resulting in deeper imaging of the white matter layer with the former.Our results indicate that 3PF microscopy is a promising technology for white matter imaging in the deep brain in vivo.
基金supported by National Natural Science Foundation of China(61975172,82001874 and 61735016).
文摘Fluorescence imaging in the second near-infrared window(NIR-II,900–1880 nm)with less scattering background in biological tissues has been combined with the confocal microscopic system for achieving deep in vivo imaging with high spatial resolution.However,the traditional NIR-IIfluorescence confocal microscope with separate excitation focus and detection pinhole makes it possess low confocal e±ciency,as well as di±cultly to adjust.Two types of upgraded NIR-IIfluorescence confocal microscopes,sharing the same pinhole by excitation and emission focus,leading to higher confocal e±ciency,are built in this work.One type is-ber-pinhole-based confocal microscope applicable to CW laser excitation.It is constructed forfluorescence intensity imaging with large depth,high stabilization and low cost,which could replace multiphotonfluorescence microscopy in some applications(e.g.,cerebrovascular and hepatocellular imaging).The other type is air-pinhole-based confocal microscope applicable to femtosecond(fs)laser excitation.It can be employed not only for NIR-IIfluorescence intensity imaging,but also for multi-channelfluorescence lifetime imaging to recognize different structures with similarfluorescence spectrum.Moreover,it can be facilely combined with multiphotonfluorescence microscopy.A single fs pulsed laser is utilized to achieve up-conversion(visible multiphotonfluorescence)and down-conversion(NIR-II one-photonfluorescence)excitation simultaneously,extending imaging spectral channels,and thus facilitates multi-structure and multi-functional observation.
基金This work was supported by the Innovation Fund of WNLO(2018WNLOKF023)the Start-up Fund of Hainan University(KYQD(ZR)-20077).
文摘Hematologic malignancies are one of the most common malignant tumors caused by the clonal proliferation and differentiation of hematopoietic and lymphoid stem cells.The examination of bone marrow cells combined with immunodeficiency typing is of great significance to the diagnostic type,treatment and prognosis of hematologic malignancies.Super-resolution fluorescence microscopy(SRM)is a special kind of optical microscopy technology,which breaks the resolution limit and was awarded the Nobel Prize in Chemistry in 2014.With the development of SRM,many related technologies have been applied to the diagnosis and treatment of clinical diseases.It was reported that a major type of SRM technique,single molecule localization microscopy(SMLM),is more sensitive than flow cytometry(FC)in detecting cell membrane antigens'expression,thus enabling better chances in detecting antigens on hematopoietic cells than traditional analytic tools.Furthermore,SRM may be applied to clinical pathology and may guide precision medicine and personalized medicine for clone hematopoietic cell diseases.In this paper,we mainly discuss the application of SRM in clone hematological malignancies.
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.
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.
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.
文摘The interphase nuclei of parenchyma cells and epidermal cells of garlic ( Allium sativum L.) clove were labelled with rabbit anti_actin antibody and FITC_conjugated goat anti_rabbit IgG antibody. The authors observed results with fluorescence microscopy and confocal laser scanning microscopy. The nuclei showed prominent green_yellow fluorescence, indicating the presence of actin in the nuclei. Fluorescence examination with TRITC_phalloidin showed distinctive red fluorescence in the nuclei, indicating that F_actin is present in the nuclei. Confocal laser scanning microscopy indicated the presence of F_actin containing network structures in the nuclei, but the network structures were absent and the nuclei still showed red fluorescence when the cells were treated with cytochalasin D before fixation; the red fluorescence in the nuclei was hard to be observed when the cells were treated with unlabelled phalloidin before the cells were stained with TRITC_phalloidin. These results indicate that F_actin is in the nuclei and forms network structures in the nuclei of garlic cells.
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.
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 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 National Natural Science Foundation of China(52122008,52270008,52370003,62025502)Guangdong Introducing Innovative and Entrepreneurial Teams of“The Pearl River Talent Recruitment Program”(2021ZT09X044)Shenzhen Technology University under Grant JSZZ202301010.
文摘Time-resolved volumetric fluorescence imaging over an extended duration with high spatial/temporal resolution is a key driving force in biomedical research for investigating spatial-temporal dynamics at organism-level systems,yet it remains a major challenge due to the trade-off among imaging speed,light exposure,illumination power,and image quality.Here,we present a deep-learning enhanced light sheet fluorescence microscopy(LSFM)approach that addresses the restoration of rapid volumetric time-lapse imaging with less than 0.03%light exposure and 3.3%acquisition time compared to a typical standard acquisition.We demonstrate that the convolutional neural network(CNN)-transformer network developed here,namely U-net integrated transformer(UI-Trans),successfully achieves the mitigation of complex noise-scattering-coupled degradation and outperforms state-of-the-art deep learning networks,due to its capability of faithfully learning fine details while comprehending complex global features.With the fast generation of appropriate training data via flexible switching between confocal line-scanning LSFM(LS-LSFM)and conventional LSFM,this method achieves a three-to five-fold signal-to-noise ratio(SNR)improvement and~1.8 times contrast improvement in ex vivo zebrafish heart imaging and long-term in vivo 4D(3D morphology+time)imaging of heartbeat dynamics at different developmental stages with ultra-economical acquisitions in terms of light dosage and acquisition time.
文摘Background: Confirmatory diagnosis of Trypanosoma brucei gambiense human African trypanosomiasis (HAT) is based on demonstration of parasites by microscopy. However, the sensitivity of routine microscopy methods is very low, and many cases are missed and left untreated. A clinical study was conducted in the Democratic Republic of the Congo to evaluate the accuracy of improved microscopy methods in diagnosis of HAT. These included examination by fluorescence microscopy (FM) of acridine orange (AO) stained smears of whole blood and smears made following a new procedure for concentrating trypanosomes by selective lysis of red blood cells (RBC). Methodology/Principal Findings: Venous blood was collected from 213 HAT cases, 101 HAT suspects and 95 controls and used to determine the accuracy of four microscopy methods: bright field microscopy of Giemsa-stained thick blood smears, FM of AO-stained thick blood smears, FM of AO-stained thick blood smears prepared after RBC lysis and concentration, and FM of AO-stained thin blood smears prepared after RBC lysis and concentration. The sensitivity of FM using thick blood smears stained with AO was 3 times higher than bright field microscopy using Giemsa-stained thick blood smears [19.7% (95% CI: 14.9% - 25.6%) versus 6.1% (95% CI: 3.6% - 10.2%)]. When the RBC lysis and concentration procedure was included, sensitivity of the test was further enhanced to 23.0% (95% CI: 17.9% - 29.1%) with thick blood smears and 34.3% (95% CI: 28.2% - 40.9%) with thin blood smears. Specificity of all four microscopy methods was 100% (95% CI: 96.1% - 100.0%). However, the miniature anion exchange chromatography technique (mAECT) and capillary tube centrifugation (CTC) method remained more sensitive. Conclusions: These new methods have practical advantages, including shorter staining time, ease of demonstration of parasites, and the possibility of archiving slides. They could, therefore, be alternative methods to improve case detection where concentration procedures such as mAECT or CTC are not performed.
文摘Diagnosis of Trypanosoma brucei rhodesiense human African trypanosomiasis requires demonstration of parasites in body fluids by microscopy. The microscopy methods that are routinely used are difficult to deploy in resource-limited settings due to practical challenges, including lengthy and tedious procedures, and the need for specific equipment to centrifuge samples in glass capillary tubes. We report here on a study that was conducted in a rural region of eastern Uganda to evaluate new methods that take advantage of a field-deployable LED fluorescence microscope. Examination of acridine orange-stained blood smears by LED fluorescence microscopy resulted in a diagnostic accuracy that was similar to that of routine methods, while the time needed to identify parasites was shortened significantly. These findings make these new microscopy methods attractive alternatives to procedures that are currently used for diagnosis of T. b. rhodesiense human African trypanosomiasis.
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 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.
