The fluorescence from the out-of-focus region excited by the sidelobes of a Bessel beam is the major concern for light-sheet fluorescence microscopy （LSFM） with Bessel beam plane illumination. Here, we propose a met...The fluorescence from the out-of-focus region excited by the sidelobes of a Bessel beam is the major concern for light-sheet fluorescence microscopy （LSFM） with Bessel beam plane illumination. Here, we propose a method of applying the subtractive imaging to overcome the limitation of the conventional LSFM with Bessel beam plane illumination. In the proposed method, the sample is imaged twice by line scanning using the extended solid Bessel beam and the ring-like Bessel beam. By subtracting between the two images with similar out-of-focus blur, the improved image quality with the suppression of the Bessel beam sidelobes and enhanced sectioning ability with improved contrast are demonstrated.展开更多
Traumatic penumbra(TP)is a region with recoverable potential around the primary lesion of brain injury.Rapid and accurate imaging for identifying TP is essential for treating traumatic brain injury(TBI).In this study,...Traumatic penumbra(TP)is a region with recoverable potential around the primary lesion of brain injury.Rapid and accurate imaging for identifying TP is essential for treating traumatic brain injury(TBI).In this study,we first established traumatic brain injuries(TBIs)in rats using a modified Feeney method,followed by label-free imaging of brain tissue sections with multiphoton fluorescence microscopy.The results showed that the technique effectively imaged normal and traumatic brain tissues,and revealed pathological features such as extracellular matrix changes,vascular cell proliferation,and intracellular edema in the traumatic penumbra.Compared with normal brain tissue,the extracellular matrix in the TP was sparse,cells were disorganized,and hyperplastic vascular cells emitted higher two-photon excited fluorescence(TPEF)signals.Our research demonstrates the potential of multiphoton fluorescence technology in the rapid diagnosis and therapeutic evaluation of TBI.展开更多
Fluorescence microscopy is indispensable in life science research,yet denoising remains challenging due to varied biological samples and imaging conditions.We introduce a wavelet-enhanced transformer based on DnCNN th...Fluorescence microscopy is indispensable in life science research,yet denoising remains challenging due to varied biological samples and imaging conditions.We introduce a wavelet-enhanced transformer based on DnCNN that fuses wavelet preprocessing with a dual-branch transformer-convolutional neural network(CNN)architecture.Wavelet decomposition separates highand low-frequency components for targeted noise reduction;the CNN branch restores local details,whereas the transformer branch captures global context;and an adaptive loss balances quantitative fidelity with perceptual quality.On the fluorescence microscopy denoising benchmark,our method surpasses leading CNNand transformer-based approaches,improving peak signal-to-noise ratio by 2.34%and 0.88%and structural similarity index measure by 0.53%and 1.07%,respectively.This framework offers enhanced generalization and practical gains for fluorescence image denoising.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
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
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
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
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
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
Single-particle microbeam as a powerful tool can open a research field to find answers to many enigmas in radiobiology. A single-particle microbeam facility has been constructed at the Key Laboratory of Ion Beam Bioen...Single-particle microbeam as a powerful tool can open a research field to find answers to many enigmas in radiobiology. A single-particle microbeam facility has been constructed at the Key Laboratory of Ion Beam Bioengineering （LIBB）, Chinese Academy of Sciences （CAS）, China. However there has been less research activities in this field concerning the original process of the interaction between low-energy ions and complicated organisms. To address this challenge, an in situ multi-dimensional quantitative fluorescence microscopy system combined with the CAS-LIBB single-particle microbeam II endstation is proposed. In this article, the rationale, logistics and development of many aspects of the proposed system are discussed.展开更多
We have developed and tested transparent microelectrode arrays capable of simultaneous amperometric measurement of oxidizable molecules and fluorescence imaging through the electrodes. Surface patterned microelectrode...We have developed and tested transparent microelectrode arrays capable of simultaneous amperometric measurement of oxidizable molecules and fluorescence imaging through the electrodes. Surface patterned microelectrodes were fabricated from three different conducting materials: Indium-tin-oxide (ITO), nitrogen-doped diamond-like carbon (DLC) deposited on top of ITO, or very thin (12 - 17 nm) gold films on glass substrates. Chromaffin cells loaded with lysotracker green or acridine orange dye were placed atop the electrodes and vesicle fluorescence imaged with total internal reflection fluorescence (TIRF) microscopy while catecholamine release from single vesicles was measured as amperometric spikes with the surface patterned electrodes. Electrodes fabricated from all three materials were capable of detecting amperometric signals with high resolution. Unexpectedly, amperometric spikes recorded with ITO electrodes had only about half the amplitude and about half as much charge as those detected with DLC or gold electrodes, indicating that the ITO electrodes are not as sensitive as gold or DLC electrodes for measurement of quantal catecholamine release. The lower sensitivity of ITO electrodes was confirmed by chronoamperometry measurements comparing the currents in the presence of different analytes with the different electrode materials.展开更多
In this paper,we propose a new fluorescence emission difference microscopy(FED)technique based on polarization modulation.An electro-optical modulator(EOM)is used to switch the excitation beam between the horizontal a...In this paper,we propose a new fluorescence emission difference microscopy(FED)technique based on polarization modulation.An electro-optical modulator(EOM)is used to switch the excitation beam between the horizontal and vertical polarization states at a high frequency,which leads to solid-and donut-shaped beams after spatial light modulation.Experiment on the fluorescent nanoparticles demonstrates that the proposed method can achieve~λ=4 spatial resolution.Using the proposed system,the dynamic imaging of subcellular structures in living cells over time is achieved.展开更多
By laser scanning fluorescence microscopy for quan-titative measurement of fluorescence intensity changes on egg surface stained with fluorescein isothiocyanate duxing cleavage furrow extending forward, it was found t...By laser scanning fluorescence microscopy for quan-titative measurement of fluorescence intensity changes on egg surface stained with fluorescein isothiocyanate duxing cleavage furrow extending forward, it was found that in area of presumptive cleavage furrow the scanning curve became ∨ shape, indicating dark stripe appeared in that place. Then the fluorescence intensity increased at the place where the botton of ∨ shape had located, and the scanning curve tuxned to ∧ shape, indicating single stripe was formed. While enhanced fluorescence appeared on the borders of ∧ shape, an M shape curve was found, show-ing double stripe occurred. During the distance between two borders of M shape incresing from 50 μm to 100μm,a fluorescence peak came to sight in the middle of the M shape, which being the cleavge furrow bottom. The two lateral sides of furrow bottom with decreasing fluorescence were nascent membrane. At that time the curve became W shape. By the sides of cleavage furrow the the stress folds became conspicous after double stripe stage, showing the stretching of the egg surface being increased. With our[31, 33] and others[32] reports that polylysine could induce the appearance of nascent membrane and phyto-hemagglutinins could decrease or prevent the appearance of nascent membrane, we believed the idea of Schroeder[25] that increasing mechanical stress could initiate nascent membrane formation and thought that the stress lay to the outsides of cleavage furrow.展开更多
基金supported by the National Natural Science Foundation of China(Nos.61665006,61661028,61565012,and 61378062)the Natural Science Foundation of Jiangxi Province(Nos.20161BAB212041,20162BCB23012,and 20171ACB21018)
文摘The fluorescence from the out-of-focus region excited by the sidelobes of a Bessel beam is the major concern for light-sheet fluorescence microscopy （LSFM） with Bessel beam plane illumination. Here, we propose a method of applying the subtractive imaging to overcome the limitation of the conventional LSFM with Bessel beam plane illumination. In the proposed method, the sample is imaged twice by line scanning using the extended solid Bessel beam and the ring-like Bessel beam. By subtracting between the two images with similar out-of-focus blur, the improved image quality with the suppression of the Bessel beam sidelobes and enhanced sectioning ability with improved contrast are demonstrated.
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 Natural Science Foundation of China(Grant No.62275210)the National Leading Talent Program,the National Young Talent Program,the Key Research and Development Program of Shaanxi(Grant No.2024SF2-GJHX-25)+5 种基金the Scientific Research Program Funded by the Education Department of Shaanxi Provincial Government(Grant No.24JS016)the Xidian University Specially Funded Project for Interdisciplinary Exploration(Grant No.TZJHF202523)the Fundamental Research Funds for Central Universities(Grant No.YJSJ25014)the Guangdong Provincial General Colleges and Universities Young Innovative Talents Research Project(Grant No.2024KQNCX172)the Shenzhen Science and Technology Program(Grant No.GJHZ20210705141805015)the Key Research Areas Support Science and Technology Project of Shenzhen Institute of Information Technology(Grant No.SZIIT2024KJ056).
文摘Fluorescence microscopy is indispensable in life science research,yet denoising remains challenging due to varied biological samples and imaging conditions.We introduce a wavelet-enhanced transformer based on DnCNN that fuses wavelet preprocessing with a dual-branch transformer-convolutional neural network(CNN)architecture.Wavelet decomposition separates highand low-frequency components for targeted noise reduction;the CNN branch restores local details,whereas the transformer branch captures global context;and an adaptive loss balances quantitative fidelity with perceptual quality.On the fluorescence microscopy denoising benchmark,our method surpasses leading CNNand transformer-based approaches,improving peak signal-to-noise ratio by 2.34%and 0.88%and structural similarity index measure by 0.53%and 1.07%,respectively.This framework offers enhanced generalization and practical gains for fluorescence image denoising.
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.
文摘INSULIN secretion was traditionally measured with biochemical and immunological methods such as enzyme linked immunosorbant assay and radioimmunoassay. However, these methods can only tell the amount of insulin secreted; they give no information about the secretion process or mechanism of exocytosis. In recent years, an imaging technique known as total internal reflection fluorescence （TIRF） microscopy has been employed to study insulin secretion.
基金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.
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.
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.
文摘Single-particle microbeam as a powerful tool can open a research field to find answers to many enigmas in radiobiology. A single-particle microbeam facility has been constructed at the Key Laboratory of Ion Beam Bioengineering （LIBB）, Chinese Academy of Sciences （CAS）, China. However there has been less research activities in this field concerning the original process of the interaction between low-energy ions and complicated organisms. To address this challenge, an in situ multi-dimensional quantitative fluorescence microscopy system combined with the CAS-LIBB single-particle microbeam II endstation is proposed. In this article, the rationale, logistics and development of many aspects of the proposed system are discussed.
文摘We have developed and tested transparent microelectrode arrays capable of simultaneous amperometric measurement of oxidizable molecules and fluorescence imaging through the electrodes. Surface patterned microelectrodes were fabricated from three different conducting materials: Indium-tin-oxide (ITO), nitrogen-doped diamond-like carbon (DLC) deposited on top of ITO, or very thin (12 - 17 nm) gold films on glass substrates. Chromaffin cells loaded with lysotracker green or acridine orange dye were placed atop the electrodes and vesicle fluorescence imaged with total internal reflection fluorescence (TIRF) microscopy while catecholamine release from single vesicles was measured as amperometric spikes with the surface patterned electrodes. Electrodes fabricated from all three materials were capable of detecting amperometric signals with high resolution. Unexpectedly, amperometric spikes recorded with ITO electrodes had only about half the amplitude and about half as much charge as those detected with DLC or gold electrodes, indicating that the ITO electrodes are not as sensitive as gold or DLC electrodes for measurement of quantal catecholamine release. The lower sensitivity of ITO electrodes was confirmed by chronoamperometry measurements comparing the currents in the presence of different analytes with the different electrode materials.
基金supported in part by the National Natural Science Foundation of China(61827825,62125504,and 61735017)Major Program of the Natural Science Foundation of Zhejiang Province(LD21F050002)+2 种基金Key Research and Development Program of Zhejiang Province(2020C01116)Zhejiang Lab(2020MC0AE01)China Postdoctoral Science Foundation(BX2021272).
文摘In this paper,we propose a new fluorescence emission difference microscopy(FED)technique based on polarization modulation.An electro-optical modulator(EOM)is used to switch the excitation beam between the horizontal and vertical polarization states at a high frequency,which leads to solid-and donut-shaped beams after spatial light modulation.Experiment on the fluorescent nanoparticles demonstrates that the proposed method can achieve~λ=4 spatial resolution.Using the proposed system,the dynamic imaging of subcellular structures in living cells over time is achieved.
文摘By laser scanning fluorescence microscopy for quan-titative measurement of fluorescence intensity changes on egg surface stained with fluorescein isothiocyanate duxing cleavage furrow extending forward, it was found that in area of presumptive cleavage furrow the scanning curve became ∨ shape, indicating dark stripe appeared in that place. Then the fluorescence intensity increased at the place where the botton of ∨ shape had located, and the scanning curve tuxned to ∧ shape, indicating single stripe was formed. While enhanced fluorescence appeared on the borders of ∧ shape, an M shape curve was found, show-ing double stripe occurred. During the distance between two borders of M shape incresing from 50 μm to 100μm,a fluorescence peak came to sight in the middle of the M shape, which being the cleavge furrow bottom. The two lateral sides of furrow bottom with decreasing fluorescence were nascent membrane. At that time the curve became W shape. By the sides of cleavage furrow the the stress folds became conspicous after double stripe stage, showing the stretching of the egg surface being increased. With our[31, 33] and others[32] reports that polylysine could induce the appearance of nascent membrane and phyto-hemagglutinins could decrease or prevent the appearance of nascent membrane, we believed the idea of Schroeder[25] that increasing mechanical stress could initiate nascent membrane formation and thought that the stress lay to the outsides of cleavage furrow.
