The subcellular localization of human proteins is vital for understanding the structure of human cells.Proteins play a significant role within human cells,as many different groups of proteins are located in a specific...The subcellular localization of human proteins is vital for understanding the structure of human cells.Proteins play a significant role within human cells,as many different groups of proteins are located in a specific location to perform a particular function.Understanding these functions will help in discoveringmany diseases and developing their treatments.The importance of imaging analysis techniques,specifically in proteomics research,is becoming more prevalent.Despite recent advances in deep learning techniques for analyzing microscopy images,classification models have faced critical challenges in achieving high performance.Most protein subcellular images have a significant class imbalance.We use oversampling and under sampling techniques in this research to overcome this issue.We have used a Convolutional Neural Network(CNN)model called GapNet-PL for the multi-label classification task on the Human Protein Atlas Classification(HPA)Dataset.Authors have found that the ParametricRectified LinearUnit(PreLU)activation function is better than the Scaled Exponential LinearUnit(SeLU)activation function in the GapNet-PL model in most classification metrics.The results showed that the GapNet-PL model with the PReLU activation function achieved an area under the ROC curve(AUC)equal to 0.896,an F1 score of 0.541,and a recall of 0.473.展开更多
A novel mathematical morphological approach for region of interest(ROI) automatic determination and JPEG2000-based coding of microscopy image compression is presented.The algorithm is very fast and requires lower comp...A novel mathematical morphological approach for region of interest(ROI) automatic determination and JPEG2000-based coding of microscopy image compression is presented.The algorithm is very fast and requires lower computing power,which is particularly suitable for some irregular region-based cell microscopy images with poor qualities.Firstly,an active threshold-based method is discussed to create a rough mask of regions of interest(cells).And then some morphological operations are designed and applied to achieve the segmentation of cells.In addition,an extra morphological operation,dilation,is applied to create the final mask with some redundancies to avoid the"edge effect"after removing false cells.Finally,ROI and region of background(ROB) are obtained and encoded individually in different compression ratio flexibly based on the JPEG2000,which can adjust the quality between ROI and ROB without coding for ROI shape.The experimental results certify the effectiveness of the proposed algorithm,and compared with JPEG2000,the proposed algorithm has better performance in both subjective quality and objective quality at the same compression ratios.展开更多
The high-content image-based assay is commonly leveraged for identifying the phenotypic impact of genetic perturbations in biology field.However,a persistent issue remains unsolved during experiments:the interferentia...The high-content image-based assay is commonly leveraged for identifying the phenotypic impact of genetic perturbations in biology field.However,a persistent issue remains unsolved during experiments:the interferential technical noises caused by systematic errors(e.g.,temperature,reagent concentration,and well location)are always mixed up with the real biological signals,leading to misinterpretation of any conclusion drawn.Here,we reported a mean teacher-based deep learning model(Deep Noise)that can disentangle biological signals from the experimental noises.Specifically,we aimed to classify the phenotypic impact of 1108 different genetic perturbations screened from 125,510 fluorescent microscopy images,which were totally unrecognizable by the human eye.We validated our model by participating in the Recursion Cellular Image Classification Challenge,and Deep Noise achieved an extremely high classification score(accuracy:99.596%),ranking the 2nd place among 866 participating groups.This promising result indicates the successful separation of biological and technical factors,which might help decrease the cost of treatment development and expedite the drug discovery process.The source code of Deep Noise is available at https://github.com/Scu-sen/Recursion-Cellular-Image-Classification-Challenge.展开更多
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
By using a vectorial approach, the validity of paraxial approximation in second harmonic generation （SHG） microscopy under low numerical aperture （NA） is examined when the sample is a collagen fibril. Due to the l...By using a vectorial approach, the validity of paraxial approximation in second harmonic generation （SHG） microscopy under low numerical aperture （NA） is examined when the sample is a collagen fibril. Due to the larger value of dzzz and tensorial nature of SHG, the component Ez of the focused fieM may have strong effect on the radiation pattern of SHG. Numerical results indicate that when the value of NA exceeds 0.3, the effect of Ez can not be neglected, which results in the invalidation of paraxial approximation in SHG microscopy despite the fact that SHG microscopy is still under low NA focusing.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
Fluorescence microscopy, as a sensitive method to detect microenvironment of molecules, is widely used in protein conformation and dynamic studies in live cells. Fluorescence lifetime imaging microscopy（FLIM）, which...Fluorescence microscopy, as a sensitive method to detect microenvironment of molecules, is widely used in protein conformation and dynamic studies in live cells. Fluorescence lifetime imaging microscopy（FLIM）, which is independent of fluorophore concentrations, scattering and bleaching, is a suitable tool to analyze membrane proteins in a single cell. Ferroportin（FPN）, a multi-ion exporter in vertebrates, was modulated by metal ions with unknown mechanism. Herein, we fused green fluorescence protein on Cterminal of FPN（FPN-eGFP） and applied fluorescence lifetime to monitor conformation changes of FPN in a live cell. The fluorescence lifetime distribution showed a shift to shorter lifetime upon Mn^（2＋） treatment,suggesting a preference conformation of FPN in Mn^（2＋） exposure. It is also observed that the lifetime（rather than intensity） measurement was not strongly influenced by laser power. The observed fluorescence lifetime changes of FPN-eGFP upon Mn^（2＋） treatments indicated that extracellular metal ions can modulate FPN through conformation exchanges between several different states.展开更多
Recently,photothermal therapy(PTT)has been proved to have great potential in tumor therapy.In the last several years,MoS_(2),as one novel member of nanomaterials,has been applied into PTT due to its excellent photothe...Recently,photothermal therapy(PTT)has been proved to have great potential in tumor therapy.In the last several years,MoS_(2),as one novel member of nanomaterials,has been applied into PTT due to its excellent photothermal conversion efficacy.In this work,we applied fuorescence lifetime imaging microscopy(FLIM)techniques into monitoring the PPT-triggered cell death under MoS_(2) nanosheet treatment.Two types of MoS_(2) nanosheets(single layer nanosheets and few layer nanosheets)were obtained,both of which exhibited presentable photothermal conversion fficacy,leading to high cell death rates of 4T1 cells(mouse breast cancer cells)under PTT.Next,live cell images of 4T1 cells were obtained via directly labeling the mitochondria with Rodamine123,which were then continuously observed with FLIM technique.FLIM data showed that the fuorescence lifetimes of mitochondria targeting dye in cells treated with each type of MoS_(2) nanosheets significantly increased during PTT treatment.By contrast,the fuorescence lifetime of the same dye in control cells(without nanomaterials)remained constant after laser irradiation.These findings suggest that FLIM can be of great value in monitoring cell death process during PTT of cancer cells,which could provide dynamic data of the cellular microenvironment at single cell level in multiple biomedical applications.展开更多
Near infrared microscopy imaging fers the opportunity to explore not only what lkinds ofchemical species are present at micro-scale level but also where the chemical species would bepr esent.By revealing the spectral ...Near infrared microscopy imaging fers the opportunity to explore not only what lkinds ofchemical species are present at micro-scale level but also where the chemical species would bepr esent.By revealing the spectral and spatial information,the technique can identify and localizeany interested component.This study investigates the feasibility of using Near infrared mi.croscopy imaging to detect melamine in soybean meal.The results showed that 6805 cm^(-1) is verysensitive for melamine but not for soybean meal,so can be used for univariate analysis,Singlewavelength image and peak integr ation image at 6805 cm^(-1) are simple and efective met hods todetect the melamine in soybean meal.Furthermore,Principal Component Analysis is applied todetect the melamine in soybean meal.展开更多
In light-sheet fluorescence microscopy,the axial resolution and field of view are mutually constrained.Axially swept light-sheet microscopy(ASLM)can decouple the trade-off,but the confocal detection scheme using a rol...In light-sheet fluorescence microscopy,the axial resolution and field of view are mutually constrained.Axially swept light-sheet microscopy(ASLM)can decouple the trade-off,but the confocal detection scheme using a rolling shutter also rejects fluorescence signals from the specimen in the field of interest,which sacrifices the photon efficiency.Here,we report a laterally swept light-sheet microscopy(LSLM)scheme in which the focused beam is first scanned along the axial direction and subsequently laterally swept with the rolling shutter.We show that LSLM can obtain a higher photon efficiency when similar axial resolution and field of view can be achieved.Moreover,based on the principle of image scanning microscopy,applying the pixel reassignment to the LSLM images,hereby named iLSLM,improves the optical sectioning.Both simulation and experimental results demonstrate the higher photon efficiency with similar axial resolution and optical sectioning.Our proposed scheme is suitable for volumetric imaging of specimens that are susceptible to photobleaching or phototoxicity.展开更多
Skin scar is unique to humans,the major significant negative outcome sustained after thermal injuries,traumatic injuries,and surgical procedures.Hypertrophic scar in human skin is investigated using non-linear spectra...Skin scar is unique to humans,the major significant negative outcome sustained after thermal injuries,traumatic injuries,and surgical procedures.Hypertrophic scar in human skin is investigated using non-linear spectral imaging microscopy.The high contrast images and spectroscopic intensities of collagen and elastic fibers extracted from the spectral imaging of normal skin tissue,and the normal skin near and far away from the hypertrophic scar tissues in a 10-year-old patient case are obtained.The results show that there are apparent differences in the morphological structure and spectral characteristics of collagen and elastic fibers when comparing the normal skin with the hypertrophic scar tissue.These differences can be good indicators to differentiate the normal skin and hypertrophic scar tissue and demonstrate that non-linear spectral imaging microscopy has potential to noninvasively investigate the pathophysiology of human hypertrophic scar.展开更多
Image scanning microscopy(ISM)is a promising imaging technique that offers sub-diffraction-limited resolution and optical sectioning.Theoretically,ISM can improve the optical resolution by a factor of two through pixe...Image scanning microscopy(ISM)is a promising imaging technique that offers sub-diffraction-limited resolution and optical sectioning.Theoretically,ISM can improve the optical resolution by a factor of two through pixel reassignment and deconvolution.Multifocal array illumination and scanning have been widely adopted to implement ISM because of their simplicity.Conventionally,digital micromirror devices(DMDs)1 and microlens arrays(MLAs)2,3 have been used to generate dense and uniform multifocal arrays for ISM,which are critical for achieving fast imaging and high-quality ISM reconstruction.However,these approaches have limitations in terms of cost,numerical aperture(NA),pitch,and uniformity,making it challenging to create dense and high-quality multifocal arrays at high NA.To overcome these limitations,we introduced a novel multifocal metalens design strategy called the hybrid multiplexing method,which combines two conventional multiplexing approaches:phase addition and random multiplexing.Through numerical simulations,we demonstrate that the proposed method generates more uniform and denser multifocal arrays than conventional methods,even at small pitches.As a proof of concept,we fabricated a multifocal metalens generating 40×40 array of foci with a 3μm pitch and NA of 0.7 operating at a wavelength of 488 nm and then constructed the multifocal metalens-based ISM(MMISM).We demonstrated that MMISM successfully resolved sub-diffraction-limited features in imaging of microbead samples and forebrain organoid sections.The results showed that MMISM imaging achieved twice the diffraction-limited resolution and revealed clearer structural features of neurons compared to wide-field images.We anticipate that our novel design strategy can be widely applied to produce multifunctional optical elements and replace conventional optical elements in specialized applications.展开更多
Theα＋βtitanium alloy, Ti-6Al-4V, was welded by friction stir welding using a W-Re pin tool, and the defect-free weld was produced with proper welding parameters. Texture of the Ti-6Al-4V friction stir weld was stud...Theα＋βtitanium alloy, Ti-6Al-4V, was welded by friction stir welding using a W-Re pin tool, and the defect-free weld was produced with proper welding parameters. Texture of the Ti-6Al-4V friction stir weld was studied by orientation imaging microscopy. The as-received Ti-6Al-4V sheet mill annealed was composed of elongated primary α and transformed β. A typical rolling texture was observed in the base material. The microstructure of the stir zone was significantly different from that of the base material. The stir zone was characterized by the presence of considerable amount of equiaxed dynamically recrystallized grains and a texture around{Ф1=30°,φ=62°,Ф2=30°}was developed during the friction stir welding.展开更多
Compared with visible light,near infrared(NIR)light has deeper penetration in biological tisues.Three-photon fuorescence microscopy(3PFM)can effectively utilize the NIR excitation to obtain high-contrast images in the...Compared with visible light,near infrared(NIR)light has deeper penetration in biological tisues.Three-photon fuorescence microscopy(3PFM)can effectively utilize the NIR excitation to obtain high-contrast images in the deep tisue.However,the weak three photon fluorescence signals may be not well presented in the traditional fuorescence intensity imaging mode.Fluorescence lifetime of certain probes is insensitive to the intensity of the excitation laser.Moreover,fluorescence lifetimne imaging microscopy(FLIM)can detect weak signals by utilizing time correlated single photon counting(TCSPC)technique.Thus,it would be an improved strategy to combine the 3PFM imaging with the FLIM together.Herein,DCDPP-2TPA,a novel agegation-induced emission luminogen(AIEgen),was adopted as the fluorescent probes.The three-photon absorption cros-section of the AlEgen,which has a deep-red fluorescence emission,was proved to be large.DCDPP-2TPA nanoparticles were synthesized,and the three photon fluorescence lifetime of which was measured in water.Moreover,in vrivo thre-photon fuorescence lifetime microscopic imaging of a craniotomy mouse was conducted via a home made optical system.High contrast cerebrovascular images of different vertical depths were obtained and the maximun depth was about 600 pumn.Even reaching the depth of 600 pum,tiny capillary vessels as small as 1.9 pum could still be distinguished.The three photon fuorescence lifetimes of the capillaries in some representative images were in accord with that of DCDPP-2TPA nanoparticles in water.A vivid 3D reconstruction was further organized to present a wealth of lifetime information.In the future,the combination strategy of 3PFM and FLIM could be further applied in the brain functional imaging.展开更多
Fluorescence lifetime is not only associated with the molecular structure f fuorophores,but alsostrongly depends on the environment around them,which llows fuorescence lifetime imagingmicroscopy(FLIM)to be used as a t...Fluorescence lifetime is not only associated with the molecular structure f fuorophores,but alsostrongly depends on the environment around them,which llows fuorescence lifetime imagingmicroscopy(FLIM)to be used as a tool for precise measurement of the cell or tisue microenvironment,This review introduces the basic principle of fuorescence lifetime imagingtechnology and its application in clinical medicine,including research and diagnosis of diseases inskin,brain,eyes,mouth,bone,blood vessels and cavity organs,and drug evaluation.As anoninvasive,nontoxic and nonionizing radiation technique,FLIM demonstrates excellent per-formance with high sensitivity and specificity,which allows to determine precise position of thelesion and,thus,has good potential for application in biomedical research and clinical diagnosis.展开更多
With the development of the new detection methods and the function of fluorescent molecule,researchers hope to further explore the internal mechanisms of organisms,monitor changes in the intracellular microenvironment...With the development of the new detection methods and the function of fluorescent molecule,researchers hope to further explore the internal mechanisms of organisms,monitor changes in the intracellular microenvironment,and dynamic processes of molecular interactions in cells.Fluo-rescence resonance energy transfer(FRET)describes the energy transfer process between donor fluorescent molecules and acceptor fluorescent molecules.It is an important means to detect protein-protein interactions and protein conformation changes in cells.Fluorescence lifetime imaging microscopy(FLIM)enables noninvasive measurement of the fAuorescence lifetime of fluorescent particles in vivo.The FRET-FLIM technology,which is use FLIM to quantify and analyze FRET,enables real-time monitoring of dynamic changes of proteins in biological cells and analysis of protein interaction mechanisms.The distance between donor and acceptor and their respective fAuorescent lifetime,which are of great importance for studying the mechanism of intracellular activity can be obtained by data analysis and algorithm ftting.展开更多
Fluorescence lifetime imaging microscopy(FLIM)is increasingly used in biomedicine,material science,chemistry,and other related research fields,because of its advantages of high specificity and sensitivity in monitorin...Fluorescence lifetime imaging microscopy(FLIM)is increasingly used in biomedicine,material science,chemistry,and other related research fields,because of its advantages of high specificity and sensitivity in monitoring cellular microenvironments,studying interaction between proteins,metabolic state,screening drugs and analyzing their efficacy,characterizing novel materials,and diagnosing early cancers.Understandably,there is a large interest in obtaining FLIM data within an acquisition time as short as possible.Consequently,there is currently a technology that advances towards faster and faster FLIM recording.However,the maximum speed of a recording technique is only part of the problerm.The acquisition time of a FLIM image is a complex function of many factors.These include the photon rate that can be obtained from the sample,the amount of information a technique extracts from the decay functions,the fficiency at which it determines fluorescence decay parameters from the recorded photons,the demands for the accuracy of these parameters,the number of pixels,and the lateral and axial resolutions that are obtained in biological materials.Starting from a discussion of the parameters which determine the acquisition time,this review will describe existing and emerging FLIM techniques and data analysis algo-rithms,and analyze their performance and recording speed in biological and biomedical applications.展开更多
The effect of cooling rate on the microstructure and transformation textures of high strength hot-rolled steels was investigated.Heat treated samples subjected to different cooling conditions were characterized by opt...The effect of cooling rate on the microstructure and transformation textures of high strength hot-rolled steels was investigated.Heat treated samples subjected to different cooling conditions were characterized by optical and scanning electron microscopes using orientation imaging microscopy（OIM）.The experimental results demonstrate that there is a significant effect of cooling rate on microstructures and textures resulting from phase transformation.Slow cooling rates lead to the appearance of the cube（001）[010],rotated cube（001）[110]/（001）[110],Goss（110）[001]and rotated Goss（110）[110]components.In contrast,textures developed at rapid cooling rates are preferably of Cu（112）[111],Br（110）[112],transformed Cu（113）[110]and transformed Br（332）[113]/（112）[131].These texture changes are attributed to the selective character of the phase transformation.The OIM technique was used to have a better understanding of the formation of phases and their relationship between microstructure and processing conditions.The volume fraction of micro-constituents resulting from phase transformation such as bainite,martensite and different types of ferrite,can be measured satisfactorily by this technique correlating image quality of EBSD patterns to specific phases.展开更多
Fluorescence lifetime imaging microscopy(FLIM)has been rapidly developed over the past 30 years and widely applied in biomedical engineering.Recent progress in fluorophore-dyed probe design has widened the application...Fluorescence lifetime imaging microscopy(FLIM)has been rapidly developed over the past 30 years and widely applied in biomedical engineering.Recent progress in fluorophore-dyed probe design has widened the application prospects of fluorescence.Because fluorescence lifetime is sensitive to microenvironments and molecule alterations,FLIM is promising for the detection of pathological conditions.Current cancer-related FLIM applications can be divided into three main categories:(i)FLIM with autofluorescence molecules in or out of a cell,especially with reduced form of nicotinamide adenine dinucleotide,and flavin adenine dinucleotide for cellular metabolism research;(ii)FLIM with Förster resonance energy transfer for monitoring protein interactions;and(iii)FLIM with fluorophore-dyed probes for specific aberration detection.Advancements in nanomaterial production and efficient calculation systems,as well as novel cancer biomarker discoveries,have promoted FLIM optimization,offering more opportunities for medical research and applications to cancer diagnosis and treatment monitoring.This review summarizes cutting-edge researches from 2015 to 2020 on cancer-related FLIM applications and the potential of FLIM for future cancer diagnosis methods and anti-cancer therapy development.We also highlight current challenges and provide perspectives for further investigation.展开更多
A new two-photon fluorescent probe, ADNO, for nitric oxide （NO） based on intramolecular photoinduced electron transfer （PET） mechanism d/splays a rapid response to NO with a remarkable fluorescent enhancement in P...A new two-photon fluorescent probe, ADNO, for nitric oxide （NO） based on intramolecular photoinduced electron transfer （PET） mechanism d/splays a rapid response to NO with a remarkable fluorescent enhancement in PBS buffer. The excellent chemoselectivity of ADNO for NO over other ROS/RNS （reactive oxygen species or nitrogen species） and common metal ions was observed. Moreover, ADNO has been successfully applied in fluorescence imaging of NO of living cells using both one-photon microscopy （OPM） and two-~hoton microscopy （TPM）,展开更多
文摘The subcellular localization of human proteins is vital for understanding the structure of human cells.Proteins play a significant role within human cells,as many different groups of proteins are located in a specific location to perform a particular function.Understanding these functions will help in discoveringmany diseases and developing their treatments.The importance of imaging analysis techniques,specifically in proteomics research,is becoming more prevalent.Despite recent advances in deep learning techniques for analyzing microscopy images,classification models have faced critical challenges in achieving high performance.Most protein subcellular images have a significant class imbalance.We use oversampling and under sampling techniques in this research to overcome this issue.We have used a Convolutional Neural Network(CNN)model called GapNet-PL for the multi-label classification task on the Human Protein Atlas Classification(HPA)Dataset.Authors have found that the ParametricRectified LinearUnit(PreLU)activation function is better than the Scaled Exponential LinearUnit(SeLU)activation function in the GapNet-PL model in most classification metrics.The results showed that the GapNet-PL model with the PReLU activation function achieved an area under the ROC curve(AUC)equal to 0.896,an F1 score of 0.541,and a recall of 0.473.
文摘A novel mathematical morphological approach for region of interest(ROI) automatic determination and JPEG2000-based coding of microscopy image compression is presented.The algorithm is very fast and requires lower computing power,which is particularly suitable for some irregular region-based cell microscopy images with poor qualities.Firstly,an active threshold-based method is discussed to create a rough mask of regions of interest(cells).And then some morphological operations are designed and applied to achieve the segmentation of cells.In addition,an extra morphological operation,dilation,is applied to create the final mask with some redundancies to avoid the"edge effect"after removing false cells.Finally,ROI and region of background(ROB) are obtained and encoded individually in different compression ratio flexibly based on the JPEG2000,which can adjust the quality between ROI and ROB without coding for ROI shape.The experimental results certify the effectiveness of the proposed algorithm,and compared with JPEG2000,the proposed algorithm has better performance in both subjective quality and objective quality at the same compression ratios.
文摘The high-content image-based assay is commonly leveraged for identifying the phenotypic impact of genetic perturbations in biology field.However,a persistent issue remains unsolved during experiments:the interferential technical noises caused by systematic errors(e.g.,temperature,reagent concentration,and well location)are always mixed up with the real biological signals,leading to misinterpretation of any conclusion drawn.Here,we reported a mean teacher-based deep learning model(Deep Noise)that can disentangle biological signals from the experimental noises.Specifically,we aimed to classify the phenotypic impact of 1108 different genetic perturbations screened from 125,510 fluorescent microscopy images,which were totally unrecognizable by the human eye.We validated our model by participating in the Recursion Cellular Image Classification Challenge,and Deep Noise achieved an extremely high classification score(accuracy:99.596%),ranking the 2nd place among 866 participating groups.This promising result indicates the successful separation of biological and technical factors,which might help decrease the cost of treatment development and expedite the drug discovery process.The source code of Deep Noise is available at https://github.com/Scu-sen/Recursion-Cellular-Image-Classification-Challenge.
基金supported in part by the National Key R&D Program of China(2017YFA0700402)National Natural Science Foundation of China(61961136005/61935012/62175163/61835009)+1 种基金Shenzhen Key projects(JCYJ20200109105404067)Shenzhen International Cooperation Project(GJHZ 20190822095420249).
文摘Apoptosis is very important for the maintenance of cellular homeostasis and is closely related to the occurrence and treatment of many diseases.Mitochondria in cells play a crucial role in programmed cell death and redox processes.Nicotinamide adenine dinucleotide(NAD(P)H)is the primary producer of energy in mitochondria,changing NAD(P)H can directly reflect the physiological state of mitochondria.Therefore,NAD(P)H can be used to evaluate metabolic response.In this paper,we propose a noninvasive detection method that uses two-photon fluorescence lifetime imaging microscopy(TP-FLIM)to characterize apoptosis by observing the binding kinetics of cellular endogenous NAD(P)H.The result shows that the average fluorescence lifetime of NAD(P)H and the fluorescence lifetime of protein-bound NAD(P)H will be affected by the changing pH,serum content,and oxygen concentration in the cell culture environment,and by the treatment with reagents such as H2O2 and paclitaxel.Taxol(PTX).This noninvasive detection method realized the dynamic detection of cellular endogenous substances and the assessment of apoptosis.
基金Supported by the National Natural Science Foundation of China under Grant Nos 10704043 and 60772105.
文摘By using a vectorial approach, the validity of paraxial approximation in second harmonic generation （SHG） microscopy under low numerical aperture （NA） is examined when the sample is a collagen fibril. Due to the larger value of dzzz and tensorial nature of SHG, the component Ez of the focused fieM may have strong effect on the radiation pattern of SHG. Numerical results indicate that when the value of NA exceeds 0.3, the effect of Ez can not be neglected, which results in the invalidation of paraxial approximation in SHG microscopy despite the fact that SHG microscopy is still under low NA focusing.
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 (Nos. 2016YFA0400900, 2017YFA0505300)the Instrument Developing Project of the Chinese Academy of Sciences (No. YZ201564)
文摘Fluorescence microscopy, as a sensitive method to detect microenvironment of molecules, is widely used in protein conformation and dynamic studies in live cells. Fluorescence lifetime imaging microscopy（FLIM）, which is independent of fluorophore concentrations, scattering and bleaching, is a suitable tool to analyze membrane proteins in a single cell. Ferroportin（FPN）, a multi-ion exporter in vertebrates, was modulated by metal ions with unknown mechanism. Herein, we fused green fluorescence protein on Cterminal of FPN（FPN-eGFP） and applied fluorescence lifetime to monitor conformation changes of FPN in a live cell. The fluorescence lifetime distribution showed a shift to shorter lifetime upon Mn^（2＋） treatment,suggesting a preference conformation of FPN in Mn^（2＋） exposure. It is also observed that the lifetime（rather than intensity） measurement was not strongly influenced by laser power. The observed fluorescence lifetime changes of FPN-eGFP upon Mn^（2＋） treatments indicated that extracellular metal ions can modulate FPN through conformation exchanges between several different states.
基金supported by the National Key R&D Program of China(2018YFC0910602)the National Natural Science Foundation of China(Grant Nos.31771584/61775145/61605121,61620106016/61525503/61835009/81727804)+2 种基金Guangdong Natural Science Foundation Innovation Team(2014A030312008)Shenzhen Basic Research Project(JCYJ20170818100153423/JCYJ20170412110212234/JCYJ20160328144746940/JCYJ20170412105003520/JCYJ20170302142902581)Science Foundation of SZU(Grant No.000193).
文摘Recently,photothermal therapy(PTT)has been proved to have great potential in tumor therapy.In the last several years,MoS_(2),as one novel member of nanomaterials,has been applied into PTT due to its excellent photothermal conversion efficacy.In this work,we applied fuorescence lifetime imaging microscopy(FLIM)techniques into monitoring the PPT-triggered cell death under MoS_(2) nanosheet treatment.Two types of MoS_(2) nanosheets(single layer nanosheets and few layer nanosheets)were obtained,both of which exhibited presentable photothermal conversion fficacy,leading to high cell death rates of 4T1 cells(mouse breast cancer cells)under PTT.Next,live cell images of 4T1 cells were obtained via directly labeling the mitochondria with Rodamine123,which were then continuously observed with FLIM technique.FLIM data showed that the fuorescence lifetimes of mitochondria targeting dye in cells treated with each type of MoS_(2) nanosheets significantly increased during PTT treatment.By contrast,the fuorescence lifetime of the same dye in control cells(without nanomaterials)remained constant after laser irradiation.These findings suggest that FLIM can be of great value in monitoring cell death process during PTT of cancer cells,which could provide dynamic data of the cellular microenvironment at single cell level in multiple biomedical applications.
基金funded by the European Commissionunder the Seventh Framework Programme(Qualityand safety of feeds and food for Europe(QSAFFE),Contract No.FP7-KBBE-2010-4)Program forNew Century Excellent Talents in University(NCET-10-0785).
文摘Near infrared microscopy imaging fers the opportunity to explore not only what lkinds ofchemical species are present at micro-scale level but also where the chemical species would bepr esent.By revealing the spectral and spatial information,the technique can identify and localizeany interested component.This study investigates the feasibility of using Near infrared mi.croscopy imaging to detect melamine in soybean meal.The results showed that 6805 cm^(-1) is verysensitive for melamine but not for soybean meal,so can be used for univariate analysis,Singlewavelength image and peak integr ation image at 6805 cm^(-1) are simple and efective met hods todetect the melamine in soybean meal.Furthermore,Principal Component Analysis is applied todetect the melamine in soybean meal.
基金supported by the National Natural Science Foundation of China(62005116 and 51720105015)the Science and Technology Innovation Commission of Shenzhen(KQTD20170810110913065 and 20200925174735005).
文摘In light-sheet fluorescence microscopy,the axial resolution and field of view are mutually constrained.Axially swept light-sheet microscopy(ASLM)can decouple the trade-off,but the confocal detection scheme using a rolling shutter also rejects fluorescence signals from the specimen in the field of interest,which sacrifices the photon efficiency.Here,we report a laterally swept light-sheet microscopy(LSLM)scheme in which the focused beam is first scanned along the axial direction and subsequently laterally swept with the rolling shutter.We show that LSLM can obtain a higher photon efficiency when similar axial resolution and field of view can be achieved.Moreover,based on the principle of image scanning microscopy,applying the pixel reassignment to the LSLM images,hereby named iLSLM,improves the optical sectioning.Both simulation and experimental results demonstrate the higher photon efficiency with similar axial resolution and optical sectioning.Our proposed scheme is suitable for volumetric imaging of specimens that are susceptible to photobleaching or phototoxicity.
基金supported by the National Natural Science Foundation of China(No.60508017)the Natural Science Foundation of Fujian Province of China(2007J0007,C0720001)+1 种基金the Science and Technology Planning Key Program of Fujian Province(2008Y0037)the Program for New Century Excellent Talents in University(NCET-07-0191).
文摘Skin scar is unique to humans,the major significant negative outcome sustained after thermal injuries,traumatic injuries,and surgical procedures.Hypertrophic scar in human skin is investigated using non-linear spectral imaging microscopy.The high contrast images and spectroscopic intensities of collagen and elastic fibers extracted from the spectral imaging of normal skin tissue,and the normal skin near and far away from the hypertrophic scar tissues in a 10-year-old patient case are obtained.The results show that there are apparent differences in the morphological structure and spectral characteristics of collagen and elastic fibers when comparing the normal skin with the hypertrophic scar tissue.These differences can be good indicators to differentiate the normal skin and hypertrophic scar tissue and demonstrate that non-linear spectral imaging microscopy has potential to noninvasively investigate the pathophysiology of human hypertrophic scar.
基金supported by the Samsung Research Funding&Incubation Center of Samsung Electronics under Project Number SRFC-IT2401-01 and by National Research Foundation(NRF)grants(RS-2024-00462912,RS-2023-00266110,and RS-2020-NR049544)funded by the Ministry of Science and ICT(MSIT)of the Korean governmentI.K.acknowledges the NRF Sejong Science Fellowship(RS-2021-NR061797)funded by the MSIT of the Korean government.We would like to express our sincere gratitude to Yangkyu Kim for his invaluable assistance in correcting the mathematical errors in this paper.
文摘Image scanning microscopy(ISM)is a promising imaging technique that offers sub-diffraction-limited resolution and optical sectioning.Theoretically,ISM can improve the optical resolution by a factor of two through pixel reassignment and deconvolution.Multifocal array illumination and scanning have been widely adopted to implement ISM because of their simplicity.Conventionally,digital micromirror devices(DMDs)1 and microlens arrays(MLAs)2,3 have been used to generate dense and uniform multifocal arrays for ISM,which are critical for achieving fast imaging and high-quality ISM reconstruction.However,these approaches have limitations in terms of cost,numerical aperture(NA),pitch,and uniformity,making it challenging to create dense and high-quality multifocal arrays at high NA.To overcome these limitations,we introduced a novel multifocal metalens design strategy called the hybrid multiplexing method,which combines two conventional multiplexing approaches:phase addition and random multiplexing.Through numerical simulations,we demonstrate that the proposed method generates more uniform and denser multifocal arrays than conventional methods,even at small pitches.As a proof of concept,we fabricated a multifocal metalens generating 40×40 array of foci with a 3μm pitch and NA of 0.7 operating at a wavelength of 488 nm and then constructed the multifocal metalens-based ISM(MMISM).We demonstrated that MMISM successfully resolved sub-diffraction-limited features in imaging of microbead samples and forebrain organoid sections.The results showed that MMISM imaging achieved twice the diffraction-limited resolution and revealed clearer structural features of neurons compared to wide-field images.We anticipate that our novel design strategy can be widely applied to produce multifunctional optical elements and replace conventional optical elements in specialized applications.
基金Project(2010CB731704)supported by the National Basic Research Program of ChinaProject(AWJ-M13-11)supported by State Key Laboratory of Advanced Welding and Joining,Harbin Institute of Technology,ChinaProject(2012M511470)supported by China Postdoctoral Science Foundation
文摘Theα＋βtitanium alloy, Ti-6Al-4V, was welded by friction stir welding using a W-Re pin tool, and the defect-free weld was produced with proper welding parameters. Texture of the Ti-6Al-4V friction stir weld was studied by orientation imaging microscopy. The as-received Ti-6Al-4V sheet mill annealed was composed of elongated primary α and transformed β. A typical rolling texture was observed in the base material. The microstructure of the stir zone was significantly different from that of the base material. The stir zone was characterized by the presence of considerable amount of equiaxed dynamically recrystallized grains and a texture around{Ф1=30°,φ=62°,Ф2=30°}was developed during the friction stir welding.
基金supported by National Natural Science Foundation of China(61735016)Zhejiang Provincial Natural Science Foundation of China(LR17F050001).
文摘Compared with visible light,near infrared(NIR)light has deeper penetration in biological tisues.Three-photon fuorescence microscopy(3PFM)can effectively utilize the NIR excitation to obtain high-contrast images in the deep tisue.However,the weak three photon fluorescence signals may be not well presented in the traditional fuorescence intensity imaging mode.Fluorescence lifetime of certain probes is insensitive to the intensity of the excitation laser.Moreover,fluorescence lifetimne imaging microscopy(FLIM)can detect weak signals by utilizing time correlated single photon counting(TCSPC)technique.Thus,it would be an improved strategy to combine the 3PFM imaging with the FLIM together.Herein,DCDPP-2TPA,a novel agegation-induced emission luminogen(AIEgen),was adopted as the fluorescent probes.The three-photon absorption cros-section of the AlEgen,which has a deep-red fluorescence emission,was proved to be large.DCDPP-2TPA nanoparticles were synthesized,and the three photon fluorescence lifetime of which was measured in water.Moreover,in vrivo thre-photon fuorescence lifetime microscopic imaging of a craniotomy mouse was conducted via a home made optical system.High contrast cerebrovascular images of different vertical depths were obtained and the maximun depth was about 600 pumn.Even reaching the depth of 600 pum,tiny capillary vessels as small as 1.9 pum could still be distinguished.The three photon fuorescence lifetimes of the capillaries in some representative images were in accord with that of DCDPP-2TPA nanoparticles in water.A vivid 3D reconstruction was further organized to present a wealth of lifetime information.In the future,the combination strategy of 3PFM and FLIM could be further applied in the brain functional imaging.
基金funded by the Science and Technology Planning Fundamental Research Project of Shenzhen(No.JCYJ20150324140036853)National Natural Science Foundation of China(No.61378091)+1 种基金Ningbo Natural Science Foundation Project(No.2016A610032)the Central University Basic Scientic Research Business Expenses Project(No.NSIY051405).
文摘Fluorescence lifetime is not only associated with the molecular structure f fuorophores,but alsostrongly depends on the environment around them,which llows fuorescence lifetime imagingmicroscopy(FLIM)to be used as a tool for precise measurement of the cell or tisue microenvironment,This review introduces the basic principle of fuorescence lifetime imagingtechnology and its application in clinical medicine,including research and diagnosis of diseases inskin,brain,eyes,mouth,bone,blood vessels and cavity organs,and drug evaluation.As anoninvasive,nontoxic and nonionizing radiation technique,FLIM demonstrates excellent per-formance with high sensitivity and specificity,which allows to determine precise position of thelesion and,thus,has good potential for application in biomedical research and clinical diagnosis.
基金supported by the National Natural Science Foundation of China(61722508/61525503/61620106016/81727804)The National Key Research and Development Program of China(2017YFA0700402)+1 种基金Guangdong Natural Science Foundation Innovation Team(2014A030312008)Shenzhen Basic Research Project(JCYJ20150930104948169/JCYJ20160328144746940/GJHZ20160226202139185/JCYJ20170412105003520)。
文摘With the development of the new detection methods and the function of fluorescent molecule,researchers hope to further explore the internal mechanisms of organisms,monitor changes in the intracellular microenvironment,and dynamic processes of molecular interactions in cells.Fluo-rescence resonance energy transfer(FRET)describes the energy transfer process between donor fluorescent molecules and acceptor fluorescent molecules.It is an important means to detect protein-protein interactions and protein conformation changes in cells.Fluorescence lifetime imaging microscopy(FLIM)enables noninvasive measurement of the fAuorescence lifetime of fluorescent particles in vivo.The FRET-FLIM technology,which is use FLIM to quantify and analyze FRET,enables real-time monitoring of dynamic changes of proteins in biological cells and analysis of protein interaction mechanisms.The distance between donor and acceptor and their respective fAuorescent lifetime,which are of great importance for studying the mechanism of intracellular activity can be obtained by data analysis and algorithm ftting.
基金support from the National Key R&D Program of China(2017YFA0700500)National Natural Science Foundation of China(61775144/61525503/61620106016/61835009/81727804)+2 种基金(Key)Project of Department of Education of Guangdong Province(2015KGJHZ002/2016KCXTD007)Guangdong Natural Science Foundation(2014A030312008,2017A030310132,2018A030313362)Shenzhen Basic Research Project(JCYJ20170818144012025/JCYJ20170818141701667/JCYJ20170412105003520/JCYJ20150930104948169).
文摘Fluorescence lifetime imaging microscopy(FLIM)is increasingly used in biomedicine,material science,chemistry,and other related research fields,because of its advantages of high specificity and sensitivity in monitoring cellular microenvironments,studying interaction between proteins,metabolic state,screening drugs and analyzing their efficacy,characterizing novel materials,and diagnosing early cancers.Understandably,there is a large interest in obtaining FLIM data within an acquisition time as short as possible.Consequently,there is currently a technology that advances towards faster and faster FLIM recording.However,the maximum speed of a recording technique is only part of the problerm.The acquisition time of a FLIM image is a complex function of many factors.These include the photon rate that can be obtained from the sample,the amount of information a technique extracts from the decay functions,the fficiency at which it determines fluorescence decay parameters from the recorded photons,the demands for the accuracy of these parameters,the number of pixels,and the lateral and axial resolutions that are obtained in biological materials.Starting from a discussion of the parameters which determine the acquisition time,this review will describe existing and emerging FLIM techniques and data analysis algo-rithms,and analyze their performance and recording speed in biological and biomedical applications.
文摘The effect of cooling rate on the microstructure and transformation textures of high strength hot-rolled steels was investigated.Heat treated samples subjected to different cooling conditions were characterized by optical and scanning electron microscopes using orientation imaging microscopy（OIM）.The experimental results demonstrate that there is a significant effect of cooling rate on microstructures and textures resulting from phase transformation.Slow cooling rates lead to the appearance of the cube（001）[010],rotated cube（001）[110]/（001）[110],Goss（110）[001]and rotated Goss（110）[110]components.In contrast,textures developed at rapid cooling rates are preferably of Cu（112）[111],Br（110）[112],transformed Cu（113）[110]and transformed Br（332）[113]/（112）[131].These texture changes are attributed to the selective character of the phase transformation.The OIM technique was used to have a better understanding of the formation of phases and their relationship between microstructure and processing conditions.The volume fraction of micro-constituents resulting from phase transformation such as bainite,martensite and different types of ferrite,can be measured satisfactorily by this technique correlating image quality of EBSD patterns to specific phases.
基金This work was partially supported by the National Natural Science Foundation of China(Grant No.61775241)the Hunan Science Fund for Distinguished Young Scholar(2020JJ2059)+3 种基金Youth Innovation Team(Grant No.2019012)of CSU,Hunan province key research and development project(Grant No.2019GK2233,Grant 2020SK2053)Hunan Province Graduate Research and Innovation Project(Grant No.CX20190177)the Science and Technology Innovation Basic Research Project of Shenzhen(Grant No.JCYJ20180307151237242)Also,YPL acknowledges the support by the Project of State Key Laboratory of High-Performance Complex Manufacturing,Central South University(Grant No.ZZYJKT2020-12).Besides,we acknowledge the art work from Servier Medical Art.Y.Z.O and Y.P.L contributed equally to this work.
文摘Fluorescence lifetime imaging microscopy(FLIM)has been rapidly developed over the past 30 years and widely applied in biomedical engineering.Recent progress in fluorophore-dyed probe design has widened the application prospects of fluorescence.Because fluorescence lifetime is sensitive to microenvironments and molecule alterations,FLIM is promising for the detection of pathological conditions.Current cancer-related FLIM applications can be divided into three main categories:(i)FLIM with autofluorescence molecules in or out of a cell,especially with reduced form of nicotinamide adenine dinucleotide,and flavin adenine dinucleotide for cellular metabolism research;(ii)FLIM with Förster resonance energy transfer for monitoring protein interactions;and(iii)FLIM with fluorophore-dyed probes for specific aberration detection.Advancements in nanomaterial production and efficient calculation systems,as well as novel cancer biomarker discoveries,have promoted FLIM optimization,offering more opportunities for medical research and applications to cancer diagnosis and treatment monitoring.This review summarizes cutting-edge researches from 2015 to 2020 on cancer-related FLIM applications and the potential of FLIM for future cancer diagnosis methods and anti-cancer therapy development.We also highlight current challenges and provide perspectives for further investigation.
基金the National Natural Science Foundation of China(Nos.21102148 and 21125205)National Basic Research Program of China(No.2011CB935800)the State Key Laboratory of Fine Chemicals,Department of Chemical Engineering,Dalian University of Technology for financial supports
文摘A new two-photon fluorescent probe, ADNO, for nitric oxide （NO） based on intramolecular photoinduced electron transfer （PET） mechanism d/splays a rapid response to NO with a remarkable fluorescent enhancement in PBS buffer. The excellent chemoselectivity of ADNO for NO over other ROS/RNS （reactive oxygen species or nitrogen species） and common metal ions was observed. Moreover, ADNO has been successfully applied in fluorescence imaging of NO of living cells using both one-photon microscopy （OPM） and two-~hoton microscopy （TPM）,
