Optical microscopy is an essential tool for exploring the structures and activities of cells and tissues.To break the limit of resolution caused by diffraction,researchers have made continuous advances and innovations...Optical microscopy is an essential tool for exploring the structures and activities of cells and tissues.To break the limit of resolution caused by diffraction,researchers have made continuous advances and innovations to improve the resolution of optical microscopy since the 1990s.These contributions,however,still make sub-10nm imaging an obstacle.Here,we name a series of technologies as modulated illumination localization microscopy(MILM),which makes ultra-high-resolution imaging practical.Besides,we review the recent progress since 2017 when MINFLUX was proposed and became the inspiration and foundation for the follow-up devel-opment of MILM.This review divides MILM into two types:point-scanning and wide-field.The schematics,principles and future research directions of MILM are discussed elaborately.展开更多
Quantitative data analysis in single-molecule localization microscopy(SMLM)is crucial for studying cellular functions at the biomolecular level.In the past decade,several quantitative methods were developed for analyz...Quantitative data analysis in single-molecule localization microscopy(SMLM)is crucial for studying cellular functions at the biomolecular level.In the past decade,several quantitative methods were developed for analyzing SMLM data;however,imaging artifacts in SMLM experiments reduce the accuracy of these methods,and these methods were seldom designed as user-friendly tools.Researchers are now trying to overcome these di±culties by developing easyto-use SMLM data analysis software for certain image analysis tasks.But,this kind of software did not pay su±cient attention to the impact of imaging artifacts on the analysis accuracy,and usually contained only one type of analysis task.Therefore,users are still facing di±culties when they want to have the combined use of different types of analysis methods according to the characteristics of their data and their own needs.In this paper,we report an ImageJ plug-in called DecodeSTORM,which not only has a simple GUI for human–computer interaction,but also combines artifact correction with several quantitative analysis methods.DecodeSTORM includes format conversion,channel registration,artifact correction(drift correction and localization¯ltering),quantitative analysis(segmentation and clustering,spatial distribution statistics and colocalization)and visualization.Importantly,these data analysis methods can be combined freely,thus improving the accuracy of quantitative analysis and allowing users to have an optimal combination of methods.We believe DecodeSTORM is a user-friendly and powerful ImageJ plug-in,which provides an easy and accurate data analysis tool for adventurous biologists who are looking for new imaging tools for studying important questions in cell biology.展开更多
Fluorescent dye （YOYO-I） intercalated with single DNA molecules were investigated via bindingactivated localization microscopy （BALM） at sub-diffraction limit resolutions. Various dye-to-DNA base pair （bp） ratio...Fluorescent dye （YOYO-I） intercalated with single DNA molecules were investigated via bindingactivated localization microscopy （BALM） at sub-diffraction limit resolutions. Various dye-to-DNA base pair （bp） ratios were imaged using the blinking property of YOYO-1 dye under optimum BALM switching buffer conditions. Individual DNA molecules exhibited regular/irregular intercalating phenomena with respect to dye-to-DNA bp ratio. The acquired images were reconstructed into super-resolution images by applying a Gaussian fit to the centroid of the point spread function. The YOYO-1 intercalated with λ-DNA possessed a non-homogeneous region due to the different binding modes of YOYO-1 with λ-DNA. Each binding mode was imaged at the sub-diffraction limit super-resolution. The distance between homogenously localized intercalating dyes within the DNA molecules was measured to be 34nm （n= 10; dye：DNAbp= 1：100） without photocleavage in 50mmol/L β-mercaptoethylamine buffer. The results were similar to those of the theoretical values without photocleavage in the base pairs of single DNA molecules below the diffraction limit. The results paved the way for an in-depth microscopic analysis of molecular variation with single λ-DNA molecules. With this method, it should be possible to analyze the exact base pair breakdown during various stages of cell apoptosis.展开更多
Super-resolution microscopy has revolutionized our ability to visualize structures below the diffraction limit of conventional optical microscopy and is particularly useful for investigating complex biological targets...Super-resolution microscopy has revolutionized our ability to visualize structures below the diffraction limit of conventional optical microscopy and is particularly useful for investigating complex biological targets like chromatin.Chromatin exhibits a hierarchical organization with structural compartments and domains at different length scales,from nanometers to micrometers.Single molecule localization microscopy(SMLM)methods,such as STORM,are essential for studying chromatin at the supra-nucleosome level due to their ability to target epigenetic marks that determine chromatin organization.Multi-label imaging of chromatin is necessary to unpack its structural complexity.However,these efforts are challenged by the high-density nuclear environment,which can affect antibody binding affinities,diffusivity and non-specific interactions.Optimizing buffer conditions,fluorophore stability,and antibody specificity is crucial for achieving effective antibody conjugates.Here,we demonstrate a sequential immunolabeling protocol that reliably enables three-color studies within the dense nuclear environment.This protocol couples multiplexed localization datasets with a robust analysis algorithm,which utilizes localizations from one target as seed points for distance,density and multi-label joint affinity measurements to explore complex organization of all three targets.Applying this multiplexed algorithm to analyze distance and joint density reveals that heterochromatin and euchromatin are not-distinct territories,but that localization of transcription and euchromatin couple with the periphery of heterochromatic clusters.This work is a crucial step in molecular imaging of the dense nuclear environment as multi-label capacity enables for investigation of complex multi-component systems like chromatin with enhanced accuracy.展开更多
Single-molecule localization microscopy(SMLM)has pushed resolution to sub-40 nm.Combined with structured illumination,lateral resolution can be doubled or the axial resolution can be improved fourfold.However,current ...Single-molecule localization microscopy(SMLM)has pushed resolution to sub-40 nm.Combined with structured illumination,lateral resolution can be doubled or the axial resolution can be improved fourfold.However,current techniques are challenging in balancing the lateral and axial resolutions.Here,we report a new modulated illumination single-molecule localization modality,isoFLUX.Utilizing two objective lenses to form interference patterns along the x-z and y-z directions,the lateral and axial resolutions can be improved simultaneously.Compared to SMLM,isoFLUX maintains a twofold average enhancement in both lateral and axial resolutions under an astigmatic point spread function(PSF),1.5-fold in the lateral resolution and 2.5-fold in the axial resolution under the saddle-point PSF.展开更多
Single-molecule localization microscopy(SMLM)surpasses the diffraction limit by randomly switching fluorophores between fluorescent and dark states,precisely pinpointing the resulted isolated emission patterns,thereby...Single-molecule localization microscopy(SMLM)surpasses the diffraction limit by randomly switching fluorophores between fluorescent and dark states,precisely pinpointing the resulted isolated emission patterns,thereby reconstructing the superresolution images based on the accumulated locations of thousands to millions of single molecules.This technique achieves a ten-fold improvement in resolution,unveiling the intricate details of molecular activities and structures in cells and tissues.Multicolor SMLM extends this capability by imaging distinct protein species labeled with various fluorescent probes,providing insights into structural intricacies and spatial relationships among different targets.This review explores recent advancements in multicolor SMLM,evaluates the strengths and limitations of each variant,and discusses the future prospects.展开更多
The 3D location and dipole orientation of light emitters provide essential information in many biological,chemical,and physical systems.Simultaneous acquisition of both information types typically requires pupil engin...The 3D location and dipole orientation of light emitters provide essential information in many biological,chemical,and physical systems.Simultaneous acquisition of both information types typically requires pupil engineering for 3D localization and dual-channel polarization splitting for orientation deduction.Here we report a geometric phase helical point spread function for simultaneously estimating the 3D position and dipole orientation of point emitters.It has a compact and simpler optical configuration compared to polarization-splitting techniques and yields achromatic phase modulation in contrast to pupil engineering based on dynamic phase,showing great potential for single-molecule orientation and localization microscopy.展开更多
Stochastic optical reconstruction microscopy(STORM),as a typical technique of single-molecule localization microscopy(SMLM),has overcome the diffraction limit by randomly switching fluorophores between fluorescent and...Stochastic optical reconstruction microscopy(STORM),as a typical technique of single-molecule localization microscopy(SMLM),has overcome the diffraction limit by randomly switching fluorophores between fluorescent and dark states,allowing for the precise localization of isolated emission patterns and the super-resolution reconstruction from millions of localized positions of single fluorophores.A critical factor influencing localization precision is the photo-switching behavior of fluorophores,which is affected by the imaging buffer.The imaging buffer typically comprises oxygen scavengers,photo-switching reagents,and refractive index regulators.Oxygen scavengers help prevent photobleaching,photo-switching reagents assist in facilitating the conversion of fluorophores,and refractive index regulators are used to adjust the refractive index of the solution.The synergistic interaction of these components promotes stable blinking of fluorophores,reduces irreversible photobleaching,and thereby ensures high-quality super-resolution imaging.This review provides a comprehensive overview of the essential compositions and functionalities of imaging buffers used in STORM,serving as a valuable resource for researchers seeking to select appropriate imaging buffers for their experiments.展开更多
Monitoring the dynamics of cellular pseudopodia at nanoscale has become essential for understanding their diverse and complex functions in living cells.This is made possible by combining single-molecule localization m...Monitoring the dynamics of cellular pseudopodia at nanoscale has become essential for understanding their diverse and complex functions in living cells.This is made possible by combining single-molecule localization microscopy(SMLM)with self-blinking dyes.However,existing self-blinking dyes often face limitations,such as nonspecific blinking and low photostability,which can bring background noise and yield erroneous localization signals,hindering their effectiveness for nanoscale visualization.Here,we present a method for long-term SMLM imaging of cellular pseudopodia dynamics using a blinkogenic probe that exhibits self-blinking activation upon molecular recognition.This approach enabled the precise tracking of various pseudopodia structures,including filopodia,lamellipodia,and(tunneling nanotubes)-nanoscale(TNTs),in living cells.We monitored the growth and fusion of filopodia,as well as the extension and shrinkage of lamellipodia,in real-time.Additionally,we identified two distinct fusion modes between filopodia and lamellipodia and captured the formation of TNTs and their interactions with filopodia,demonstrating the probe's utility in visualizing real-time pseudopodia dynamics at nanoscale.展开更多
Hematologic malignancies are one of the most common malignant tumors caused by the clonal proliferation and differentiation of hematopoietic and lymphoid stem cells.The examination of bone marrow cells combined with i...Hematologic malignancies are one of the most common malignant tumors caused by the clonal proliferation and differentiation of hematopoietic and lymphoid stem cells.The examination of bone marrow cells combined with immunodeficiency typing is of great significance to the diagnostic type,treatment and prognosis of hematologic malignancies.Super-resolution fluorescence microscopy(SRM)is a special kind of optical microscopy technology,which breaks the resolution limit and was awarded the Nobel Prize in Chemistry in 2014.With the development of SRM,many related technologies have been applied to the diagnosis and treatment of clinical diseases.It was reported that a major type of SRM technique,single molecule localization microscopy(SMLM),is more sensitive than flow cytometry(FC)in detecting cell membrane antigens'expression,thus enabling better chances in detecting antigens on hematopoietic cells than traditional analytic tools.Furthermore,SRM may be applied to clinical pathology and may guide precision medicine and personalized medicine for clone hematopoietic cell diseases.In this paper,we mainly discuss the application of SRM in clone hematological malignancies.展开更多
The MINFLUX concept significantly improves the localization properties of single-molecule localization microscopy(SMLM)by overcoming the limit imposed by the fluorophore's photon counts.Typical MINFLUX microscopes...The MINFLUX concept significantly improves the localization properties of single-molecule localization microscopy(SMLM)by overcoming the limit imposed by the fluorophore's photon counts.Typical MINFLUX microscopes localize the target molecule by scanning a zero-intensity focus around the molecule in a circular trajectory,with smaller trajectory diameters yielding better localization uncertainties for a given number of photons.Since this approach requires the molecule to be within the scanned trajectory,MINFLUX typically relies on an iterative scheme with decreasing trajectory diameters.This iterative approach is prone to misplacements of the trajectory and increases the system's complexity.In this work,we introduce ISM-FLUX,a novel implementation of MINFLUX using image-scanning microscopy(ISM)with a single-photon avalanche diode array detector.ISM-FLUX provides a precise MINFLUX localization within the trajectory while maintaining a conventional photon-limited uncertainty outside it.The robustness of ISM-FLUX localization results in a larger localization range and greatly simplifies the architecture,which may facilitate broader adoption of MINFLUX.展开更多
Work function plays a significant role in surface chemistry. Local work function provides the information of local d/pole-d/pole interaction and charge distribution between adsorbates and substrate, highlighting the l...Work function plays a significant role in surface chemistry. Local work function provides the information of local d/pole-d/pole interaction and charge distribution between adsorbates and substrate, highlighting the local charge effect of the targeted spot which is normally smeared out in conventional average work function measurements. Chloroaluminum phthalocyanine （CIA1Pc）, an important optoelectronic molecule with a permanent dipole moment pointing from the Pc ring to the ending CI atom, adsorbed on Au（111） in either Cl-up or Cl-down configuration. Scanning tunneling microscopy/spectroscopy measurements revealed that at the centers of Cl-up and CI-down molecules, the local work functions changed oppositely with respect to the Au（111） substrate. At their Pc lobes, however, the local work functions unanimously increased due to charging effect of the indole lobes in the CIAIPc molecule.展开更多
This article reviews recent super-resolution(SR)optical imaging techniques for cellular study,encompassing structured illumination microscopy(SIM),point-scanning super-resolution(PS-SR)microscopy,single-molecule local...This article reviews recent super-resolution(SR)optical imaging techniques for cellular study,encompassing structured illumination microscopy(SIM),point-scanning super-resolution(PS-SR)microscopy,single-molecule localization microscopy(SMLM),mathematical and deep learning(DL)SR algorithms.Historically,the resolution of traditional far-field optical imaging was constrained by the diffraction limit.The emergence of SR imaging techniques and image processing algorithms has propelled biological research into nanoscale realm.SIM enhances resolution by manipulating spatial frequency content,effectively doubling the resolution capacity of traditional microscopy.PS-SR imaging,on the other hand,offers superior optical sectioning and a high signal-to-noise ratio.SMLM has achieved a remarkable spatial resolution of approximately 20 nm and supports multi-color,wide-field-of-view(FOV),automated 3D high-throughput imaging,thus broadening the horizons for advanced biomedical investigations.Additionally,both mathematical and DL-based SR algorithms have significantly advanced the conversion of low-resolution images to high-resolution counterparts,extending the capabilities of conventional microscopes.This review underscores the principles,recent developments,and diverse applications of these cutting-edge SR imaging methodologies in biological research.展开更多
Single-molecule localization microscopy(SMLM)provides nanoscale imaging,but pixel integration of acquired SMLM images limited the choice of sampling rate,which restricts the information content conveyed within each im...Single-molecule localization microscopy(SMLM)provides nanoscale imaging,but pixel integration of acquired SMLM images limited the choice of sampling rate,which restricts the information content conveyed within each image.We propose an upsampled point spread function(PSF)inverse modeling method for large-pixel singlemolecule localization,enabling precise three-dimensional superresolution imaging with a sparse sampling rate.展开更多
Endogenous biomolecules in cells are the basis of all life activities.Directly visualizing the structural characteristics and dynamic behaviors of cellular biomolecules is signiffcant for understanding the molecular m...Endogenous biomolecules in cells are the basis of all life activities.Directly visualizing the structural characteristics and dynamic behaviors of cellular biomolecules is signiffcant for understanding the molecular mechanisms in various biological processes.Singlemolecule localization microscopy(SMLM)can circumvent the optical diffraction limit,achieving analysis of the ffne structures and biological processes in living cells with nanoscale resolution.However,the large size of traditional imaging probes prevents SMLM from accurately locating ffne structures and densely distributed biomolecules within cells.In recent years,nucleic acid probes have emerged as potential tools to replace conventional SMLM probes by virtue of their small size and high speciffcity.In addition,due to their programmability,nucleic acid probes with different conformations can be constructed via sequence design,further extending the application of SMLM in bioanalysis.Here,we discuss the design concepts of different conformational nucleic acid probes for SMLM and summarize the application of SMLM based on nucleic acid probes in the ffeld of biomolecules.Furthermore,we provide a summary and future perspectives of the nucleic acid probe-based SMLM technology,aiming to provide guidance for the acquisition of nanoscale information about cellular biological processes.展开更多
Acentral theme of chemical imaging is the visualization of chemistry in situ in functional materials and in vivo in a living system.To achieve this,technologies with exceptional detection sensitivity and spatial resol...Acentral theme of chemical imaging is the visualization of chemistry in situ in functional materials and in vivo in a living system.To achieve this,technologies with exceptional detection sensitivity and spatial resolution are essential for resolving specific chemicals at the nanoscale.As we celebrate the 10th anniversary of the Nobel Prize in Chemistry awarded for super-resolved fluorescence microscopy,the field’s remarkable advancements become clear.Over the past 10 years,novel microscopies that break light diffraction limits continue to be invented.These include,for example,midinfrared photothermal microscopy that breaks the diffraction limit in IR imaging,and expansion microscopy that allows super-resolution fluorescence imaging on a conventional microscope.Meanwhile,researchers have much increased the speed of structural illumination microscopy(SIM)and single molecule localization microscopy(SMLM),and pushed the resolution limit of stimulated emission depletion(STED)microscopy to angstrom scale.In parallel,data science and artificial intelligence(AI)are elegantly applied to break the diffraction limits of images recorded on a conventional microscope.These innovations have enabled very exciting applications,including the study of single particle catalysis,biomolecule trafficking inside a live cell,dynamic imaging of cellular organelles,and many others.展开更多
There is an increasing demand for advanced optical imaging techniques that can detect and resolve nanosize objects at a spatial resolution below the optical diffraction limit, especially in three-dimensional （3D） ce...There is an increasing demand for advanced optical imaging techniques that can detect and resolve nanosize objects at a spatial resolution below the optical diffraction limit, especially in three-dimensional （3D） cellular environments. In this study, using a polarization-activated localization scheme based on the orientation-dependent properties of anisotropic plasmonic metal nanoparticles （MNPs）, ＂photoswitchable＂ imaging of single gold nanorods （AuNRs） was accomplished not only in two dimensions but also in three dimensions. Moreover, the Rayleigh scattering background arising from the congested subcellular structures was efficiently suppressed. Thus, we obtained the 3D distributions of both the position and the orientation of the AuNRs inside the cells and investigated their intemalization kinetics. To our knowledge, this is the first demonstration of the confocal-like 3D imaging of non-fluorescence nanoparticles with a high resolution and almost zero background. This technique is easy to implement and should greatly facilitate MNP studies and applications in biomedicine and biology.展开更多
Quantum dots(QDs)can be used as fluorescent probes in single molecule localization microscopy to achieve subdiffraction limit resolution(super-resolution fluorescence imaging).However,the toxicity of Cd in the prototy...Quantum dots(QDs)can be used as fluorescent probes in single molecule localization microscopy to achieve subdiffraction limit resolution(super-resolution fluorescence imaging).However,the toxicity of Cd in the prototypical CdSe-based QDs can limit their use in biological applications.Furthermore,commercial CdSe QDs are usually modified with relatively thick shells of both inorganic and organic materials to render them in the 10−20 nm size range,which is relatively large for biological labels.In this report,we present compact(4−6 nm)CuInS2/ZnS(CIS/ZnS)and compare them to commercially sourced CdSe/ZnS QDs for their blinking behavior,localization precision and super-resolution imaging.Although commercial CdSe/ZnS QDs are brighter than the more compact Cd-free CIS/ZnS QD,both give comparable results of 4.5−5.0-fold improvement in imaging resolution over conventional TIRF imaging of actin filaments.This likely results from the fact that CIS/ZnS QDs show very short on-times and long off times which leads to less overlap in the point spread functions of emitting CIS/ZnS QD labels on the actin filaments at the same labeling density.These results demonstrate that CIS/ZnS QDs are an excellent candidate to complement and perhaps even replace the larger and more toxic CdSe-based QDs for robust single-molecule super-resolution imaging.展开更多
Single-molecule localization microscopy circumvents the diffraction limit of traditional fluorescence microscopy by detecting the photoemission signals of individual fluorescent molecules.The accurate recognitions of ...Single-molecule localization microscopy circumvents the diffraction limit of traditional fluorescence microscopy by detecting the photoemission signals of individual fluorescent molecules.The accurate recognitions of fluorescence molecules/events are critical to single-molecule/super-resolution imaging experiments,which determine the precision of molecular localizations and the quality of the image reconstruction.Herein,we presented a single-molecule detection method which relied on the temporal pixel intensity fluctuation.The method was capable of quickly determining the approximate localizations of fluorescence events with high sensitivity.We evaluated the performance of the method under a series of signal-to-noise ratios(SNR)and discussed the criterion of setting the temporal fluctuation threshold to achieve the optimized spots recognition results.展开更多
基金This work was financially sponsored by National Natural Science Foundation of China(61735017,61827825)Major Program of the Natural Science Foundation of Zhejiang Province(LD21F050002)+1 种基金Key Research and Development Program of Zhejiang Province(2020C01116)Fundamental Research Funds for the Central Universities(K20200132),Zhejiang Lab(2020MC0AE01)and Zhejiang Provincial Ten Thousand Plan for Young Top Talents(2020R52001).Y.S.and L.Y.contributed equally to this work.
文摘Optical microscopy is an essential tool for exploring the structures and activities of cells and tissues.To break the limit of resolution caused by diffraction,researchers have made continuous advances and innovations to improve the resolution of optical microscopy since the 1990s.These contributions,however,still make sub-10nm imaging an obstacle.Here,we name a series of technologies as modulated illumination localization microscopy(MILM),which makes ultra-high-resolution imaging practical.Besides,we review the recent progress since 2017 when MINFLUX was proposed and became the inspiration and foundation for the follow-up devel-opment of MILM.This review divides MILM into two types:point-scanning and wide-field.The schematics,principles and future research directions of MILM are discussed elaborately.
基金supported by the National Natural Science Foundation of China(82160345)Key research and development project of Hainan province(ZDYF2021GXJS017)+2 种基金Key Science and Technology Plan Project of Haikou(2021-016)the Start-up Fund from Hainan University(KYQD(ZR)-20022 and KYQD(ZR)-20077)the Student Innovation and Entrepreneurship Project of Biomedical Engineer-ing School,Hainan University(BMECF2D2021001).
文摘Quantitative data analysis in single-molecule localization microscopy(SMLM)is crucial for studying cellular functions at the biomolecular level.In the past decade,several quantitative methods were developed for analyzing SMLM data;however,imaging artifacts in SMLM experiments reduce the accuracy of these methods,and these methods were seldom designed as user-friendly tools.Researchers are now trying to overcome these di±culties by developing easyto-use SMLM data analysis software for certain image analysis tasks.But,this kind of software did not pay su±cient attention to the impact of imaging artifacts on the analysis accuracy,and usually contained only one type of analysis task.Therefore,users are still facing di±culties when they want to have the combined use of different types of analysis methods according to the characteristics of their data and their own needs.In this paper,we report an ImageJ plug-in called DecodeSTORM,which not only has a simple GUI for human–computer interaction,but also combines artifact correction with several quantitative analysis methods.DecodeSTORM includes format conversion,channel registration,artifact correction(drift correction and localization¯ltering),quantitative analysis(segmentation and clustering,spatial distribution statistics and colocalization)and visualization.Importantly,these data analysis methods can be combined freely,thus improving the accuracy of quantitative analysis and allowing users to have an optimal combination of methods.We believe DecodeSTORM is a user-friendly and powerful ImageJ plug-in,which provides an easy and accurate data analysis tool for adventurous biologists who are looking for new imaging tools for studying important questions in cell biology.
基金supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology(No. 2015R1A2A2A01003839)
文摘Fluorescent dye （YOYO-I） intercalated with single DNA molecules were investigated via bindingactivated localization microscopy （BALM） at sub-diffraction limit resolutions. Various dye-to-DNA base pair （bp） ratios were imaged using the blinking property of YOYO-1 dye under optimum BALM switching buffer conditions. Individual DNA molecules exhibited regular/irregular intercalating phenomena with respect to dye-to-DNA bp ratio. The acquired images were reconstructed into super-resolution images by applying a Gaussian fit to the centroid of the point spread function. The YOYO-1 intercalated with λ-DNA possessed a non-homogeneous region due to the different binding modes of YOYO-1 with λ-DNA. Each binding mode was imaged at the sub-diffraction limit super-resolution. The distance between homogenously localized intercalating dyes within the DNA molecules was measured to be 34nm （n= 10; dye：DNAbp= 1：100） without photocleavage in 50mmol/L β-mercaptoethylamine buffer. The results were similar to those of the theoretical values without photocleavage in the base pairs of single DNA molecules below the diffraction limit. The results paved the way for an in-depth microscopic analysis of molecular variation with single λ-DNA molecules. With this method, it should be possible to analyze the exact base pair breakdown during various stages of cell apoptosis.
基金supported by NIH grants U54CA268084,U54CA261694,and R01CA228272National Science Foundation grants EFMA-1830961 and CBET-2430743+1 种基金philanthropic support from Rob and Kristin Goldman,Mr.David Sachsthe Christina Carinato Charitable Foundation.
文摘Super-resolution microscopy has revolutionized our ability to visualize structures below the diffraction limit of conventional optical microscopy and is particularly useful for investigating complex biological targets like chromatin.Chromatin exhibits a hierarchical organization with structural compartments and domains at different length scales,from nanometers to micrometers.Single molecule localization microscopy(SMLM)methods,such as STORM,are essential for studying chromatin at the supra-nucleosome level due to their ability to target epigenetic marks that determine chromatin organization.Multi-label imaging of chromatin is necessary to unpack its structural complexity.However,these efforts are challenged by the high-density nuclear environment,which can affect antibody binding affinities,diffusivity and non-specific interactions.Optimizing buffer conditions,fluorophore stability,and antibody specificity is crucial for achieving effective antibody conjugates.Here,we demonstrate a sequential immunolabeling protocol that reliably enables three-color studies within the dense nuclear environment.This protocol couples multiplexed localization datasets with a robust analysis algorithm,which utilizes localizations from one target as seed points for distance,density and multi-label joint affinity measurements to explore complex organization of all three targets.Applying this multiplexed algorithm to analyze distance and joint density reveals that heterochromatin and euchromatin are not-distinct territories,but that localization of transcription and euchromatin couple with the periphery of heterochromatic clusters.This work is a crucial step in molecular imaging of the dense nuclear environment as multi-label capacity enables for investigation of complex multi-component systems like chromatin with enhanced accuracy.
基金supported by the National Natural Science Foundation of China(No.62305320)。
文摘Single-molecule localization microscopy(SMLM)has pushed resolution to sub-40 nm.Combined with structured illumination,lateral resolution can be doubled or the axial resolution can be improved fourfold.However,current techniques are challenging in balancing the lateral and axial resolutions.Here,we report a new modulated illumination single-molecule localization modality,isoFLUX.Utilizing two objective lenses to form interference patterns along the x-z and y-z directions,the lateral and axial resolutions can be improved simultaneously.Compared to SMLM,isoFLUX maintains a twofold average enhancement in both lateral and axial resolutions under an astigmatic point spread function(PSF),1.5-fold in the lateral resolution and 2.5-fold in the axial resolution under the saddle-point PSF.
基金funded by the Natural Science Foundation of Liaoning Province(2023-MS-103),and the National Natural Science Foundation of China(62305041).
文摘Single-molecule localization microscopy(SMLM)surpasses the diffraction limit by randomly switching fluorophores between fluorescent and dark states,precisely pinpointing the resulted isolated emission patterns,thereby reconstructing the superresolution images based on the accumulated locations of thousands to millions of single molecules.This technique achieves a ten-fold improvement in resolution,unveiling the intricate details of molecular activities and structures in cells and tissues.Multicolor SMLM extends this capability by imaging distinct protein species labeled with various fluorescent probes,providing insights into structural intricacies and spatial relationships among different targets.This review explores recent advancements in multicolor SMLM,evaluates the strengths and limitations of each variant,and discusses the future prospects.
基金supported by the National Natural Science Foundation of China(Nos.62105368,62275268,and 62375284)the Science and Technology Innovation Program of Hunan Province(No.2023RC3010)。
文摘The 3D location and dipole orientation of light emitters provide essential information in many biological,chemical,and physical systems.Simultaneous acquisition of both information types typically requires pupil engineering for 3D localization and dual-channel polarization splitting for orientation deduction.Here we report a geometric phase helical point spread function for simultaneously estimating the 3D position and dipole orientation of point emitters.It has a compact and simpler optical configuration compared to polarization-splitting techniques and yields achromatic phase modulation in contrast to pupil engineering based on dynamic phase,showing great potential for single-molecule orientation and localization microscopy.
基金funded by the National Natural Science Foundation of China(No.62305041)the Natural Science Foundation of Liaoning Province(No.2023-MS-103)。
文摘Stochastic optical reconstruction microscopy(STORM),as a typical technique of single-molecule localization microscopy(SMLM),has overcome the diffraction limit by randomly switching fluorophores between fluorescent and dark states,allowing for the precise localization of isolated emission patterns and the super-resolution reconstruction from millions of localized positions of single fluorophores.A critical factor influencing localization precision is the photo-switching behavior of fluorophores,which is affected by the imaging buffer.The imaging buffer typically comprises oxygen scavengers,photo-switching reagents,and refractive index regulators.Oxygen scavengers help prevent photobleaching,photo-switching reagents assist in facilitating the conversion of fluorophores,and refractive index regulators are used to adjust the refractive index of the solution.The synergistic interaction of these components promotes stable blinking of fluorophores,reduces irreversible photobleaching,and thereby ensures high-quality super-resolution imaging.This review provides a comprehensive overview of the essential compositions and functionalities of imaging buffers used in STORM,serving as a valuable resource for researchers seeking to select appropriate imaging buffers for their experiments.
基金supported by the National Natural Science Foundation of China(Nos.22225806,22078314,22278394,22378385)Dalian Institute of Chemical Physics(Nos.DICPI202227,DICPI202436)。
文摘Monitoring the dynamics of cellular pseudopodia at nanoscale has become essential for understanding their diverse and complex functions in living cells.This is made possible by combining single-molecule localization microscopy(SMLM)with self-blinking dyes.However,existing self-blinking dyes often face limitations,such as nonspecific blinking and low photostability,which can bring background noise and yield erroneous localization signals,hindering their effectiveness for nanoscale visualization.Here,we present a method for long-term SMLM imaging of cellular pseudopodia dynamics using a blinkogenic probe that exhibits self-blinking activation upon molecular recognition.This approach enabled the precise tracking of various pseudopodia structures,including filopodia,lamellipodia,and(tunneling nanotubes)-nanoscale(TNTs),in living cells.We monitored the growth and fusion of filopodia,as well as the extension and shrinkage of lamellipodia,in real-time.Additionally,we identified two distinct fusion modes between filopodia and lamellipodia and captured the formation of TNTs and their interactions with filopodia,demonstrating the probe's utility in visualizing real-time pseudopodia dynamics at nanoscale.
基金This work was supported by the Innovation Fund of WNLO(2018WNLOKF023)the Start-up Fund of Hainan University(KYQD(ZR)-20077).
文摘Hematologic malignancies are one of the most common malignant tumors caused by the clonal proliferation and differentiation of hematopoietic and lymphoid stem cells.The examination of bone marrow cells combined with immunodeficiency typing is of great significance to the diagnostic type,treatment and prognosis of hematologic malignancies.Super-resolution fluorescence microscopy(SRM)is a special kind of optical microscopy technology,which breaks the resolution limit and was awarded the Nobel Prize in Chemistry in 2014.With the development of SRM,many related technologies have been applied to the diagnosis and treatment of clinical diseases.It was reported that a major type of SRM technique,single molecule localization microscopy(SMLM),is more sensitive than flow cytometry(FC)in detecting cell membrane antigens'expression,thus enabling better chances in detecting antigens on hematopoietic cells than traditional analytic tools.Furthermore,SRM may be applied to clinical pathology and may guide precision medicine and personalized medicine for clone hematopoietic cell diseases.In this paper,we mainly discuss the application of SRM in clone hematological malignancies.
基金supported by the European Research Council,BrightEyes No.818699(E.S.,S.P.,M.O.H.,G.V.)the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie Grant Agreement No.890923(SMSPAD)(E.S.).
文摘The MINFLUX concept significantly improves the localization properties of single-molecule localization microscopy(SMLM)by overcoming the limit imposed by the fluorophore's photon counts.Typical MINFLUX microscopes localize the target molecule by scanning a zero-intensity focus around the molecule in a circular trajectory,with smaller trajectory diameters yielding better localization uncertainties for a given number of photons.Since this approach requires the molecule to be within the scanned trajectory,MINFLUX typically relies on an iterative scheme with decreasing trajectory diameters.This iterative approach is prone to misplacements of the trajectory and increases the system's complexity.In this work,we introduce ISM-FLUX,a novel implementation of MINFLUX using image-scanning microscopy(ISM)with a single-photon avalanche diode array detector.ISM-FLUX provides a precise MINFLUX localization within the trajectory while maintaining a conventional photon-limited uncertainty outside it.The robustness of ISM-FLUX localization results in a larger localization range and greatly simplifies the architecture,which may facilitate broader adoption of MINFLUX.
基金supported by National Natural Science Foundation of China(Nos. 91527303, 21333001,21373020, 61321001)MOST(Nos.2013CB933404,2014CB239302),China
文摘Work function plays a significant role in surface chemistry. Local work function provides the information of local d/pole-d/pole interaction and charge distribution between adsorbates and substrate, highlighting the local charge effect of the targeted spot which is normally smeared out in conventional average work function measurements. Chloroaluminum phthalocyanine （CIA1Pc）, an important optoelectronic molecule with a permanent dipole moment pointing from the Pc ring to the ending CI atom, adsorbed on Au（111） in either Cl-up or Cl-down configuration. Scanning tunneling microscopy/spectroscopy measurements revealed that at the centers of Cl-up and CI-down molecules, the local work functions changed oppositely with respect to the Au（111） substrate. At their Pc lobes, however, the local work functions unanimously increased due to charging effect of the indole lobes in the CIAIPc molecule.
基金supported by the National Natural Science Foundation of China(62125504,61827825,62205288)China Postdoctoral Science Foundation(2021TQ0275,2022M712734)。
文摘This article reviews recent super-resolution(SR)optical imaging techniques for cellular study,encompassing structured illumination microscopy(SIM),point-scanning super-resolution(PS-SR)microscopy,single-molecule localization microscopy(SMLM),mathematical and deep learning(DL)SR algorithms.Historically,the resolution of traditional far-field optical imaging was constrained by the diffraction limit.The emergence of SR imaging techniques and image processing algorithms has propelled biological research into nanoscale realm.SIM enhances resolution by manipulating spatial frequency content,effectively doubling the resolution capacity of traditional microscopy.PS-SR imaging,on the other hand,offers superior optical sectioning and a high signal-to-noise ratio.SMLM has achieved a remarkable spatial resolution of approximately 20 nm and supports multi-color,wide-field-of-view(FOV),automated 3D high-throughput imaging,thus broadening the horizons for advanced biomedical investigations.Additionally,both mathematical and DL-based SR algorithms have significantly advanced the conversion of low-resolution images to high-resolution counterparts,extending the capabilities of conventional microscopes.This review underscores the principles,recent developments,and diverse applications of these cutting-edge SR imaging methodologies in biological research.
基金National Key Research and Development Program of China(2024YFF0726003)Shenzhen Medical Research Fund(B2302038)+8 种基金National Natural Science Foundationof China(62375116)Key,Technology Research and Development Program of Shandong Province(2021CXGC010212)Shenzhen Science and Technology Innovation Program JCYJ20220818100416036,KQTD20200820113012029)BasicandApplied Basic Research Foundation of Guangdong Province(2024A1515011565)Postdoctoral Fellowship Program of CPSF(GZC20240651)GuangdongProvincial Key Laboratory of Advanced Biomaterials(2022B1212010003)Startup Grant from Southern University of Science and TechnologyCenter for Computational Science and Engineering of Southern University of Science and Technology。
文摘Single-molecule localization microscopy(SMLM)provides nanoscale imaging,but pixel integration of acquired SMLM images limited the choice of sampling rate,which restricts the information content conveyed within each image.We propose an upsampled point spread function(PSF)inverse modeling method for large-pixel singlemolecule localization,enabling precise three-dimensional superresolution imaging with a sparse sampling rate.
基金supported by the Natural Science Foundation of Hunan Province(2022JJ20005)National Natural Science Foundation of China(22174038,21925401,and 52221001),and Tencent Foundation.
文摘Endogenous biomolecules in cells are the basis of all life activities.Directly visualizing the structural characteristics and dynamic behaviors of cellular biomolecules is signiffcant for understanding the molecular mechanisms in various biological processes.Singlemolecule localization microscopy(SMLM)can circumvent the optical diffraction limit,achieving analysis of the ffne structures and biological processes in living cells with nanoscale resolution.However,the large size of traditional imaging probes prevents SMLM from accurately locating ffne structures and densely distributed biomolecules within cells.In recent years,nucleic acid probes have emerged as potential tools to replace conventional SMLM probes by virtue of their small size and high speciffcity.In addition,due to their programmability,nucleic acid probes with different conformations can be constructed via sequence design,further extending the application of SMLM in bioanalysis.Here,we discuss the design concepts of different conformational nucleic acid probes for SMLM and summarize the application of SMLM based on nucleic acid probes in the ffeld of biomolecules.Furthermore,we provide a summary and future perspectives of the nucleic acid probe-based SMLM technology,aiming to provide guidance for the acquisition of nanoscale information about cellular biological processes.
文摘Acentral theme of chemical imaging is the visualization of chemistry in situ in functional materials and in vivo in a living system.To achieve this,technologies with exceptional detection sensitivity and spatial resolution are essential for resolving specific chemicals at the nanoscale.As we celebrate the 10th anniversary of the Nobel Prize in Chemistry awarded for super-resolved fluorescence microscopy,the field’s remarkable advancements become clear.Over the past 10 years,novel microscopies that break light diffraction limits continue to be invented.These include,for example,midinfrared photothermal microscopy that breaks the diffraction limit in IR imaging,and expansion microscopy that allows super-resolution fluorescence imaging on a conventional microscope.Meanwhile,researchers have much increased the speed of structural illumination microscopy(SIM)and single molecule localization microscopy(SMLM),and pushed the resolution limit of stimulated emission depletion(STED)microscopy to angstrom scale.In parallel,data science and artificial intelligence(AI)are elegantly applied to break the diffraction limits of images recorded on a conventional microscope.These innovations have enabled very exciting applications,including the study of single particle catalysis,biomolecule trafficking inside a live cell,dynamic imaging of cellular organelles,and many others.
基金Acknowledgements This work was supported by the National Natural Sdence Foundation of China （Nos. 91027037, 21127009, 21425519 and 21221003）, Hunan University 985 fund, Tsinghua University Startup fund, the Natural Science Foundation of Zhejiang Province （No. LY16B050006） and Wenzhou Medical University Setup fund （No. QTJ15022）.
文摘There is an increasing demand for advanced optical imaging techniques that can detect and resolve nanosize objects at a spatial resolution below the optical diffraction limit, especially in three-dimensional （3D） cellular environments. In this study, using a polarization-activated localization scheme based on the orientation-dependent properties of anisotropic plasmonic metal nanoparticles （MNPs）, ＂photoswitchable＂ imaging of single gold nanorods （AuNRs） was accomplished not only in two dimensions but also in three dimensions. Moreover, the Rayleigh scattering background arising from the congested subcellular structures was efficiently suppressed. Thus, we obtained the 3D distributions of both the position and the orientation of the AuNRs inside the cells and investigated their intemalization kinetics. To our knowledge, this is the first demonstration of the confocal-like 3D imaging of non-fluorescence nanoparticles with a high resolution and almost zero background. This technique is easy to implement and should greatly facilitate MNP studies and applications in biomedicine and biology.
基金support by the NSF(CHE-1255440)the NIH(COBRE P30 GM103450)。
文摘Quantum dots(QDs)can be used as fluorescent probes in single molecule localization microscopy to achieve subdiffraction limit resolution(super-resolution fluorescence imaging).However,the toxicity of Cd in the prototypical CdSe-based QDs can limit their use in biological applications.Furthermore,commercial CdSe QDs are usually modified with relatively thick shells of both inorganic and organic materials to render them in the 10−20 nm size range,which is relatively large for biological labels.In this report,we present compact(4−6 nm)CuInS2/ZnS(CIS/ZnS)and compare them to commercially sourced CdSe/ZnS QDs for their blinking behavior,localization precision and super-resolution imaging.Although commercial CdSe/ZnS QDs are brighter than the more compact Cd-free CIS/ZnS QD,both give comparable results of 4.5−5.0-fold improvement in imaging resolution over conventional TIRF imaging of actin filaments.This likely results from the fact that CIS/ZnS QDs show very short on-times and long off times which leads to less overlap in the point spread functions of emitting CIS/ZnS QD labels on the actin filaments at the same labeling density.These results demonstrate that CIS/ZnS QDs are an excellent candidate to complement and perhaps even replace the larger and more toxic CdSe-based QDs for robust single-molecule super-resolution imaging.
文摘Single-molecule localization microscopy circumvents the diffraction limit of traditional fluorescence microscopy by detecting the photoemission signals of individual fluorescent molecules.The accurate recognitions of fluorescence molecules/events are critical to single-molecule/super-resolution imaging experiments,which determine the precision of molecular localizations and the quality of the image reconstruction.Herein,we presented a single-molecule detection method which relied on the temporal pixel intensity fluctuation.The method was capable of quickly determining the approximate localizations of fluorescence events with high sensitivity.We evaluated the performance of the method under a series of signal-to-noise ratios(SNR)and discussed the criterion of setting the temporal fluctuation threshold to achieve the optimized spots recognition results.
