Since the 1990s,continuous technical and scientific advances have defied the diffraction limit in microscopy and enabled three-dimensional(3D)super-resolution imaging.An important milestone in this pursuit is the cohe...Since the 1990s,continuous technical and scientific advances have defied the diffraction limit in microscopy and enabled three-dimensional(3D)super-resolution imaging.An important milestone in this pursuit is the coherent utilization of two opposing objectives(4Pi geometry)and its combination with superresolution microscopy.Herein,we review the recent progress in 4Pi nanoscopy,which provides a 3D,non-invasive,diffraction-unlimited,and isotropic resolution in transparent samples.This review includes both the targeted and stochastic switching modalities of 4Pi nanoscopy.The schematics,principles,applications,and future potential of 4Pi nanoscopy are discussed in detail.展开更多
The MINimal emission FLUXes(MINFLUX)technique in optical microscopy,widely recognized as the next innovative fluorescence microscopy method,claims a spatial resolution of 1-3 nm in both dead and living cells.To make u...The MINimal emission FLUXes(MINFLUX)technique in optical microscopy,widely recognized as the next innovative fluorescence microscopy method,claims a spatial resolution of 1-3 nm in both dead and living cells.To make use of the full resolution of the MINFLUX microscope,it is important to select appropriate fluorescence probes and labeling strategies,especially in living-cell imaging.This paper mainly focuses on recent applications and developments of fluorescence probes and the relevant labeling strategy for MINFLUX microscopy.Moreover,we discuss the deficiencies that need to be addressed in the future and a plan for the possible progression of MINFLUX to help investigators who have been involved in or are just starting in the field of super-resolution imaging microscopy with theoretical support.展开更多
A thorough understanding of biological species and emerging nanomaterials requires,among other efforts,their in-depth characterization through optical techniques capable of nanoresolution.Nanoscopy techniques based on...A thorough understanding of biological species and emerging nanomaterials requires,among other efforts,their in-depth characterization through optical techniques capable of nanoresolution.Nanoscopy techniques based on tip-enhanced optical effects have gained tremendous interest over the past years,given their potential to obtain optical information with resolutions limited only by the size of a sharp probe interacting with focused light,irrespective of the illumination wavelength.Although their popularity and number of applications is rising,tip-enhanced nanoscopy(TEN)techniques still largely rely on probes that are not specifically developed for such applications,but for atomic force microscopy.This limits their potential in many regards,e.g.,in terms of signal-to-noise ratio,attainable image quality,or extent of applications.We take the first steps toward next-generation TEN by demonstrating the fabrication and modeling of specialized TEN probes with known optical properties.The proposed framework is highly flexible and can be easily adjusted to be used with diverse TEN techniques,building on various concepts and phenomena,significantly augmenting their function.Probes with known optical properties could potentially enable faster and more accurate imaging via different routes,such as direct signal enhancement or facile and ultrafast optical signal modulation.We consider that the reported development can pave the way for a vast number of novel TEN imaging protocols and applications,given the many advantages that it offers.展开更多
Minimal photon fluxes(MINFLUX)nanoscopy has emerged as a transformative advancement in superresolution imaging,enabling unprecedented nanoscale observations across diverse biological scenarios.In this work,we propose,...Minimal photon fluxes(MINFLUX)nanoscopy has emerged as a transformative advancement in superresolution imaging,enabling unprecedented nanoscale observations across diverse biological scenarios.In this work,we propose,for the first time,that employing high-order vortex beams can significantly enhance the performance of MINFLUX,surpassing the limitations of the conventional MINFLUX using the first-order vortex beam.Our theoretical analysis indicates that,for standard MINFLUX,high-order vortex beams can improve the maximum localization precision by a factor corresponding to their order,which can approach a sub-nanometer scale under optimal conditions,and for raster scan MINFLUX,high-order vortex beams allow for a wider field of view while maintaining enhanced precision.These findings underscore the potential of high-order vortex beams to elevate the performance of MINFLUX,paving the way towards ultra-high resolution imaging for a broad range of applications.展开更多
Surface plasmons are collective excitations of conduction electrons situated at the metal-dielectric interface,resulting in markedly enhanced light–matter interactions.The high local field intensity has enabled a wid...Surface plasmons are collective excitations of conduction electrons situated at the metal-dielectric interface,resulting in markedly enhanced light–matter interactions.The high local field intensity has enabled a wide range of novel physical phenomena and innovative applications.However,the small mode volume and the femtosecond dynamics necessitate rigorous experimental conditions for complete characterizations.The demand for subwavelength resolution has outpaced the capabilities of conventional methods,prompting the development of novel characterization instruments.These instruments utilize two categories of probes with exceptional resolution:nanoscale tips and electron beams.The former has led to the emergence of scanning near-field optical microscopies,while the latter has resulted in electron nanoscopies.These technologies offer ultrahigh spatiotemporal resolutions in the multi-dimensional characterization of surface plasmons.Although advanced characterization technologies have promoted multi-dimensional manipulations of surface plasmons,quantum detection is still a challenge for them.This review article provides a comprehensive overview of the recent advances in plasmonics from the perspectives of near-field optics and electron nanoscopy.It introduces the latest characterization technologies and the manipulation of surface plasmons,including their spatial distribution,energy,momentum,and polarization.Additionally,the article describes advances and challenges in quantum plasmonics and the upgrade of characterization as a potential technical solution.Keywords:surface plasmons;plasmonics;electron nanoscopy;cathodoluminescence.展开更多
Deterministic three-dimensional(3D)super-resolution microscopy can achieve light-matter interaction in a small volume,but usually with the axial extension distinctly more elongated than the lateral one.The isoSTED met...Deterministic three-dimensional(3D)super-resolution microscopy can achieve light-matter interaction in a small volume,but usually with the axial extension distinctly more elongated than the lateral one.The isoSTED method combining two opposing objectives and multiple laser beams can offer high axial extension atλ/12 level,but at the cost of optical system complexity and inherent sidelobes.The high-order nonlinear effect by multiphoton excitation would benefit to achieve a sub-diffraction resolution as well as to suppress the sidelobes.Herein,to achieve an easyto-use,sidelobe-free deterministic 3D nanoscopy with high axial resolution,we developed a purely physical deterministic strategy(UNEx-4Pi)by fusion of ultrahighly nonlinear excitation(UNEx)of photon avalanching nanoparticles and mirror-based bifocal vector field modulation(4Pi).The theoretical studies of UNEx-4Pi concept showed that the main peak of fluorescence spot became sharper and its large sidelobe height was suppressed with the increasing optical nonlinearity.In addition,the simplicity and robustness of UNEx-4Pi system were demonstrated utilizing a mirror-assisted single-objective bifocal self-interference strategy.Experimentally,UNEx-4Pi realized an extremely constringent focal spot without sidelobes observed,achieving an axial resolution up toλ/33(26 nm)using one low-power CW beam.We also demonstrated the super-resolution ability of the UNEx-4Pi scheme to bioimaging and nuclear envelope of BSC-1 cells were stained and imaged at an axial resolution of 32 nm.The proposed UNEx-4Pi method will pave the way for achieving light-matter interaction in a highly confined space,thereby advancing cuttingedge technologies like deterministic super-resolution sensing,imaging,lithography,and data storage.展开更多
Stimulated emission depletion microscopy(STED)holds great potential in biological science applications,especially in studying nanoscale subcellular structures.However,multi-color STED imaging in live-cell remains chal...Stimulated emission depletion microscopy(STED)holds great potential in biological science applications,especially in studying nanoscale subcellular structures.However,multi-color STED imaging in live-cell remains challenging due to the limited excitation wavelengths and large amount of laser radiation.Here,we develop a multiplexed live-cell STED method to observe more structures simultaneously with limited photo-bleaching and photo-cytotoxicity.By separating live-cell fluorescent probes with similar spectral properties using phasor analysis,our method enables five-color live-cell STED imaging and reveals long-term interactions between different subcellular structures.The results here provide an avenue for understanding the complex and delicate interactome of subcellular structures in live-cell.展开更多
Fibroblasts support a broad range of essential organ functions via microarchitectural,biomechanical,and biochemical cues.Despite great advances in fluorescence,photoacoustic conversion,and Raman scattering over the pa...Fibroblasts support a broad range of essential organ functions via microarchitectural,biomechanical,and biochemical cues.Despite great advances in fluorescence,photoacoustic conversion,and Raman scattering over the past decades,their invasiveness and limited spatial resolution hinder the characterization of fibroblasts in a single cell.Here,taking mouse embryonic fibroblasts(MEFs)as an example,we propose a novel noninvasive approach to investigate the compositional distribution of MEFs at the single-cell scale via terahertz(THz)nanos⁃copy.Compared to the topological morphology,THz nano-imaging enables the component-based visualization of MEFs,such as the membrane,cytoplasm,nucleus,and extracellular vesicles(EVs).Notably,we demonstrate the real-space observation of the influence of rapamycin treatment on the increase of EVs in MEFs.Moreover,the line-cut and area-statistical analysis establishes the relationship between the topological morphology and the THz near-field amplitudes for different cellular components of MEFs.This work provides a new pathway to char⁃acterize the effects of pharmaceutical treatments,with potential applications in disease diagnosis and drug devel⁃opment.展开更多
The resolution of conventional optical equipment is always restricted by the diffraction limit,and improving on this was previously considered improbable.Optical super-resolution imaging,which has recently experienced...The resolution of conventional optical equipment is always restricted by the diffraction limit,and improving on this was previously considered improbable.Optical super-resolution imaging,which has recently experienced rapid growth and attracted increasing global interest,will result in applications in many domains,benefiting fields such as biology,medicine and material research.This review discusses the contributions of different researchers who identified the diffractive barrier and attempted to realize optical super-resolution.This is followed by a personal viewpoint of the development of optical nanoscopy in recent decades and the road towards the next generation of optical nanoscopy.展开更多
Many kinds of nano particles and organic dyes as fluorescent probes have been used in the stimulated emission depletion(STED)nanoscopy.Due to high toxicity,photobleaching and non-water solubility,these fluorescent pro...Many kinds of nano particles and organic dyes as fluorescent probes have been used in the stimulated emission depletion(STED)nanoscopy.Due to high toxicity,photobleaching and non-water solubility,these fluorescent probes are hard to apply in living cell imaging.Here,we reporta new fluorescence carbon dots(FNCDs)with high photoluminescence quantum yield(56%),low toxicity,anti-photobleaching and goodwater-solubility that suitable for live-cell imaging can be obtained by doping fluorine element.Moreover,the FNCDs can stain the nucleolusand tunneling nanotubes(TNTs)in the living cell.More importantly,for STED nanoscopy imaging,the FNCDs effectively depleted backgroundsignals and improved imaging resolution.Furthermore,the lateral resolution of single FNCDs size under the STED nanoscopy is up to 22.1 nm for FNCDs deposited on a glass slide was obtained.And because of their good water dispersibility,the higher resolution of single FNCDs sizein the nucleolus of a living cell can be up to 19.7 nm.After the image optimizati on steps,the fine fluoresce nee images of TNTs diameter with ca.75 nm resolution is obtained living cell,yielding a threefold enhancement compared with that in confocal imaging.Additionally,the FNCDs show excellent photobleaching resistance after 1,000 scan cycles in the STED model.All results show that FNCDs have significant potentialfor application in STED nanoscopy.展开更多
Black phosphorus(BP)is an emerging two-dimensional material with intriguing physical properties.It is highly anisotropic and highly tunable by means of both the number of monolayers and surface doping.Here,we experime...Black phosphorus(BP)is an emerging two-dimensional material with intriguing physical properties.It is highly anisotropic and highly tunable by means of both the number of monolayers and surface doping.Here,we experimentally investigate and theoretically interpret the near-field properties of a-few-atomic-monolayer nanoflakes of BP.We discover near-field patterns of bright outside fringes and a high surface polarizability of nanofilm BP consistent with its surface-metallic,plasmonic behavior at mid-infrared frequencies o1176 cm−1.We conclude that these fringes are caused by the formation of a highly polarizable layer at the BP surface.This layer has a thickness of~1 nm and exhibits plasmonic behavior.We estimate that it contains free carriers in a concentration of n≈1.1×10^(20) cm^(−3).Surface plasmonic behavior is observed for 10–40 nm BP thicknesses but absent for a 4-nm BP thickness.This discovery opens up a new field of research and potential applications in nanoelectronics,plasmonics and optoelectronics.展开更多
Fluorescence nanoscopy has become increasingly powerful for biomedical research,but it has historically afforded a small field-ofview(FOV)of around 50μm×50μm at once and more recently up to∼200μm×200μm....Fluorescence nanoscopy has become increasingly powerful for biomedical research,but it has historically afforded a small field-ofview(FOV)of around 50μm×50μm at once and more recently up to∼200μm×200μm.Efforts to further increase the FOV in fluorescence nanoscopy have thus far relied on the use of fabricated waveguide substrates,adding cost and sample constraints to the applications.Here we report PRism-Illumination and Microfluidics-Enhanced DNA-PAINT(PRIME-PAINT)for multiplexed fluorescence nanoscopy across millimeter-scale FOVs.Built upon the well-established prism-type total internal reflection microscopy,PRIME-PAINT achieves robust singlemolecule localization with up to∼520μm×520μm single FOVs and 25−40 nm lateral resolutions.Through stitching,nanoscopic imaging over mm^(2)sample areas can be completed in as little as 40 min per target.An on-stage microfluidics chamber facilitates probe exchange for multiplexing and enhances image quality,particularly for formalin-fixed paraffin-embedded(FFPE)tissue sections.We demonstrate the utility of PRIME-PAINT by analyzing∼106 caveolae structures in∼1,000 cells and imaging entire pancreatic cancer lesions from patient tissue biopsies.By imaging from nanometers to millimeters with multiplexity and broad sample compatibility,PRIMEPAINT will be useful for building multiscale,Google-Earth-like views of biological systems.展开更多
Myasthenia gravis is a rare and invalidating disease affecting the neuromuscular junction of voluntary muscles.The classical form of this autoimmune disease is characterized by the presence of antibodies against the m...Myasthenia gravis is a rare and invalidating disease affecting the neuromuscular junction of voluntary muscles.The classical form of this autoimmune disease is characterized by the presence of antibodies against the most abundant protein in the neuromuscular junction,the nicotinic acetylcholine receptor.Other variants of the disease involve autoimmune attack of non-receptor scaffolding proteins or enzymes essential for building or maintaining the integrity of this peripheral synapse.This review summarizes the participation of the above proteins in building the neuromuscular junction and the destruction of this cholinergic synapse by autoimmune aggression in myasthenia gravis.The review also covers the application of a powerful biophysical technique,superresolution optical microscopy,to image the nicotinic receptor in live cells and follow its motional dynamics.The hypothesis is entertained that anomalous nanocluster formation by antibody crosslinking may lead to accelerated endocytic internalization and elevated turnover of the receptor,as observed in myasthenia gravis.展开更多
Cholesterol is a major lipid in biological membranes.It not only plays a structural role but also modulates a wide range of functional properties of neurotransmitter and hormone receptors and ion channels.The membrane...Cholesterol is a major lipid in biological membranes.It not only plays a structural role but also modulates a wide range of functional properties of neurotransmitter and hormone receptors and ion channels.The membraneembedded segments of the paradigm neurotransmitter receptor for acetylcholine(nAChR)contain linear sequences of amino acids with the capacity to recognize cholesterol.These cholesterol consensus domains have been designated as“CARC”and its mirror sequence“CRAC”.CARC preferentially occurs in the exoplasmic-facing membrane leaflet,and CRAC,in the cytoplasmic-facing hemilayer.Both motifs are highly conserved among ion-channel and neurotransmitter receptor proteins in vertebrate nervous systems,where they recognize cholesterol,and in prokaryotic homologues in bacteria,where they recognize hopanoids.This phylogenetically conserved trait is an indication that the hopanoids in some bacteria and cholesterol in eukaryotes subserve analogous functions,probably contributing to the stability of membrane-embedded protein domains.Structural studies from our laboratory using superresolution optical microscopy(“nanoscopy”)have disclosed other interrelated functional and structural properties exerted by cholesterol on the nAChR.The neutral lipid content at the cell surface influences both the macromolecular organization of the receptor and its translational mobility(diffusion)in the plane of the membrane.展开更多
A discrete model of the Differential Evanescent Light Intensity (DELI) technique was developed to calculate and map 3D nanolayers thicknesses from the evanescent light intensity captured from optical waveguides. The m...A discrete model of the Differential Evanescent Light Intensity (DELI) technique was developed to calculate and map 3D nanolayers thicknesses from the evanescent light intensity captured from optical waveguides. The model was used for ultra-thin Pd nanometric layers sputtered on glass substrates. The layers thickness profiles were displayed in 3D and 1D profiles plots. The total thickness profiles of the ultra-thin Pd films obtained in the range of 1-10 nm were validated using AFM measurements. Based on the model developed the evanescent photon extraction parameter of the material was estimated.展开更多
The photo-kinetics of fluorescent molecules have enabled the circumvention of the far-field optical diffraction limit.Despite its enormous potential,the necessity to label the sample may adversely influence the delica...The photo-kinetics of fluorescent molecules have enabled the circumvention of the far-field optical diffraction limit.Despite its enormous potential,the necessity to label the sample may adversely influence the delicate biology under investigation.Thus,continued development efforts are needed to surpass the far-field label-free diffraction barrier.The statistical similarity or finite coherence of the scattered light off the sample in label-free mode hinders the application of existing super-resolution methods based on incoherent fluorescence imaging.In this article,we present physics and propose a methodology to circumvent this challenge by exploiting the photoluminescence(PL)of silicon nitride waveguides for near-field illumination of unlabeled samples.The technique is abbreviated EPSLON,Evanescently decaying Photoluminescence Scattering enables Label-free Optical Nanoscopy.We demonstrate that such an illumination has properties that mimic the photo-kinetics of nano-sized fluorescent molecules,i.e.,such an illumination permits incoherence between the scattered fields from various locations on the sample plane.Thus,the illumination scheme enables the development of a far-field label-free incoherent imaging system that is linear in intensity and stable over time,thereby permitting the application of techniques like structured illumination microscopy(SIM)and intensity-fluctuation-based optical nanoscopy(IFON)in label-free mode to circumvent the diffraction limit.In this proof-of-concept work,we observed a two-point resolution of~180 nm on super-resolved nanobeads and resolution improvements between 1.9×to 2.8×over the diffraction limit,as quantified using Fourier Ring Correlation(FRC),on various biological samples.We believe EPSLON is a step forward within the field of incoherent far-field label-free super-resolution microscopy that holds a key to investigating biological systems in their natural state without the need for exogenous labels.展开更多
Localisation microscopy overcomes the diffraction limit by measuring the position of individual molecules to obtain optical images with a lateral resolution better than 30 nm. Single molecule localisation microscopy w...Localisation microscopy overcomes the diffraction limit by measuring the position of individual molecules to obtain optical images with a lateral resolution better than 30 nm. Single molecule localisation microscopy was originally demonstrated only in two dimensions but has recently been extended to three dimensions. Here we develop a new approach to three-dimensional （3D） localisation microscopy by engineering of the point-spread function （PSF） of a fluorescence microscope. By introducing a linear phase gradient between the two halves of the objective pupil plane the PSF is split into two lateral lobes whose relative position depends on defocus. Calculations suggested that the phase gradient resulting from the very small tolerances in parallelism of conventional slides made from float glass would be sufficient to generate a two-lobed PSF. We demonstrate that insertion of a suitably chosen microscope slide that occupies half the objective aperture combined with a novel fast fitting algorithm for 3D localisation estimation allows nanoscopic imaging with detail resolution well below 100 nm in all three dimensions （standard deviations of 20, 16, and 42 nm in x, y, and z directions, respectively）. The utility of the approach is shown by imaging the complex 3D distribution of microtubules in cardiac muscle cells that were stained with conventional near infrared fluorochromes. The straightforward optical setup, minimal hardware requirements and large axial localisation range make this approach suitable for many nanoscopic imaging applications.展开更多
With super-resolution microscopy,we attempt to visualize(biological)structures and processes at the sub-cellular level(i.e.,nanoscale).To obtain this information,the samples are labeled with fluorophores that have a s...With super-resolution microscopy,we attempt to visualize(biological)structures and processes at the sub-cellular level(i.e.,nanoscale).To obtain this information,the samples are labeled with fluorophores that have a stochastic on/off switching of their emissions,which help to overcome the optical diffraction limit of around 250 nm,related to the use of optical micro-scopes.However,nowadays,research focuses on the imaging of live cells and thicker samples.These investigations require a high amount of simultaneously active fluorophores(i.e.,high-density imaging)and are challenging due to the collapse of the single-molecule localization techniques and the increased background in the image.Therefore,recent efforts have shifted towards the development of new ways to process the data.This publication gives an introduction to wide-field super-resolution fluorescence microscopy,explaining the concepts of the technique,and then gives an overview of the recently developed methods to provide super-resolution images for high-density data of live cells and ways to overcome the issues related to the imaging of these samples.展开更多
基金financially supported by the grants from National Key Research and Development Program of China (2018YFA0701400 and 2018YFE0119000)the Fundamental Research Funds for the Central Universities (2019QNA5006)+2 种基金ZJU-Sunny Photonics Innovation Center (2019-01)Zhejiang Provincial Natural Science Foundation of China (LR18H180001)startup grant from Southern University of Science and Technology
文摘Since the 1990s,continuous technical and scientific advances have defied the diffraction limit in microscopy and enabled three-dimensional(3D)super-resolution imaging.An important milestone in this pursuit is the coherent utilization of two opposing objectives(4Pi geometry)and its combination with superresolution microscopy.Herein,we review the recent progress in 4Pi nanoscopy,which provides a 3D,non-invasive,diffraction-unlimited,and isotropic resolution in transparent samples.This review includes both the targeted and stochastic switching modalities of 4Pi nanoscopy.The schematics,principles,applications,and future potential of 4Pi nanoscopy are discussed in detail.
基金supported by the Science and Technology Commission of Shanghai Municipality (21DZ1100500)the Shanghai Municipal Science and Technology Major Project+1 种基金the Shanghai Frontiers Science Center Program (2021-2025 No.20)Shanghai Hong Kong,Macao,and Taiwan Cooperation Project (No.19490760900).
文摘The MINimal emission FLUXes(MINFLUX)technique in optical microscopy,widely recognized as the next innovative fluorescence microscopy method,claims a spatial resolution of 1-3 nm in both dead and living cells.To make use of the full resolution of the MINFLUX microscope,it is important to select appropriate fluorescence probes and labeling strategies,especially in living-cell imaging.This paper mainly focuses on recent applications and developments of fluorescence probes and the relevant labeling strategy for MINFLUX microscopy.Moreover,we discuss the deficiencies that need to be addressed in the future and a plan for the possible progression of MINFLUX to help investigators who have been involved in or are just starting in the field of super-resolution imaging microscopy with theoretical support.
基金support of the Romanian Executive Agency for Higher Education,Research,Development and Innovation Funding(Grant Nos.RO-NO-2019-0601 MEDYCONAI and PN-III-P1-1.1-TE-2019-1339 OPTIGAN)the support of Horizon 2020 Attract(Phase 1)via the TEFPLASNOM project.
文摘A thorough understanding of biological species and emerging nanomaterials requires,among other efforts,their in-depth characterization through optical techniques capable of nanoresolution.Nanoscopy techniques based on tip-enhanced optical effects have gained tremendous interest over the past years,given their potential to obtain optical information with resolutions limited only by the size of a sharp probe interacting with focused light,irrespective of the illumination wavelength.Although their popularity and number of applications is rising,tip-enhanced nanoscopy(TEN)techniques still largely rely on probes that are not specifically developed for such applications,but for atomic force microscopy.This limits their potential in many regards,e.g.,in terms of signal-to-noise ratio,attainable image quality,or extent of applications.We take the first steps toward next-generation TEN by demonstrating the fabrication and modeling of specialized TEN probes with known optical properties.The proposed framework is highly flexible and can be easily adjusted to be used with diverse TEN techniques,building on various concepts and phenomena,significantly augmenting their function.Probes with known optical properties could potentially enable faster and more accurate imaging via different routes,such as direct signal enhancement or facile and ultrafast optical signal modulation.We consider that the reported development can pave the way for a vast number of novel TEN imaging protocols and applications,given the many advantages that it offers.
基金supported in part by the Academic Research Fund(AcRF)-Tier 2(A-8000117-01-00)and Tier 1(A-8003279-00-00)from the Ministry of Education(MOE)of Singapore,Science and Technology Project of Jiangsu Province(BZ2022056),NUS(Suzhou)Research Institute/Biomedical and Health Technology Platform,2024 Tsinghua-NUS Joint Research Initiative Fund(A-8002557-00-00)the National Medical Research Council(NMRC)(A-0009502-01-00,and A-8001143-00-00),Singapore.
文摘Minimal photon fluxes(MINFLUX)nanoscopy has emerged as a transformative advancement in superresolution imaging,enabling unprecedented nanoscale observations across diverse biological scenarios.In this work,we propose,for the first time,that employing high-order vortex beams can significantly enhance the performance of MINFLUX,surpassing the limitations of the conventional MINFLUX using the first-order vortex beam.Our theoretical analysis indicates that,for standard MINFLUX,high-order vortex beams can improve the maximum localization precision by a factor corresponding to their order,which can approach a sub-nanometer scale under optimal conditions,and for raster scan MINFLUX,high-order vortex beams allow for a wider field of view while maintaining enhanced precision.These findings underscore the potential of high-order vortex beams to elevate the performance of MINFLUX,paving the way towards ultra-high resolution imaging for a broad range of applications.
基金supported by the National Natural Science Foundation of China(Nos.12027807 and 62225501)the National Key R&D Program of China(No.2020YFA0211300)the High-Performance Computing Platform of Peking University.
文摘Surface plasmons are collective excitations of conduction electrons situated at the metal-dielectric interface,resulting in markedly enhanced light–matter interactions.The high local field intensity has enabled a wide range of novel physical phenomena and innovative applications.However,the small mode volume and the femtosecond dynamics necessitate rigorous experimental conditions for complete characterizations.The demand for subwavelength resolution has outpaced the capabilities of conventional methods,prompting the development of novel characterization instruments.These instruments utilize two categories of probes with exceptional resolution:nanoscale tips and electron beams.The former has led to the emergence of scanning near-field optical microscopies,while the latter has resulted in electron nanoscopies.These technologies offer ultrahigh spatiotemporal resolutions in the multi-dimensional characterization of surface plasmons.Although advanced characterization technologies have promoted multi-dimensional manipulations of surface plasmons,quantum detection is still a challenge for them.This review article provides a comprehensive overview of the recent advances in plasmonics from the perspectives of near-field optics and electron nanoscopy.It introduces the latest characterization technologies and the manipulation of surface plasmons,including their spatial distribution,energy,momentum,and polarization.Additionally,the article describes advances and challenges in quantum plasmonics and the upgrade of characterization as a potential technical solution.Keywords:surface plasmons;plasmonics;electron nanoscopy;cathodoluminescence.
基金supported by National Key Research and Development Program of China(2023YFF0722600)the National Natural Science Foundation of China(62335008,62122028,62105106)+3 种基金Guangdong Basic and Applied Basic Research Foundation(2023B1515040018,2022A1515011395,2024A1515012073)China Postdoctoral Science Foundation(2023T160237)the Guangdong College Student Scientific and Technological Innovation‘Climbing Program’Special Fund(pdjh2024a108)the Scientific Research Innovation Project of Graduate School of South China Normal University.
文摘Deterministic three-dimensional(3D)super-resolution microscopy can achieve light-matter interaction in a small volume,but usually with the axial extension distinctly more elongated than the lateral one.The isoSTED method combining two opposing objectives and multiple laser beams can offer high axial extension atλ/12 level,but at the cost of optical system complexity and inherent sidelobes.The high-order nonlinear effect by multiphoton excitation would benefit to achieve a sub-diffraction resolution as well as to suppress the sidelobes.Herein,to achieve an easyto-use,sidelobe-free deterministic 3D nanoscopy with high axial resolution,we developed a purely physical deterministic strategy(UNEx-4Pi)by fusion of ultrahighly nonlinear excitation(UNEx)of photon avalanching nanoparticles and mirror-based bifocal vector field modulation(4Pi).The theoretical studies of UNEx-4Pi concept showed that the main peak of fluorescence spot became sharper and its large sidelobe height was suppressed with the increasing optical nonlinearity.In addition,the simplicity and robustness of UNEx-4Pi system were demonstrated utilizing a mirror-assisted single-objective bifocal self-interference strategy.Experimentally,UNEx-4Pi realized an extremely constringent focal spot without sidelobes observed,achieving an axial resolution up toλ/33(26 nm)using one low-power CW beam.We also demonstrated the super-resolution ability of the UNEx-4Pi scheme to bioimaging and nuclear envelope of BSC-1 cells were stained and imaged at an axial resolution of 32 nm.The proposed UNEx-4Pi method will pave the way for achieving light-matter interaction in a highly confined space,thereby advancing cuttingedge technologies like deterministic super-resolution sensing,imaging,lithography,and data storage.
基金supported by the following grants:National Natural Science Foundation of China(62125504,62361166631)STI 2030-Major Projects(2021ZD0200401)+1 种基金the Fundamental Research Funds for the Central Universities(226-2022-00201)the Open Project Program of Wuhan National Laboratory for Optoelectronics(2021WNLOKF007).
文摘Stimulated emission depletion microscopy(STED)holds great potential in biological science applications,especially in studying nanoscale subcellular structures.However,multi-color STED imaging in live-cell remains challenging due to the limited excitation wavelengths and large amount of laser radiation.Here,we develop a multiplexed live-cell STED method to observe more structures simultaneously with limited photo-bleaching and photo-cytotoxicity.By separating live-cell fluorescent probes with similar spectral properties using phasor analysis,our method enables five-color live-cell STED imaging and reveals long-term interactions between different subcellular structures.The results here provide an avenue for understanding the complex and delicate interactome of subcellular structures in live-cell.
基金Supported by the National Natural Science Foundation of China(61988102,62401113,92463308)the National Safety Academic Fund(U2130113)+2 种基金the Sichuan Science and Technology Program(2022JDJQ0065)the Chengdu Science and Technology Program(2024-YF05-01803-SN)the Sichuan Provincial Administration of Traditional Chinese Medicine(2024MS512)and the from Key Laboratory of THz Technology,Ministry of Education.
文摘Fibroblasts support a broad range of essential organ functions via microarchitectural,biomechanical,and biochemical cues.Despite great advances in fluorescence,photoacoustic conversion,and Raman scattering over the past decades,their invasiveness and limited spatial resolution hinder the characterization of fibroblasts in a single cell.Here,taking mouse embryonic fibroblasts(MEFs)as an example,we propose a novel noninvasive approach to investigate the compositional distribution of MEFs at the single-cell scale via terahertz(THz)nanos⁃copy.Compared to the topological morphology,THz nano-imaging enables the component-based visualization of MEFs,such as the membrane,cytoplasm,nucleus,and extracellular vesicles(EVs).Notably,we demonstrate the real-space observation of the influence of rapamycin treatment on the increase of EVs in MEFs.Moreover,the line-cut and area-statistical analysis establishes the relationship between the topological morphology and the THz near-field amplitudes for different cellular components of MEFs.This work provides a new pathway to char⁃acterize the effects of pharmaceutical treatments,with potential applications in disease diagnosis and drug devel⁃opment.
基金This work was financially supported by grants from National Natural Science Foundation of China(Grant No.61205160)the Doctoral Fund of Ministry of Education of China(Grant Nos.20110101120061 and 20120101130006)the Scholarship Award for Excellent Doctoral Student granted by Ministry of Education.
文摘The resolution of conventional optical equipment is always restricted by the diffraction limit,and improving on this was previously considered improbable.Optical super-resolution imaging,which has recently experienced rapid growth and attracted increasing global interest,will result in applications in many domains,benefiting fields such as biology,medicine and material research.This review discusses the contributions of different researchers who identified the diffractive barrier and attempted to realize optical super-resolution.This is followed by a personal viewpoint of the development of optical nanoscopy in recent decades and the road towards the next generation of optical nanoscopy.
基金We thank X.Peng(Shenzhen University)for great assistance in tunneling nanotubes of live cell.This work was partially supported by the National Key R&D Program of China(No.2018YFC0910600)the National Natural Science Foundation of China(Nos.61975132,61775145,61525503,61620106016,61835009,and 81727804)+4 种基金China Postdoctoral Science Foundation(No.2019M650211)Guangdong Province Key Area R&D Program(No.2019B110233004)Project of Department of Education of Guangdong Province(No.2015KGJHZ002/2016KCXTD007)the Shenzhen Basic Research Project(Nos.JCYJ20170412110212234 and JCYJ20170412105003520)the Natural Science Foundation of Shenzhen University(2019108).
文摘Many kinds of nano particles and organic dyes as fluorescent probes have been used in the stimulated emission depletion(STED)nanoscopy.Due to high toxicity,photobleaching and non-water solubility,these fluorescent probes are hard to apply in living cell imaging.Here,we reporta new fluorescence carbon dots(FNCDs)with high photoluminescence quantum yield(56%),low toxicity,anti-photobleaching and goodwater-solubility that suitable for live-cell imaging can be obtained by doping fluorine element.Moreover,the FNCDs can stain the nucleolusand tunneling nanotubes(TNTs)in the living cell.More importantly,for STED nanoscopy imaging,the FNCDs effectively depleted backgroundsignals and improved imaging resolution.Furthermore,the lateral resolution of single FNCDs size under the STED nanoscopy is up to 22.1 nm for FNCDs deposited on a glass slide was obtained.And because of their good water dispersibility,the higher resolution of single FNCDs sizein the nucleolus of a living cell can be up to 19.7 nm.After the image optimizati on steps,the fine fluoresce nee images of TNTs diameter with ca.75 nm resolution is obtained living cell,yielding a threefold enhancement compared with that in confocal imaging.Additionally,the FNCDs show excellent photobleaching resistance after 1,000 scan cycles in the STED model.All results show that FNCDs have significant potentialfor application in STED nanoscopy.
基金support by the National Science Foundation CAREER award under grant no.1553251support provided by a grant from the Air Force Office of Scientific Research(AFOSR)grant no.FA9559-16-1-0172+4 种基金supported by grant no.DE-SC0007043 from the Materials Sciences and Engineering Division of the Office of the Basic Energy Sciences,Office of Science,US Department of Energysupported by MURI grant no.N00014-13-1-0649 from the US Office of Naval Researchsupported by grant no.DE-FG02-01ER15213 from the Atomic,Molecular and Optical Sciences Program,Office of the Basic Energy Sciences,Office of Science,US Department of Energysupported by DOE award no.DEFG02-07ER46376(ZL)NSF award no.1402906(SBC).
文摘Black phosphorus(BP)is an emerging two-dimensional material with intriguing physical properties.It is highly anisotropic and highly tunable by means of both the number of monolayers and surface doping.Here,we experimentally investigate and theoretically interpret the near-field properties of a-few-atomic-monolayer nanoflakes of BP.We discover near-field patterns of bright outside fringes and a high surface polarizability of nanofilm BP consistent with its surface-metallic,plasmonic behavior at mid-infrared frequencies o1176 cm−1.We conclude that these fringes are caused by the formation of a highly polarizable layer at the BP surface.This layer has a thickness of~1 nm and exhibits plasmonic behavior.We estimate that it contains free carriers in a concentration of n≈1.1×10^(20) cm^(−3).Surface plasmonic behavior is observed for 10–40 nm BP thicknesses but absent for a 4-nm BP thickness.This discovery opens up a new field of research and potential applications in nanoelectronics,plasmonics and optoelectronics.
基金supported by the Cancer Early Detection Advanced Research(CEDAR)Center of the OHSU Knight Cancer Institutesupported in part by the OHSU Knight Cancer Institute,the Damon Runyon Cancer Research Foundation,the Cancer Systems Biology Consortium from the National Cancer Institute(CSBC,grant number U54 CA209988,PI:Joe W.Gray)the National Institute of General Medical Sciences(grant number R01 GM132322,PI:X.N.).
文摘Fluorescence nanoscopy has become increasingly powerful for biomedical research,but it has historically afforded a small field-ofview(FOV)of around 50μm×50μm at once and more recently up to∼200μm×200μm.Efforts to further increase the FOV in fluorescence nanoscopy have thus far relied on the use of fabricated waveguide substrates,adding cost and sample constraints to the applications.Here we report PRism-Illumination and Microfluidics-Enhanced DNA-PAINT(PRIME-PAINT)for multiplexed fluorescence nanoscopy across millimeter-scale FOVs.Built upon the well-established prism-type total internal reflection microscopy,PRIME-PAINT achieves robust singlemolecule localization with up to∼520μm×520μm single FOVs and 25−40 nm lateral resolutions.Through stitching,nanoscopic imaging over mm^(2)sample areas can be completed in as little as 40 min per target.An on-stage microfluidics chamber facilitates probe exchange for multiplexing and enhances image quality,particularly for formalin-fixed paraffin-embedded(FFPE)tissue sections.We demonstrate the utility of PRIME-PAINT by analyzing∼106 caveolae structures in∼1,000 cells and imaging entire pancreatic cancer lesions from patient tissue biopsies.By imaging from nanometers to millimeters with multiplexity and broad sample compatibility,PRIMEPAINT will be useful for building multiscale,Google-Earth-like views of biological systems.
文摘Myasthenia gravis is a rare and invalidating disease affecting the neuromuscular junction of voluntary muscles.The classical form of this autoimmune disease is characterized by the presence of antibodies against the most abundant protein in the neuromuscular junction,the nicotinic acetylcholine receptor.Other variants of the disease involve autoimmune attack of non-receptor scaffolding proteins or enzymes essential for building or maintaining the integrity of this peripheral synapse.This review summarizes the participation of the above proteins in building the neuromuscular junction and the destruction of this cholinergic synapse by autoimmune aggression in myasthenia gravis.The review also covers the application of a powerful biophysical technique,superresolution optical microscopy,to image the nicotinic receptor in live cells and follow its motional dynamics.The hypothesis is entertained that anomalous nanocluster formation by antibody crosslinking may lead to accelerated endocytic internalization and elevated turnover of the receptor,as observed in myasthenia gravis.
文摘Cholesterol is a major lipid in biological membranes.It not only plays a structural role but also modulates a wide range of functional properties of neurotransmitter and hormone receptors and ion channels.The membraneembedded segments of the paradigm neurotransmitter receptor for acetylcholine(nAChR)contain linear sequences of amino acids with the capacity to recognize cholesterol.These cholesterol consensus domains have been designated as“CARC”and its mirror sequence“CRAC”.CARC preferentially occurs in the exoplasmic-facing membrane leaflet,and CRAC,in the cytoplasmic-facing hemilayer.Both motifs are highly conserved among ion-channel and neurotransmitter receptor proteins in vertebrate nervous systems,where they recognize cholesterol,and in prokaryotic homologues in bacteria,where they recognize hopanoids.This phylogenetically conserved trait is an indication that the hopanoids in some bacteria and cholesterol in eukaryotes subserve analogous functions,probably contributing to the stability of membrane-embedded protein domains.Structural studies from our laboratory using superresolution optical microscopy(“nanoscopy”)have disclosed other interrelated functional and structural properties exerted by cholesterol on the nAChR.The neutral lipid content at the cell surface influences both the macromolecular organization of the receptor and its translational mobility(diffusion)in the plane of the membrane.
文摘A discrete model of the Differential Evanescent Light Intensity (DELI) technique was developed to calculate and map 3D nanolayers thicknesses from the evanescent light intensity captured from optical waveguides. The model was used for ultra-thin Pd nanometric layers sputtered on glass substrates. The layers thickness profiles were displayed in 3D and 1D profiles plots. The total thickness profiles of the ultra-thin Pd films obtained in the range of 1-10 nm were validated using AFM measurements. Based on the model developed the evanescent photon extraction parameter of the material was estimated.
文摘The photo-kinetics of fluorescent molecules have enabled the circumvention of the far-field optical diffraction limit.Despite its enormous potential,the necessity to label the sample may adversely influence the delicate biology under investigation.Thus,continued development efforts are needed to surpass the far-field label-free diffraction barrier.The statistical similarity or finite coherence of the scattered light off the sample in label-free mode hinders the application of existing super-resolution methods based on incoherent fluorescence imaging.In this article,we present physics and propose a methodology to circumvent this challenge by exploiting the photoluminescence(PL)of silicon nitride waveguides for near-field illumination of unlabeled samples.The technique is abbreviated EPSLON,Evanescently decaying Photoluminescence Scattering enables Label-free Optical Nanoscopy.We demonstrate that such an illumination has properties that mimic the photo-kinetics of nano-sized fluorescent molecules,i.e.,such an illumination permits incoherence between the scattered fields from various locations on the sample plane.Thus,the illumination scheme enables the development of a far-field label-free incoherent imaging system that is linear in intensity and stable over time,thereby permitting the application of techniques like structured illumination microscopy(SIM)and intensity-fluctuation-based optical nanoscopy(IFON)in label-free mode to circumvent the diffraction limit.In this proof-of-concept work,we observed a two-point resolution of~180 nm on super-resolved nanobeads and resolution improvements between 1.9×to 2.8×over the diffraction limit,as quantified using Fourier Ring Correlation(FRC),on various biological samples.We believe EPSLON is a step forward within the field of incoherent far-field label-free super-resolution microscopy that holds a key to investigating biological systems in their natural state without the need for exogenous labels.
文摘Localisation microscopy overcomes the diffraction limit by measuring the position of individual molecules to obtain optical images with a lateral resolution better than 30 nm. Single molecule localisation microscopy was originally demonstrated only in two dimensions but has recently been extended to three dimensions. Here we develop a new approach to three-dimensional （3D） localisation microscopy by engineering of the point-spread function （PSF） of a fluorescence microscope. By introducing a linear phase gradient between the two halves of the objective pupil plane the PSF is split into two lateral lobes whose relative position depends on defocus. Calculations suggested that the phase gradient resulting from the very small tolerances in parallelism of conventional slides made from float glass would be sufficient to generate a two-lobed PSF. We demonstrate that insertion of a suitably chosen microscope slide that occupies half the objective aperture combined with a novel fast fitting algorithm for 3D localisation estimation allows nanoscopic imaging with detail resolution well below 100 nm in all three dimensions （standard deviations of 20, 16, and 42 nm in x, y, and z directions, respectively）. The utility of the approach is shown by imaging the complex 3D distribution of microtubules in cardiac muscle cells that were stained with conventional near infrared fluorochromes. The straightforward optical setup, minimal hardware requirements and large axial localisation range make this approach suitable for many nanoscopic imaging applications.
基金C.R.and M.S acknowledge the financial support of the Agence National de la Recherche(ANR-14-CE08-0015-01 Ultrafast Nanoscopy).
文摘With super-resolution microscopy,we attempt to visualize(biological)structures and processes at the sub-cellular level(i.e.,nanoscale).To obtain this information,the samples are labeled with fluorophores that have a stochastic on/off switching of their emissions,which help to overcome the optical diffraction limit of around 250 nm,related to the use of optical micro-scopes.However,nowadays,research focuses on the imaging of live cells and thicker samples.These investigations require a high amount of simultaneously active fluorophores(i.e.,high-density imaging)and are challenging due to the collapse of the single-molecule localization techniques and the increased background in the image.Therefore,recent efforts have shifted towards the development of new ways to process the data.This publication gives an introduction to wide-field super-resolution fluorescence microscopy,explaining the concepts of the technique,and then gives an overview of the recently developed methods to provide super-resolution images for high-density data of live cells and ways to overcome the issues related to the imaging of these samples.
