Unlike ensemble-averaging measurements,single-molecule tracking provides quantitative information on the kinetics of individual molecules within living cells in real time and may provide insight into the respective mo...Unlike ensemble-averaging measurements,single-molecule tracking provides quantitative information on the kinetics of individual molecules within living cells in real time and may provide insight into the respective molecular interactions behind that.The advancement of single-molecule tracking has been signi-cantly boosted by the development of high-resolution microscopy techniques.In this review,we will discuss this aspect with a particular focus on their recent advance in MINFLUX nanoscopy with feedback approaches where tracking is performed in real time.MINFLUX localization requires fewer than 100 photons from a-1 nm-sized°uorophore,enabling precise tracking.This approach,which demands over an order of magnitude fewer photons than other localization-based techniques(such as STORM,PLAM),allows molecular tracking with single-digit nanometer accuracy in less than 1 ms—an achievement previously unattainable.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
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
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
Achieving localization with molecular precision has been of great interest for extending fluorescence microscopy to nanoscopy.MINFLUX pioneers this transition through point spread function(PSF)engineering,yet its perf...Achieving localization with molecular precision has been of great interest for extending fluorescence microscopy to nanoscopy.MINFLUX pioneers this transition through point spread function(PSF)engineering,yet its performance is primarily limited by the signal-to-background ratio.Here we demonstrate theoretically that two-photon MINFLUX(2p-MINFLUX)could double its localization precision through PSF engineering by nonlinear effect.Cramér-Rao Bound(CRB)is studied as the maximum localization precision,and CRB of two-photon MINFLUX is halved compared to single-photon MINFLUX(1p-MINFLUX)in all three dimensions.Meanwhile,in order to achieve same localization precision with 1p-MINFLUX,2p-MINFLUX requires only 1/4 of fluorescence photons.Exploiting simultaneous two-photon excitation of multiple fluorophore species,2p-MINFLUX may have the potential for registration-free nanoscopy and multicolor tracking.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
基金supported by the Science and Technology Commission of Shanghai Municipality(21DZ1100500)the Shanghai Municipal Science and Technology Major Project+2 种基金the Shanghai Frontiers Science Center Program(2021–2025 No.20)The National Natural Science Foundation of China(32471545)the Natural Science Foundation of Shanghai(24ZR1454300).
文摘Unlike ensemble-averaging measurements,single-molecule tracking provides quantitative information on the kinetics of individual molecules within living cells in real time and may provide insight into the respective molecular interactions behind that.The advancement of single-molecule tracking has been signi-cantly boosted by the development of high-resolution microscopy techniques.In this review,we will discuss this aspect with a particular focus on their recent advance in MINFLUX nanoscopy with feedback approaches where tracking is performed in real time.MINFLUX localization requires fewer than 100 photons from a-1 nm-sized°uorophore,enabling precise tracking.This approach,which demands over an order of magnitude fewer photons than other localization-based techniques(such as STORM,PLAM),allows molecular tracking with single-digit nanometer accuracy in less than 1 ms—an achievement previously unattainable.
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 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Beijing Natural Science Foundation(JQ18019)National Natural Science Foundation of China(62025501,31971376,61729501)+1 种基金State Key Research Development Program of China(2017YFC0110202)Shenzhen Science and Technology Program(KQTD20170810110913065)。
文摘Achieving localization with molecular precision has been of great interest for extending fluorescence microscopy to nanoscopy.MINFLUX pioneers this transition through point spread function(PSF)engineering,yet its performance is primarily limited by the signal-to-background ratio.Here we demonstrate theoretically that two-photon MINFLUX(2p-MINFLUX)could double its localization precision through PSF engineering by nonlinear effect.Cramér-Rao Bound(CRB)is studied as the maximum localization precision,and CRB of two-photon MINFLUX is halved compared to single-photon MINFLUX(1p-MINFLUX)in all three dimensions.Meanwhile,in order to achieve same localization precision with 1p-MINFLUX,2p-MINFLUX requires only 1/4 of fluorescence photons.Exploiting simultaneous two-photon excitation of multiple fluorophore species,2p-MINFLUX may have the potential for registration-free nanoscopy and multicolor tracking.
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.
