Recent advances in fluorescence nanoscopy have pushed resolution to the 1–10 nm range,enabling the direct visualization of individual molecules even in crowded biological environments.Achieving this level of precisio...Recent advances in fluorescence nanoscopy have pushed resolution to the 1–10 nm range,enabling the direct visualization of individual molecules even in crowded biological environments.Achieving this level of precision requires rigorous sample drift control.Techniques such as MINFLUX and RASTMIN,which rely on keeping the sample fixed within an excitation pattern,demand active drift correction to achieve their theoretical nanometer-scale resolution limits.Here,we present an active stabilization system for super-resolution microscopy that delivers sub-nm precision for hours.Featuring a simple optical design,the system can be added as a separate module to any fluorescence microscope.We also provide an open-source control software including a user-friendly graphical interface readily adaptable to different setups.We demonstrate the adaptability and performance of the stabilization system with p-MINFLUX and RASTMIN measurements performed in two different setups,reaching the theoretical Cramér-Rao Bound and resolving~10 nm distances within DNA origami structures.展开更多
Using sequential excitation with a minimum of light to localize single fluorescent molecules represented a breakthrough because it delivers 1-2 nm precision with moderate photon counts,enabling tracking and superresol...Using sequential excitation with a minimum of light to localize single fluorescent molecules represented a breakthrough because it delivers 1-2 nm precision with moderate photon counts,enabling tracking and superresolution imaging with true molecular resolution.Expanding this concept to multi-photon regimes may be a useful complement to reach even higher localization precision and get deeper into biological specimens.展开更多
Localization of single fluorescent emitters is key for physicochemical and biophysical measurements at the nanoscale and beyond ensemble averaging.Examples include single-molecule tracking and super-resolution imaging...Localization of single fluorescent emitters is key for physicochemical and biophysical measurements at the nanoscale and beyond ensemble averaging.Examples include single-molecule tracking and super-resolution imaging by single-molecule localization microscopy.Among the numerous localization methods available,MINFLUX outstands for achieving a~10-fold improvement in resolution over wide-field camera-based approaches,reaching the molecular scale at moderate photon counts.Widespread application of MINFLUX and related methods has been hindered by the technical complexity of the setups.Here,we present RASTMIN,a single-molecule localization method based on raster scanning a light pattern comprising a minimum of intensity.RASTMIN delivers~1-2nm localization precision with usual fluorophores and is easily implementable on a standard confocal microscope with few modifications.We demonstrate the performance of RASTMIN in localization of single molecules and super-resolution imaging of DNA origami structures.展开更多
基金funded by Consejo Nacional de Investigaciones Científicas y Técnicas(CONICET)Swiss National Science Foundation project 200021_184687(G.P.A.)+1 种基金Agencia Nacional de Promoción Científica y Tecnológica(ANPCYT),projects PICT-2017-0870(F.D.S.),and PICT-2021-01216(F.D.S.)funded by Warsaw University of Technology within the Excellence Initiative:Research University(IDUB)programme.
文摘Recent advances in fluorescence nanoscopy have pushed resolution to the 1–10 nm range,enabling the direct visualization of individual molecules even in crowded biological environments.Achieving this level of precision requires rigorous sample drift control.Techniques such as MINFLUX and RASTMIN,which rely on keeping the sample fixed within an excitation pattern,demand active drift correction to achieve their theoretical nanometer-scale resolution limits.Here,we present an active stabilization system for super-resolution microscopy that delivers sub-nm precision for hours.Featuring a simple optical design,the system can be added as a separate module to any fluorescence microscope.We also provide an open-source control software including a user-friendly graphical interface readily adaptable to different setups.We demonstrate the adaptability and performance of the stabilization system with p-MINFLUX and RASTMIN measurements performed in two different setups,reaching the theoretical Cramér-Rao Bound and resolving~10 nm distances within DNA origami structures.
文摘Using sequential excitation with a minimum of light to localize single fluorescent molecules represented a breakthrough because it delivers 1-2 nm precision with moderate photon counts,enabling tracking and superresolution imaging with true molecular resolution.Expanding this concept to multi-photon regimes may be a useful complement to reach even higher localization precision and get deeper into biological specimens.
基金funded by ConsejoNacional de Investigaciones CientificasyTecnicas(CONiCET),Swiss National Science Foundation project 200021_184687(G.P.A.)National Center of Competence in Research Bio-lnspired Materials NCCR,project 51NF40_182881(G.P.A.),and Agencia Nacional de Promocion Cientifica y Tecnologica(ANPCYT),projects PICT-2017-0870(F.D.S.),and PICT-2014-3729(F.D.S.).
文摘Localization of single fluorescent emitters is key for physicochemical and biophysical measurements at the nanoscale and beyond ensemble averaging.Examples include single-molecule tracking and super-resolution imaging by single-molecule localization microscopy.Among the numerous localization methods available,MINFLUX outstands for achieving a~10-fold improvement in resolution over wide-field camera-based approaches,reaching the molecular scale at moderate photon counts.Widespread application of MINFLUX and related methods has been hindered by the technical complexity of the setups.Here,we present RASTMIN,a single-molecule localization method based on raster scanning a light pattern comprising a minimum of intensity.RASTMIN delivers~1-2nm localization precision with usual fluorophores and is easily implementable on a standard confocal microscope with few modifications.We demonstrate the performance of RASTMIN in localization of single molecules and super-resolution imaging of DNA origami structures.
