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
Structured illumination microscopy(SIM)achieves super-resolution(SR)by modulating the high-frequency information of the sample into the passband of the optical system and subsequent image reconstruction.The traditiona...Structured illumination microscopy(SIM)achieves super-resolution(SR)by modulating the high-frequency information of the sample into the passband of the optical system and subsequent image reconstruction.The traditional Wiener-filtering-based reconstruction algorithm operates in the Fourier domain,it requires prior knowledge of the sinusoidal illumination patterns which makes the time-consuming procedure of parameter estimation to raw datasets necessary,besides,the parameter estimation is sensitive to noise or aberration-induced pattern distortion which leads to reconstruction artifacts.Here,we propose a spatial-domain image reconstruction method that does not require parameter estimation but calculates patterns from raw datasets,and a reconstructed image can be obtained just by calculating the spatial covariance of differential calculated patterns and differential filtered datasets(the notch filtering operation is performed to the raw datasets for attenuating and compensating the optical transfer function(OTF)).Experiments on reconstructing raw datasets including nonbiological,biological,and simulated samples demonstrate that our method has SR capability,high reconstruction speed,and high robustness to aberration and noise.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
Imaging three-dimensional,subcellular structures with high axial resolution has always been the core purpose of fluorescence microscopy.However,trade-offs exist between axial resolution and other important technical i...Imaging three-dimensional,subcellular structures with high axial resolution has always been the core purpose of fluorescence microscopy.However,trade-offs exist between axial resolution and other important technical indicators,such as temporal resolution,optical power density,and imaging process complexity.We report a new imaging modality,fluorescence interference structured illumination microscopy(FI-SIM),which is based on three-dimensional structured illumination microscopy for wide-field lateral imaging and fluorescence interference for axial reconstruction.FI-SIM can acquire images quickly within the order of hundreds of milliseconds and exhibit even 30 nm axial resolution in half the wavelength depth range without z-axis scanning.Moreover,the relatively low laser power density relaxes the requirements for dyes and enables a wide range of applications for observing fixed and live subcellular structures.展开更多
Single-molecule localization microscopy(SMLM)gradually plays an important role in deep tissue imaging.However,current SMLM methods primarily rely on fiducial marks,neglecting aberrations introduced by thick samples,th...Single-molecule localization microscopy(SMLM)gradually plays an important role in deep tissue imaging.However,current SMLM methods primarily rely on fiducial marks,neglecting aberrations introduced by thick samples,thereby resulting in decreased image quality in deep tissues.Here,we introduce vectorial in situ point spread function(PSF)retrieval(VISPR),a method that retrieves a precise PSF model considering both systemand sample-induced aberrations under SMLM conditions.By employing the theory of vectorial PSF model and maximum likelihood estimation(MLE)phase retrieval,VISPR is capable of reconstructing an accurate in situ 3D PSF model achieving the theoretically minimum uncertainty and accurately reflecting three-dimensional information of single molecules.This capability enables accurate 3D super-resolution reconstruction in deep regions away from the coverslips.Additionally,VISPR demonstrates applicability in low signal-to-noise ratio scenarios and compatibility with various SMLM microscope modalities.From both simulations and experiments,we verified the superiority and effectiveness of VISPR.We anticipate that VISPR will become a pivotal tool for advancing deep tissue SMLM imaging.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funded by the National Natural Science Foundation of China(62125504,61827825,and 31901059)Zhejiang Provincial Ten Thousand Plan for Young Top Talents(2020R52001)Open Project Program of Wuhan National Laboratory for Optoelectronics(2021WNLOKF007).
文摘Structured illumination microscopy(SIM)achieves super-resolution(SR)by modulating the high-frequency information of the sample into the passband of the optical system and subsequent image reconstruction.The traditional Wiener-filtering-based reconstruction algorithm operates in the Fourier domain,it requires prior knowledge of the sinusoidal illumination patterns which makes the time-consuming procedure of parameter estimation to raw datasets necessary,besides,the parameter estimation is sensitive to noise or aberration-induced pattern distortion which leads to reconstruction artifacts.Here,we propose a spatial-domain image reconstruction method that does not require parameter estimation but calculates patterns from raw datasets,and a reconstructed image can be obtained just by calculating the spatial covariance of differential calculated patterns and differential filtered datasets(the notch filtering operation is performed to the raw datasets for attenuating and compensating the optical transfer function(OTF)).Experiments on reconstructing raw datasets including nonbiological,biological,and simulated samples demonstrate that our method has SR capability,high reconstruction speed,and high robustness to aberration and noise.
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sponsored by the National Natural Science Foundation of China(Grant Nos.62125504,61827825,and 31901059)STI 2030—Major Projects(Grant No.2021ZD0200401)+3 种基金Major Program of the Natural Science Foundation of Zhejiang Province(Grant No.LD21F050002)Zhejiang Provincial Ten Thousand Plan for Young Top Talents(Grant No.2020R52001)Croucher Foundation(Grant No.CM/CT/CF/CIA/0688/19ay)Hong Kong Innovation and Technology Fund(ITS/178/20FP and ITS/148/20).
文摘Imaging three-dimensional,subcellular structures with high axial resolution has always been the core purpose of fluorescence microscopy.However,trade-offs exist between axial resolution and other important technical indicators,such as temporal resolution,optical power density,and imaging process complexity.We report a new imaging modality,fluorescence interference structured illumination microscopy(FI-SIM),which is based on three-dimensional structured illumination microscopy for wide-field lateral imaging and fluorescence interference for axial reconstruction.FI-SIM can acquire images quickly within the order of hundreds of milliseconds and exhibit even 30 nm axial resolution in half the wavelength depth range without z-axis scanning.Moreover,the relatively low laser power density relaxes the requirements for dyes and enables a wide range of applications for observing fixed and live subcellular structures.
基金National Natural Science Foundation of China(62125504,62361166631)STI 2030—Major Projects(2021ZD0200401)+1 种基金Fundamental Research Funds for the Central Universities(2022FZZX01-20)Open Project Program of Wuhan National Laboratory for Optoelectronics(2021WNLOKF007)。
文摘Single-molecule localization microscopy(SMLM)gradually plays an important role in deep tissue imaging.However,current SMLM methods primarily rely on fiducial marks,neglecting aberrations introduced by thick samples,thereby resulting in decreased image quality in deep tissues.Here,we introduce vectorial in situ point spread function(PSF)retrieval(VISPR),a method that retrieves a precise PSF model considering both systemand sample-induced aberrations under SMLM conditions.By employing the theory of vectorial PSF model and maximum likelihood estimation(MLE)phase retrieval,VISPR is capable of reconstructing an accurate in situ 3D PSF model achieving the theoretically minimum uncertainty and accurately reflecting three-dimensional information of single molecules.This capability enables accurate 3D super-resolution reconstruction in deep regions away from the coverslips.Additionally,VISPR demonstrates applicability in low signal-to-noise ratio scenarios and compatibility with various SMLM microscope modalities.From both simulations and experiments,we verified the superiority and effectiveness of VISPR.We anticipate that VISPR will become a pivotal tool for advancing deep tissue SMLM imaging.
