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.
