High-resolution Three-dimensional Fluorescence Microscopy Based On Double-helix Point Spread Function

Fluorescence microscopy has found widespread applications in modern biomedical imaging for its non-contact,high contrast/resolution,and specific labelling.However,with the rapid development of life science,the conventional fluorescence microscope is unable to meet the ever-increasing requirements of biomedical imaging.For instance,studying the dynamic process of bio-molecules and organelle in three-dimension(3D)not only needs high resolution,but also requires excellent 3D imaging ability and imaging speed.Therefore,developing fast and high-resolution 3D fluorescence microscopy is of great significance for the development of life science.In this thesis,based on the double-helix point spread function(DH-PSF),we first develop a novel single-shot 3D fluorescence microscope.In addition,to eliminate the impact of system aberration,we propose a new aberration correction method based on double-helix PSF.In order to further improve the resolution of the 3D microscopy,we also conduct the researches on super-resolution 3D fluorescence microscopy.The majority of the researches is outlined as below:1.A single-shot 3D fluorescence microscopy based on double-helix PSF is proposed and established.For conventional microscope,we need to axially scan the focal plane of the system throughout the specimen to obtain the interested information in 3D,which dramatically impedes the imaging speed.In this thesis,we constructed a wide-field fluorescence microscope with a double-helix point spread function which is able to obtain the specimen's 3D distribution within a single snapshot.To make the depth-of-field of the microscope adjustable,spiral-phase-based computer-generated holograms(CGHs)are adopted.Besides,a modified cepstrum-based reconstruction scheme is promoted in accordance with the properties of a new double-helix PSF,which can recover the extended depth-of-field image and the depth distribution of the sample without sacrificing the lateral resolution.This three-dimensional fluorescence microscope with a framerate-rank time resolution and an axial localization precision of 23.4 nm is suitable for studying the fast developing process of thin and sparsely distributed micron-scale cells in extended depth-of-field.2.An iteration-free aberration correction method based on double-helix PSF is proposed.Traditional aberration correction methods are mostly based on iterative algorithms,which are time-consuming(longer than 1 minute)and may not converge sometimes.In this paper,we present a novel aberration correction method by using a spiral-phase-based double-helix PSF as an aberration indicator,which is proved to be sensitive and quantitatively correlated to the aberrations.Superior to the routine iteration-based correction methods,the presented approach is iteration-free and the aberration coefficients can be directly calculated from the measured parameters within 4 seconds.This iteration-free correction method has a potential application in PSF engineering systems equipped with a spatial light modulator.3.The super-resolution principle of multifocal structured illumination microscopy(MSIM)is investigated,and the performance of two reconstruction algorithms of MSIM is compared through simulations and experiments.A newly-proposed super-resolution microscopy,image scanning microscopy(ISM),can achieve a resolution of twice the diffraction limit.Unfortunately,the imaging speed of this method is limited by its requirement for point-by-point scanning and sensor-array detection.MSIM solves this problem through parallel excitation and detection.In this thesis,we investitage the super-resolution principle of MSIM,and introduce two routine super-resolution reconstruction algorithms of MSIM.Further,the applicable scenarios of the two algorithms are analyzed by comparing the advantages and disadvantages of these two reconstruction algorithms through numerical simulation and experiment,which lays a foundation for the following work.4.A super-resolution 3D fluorescence microscope is proposed by introducing the double-helix PSF in the detection path of MSIM.Existing 3D super-resolution SIM can improve the z-resolution to 300 nm.However,it still can not recognize the depth difference of the specimen within 300 nm.In this thesis,we develop a super-resolution 3D fluorescence microscope by introducing the double-helix PSF into the detection path of MSIM.Eventually,the axial localization precision of the system can be highly improved(~20 nm)while retaining the lateral superresolution ability(~136 nm).This method will be a promising tool for observing high-precision 3D spatial distribution of biological specimens.