| Cells are the smallest unit of life.The understanding of complex life often starts with cells,and understanding the life activities within cells can help people understand human life activities.Due to the existence of the diffraction limit,traditional optical microscopy methods have been unable to meet the needs of biological research.Under this requirement,scientists have proposed many super-resolution imaging methods that break the resolution limit,which improve the resolution to the nanometer magnitude.Spatial resolution and temporal resolution are mutually restricted,and higher spatial resolution often takes more time.To achieve intracellular multiple particle tracking imaging,it is required to be able to quickly detect the dynamic process of particles while maintaining high spatial resolution.The single particle tracking method meets this requirement,it can track multiple particles in the cell at the same time,which provides a very good optical tool for the research of intracellular biological particles.In recent years,many single particle tracking methods have emerged in succession,and have achieved three-dimensional localization in a certain depth range,but their depth of field is far from sufficient for the thickness of the entire cell.To obtain dynamic information in the full range of living cells,a single particle tracking method and a depth-of-field extension method need to be combined.Based on this background,this paper proposes a combination of a distorted grating with depth-of-field extension capability and a dual-objective bifocal plane method with three-dimensional localization capability to achieve three-dimensional localization tracking across the entire cell range,which can simultaneously track intracellular multiple particle.The main work of this paper is as follows:1.A three-dimensional nano-localization tracking system based on the bifocal plane method was designed.The basic principle of the bifocal plane method is to use two detection paveswith stagger focal planes for imaging to obtain two images with different defocus,which is a bright ring with Gaussian distribution,and the intensity of the bright ring changes with the defocus of the particle.This change in two images can be used to locate.In this paper,matlab is used to simulate the bifocal plane imaging system,and perform the localization analysis based on the bifocal plane localization algorithm to obtain the localization precision within 5nm and 20 nm in the lateral and axial directions.2.Designed and optimized the distorted grating with depth of field extension ability,and built a 3D nano-localization and tracking system with large depth of field.The bifocal plane method can achieve axial positioning in the range of about 2microns,which is not sufficient for cell research.Therefore,the bifocal plane method needs to be combined with the method of extension the depth of field.Actually,distorted grating is an off-axis Fresnel zone plate,which is able to simultaneously imaging different diffraction orders of three different focal planes on the same image plane.In this paper,the imaging method of the distorted grating is simulated,the distorted grating is optimized in combination with the system parameters,and the feasibility of three-dimensional nano-localization tracking within the general cell thickness(10 microns)was verified.3.Theoretically,the three-dimensional localization capability of the method proposed in this paper is compared with the traditional bifocal plane method and the distorted grating combined single-objective bifocal plane method.The dual-objective is used to realize dual detection channels,and the source image required by bifocal plane method is acquired,which combine one-dimensional distorted grating to realize simultaneous imaging of 6 focal planes.In addition,because the fluorescence is radiated from all directions,a single objective can only collect half of the fluorescence.While using two objectives to achieve dual detection channels,the photon utilization is improved,and the photon loss caused by the beam splitting by the deformed grating is also compensated.Theoretically,the three-position localization ability of the method proposed in this paper is comparedwith the traditional bifocal plane method and the distorted grating combined with the single objective bifocal plane method.The innovation of this paper is to propose a three-dimensional nano-localization tracking method with large depth of field.The combination of parallel imaging of multiple focal planes of a deformed grating and bifocal three-dimensional nano-localization methods can break through depth of field of the existing optical microscope system,and locate and trace the moving targets within the thickness of the cell.In addition,the light splitting ability of the distorted grating will decrease the number of photons assigned per plane,which will affect the localization accuracy of the system.For this,considering that a single objective collects only half photons compared with dual objective,this paper adopts dual objective to make up and make full use of the fluorescence signal. |
