Research On Three-dimensional Scanning Microscope Imaging System

The three-dimensional(3D)scanning microscope can carry out 3D analysis of thick samples.It is an important technical means in the fields of cell biology,molecular biology,and medicine.Besides,it is also an important research platform for super-resolution imaging.Based on the scanning microscope platform,it can be constructed into polarization modulation super-resolution microscopy or the image scanning microscope.By adding a doughnut-shaped beam,it can be upgraded to super-resolution microscope such as stimulated emission depletion microscope or the fluorescence emission difference microscope.The improvement is beneficial to promote research in related fields at the sub-diffraction scale.Therefore,the construction of a 3D scanning microscope system is of great significance for the development of scientific research on super-resolution imaging.In this paper,the research on the three-dimensional scanning microscope imaging system was carried out.Based on the combination of the transverse beam scanning with the galvanometer scanner and the axial scanning with the piezoelectric nano-translation stage,a three-dimensional scanning confocal imaging system was developed,including the hardware system design,construction of optical path system and programming of software control system.The main work is shown as follows:(1)According to the principle of three-dimensional laser scanning confocal imaging system,the three-dimensional scanning is realized.In this system,a galvanometer scanner is used to realize two-dimensional scanning imaging of a single layer.Combined with the axial movement of the piezoelectric nano-translation stage,the sample is scanned layer by layer.Using Image J software,these two-dimensional image sequences are combined into a whole to form a three-dimensional image.During the development process,the relevant principles,circuits and control functions of hardware equipments such as galvanometer oscillating mirror,piezoelectric nano-translation stage,high-sensitivity single-photon photodetector,time-correlated single-photon counting card,were sorted out and analyzed.And the hardware system was designed.A laser confocal imaging optical system has been constructed by using the confocal scheme where a focal point excitation is realized with high numerical aperture objective and the point detection is realized by using the multimode optical fiber as the pinhole.(2)A three-dimensional scanning imaging software system is developed,in which the functions of two-dimensional scanning and three-dimensional sectioning were realized through the control of various hardware devices.The software system of the system is developed based on MFC programming which has the functions like the horizontal scanning of galvanometer scanner controlled by the FPGA,the control and position display of the piezoelectric nano-translation stage,the synchronous control between the point-by-point scanning and the collection of photon numbers,the real-time display of photon numbers,image drawing,and data storage.(3)A three-dimensional scanning was demonstrated with the samples of uniformly distributed gold nanoparticles and fluorescent beads.In this thesis,the experiments of two-dimensional and three-dimensional point-by-point scanning imaging were carried out.And the data was analyzed,which shows that the resolution of the system is about 365 nm,and three-dimensional image construction is realized based on pixel information.The system is stable and has good compatibility and scalability,which is expected to be further applied to fluorescence lifetime imaging microscope and super-resolution microscope construction.