Investigation On Stuctured Light Illumination Fluorescent Digital Holography Optical Sectioning

Microscopic imaging of fluorescently labeled biological samples is one of the important means to visualize the information to be observed in the fields of biology and medicine.The continuous development of fluorescence microscopy in recent years has helped people to observe the labeled structural information of biological samples at the micro-nano scale.Fluorescent digital holography provides a non-scanning fast three-dimensional microscopic imaging method,which can realize the recording and three-dimensional reconstruction of the fluorescent sample hologram,and the recording process does not need to be scanned.Fluorescent digital holography uses the spatial self-coherence characteristics of the fluorescence emitted from the same point of the sample to realize the recording of the point source hologram.All the point-source holograms are superposed incoherently to form the hologram of the fluorescent sample.The advantage of fluorescent digital holographic microscopy imaging technology is that imaging speed is fast,the lateral resolution of imaging is higher than the resolution of direct imaging of the system under the same structural parameters,but the axial resolution of the technology is lower than that of the same structural parameters.The resolution of direct imaging,for 3D biological samples or thick biological samples,cannot achieve 3D optical sectioning.In this paper,we combined with structured light illumination technology and fluorescent digital holography technology to investigation the three-dimensional tomographic characteristics of fluorescent digital holography with structured light illumination.The main research works of the thesis is summarized as follows:1.The basic principle of structured light illumination fluorescent digital holography imaging is illustrated.The basic process of fluorescent radiation and the basic principle of fluorescence microscopy are given.The recording process of fluorescent digital holography in the case of two-dimensional and three-dimensional structured light illumination and the reconstruction method of direct wide field reconstruction,two-dimensional super-resolution reconstruction and three-dimensional optical sectioning reconstruction are analyzed.2.The incoherent digital holographic optical sectioning characteristics of two-dimensional structured light illumination are investigated.The effects of two-dimensional structured light modulation,normalized spatial frequency and axial distance of different object planes on system optical sectioning ability were investigated and verified,which are verified by Matlab simulation and experiment.A fluorescent digital holographic microscopic imaging system with two-dimensional structured light illumination was designed and explore the imaging axial resolution and optical sectioning characteristics of the fluorescent digital holographic microscopy system under the two-dimensional structured light illumination generated by the two-beam interference.3.The imaging characteristics of fluorescent digital holographic microscopy of three-dimensional structured light illumination were investigated.The lateral distribution and axial distribution of three-dimensional structured light are explored by Matlab simulation.And using this three-dimensional structured light as the illumination source to investigate the influence of the axial distance of the fluorescent sample on the chromatographic ability.The influence of the axial distance of the fluorescent sample on the optical sectioning ability is explored.A three-dimensional structured light-emitting fluorescent digital holographic imaging system was constructed to obtain the distribution characteristics of three-dimensional structured light.Three-dimensional fluorescence samples of different thicknesses were fabricated,and three-dimensional super-resolution reconstruction and complex biological cell tomography reconstruction were performed on simple fluorescent samples.