High-Speed 3D Imaging Technology For Fluorescent Pathological Sections

Fluorescent in situ hybridization(FISH)is a new gene detection technology in recent years.It mainly uses fluorescent labeled probes to hybridize with target DNA to show the location of specific sequences to detect whether gene mutation occurs.Fish has high sensitivity and simple operation,it is often used in the pathological diagnosis of tumors.With the development of probe technology,various fluorescent probes for duplications,chromosome translocation,deletion,and inversion have emerged.The discrimination of gene mutation is no longer limited to the type of probes counting,but also involves multiple methods such as co-localization of fluorescent sites.Therefore,there is a corresponding demand for precise localization of fluorescent sites.However,the current fluorescence microscope or pathological slide scanner can only perform two-dimensional imaging,and three-dimensional imaging requires a mechanically moving sample stage or microscope objective,which cannot meet the needs of large-scale rapidly imaging of pathological slides,and the positioning accuracy of fluorescence sites is limited by the optical diffraction limit.This thesis focuses on the autofocus system,the z-axial localization method of fluorescence site,and the three-dimensional imaging instrument system in FISH pathological slides scanning.Andfocuses on the application of the method of single-molecule localization super-resolution microscopy(SMLM)with double-helix point spread function(DH-PSF)to the three-dimensional imaging of FISH pathological sections,and realizes the threedimensional localization of fluorescent sites in a single frame.The main contents of this thesis as follows:(1)Aimed at the axial drift problem of fluorescence pathological whole slide imaging,we design a method for focal-plane drift measurement by detecting the reflected spot position of a non-symmetric beam.With the simulation of ZEMAX,the shapes of the reflected spots are simulated with the sample surface at different defocus positions.An integrated focusplane drift measurement module is constructed and tested on a commercial upright microscope.The results show that this module has a drift measurement precision of 250 nm and a response time of less than 500 ms,satisfying the demand for long-term and highresolution imaging.(2)For the three-dimensional imaging of fluorescence pathological sections,this thesis uses Wirtinger Flow phase retrieval method transforms the problem of DH-PSF design into a non-convex optimization problem.The imaging system model is constructed by scalar diffraction,and the model loss function is calculated by the mean square error between the target modulation result and the system model output.The phase information of the double helix point spread function was designed by applying the gradient descent method to optimize the phase.Finally,the z-axial information of the fluorescence site is encoded into the angle information of two rotation points in the two-dimensional plane.(3)In this thesis,a single-frame three-dimensional imaging scanner system for fluorescence pathological slides was further constructed.The scanning and stitch process of the whole slide is realized by invoking the XY axis electric moving stages.The effect of the autofocus system on the fluorescence slide scanner was verified by experiments.The axial localization of DH-PSF was compared with the axial localization of layer by layer scanning.The results show that the use of DH-PSF for axial localization can basically meet the requirements.