Research And Application Of High-throughput Light Sheet Fluorescence Microscopy

With its advantages in optical tomography and phototoxicity,light-sheet illumination fluorescence microscopy has become one of the most important threedimensional imaging tools in the field of life sciences.It is widely used in various spatial structures of biological tissues,organs and embryos.Although light-sheet illumination fluorescence microscopy has evolved a structural form suitable for various application scenarios such as ultra-high spatial resolution,ultra-large field of view,and high-speed imaging based on the original structure according to specific imaging requirements.However,the current light-sheet forms still cannot meet the highthroughput imaging requirements in large-scale genetic development,drug screening,and other studies.In response to this requirement,this dissertation proposes a highthroughput light-sheet imaging system and makes the following research.First,a high-throughput light sheet flow imaging technique was proposed by combining flow cytometry with light sheet fluorescence imaging.The focus is on the research and design of the automatic sample loading and unloading system for zebrafish embryos.The light-sheet illumination and imaging system was designed and coupled with the liquid flow system,the control timing design and control software development were carried out,and the setup of light-sheet flow imaging system was built.The throughput can reach 200 embryos/hour.Afterwards,the image reconstruction algorithm research of high-throughput flow microcopy was carried out.Firstly,a velocity measurement and correction algorithm for large particle samples under flow conditions is designed to solve the problem of motion artifacts in flow imaging.On this basis,the design of the 3D image reconstruction algorithm for non-uniform sampling under inclined sampling conditions was completed,and the automatic calibration of the typical pose of the zebrafish sample was realized.An image registration algorithm was designed for dual-channel 3D images,and a high-throughput 3D image reconstruction software was developed.Furthermore,a high-throughput light-sheet flow imaging system was used to study the vascular development of zebrafish embryos.In this part of the study,a total of 385 groups of full-size 3D images of zebrafish embryos were collected,and a set of image segmentation,refinement and statistical analysis processes were designed for the collected large-scale 3D vascular images.Based on the extracted three-dimensional structure information of blood vessels,the developmental heterogeneity of intersegmental vessels located in the trunk and vitreous vessels located in the head of zebrafish was studied.The application demonstration of developmental research using high-throughput flow cytometry was carried out.Finally,a high-throughput light-sheet flow imaging system was used to study the drug response of zebrafish.Zebrafish embryos were treated with a typical kind of vascular inhibition drug,and the effects of drug on the morphology of zebrafish trunk vessels,the degree of inhibition of the length and number of intersegmental vessels,and the developmental status of head vessels were quantitatively analyzed.It demonstrates the application prospect of high-throughput light-sheet flow imaging system in large-scale drug screening.In this dissertation,from basic principles to optics,flow control,and reconstruction algorithms and software,the research and system development of high-throughput light-sheet flow microscopy were systematically carried out.The application prospects of high-throughput light-sheet flow imaging system in biology were demonstrated in vascular development and drug response.