Cryo-line-scanning Micro-optical Sectioning Tomography System

Biomedical imaging techniques provide powerful tools for the study of biological tissue structure and disease mechanisms.The optical microscopy can reach subcellular resolution,and combined with increasingly sophisticated labeling techniques,is suitable for studying the biology of centimeter-scale large-volume samples with micron resolution.The cyro-micro-optical sectioning tomography system developed by the author’s research group uses liquid nitrogen to create a cryogenic environment of-196℃,combines with mechanical milling to break the limit of imaging depth,and obtains three-dimensional structural and metabolic information of the sample in the frozen environment by optical imaging.The fixation and embedding method in the cyrogenic environment keep the original shape and metabolic state of the biological sample well.However,the existing wide-field illumination deep cryo-microscopic optical section tomography system still has some shortcomings,mainly the lack of optical lamination capability leading to high background fluorescence interference and poor image quality,as well as the low operating efficiency of the system and the need for manual intervention by the experimenter throughout the whole process,resulting in the inability to image continuously for long periods of time.This greatly limits the system’s ability to perform high-resolution axial sequential acquisitions of intact organs,or even the whole body of small animals.Responding to the existing problems,this paper improves the system the system in terms of both hardware and software,realizes the automation of system,and improves the ability of continuous data collection of the system.In order to improve image quality,the existing wide-field illumination imaging system has been modified to a line-scan imaging system,which improves the optical-sectioning capability and resolution of the system.Firstly,the main hardware modules,such as the translation stage and illumination light path,were improved to meet the requirements of line-scanning imaging.The system was tested for its ability to image different tissues and organs of mice at room temperature and cyrogenic environment.Afterwards,two opticalsectioning algorithms,line illumination modulated microscopy and digital structured light illumination modulated microscopy,were systematically derived by spatial domain analysis of the linear system,and their application to this system was experimentally verified.In order to improve the data acquisition efficiency of the system,this paper designs and implements a complete control program to improve the data acquisition capability of the system.Based on the function of each hardware and the connection to the workstation,the program functions are modularised and the UI interface is designed.An automatic liquid nitrogen level control sub-module,exploiting temperature changes caused by changes in liquid nitrogen level,was developed to achieve automatic control of the different liquid nitrogen levels required for milling and imaging.Different solutions are designed for milling,imaging and cleaning according to the characteristics of the two types of samples,mouse whole body and mouse organs,and the automated data acquisition module is implemented.Finally,this paper demonstrates the potential of a cryo-line-scan micro-optical section tomography system for acquiring three-dimensional structural information on the whole body and intact organs of mice by performing three-dimensional automated imaging of the whole body of B6-GFP transgenic mice and the lungs of C57BL/6 mice.A method relying on the original line illumination intensity distribution aimed to correct the brightness of strips was developed to mitigate strip stitching traces.The experimental process of largevolume samples for three-dimensional continuous cryogenic imaging has been improved.The whole-body imaging results of cryo-fixed B6-GFP transgenic mice were collected,and the whole-body organs of the mice were clearly distinguished,indicating that the system is capable of imaging biological tissues of different textures and can switch between different resolutions during the imaging process to ensure the overall imaging efficiency while acquiring fine information on the region of interest at high resolution.The label-free threedimensional imaging of the lungs of cryo-fixed C57BL/6 mice demonstrated the results of lung tracheal reconstruction,demonstrating the technical advantages of the system in terms of autofluorescence of biological tissues and preservation of the morphology of intact organs with cavity structure.