Research On Dual-color 4D Fluorescence Microscopic Imaging System Based On Non-diffraction Light Sheet Illumination

High-resolution recording of the dynamic biological processes of organelles in living cells is essential for understanding life.How to make real-time high-spatial-temporal resolution imaging of living cells in a non-invasive way has always been the focus of scientists' research.Compared with traditional wide-field and confocal imaging techniques,light sheet fluorescent microscopy has become the preferred technology for achieving this goal with its high temporal and spatial resolution,low damage and long-term imaging capabilities.However,limited by the resolution and field of view of the traditional Gaussian light sheet,how to achieve a larger field of view in the direction of light propagation without losing axial resolution is an urgent problem to be solved in light sheet fluorescence microscopy.This paper demonstrates a simple and effective way to generate a color-free,low sidelobe ultrathin static Bessel light sheet using a combination of a line beam and a dual-ring mask,which can achieve long-term non-invasive three-dimensional two-color imaging of living cells with a speed of 1 vol/s and a lateral/axial resolution of 280 nm/500 nm.The specific work is as follows:1.Complete the theoretical analysis and optical simulation of light sheet generation based on the Fresnel diffraction theory,and optimize the mask geometric parameters to generate a low sidelobe thin light sheet that can cover a whole cell.2.Designed and built a set of fluorescence microscopy imaging system based on the above ideas,the system control program is written,and the performance of the system is tested.The system structure is further optimized according to the actual application.3.Using the built-up light sheet microscopic imaging system to observe the different labeled organelles and their interactions,and the imaging results showed the great potential of the system in biological research.The design of the system is based on the technical requirements for high-resolution and long-term imaging of 3D live cells in the life sciences field,and aims to provide researchers in related biomedical fields with a low-cost,high-performance,simple structure and easy-to-use imaging tools.It is hoped that the continuous iterative improvement of the system will eventually be widely used.