基于液体变焦透镜和振镜的三维光片显微成像系统

Three-Dimensional Light-Sheet Microscopy Imaging System with Liquid Zoom Lens and Galvanometer Mirror

搭建了一种基于液体变焦透镜和振镜的三维光片显微成像系统,设计了振镜、液体变焦透镜、相机的同步控制采集成像系统,通过调谐振镜和液体变焦透镜,使得光片激发样品和成像同步,获得样品不同切面的图像堆栈并实现样品的三维重建。当采用数值孔径为0.3、放大倍率为10的成像物镜时,该系统的轴向扫描范围为507μm,横向视场达到1970μm×1300μm,横向分辨率为1.32μm,轴向分辨率可达12.75μm。在轴向扫描过程中,系统的放大倍率保持恒定,可以用于对一定尺寸生物样品的成像实验和相关研究,并通过对斑马鱼胚胎进行成像验证所提系统对厚生物样品成像的可行性。

Objective With the advancement of science,the research objects of life science have changed from monolayers of cells to organs and even in-situ measurement of animals.The study of biological samples relies heavily on three-dimensional(3D)volumetric imaging to provide structural and functional information about the samples.At present,there are mainly two methods to obtain the structure of biological tissue at different levels for 3D imaging of thick biological samples,i.e.,the movement of samples and the simultaneous movement of the light sheet and the detection objective lens.The former can cause instability problems,while for the latter,the imaging speed is limited by inertia,and the magnification of the system changes during axial scanning.To capture the dynamic state of samples and avoid distortion of recorded data,we should ensure the volumetric imaging is performed in parallel at tremendously high speeds or by multiple planes sequentially,ideally without any sample movement.Therefore,we set up a 3D light-sheet microscopy imaging system based on the cooperation of the liquid zoom lens and the galvanometer mirror and design a synchronous control acquisition imaging system for the galvanometer mirror,the liquid zoom lens,and the camera to enable 3D imaging of the entire samples without sample movement.We use microspheres and zebrafish embryos to demonstrate the feasibility of the system to image thick biological samples.This study provides a feasible method for the 3D topographical observation of thick biological samples.Methods In this paper,we set up a 3D light-sheet microscopy imaging system based on the cooperation of the liquid zoom lens and the galvanometer mirror and design a synchronous control acquisition imaging system for the galvanometer mirror,the liquid zoom lens,and the camera.First,we use a double telecentric optical path as the imaging optical path,and thus the displacement of the principal plane of the system changes linearly with the focal length of the liquid zoom lens,while the magnification of the system remains constant.Then,we analyze the relationship between the power control range of the liquid zoom lens and the axial scanning range of the system and the relationship between the applied voltage of the galvanometer mirror and the scanning range of the light sheet.Next,the control timing relationship among the galvanometer mirror,the liquid zoom lens,and the camera is obtained by the imaging of the fluorescent microspheres.Afterward,the field of view and the resolution of the system are analyzed by the imaging of the standard fluorescent microsphere samples.Finally,the 3D imaging performance of the system is evaluated by the imaging results of microsphere samples and zebrafish embryos.Results and Discussions The constructed 3D light-sheet microscopy imaging system based on the cooperation of the liquid zoom lens and the galvanometer mirror has good 3D imaging performance.As the drive current of the liquid zoom lens increases,the magnification of the system remains unchanged(Fig.4).The displacement distance of the object-side principal plane has a linear relationship with the current applied to the liquid zoom lens(Fig.5),and the axial position of the scanning light sheet has a linear relationship with the applied voltage of the galvanometer mirror(Fig.6).For every 1 mV increase in the applied voltage of the galvanometer mirror,the liquid zoom is required.Specifically,when the applied voltage of the galvanometer mirror increases by 1 mV,the applied current of the liquid zoom lens needs to be increased by 1.73 mA.The position of the light sheet is moved by 3.00μm,and the axial scanning range of the system,namely,the moving range of the object-side principal plane,can reach 507μm(Fig.7).The imaging experiments of fluorescent microspheres demonstrate that the lateral field of view of the system can reach 1970μm×1300μm(Fig.9),and the actual detection field of view is 1970μm×1300μm×507μm;the lateral resolution of the system is 1.32μm,and the axial resolution is 12.75μm(Fig.10).The fluorescent microsphere samples are imaged by the 3D light-sheet microscopy imaging system to verify the correctness of the synchronous control timing of the liquid zoom lens,the galvanometer mirror,and the camera.The 3D reconstruction of the obtained light-sheet image is used to verify the feasibility of the system for 3D imaging of actual samples(Fig.11).This paper employs the imaging of zebrafish embryos to verify the feasibility of the device for 3D imaging of thick biological samples(Figs.12 and 13).Conclusions We set up a 3D light-sheet microscopy imaging system based on the cooperation of the liquid zoom lens and the galvanometer mirror and design a synchronous control acquisition imaging system of the galvanometer,the liquid zoom lens,and the camera.By the adjustment of the galvanometer mirror and the liquid zoom lens,the sample excitation by the light sheet is synchronized with the imaging to obtain the sample image stacks of different sections for 3D reconstruction and 3D imaging of the samples.When the imaging objective lens with a magnification of 10 and an NA of 0.3 is used,the axial scanning range of the system is 507μm,and the lateral field of view reaches 1970μm×1300μm;the lateral resolution is 1.32μm,and the axial resolution can reach 12.75μm.The magnification of the imaging system remains constant during the axial scanning process,which can meet the requirements of the imaging experiments and related studies of a certain size of biological samples,and the imaging of zebrafish embryos demonstrates the imaging feasibility of thick biological samples.It is expected that liquid zoom lenses with larger apertures and better focusing performance will emerge soon,and with high-speed cameras,high-speed volumetric imaging of large biological samples can be achieved,or with line scanning for the suppression of sample scattering,the imaging performance of large biological samples can be improved.