CN-121253497-B - Method for super-resolution large-depth three-dimensional scanning light sheet microscope

Abstract

The invention discloses a method for a super-resolution large-depth three-dimensional scanning light sheet microscope, which belongs to the technical field of fluorescence imaging, and the method comprises the steps of processing excitation light through a cylindrical lens, and obtaining a longer working distance of an excitation objective lens by utilizing secondary convergence of a generated initial light sheet, so that the space conflict between the excitation objective lens and a sample is reduced; the configuration determines the imaging depth of the collection objective by placing the collection objective at a small angle of inclination and matching a liquid chamber with an optical window having the same angle of inclination, and then selecting the collection objective and imaging buffer solution matching the refractive index of the tissue-permeabilized sample, which can avoid optical aberrations to achieve high resolution imaging. The invention can realize both high-resolution imaging and depth imaging, and simultaneously can improve the lateral range of imaging compared with the prior art, thereby providing powerful support for the research of bioscience and clinical medicine.

Inventors

• ZHAN YIFEI
• YE QING
• TIAN JIANGUO

Assignees

• 南开大学

Dates

Publication Date

20260512

Application Date

20251203

Claims (1)

1. 1. A method for a super-resolution large-depth three-dimensional scanning light sheet microscope is characterized by comprising the following steps: S1, generating a secondary convergence excitation light sheet: the excitation laser beam is converged before the back focal plane of the excitation objective lens through the action of the cylindrical lens, the converged light beam formed by the excitation objective lens generates a primary light sheet on the focal plane of the excitation objective lens, and simultaneously, a secondary converged excitation light sheet is generated outside the focal plane; s2, setting an optical path of a collecting objective lens: placing the collecting objective lens at a small inclination angle, and matching with an optically transparent liquid chamber with the same inclination angle, so that the excitation light plane can coincide with the imaging plane of the collecting objective lens; s3, sample transparentizing treatment: The sample to be observed is subjected to tissue transparentization treatment, so that the uniform refractive index is realized to achieve optical transparentization; s4, executing three-dimensional scanning imaging: Selecting an imaging buffer solution matched with the transparent sample refractive index and a collecting objective lens, placing the sample into a liquid chamber, injecting the imaging buffer solution, carrying out surface excitation on the sample by a secondary convergence excitation light sheet, collecting fluorescent signals emitted by a sample plane by the collecting objective lens, controlling the operation of a camera and a displacement table, and realizing high-resolution large-depth three-dimensional scanning imaging on the sample; s5, image data processing: processing the image data acquired in the step S4 by image data to obtain a three-dimensional reconstruction image and a high-resolution sample single image; in the step S1, the excitation distance of the secondary convergence excitation light sheet is 20-100 mm; In the step S2, the small inclination angle is 5-30 degrees; In the step S5, the resolution of the high-resolution sample single image is 100-300 nm.

Description

Method for super-resolution large-depth three-dimensional scanning light sheet microscope Technical Field The invention belongs to the technical field of fluorescence imaging, and particularly relates to a method for a super-resolution large-depth three-dimensional scanning light sheet microscope. Background In recent years, three-dimensional high-resolution imaging of biological samples can provide rich information in life sciences and clinical medicine research. The light sheet microscope is a main technical platform for rapid high-resolution three-dimensional fluorescence imaging of transparent large tissues because of the low photobleaching property (photobleaching is a process that fluorescent groups cause covalent bond fracture or non-specific chemical reaction due to illumination, so that permanent fluorescence is disabled, and fluorescent signals are continuously attenuated in illumination in the imaging process, so that the signal to noise ratio is reduced, the resolution is reduced) and the advantage of high-speed imaging. The light sheet microscope is a microscope based on the fluorescent imaging principle, excitation light is modulated into a sheet structure through a light path, and only fluorescent dye near an imaging plane is excited, so that signal attenuation caused by the photo-bleaching is avoided, interference of signals in a non-imaging area is avoided, the signal to noise ratio is improved, and the light sheet microscope is suitable for imaging thick samples. With the continuous and intensive research, higher requirements are placed on the spatial resolution and imaging range of the light sheet microscope. Because of the spatial conflict of the excitation objective, the detection objective and the sample to be observed, the existing light sheet microscope is difficult to realize high resolution, large depth imaging and compatibility of various samples at the same time, and is difficult to meet the demands in life science and clinical medical research. For this core problem, in most cases only one of the critical properties is selected and the other is sacrificed. Some light sheet microscopes choose to use low numerical aperture objectives to reduce imaging resolution in exchange for large imaging depths, and others use high numerical aperture objectives but limited imaging depths, both of which are difficult to achieve at the same time. Disclosure of Invention The invention aims to provide a method for a super-resolution large-depth three-dimensional scanning light sheet microscope. In order to solve the technical problems, the technical scheme adopted by the invention is that the method for the super-resolution large-depth three-dimensional scanning light sheet microscope comprises the following steps: S1, generating a secondary convergence excitation light sheet: The excitation laser beam is converged before the back focal plane of the excitation objective lens through the cylindrical lens, the converged light beam formed by the excitation objective lens generates a primary light sheet on the focal plane of the excitation objective lens, and simultaneously, a secondary converged excitation light sheet with longer excitation distance is generated outside the focal plane. The secondary converging excitation light sheet reserves more free space for the sample and the collection objective, so that the collection objective can be placed at a smaller inclination angle. S2, setting an optical path of a collecting objective lens: The collecting objective lens is placed at a small inclination angle and matched with an optically transparent liquid chamber with the same inclination angle, so that the excitation light plane can coincide with the imaging plane of the collecting objective lens, and aberration (the deviation between actual optical system imaging and ideal Gaussian optical imaging is avoided, and the smaller the aberration is, the better the imaging effect is). S3, sample transparentizing treatment: the sample to be observed is subjected to tissue transparentization treatment, so that the uniform refractive index is realized, and the optical transparentization is realized. S4, executing three-dimensional scanning imaging: Selecting an imaging buffer solution matched with the transparent sample refractive index and a collecting objective lens, placing the sample into a liquid chamber, injecting the imaging buffer solution, carrying out surface excitation on the sample by a secondary converging excitation light sheet, collecting fluorescent signals emitted by the plane by the collecting objective lens, controlling the camera and a displacement table to run successively, and realizing high-resolution large-depth three-dimensional scanning imaging on the sample. S5, image data processing: And (3) processing the image data acquired in the step (S4) through the image data to obtain a three-dimensional reconstructed image and a high-resolution sample single image. Further, in the step S1,