Optical sheet illumination microscopic method and device based on differential

Abstract

The invention discloses an optical sheet illumination microscopic method based on differential; the method comprises the following steps: 1, a lighting beam after beam expanding is converted into parallel polarized light and vertical polarized light; 2, the parallel polarized light is individually used, the parallel polarized light with a light spot as a solid center is regarded as a first illumination light converted to a first circular polarized illumination light, the first circular polarized illumination light is focused to a surface of a fluorescence sample through a focusing module, and the fluorescence emitted by the fluorescence sample is collected to obtain a first photo; 3, the vertical polarized light is individually used and modulated into a second illumination light having a hollow light spot, the second illumination light is converted into a second circular polarized illumination light and focused to a surface of the fluorescence sample through the focusing module, and the fluorescence emitted by the fluorescence sample is collected to obtain a second photo; 4, a computer carries out difference treatment for the two photos in the step 2and the step 3, thereby completing scanning of the sample. The invention also discloses an optical sheet illumination microscopic device based on differential.

Description

technical field [0001] The invention belongs to the field of light sheet illumination microscopy, in particular to a method and device for light sheet illumination microscopy based on difference. Background technique [0002] Light sheet illumination microscopy uses a layer of light beam to excite the fluorescent sample from the side, and the fluorescent light is received by the detector in the vertical direction of the incident illumination light path to realize imaging. By moving the sample, the incident light path excites different planes, and the excitation beam can be incident on the sample from left and right directions, and the angle of the beam can be changed. Using the light sheet illumination technology, we can easily obtain the image of the whole tissue without slicing the sample. 3D images. Since the plane of excitation of the sample is the imaging plane, photobleaching and optical damage can be minimized. Therefore, the light sheet illumination microscope has ...

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Problems solved by technology

[0004] However, because the resolution of the microscope is related to the numerical aperture of the objective lens, the larger the numerical aperture, the smaller the spot focused on the sample surface, and the higher the resolution of the microscope. At the same time, the imaging depth is inversely proportional to the numerical aperture. The larger the imaging depth, the smaller the imaging depth, so the traditional light-sheet illumination microscope can only observe the living dynamics of animals and plants at the subcellular level, and cannot achieve super-resolution microscopic imaging while ensuring the imaging depth

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