阅读说明：本技术 一种阵列结构光照明的高信噪比显微成像系统 (High signal-to-noise ratio microscopic imaging system illuminated by array structure light ) 是由 匡登峰 闫超 于 2021-09-07 设计创作，主要内容包括：本发明涉及一种阵列结构光照明的高信噪比显微成像系统,主要由照明系统和成像系统两部分组成。其中照明系统的特点在于使高斯光束经过加载有正多边棱锥阵列结构相位图的空间光调制器后产生阵列密集排布的光场,光利用率大大提高；成像系统的特点在于由显微物镜、成像透镜和相机搭建而成的显微系统实现放大成像,同时能够在计算机中实时成像,系统装置结构简易,自组搭建设计,光学放大倍率可调。本发明相比传统器件调制照明的方法简易、快捷,拥有更多的调制选择性,成像方面图像信噪比提升,拥有更大的成像范围,更小的光漂白性和光毒性。(The invention relates to a high signal-to-noise ratio microscopic imaging system illuminated by array structured light, which mainly comprises an illumination system and an imaging system. The illumination system is characterized in that after Gaussian beams pass through a spatial light modulator loaded with a phase diagram of a regular polygonal pyramid array structure, light fields in array dense arrangement are generated, and the light utilization rate is greatly improved; the imaging system is characterized in that the microscopic system constructed by the microscopic objective, the imaging lens and the camera realizes magnification imaging, and simultaneously can realize real-time imaging in a computer, the system device has simple structure, is constructed and designed by self-assembly, and has adjustable optical magnification. Compared with the traditional device, the method for modulating the illumination is simple and rapid, has more modulation selectivity, improves the image signal-to-noise ratio in the imaging aspect, and has larger imaging range, smaller photobleaching property and phototoxicity.)

1. A high signal-to-noise ratio microscopic imaging system illuminated by array structure light is characterized by comprising an illumination system and an imaging system, wherein illumination and imaging light paths are perpendicular to each other. The illumination system converts Gaussian beams emitted by the laser into densely-arranged array light in a regular quadrilateral and regular hexagon through the spatial light modulator, and light sheet lattice array illumination is formed in the sample cell through the adjustment of the lens group and the focusing of the microscope objective. The imaging light path utilizes the microscope objective to collect sample signals vertical to the illumination direction, and the sample signals are collected by the high-speed camera to be imaged after being adjusted by the lens. The final imaging resolution is in micron order, and high-speed imaging on the living sample cell level can be realized.

2. The arrayed structured light illuminated high signal-to-noise ratio microscopic imaging system according to claim 1, wherein the structured devices are respectively formed by array close-joint arrangement of regular quadrangular pyramids and regular hexagonal pyramids, and the maximum size setting range of the bottom surfaces of the single regular polygonal pyramids is 10 micrometers to 30 micrometers. Height of each cone

Where λ is the laser operating wavelength and n is the material refractive index of the device.

3. The arrayed structure illuminated high signal-to-noise ratio microscopic imaging system according to claim 2, wherein a phase diagram is obtained through simulating a device structure, the phase diagram is loaded on a spatial light modulator to realize modulation of illumination light, incident light passes through the spatial light modulator after being transmitted, the phase diagram of the regular polygonal pyramid of each unit can be modulated to generate Bessel-like light sheets with working distances of millimeter magnitude and full widths at half maximum within the working distances of micrometer magnitude, and the working distance w of each light sheet is_dAnd the thickness q can be expressed as

Wherein h is₀Is the height of the regular polygonal pyramid, n is the refractive index of the material of the device, and D is the half width of the bottom surface of the regular polygonal pyramid. Compared with the traditional device regulation and control method, the illumination mode greatly reduces the photobleaching and phototoxicity of the illumination light on the living biological sample, and simultaneously improves the imaging quality.

4. The arrayed structured light illuminated high signal-to-noise ratio microscopic imaging system according to claim 1, wherein the regular polygonal pyramid array can improve the utilization rate of incident light compared with the traditional circular micro axicon array, wherein the regular rectangular pyramid array arrangement can improve the lens occupancy by 21.5%, the regular hexagonal pyramid array arrangement can improve the lens occupancy by 27.3%, and the acquisition frequency in light field imaging is further improved.

5. The arrayed, structured-light illuminated high signal-to-noise ratio microscopy imaging system of claim 1, wherein a square capillary tube is used to carry the sample while ensuring that structured light is incident normally on the capillary tube wall to reduce scattering and refraction of the incident light on the capillary tube wall. If a circular capillary tube is adopted, the scattering and refraction noises of the tube wall are too large, and the imaging quality is seriously influenced.

6. An arrayed structured light illuminated high signal to noise ratio microscopy imaging system as claimed in claim 1 wherein the spatial light modulator in the illumination system is transmissive.

7. The array structured light illuminated high signal-to-noise ratio microscopic imaging system according to claim 1, wherein the number of layers of structured light sheets can be changed by changing the arrangement period of the regular polygonal pyramid array phase diagram, so that the purpose of structured light illuminating different samples in depth is achieved, and three-dimensional structure information of the samples is obtained by reconstructing two-dimensional images.

8. The arrayed structured light illuminated high signal to noise ratio microscopic imaging system of claim 1, wherein the imaging system is a coaxial optical system built by a microscope objective, an imaging lens and a high speed camera, wherein the imaging microscope objective can be freely focused, and the camera is placed on a back focal plane of the imaging lens to collect sample signals and display the signals in real time in a computer.

Technical Field

The invention relates to micro-optical devices and biological living body microscopic imaging, in particular to a high signal-to-noise ratio microscopic imaging system illuminated by array structured light, which is mainly characterized in that structured light of a lattice array of regular polygonal light sheets which are closely arranged is used for illuminating and real-time imaging of a biological living body.

Background

The fluorescent microscope is a new microscopic imaging technology in the rise of this century. Unlike conventional fluorescence microscopes, it uses sheet light to laterally illuminate transparent biological tissue, thereby enabling microscopic imaging of biological tissue at different depths. The basic working principle of the device is that a beam of sheet-shaped light source for illuminating a sample from the side is utilized, the thickness of the sheet of light is generally in the order of micrometers, then a biological fluorescence signal is acquired from the direction vertical to an illumination light path, and the fluorescence image acquired by a wide field is utilized to rapidly acquire three-dimensional space information of the sample, thereby providing help. At present, most of the used technologies mainly excite different biological tissue planes by moving a sample to enable an incident light surface to excite the different biological tissue planes, and since the excited plane of the sample is an imaging plane, a two-dimensional image sequence can be obtained to reconstruct three dimensions by scanning the whole sample, so that the resolution at the cell level can be achieved. Compared with the traditional microscopic technology, the light sheet microscope has lower phototoxicity and photobleaching property, higher axial resolution and faster imaging speed. However, because the three-dimensional image obtained by scanning the biological sample with the optical sheet is not captured at the same time, the real-time performance is not good. In addition, repeated scanning of the sample can cause damage to living organisms, and thus this technique is not well suited for studies requiring long-term observation. Therefore, the system modulates the light beam by utilizing the phase diagram of the closely-arranged regular polygonal pyramid array, improves the illumination depth of light sheet microscopic imaging, realizes the integral illumination of organisms, simultaneously uses the improved light field imaging system, has less photobleaching and phototoxicity, improves the signal to noise ratio of the image, increases the illumination depth, can obtain an integral three-dimensional image at a certain moment, and is suitable for the body imaging of a living organism sample observed for a long time.

Disclosure of Invention

Aiming at the technical defects in the prior art, the invention aims to provide a method for modulating an illumination light field by utilizing a phase diagram of a closely-packed regular polygonal pyramid array, improving the image resolution and solving the problem of imaging quality of a light-sheet illumination microscope. Meanwhile, the invention has the characteristics of compact system structure, easy construction, low implementation cost, high image quality and the like.

In order to achieve the purpose, the technical scheme adopted by the invention is to provide a high signal-to-noise ratio microscopic imaging system illuminated by array structure light, the system consists of an illumination system and an imaging system, and the illumination and imaging light paths are perpendicular to each other. The illumination system converts Gaussian beams emitted by the laser into densely-arranged array light in a regular quadrilateral and regular hexagon through the spatial light modulator, and light sheet lattice array illumination is formed in the sample cell through the adjustment of the lens group and the focusing of the microscope objective. The imaging light path utilizes the microscope objective to collect sample signals vertical to the illumination direction, and the sample signals are collected by the high-speed camera to be imaged after being adjusted by the lens. The final imaging resolution is in micron order, and high-speed imaging on the living sample cell level can be realized.

In the invention, the array structure light is respectively formed by the array close-joint arrangement of a regular quadrilateral pyramid and a regular hexagonal pyramid, and the maximum size setting range of the bottom surface of a single regular polygonal pyramid is 10-30 micrometers. Height of each cone

Where λ is the laser operating wavelength and n is the material refractive index of the device.

In the array structure light illuminated high signal-to-noise ratio microscopic imaging system, a phase diagram can be obtained through a simulation device structure and loaded on a spatial light modulator to realize modulation of illumination light, incident light passes through the spatial light modulator after being transmitted, the regular polygonal pyramid phase diagram of each unit can be modulated to generate Bessel-like light sheets with working distance of millimeter magnitude and full width at half maximum within working distance of micrometer magnitude, and the working distance w of each light sheet_dAnd the thickness q can be expressed as

Wherein h is₀Is the height of the regular polygonal pyramid, n is the refractive index of the material of the device, and D is the half width of the bottom surface of the regular polygonal pyramid. The lighting mode is adjusted relative to the traditional deviceThe control method greatly reduces the photobleaching and phototoxicity of the illuminating light to the living biological sample, and simultaneously improves the imaging quality.

The high signal-to-noise ratio microscopic imaging system illuminated by the array structure light is characterized in that compared with the traditional circular micro-axicon array, the regular polygonal pyramid array can improve the utilization rate of incident light, wherein the regular rectangular pyramid array arrangement can improve the lens proportion by 21.5%, the regular hexagonal pyramid array arrangement can improve the lens proportion by 27.3%, and the acquisition frequency in light field imaging is further improved.

The array structured light illuminated high signal-to-noise ratio microscopic imaging system is characterized in that a square capillary is used for carrying a sample, and meanwhile, the normal incidence of structured light on the wall of the capillary is ensured, so that the scattering and refraction of incident light on the wall of the capillary are reduced. If a circular capillary tube is adopted, the scattering and refraction noises of the tube wall are too large, and the imaging quality is seriously influenced.

The array structured light illuminated high signal-to-noise ratio microscopic imaging system is characterized in that the number of layers of structured light sheets can be changed by changing the arrangement period of a regular polygonal pyramid array phase diagram, so that the purpose of deeply illuminating different samples by structured light is achieved, and three-dimensional structure information of the samples is obtained by reconstructing two-dimensional images.

The high-signal-to-noise-ratio microscopic imaging system illuminated by the array structured light is characterized in that the imaging system is a coaxial optical system built by a microscope objective, an imaging lens and a high-speed camera, wherein the imaging microscope objective can be freely focused, and the camera is placed on a back focal plane of the imaging lens to collect a sample signal and display the sample signal in real time in a computer. At present, the high-speed camera can reach 100fps, which means that the target sample can be observed and recorded 100 times per second, so that real-time imaging can be realized. Compared with the prior art, the invention has the technical effects that:

the invention provides a high signal-to-noise ratio microscopic imaging system illuminated by array structure light, which is simple in structure, self-assembled in design and suitable for in-vivo biological sample experiments with the spatial resolution at the micron level. The closely-arranged regular polygonal pyramid array phase diagram is loaded on the spatial light modulator to generate light sheet lattice array illumination with long working distance, uniform intensity distribution and flat light sheet shape, wherein the regularly polygonal pyramid array in close contact arrangement can improve the utilization rate of incident light, improve the acquisition frequency by 21.5-26%, realize large-range deep illumination and simultaneously improve the signal-to-noise ratio, and greatly reduce photobleaching and phototoxicity.

Drawings

FIG. 1 is a light path of a regular polygonal array light sheet lattice illumination microscopic imaging system provided by the present invention, wherein 1 is a laser; 2 is an attenuation sheet; 3 is a light beam collimating objective lens; 4 is a lens 1; 5 is a polarizing plate; 6 is a spatial light modulator; 7 is a polarizing plate; 8 is an illumination microscope objective; 9 is a square capillary tube; 10 is a detection microscope objective; 11 is a lens 2; 12 is a high speed camera.

FIG. 2 is a schematic diagram of the optical path of a detection imaging system, wherein 1 is a square capillary tube filled with a zebra fish sample; 2 is a detection microscope objective; 3 is a lens; 4 is a high-speed camera; 5 is a capillary clamping device and a fixed table; 6 is a cylinder.

Fig. 3 is a schematic diagram of the light path of a light beam passing through a regular polygonal pyramid.

FIG. 4 is a view of a regular polygonal pyramid structure and its phase, wherein (a) is a regular rectangular pyramid; (b) is a regular rectangular pyramid array; (c) is a regular rectangular pyramid phase mask diagram; (d) is a phase mask diagram of a regular rectangular pyramid array; (e) is a regular hexagonal pyramid; (f) is a regular hexagonal pyramid array; (g) is a regular hexagonal pyramid phase mask diagram; (h) is a phase mask diagram of a regular rectangular hexagonal pyramid array.

FIG. 5 is a structured light field modulated by a phase diagram of a close-packed regular polygonal array, where (a) is a regular rectangular pyramid; (b) is a regular rectangular pyramid array; (c) is a regular hexagonal pyramid; (d) is a regular hexagonal pyramid array.

Fig. 6 is the imaging results of zebra fish samples under the illumination conditions of structured light and gaussian light, respectively, wherein (a) the zebra fish head illuminated by the structured light in the regular hexagonal array is imaged; (b) imaging the zebra fish head illuminated by Gaussian light; (c) the result of gray scale analysis of (a) and (b) is an enlargement of the zebra fish eye outline (the portion enclosed by the yellow dotted line).

Detailed Description

Example 1

As shown in FIG. 1, an array structured light illuminated high signal-to-noise ratio microscopic imaging system is used for observation, wherein 1 is a laser; 2 is an attenuation sheet; 3 is a light beam collimating objective lens; 4 is a lens 1; 5 is a polarizing plate; 6 is a spatial light modulator; 7 is a polarizing plate; 8 is an illumination microscope objective; 9 is a square capillary tube; 10 is a detection microscope objective; 11 is a lens 2; 12 is a high speed camera.

FIG. 2 is a schematic diagram of the optical path of a detection imaging system, wherein 1 is a square capillary tube filled with a zebra fish sample; 2 is a detection microscope objective; 3 is a lens; 4 is a high-speed camera; 5 is a capillary clamping device and a fixed table; 6 is a cylinder.

The phase mask pattern of the array structured light proposed in the present invention was first tested. The experimental procedure was as follows:

1. the part of the lighting system as shown in fig. 1 is first built up.

2. A high-speed camera is placed near the focal plane of the illumination microscope objective.

3. And turning on a power supply, loading a mask pattern of an array structure on the spatial light modulator, and simultaneously adjusting the two polaroids to enable emergent light to be structured light as far as possible.

4. The high speed camera position was adjusted and a light field picture was taken, the result of which is shown in fig. 5.

And (3) performing in-vivo biological sample real test by using an array structured light illuminated microscopic imaging system. The experimental procedure was as follows:

1. on the basis of a structural light field detection experimental system, a detection imaging system is set up as shown in fig. 2.

2. Live zebra fish larvae are placed in a square capillary tube, and the capillary tube is fixed by a clamping device.

3. The power is turned on and the square capillary is fine-tuned to the focal plane of the illumination microscope objective. At the same time, the 4 x observation objective is used to adjust the imaging to be clear.

4. And selecting Gaussian light illumination as a control group and regular hexagonal array structured light as an experimental group, sequentially testing and repeating a plurality of groups, and shooting an imaging result by using a high-speed camera.

5. The imaging results of the gaussian light and the regular hexagonal array structured light illumination are analyzed and compared, and the results are shown in fig. 6. Wherein (a) is zebra fish head imaging illuminated by regular hexagonal array structured light; (b) imaging the zebra fish head illuminated by Gaussian light; (c) the result of gray scale analysis of (a) and (b) is an enlargement of the zebra fish eye outline (the portion enclosed by the yellow dotted line).

FIG. 6 shows that under the Gaussian illumination condition, the illumination brightness is not uniform, an overexposure phenomenon exists in a local range, the number of overexposure noise pixels is equivalent to that of actual image signal pixels, and the image signal-to-noise ratio is low; under the condition of regular hexagonal array structured light illumination, the zebra fish sample can be seen to have uniform overall imaging brightness, only a small part of the zebra fish sample has an overexposure phenomenon, the contrast is high, the signal-to-noise ratio and the definition are greatly improved, and the phototoxicity and photobleaching on organisms can be correspondingly greatly reduced.