阅读说明：本技术 基于细胞旋转主动光操控技术的光片荧光显微方法和系统 (Optical sheet fluorescence microscopy method and system based on cell rotation active light control technology ) 是由 尹君 陈宏宇 于凌尧 王少飞 贾源 胡徐锦 苑立波 于 2021-07-12 设计创作，主要内容包括：本发明提供的是一种基于细胞旋转主动光操控技术的光片荧光显微方法和系统。其特征是：一台激光器输出的激光耦合进显微物镜稳定捕获待测细胞。另一台激光器输出的激光经声光偏转器可偏转不同角度,由显微物镜聚焦交替照射到被捕获细胞两端,实现细胞旋转角度的精准主动光操控。当被捕获细胞旋转至特定角度并稳定后,第三台激光器输出的激光经扩束整形形成片状光,激发照明层面内的荧光团,产生的荧光信号经与片状光垂直的显微物镜收集。通过对细胞旋转的精准主动光操控,获取细胞三维结构的高空间分辨率荧光层析图像。本发明提供的方法和系统具有非接触、光致损伤小、灵活性高等特点,在生物学、医学和生命科学等研究领域中具有广泛的应用前景。(The invention provides a light sheet fluorescence microscopy method and a system based on a cell rotation active light control technology. The method is characterized in that: laser output by one laser is coupled into the microscope objective to stably capture cells to be detected. The laser output by the other laser can deflect at different angles through the acousto-optic deflector, and the two ends of the captured cell are alternately irradiated by focusing of the micro objective lens, so that accurate active light control of the cell rotation angle is realized. When the captured cell rotates to a specific angle and is stable, laser output by the third laser is expanded and shaped to form sheet light, a fluorophore in the illumination layer surface is excited, and a generated fluorescence signal is collected through a microscope objective perpendicular to the sheet light. And acquiring a high-spatial-resolution fluorescence tomography image of a three-dimensional structure of the cell by accurate active light control on cell rotation. The method and the system provided by the invention have the characteristics of non-contact, small photoinduced damage, high flexibility and the like, and have wide application prospects in the research fields of biology, medicine, life science and the like.)

1. The invention provides a light sheet fluorescence microscopic imaging method and a system based on a cell rotation active light control technology. The method is characterized in that: it consists of laser light sources 1, 13, 18; lenses 2, 3, 14, 15, 20, 21; the mirrors 4, 22; a cylindrical lens 5; apochromatic microobjectives 6, 9, 17; an optical filter 10; an imaging lens 11; a CMOS camera 12; a dichroic mirror 16; an acousto-optic deflector 19; a sheet-like illumination beam 7; and a single cell 8 of the living body to be detected. After the laser output by the laser 13 is expanded by the lenses 14 and 15, the laser is reflected and coupled into the apochromatic microscope objective 17 by the dichroic mirror 16, and acts on the living body single cell 8 to be detected to form a focused light field, so that the living body single cell to be detected is stably captured. The laser light output from the laser 18 is coupled into the acousto-optic deflector 19 to generate a laser beam with a certain deflection angle. Coupled into apochromatic microscope objective 17 via lenses 20, 21. By changing the modulation frequency of the acousto-optic deflector alternately, the light beams are focused on two ends of the living body unicell 8 to be detected alternately and are respectively used as rotating light for pushing the cell to rotate and braking light for stopping the cell to rotate, so that accurate active light control on the cell rotating angle is realized. When the cell rotates to an angle and reaches a stable state, the laser output by the laser source 1 is expanded by the lenses 2 and 3, reflected by the reflector 4, shaped by the cylindrical lens 5 and coupled into the apochromatic microscope objective 6 to generate a sheet-shaped illuminating beam 7 to illuminate one layer of the cell 8 to be detected. The fluorophores in the illumination area of the single cell 8 of the living body to be detected are excited, and the generated fluorescence signals are collected by an apochromatic microscope objective 9 with the optical axis vertical to the plane of the sheet-shaped illumination light beam 7. The fluorescence signal is detected and received by a CMOS camera 12 after passing through an optical filter 10 and an imaging lens 11, and a fluorescence tomography image of the sheet-shaped illumination area is obtained. Under the alternate action of the pushing light and the braking light, the living body single cell 8 to be detected continuously rotates, the rapid scanning of the sheet-shaped illumination light beam 7 in the cell is realized, and thus the high-spatial resolution fluorescence tomography image of the three-dimensional structure in the cell is obtained.

2. The light manipulation device of claim 1: the light manipulation section consists of the capture light from the laser 13 and the deflectable light from the laser 18 controlled by the acousto-optic deflector 19. The capture light serves to stably capture the cells to be tested, so that the cells are fixed in a position for imaging and rotating around a fixed axis. Laser light emitted by the laser 18 is coupled into the acousto-optic deflector 19, the modulation frequency of the acousto-optic deflector 19 is changed so as to change the deflection angle of the laser light, and the cell is pushed by focusing on one side of the captured cell through the microscope objective lens 17. When the cell rotates a certain angle, the laser is focused on the other end of the cell through the acousto-optic deflector 19, and the rotating cell is braked. When the cell is at rest, the CMOS camera again collects structural images of the cell at different angles. Under the combined action of the capture light, the pushing light and the braking light, the cell continuously rotates around a specific rotating shaft, so that the sheet-shaped illumination light is rapidly scanned inside the cell, and the three-dimensional structural image of the cell is finally acquired.

3. A sheet light imaging system according to claim 1. The method is characterized in that: the light sheet microscope is characterized in that laser emitted by a laser light source 1 is expanded by a beam expanding system consisting of lenses 2 and 3, reflected by a reflector 4, shaped by a cylindrical lens 5 and then coupled into an apochromatic microscope objective 6, and a sheet-shaped illuminating light beam 7 is generated to illuminate one layer of a cell 8 to be detected. A certain layer of the cell 8 to be detected is excited by the beam waist part of the sheet light, the structure with the fluorescent dye mark in the cell is excited to emit fluorescence, a fluorescence signal is collected through an apochromatism microscope objective 9, background noise is eliminated through an optical filter 10, and a fluorescence image is obtained through recording by a CMOS camera 12. And rotating the cells by a certain angle through the optical control system, shooting fluorescence images of the cells at different angles after the cells are stabilized, and finally obtaining a complete three-dimensional structural image of the cells through image processing.

(I) technical field

The invention provides a light sheet fluorescence microscopic imaging method and a light sheet fluorescence microscopic imaging system based on a cell rotation active light control technology, which change the position of a focused laser beam through an acousto-optic deflector and carry out accurate active light control on the rotation angle of a living body single cell, thereby realizing the rapid scanning of the sheet beam in the cell and obtaining the fluorescence tomography image in the living body single cell, and belongs to the field of light control and optical microscopic imaging.

(II) background of the invention

The 21 st century is the century of life sciences. The understanding of life is gradually developed from the structure, from the macro to the micro, from the whole to the local, from the individual to the organ, tissue, cell, organelle, or even the basic substance-molecule that constitutes life. The cell is used as a basic unit of the vital structure and function, and the intensive research on the cell is the key for revealing the phenomenon of life, conquering diseases and modifying life. With the progress of research, the challenge is to use large environmental living cells for developing life activities as 'test tubes', and to obtain sub-cell fine structure image information of living unicells in a non-contact and non-destructive manner on the premise of avoiding influencing the properties of the cells and the microenvironment where the cells are located as much as possible, spatial distribution and change information of different types of biomolecules in the life process of the cells, and functional information of interaction processes between organelles, biomolecules and different types of biomolecules, thereby providing reliable scientific basis for revealing the essence and basic rules of the life activities at a deeper level.

With the increasingly intensive exploration of the nature of life activities, it is desirable to gain a clearer understanding of the structure and function of cells through more detailed observation of the internal structure of cells. From the first discovery of cellular structures by Levensoke with the aid of the first microscope to the present invention of several types of novel microscopic methods and systems, the present inventors have gradually opened the door to the micro world with the aid of these modern instruments and advanced technology. The appearance of the microscope enables researchers to have a completely new understanding of the micro world, particularly the cells, and the understanding of organisms is virtually expressed.

The requirements of current medical research on the resolution and precision of a microscope are high, more complex research needs higher-performance experimental instruments, and in recent years, the field of biological imaging always makes research popular, and new technology which is changed day by day makes experimental methods diversified.

With the development of fluorescence labeling techniques, laser techniques, weak signal detection techniques, and computer techniques, modern microscopic imaging techniques are able to record spatiotemporal information of biological systems with unprecedented temporal and spatial resolutions, changing the way we see, record, interpret, and understand biological events. Especially, the wide-field fluorescence microscopy technology based on autofluorescence or providing high chemical specificity and imaging contrast through fluorescent labeling becomes an important research tool for researching living single cells, acquiring fine three-dimensional structures and quantitative functional information in the living single cells.

In recent years, light sheet fluorescence microscopy using a laminar laser beam has proven to be one of the first techniques to achieve this challenging goal. The light sheet fluorescence microscope comprises two main components of an excitation light path and a detection light path. A thin sheet-like laser beam, commonly referred to as a "light sheet", is used in the excitation light path as excitation light to excite fluorophores in the sheet-like illumination area. The detection light path adopts a wide-field fluorescent signal parallel detection mode, and the generated fluorescent signals are collected in the direction vertical to the plane of the flaky excitation light. And acquiring a three-dimensional structure chromatographic image with high spatial resolution of the biological sample to be detected by rapidly scanning the sheet exciting light. The efficient excitation and detection mode of the light sheet fluorescence microscope not only effectively avoids the problems of photobleaching, photodamage and the like of fluorophores and endogenous organic molecules outside a sheet illumination area, reduces the influence of phototoxicity on the whole sample, ensures the vitality of a living biological sample to be tested in a long-time research process, but also effectively avoids the interference of defocused fluorescence signals, and greatly improves the signal-to-noise ratio of the system.

The optical tweezers technology utilizes an optical trap formed by a focusing optical field to generate a mechanical effect, and can stably capture, accurately control and rapidly screen single viruses, cells and even biomacromolecules in a non-mechanical contact and non-damage mode under the condition of not influencing the interior of cells and the microenvironment where the cells are located. In addition, the optical tweezers can not only control the particles, but also measure the tiny force. As a quantitative analysis tool, the optical tweezers technology can apply a calibrated piconiu-level optical trapping force to a system of interest, so that the displacement of a target system caused by the action of the optical trapping force can be measured with high precision and high sensitivity. At present, in the research fields of molecular biology, biochemistry, biophysics and the like, the optical tweezers analysis technology has been developed into a powerful tool for controlling and analyzing cells at the molecular level, and is widely applied to numerous research fields of biomechanics, biopolymers, biomacromolecules, molecular motors and the like. Meanwhile, the proposal and development of the optical tweezers technology opens the door for observing living cells in a liquid environment in a non-mechanical contact and non-destructive manner for a long time to obtain the internal structure and the physical and chemical properties of the living cells, and further deeply researching the biological regulation and control mechanism and the like of the cell life activity process. The light sheet fluorescence microscopy technology and the optical tweezers technology are organically combined together, so that researchers change from passive observation to active control of living cells, and an effective way is provided for solving the problems.

The development of the light sheet fluorescence microscope for many years, the imaging speed and resolution meet most experimental requirements, and the mode of controlling and fixing the sample is not ignored. The fixation of the sample also has a great influence on imaging, and in a light sheet fluorescence microscopic imaging system, in order to acquire a complete three-dimensional structural image of a cell, the cell needs to be rotated or moved so as to acquire complete information of different layers of the cell. The most used technology of the prior art is to add a micro-displacement table to control the axial movement of cells to be detected; or moving the light sheet to realize the tomography of the cells by scanning the sample cells.

The invention discloses a light sheet fluorescence microscopic imaging method and a system based on a cell rotation angle active light control technology, which can be widely applied to obtaining three-dimensional structural images with high spatial resolution of cells or microorganisms. According to the design, a Gaussian beam is expanded and shaped to generate sheet light, the sheet light is focused in a cell to be detected through an objective lens to excite fluorescent molecules to generate a fluorescent signal, and the fluorescent signal is collected through the objective lens and recorded by a CMOS camera to obtain a chromatographic image of the cell. The cell rotation is controlled through an optical control system, the chromatographic images of the cells at different angles are obtained, and the three-dimensional structural images of the cells are obtained through image reconstruction. The light control system realizes the purpose of accurately and actively controlling the cell rotation angle by controlling the acousto-optic deflector, has the characteristics of simple structure, high flexibility, easy operation, low cost and the like, and has wide application prospect in a plurality of research fields such as biology, medicine, life science and the like.

Disclosure of the invention

The invention aims to provide a light control light sheet fluorescence microscopic imaging method and a light control light sheet fluorescence microscopic imaging system which are simple in structure, convenient to control and free of contact with cells and based on a cell rotation accurate active light control technology.

The purpose of the invention is realized as follows:

it consists of laser light sources 1, 13, 18; lenses 2, 3, 14, 15, 20, 21; the mirrors 4, 22; a cylindrical lens 5; apochromatic microobjectives 6, 9, 17; an optical filter 10; an imaging lens 11; a CMOS camera 12; a dichroic mirror 16; an acousto-optic deflector 19; a sheet-like illumination beam 7; and a single cell 8 of the living body to be detected. After the laser output by the laser 13 is expanded by the lenses 14 and 15, the laser is reflected and coupled into the apochromatic microscope objective 17 by the dichroic mirror 16, and acts on the living body single cell 8 to be detected to form a focused light field, so that the living body single cell to be detected is stably captured. The laser light output from the laser 18 is coupled into the acousto-optic deflector 19 to generate a laser beam with a certain deflection angle. Coupled into apochromatic microscope objective 17 via lenses 20, 21. By changing the modulation frequency of the acousto-optic deflector alternately, the light beams form focused light fields alternately at two ends of the living body single cell 8 to be detected, and the focused light fields are respectively used as rotating light for pushing the cell to rotate and brake light for stopping the cell to rotate, so that accurate active light control on the cell rotating angle is realized. When the cell rotates to an angle and reaches a stable state, the laser output by the laser source 1 is expanded by the lenses 2 and 3, reflected by the reflector 4, shaped by the cylindrical lens 5 and coupled into the apochromatic microscope objective 6 to generate a sheet-shaped illuminating beam 7 to illuminate one layer of the cell 8 to be detected. The fluorophores in the illumination area of the single cell 8 of the living body to be detected are excited, and the generated fluorescence signals are collected by an apochromatic microscope objective 9 with the optical axis vertical to the plane of the sheet-shaped illumination light beam 7. The fluorescence signal is detected and received by a CMOS camera 12 after passing through an optical filter 10 and an imaging lens 11, and a fluorescence tomography image of the sheet-shaped illumination area is obtained. Under the alternate action of the pushing light and the braking light, the living body single cell 8 to be detected continuously rotates, the rapid scanning of the sheet-shaped illumination light beam 7 in the cell is realized, and thus the high-spatial resolution fluorescence tomography image of the three-dimensional structure in the cell is obtained.

When the light sheet fluorescence microscope works, a laser source emits laser, a beam expanding system expands an incident light beam, the beam is shaped by a cylindrical lens, and finally the light beam irradiates a sample to be measured in a sheet light mode. The resolution of a light sheet microscope is mainly determined by the thickness of the light sheet and can be expressed as:

wherein f is the focal length; λ is the light source wavelength; d_lThe length is Reuli length. And the length b of the light sheet determines the field of view of the light sheet fluorescence microscope and can be expressed as:

when a sample to be detected is irradiated by the light sheet, the fluorescent marking dye in the cell is excited and emits fluorescence, and the fluorescence is collected by the detection objective lens and is transmitted to the CMOS camera, so that the effect of imaging a thin layer of the cell is realized.

In order to realize the fixation and control of the cells, the invention is added with a light control system which can realize the precise and active control of the rotation angle of the cells, and the light control device is mainly completed by an acousto-optic deflector 19.

The acousto-optic deflector 19 alternately changes the modulation frequency of the acousto-optic deflector to enable the light beams to alternately focus at two ends of the living body single cell 8 to be detected, and the light beams are respectively used as rotating light for pushing the cell to rotate and braking light for stopping the cell to rotate, so that accurate active light control of the cell rotation angle is realized, and the cell rotation is controlled. The laser light output by the laser 13 finally reaches the cell through the designed light path, and is used for capturing the cell. FIG. 2 is a schematic view of a light-manipulated cell.

After being expanded by the lenses 14 and 15, the laser emitted by the laser source 13 is reflected and coupled into the apochromatic microscope objective 17 by the dichroic mirror 16, and acts on the living body single cell 8 to be detected to form a focused light field, so that the living body single cell to be detected is stably captured. The laser light output from the laser 18 is coupled into the acousto-optic deflector 19 to generate a laser beam with a certain deflection angle. Coupled into apochromatic microscope objective 17 via lenses 20, 21. By changing the modulation frequency of the acousto-optic deflector alternately, light beams form light fields alternately at two ends of the living body single cell 8 to be detected, and the light fields are respectively used as rotating light for pushing the cell to rotate and brake light for stopping the cell to rotate, so that accurate active light control on the cell rotating angle is realized.

The optical power during pushing is slightly larger than that during braking, so as to convert the angle of the laser by the AOD, the optical power should be changed accordingly, in order to give a certain time for the cell to rotate, and the timing diagram of the power conversion is shown in fig. 3. As shown in fig. 2, taking the clockwise rotation of the cell as an example, the laser irradiates the left side of the cell through the apochromatic microscope objective 17 to push the cell to rotate, the AOD changes the light deflection angle after the cell rotates a certain angle, the laser irradiates the right side of the cell at this time to brake the rotation of the cell, the output of the control light is not performed within a period of time after the cell stops, the cell is in a static state, the period of time is used for imaging the cell, and the above process is repeated after a period of time.

After the cells are controlled to rotate once and are stabilized, the CMOS camera acquires an image of one layer of the cells once, and after the cells are rotated for a plurality of times, a structure of a plurality of layers of the cells can be obtained, as shown in fig. 4.

(IV) description of the drawings

Fig. 1 is a schematic structural diagram of a light-operated light sheet fluorescence microscopic imaging method and system based on a cell rotation precise active light-operated technology.

FIG. 2 is a schematic view of a light manipulation section consisting of a fixed beam of light and an alternating laser beam to fix and manipulate cells, showing the structure of the capturing and manipulating light.

FIG. 3 is a power diagram of light for manipulating cell rotation and fixation.

Fig. 4 is a schematic view of a method of imaging a cell, in which different layers of the cell are imaged by fixing and rotating the light manipulation part of fig. 2 a plurality of times by a certain angle.

Description of reference numerals: 1-a laser light source; 2-a lens; 3-a lens; 4-a mirror; 5-cylindrical lens; 6-apochromatic microobjective; 7-a sheet-like illumination beam; 8-a cell to be tested; 9-apochromatic microobjective; 10-an optical filter; 11-an imaging lens; 12-a CMOS camera; 13-a laser light source; 14-a lens; 15-a lens; 16-a dichroic mirror; 17-apochromatic microobjective; 18-a laser light source; 19-an acousto-optic deflector; 20-a lens; 21-a lens; 22-mirror.

(V) detailed description of the preferred embodiments

The present invention is further described in detail below with reference to examples to enable those skilled in the art to practice the invention with reference to the description.

It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

A light control optical sheet fluorescence microscopic imaging method and system based on a cell rotation angle light control technology. The method is characterized in that: it consists of laser light sources 1, 13, 18; lenses 2, 3, 14, 15, 20, 21; the mirrors 4, 22; a cylindrical lens 5; apochromatic microobjectives 6, 9, 17; an optical filter 10; an imaging lens 11; a CMOS camera 12; a dichroic mirror 16; an acousto-optic deflector 19; a sheet-like illumination beam 7; and a single cell 8 of the living body to be detected. After the laser output by the laser 13 is expanded by the lenses 14 and 15, the laser is reflected and coupled into the apochromatic microscope objective 17 by the dichroic mirror 16, and acts on the living body single cell 8 to be detected to form a focused light field, so that the living body single cell to be detected is stably captured. The laser light output from the laser 18 is coupled into the acousto-optic deflector 19 to generate a laser beam with a certain deflection angle. Coupled into apochromatic microscope objective 17 via lenses 20, 21. By changing the modulation frequency of the acousto-optic deflector alternately, light beams form light fields alternately at two ends of the living body single cell 8 to be detected, and the light fields are respectively used as rotating light for pushing the cell to rotate and brake light for stopping the cell to rotate, so that accurate active light control on the cell rotating angle is realized. When the cell rotates to an angle and reaches a stable state, the laser output by the laser source 1 is expanded by the lenses 2 and 3, reflected by the reflector 4, shaped by the cylindrical lens 5 and coupled into the apochromatic microscope objective 6 to generate a sheet-shaped illuminating beam 7 to illuminate one layer of the cell 8 to be detected. The fluorophores in the illumination area of the single cell 8 of the living body to be detected are excited, and the generated fluorescence signals are collected by an apochromatic microscope objective 9 with the optical axis vertical to the plane of the sheet-shaped illumination light beam 7. The fluorescence signal is detected and received by a CMOS camera 12 after passing through an optical filter 10 and an imaging lens 11, and a fluorescence tomography image of the sheet-shaped illumination area is obtained. Under the alternate action of the pushing light and the braking light, the living body single cell 8 to be detected continuously rotates, the rapid scanning of the sheet-shaped illumination light beam 7 in the cell is realized, and thus the high-spatial resolution fluorescence tomography image of the three-dimensional structure in the cell is obtained.

When the sample to be detected is irradiated by the light sheet, the fluorescent dye in the cell is excited and emits a fluorescent signal, and the fluorescent light is collected by the detection objective lens and transmitted to the CMOS camera to achieve the effect of imaging a thin layer of the cell. In order to realize the fixation and the control of the cells, the design adds a light control system. The cell is fixed by the laser emitted by the laser source 13, after the laser source 13 is collimated and expanded by the lens 14 and the lens 15, a focusing light field is formed by the reflector 16 and the apochromatic microscope objective 17, and the cell is stably captured. Meanwhile, the power of the captured light is required to be larger than that of the light for controlling the rotation of the cell, so that the captured light can stably catch the cell and cannot be pushed to other places by the controlled light when the controlled cell rotates. The rotation of the cell is mainly accomplished by an acousto-optic deflector, and the laser output by the laser 18 is coupled into the acousto-optic deflector 19 to generate a laser beam with a certain deflection angle. The light beams are coupled into an apochromatic microobjective 17 through lenses 20 and 21 and irradiate the left side of the cell to push the cell to rotate, after the cell rotates for a certain angle, the modulation frequency of an acousto-optic deflector is changed alternately, the laser irradiates the right side of the cell at the moment to brake the rotation of the cell, and the light beams form light fields alternately at two ends of a single cell 8 of a living body to be detected under the action of AOD (acousto-optic modulator) and are respectively used as rotating light for pushing the cell to rotate and brake light for stopping the cell to rotate, so that accurate active light control on the rotating angle of the cell is realized. And after the cell stops, no control light is output within a period of time, the cell is in a static state, the period of time is used for cell imaging, the light-sheet microscope starts to work at the moment, a layer of the cell is imaged, and after the CMOS camera acquires a chromatographic image of the cell, the imaging process is repeated to acquire a complete cell structure.

For the same kind of cells, the intensity of the capture light is kept unchanged and is higher than that of the push light, so that the cells are ensured to be fixed and cannot be pushed out of the field of view under the action of the push light. The rotation of the cells in the culture solution is relatively slow, so the optical power when the cells are pushed is slightly larger than the optical power when the cells are braked, a short time interval exists between braking and pushing, the cells rotate under the inertia effect in the short time interval, the braking is started after the cells rotate by an angle, the cells are slowly stopped, the sum of the rotating angles of the cells in the pushing light pushing stage, the inertia rotating stage and the braking stage is the rotating angle of the imaging cells every time, and the rotating angle can be controlled by controlling the size of the optical power and the length of the time interval, so that the rotating angle can be accurately controlled for the cells with different sizes and different qualities.

After the cell rotates once and is stable, the CMOS camera can obtain a layer of structural image of the cell at the angle, and when the cell rotates for multiple times, a complete three-dimensional structural image of the cell can be obtained.

The above examples are provided for the purpose of describing the invention only, and are not intended to limit the scope of the invention. The scope of the invention is defined by the appended claims. Various equivalent substitutions and modifications can be made without departing from the spirit and principles of the invention, and are intended to be within the scope of the invention.