阅读说明：本技术 基于四芯光纤主动光操控的光片荧光显微成像方法及装置 (Light sheet fluorescence microscopic imaging method and device based on four-core optical fiber active light control ) 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四芯光纤光操控的光片荧光显微成像方法和系统。其特征是：该装置由激光器输出的激光光束经一根输出端加工成特定角度锥台的四芯光纤的纤芯传输后,在水平位置的两根纤芯的输出端形成贝塞尔光场,捕获活体单细胞。经调制的激光脉冲经另外两根垂直位置的纤芯传输后,在输出端形成推动和制动光场,来对细胞的精准主动光操控。当细胞旋转后并达到稳定状态,对细胞进行层析成像。通过驱动细胞连续转动获取整个细胞内部的三维结构荧光图像。本发明构建的方法和系统可对活体单细胞进行高时间和空间分辨率三维层析成像,具有光损伤小、空间分辨率高、操作灵活、成本低等特点,在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 an optical sheet fluorescence microscopic imaging method and system based on four-core optical fiber optical manipulation. The method is characterized in that: in the device, laser beams output by a laser are transmitted through fiber cores of four-core optical fibers processed into frustum with a specific angle by an output end, and Bessel optical fields are formed at the output ends of two fiber cores in a horizontal position to capture living unicells. After the modulated laser pulse is transmitted by the other two fiber cores in the vertical positions, a pushing and braking light field is formed at the output end to accurately and actively control the cells. After the cells had spun and reached a steady state, the cells were subjected to tomography. And acquiring a three-dimensional structure fluorescence image of the whole cell interior by driving the cell to rotate continuously. The method and the system constructed by the invention can carry out high-time and spatial resolution three-dimensional tomography on living unicells, have the characteristics of small optical damage, high spatial resolution, flexible operation, low cost and the like, and have wide application prospects in the research fields of medicine, life science and the like.)

1. The invention provides an optical sheet fluorescence microscopic imaging method and system based on four-core optical fiber optical manipulation. The method is characterized in that: it consists of laser light sources 1, 24; single mode optical fibers 2, 4, 5, 8, 9, 14, 15; beam splitters 3, 7, 13; a frequency modulator 6; a time delayer 10; intensity modulators 11, 12; an optical fiber combiner 16; a four-core optical fiber 17; lenses 25, 26; a cylindrical lens 27; apochromatic microobjectives 20, 28; an optical filter 21; an imaging lens 22; a CMOS camera 23; and a single cell 18 of the living body to be detected. The laser beam output by the laser 1 is coupled into each fiber core of a four-core optical fiber 17 of which the output end is processed into a frustum with a specific angle. The four-core optical fiber 17 is fixed in a culture dish for placing cells to be detected, wherein two laser beams form a Bessel optical field at the output end of the processed frustum after being transmitted by two fiber cores in the horizontal position, and living single cells 18 are stably captured. The other two laser beams are modulated to form two laser pulses with a certain time interval and repetition frequency. After the two laser pulses are transmitted by the two fiber cores at the vertical positions, the light field formed at the output end acts on the two ends of the captured cell respectively, and the periodic light driving force and the periodic light braking force in opposite directions are applied, so that the stable and accurate active light control on the rotation angle of the captured living unicell 18 is realized. When the living single cell 18 rotates to an angle and is stable, the laser beam output by the laser source 24 is expanded and shaped, and then is coupled into the apochromatic microscope objective lens 28 to form a sheet-shaped light beam 19 to irradiate the captured living single cell 18. The fluorescence signal generated by the fluorescent substance in the illumination layer of the laser sheet is collected by an apochromatic microscope objective lens 20, and a fluorescence tomographic image is recorded by a CMOS camera 23. Through continuous and accurate active light control of the cell rotation angle, the rapid scanning of the light sheet in the cell is realized, and the high spatial resolution three-dimensional structural image of the cell is obtained.

2. The light manipulation device of claim 1. The method is characterized in that: the optical control device is a four-core optical fiber 17 which is fixed in a culture dish for placing cells to be detected, and the output end of the optical control device is processed into a frustum with a specific angle. Two of the fiber cores are parallel to the horizontal plane, and the other two fiber cores are perpendicular to the horizontal plane. The laser beam output by the laser source 1 is transmitted by the optical fiber 2 and then is divided into two beams by the beam splitter 3 according to a certain intensity ratio. One of the laser beams is transmitted through the single-mode fiber 5 and then split into two beams with equal intensity through the beam splitter 13. After being transmitted by single-mode fibers 14 and 15, the two beams of laser are coupled into an output end by a fiber coupler 16 and processed into two fiber cores at horizontal positions in a four-core fiber of a frustum with a specific angle. And a capture optical field is formed at the output end after the transmission of the fiber core and is used as capture light to stably capture living single cells 18. The other beam of laser is transmitted through the single-mode fiber 4 and modulated by the frequency modulator 6 to generate laser pulses with a certain repetition frequency. The laser pulse is divided into two beams by a beam splitter 7 according to a certain proportion, and the two beams are respectively coupled into single mode fibers 8 and 9. The intensity of the two laser pulses is adjusted by intensity modulators 11, 12 and the time delay 10 adjusts the time interval between the two laser pulses. Two laser pulses are coupled into two fiber cores at the vertical position of the four-core optical fiber through an optical fiber coupler 16, and a Bessel optical field is formed at the output end of the optical fiber processed into a frustum with a specific angle after the two laser pulses are transmitted through the fiber cores. The laser pulses with a certain time delay respectively act on two ends of the captured living single cell 18 and respectively serve as a pushing light for pushing the cell 18 to rotate and a braking light for stopping the cell from rotating. Since the two core-to-fiber center distances in the horizontal position are greater than the two core-to-fiber center distances in the vertical position. Thus, the captured live single cell 18 is rotated about the axis of rotation created by the captured light by the pushing light pulse acting on one end of the cell. When rotated through an angle, a braking light pulse with a certain time delay acts on the other end of the cell, thereby applying a braking force to the rotation of the cell. By changing the intensity of the pushing light pulse and the braking light pulse and the time delay between the pushing light pulse and the braking light pulse, the cell rotation angle is stably and accurately controlled actively.

3. The light sheet fluorescence microscope system of claim 1. The method is characterized in that: in the light sheet microscope system, when the living body single cell 18 rotates to an angle and is stable under the drive of the light control device, a laser beam output by a laser light source 24 is expanded by lenses 25 and 26, and then is coupled into an apochromatic microscope objective lens 28 after passing through a cylindrical lens 27 to form sheet light 19 which irradiates on one layer of the captured living body single cell 18, so as to excite a fluorescent substance on the layer, a generated fluorescent signal is collected by a detection objective lens 20 with an optical axis vertical to the plane of an illumination light sheet, and a CMOS camera 23 records a fluorescent tomography image through a filter 21 and an imaging lens 22. By the accurate active light control of the cell rotation angle, the continuous and rapid scanning of the sheet light 19 in the cell is realized, and the high spatial resolution three-dimensional structural image of the cell is obtained.

(I) technical field

The invention provides an optical sheet fluorescence microscopic imaging method and device based on four-core optical fiber active light control, which can stably capture and accurately control the rotation angle of a living body single cell through one four-core optical fiber, realize the rapid scanning of a sheet light beam in a cell and acquire a cell three-dimensional structure chromatographic image with high time and spatial resolution, and belongs to the field of light control and optical microscopic imaging.

(II) background of the invention

Cells are the basic unit of life structure and function, and no cell has a complete life, and the deep research on the cells is the key for exploring the secret of life, conquering diseases and modifying life. Since the discovery of cells, researchers have conducted extensive and intensive research on their physical, chemical, and biological properties, revealing a lot of secrets about cells.

It has long been generally accepted that the biological properties of individual cells that make up different populations of cell types are substantially uniform. Based on this knowledge, the general life science research is mainly performed on a population of cells consisting of a large number of cells of the same type. In recent years, it has been increasingly recognized that there is significant microscopic heterogeneity, i.e., heterogeneity character, among individual cells that constitute a homogeneous population of cells. It has been proved that even though there are significant differences in gene transcription and translation, protein activity, and multiple levels in the same population of cells, it is difficult to reflect the life activity rule at the single cell level based on the results of a large number of cells. Therefore, the single cell-based analysis technology can reveal the essence and basic rules of life activities at a deeper level, and provides more reliable scientific basis for exploring the cause, development and treatment of serious diseases.

Optical microscopy plays a crucial role in the discovery of cell structures, as well as in the study of their function. With the deep research on cell structure and function, the challenge is to use large environmental living cells for developing life activities as 'test tubes', 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, obtain the spatial distribution and change information of different types of biomolecules in the cell life process and the functional information of the interaction process between organelles, biomolecules and different types of biomolecules, and provide reliable scientific basis for revealing the essence and basic rules of life activities at a deeper level.

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. Particularly, Laser Scanning Confocal Fluorescence Microscopy (LSCM), which combines Laser beam Scanning and Confocal detection, provides high chemical specificity and imaging contrast based on autofluorescence or by Fluorescence labeling, and effectively eliminates the influence of defocused Fluorescence signals by using a Confocal diaphragm installed at a conjugate position of a photodetector, thereby realizing three-dimensional optical tomography with high spatial resolution.

However, in the LSCM system, fluorophores in the region near the focal plane of the sample to be measured along the excitation light transmission path are excited, which greatly reduces the excitation efficiency. Second, many endogenous fluorescent and non-fluorescent organic components in the sample will also be excited. Phototoxicity, photodamage, photobleaching and the like caused by continuous long-time irradiation are inevitable problems in long-time imaging of a living biological sample to be measured. In addition, the use of laser beam scanning in the system and the installation of a confocal diaphragm in front of the detector results in a complex system structure, high cost and long imaging time. Therefore, the researchers are always dedicated to develop a microscopic imaging method capable of rapidly acquiring a tomography image of a living single cell with a large volume and a high spatial-temporal resolution three-dimensional structure within a long observation time.

In recent years, Light Sheet Fluorescence Microscopy (LSFM) using a laminar laser beam has proven to be one of the first techniques to achieve this challenging goal. The LSFM comprises two main components, 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 longitudinally and rapidly scanning the sheet-shaped excitation light in the cell to obtain a three-dimensional structural chromatographic image with high spatial resolution of the cell sample to be detected. The LSFM high-efficiency excitation and detection mode not only effectively avoids the problems of photobleaching, photodamage and the like of fluorophores and endogenous organic molecules outside a sheet-shaped illumination area, reduces the phototoxicity influence on the whole sample, ensures the vitality of a biological sample of a living body to be detected 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. Due to the advantages, the LSFM is brought forward and draws wide attention of domestic and foreign researchers.

When using LSFM to acquire images of the complete three-dimensional structure of cells, it is desirable to achieve rapid scanning of the sheet beam inside the cell. The technology commonly used at present is to add a special fixing device to the system for treating the cells to be tested. The longitudinal translation or axial rotation of the cell to be detected is controlled by longitudinal scanning of the sheet-shaped light beam or by using a micro-displacement platform, so that the tomography of the cell is realized. This makes it impossible to avoid the effect on the activity of the sample during its preparation and the effect of the disturbance of the liquid environment on the imaging quality during long-term imaging studies. In addition, the system is additionally provided with a light beam scanning device or a micro-displacement platform, so that the complexity and the cost of the system are greatly increased, and the operability and the flexibility of the system are reduced.

The LSFM and Optical Tweezers (OTs) technologies 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 OTs technology utilizes the mechanical effect of the optical field, can stably capture, accurately control and rapidly screen single viruses, cells and even biomacromolecules in a non-contact and non-destructive manner under the condition that the interior and the surrounding environment of the cells are not affected, and opens the door for observing living cells in a liquid environment for a long time to obtain the internal structure of the living cells and further deeply researching the biological regulation and control mechanism and the like of the cell life activity process.

The traditional OTs system not only needs to use a large-numerical-aperture objective lens and a complex optical path system, but also is limited by many factors such as working distance and substrate compatibility during the use process, and the system is large in size, expensive in manufacturing cost and poor in flexibility. The Optical Fiber Optical Tweezers (OFTs) technology realized based on the Optical Fiber can accurately control living unicells in a liquid environment, such as capture, stretching, moving, rotating and the like, can flexibly move in a medium at will, and has the advantages of simple system structure, small volume, strong operability, high integratability and flexibility.

The invention discloses a light sheet fluorescence microscopic imaging method and a light sheet fluorescence microscopic imaging system based on four-core optical fiber optical control, which can be widely applied to stably capturing and accurately controlling living body single cells in a non-contact and non-destructive mode, so that a high-spatial-resolution three-dimensional structural image of the living body single cells is rapidly acquired. The design stably captures living unicells through an optical control system based on the four-core optical fiber and carries out accurate optical control on the rotation angle of the living unicells. After the cell has been rotated through an angle to stabilize, the fluorophore in the plane of the illumination layer is excited by the sheet beam. The resulting fluorescence signal is collected by a microscope objective with its optical axis perpendicular to the illumination plane and recorded by a CMOS camera to obtain a fluorescence tomographic image of the cell. Through the accurate optical control of the rotating angle in the continuous rotating process of the cells, the rapid scanning of the sheet-shaped light beam in the cells is realized, and thus the three-dimensional structure fluorescence image of the living unicells is obtained. The method and the system realized by the design not only can stably capture and accurately control the living unicells in a non-contact mode and a non-destructive mode, but also can quickly acquire the high-spatial-resolution three-dimensional structural image of the living unicells. The design effectively overcomes the problems of photodamage and phototoxicity of the fluorescence microscopy technology, 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 of biology, medicine, life science and the like.

Disclosure of the invention

The invention aims to provide an optical sheet fluorescence microscopic imaging method and device system based on four-core optical fiber active light control. The method can not only stably capture and accurately control the living body single cells in a non-contact mode and a non-damage mode, but also quickly acquire the high-spatial-resolution three-dimensional structural image of the living body single cells. Effectively overcomes the problems of light damage and phototoxicity of the fluorescence microscopy, and has the characteristics of simple structure, high flexibility, easy operation, low cost and the like.

The purpose of the invention is realized as follows:

it consists of laser light sources 1, 24; single mode optical fibers 2, 4, 5, 8, 9, 14, 15; beam splitters 3, 7, 13; a frequency modulator 6; a time delayer 10; intensity modulators 11, 12; a fiber coupler 16; a four-core optical fiber 17; lenses 25, 26; a cylindrical lens 27; apochromatic microobjectives 20, 28; an optical filter 21; an imaging lens 22; a CMOS camera 23; and a single cell 18 of the living body to be detected.

The optical control device is a four-core optical fiber 17 which is fixed in a culture dish for placing cells to be detected, and the output end of the optical control device is processed into a frustum with a specific angle. Two of the fiber cores are parallel to the horizontal plane, and the other two fiber cores are perpendicular to the horizontal plane. The end face structure of the four-core fiber optical tweezers for realizing the accurate active optical control of the cell rotation angle is shown in fig. 2-a. The laser beam output by the laser source 1 is transmitted by the optical fiber 2 and then is divided into two beams by the beam splitter 3 according to a certain intensity ratio. One of the laser beams is transmitted through the single-mode fiber 5 and then split into two beams with equal intensity through the beam splitter 13. After being transmitted by single-mode fibers 14 and 15, the two beams of laser are coupled into an output end by a fiber coupler 16 and processed into two fiber cores at horizontal positions in a four-core fiber of a frustum with a specific angle. And after being transmitted by the fiber core, a Bessel optical field is formed at the output end and is used as capture light to stably capture living single cells 18.

The other beam of laser is transmitted through the single-mode fiber 4 and modulated by the frequency modulator 6 to generate laser pulses with a certain repetition frequency. The laser pulse is divided into two beams by a beam splitter 7 according to a certain proportion, and the two beams are respectively coupled into single mode fibers 8 and 9. The intensity of the two laser pulses is adjusted by intensity modulators 11, 12 and the time delay 10 adjusts the time interval between the two laser pulses. Two laser pulses are coupled into two fiber cores at the vertical position of the four-core optical fiber through an optical fiber coupler 16, and a Bessel optical field is formed at the output end of the optical fiber processed into a frustum with a specific angle after the two laser pulses are transmitted through the fiber cores. The laser pulses with a certain time delay respectively act on two ends of the captured living single cell 18 and respectively serve as a pushing light for pushing the cell 18 to rotate and a braking light for stopping the cell from rotating. Since the two core-to-fiber center distances in the horizontal position are greater than the two core-to-fiber center distances in the vertical position. The cross-sectional structure of the end face of the four-core optical fiber whose output end is processed into a frustum of a specific angle is shown in fig. two-b. Thus, the captured live single cell 18 is rotated about the axis of rotation created by the captured light by the pushing light pulse acting on one end of the cell. When rotated through an angle, a braking light pulse with a certain time delay acts on the other end of the cell, thereby applying a braking force to the rotation of the cell. By changing the intensity of the pushing light pulse and the braking light pulse and the time delay between the pushing light pulse and the braking light pulse, the cell rotation angle is stably and accurately controlled actively.

When the living single cell 18 rotates to an angle and is stable, the laser beam output by the laser source 24 is expanded by the lenses 25 and 26, and then is coupled into the apochromatic microscope objective lens 28 through the cylindrical lens 27 to form the sheet-shaped light 19.

In the light sheet fluorescence microscope, laser emitted by a laser source is expanded by a beam expanding system, is shaped by a cylindrical lens, and finally forms light sheet illumination on a sample to be detected through a microscope objective. Thickness omega of beam waist part of optical sheet₀The spatial resolution of a light sheet fluorescence microscope is determined 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 the light sheet 19 irradiates on the living body single cell 18 to be detected, the fluorophore in the illumination range in the cell is excited, and the fluorescence signal generated by excitation is collected through the detection objective lens 20 with the optical axis vertical to the plane of the light sheet and is detected and received by the CMOS camera 23.

The push pulses and the brake pulses in the two cores in the vertical position in the four-core fiber are generated alternately, and the delay of the brake pulses through the time delayer 10 slightly lags behind the push pulses in order to give the cells a certain time to rotate. The pushing pulse in the fiber core 8 makes the cell rotate, the pushing pulse stops after the cell rotates for a certain angle, the fiber core 9 generates an artery making pulse to stop the cell rotating, no pulse light is input within a period of time after the cell stops, the cell is in a static state, the period of time is used for cell imaging, and the process is continuously repeated after a period of time. The timing diagram of the laser pulses acting on the cells to achieve precise active light manipulation of the angle of rotation of the cells is shown in fig. 3.

After each control cell is brought to an angle and stabilized, the CMOS camera 23 records a tomographic fluorescence image of the cell at that angle. By the accurate active light control of the cell rotation angle, the continuous and rapid scanning of the sheet light 19 in the cell is realized, and the high spatial resolution three-dimensional structural image of the cell is obtained. In the cell rotation process, tomography is realized by optical sheet scanning, and the process of obtaining the three-dimensional structural image of the cell is shown in fig. 4.

(IV) description of the drawings

Fig. 1 is a schematic system structure diagram of an optical sheet fluorescence microscopic imaging device based on four-core optical fiber active light control.

Fig. 2 is a schematic structural diagram of a system of a light manipulation part, which shows a side view and a perspective view of an optical fiber, and the light manipulation part is coupled to a four-core optical fiber by four single-mode optical fibers, and is used for stably capturing a cell to be measured, and performing precise active light manipulation and cell manipulation on a rotation angle of the cell.

Fig. 3 is a timing diagram of laser pulses acting on a cell to achieve precise active light manipulation of the angle of rotation of the cell.

FIG. 4 is a process of obtaining three-dimensional structural images of cells by tomographic imaging with optical scanning during cell rotation.

Description of reference numerals: 1-a laser light source; 2-single mode fiber; 3-a beam splitter; 4-single mode fiber; 5-single mode fiber; 6-a frequency modulator; 7-a beam splitter; 8-single mode fiber; 9-single mode fiber; 10-a time delay; 11-an intensity modulator; 12-an intensity modulator; 13-a beam splitter; 14-a single mode optical fiber; 15-single mode fiber; 16-a light coupler; 17-a four-core optical fiber; 18-a test cell; 19-sheet light; 20-apochromatic microobjective; 21-an optical filter; 22-an imaging lens; 23-a CMOS camera; 24-a laser light source; 25-a lens; 26-a lens; 27-cylindrical lens; 28-apochromatic microobjective.

(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.

An optical sheet fluorescence microscopic imaging method and system based on four-core optical fiber optical control. The method is characterized in that: it consists of laser light sources 1, 24; single mode optical fibers 2, 4, 5, 8, 9, 14, 15; beam splitters 3, 7, 13; a frequency modulator 6; a time delayer 10; intensity modulators 11, 12; a fiber coupler 16; a four-core optical fiber 17; lenses 25, 26; a cylindrical lens 27; apochromatic microobjectives 20, 28; an optical filter 21; an imaging lens 22; a CMOS camera 23; and test cells 18.

The laser beam emitted by the laser source 1 is transmitted to the beam splitter 3 through the optical fiber 2 and is divided into two beams which are respectively coupled into the single mode optical fibers 4 and 5. The laser beam in the single-mode fiber 5 is divided into two uniform beams by the beam splitter 13, and after being transmitted by the single-mode fibers 14 and 15, the two uniform beams are coupled into two fiber cores at horizontal positions in the frustum four-core fiber with specific angles by the fiber coupler 16, and a Bessel optical field is formed at the output end to stably capture the cell 18 to be detected. The laser beam in the single mode fiber 4 is modulated by the frequency modulator 6 to generate laser pulse, and is divided into two beams by the beam splitter 7 to be coupled into the single mode fibers 8 and 9. The laser pulses in the single mode fiber 9 are intensity modulated by an intensity modulator 12 to produce laser pulses of suitable intensity. The laser pulse transmitted by the single-mode fiber is coupled into one of two fiber cores at a vertical position in the four-core fiber of which the output end face is processed into a frustum with a specific angle through the fiber coupler 16. Since the two core-to-fiber center distances in the horizontal position are greater than the two core-to-fiber center distances in the vertical position. The laser pulse output by the laser pulse at the output end acts on one end of the cell and is used as pushing light for pushing the cell to rotate. The laser pulse in the single mode fiber 8 is intensity-modulated by an intensity modulator 11 and a time-delayed laser pulse is generated by a time delay 10. The laser pulse transmitted by the single-mode fiber is coupled into the other of the two fiber cores at the vertical position in the four-core fiber of which the output end face is processed into the frustum with a specific angle through the fiber coupler 16. The laser pulse output at the output end acts on the other end of the cell as a braking light for stopping the cell from rotating.

When the cell to be detected rotates to a certain angle around a specific rotating shaft and is stable, laser emitted by the laser source 24 is expanded by the lenses 25 and 26 and shaped by the cylindrical lens 27, the sheet-shaped light 19 is generated by the apochromatic microscope 28, and the sheet-shaped light 19 irradiates the cell to be detected 18. The sheet light illuminates a layer in the cell, excites fluorescent substances in the cell in the layer to generate a fluorescent signal, the fluorescent signal is collected by an apochromatism microscope objective lens 20, and is detected and received by a CMOS camera 23 through an optical filter 21 and an imaging lens 22 to obtain a tomography fluorescent image of an illumination area of the sheet light.

During the process of acquiring the three-dimensional structural tomographic image of a specific kind of living single cell, the intensity of the captured light is kept constant. For different kinds of cells, the laser intensity needs to be adjusted to realize stable capture of the cells. The laser beam generates laser pulse through a frequency modulator, and the intensity of the push light pulse is modulated by an intensity modulator to be slightly larger than that of the brake light. The captured cell starts to rotate under the action of the driving light pulse, and when the laser pulse disappears, the cell also rotates due to the action of inertia. At this time, in order to realize accurate active control of the cell rotation angle, a braking light pulse with a certain time delay is required to act on the other end of the cell to generate a braking force opposite to the cell rotation direction, so as to accurately control the cell rotation angle. Under the continuous action of the pushing and braking light pulses with certain repetition frequency, the cells continuously rotate around the capture axis, so that the illumination light sheet can rapidly scan in the cells, and a three-dimensional structure chromatography fluorescence image of the cells is obtained.

In order for a CMOS camera to be able to record the fluorescence signal in time, the time interval between the pushing of the light pulses is slightly larger than the exposure time of the camera. The sum of the rotating angles of the captured cell in the pushing phase of the pushing light pulse, the inertial rotating phase and the braking phase of the braking pulse is the rotating angle of the cell in each rotation. The rotation angle of the cell can be accurately controlled by controlling the intensity of the laser pulse and the time interval between pulses, so that the requirement of carrying out accurate active light control on the rotation angle of the cell with different volume and quality is met.

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.