CN117007567A - Digital scanning light sheet fluorescence microscopic imaging system based on five-core optical fiber active light control - Google Patents
Digital scanning light sheet fluorescence microscopic imaging system based on five-core optical fiber active light control Download PDFInfo
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Abstract
The invention provides a digital scanning light sheet fluorescence microscopic imaging system based on five-core optical fiber active light control. The method is characterized in that: the multi-core optical fiber optical tweezers are used for conducting stable and accurate active light control on the rotation angle of a living single cell in a liquid state environment around a specific rotation axis in a non-contact and non-damaging mode. In the cell rotation process, based on a holographic wave-front shaping method, bessel light beams transmitted by a multi-core optical fiber are optimized, a virtual light sheet is formed by rapid scanning in a cell, fluorescence signals generated by excitation are collected by a microscope objective perpendicular to the excitation light plane of the virtual light sheet, and fluorescent chromatographic images of different angles of living single cells are recorded by a CMOS camera. The method can realize the real-time in-situ acquisition of the high-resolution fluorescence chromatographic image of the three-dimensional structure inside the living single cell, has the characteristics of high precision, high resolution, high reliability, high flexibility, strong applicability and the like, and has wide application prospects in a plurality of research fields such as biology, medicine, life science and the like.
Description
Technical Field
The invention provides a digital scanning light sheet fluorescence microscopic imaging system based on five-core optical fiber active light control. The four cores of the five-core optical fiber are used for stably capturing cells and precisely controlling the rotation angle of living cells, the middle fiber core is used for realizing the rapid scanning of sheet-shaped light beams on the cells, and further obtaining the three-dimensional structure tomographic images of the cells with high time and spatial resolution, and the three-dimensional structure tomographic images belong to the fields of light control and optical microscopic imaging. .
Background
The research on cells is continuous and advanced along with the law of scientific development, and different research levels such as cell level, subcellular level and molecular level are experienced. Along with the development, the challenge faced by the present invention is to take the living cells in the large environment for developing the life activities as a test tube, and obtain the subcellular fine structure image information of the living single cells in a non-contact and non-damaging way on the premise of avoiding influencing the properties of the cells and the microenvironment where the living single cells are located, so as to provide reliable scientific basis for revealing the nature and basic rules of the life activities in a deeper level. To achieve this, detection means are required to have a molecular level discrimination capability, a spatial resolution of submicron or even nanometer scale, a high detection sensitivity, and a temporal resolution of millisecond or more.
Therefore, the research and development of the light-sheet fluorescence microscopic tomography method and the quantitative analysis research platform realize real-time in-situ acquisition of the three-dimensional structure fluorescence tomography image with high time and spatial resolution in living single cell and long-time dynamic quantitative monitoring of the vital activity process of the living single cell by accurately and actively controlling the cell gesture in a non-contact and non-damage mode on the premise of not affecting the property of the cell and the microenvironment of the cell, and have important significance in researching the property, the function, the interaction dynamic process and the like of the biomolecules in the vital activity process of the cell.
With the development of fluorescent labeling technology, laser technology, weak signal detection technology and computer technology, modern microscopic imaging technology can record the time-space information of biological systems with unprecedented time and spatial resolution, changing the way we see, record, explain and understand biological events. In particular, wide-field fluorescence microscopy (Wide-field Fluorescence Microscopy, WFM), which provides high chemical specificity and imaging contrast based on autofluorescence or through fluorescent labeling, is an important research tool for studying living single cells and obtaining information on the fine three-dimensional structure and quantitative functions inside them. However, since the image obtained by WFM contains a large amount of out-of-focus fluorescent signals, the image contrast is greatly reduced, and thus a three-dimensional structure fluorescent tomographic image with high spatial resolution cannot be obtained. Laser scanning confocal fluorescence microscopy (Laser Scanning Confocal Fluorescence Microscopy, LSCM) combining laser beam scanning and confocal detection effectively eliminates the effects of out-of-focus fluorescence signals using confocal pinholes mounted at the conjugate locations of photodetectors, thereby achieving three-dimensional optical tomography with high spatial resolution.
In the LSCM system, fluorophores in the area near the focal plane of the excitation light transmission light path of the sample to be detected are excited, so that the excitation efficiency is greatly reduced. Second, many endogenous fluorescent and non-fluorescent organic components in the sample are also excited. Phototoxicity, photodamage, photobleaching and the like caused by continuous long-time irradiation are unavoidable problems in the long-time imaging process of a biological sample of a living body to be detected. In addition, the use of laser beam scanning and the installation of confocal pinholes in front of the detector in the system results in complex system architecture, high cost and long imaging times. Therefore, a great deal of scientific researchers have been devoted to develop a microscopic imaging method capable of rapidly acquiring a fluorescence tomographic image of a three-dimensional structure with high space-time resolution of a large volume of living single cells in a long observation time.
In recent years, light sheet fluorescence microscopy (Light Sheet Fluorescence Microscopy, LSFM) using laminar laser beams has proven to be one of the preferred techniques for achieving this challenging goal. LSFM comprises two main components of the excitation and detection light paths. A laminar laser beam, commonly referred to as a "Light Sheet" (LS), is used in the excitation Light path as excitation Light to excite fluorophores in the laminar illumination region. The detection light path uses a wide-field fluorescence signal parallel detection mode to collect generated fluorescence signals in a direction perpendicular to the sheet-shaped excitation light plane. And obtaining a three-dimensional structure fluorescence chromatographic image with high spatial resolution of the biological sample to be detected through the rapid scanning of the sheet-like excitation light. 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 illumination area, reduces the influence of phototoxicity on the whole sample, ensures the activity of a living biological sample to be detected in a long-time research process, effectively avoids the interference of defocused fluorescent signals, and greatly improves the signal-to-noise ratio and the axial resolution of the system. Due to the advantages, LSFM has attracted extensive attention from domestic and foreign scientific researchers after being proposed. Many well-known scientific institutions in the world have conducted extensive and intensive research work on the LSFM method and its applications, such as: european molecular biology laboratory E H K Stelzer task group, howland House medical institute E Betzig task group, U.S.A. In recent years, there are many domestic scientific institutions such as: various characteristic research works are carried out on LSFM methods and applications of the Beijing university Chen Liangyi subject group, the Shenzhen university Niu Han medical institution subject group and the like, and a great research result is obtained. Currently, LSFM has been widely used in many research fields of developmental biology, cell biology, brain function imaging, neurobiology, and the like.
When the LSFM technology is used for obtaining the three-dimensional structure image of the cell, the light sheet is controlled to move rapidly in the direction of the detection axis so as to realize the tomography of the light sheet on the cell. There are two common means, namely, a fast moving light sheet is used for scanning, and a micro displacement table is used for controlling the longitudinal translation or axial rotation of the cells to be detected. Therefore, a light beam scanning device or a micro-displacement table is added in the system, 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 advent of Optical Tweezers (OTs) technology provides a solution path to the above-mentioned problems. The core of OTs is the interaction between light and matter, which is a tool that uses light to capture and manipulate tiny objects. The OTs technology has the remarkable advantages that the OTs technology can stably capture and accurately control cells or other macromolecular structures in a non-contact and non-invasive mode, and can perform actions such as waking, rotating, stretching and the like on the cells for a long time in a liquid environment. However, the conventional OTs system is limited by many factors such as working distance in the use process, and the system has a complex structure, high price and poor flexibility.
If the LSFM and the OTs technology can be combined, the complexity and cost of the LSFM system can be greatly reduced, and the OTs technology can be applied more flexibly. The innovation fills the defects in the aspects of cost and operability by utilizing the technical advantages of LSFM and OTs, and provides great convenience for scientific researchers to study the microscopic field.
The invention provides a digital scanning light sheet fluorescence microscopic imaging system based on five-core optical fiber active light control. The invention utilizes four outer cores in the five-core optical fiber to stably capture cells and precisely control the rotation angle of the cells. After the cells are controlled to rotate for a certain angle and are stabilized, the scanning light sheet emitted from the middle core of the five-core optical fiber excites fluorescent substances on the illumination layer. The microscope objective with the optical axis perpendicular to the illumination plane collects the fluorescence signal of the focal plane (illumination plane). The collected fluorescence signals are recorded and stored by a CMOS camera, and a three-dimensional image of the cell is obtained after the computer processing. The system can stably capture and accurately control the cells in a non-contact and non-invasive mode, can rapidly acquire three-dimensional structure images with high spatial resolution of the cells, and greatly relieves the problems of photobleaching and phototoxicity of the fluorescence microscopy technology on living cells. The system has the characteristics of high flexibility, easy operation, low cost and the like, and has bright application prospect in the fields of biomedicine, life science and the like.
Disclosure of Invention
The invention provides a digital scanning light sheet fluorescence microscopic imaging system based on five-core optical fiber active light control. The system can stably capture and accurately control the cells in a non-contact and non-invasive mode, and can also rapidly acquire three-dimensional structure images with high spatial resolution of the cells, so that the problems of photobleaching and phototoxicity of the fluorescence microscopy technology on living cells are greatly relieved. Compared with other fluorescence microscopy systems, the system has the characteristics of high flexibility, easiness in operation, low cost and the like.
The purpose of the invention is realized in the following way:
the method is characterized in that: the device consists of lasers 1 and 19, optical coupling systems 2 and 23, beam splitters 4 and 7 and 8, single-mode optical fibers 3 and 5 and 6 and 12 and 15, single-mode optical fibers 16 and 17 and 24, frequency modulators 9 and 13, intensity modulators 10 and 14, a time delay 11, a coupler 18, a relay optical system 20, a microstructure mask plate 21, a spatial light modulator 22, a five-core optical fiber 25, cells 26, a cell culture dish 27, a microscope objective 28, a sampling mirror 29, a band-pass filter 30, a CMOS camera 31, a condenser 32, an illumination light source 33, data connecting wires 34 and 36 and a computer 35; .
The light control part of the cells is realized by four outer cores of five-core optical fibers (25) which are fixed in a culture dish for placing the cells to be tested and the output ends of which are processed into specific angles; the laser beam output by the laser 1 is coupled into a single-mode fiber 3 through an optical coupling system 2, and is split into two beams according to a certain proportion through an optical fiber beam splitter 4; one of the continuous lasers is transmitted through a single-mode fiber 6 and then is split into two beams by an optical fiber beam splitter 8, the two beams are respectively coupled into two fiber cores parallel to the horizontal plane on the periphery of a five-core optical fiber 25 through an optical fiber coupler 28, and a focused light field is formed at the output end of the optical fiber processed into a frustum with a specific angle after the transmission through the fiber cores and is used as capturing light for capturing cells; the other beam of continuous laser is divided into two beams by an optical fiber beam splitter 7, two beams of laser pulses with certain repetition frequency f and a period of T1 and T2 are respectively modulated by optical fiber frequency modulators 9 and 13, the two beams of laser pulses are respectively subjected to intensity modulation by optical fiber intensity modulators 10 and 14, an adjustable time delay delta T is introduced between the two beams of laser pulses by using an optical fiber time delay 11, the two beams of laser pulses are coupled into two fiber cores which are perpendicular to the horizontal plane at the periphery of a five-core optical fiber 25 by an optical fiber coupler 18, and a focusing light field is formed at the output end of the optical fiber processed into a specific angle frustum after being transmitted by the fiber cores and is respectively used as pushing light for pushing the captured cells to rotate and braking light for stopping the rotation of the cells; the output end of the five-core optical fiber 25 is arranged on an optical fiber clamping seat and fixed in a cell culture dish 27, a potential well is formed near the output end face of the five-core optical fiber by capturing living single cells 26 suspended in the culture dish, laser pulses with a certain time delay are applied to captured cells at the output end of the optical fiber by periodic light pushing force and braking force with opposite directions, and the rotation angle of the captured cells is controlled by stable and accurate active light.
An optical sheet fluorescence microscope part, characterized in that: the femtosecond laser pulse output by the other laser 19 is coupled into a Bessel beam generating system and a holographic wave front pre-shaping and optimizing system which are formed by a microstructure mask plate 21 and a spatial light modulator 22 after being expanded and shaped by a relay optical system 20, the beam output by the wave front pre-shaping and optimizing system is coupled into a single-mode optical fiber 24 by an optical coupling system 23, the femtosecond laser pulse transmitted by the single-mode optical fiber 24 is coupled into the central fiber core of a five-core optical fiber 25 by an optical fiber coupler 18, and the Bessel beam meeting the requirement is output at the output end of the five-core optical fiber 25; the angle of the Bessel light beam output by the output end of the five-core optical fiber 25 is adjusted, and the Bessel light beam is rapidly scanned in the cell to be detected to form a virtual light sheet; in the process of driving the cells to rotate around a specific rotation axis by the light field, exciting fluorophores in an illumination area, collecting generated fluorescent signals by a micro objective lens 28 perpendicular to the excitation light plane of the virtual light sheet, filtering stray light by a liquid crystal tunable bandpass filter 30, and detecting and receiving by a CMOS camera 31; based on the spatial attitude of the cells and the characteristic mark points on the rotating shaft, a three-dimensional structure fluorescence tomographic image reconstruction algorithm of the rotating cells is established, and a high spatial resolution three-dimensional structure fluorescence image of the living single cells is obtained by analyzing fluorescence tomographic images of different angles of the cells recorded by the CMOS camera 31.
Push pulses and brake pulses in two outer cores in vertical positions in a five-core fiber are alternately generated. The braking pulse passes through a time delay 11 to slightly lag the braking period behind the pushing pulse in order to give the cell a certain rotational time. The pushing pulse rotates the cells, and the pushing pulse stops after the cells rotate a certain angle. The cell will not stop rotating due to the stop of the push pulse due to inertia. The braking pulse is used to brake the rotation of the cell due to inertia. No pulsed light is input for a period of time after the cell stops spinning, and the cell is in a resting state, which is used for cell imaging. And repeating the process after a period of time.
After each laser controls the cell to an angle and stabilizes, the CMOS camera 31 records a tomogram of the cell at that angle. Through the accurate control of the cell rotation angle, the continuous and rapid illumination of the scanning light sheet in the cell is realized, fluorescence signals are excited, and a high-spatial-resolution three-dimensional structure image of the cell is obtained. In the process of cell rotation, the optical sheet scanning realizes tomography, and the process of obtaining the three-dimensional structure image of the cell is shown in a fifth figure.
The exit end face of the five-core optical fiber 25 is shown in fig. 2. The critical angle at which light is totally reflected, depending on the total reflection conditions of the light, is:
wherein n is 1 Refractive index n of four outer cores of five-core optical fiber 25 2 Is the refractive index of water. Due to refractive index n of water 2 About 1.33, refractive index n of single mode optical fiber 1 ≈[1.463,1.467]In order for total reflection not to occur at the exit end face, the exit angle must be smaller than the critical angle θ C 。
For example, if the core refractive index n 1 Taking 1.467, the refractive index of water is 1.33, then:
in order for two outer cores in the horizontal direction to form a bessel beam at the output end face, the exit angle must be greater than 0 °, so:
α∈(0°,65°] (3)
beta epsilon [25 DEG, 90 DEG), (4) wherein alpha is the angle between the emergent light and the normal, and beta is the angle of the cutting end face. The optimal value of alpha and beta in the respective angle range depends on the diameter of the object to be measured and the loss degree of the emergent light. For the cells to be tested, which do not exceed the diameter of the optical fiber, the closer the cells are to the end face, the smaller the optical loss is, and the better the imaging quality is.
Drawings
Fig. 1 is a schematic structural diagram of a digital scanning light sheet fluorescence microscopic imaging system based on five-core optical fiber active light control.
Fig. 2 is a side view of a five-core optical fiber showing the end face configuration of the output end of the five-core optical fiber.
Fig. 3 is a scanning light sheet forming process.
Fig. 4 is a schematic diagram of light manipulation.
Fig. 5 is a timing diagram of the pushing light and the braking light.
Reference numerals illustrate: 1-a laser; 2-an optical coupling system; 3-single mode optical fiber; 4-beam splitters; 5-single mode optical fiber; 6-single mode optical fiber; 7-beam splitters; 8-beam splitter; a 9-frequency modulator; 10-intensity modulator; 11-a time delay; 12-single mode optical fiber; 13-a frequency modulator; 14-an intensity modulator; 15-single mode optical fiber; 16-single mode optical fiber; 17-single mode optical fiber; an 18-coupler; 19-a laser; a 20-relay optical system; 21-a microstructure mask plate; 22-spatial light modulator; a 23-optical coupling system; 24-single mode optical fiber; 25-five-core optical fiber; 26-living single cells; 27-cell culture dish; 28-a microobjective; 29-a sampling mirror; 30-bandpass filters; 31-CMOS camera; 32-a condenser; 33-an illumination source; 34-data connection lines; 35-a computer; 36-data connection lines.
Detailed Description
The present invention is described in further detail below with reference to examples to enable those skilled in the art to practice the same and to refer 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 digital scanning light sheet fluorescence microscopic imaging system based on five-core optical fiber active light control. The method is characterized in that: the device consists of lasers 1 and 19, optical coupling systems 2 and 23, beam splitters 4 and 7 and 8, single-mode optical fibers 3 and 5 and 6 and 12 and 15, single-mode optical fibers 16 and 17 and 24, frequency modulators 9 and 13, intensity modulators 10 and 14, a time delay 11, a coupler 18, a relay optical system 20, a microstructure mask plate 21, a spatial light modulator 22, a five-core optical fiber 25, cells 26, a cell culture dish 27, a microscope objective 28, a sampling mirror 29, a band-pass filter 30, a CMOS camera 31, a condenser 32, an illumination light source 33, data connecting wires 34 and 36 and a computer 35;
the laser beam output by the laser 1 is coupled into a single-mode fiber 3 through an optical coupling system 2, and is split into two beams according to a certain proportion through an optical fiber beam splitter 4; one of the continuous lasers is transmitted by a single-mode fiber 6 and then is split into two beams by an optical fiber beam splitter 8, the two beams are respectively coupled into two fiber cores parallel to the horizontal plane on the periphery of a five-core optical fiber 25 by an optical fiber coupler 18, and a focused light field is formed at the output end of the optical fiber processed into a frustum with a specific angle after the transmission of the fiber cores and is used as capturing light for capturing cells; the other beam of continuous laser is divided into two beams by an optical fiber beam splitter 7, intensity modulation is respectively carried out by optical fiber frequency modulators 9 and 13 and optical fiber intensity modulators 10 and 14, an adjustable time delay delta t is introduced between the two beams of laser pulses by an optical fiber time delay device 11, the two beams of laser pulses are coupled into two fiber cores which are perpendicular to the horizontal plane at the periphery of a five-core optical fiber 25 by an optical fiber coupler 18, and focused light fields are formed at the output ends of the optical fibers processed into a frustum with a specific angle after being transmitted by the fiber cores and are respectively used as pushing light for pushing the captured cells to rotate and brake light for stopping the rotation of the cells; the output end of the five-core optical fiber 25 is arranged on an optical fiber clamping seat and is fixed in a cell culture dish 27.
The femtosecond laser pulse output by the other laser 16 is coupled into a Bessel beam generation system and a holographic wavefront pre-shaping and optimizing system which are formed by a microstructure mask plate 21 and a spatial light modulator 22 after beam expansion and shaping by a relay optical system 20, then is coupled into a single-mode fiber 24, is coupled into the central fiber core of a five-core optical fiber 25, and outputs the Bessel beam meeting the requirement at the output end of the five-core optical fiber 25; the capturing light captures the cells, the braking light controls the cells, the Bessel light beam forms a virtual light sheet, fluorophores in an illumination area are excited, generated fluorescent signals are collected by a microscope objective 28 perpendicular to the excitation light plane of the virtual light sheet, and fluorescent tomographic images of different angles of the cells recorded by a CMOS camera 31 are analyzed to obtain high-spatial-resolution three-dimensional structure fluorescent images of living single cells.
The intensity of the captured light remains unchanged during manipulation of the cells. Cells of different sizes require adjustment of the laser intensity to achieve stable capture of the cells. The laser beam generates pushing light and braking light via fiber frequency modulators 9, 13, fiber intensity modulators 10, 14 and fiber time delay 11, respectively. The pushing light pulse acts on one end of the cell to push the cell to rotate. When the pushing light pulse disappears, the cell will continue to rotate slowly due to inertia, and if the braking light pulse with the intensity weaker than that of the pushing light pulse acts on the other end of the cell at the moment, the cell can be braked by generating braking force opposite to the rotation direction of the cell, so that accurate control of the cell is realized. Under the continuous action of the pushing light pulse and the braking light pulse, the cells continuously rotate around the capturing shaft, so that the rapid scanning of the light sheet in the cells is realized, and the three-dimensional structure chromatography fluorescence image of the cells is obtained.
Considering the exposure time of the CMOS camera 31, the time interval between the pushing light pulses is slightly longer than the exposure time of the camera in order to achieve tomographic imaging of the cells. The fluorescence image collected by the CMOS camera 31 is the image of the same position of the cell, so that the disorder of fluorescence signals caused by the multiple rotations of the cell during the exposure time of the camera is avoided. The angle of rotation of the cell per rotation is the sum of the angles through which the cell rotates in the pushing phase, the inertial rotation phase and the braking phase. The rotation angle of the cell can be controlled by controlling the intensity of the laser pulses and the time interval between pulses.
The above examples are provided for the purpose of describing the present invention only and are not intended to limit the scope of the present invention. The scope of the invention is defined by the appended claims. Various equivalent substitutions and modifications may be made without departing from the spirit and principles of the present invention, and it is intended to be within the scope of the present invention. .
Claims (3)
1. The invention provides a digital scanning light sheet fluorescence microscopic imaging system based on five-core optical fiber active light control, which is characterized in that: the device consists of lasers 1 and 19, optical coupling systems 2 and 23, beam splitters 4 and 7 and 8, single-mode optical fibers 3 and 5 and 6 and 12 and 15, single-mode optical fibers 16 and 17 and 24, frequency modulators 9 and 13, intensity modulators 10 and 14, a time delay 11, a coupler 18, a relay optical system 20, a microstructure mask plate 21, a spatial light modulator 22, a five-core optical fiber 25, cells 26, a cell culture dish 27, a microscope objective 28, a sampling mirror 29, a band-pass filter 30, a CMOS camera 31, a condenser 32, an illumination light source 33, data connecting wires 34 and 36 and a computer 35; the laser beam output by the laser 1 is coupled into a single-mode fiber 3 through an optical coupling system 2, and is split into two beams according to a certain proportion through an optical fiber beam splitter 4; one of the continuous lasers is transmitted by a single-mode fiber 6 and then is split into two beams by an optical fiber beam splitter 8, the two beams are respectively coupled into two fiber cores parallel to the horizontal plane on the periphery of a five-core optical fiber 25 by an optical fiber coupler 18, and a focused light field is formed at the output end of the optical fiber processed into a frustum with a specific angle after the transmission of the fiber cores and is used as capturing light for capturing cells; the other beam of continuous laser is divided into two beams by an optical fiber beam splitter 7, intensity modulation is respectively carried out by optical fiber frequency modulators 9 and 13 and optical fiber intensity modulators 10 and 14, an adjustable time delay delta t is introduced between the two beams of laser pulses by an optical fiber time delay device 11, the two beams of laser pulses are coupled into two fiber cores which are perpendicular to the horizontal plane at the periphery of a five-core optical fiber 25 by an optical fiber coupler 18, and focused light fields are formed at the output ends of the optical fibers processed into a frustum with a specific angle after being transmitted by the fiber cores and are respectively used as pushing light for pushing the captured cells to rotate and brake light for stopping the rotation of the cells; the output end of the five-core optical fiber 25 is arranged on an optical fiber clamping seat and is fixed in a cell culture dish 27; the femtosecond laser pulse output by the other laser 16 is coupled into a Bessel beam generation system and a holographic wavefront pre-shaping and optimizing system which are formed by a microstructure mask plate 21 and a spatial light modulator 22 after beam expansion and shaping by a relay optical system 20, then is coupled into a single-mode fiber 24, is coupled into the central fiber core of a five-core optical fiber 25, and outputs the Bessel beam meeting the requirement at the output end of the five-core optical fiber 25; the capturing light captures the cells, the braking light controls the cells, the Bessel light beam forms a virtual light sheet, fluorophores in an illumination area are excited, generated fluorescent signals are collected by a microscope objective 28 perpendicular to the excitation light plane of the virtual light sheet, and fluorescent tomographic images of different angles of the cells recorded by a CMOS camera 31 are analyzed to obtain high-spatial-resolution three-dimensional structure fluorescent images of living single cells.
2. The light manipulation section according to claim 1, wherein: the light control part is realized by four outer cores of five-core optical fibers (25) which are fixed in a culture dish for placing cells to be tested and the output ends of which are processed into specific angles; the laser beam output by the laser 1 is coupled into a single-mode fiber 3 through an optical coupling system 2, and is split into two beams according to a certain proportion through an optical fiber beam splitter 4; one of the continuous lasers is transmitted through a single-mode fiber 6 and then is split into two beams by an optical fiber beam splitter 8, the two beams are respectively coupled into two fiber cores parallel to the horizontal plane on the periphery of a five-core optical fiber 25 through an optical fiber coupler 28, and a focused light field is formed at the output end of the optical fiber processed into a frustum with a specific angle after the transmission through the fiber cores and is used as capturing light for capturing cells; the other beam of continuous laser is divided into two beams by an optical fiber beam splitter 7, two beams of laser pulses with certain repetition frequency f and a period of T1 and T2 are respectively modulated by optical fiber frequency modulators 9 and 13, the two beams of laser pulses are respectively subjected to intensity modulation by optical fiber intensity modulators 10 and 14, an adjustable time delay delta T is introduced between the two beams of laser pulses by using an optical fiber time delay 11, the two beams of laser pulses are coupled into two fiber cores which are perpendicular to the horizontal plane at the periphery of a five-core optical fiber 25 by an optical fiber coupler 18, and a focusing light field is formed at the output end of the optical fiber processed into a specific angle frustum after being transmitted by the fiber cores and is respectively used as pushing light for pushing the captured cells to rotate and braking light for stopping the rotation of the cells; the output end of the five-core optical fiber 25 is arranged on an optical fiber clamping seat and fixed in a cell culture dish 27, a potential well is formed near the output end face of the five-core optical fiber by capturing living single cells 26 suspended in the culture dish, laser pulses with a certain time delay are applied to captured cells at the output end of the optical fiber by periodic light pushing force and braking force with opposite directions, and the rotation angle of the captured cells is controlled by stable and accurate active light.
3. The light sheet fluorescence microscope portion of claim 1, wherein: the femtosecond laser pulse output by the other laser 19 is coupled into a Bessel beam generating system and a holographic wave front pre-shaping and optimizing system which are formed by a microstructure mask plate 21 and a spatial light modulator 22 after being expanded and shaped by a relay optical system 20, the beam output by the wave front pre-shaping and optimizing system is coupled into a single-mode optical fiber 24 by an optical coupling system 23, the femtosecond laser pulse transmitted by the single-mode optical fiber 24 is coupled into the central fiber core of a five-core optical fiber 25 by an optical fiber coupler 18, and the Bessel beam meeting the requirement is output at the output end of the five-core optical fiber 25; the angle of the Bessel light beam output by the output end of the five-core optical fiber 25 is adjusted, and the Bessel light beam is rapidly scanned in the cell to be detected to form a virtual light sheet; in the process of driving the cells to rotate around a specific rotation axis by the light field, exciting fluorophores in an illumination area, collecting generated fluorescent signals by a micro objective lens 28 perpendicular to the excitation light plane of the virtual light sheet, filtering stray light by a liquid crystal tunable bandpass filter 30, and detecting and receiving by a CMOS camera 31; based on the spatial attitude of the cells and the characteristic mark points on the rotating shaft, a three-dimensional structure fluorescence tomographic image reconstruction algorithm of the rotating cells is established, and a high spatial resolution three-dimensional structure fluorescence image of the living single cells is obtained by analyzing fluorescence tomographic images of different angles of the cells recorded by the CMOS camera 31.
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