Flow cytometer based on multi-focus confocal microscopic imaging

Abstract

The invention provides a multi-focus excitation confocal fluorescence microscopic imaging flow cytometry screening method. The method is characterized in that a laser beam generates three laser beams through a spatial light modulator, and the three laser beams are coherently superposed in a focal plane of a microscope objective to generate a two-dimensional optical lattice light field. And after the other laser beam is shaped by the cylindrical lens, linear laser optical tweezers are realized through the microscope objective, cells to be detected in an imaging detection area of the micro-flow chip are captured, and accurate active optical control on the rotation angle of the cells to be detected is realized. In a cell rotation process, a two-dimensional optical lattice light field rapidly scans in a focal plane at different angles, and a fluorophore in the cell is excited to generate a fluorescence signal. The confocal pinhole array installed at the position conjugate with the focal plane eliminates background fluorescence, a high-spatial-resolution fluorescence image of a three-dimensional structure in the cell is obtained, and cell screening is achieved. The method provided by the invention has the characteristics of high detection rate, high accuracy and the like, and has wide application prospects in the research fields of biology, medicine and life science.

Description

TECHNICAL FIELD

[0001] The application provides a multi-focus excitation confocal fluorescence microscopic imaging flow cytometry screening method, which generates three laser beams to form a two-dimensional optical lattice light field by using a spatial light modulator, and combines a line laser optical tweezer to accurately manipulate the rotation of cells in a microfluidic chip. The fluorescence of the cells is excited by scanning the optical lattice light field, the background is eliminated by a confocal pinhole array, high-resolution three-dimensional fluorescence images are obtained, and cell screening is realized. The method has high detection rate and good accuracy, and has wide application prospects in the field of biomedicine. It belongs to the field of biophotonics. BACKGROUND

[0002] Imaging flow cytometry (IFC) is an advanced analysis tool combining flow cytometry and digital microscopic imaging technology, which can perform multi-parameter optical detection and image acquisition on single cells under high-speed flow conditions. As an important analysis means in modern biomedical research, it plays an irreplaceable role in the field of multi-parameter detection at the single-cell level. Traditional flow cytometers can analyze the scattering light and fluorescence signals of cell populations at high throughput, but they mostly use wide-field illumination or single-point scanning, which makes it difficult to obtain three-dimensional morphological information of cells, limiting their application in cell mechanics or substructure analysis. However, they cannot provide cell morphological and subcellular structure information. And the existing technology relies on fluid dynamic focusing or mechanical fixation, lacking non-contact manipulation ability for target cells, making it difficult to realize real-time separation or three-dimensional positioning of specific cells, lacking dynamic manipulation. With the in-depth development of cell biology research, researchers have higher requirements for multi-dimensional characteristic parameters, high-efficiency high-throughput detection, and real-time detection.

[0003] Optical lattice is a periodic light field distribution structure formed by coherent laser interference, and its multi-dimensional controllable characteristics provide a new technology for high-throughput optical detection. In the field of fluorescence microscopy, optical lattice has become an effective solution to realize parallel multi-focus excitation due to its unique spatial light field modulation capability. By adjusting the wavefront phase of the incident laser using a spatial light modulator, an excitation light field array with specific symmetry can be generated, forming a high-density, low-crosstalk multi-focus excitation light field in the focal plane. Traditional multi-focus excitation techniques, such as Nipkow disk or microlens array, are limited by mechanical scanning speed or optical efficiency, making it difficult to balance throughput and resolution. Optical lattice technology can flexibly adjust the required multi-focus light field, enabling multi-focus excitation at the diffraction limit scale, significantly improving the spatial and temporal resolution of the system. In flow cytometry detection, this characteristic is particularly suitable for parallel imaging of cell populations under high-speed flow conditions, providing a physical basis for solving the throughput bottleneck of single-focus scanning systems.

[0004] Since its invention by Arthur Ashkin in 1986, optical tweezers have become a core tool for single-cell manipulation. They achieve non-contact capture of tiny particles through gradient forces generated by a highly focused laser beam, demonstrating unique advantages in biomedical research. While traditional imaging flow cytometry can provide two-dimensional cell images, it cannot acquire three-dimensional morphological information. However, by combining optical tweezers with the three-dimensional manipulation capabilities of optical tweezers, cells can be actively rotated during imaging, thus overcoming the limitations of traditional two-dimensional imaging.

[0005] This invention proposes a flow cytometry cell screening method based on multifocal excitation confocal fluorescence microscopy. The multifocal confocal microscopy system, through a spatially parallel excitation and detection mechanism, significantly improves imaging throughput and spatial resolution, effectively overcoming the technical bottleneck of detection rate in traditional single-point scanning modes, and providing a new solution for dynamic observation of live cells and high-throughput cell screening. Meanwhile, optical tweezers technology, with its unique advantages of non-contact operation and low optical damage, demonstrates significant value in the field of precise single-cell manipulation. This technology can achieve high-precision capture, three-dimensional rotation, and spatial displacement control of target cells at the micrometer scale, thereby overcoming the cell disturbance problem caused by traditional hydrodynamic focusing, significantly reducing the false negative rate and improving detection and imaging reliability. Furthermore, through the optical tomography capabilities of the multifocal confocal system, this research method can acquire high-resolution three-dimensional morphological information of cells, overcoming the inherent limitation of traditional flow cytometry technology, which can only provide two-dimensional imaging data, and providing more comprehensive technical support for cell substructure analysis and functional characterization. Summary of the Invention

[0006] The purpose of this invention is to provide a multifocal excitation confocal fluorescence microscopy flow cytometry method for cell screening. The technical method is as follows:

[0007] The system uses a linearly polarized continuous laser 1 in the visible light band as an excitation source, which outputs a linearly polarized laser beam of a specific wavelength. The laser polarization direction is precisely controlled by a λ / 2 waveplate 2. The laser beam is collimated and expanded by a collimation and beam-expanding system composed of lenses 3 and 4, ensuring its diameter reaches the effective working aperture of the spatial light modulator 5. The expanded beam is easier to modulate in subsequent light field operations. The expanded linearly polarized light is reflected to a λ / 4 waveplate 6. A polarizing beam splitter separates the beam according to the polarization direction, reflecting light with a specific polarization direction. This converts the linearly polarized light into circularly polarized light. The circularly polarized light is incident perpendicularly onto the spatial light modulator 5, ensuring the uniformity of the light field modulation. A hologram generated by computer 31 is loaded, and the phase of the light field is periodically modulated in two dimensions. The phase distribution of the light can be dynamically adjusted through programming to generate the light field pattern required for the experiment. The modulated outgoing light passes through the λ / 4 waveplate 6 again, converting the circularly polarized light back into linearly polarized light, which can then be transmitted through a polarizing beam splitter 7. The polarizing beam splitter 7 ensures that only the modulated light enters the subsequent optical path. The transmitted light is rapidly switched and scanned through the laser beam scanning system 8 to achieve complex light field patterns, thereby improving excitation efficiency. Then, the secondary collimation and beam expansion system composed of lenses 9 and 10 filters out unnecessary stray light or separates light signals of different wavelengths through the dichroic mirror 15, and finally focuses the light onto the detection area of ​​the microfluidic chip 19 through the microscope objective 16 to form a two-dimensional optical lattice light field.

[0008] The optical lattice light field, used as the excitation field in multifocal microscopes, is theoretically based primarily on the interference principle of coherent waves, lattice symmetry manipulation, and polarization optimization. Specifically, by carefully designing the wave vector set {Kn} of multiple coherent plane waves, a periodically distributed array of excitation points can be formed in space, thereby achieving efficient and high-resolution parallel excitation.

[0009] An optical lattice is generated by the interference of N coherent plane waves, and its electric field distribution can be expressed as: , Where En is the complex amplitude of the nth wave, kn is the wave vector, and ω is the angular frequency. Phase is used to control interference patterns. By adjusting the geometric arrangement of the wave vectors, lattice structures with different symmetries (such as hexagonal, cubic, etc.) can be constructed to meet the needs of specific applications.

[0010] The periodicity of the lattice is determined by the direct lattice basis vectors {a1,a2,a3}, and their corresponding reciprocal lattice vectors {b1,b2,b3} satisfy: The relationship between the reciprocal lattice vector matrix B and the direct lattice matrix A is as follows: By adjusting A, i.e., selecting different lattice symmetries, the period and spatial distribution of the optical lattice can be controlled. Lattices with a period much larger than the wavelength λ are called sparse lattices. These lattices allow excitation points to be sufficiently separated in space, facilitating individual detection. Based on sparse lattices, the wave vector set is expanded by applying symmetry operations (such as rotation and reflection) to form composite lattices (N>D+1, where D is the dimension). Composite lattices significantly enhance the locality and contrast of the excitation points by increasing the number of interference waves, bringing them close to the diffraction limit.

[0011] Composite lattices, by increasing the number of wave vectors, form multiple high-contrast excitation points in real space. Their intensity distribution is |e(x)|. 2 The locality is determined by the spatial frequency of the wave vector: Highly symmetric lattices can achieve diffraction-limited localization.

[0012] Based on the above theory, the target lattice type and wave vector {kn} arrangement are first determined in the experiment. Multiple plane waves are generated using a spatial light modulator, and a high numerical aperture objective lens is used to focus the beam onto the sample plane to simulate plane wave interference. The phase of each beam is adjusted using an SLM or liquid crystal phase modulator. To ensure the stability of the interference pattern, the polarization direction is controlled by a polarization beam splitter and a waveplate to optimize the localization of the excitation point. The resulting composite optical lattice light field strikes the cell flow chamber detection area in the imaging flow cytometer, simultaneously performing multi-point excitation.

[0013] In this system, a near-infrared continuous laser 23 serves as the light source, and its wavelength selection must balance cell viability and optical tweezers efficiency. Its output beam is pulse-modulated by a computer 31 through programming, precisely coordinating the timing of the laser's capture, rotation, and release phases. The capture phase involves continuous laser output to form a stable optical trap for cell capture. The rotation phase involves dynamically adjusting the optical field by modulating the pulse frequency in conjunction with the rotation of a cylindrical mirror. The release phase involves reducing the laser power or shutting off the beam to release the cells. The laser beam first passes through a collimation and beam-expanding system composed of lenses 24 and 25. A cylindrical lens 26 rotates around the optical axis, converting the circular Gaussian beam into an elliptical line laser. The tilt direction of the line laser is changed by adjusting the rotation angle. The tilted line laser forms an intensity gradient distribution on the imaging plane, generating an asymmetric optical gradient force that provides torque for cell rotation. The shaped beam then passes through a secondary collimation and beam-expanding system composed of mirrors 27 and 28 to ensure beam quality meets the requirements for high-precision control. Finally, the light is reflected by the total reflection mirror 29 and focused by the microscope objective 30, forming a line laser optical tweezers in the imaging and detection area of ​​the microfluidic chip 19.

[0014] Optical tweezers use the gradient force generated by a highly focused laser beam to capture and manipulate tiny particles. The force of optical tweezers is mainly divided into gradient force and scattering force.

[0015] Gradient force points towards the point of highest light intensity and is used to capture particles. Where α is the polarizability of the particle, and I(r) is the light intensity distribution.

[0016] Scattering force refers to the force along the direction of light propagation that can disrupt capture stability. Where Qgrad and Qscat are dimensionless efficiency factors, and c is the speed of light.

[0017] When the gradient force is greater than the scattering force, a potential well is formed, and the particle is pulled towards the point of maximum light intensity, that is, the particle is captured.

[0018] Then, by introducing an orbital angular momentum beam to form an asymmetric light field, the spatial symmetry of the optical force is broken, generating torque to drive the rotation of the captured particles: τ=r×F grad.

[0019] Based on the above theory, a precision peristaltic pump 20 draws sheath fluid from the sheath fluid pool 21 and a sample solution containing the cells to be tested from the sample pool 22, injecting it into the detection chamber of the microfluidic chip 19 in laminar flow mode. The laser beam output from the visible-band linearly polarized continuous laser 1 is modulated by the spatial light modulator 5 and, after a series of optical path optimizations, focused onto the detection area of ​​the microfluidic chip 19 by the microscope objective 16, forming a two-dimensional optical lattice multifocal excitation light field that covers the cell flow path. The beam output from the near-infrared continuous laser 23 is collimated and expanded by lens groups 24 and 25, and then shaped into a linear light field by a rotating cylindrical mirror 26. This adjusts the angle of the linear light field, enabling active manipulation of the cell's three-dimensional posture. This is combined with white light illumination 17 and 18 to observe the cell's rotational state. The multifocal laser excites fluorophores within the cells, and the resulting fluorescence signal is collected by the microscope objective 16. Stray light is filtered out by a dichroic mirror 15 and a long-pass filter 14. Fluorescence is focused by imaging lens 13 onto a confocal pinhole array modulated by intensity-type spatial light modulator 12, allowing only focal plane signals to pass through, significantly improving axial resolution; EMCCD camera 11 acquires high signal-to-noise ratio three-dimensional fluorescence images. Computer 31 controls the peristaltic pump of the microfluidic system in real time to precisely regulate cell flow rate, ensuring it matches the image acquisition frame rate; simultaneously, it controls the rotating cylindrical mirror to adjust the optical tweezers angle, achieving multi-directional cell imaging; and by synchronously controlling the timing of laser excitation, optical tweezers manipulation, and EMCCD exposure, it ensures the spatiotemporal consistency of multimodal data. Attached Figure Description

[0020] Figure 1This is a schematic diagram of the overall structure of a flow cytometer based on multifocal confocal microscopy.

[0021] Figure 2 This is a schematic diagram of the incident light path of microscope objective 16 in the optical lattice light field system.

[0022] Figure 3 This is a schematic diagram of a two-dimensional optical lattice light field simulation.

[0023] Figure 4 This is a schematic diagram of the specific design of a flow cytometer for microscopic imaging.

[0024] Figure labeling: 1. Visible band linearly polarized continuous laser; 2. λ / 2 waveplate; 3, 4, 9, 10, 24, 25, 27, 28; 5. Phase-type spatial light modulator; 6. λ / 4 waveplate; 7. Polarizing beam splitter; 8. Laser beam scanning system; 11. EMCCD camera; 12. Intensity-type spatial light modulator; 13. Imaging lens; 14. Long-pass filter; 15. Dichroic mirror; 16, 30. Microscope objectives; 17. White light source; 18. Condenser lens; 19. Microfluidic chip; 20. Peristaltic pump; 21. Sheath fluid cell; 22. Sample cell; 23. Near-infrared band continuous laser; 16. Cylindrical lens; 29. ​​Total internal reflection mirror; 31. Computer. Detailed Implementation

[0025] The present invention will now be described in further detail with reference to examples, so that those skilled in the art can implement it based on the description.

[0026] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0027] A multifocal excitation confocal fluorescence microscopy flow cytometry method for cell screening. Its features include: the system is composed of a two-dimensional optical lattice light field system, a line laser optical tweezers manipulation system, and a microscopic imaging flow cytometer; the system comprises a visible-band polarized continuous-wave laser 1; a λ / 2 waveplate 2; lenses 3, 4, 11, 13, 14, 23, 24, 26, 27; a spatial light modulator 5; a λ / 4 waveplate 6; a polarizing beam splitter 7; a dichroic mirror 8; an EMCCD camera 9; a mask 10; a long-pass filter 12; microscope objectives 15, 29; a white light source 16; a condenser lens 17; a microfluidic chip 18; a peristaltic pump 19; a sheath fluid reservoir 20; a sample solution reservoir 21; a near-infrared continuous-wave tunable laser pulse 22; a cylindrical lens 25; a reflector 28; and a computer 30.

[0028] In the system, the laser beam output from the visible-band polarized continuous-wave laser 1 is sequentially polarized by a λ / 2 waveplate 2. After passing through a collimation and beam-expanding system composed of lenses 3 and 4, it is reflected by a polarizing beam splitter 7 and passes through a λ / 4 waveplate 6 to generate circularly polarized light, which is then incident on the spatial light modulator 5. The modulated outgoing light passes through the λ / 4 waveplate 6 again and is transmitted through the polarizing beam splitter 7 and the laser beam scanning system 8. The collimation and beam-expanding system composed of lenses 9 and 10 collimates and expands the beam, and finally focuses it onto the detection area of ​​the microfluidic chip 19 through a dichroic mirror 15 and a microscope objective 16. The sheath fluid pool 21 and the sample pool 22 are injected into the microfluidic chip 19 through a precision peristaltic pump 20 to achieve laminar flow. The generated multifocal excitation light field illuminates the cell flow chamber detection area in the microfluidic chip 19, working in conjunction with the white light illumination system consisting of the white light source 17 and the condenser lens 18. With the help of optical tweezers, the cell is rotated at different angles, and the two-dimensional optical lattice light field is rapidly scanned within the focal plane, exciting the fluorophores in the cell to generate fluorescence signals. The fluorescence signal collected by the microscope objective 16 is reflected by the dichroic mirror 15, and then spectrally purified by the long-pass filter 14. It is then focused by the imaging lens 13 onto the confocal pinhole array formed at the conjugate position of the intensity spatial light modulator 12 at the focal plane to eliminate background fluorescence. The high spatial resolution fluorescence image of the three-dimensional structure inside the cell is acquired by the EMCCD camera 11. The line laser optical tweezers manipulation system uses a near-infrared continuous wave tunable laser pulse 22 with an externally modulated signal. After passing through the optical path collimation and beam expansion system composed of lenses 24 and 25, the beam is shaped by a rotating cylindrical lens 26, and then passes through the optical path collimation and beam expansion system composed of lenses 27 and 28. Finally, it is focused by a total reflection mirror 29 onto a microscope objective 30 to realize line laser optical tweezers, capture the cells to be tested in the imaging and detection area of ​​the microfluidic chip 19, and achieve precise active optical control over their rotation angle.

[0029] In a multifocal excitation fluorescence microscopy system with a two-dimensional optical lattice light field, a linearly polarized continuous laser 1 in the visible light band outputs a linearly polarized laser beam of a specific wavelength as the excitation source. The polarization direction of the linearly polarized laser beam is controlled by a λ / 2 waveplate 2. After being collimated and expanded by a beam-expanding system composed of lenses 3 and 4, the beam diameter reaches the effective working aperture of the spatial light modulator 5. The beam is reflected by a polarizing beam splitter 7 and passes through a λ / 4 waveplate 6 to generate circularly polarized light that is perpendicularly incident on the spatial light modulator 5. A hologram generated by a computer 31 is loaded, and the phase of the light field is periodically modulated in two dimensions. The modulated outgoing light passes through the λ / 4 waveplate 6 again and is converted into an orthogonal linearly polarized state. The modulated linearly polarized light is transmitted through the polarizing beam splitter 7, and the transmitted light passes through a laser beam scanning system 8 to achieve rapid switching and scanning of complex light field patterns, thereby improving excitation efficiency. A secondary collimation and beam-expanding system composed of lenses 9 and 10 and a microscope objective 16 are then focused on the detection area of ​​a microfluidic chip 19 to form a two-dimensional optical lattice light field.

[0030] In the online laser optical tweezers active optical manipulation system, the laser beam output from the near-infrared continuous laser 23 first passes through a collimation and beam-expanding system composed of lenses 24 and 25. The Gaussian beam is then shaped by a cylindrical lens 26 on a rotatable frame. The shaped beam then passes through a secondary collimation and beam-expanding system composed of lenses 27 and 28 to ensure beam quality meets high-precision manipulation requirements. Finally, it is reflected by a total reflection mirror 29 and focused by a microscope objective 30, forming a line laser optical tweezer within the imaging detection area of ​​the microfluidic chip 19. By adjusting the rotation angle of the cylindrical lens, the system can generate a line laser light field with a controllable tilt angle within the imaging plane, thereby applying an asymmetric optical gradient force to the target cell and achieving precise continuous rotation of the cell in three-dimensional space. The computer 31 uses an external interface of the near-infrared continuous laser 22 for programming control to achieve pulse modulation, precisely coordinating the timing of the laser's capture, rotation, and release phases.

[0031] The microfluidic cell screening system primarily utilizes a precision peristaltic pump 20 to extract sheath fluid from the sheath fluid reservoir 21 and a sample solution containing test cells from the sample reservoir 22, injecting this solution into the detection chamber of the microfluidic chip 19 in laminar flow mode. Within the imaging detection area of ​​the microfluidic chip 19, a line laser optical tweezers active light manipulation system is used to capture and precisely manipulate the test cells. A multi-focal excitation fluorescence microscopy imaging system based on a two-dimensional optical lattice light field is used to acquire three-dimensional structural fluorescence tomography images of the test cells. Throughout the entire microscopic imaging flow cytometer system, a computer 31 controls and coordinates the flow rate adjustment of the microfluidic system, the precise control of the rotation angle of the line laser optical tweezers, and the image acquisition timing, ensuring the spatiotemporal synchronization of data acquisition from each module. Cell screening is then performed based on the acquired three-dimensional structural fluorescence tomography images of the cells.

[0032] The examples provided above are merely for illustrative purposes and are not intended to limit the scope of the invention. The scope of the invention is defined by the appended claims. All equivalent substitutions and modifications made without departing from the spirit and principles of the invention should be covered within its scope.

Claims

1. This invention provides a multifocal excitation confocal fluorescence microscopy flow cytometry cell screening method; characterized in that: The system consists of three parts: a multifocal excitation fluorescence microscopy imaging system based on a two-dimensional optical lattice light field, a linear laser optical tweezers active light manipulation system, and a microfluidic cell screening system. The system mainly comprises: a visible-band linearly polarized continuous laser 1; a λ / 2 waveplate 2; lenses 3, 4, 9, 10, 24, 25, 27, 28; a phase-type spatial light modulator 5; a λ / 4 waveplate 6; a polarizing beam splitter 7; a laser beam scanning system 8; an EMCCD camera 11; an intensity-type spatial light modulator 12; an imaging lens 13; a long-pass filter 14; a dichroic mirror 15; microscope objectives 16, 30; a white light source 17; a condenser lens 18; a microfluidic chip 19; and a peristaltic cell screening system. The system comprises a pump 20; a sheath fluid reservoir 21; a sample cell 22; a near-infrared continuous-wave laser 23; a cylindrical lens 16; a total reflection mirror 29; and a computer 31. The laser beam output from the visible-band polarized continuous-wave laser 1 is sequentially polarized by a λ / 2 waveplate 2. After passing through a collimation and beam-expanding system composed of lenses 3 and 4, it is reflected by a polarizing beam splitter 7, passes through a λ / 4 waveplate 6 to generate circularly polarized light, and then enters a spatial light modulator 5. The modulated outgoing light passes through the λ / 4 waveplate 6 again and is transmitted through the polarizing beam splitter 7 and a laser beam scanning system 8. The collimation and beam-expanding system composed of lenses 9 and 10 collimates and expands the beam, finally passing through a dichroic mirror 15 and a microscope. Objective lens 16 focuses on the detection area of ​​microfluidic chip 19; sheath fluid pool 21 and sample pool 22 are laminarly injected into microfluidic chip 19 via precision peristaltic pump 20; the generated multifocal excitation light field irradiates the cell flow chamber detection area in microfluidic chip 19, working in conjunction with a white light illumination system consisting of white light source 17 and condenser lens 18, and with the help of optical tweezers, the cells are rotated at different angles to rapidly scan the two-dimensional optical lattice light field within the focal plane, exciting the fluorophores in the cells to generate fluorescence signals; the fluorescence signal collected by microscope objective lens 16 is reflected by dichroic mirror 15, spectrally purified by long-pass filter 14, and then focused by imaging lens 13 onto intensity spatial light modulator.

12. A confocal pinhole array formed at the conjugate position of the focal plane eliminates background fluorescence, and a high spatial resolution fluorescence image of the three-dimensional structure inside the cell is acquired by an EMCCD camera 11. The line laser optical tweezers manipulation system uses a near-infrared continuous wave tunable laser pulse 22 with an externally modulated signal. After passing through the optical path collimation and beam expansion system composed of lenses 24 and 25, the beam is shaped by a rotating cylindrical lens 26, and then passes through the optical path collimation and beam expansion system composed of lenses 27 and 28. The beam is focused by a total reflection mirror 29 onto a microscope objective 30 to realize line laser optical tweezers, capture the cell to be tested in the imaging detection area of ​​the microfluidic chip 19, and achieve precise active light control of its rotation angle.

2. The multifocal excitation fluorescence microscopy imaging system for a two-dimensional optical lattice light field according to claim 1 mainly comprises: a visible light band linearly polarized continuous laser 1; a λ / 2 waveplate 2; lenses 3, 4, 9, 10; a phase-type spatial light modulator 5; a λ / 4 waveplate 6; a polarizing beam splitter 7; a laser beam scanning system 8; an EMCCD camera 11; a dichroic mirror 15; a microscope objective 16; a microfluidic chip 19; and a computer 31; characterized in that: A linearly polarized continuous laser 1 in the visible light band outputs a linearly polarized laser beam of a specific wavelength as an excitation source. The polarization direction of the linearly polarized laser beam is controlled by a λ / 2 waveplate 2. After being collimated and expanded by a collimation and beam-expanding system composed of lenses 3 and 4, the beam diameter reaches the effective working aperture of the spatial light modulator 5. The beam is reflected by a polarizing beam splitter 7 and passes through a λ / 4 waveplate 6 to generate circularly polarized light that is perpendicularly incident on the spatial light modulator 5. A hologram generated by a computer 31 is loaded to perform two-dimensional periodic modulation of the phase of the light field. The modulated outgoing light passes through the λ / 4 waveplate 6 again and is converted into an orthogonal linearly polarized state. The modulated linearly polarized light is transmitted through the polarizing beam splitter 7. The transmitted light passes through a laser beam scanning system 8 to achieve rapid switching and scanning of complex light field patterns, thereby improving excitation efficiency. Then, a secondary collimation and beam-expanding system composed of lenses 9 and 10 and a microscope objective 16 focus the light onto the detection area of ​​a microfluidic chip 19 to form a two-dimensional optical lattice light field.

3. The line laser optical tweezers active optical manipulation system according to claim 1 mainly consists of a near-infrared continuous laser 23; lenses 24, 25, 27, 28; a cylindrical lens 26; a total reflection mirror 29; a microscope objective 30; a microfluidic chip 19; and a computer 31; characterized in that: The laser beam output from the near-infrared continuous laser 23 first passes through a collimation and beam-expanding system composed of lenses 24 and 25. The Gaussian beam is shaped by a cylindrical lens 26 on a rotatable frame. The shaped beam then passes through a secondary collimation and beam-expanding system composed of lenses 27 and 28 to ensure that the beam quality meets the requirements of high-precision control. Finally, it is reflected by a total reflection mirror 29 and focused by a microscope objective 30, forming a line laser optical tweezers in the imaging and detection area of ​​the microfluidic chip 19. By adjusting the rotation angle of the cylindrical lens, the system can generate a line laser light field with a controllable tilt angle in the imaging plane, thereby applying an asymmetric optical gradient force to the target cell and achieving precise continuous rotation of the cell in three-dimensional space. The computer 31 uses the external interface of the near-infrared continuous laser 22 to program and control pulse modulation, precisely coordinating the timing of the laser's capture, rotation, and release.

4. The microfluidic cell screening system according to claim 1 mainly comprises an EMCCD camera 11; an intensity-type spatial light modulator 12; an imaging lens 13; a long-pass filter 14; a dichroic mirror 15; a microscope objective 16; a white light source 17; a condenser lens 18; a microfluidic chip 19; a peristaltic pump 20; a sheath fluid pool 21; a sample cell 22; and a computer 31; characterized in that: A precision peristaltic pump 20 draws sheath fluid from the sheath fluid pool 21 and a sample solution containing test cells from the test sample pool 22, injecting it into the detection chamber of the microfluidic chip 19 in laminar flow mode. Within the imaging detection area of ​​the microfluidic chip 19, a line laser optical tweezers active light manipulation system captures and precisely manipulates the test cells. A multi-focal excitation fluorescence microscopy imaging system based on a two-dimensional optical lattice light field acquires three-dimensional structural fluorescence tomography images of the test cells. Throughout the entire microscopic imaging flow cytometer system, a computer 31 controls and coordinates the flow rate adjustment of the microfluidic system, the precise control of the rotation angle of the line laser optical tweezers, and the image acquisition timing, ensuring the spatiotemporal synchronization of data acquisition from each module. Cells are then screened based on the acquired three-dimensional structural fluorescence tomography images.

Images