WO2023274422A1 - 一种远场光学超薄片层成像系统及方法 - Google Patents

一种远场光学超薄片层成像系统及方法 Download PDF

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WO2023274422A1
WO2023274422A1 PCT/CN2022/114392 CN2022114392W WO2023274422A1 WO 2023274422 A1 WO2023274422 A1 WO 2023274422A1 CN 2022114392 W CN2022114392 W CN 2022114392W WO 2023274422 A1 WO2023274422 A1 WO 2023274422A1
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microfluidic
objective lens
microfluidic valve
valve
liquid inlet
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PCT/CN2022/114392
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English (en)
French (fr)
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赵伟
张策
朱月强
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西北大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/84—Systems specially adapted for particular applications
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/4833—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/12—Specific details about manufacturing devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N2021/0106—General arrangement of respective parts
    • G01N2021/0112—Apparatus in one mechanical, optical or electronic block
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/06—Illumination; Optics
    • G01N2201/063—Illuminating optical parts
    • G01N2201/0636—Reflectors

Definitions

  • the invention relates to the technical field of optics and biomedical equipment, and more specifically relates to a far-field optical ultra-thin slice imaging system and method.
  • the 3D structure of biological samples directly determines its biomechanical properties and functions in life phenomena. Rapid and high-resolution measurement of the 3D structure of biological samples is the frontier and hotspot of research in the fields of biomedicine and optical technology.
  • optical technologies capable of high-resolution measurement of the 3D structure of biological samples mainly include: Confocal Microscope, Stimulated Emission Depletion Microscope, Saturated Structured Illumination Microscopy, and Saturated Structured Illumination Microscopy. , random positioning imaging microscope, light sheet imaging technology, etc.
  • the biological cells are usually scanned row by row and column by column, due to the fluorescent spot that the laser converges through the microscope objective lens Usually there is only one fluorescent spot, so the scanning time of row-by-row and column-by-column scanning of biological cells is longer and the imaging speed is slower, which is very unfavorable for live cell imaging and biological cells with poor bleaching resistance.
  • these techniques due to the depth of focus, whether confocal microscopy, stimulated radiation depletion microscopy, saturated structured light microscopy or random positioning imaging microscopy, these techniques have low spatial resolution along the optical axis and imaging direction. , resulting in a low ability to identify the structure of the biological sample in the direction of the optical axis.
  • the present invention provides a far-field optical ultra-thin slice imaging system and method, which can perform continuous, high-resolution, high-throughput slice imaging and 3D reconstruction analysis.
  • the present invention provides the following technical solutions:
  • a far-field optical ultra-thin slice imaging system including: a laser, a beam modulation module, an objective lens, a microfluidic chip control box, a microfluidic chip, a narrowband filter, an imaging lens, and an imaging device;
  • the laser emits a single laser beam and enters the beam modulation module; the beam modulation module modulates the incident single laser beam, and enters the rear aperture plane of the objective lens; the objective lens is incident on the rear aperture plane of the objective lens
  • the light beams on the aperture plane are focused and converged into the channel of the microfluidic chip to generate an ultra-thin light sheet; the microfluidic chip controls the flow of fluorescent dye through the microfluidic chip control box; the microfluidic chip The chip control box controls the flow of the cell sample stained with fluorescence, and the fluorescence emitted by the cells is collected on the imaging device through the narrow-band filter and the imaging lens.
  • the beam modulation module includes: a spatial light filter, a first aperture stop, a single lens, a polarizer, a beam splitter, a second aperture stop, and a phase-only reflective liquid crystal spatial light modulator , a quarter-wave plate, a first reflector, a second reflector;
  • the spatial light filter, the first aperture diaphragm and the single lens sequentially perform filtering, beam expansion and collimation operations on the single laser beam emitted by the laser, and modulate it into a linearly polarized beam with a fundamental transverse mode, which is incident on the The polarizer;
  • the polarizer modulates the polarization direction of the linearly polarized light beam to be consistent with the working direction of the liquid crystal panel of the phase-only reflective liquid crystal spatial light modulator, and enters the beam splitter;
  • the beam splitter splits the linearly polarized light beam passing through the polarizer into two and passes through the second small aperture stop, and then vertically enters the liquid crystal panel of the phase-only reflective liquid crystal spatial light modulator
  • the upper phase is modulated into a Bessel beam array (optical sheet) and reflected onto the quarter wave plate;
  • the quarter-wave plate converts the Bessel beam array into circularly polarized light, and then the circularly polarized light is incident on the rear aperture plane of the objective lens through the first reflector and the second reflector .
  • the microfluidic chip includes three three-dimensional surfaces carrying functional structures: three-dimensional surface 1, three-dimensional surface 2, and three-dimensional surface 3;
  • the three-dimensional surface 1 includes: liquid inlet S01, liquid inlet S02, liquid inlet S03, liquid inlet S04, microfluidic valve S05, microfluidic valve S06, microfluidic valve S07 controlled by elastic film , microfluidic control valve S08; the liquid inlet S01 and the liquid inlet S03 pass the sheath fluid into the pipeline, the liquid inlet S02 passes the solution loaded with cells into the pipeline, and the liquid inlet S04 is The chip peristaltic pump supplies liquid; the microfluidic valve S05 controls cells and sheath fluid to enter the microfluidic chip, and the microfluidic valve S06, microfluidic valve S07, and microfluidic valve S08 jointly control the drive of the peristaltic pump The liquid moves forward in steps;
  • the three-dimensional surface 2 includes: detection electrode S09, detection electrode S10, detection electrode S13 and ultra-thin chip excitation region S11; the detection electrode S09, detection electrode S10 and detection electrode S13 detect whether liquid flows in; the ultra-thin chip excitation Region S11 excites cells to emit fluorescence;
  • the three-dimensional surface 3 includes: a liquid outlet S12 for outputting detected cells.
  • a far-field optical ultra-thin slice imaging method characterized in that it comprises the following steps:
  • Step 1 The laser emits a single laser beam and enters the beam modulation module
  • Step 2 the beam modulation module modulates the incident single laser beam, and injects it into the rear aperture plane of the objective lens;
  • Step 3 The objective lens focuses the light beam incident on the rear aperture plane, and gathers it into the channel of the microfluidic chip to generate an ultra-thin light sheet;
  • Step 4 The microfluidic chip controls the flow of the fluorescently stained cell sample through the microfluidic chip control box; the fluorescent light emitted by the cells is collected on the imaging device through a narrow-band filter and an imaging lens.
  • modulating the incident single laser beam includes the following steps:
  • Step 201 the spatial light filter, the first aperture diaphragm and the single lens sequentially filter, expand and collimate the single laser beam emitted by the laser and modulate it into a linearly polarized beam with a fundamental transverse mode, which is incident on the polarizer device;
  • Step 202 the polarizer modulates the polarization direction of the linearly polarized light beam to be consistent with the working direction of the liquid crystal panel of the phase-only reflective liquid crystal spatial light modulator, and enters the beam splitter;
  • Step 203 the beam splitter divides the linearly polarized light beam passing through the polarizer into two, and after passing through the second aperture diaphragm, the beam is vertically incident on the liquid crystal panel of the pure-phase reflective liquid crystal spatial light modulator, and the phase is modulated into a shell. Searle beam array (light sheet), and reflected to a quarter wave plate;
  • Step 204 the quarter-wave plate converts the Bessel beam into circularly polarized light, and then the circularly polarized light enters the rear aperture plane of the objective lens through the first reflector and the second reflector.
  • generating an ultra-thin light sheet inside the channel of the microfluidic chip includes the following steps:
  • Step 301 Divide the rear aperture plane of the objective lens into P strip-shaped areas equally with the diameter R, and change the uniformity of the light spot by adjusting the size of P;
  • Step 302 each strip area is further divided into Q small strip areas
  • Step 303 fill the phase distribution into the small strip area sequentially, and change the value of the light spot by adjusting the size of Q, the phase distribution is:
  • NA is the numerical aperture of the objective lens
  • ⁇ is the wavelength emitted by the laser
  • R is the radius of the rear aperture of the objective lens
  • n t is the refractive index of the objective lens
  • x', y' are the rectangular coordinates on the plane of the rear aperture of the objective lens
  • ⁇ x and ⁇ y are the position of the focus of the spot in the focal area of the objective lens
  • ⁇ is the base angle of the axicon
  • ⁇ (x', y') is drawn as a phase diagram of the gray scale transformation from 0 to 2 ⁇ ;
  • Step 304 by designing ⁇ x and ⁇ y, the Bessel-Gaussian light is closely arranged to form an evenly distributed ultra-thin light sheet.
  • the operation process of the microfluidic chip includes:
  • Step 401 introduce the solution loaded with cells into the microfluidic chip through the liquid inlet S02, the sheath liquid through the liquid inlet S01, and the liquid inlet S03, and adjust the liquid inlet S01, the liquid inlet S02, and the liquid inlet S03
  • the pressure makes the cells enter the microfluidic chip one by one, the microfluidic valve S05 remains open, and the microfluidic valve S06, microfluidic valve S07, and microfluidic valve S08 are closed;
  • Step 402 the liquid inlet S01, the liquid inlet S02, and the liquid inlet S03 are pressurized to drive the fluid, and carry a single cell through the detection electrode S09, causing a local electrical signal change, stimulating the microfluidic chip control box to send a control signal, and closing the microfluidic
  • the control valve S05 stops the fluid movement; the control signal activates the control program, the microfluidic valve S06, the microfluidic valve S07 and the microfluidic valve S08 are switched on and off, and the microfluidic valve is controlled through the control logic sequence to drive the liquid flow.
  • the imaging process on the imaging device includes:
  • Step 403 the cells are driven by the microfluidic valve S06, microfluidic valve S07, and microfluidic valve S08, and move steppingly to the detection electrode S10.
  • the microfluidic The chip control box sends a trigger signal to the imaging device, and the imaging device starts continuous imaging;
  • Step 404 the cells are driven by the microfluidic valve S06, microfluidic valve S07, and microfluidic valve S08, and move steppingly to the detection electrode S13.
  • the detection electrode S13 detects the electrical signal generated by the cell
  • the microfluidic The chip control box sends a trigger signal to the imaging device, and the imaging device stops shooting;
  • Step 405 when the detection electrode S09 does not detect a new cell passing signal, the microfluidic valve S06, microfluidic valve S07, and microfluidic valve S08 are closed, the microfluidic valve S05 is opened, and the carrying cells leave the microfluidic chip , the microfluidic chip maintains this state until it stops running;
  • Step 406 when the detection electrode S09 has detected a new cell passing signal, the microfluidic valve S06 , the microfluidic valve S07 , and the microfluidic valve S08 continue to circulate until they stop running.
  • the present invention discloses a far-field optical ultra-thin slice imaging system and method, through spatial light modulation technology and phase division technology, a single laser beam is modulated into
  • the Searle spot array forms an ultra-thin light sheet with a large width and height, combined with microfluidic chip technology, it performs fast, high-resolution, high-throughput sheeting of biological samples such as cells that continuously pass through the light sheet in the channel Imaging can be applied to far-field and 3D structural imaging of biological samples and drug molecules in the field of biomedicine, serving clinical applications such as biophysics, biomedical basic research, cell pharmacology, and early screening of cancer cells.
  • Fig. 1 is the phase distribution schematic diagram at the objective lens rear aperture place of an embodiment
  • Fig. 2 is a schematic diagram of one of the strip sub-regions of the objective lens rear aperture phase distribution of an embodiment
  • Fig. 3 is the calculation schematic diagram of the position coordinate calculation of the objective lens focal plane focal point of an embodiment
  • Fig. 4 (a)-Fig. 4 (c) is that the two focal points of the objective lens focal area of an embodiment are close to the simulation result figure;
  • Fig. 5 is the depth of focus (objective lens uses 20X/0.5) of the focus spot Z-axis direction of an embodiment
  • FIG. 6 is a schematic diagram of the imaging system of the present invention.
  • FIG. 7 is a schematic diagram of the overall structure of the microfluidic chip of the present invention.
  • Fig. 8 is the phase diagram loaded on the liquid crystal panel of the phase-only reflective liquid crystal spatial light modulator according to the present invention.
  • Fig. 9 (a)-(b) is the experimental result of flake spot produced by 25 focusing spots in one embodiment
  • Fig. 10(a)-(b) is the experimental results of flake light spots generated by 151 focused light spots in another embodiment.
  • a far-field optical ultra-thin slice imaging method comprising the following steps:
  • Step 1 The laser emits a single laser beam and enters the beam modulation module
  • Step 2 the beam modulation module modulates the incident single laser beam, and injects it into the rear aperture plane of the objective lens;
  • Step 3 The objective lens focuses the light beam incident on the rear aperture plane, and gathers it into the channel of the microfluidic chip to generate an ultra-thin light sheet;
  • Step 4 the microfluidic chip controls the flow of the fluorescent dye through the microfluidic chip control box; the microfluidic chip control box collects the fluorescence to the camera through the narrow band filter and the imaging lens.
  • step 2 the incident single laser beam is modulated, including the following steps:
  • Step 201 the spatial light filter, the first aperture diaphragm and the single lens sequentially filter, expand and collimate the single laser beam emitted by the laser and modulate it into a linearly polarized beam with a fundamental transverse mode, which is incident on the polarizer device;
  • Step 202 the polarizer modulates the polarization direction of the linearly polarized light beam to be consistent with the working direction of the liquid crystal panel of the phase-only reflective liquid crystal spatial light modulator, and enters the beam splitter;
  • Step 203 the beam splitter divides the linearly polarized light beam passing through the polarizer into two, and after passing through the second aperture diaphragm, the beam is vertically incident on the liquid crystal panel of the pure-phase reflective liquid crystal spatial light modulator, and the phase is modulated into a shell. Searle beam array (light sheet), and reflected to a quarter wave plate;
  • Step 204 the quarter-wave plate converts the Bessel beam array into circularly polarized light, and then the circularly polarized light enters the rear aperture plane of the objective lens through the first mirror and the second mirror.
  • step three an ultra-thin light sheet is generated inside the channel of the microfluidic chip, including the following steps:
  • FIG. 1 it is a schematic diagram of the phase distribution at the rear aperture of the objective lens.
  • the rear aperture plane of the objective lens is divided into P strip-shaped areas with a diameter R, and one of the sub-areas is shown in (100).
  • the spot is changed by adjusting the size of P uniformity;
  • FIG 2 it is a schematic diagram of one of the striped sub-regions in Figure 1, and each strip-shaped region is further divided into Q small strip-shaped regions, and one of the sub-regions is as shown in Figure (200);
  • Step 303 fill the phase distribution into the small strip area sequentially, and change the value of the light spot by adjusting the size of Q, the phase distribution is:
  • NA is the numerical aperture of the objective lens
  • ⁇ is the wavelength emitted by the laser
  • R is the radius of the rear aperture of the objective lens
  • n t is the refractive index of the objective lens
  • x', y' are the rectangular coordinates on the plane of the rear aperture of the objective lens
  • ⁇ x and ⁇ y are the position of the focus of the spot in the focal area of the objective lens
  • ⁇ is the base angle of the axicon
  • ⁇ (x', y') is drawn as a phase diagram of the gray scale transformation from 0 to 2 ⁇ ;
  • Step 304 by designing ⁇ x and ⁇ y, the Bessel-Gaussian light is closely arranged to form an evenly distributed ultra-thin light sheet.
  • FIG. 3 it is a schematic diagram of calculating the position coordinates of the focus of the focal plane of the objective lens.
  • the width, thickness and height of the sheet-shaped spots can be adjusted; where, 1-8 represents eight focus spots, in the inverted In the fluorescence microscope system, the width of the light sheet is 35 ⁇ m and the thickness of the light sheet is 0.54 ⁇ m, and the height of the light sheet is 24 ⁇ m obtained by simulation.
  • Figure 4(c) shows the full width at half maximum of the lateral width of the sheet-like spot, that is, the thickness of the light sheet (the objective lens uses 20X/NA0.5) is 0.85 ⁇ m.
  • the focal depth of the focused spot in the Z-axis direction is 24 ⁇ m.
  • Step 4 the running process of the microfluidic chip includes:
  • Step 401 introduce the solution loaded with cells into the microfluidic chip through the liquid inlet S02, the sheath liquid through the liquid inlet S01, and the liquid inlet S03, and adjust the liquid inlet S01, the liquid inlet S02, and the liquid inlet S03
  • the pressure makes the cells enter the microfluidic chip one by one, the microfluidic valve S05 remains open, and the microfluidic valve S06, microfluidic valve S07, and microfluidic valve S08 are closed;
  • Step 402 the liquid inlet S01, the liquid inlet S02, and the liquid inlet S03 are pressurized to drive the fluid, and carry a single cell through the detection electrode S09, causing a local electrical signal change, stimulating the microfluidic chip control box to send a control signal, and closing the microfluidic
  • the control valve S05 stops the fluid movement; the control signal activates the control program, the microfluidic valve S06, the microfluidic valve S07 and the microfluidic valve S08 switch on and off, and pass (100,110,010,011,001) (1 means that the control valve is open and the liquid can flow in; 0 Indicates that the control valve is closed and the liquid cannot flow in) the control logic sequence controls the switch of the microfluidic valve, drives the liquid to flow, and generates a flow field.
  • the movement is driven forward in steps of 0.2 microns to 1.5 microns per cycle.
  • step four the imaging process on the camera includes:
  • Step 403 the cells are driven by the microfluidic valve S06, microfluidic valve S07, and microfluidic valve S08, and move steppingly to the detection electrode S10.
  • the detection electrode S10 detects the electrical signal generated by the cell
  • the microfluidic The chip control box sends a trigger signal to the camera, and the camera starts continuous imaging;
  • Step 404 the cells are driven by the microfluidic valve S06, microfluidic valve S07, and microfluidic valve S08, and move steppingly to the detection electrode S13.
  • the detection electrode S13 detects the electrical signal generated by the cell
  • the microfluidic The chip control box sends a trigger signal to the camera, and the camera stops shooting;
  • Step 405 when the detection electrode S09 does not detect a new cell passing signal, the microfluidic valve S06, microfluidic valve S07, and microfluidic valve S08 are closed, the microfluidic valve S05 is opened, and the carrying cells leave the microfluidic chip , the microfluidic chip maintains this state until it stops running;
  • Step 406 when the detection electrode S09 has detected a new cell passing signal, the microfluidic valve S06 , the microfluidic valve S07 , and the microfluidic valve S08 continue to circulate until they stop running.
  • FIG. 6 it is a schematic structural diagram of the middle and far field optical ultra-thin slice imaging system of the present invention, including: laser 1, beam modulation module K2, objective lens 12, microfluidic chip control box 13, microfluidic chip 14, narrowband filter Sheet 15, imaging lens 16 and camera 17;
  • the laser 1 emits a single laser beam and enters the beam modulation module K2; the beam modulation module K2 modulates the incident single laser beam, and enters the rear aperture plane of the objective lens 12; the objective lens 12 focuses the beam incident on the rear aperture plane, Converging to the inside of the channel of the microfluidic chip 14 to generate an ultra-thin light sheet; the microfluidic chip 14 controls the flow of fluorescently stained cells through the microfluidic chip control box 13; the fluorescent light emitted by the cells then passes through the narrowband filter 15 and imaging A lens 16 collects the fluorescence onto a camera 17 .
  • the beam modulation module K2 includes: a spatial light filter 2, a first aperture stop 3, a single lens 4, a polarizer 5, a beam splitter 6, a second aperture stop 7, a phase-only reflective liquid crystal Spatial light modulator 8, quarter wave plate 9, first mirror 10, second mirror 11;
  • the spatial light filter 2, the first aperture stop 3 and the single lens 4 sequentially perform filtering, beam expansion and collimation operations on the single laser beam emitted by the laser 1 and modulate it into a linearly polarized beam with a fundamental transverse mode.
  • the linearly polarized light beam of the polarizer 5 is divided into two parts and passes through the second aperture stop 7, then is vertically incident on the liquid crystal panel of the pure-phase reflective liquid crystal spatial light modulator 8 and phase-modulated into a Bessel beam, and Reflected onto the quarter-wave plate 9; the quarter-wave plate 9 converts the Bessel beam into circularly polarized light, and then the circularly polarized light is incident on the objective lens 12 through the first reflector 10 and the second reflector 11
  • FIG. 7 it is a schematic structural diagram of a microfluidic chip, including three three-dimensional surfaces carrying functional structures: three-dimensional surface 1, three-dimensional surface 2, and three-dimensional surface 3;
  • the three-dimensional surface 1 includes: liquid inlet S01, liquid inlet S02, liquid inlet S03, liquid inlet S04, microfluidic valve S05, microfluidic valve S06, microfluidic valve S07, microfluidic valve controlled by elastic film Fluid control valve S08; liquid inlet S01 and liquid inlet S03 pass the sheath fluid into the pipeline, liquid inlet S02 passes the solution loaded with cells into the pipeline, and liquid inlet S04 supplies liquid for the chip peristaltic pump; microfluidic valve S05 controls the cells and sheath fluid to enter the microfluidic chip 14, and the microfluidic valve S06, microfluidic valve S07, and microfluidic valve S08 jointly control the peristaltic pump to drive the liquid forward in a stepwise manner;
  • the three-dimensional surface 2 includes: the detection electrode S09, the detection electrode S10, the detection electrode S13 and the ultra-thin chip excitation area S11; the detection electrode S09, the detection electrode S10 and the detection electrode S13 detect whether the liquid flows in; the ultra-thin chip excitation area S11 excites the cells to emit fluorescence ;
  • the three-dimensional surface 3 includes: a liquid outlet S12, which outputs the detected cells.
  • Figure 8 shows the phase map loaded on the spatial light modulator, and the drawn phase map is saved in PNG format with 1080 ⁇ 1080 pixels.
  • Figure 9(a)-(b) the experimental results of 25 fluorescent spots with a distance of 1.2 ⁇ m between pairs are shown.
  • Figure 9(b) is the mesh image of Figure 9(a), which is obtained by simulating different lenses according to the numerical simulation results. Height, the thickness of the light sheet generated by the objective lens (10 ⁇ /NA0.2) is 1.8 ⁇ m, the width is 8 ⁇ m, and the height is 25 ⁇ m.
  • Figure 10(a)-(b) the experimental results of 151 fluorescent spots with a distance of 0.32 ⁇ m between pairs are shown.
  • Figure 10(b) is the mesh image of Figure 10(a), which is obtained by simulating different lenses according to the numerical simulation results. Height, the thickness of the light sheet generated by the objective lens (63 ⁇ /NA1.4) is 0.54 ⁇ m, the width is 35 ⁇ m, and the height is 6 ⁇ m. It should be emphasized that the width of the light sheet can be adjusted arbitrarily within 50 ⁇ m by the number of light spots.
  • the fluorescent dye used in the experiment is CY5 dye with an excitation peak at 651nm and an emission peak at 670nm.
  • Step 1 Turn on the power of the laser, spatial light modulator, and microfluidic chip control box, and load the phase map through the spatial light modulator to modulate an ultra-thin flake spot;
  • Step 2 Fix the cube chip on the light sheet excitation area and imaging platform, and connect the chip control device;
  • Step 3 Connect the 3T3 fibroblasts transfected with H2B-GFP histones to the liquid inlet S02 at a concentration of 10,000/ml, and at the same time connect the culture medium as the sheath fluid to the liquid inlet S01 and the liquid inlet S03;
  • Step 4 The cell solution is pressurized by 1psi, and the sheath liquid inlet is pressurized by 1.5psi, and the cells are input into the microfluidic chip.
  • Step 5 The system opens the microfluidic valve S05 according to the set program, and when the first cell passes the detection electrode S09, the control program is activated to perform single-cell chromosome three-dimensional structure imaging and three-dimensional reconstruction.
  • Traditional light sheet imaging technology usually generates light sheets from the side of the imaging lens.
  • a specially designed lens is often used, and the thickness of the light sheet is generally on the order of microns, and the depth of focus
  • the resolution of the direction is still low; the movement of biological samples requires the use of mechanical or piezoelectric translation stages, and the moving speed is slow, resulting in low layer-by-layer imaging speed, and 3D imaging takes a long time, which is not suitable for biomedical testing .
  • a single laser beam is modulated into a Bessel spot array to form an ultra-thin light sheet with a large thickness, width and height through spatial light modulation technology and phase division technology, combined with microfluidic chip technology, the channel Rapid, high-resolution, high-throughput slice imaging of cells and other biological samples that continuously pass through the light sheet can be applied to far-field and 3D structural imaging of biological samples and drug molecules in the field of biomedicine, serving biological Physical and biomedical basic research and clinical application fields such as cell pharmacology and early screening of cancer cells.

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Abstract

一种远场光学超薄片层成像系统及方法，涉及光学及生物医学设备技术领域，成像系统包括：激光器（1）、光束调制模块（K2）、物镜（12）、微流控芯片控制盒（13）、微流控芯片（14）、窄带滤波片（15）、成像透镜（16）和成像装置；光束调制模块（K2）对激光器（1）出射的单一激光光束进行调制后入射至物镜（12）的后孔径平面，然后汇聚到微流控芯片（14）的通道内部产生超薄光片；微流控芯片（14）通过微流控芯片控制盒（13）控制染有荧光的细胞样本的流动；细胞发出的荧光通过窄带滤波片（15）和成像透镜（16）将荧光收集到成像装置上。物镜（12）聚焦区域内的每一个贝塞尔光斑都可以被独立控制，当聚焦光斑相互靠近时干涉影响较小，可对荧光样本进行连续、高分辨率、高通量的片层成像。

Description

一种远场光学超薄片层成像系统及方法 技术领域
本发明涉及光学及生物医学设备技术领域，更具体的说是涉及一种远场光学超薄片层成像系统及方法。
背景技术
生物样本的3D结构直接决定其生物力学性能以及在生命现象中发挥的功能，对生物样本的3D结构进行快速、高分辨率的测量是目前生物医学和光学技术领域研究的前沿和热点。目前，能够对生物样本的3D结构进行高分辨率测量光学技术主要包括：共聚焦显微镜(Confocal Microscope)、受激辐射损耗显微镜(Stimulated Emission Depletion Microscope)、饱和结构光显微镜、(Saturated Structured Illumination Microscopy)、随机定位成像显微镜、光片成像技术等。
当使用扫描显微镜进行生物成像时，如采用共聚焦显微镜和受激辐射超分辨显微镜在对细胞进行生物成像时，通常是逐行和逐列扫描生物细胞，由于激光器通过显微物镜汇聚的荧光光斑通常只有一个荧光光斑，因此逐行和逐列扫描生物细胞的扫描时间较长，成像速度较慢，对于活体细胞成像和耐漂白性较差的生物细胞来说，这是非常不利的。另外，由于焦深(depth of focus)的影响，无论共聚焦显微镜、受激辐射损耗显微镜、饱和结构光显微镜还是随机定位成像显微镜，这些技术在沿光轴和成像方向的空间分辨率都较低，导致对生物样本结构在光轴方向的识别能力较低。
为了解决因焦深导致的空间分辨率低的问题并提高3D成像速度，光片成像技术应运而生。通过移动样本等方式，结合相机对每层光片照射区域的荧 光分布进行拍摄，可以得到在成像焦深方向上具有较高分辨率的生物图像，进而可以通过3D重构获得高分辨率的生物结构。然而，该技术通常从成像镜头的侧向生成光片，为兼容生成薄光片并保持长工作距离，往往要使用特殊设计的镜头，且其光片的厚度一般为微米级，焦深方向的分辨率依然较低。此外，对生物样本的移动需要使用机械或压电平移台，移动速度较慢，进而造成逐层成像速度较低，3D成像需要较长时间，不适用于生物医药检测中。因此，如何对细胞等生物样本和药物分子的3D荧光分布进行连续、高分辨率、高通量的片层成像和3D重构分析是本领域技术人员亟需解决的问题。
发明内容
有鉴于此，本发明提供了一种远场光学超薄片层成像系统及方法，可以对细胞等生物样本和药物分子的3D荧光分布进行连续、高分辨率、高通量的片层成像和3D重构分析。
为了实现上述目的，本发明提供如下技术方案：
一种远场光学超薄片层成像系统，包括：激光器、光束调制模块、物镜、微流控芯片控制盒、微流控芯片、窄带滤波片、成像透镜和成像装置；
所述激光器出射单一激光光束并入射至所述光束调制模块；所述光束调制模块对入射的所述单一激光光束进行调制，并入射至所述物镜的后孔径平面；所述物镜对入射至后孔径平面的光束进行聚焦，汇聚到所述微流控芯片的通道内部产生超薄光片；所述微流控芯片通过所述微流控芯片控制盒控制荧光染料的流动；所述微流控芯片控制盒控制染有荧光的细胞样本的流动，细胞发出的荧光通过所述窄带滤波片和所述成像透镜将荧光收集到所述成像装置上。
优选的，所述光束调制模块包括：空间光滤波器、第一小孔光阑、单透镜、起偏器、分束镜、第二小孔光阑、纯相位型反射式液晶空间光调制器、四分之一波片、第一反射镜、第二反射镜；
所述空间光滤波器、第一小孔光阑和单透镜对所述激光器出射的单一激光光束依次进行滤波、扩束和准直操作并调制为具有基横模的线偏振光束，入射至所述起偏器；
所述起偏器将所述线偏振光束的偏振方向调制为与所述纯相位型反射式液晶空间光调制器的液晶板工作方向一致，并入射至所述分束镜；
所述分束镜将所述通过起偏器的线偏振光束一分为二并通过所述第二小孔光阑后，垂直入射到所述纯相位型反射式液晶空间光调制器的液晶板上相位调制为贝塞尔光束阵列(光片)，并反射至所述四分之一波片上；
所述四分之一波片将所述贝塞尔光束阵列转成圆偏振光，然后经所述第一反射镜、第二反射镜将所述圆偏振光入射至所述物镜的后孔径平面。
优选的，所述微流控芯片包括三个载有功能结构的立体面：立体面1、立体面2、立体面3；
其中，所述立体面1包括：进液口S01、进液口S02、进液口S03、进液口S04，弹性薄膜控制的微流控阀S05、微流控阀S06、微流控阀S07、微流控阀S08；所述进液口S01和所述进液口S03将鞘液通入管道，所述进液口S02将载有细胞的溶液通入管道，所述进液口S04为芯片蠕动泵供液；所述微流控阀S05控制细胞和鞘液进入所述微流控芯片，所述微流控阀S06、微流控阀S07、微流控阀S08联动控制蠕动泵驱动液体以步进方式前行；
所述立体面2包括：检测电极S09、检测电极S10、检测电极S13和超薄芯片激发区域S11；所述检测电极S09、检测电极S10和检测电极S13检测液体是否流入；所述超薄芯片激发区域S11激发细胞发射荧光；
所述立体面3包括：出液口S12，输出检测后的细胞。
一种远场光学超薄片层成像方法，其特征在于，包括以下步骤：
步骤一、激光器出射单一激光光束，并入射至光束调制模块；
步骤二、光束调制模块对入射的单一激光光束进行调制，并入射至物镜的后孔径平面；
步骤三、物镜对入射至后孔径平面的光束进行聚焦，汇聚到微流控芯片的通道内部产生超薄光片；
步骤四、微流控芯片通过微流控芯片控制盒控制染有荧光的细胞样本的流动；细胞发出的荧光通过窄带滤波片和成像透镜将荧光收集到成像装置上。
优选的，所述步骤二中，对入射的单一激光光束进行调制，包括以下步骤：
步骤201、空间光滤波器、第一小孔光阑和单透镜对激光器出射的单一激光光束依次进行滤波、扩束和准直操作并调制为具有基横模的线偏振光束，入射至起偏器；
步骤202、起偏器将线偏振光束的偏振方向调制为与纯相位型反射式液晶空间光调制器的液晶板工作方向一致，并入射至分束镜；
步骤203、分束镜将通过起偏器的线偏振光束一分为二并通过第二小孔光阑后，垂直入射到纯相位型反射式液晶空间光调制器的液晶板上相位调制为贝塞尔光束阵列(光片)，并反射至四分之一波片上；
步骤204、四分之一波片将贝塞尔光束转成圆偏振光，然后经第一反射镜、第二反射镜将圆偏振光入射至物镜的后孔径平面。
优选的，所述步骤三中，在微流控芯片的通道内部产生超薄光片，包括以下步骤：
步骤301、将物镜的后孔径平面以直径R等分成P个条状区域，通过调整P的大小改变光斑的均匀度；
步骤302、将每个条状区域进一步划分Q个小条状区域；
步骤303、将相位分布依次填入小条状区域，通过调整Q的大小改变光斑的数值，相位分布为：
Figure PCTCN2022114392-appb-000001
其中：NA为物镜的数值孔径，λ为激光器出射的波长，R为物镜后孔径的半径，n t为物镜的折射率，x'、y'为物镜后孔径平面上的直角坐标，Δx和Δy分别为物镜聚焦区域光斑焦点的位置，α为轴棱锥底角，将φ(x',y')绘制为灰度变换从0到2π的相位图；
步骤304、通过设计Δx和Δy，使贝塞尔高斯光紧密排列形成均匀分布的超薄光片。
优选的，所述步骤四中，微流控芯片的运行过程包括：
步骤401、将载有细胞的溶液通过进液口S02、鞘液通过进液口S01、进液口S03同时导入微流控芯片中，调整进液口S01、进液口S02和进液口S03的压强，使细胞逐个进入微流控芯片，微流控阀S05保持打开状态，微流控阀S06、微流控阀S07、微流控阀S08关闭；
步骤402、进液口S01、进液口S02、进液口S03加压驱动流体，携带单个细胞通过检测电极S09，引起局部电信号变化，激发微流控芯片控制盒发出控制信号，关闭微流控阀S05，停止流体运动；控制信号激发控制程序，微流控阀S06、微流控阀S07和微流控阀S08进行开关，通过控制逻辑顺序控制微流控阀开关，驱动液体流动。
优选的，所述步骤四中，在成像装置上成像的过程包括：
步骤403、细胞由微流控阀S06、微流控阀S07、微流控阀S08驱动，步进运动至检测电极S10，检测电极S10检测到细胞经过所产生的电信号后，由微流控芯片控制盒发送触发信号给成像装置，成像装置开始连续成像；
步骤404、细胞由微流控阀S06、微流控阀S07、微流控阀S08驱动，步进运动至检测电极S13，检测电极S13检测到细胞经过所产生的电信号后，由微流控芯片控制盒发送触发信号给成像装置，成像装置停止拍摄；
步骤405、当检测电极S09没有检测到新的细胞通过信号时，微流控阀S06、微流控阀S07、微流控阀S08关闭，微流控阀S05打开，运载细胞离开微流控芯片，微流控芯片保持此状态，直至停止运行；
步骤406、当检测电极S09已经检测到新的细胞通过信号时，微流控阀S06、微流控阀S07、微流控阀S08持续循环，直至停止运行。
经由上述的技术方案可知，与现有技术相比，本发明公开提供了一种远场光学超薄片层成像系统及方法，通过空间光调制技术和相位分割技术，将单一激光光束调制为贝塞尔光斑阵列形成具有较大宽度和高度、超薄的光片，结合微流控芯片技术，对通道中持续通过光片的细胞等生物样本进行快速、高分辨率、高通量的片层成像，可以应用于生物医学领域中对生物样本和药物分子等的远场、3D结构成像，服务于生物物理、生物医学基础研究及细胞药理学、癌细胞早期筛查等临床应用领域。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1为一实施例的物镜后孔径处的相位分布示意图；
图2为一实施例的物镜后孔径相位分布其中一个条状子区域的示意图；
图3为一实施例的物镜焦平面焦点计算位置坐标计算示意图；
图4(a)-图4(c)为一实施例的物镜聚焦区域两焦点靠近仿真模拟结果图；
图5为一实施例的聚焦光斑Z轴方向的焦深(物镜使用20X/0.5)；
图6为本发明成像系统示意图；
图7为本发明微流控芯片整体结构示意图；
图8为本发明加载到纯相位型反射式液晶空间光调制器液晶板上的相位图；
图9(a)-(b)为一实施例中25个聚焦光斑产生的片状光斑实验结果；
图10(a)-(b)为另一实施例中151个聚焦光斑产生的片状光斑实验结果。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
一种远场光学超薄片层成像方法，包括以下步骤：
步骤一、激光器出射单一激光光束，并入射至光束调制模块；
步骤二、光束调制模块对入射的单一激光光束进行调制，并入射至物镜的后孔径平面；
步骤三、物镜对入射至后孔径平面的光束进行聚焦，汇聚到微流控芯片的通道内部产生超薄光片；
步骤四、微流控芯片通过微流控芯片控制盒控制荧光染料的流动；微流控芯片控制盒通过窄带滤波片和成像透镜将荧光收集到相机上。
进一步地，步骤二中，对入射的单一激光光束进行调制，包括以下步骤：
步骤201、空间光滤波器、第一小孔光阑和单透镜对激光器出射的单一激光光束依次进行滤波、扩束和准直操作并调制为具有基横模的线偏振光束，入射至起偏器；
步骤202、起偏器将线偏振光束的偏振方向调制为与纯相位型反射式液晶空间光调制器的液晶板工作方向一致，并入射至分束镜；
步骤203、分束镜将通过起偏器的线偏振光束一分为二并通过第二小孔光阑后，垂直入射到纯相位型反射式液晶空间光调制器的液晶板上相位调制为贝塞尔光束阵列(光片)，并反射至四分之一波片上；
步骤204、四分之一波片将贝塞尔光束阵列转成圆偏振光，然后经第一反射镜、第二反射镜将圆偏振光入射至物镜的后孔径平面。
进一步地，步骤三中，在微流控芯片的通道内部产生超薄光片，包括以下步骤：
如图1所示为物镜后孔径处的相位分布示意图，将物镜的后孔径平面以直径R等分成P个条状区域，其中的一个子区域如图(100)，通过调整P的大小改变光斑的均匀度；
如图2所示为图1的其中一个条状子区域的示意图，将每个条状区域进一步划分Q个小条状区域，其中的一个子区域如图(200)；
步骤303、将相位分布依次填入小条状区域，通过调整Q的大小改变光斑的数值，相位分布为：
Figure PCTCN2022114392-appb-000002
其中：NA为物镜的数值孔径，λ为激光器出射的波长，R为物镜后孔径的半径，n t为物镜的折射率，x'、y'为物镜后孔径平面上的直角坐标，Δx和 Δy分别为物镜聚焦区域光斑焦点的位置，α为轴棱锥底角，将φ(x',y')绘制为灰度变换从0到2π的相位图；
步骤304、通过设计Δx和Δy，使贝塞尔高斯光紧密排列形成均匀分布的超薄光片。
如图3所示为物镜焦平面焦点计算位置坐标计算示意图，通过调节多焦点光斑的数目和分布可调节片状光斑的宽度、厚度和高度；其中，1-8表示八个聚焦光斑，在倒置荧光显微系统中得到光片宽度35μm和光片厚度0.54μm，模拟得到光片高度24μm。
如图4(a)-图4(c)所示为物镜聚焦区域两焦点靠近仿真模拟结果图，图4(a)和图4(b)以两个光斑为例，显示的是通过调节光斑的间距将光斑逐渐融合的过程，图4(c)是片状光斑的横向宽度的半高全宽，即光片厚度(物镜使用20X/NA0.5)为0.85μm。
如图5所示为聚焦光斑Z轴方向的焦深(物镜使用20X/0.5)为24μm。
进一步地，步骤四中，微流控芯片的运行过程包括：
步骤401、将载有细胞的溶液通过进液口S02、鞘液通过进液口S01、进液口S03同时导入微流控芯片中，调整进液口S01、进液口S02和进液口S03的压强，使细胞逐个进入微流控芯片，微流控阀S05保持打开状态，微流控阀S06、微流控阀S07、微流控阀S08关闭；
步骤402、进液口S01、进液口S02、进液口S03加压驱动流体，携带单个细胞通过检测电极S09，引起局部电信号变化，激发微流控芯片控制盒发出控制信号，关闭微流控阀S05，停止流体运动；控制信号激发控制程序，微流控阀S06、微流控阀S07和微流控阀S08进行开关，通过(100,110,010,011,001)(1表示控制阀打开，液体可以流入；0表示控制阀关闭，液体不能流入)的控制逻辑顺序控制微流阀开关，驱动液体流动，产生流场，经测量细胞在尺 度为10微米×10微米到20微米×20微米的微流控芯片的通道中，以每循环0.2微米到1.5微米的步长向前驱动移动。
进一步地，步骤四中，在相机上成像的过程包括：
步骤403、细胞由微流控阀S06、微流控阀S07、微流控阀S08驱动，步进运动至检测电极S10，检测电极S10检测到细胞经过所产生的电信号后，由微流控芯片控制盒发送触发信号给相机，相机开始连续成像；
步骤404、细胞由微流控阀S06、微流控阀S07、微流控阀S08驱动，步进运动至检测电极S13，检测电极S13检测到细胞经过所产生的电信号后，由微流控芯片控制盒发送触发信号给相机，相机停止拍摄；
步骤405、当检测电极S09没有检测到新的细胞通过信号时，微流控阀S06、微流控阀S07、微流控阀S08关闭，微流控阀S05打开，运载细胞离开微流控芯片，微流控芯片保持此状态，直至停止运行；
步骤406、当检测电极S09已经检测到新的细胞通过信号时，微流控阀S06、微流控阀S07、微流控阀S08持续循环，直至停止运行。
实施例2
如图6所示为本发明中远场光学超薄片层成像系统的结构示意图，包括：激光器1、光束调制模块K2、物镜12、微流控芯片控制盒13、微流控芯片14、窄带滤波片15、成像透镜16和相机17；
激光器1出射单一激光光束并入射至光束调制模块K2；光束调制模块K2对入射的单一激光光束进行调制，并入射至物镜12的后孔径平面；物镜12对入射至后孔径平面的光束进行聚焦，汇聚到微流控芯片14的通道内部产生超薄光片；微流控芯片14通过微流控芯片控制盒13控制染有荧光的细胞的流动；细胞发出的荧光然后通过窄带滤波片15和成像透镜16将荧光收集到相机17上。
其中，光束调制模块K2包括：空间光滤波器2、第一小孔光阑3、单透镜4、起偏器5、分束镜6、第二小孔光阑7、纯相位型反射式液晶空间光调制器8、四分之一波片9、第一反射镜10、第二反射镜11；
空间光滤波器2、第一小孔光阑3和单透镜4对激光器1出射的单一激光光束依次进行滤波、扩束和准直操作并调制为具有基横模的线偏振光束，入射至起偏器5；起偏器5将线偏振光束的偏振方向调制为与纯相位型反射式液晶空间光调制器8的液晶板工作方向一致，并入射至分束镜6；分束镜6将通过起偏器5的线偏振光束一分为二并通过第二小孔光阑7后，垂直入射到纯相位型反射式液晶空间光调制器8的液晶板上相位调制为贝塞尔光束，并反射至四分之一波片9上；四分之一波片9将贝塞尔光束转成圆偏振光，然后经第一反射镜10、第二反射镜11将圆偏振光入射至物镜12的后孔径平面。
如图7所示为微流控芯片的结构示意图，包括三个载有功能结构的立体面：立体面1、立体面2、立体面3；
其中，立体面1包括：进液口S01、进液口S02、进液口S03、进液口S04，弹性薄膜控制的微流控阀S05、微流控阀S06、微流控阀S07、微流控阀S08；进液口S01和进液口S03将鞘液通入管道，进液口S02将载有细胞的溶液通入管道，进液口S04为芯片蠕动泵供液；微流控阀S05控制细胞和鞘液进入微流控芯片14，微流控阀S06、微流控阀S07、微流控阀S08联动控制蠕动泵驱动液体以步进方式前行；
立体面2包括：检测电极S09、检测电极S10、检测电极S13和超薄芯片激发区域S11；检测电极S09、检测电极S10和检测电极S13检测液体是否流入；超薄芯片激发区域S11激发细胞发射荧光；
立体面3包括：出液口S12，输出检测后的细胞。
如图8所示为加载到空间光调制器上的相位图，绘制的相位图存成1080×1080像素PNG格式。
如图9(a)-(b)所示是25个荧光光斑两两间距为1.2μm的实验结果，图9(b)是图9(a)的mesh图，根据数值模拟结果模拟不同镜头得到高度，物镜(10×/NA0.2)生成光片的厚度为1.8μm，宽度为8μm，高度为25μm。
如图10(a)-(b)所示是151个荧光光斑两两间距为0.32μm的实验结果，图10(b)是图10(a)的mesh图，根据数值模拟结果模拟不同镜头得到高度，物镜(63×/NA1.4)生成光片的厚度为0.54μm，宽度为35μm，高度为6μm。需强调的是，光片宽度可通过光斑数量在50μm内进行任意调节。
实验中用的荧光染料是CY5染料，激发峰值在651nm，发射峰值在670nm。
更进一步的本发明的其中一种具体实施例包括以下步骤：
步骤1：将激光器、空间光调制器、微流控芯片控制盒电源打开，通过空间光调制器加载相位图调制出超薄片状光斑；
步骤2：将立方体芯片固定于光片激发区域和成像平台，连接芯片控制装置；
步骤3：将转染H2B-GFP组蛋白的3T3成纤维细胞以10000个/毫升的浓度连接到进液口S02，同时将培养液作为鞘液连接到进液口S01、进液口S03；
步骤4：细胞溶液加压1psi，鞘液进液口加压1.5psi，将细胞输入到微流控芯片中。
步骤5：系统按照设定程序打开微流控阀S05，当第一个细胞通过检测电极S09时，激发控制程序进行单细胞染色体三维结构成像和三维重构。
传统的光片成像技术通常从成像镜头的侧向生成光片，为兼容生成薄光片并保持长工作距离，往往要使用特殊设计的镜头，且其光片的厚度一般为微米级，焦深方向的分辨率依然较低；对生物样本的移动需要使用机械或压电平移台，移动速度较慢，进而造成逐层成像速度较低，3D成像需要较长时间，不适用于生物医药检测中。然而，本发明中通过空间光调制技术和相位 分割技术，将单一激光光束调制为贝塞尔光斑阵列形成具有较大厚度、宽度和高度的超薄光片，结合微流控芯片技术，对通道中持续通过光片的细胞等生物样本进行快速、高分辨率、高通量的片层成像，可以应用于生物医学领域中对生物样本和药物分子等的远场、3D结构成像，服务于生物物理、生物医学基础研究及细胞药理学、癌细胞早期筛查等临床应用领域。
本说明书中各个实施例采用递进的方式描述，每个实施例重点说明的都是与其他实施例的不同之处，各个实施例之间相同相似部分互相参见即可。对于实施例公开的系统而言，由于其与实施例公开的方法相对应，所以描述的比较简单，相关之处参见方法部分说明即可。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (8)

  1. 一种远场光学超薄片层成像系统，其特征在于，包括：激光器、光束调制模块、物镜、微流控芯片控制盒、微流控芯片、窄带滤波片、成像透镜和成像装置；
    所述激光器出射单一激光光束并入射至所述光束调制模块；所述光束调制模块对入射的所述单一激光光束进行调制，并入射至所述物镜的后孔径平面；所述物镜对入射至后孔径平面的光束进行聚焦，汇聚到所述微流控芯片的通道内部产生超薄光片；所述微流控芯片通过所述微流控芯片控制盒控制荧光染料的流动；所述微流控芯片控制盒通过所述窄带滤波片和所述成像透镜将荧光收集到所述成像装置上。
  2. 根据权利要求1所述的远场光学超薄片层成像系统，其特征在于，所述光束调制模块包括：空间光滤波器、第一小孔光阑、单透镜、起偏器、分束镜、第二小孔光阑、纯相位型反射式液晶空间光调制器、四分之一波片、第一反射镜、第二反射镜；
    所述空间光滤波器、第一小孔光阑和单透镜对所述激光器出射的单一激光光束依次进行滤波、扩束和准直操作并调制为具有基横模的线偏振光束，入射至所述起偏器；
    所述起偏器将所述线偏振光束的偏振方向调制为与所述纯相位型反射式液晶空间光调制器的液晶板工作方向一致，并入射至所述分束镜；
    所述分束镜将所述通过起偏器的线偏振光束一分为二并通过所述第二小孔光阑后，垂直入射到所述纯相位型反射式液晶空间光调制器的液晶板上相位调制为贝塞尔光束阵列，并反射至所述四分之一波片上；
    所述四分之一波片将所述贝塞尔光束阵列转成圆偏振光，然后经所述第一反射镜、第二反射镜将所述圆偏振光入射至所述物镜的后孔径平面。
  3. 根据权利要求1所述的远场光学超薄片层成像系统，其特征在于，所述微流控芯片包括三个载有功能结构的立体面：立体面1、立体面2、立体面3；
    其中，所述立体面1包括：进液口S01、进液口S02、进液口S03、进液口S04，弹性薄膜控制的微流控阀S05、微流控阀S06、微流控阀S07、微流控阀S08；所述进液口S01和所述进液口S03将鞘液通入管道，所述进液口S02将载有细胞的溶液通入管道，所述进液口S04为芯片蠕动泵供液；所述微流控阀S05控制细胞和鞘液进入所述微流控芯片，所述微流控阀S06、微流控阀S07、微流控阀S08联动控制蠕动泵驱动液体以步进方式前行；
    所述立体面2包括：检测电极S09、检测电极S10、检测电极S13和超薄芯片激发区域S11；所述检测电极S09、检测电极S10和检测电极S13检测液体是否流入；所述超薄芯片激发区域S11激发细胞发射荧光；
    所述立体面3包括：出液口S12，输出检测后的细胞。
  4. 一种远场光学超薄片层成像方法，其特征在于，包括以下步骤：
    步骤一、激光器出射单一激光光束，并入射至光束调制模块；
    步骤二、光束调制模块对入射的单一激光光束进行调制，并入射至物镜的后孔径平面；
    步骤三、物镜对入射至后孔径平面的光束进行聚焦，汇聚到微流控芯片的通道内部产生超薄光片；
    步骤四、微流控芯片通过微流控芯片控制盒控制染有荧光的细胞样本的流动；细胞发出的荧光通过窄带滤波片和成像透镜将荧光收集到成像装置上。
  5. 根据权利要求4所述的远场光学超薄片层成像方法，其特征在于，所述步骤二中，对入射的单一激光光束进行调制，包括以下步骤：
    步骤201、空间光滤波器、第一小孔光阑和单透镜对激光器出射的单一激光光束依次进行滤波、扩束和准直操作并调制为具有基横模的线偏振光束，入射至起偏器；
    步骤202、起偏器将线偏振光束的偏振方向调制为与纯相位型反射式液晶空间光调制器的液晶板工作方向一致，并入射至分束镜；
    步骤203、分束镜将通过起偏器的线偏振光束一分为二并通过第二小孔光阑后，垂直入射到纯相位型反射式液晶空间光调制器的液晶板上相位调制为贝塞尔光束阵列，并反射至四分之一波片上；
    步骤204、四分之一波片将贝塞尔光束阵列转成圆偏振光，然后经第一反射镜、第二反射镜将圆偏振光入射至物镜的后孔径平面。
  6. 根据权利要求4所述的远场光学超薄片层成像方法，其特征在于，所述步骤三中，在微流控芯片的通道内部产生超薄光片，包括以下步骤：
    步骤301、将物镜的后孔径平面以直径R等分成P个条状区域，通过调整P的大小改变光斑的均匀度；
    步骤302、将每个条状区域进一步划分Q个小条状区域；
    步骤303、将相位分布依次填入小条状区域，通过调整Q的大小改变光斑的数值，相位分布为：
    Figure PCTCN2022114392-appb-100001
    其中：NA为物镜的数值孔径，λ为激光器出射的波长，R为物镜后孔径的半径，n t为物镜的折射率，x'、y'为物镜后孔径平面上的直角坐标，Δx和Δy分别为物镜聚焦区域光斑焦点的位置，α为轴棱锥底角，将φ(x',y')绘制为灰度变换从0到2π的相位图；
    步骤304、通过设计Δx和Δy，使贝塞尔高斯光紧密排列形成均匀分布的超薄光片。
  7. 根据权利要求4所述的远场光学超薄片层成像方法，其特征在于，所述步骤四中，微流控芯片的运行过程包括：
    步骤401、将载有细胞的溶液通过进液口S02、鞘液通过进液口S01、进液口S03同时导入微流控芯片中，调整进液口S01、进液口S02和进液口S03的压强，使细胞逐个进入微流控芯片，微流控阀S05保持打开状态，微流控阀S06、微流控阀S07、微流控阀S08关闭；
    步骤402、进液口S01、进液口S02、进液口S03加压驱动流体，携带单个细胞通过检测电极S09，引起局部电信号变化，激发微流控芯片控制盒发出控制信号，关闭微流控阀S05，停止流体运动；控制信号激发控制程序，微流控阀S06、微流控阀S07和微流控阀S08进行开关，通过控制逻辑顺序控制微流控阀开关，驱动液体流动。
  8. 根据权利要求7所述的远场光学超薄片层成像方法，其特征在于，在所述步骤四中，在成像装置上成像的过程包括：
    步骤403、细胞由微流控阀S06、微流控阀S07、微流控阀S08驱动，步进运动至检测电极S10，检测电极S10检测到细胞经过所产生的电信号后，由微流控芯片控制盒发送触发信号给成像装置，成像装置开始连续成像；
    步骤404、细胞由微流控阀S06、微流控阀S07、微流控阀S08驱动，步进运动至检测电极S13，检测电极S13检测到细胞经过所产生的电信号后，由微流控芯片控制盒发送触发信号给成像装置，成像装置停止拍摄；
    步骤405、当检测电极S09没有检测到新的细胞通过信号时，微流控阀S06、微流控阀S07、微流控阀S08关闭，微流控阀S05打开，运载细胞离开微流控芯片，微流控芯片保持此状态，直至停止运行；
    步骤406、当检测电极S09已经检测到新的细胞通过信号时，微流控阀S06、微流控阀S07、微流控阀S08持续循环，直至停止运行。
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