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WO2021179127A1 - 超分辨成像系统与方法、生物样品识别系统与方法、核酸测序成像系统与方法及核酸识别系统与方法 - Google Patents

超分辨成像系统与方法、生物样品识别系统与方法、核酸测序成像系统与方法及核酸识别系统与方法 Download PDF

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Publication number
WO2021179127A1
WO2021179127A1 PCT/CN2020/078444 CN2020078444W WO2021179127A1 WO 2021179127 A1 WO2021179127 A1 WO 2021179127A1 CN 2020078444 W CN2020078444 W CN 2020078444W WO 2021179127 A1 WO2021179127 A1 WO 2021179127A1
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Prior art keywords
structured light
light
super
resolution imaging
phase
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PCT/CN2020/078444
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English (en)
French (fr)
Inventor
倪洁蕾
倪鸣
周藩
苏泽宇
冀珂
魏栋
沈梦哲
梁元庆
李美
徐讯
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BGI Shenzhen Co Ltd
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BGI Shenzhen Co Ltd
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Priority to PCT/CN2020/078444 priority Critical patent/WO2021179127A1/zh
Priority to CN202080062961.9A priority patent/CN114341622A/zh
Priority to EP20924340.1A priority patent/EP4024031A4/en
Priority to US17/769,428 priority patent/US12276809B2/en
Publication of WO2021179127A1 publication Critical patent/WO2021179127A1/zh
Anticipated expiration legal-status Critical
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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
    • G01N21/62—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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • 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
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G02B21/08—Condensers
    • G02B21/14—Condensers affording illumination for phase-contrast observation
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/28—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00 for polarising
    • G02B27/286—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00 for polarising for controlling or changing the state of polarisation, e.g. transforming one polarisation state into another
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/58—Optics for apodization or superresolution; Optical synthetic aperture systems
    • 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
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B2207/00—Coding scheme for general features or characteristics of optical elements and systems of subclass G02B, but not including elements and systems which would be classified in G02B6/00 and subgroups
    • G02B2207/113—Fluorescence

Definitions

  • the present invention relates to the imaging field, in particular to super-resolution imaging systems and methods in gene sequencing, biological sample identification systems and methods, nucleic acid sequencing imaging systems and methods, and nucleic acid identification systems and methods.
  • the existing super-resolution imaging technology usually has problems such as slow imaging, which restricts its application in high-throughput sequencing technology.
  • the present application provides a super-resolution imaging system.
  • the super-resolution imaging system includes an illumination system and an imaging system.
  • the illumination system includes an excitation light source and a structured light generation and modulation device
  • the excitation light source is used to output the excitation light
  • the structured light generation and modulation device is used
  • the structured light generation and modulation device includes a structured light control device
  • the structured light control device is used to control the structured light generation
  • a modulation device to change the phase of the structured light output by the structured light generation and modulation device and the direction of the illumination pattern projected on the biological sample by the structured light
  • the imaging system is used to match the structured light phase and /Or the change of the direction of the illumination pattern captures at least a laser-induced image excited by at least one wavelength of excitation light in the imaging field of view.
  • the structured light generating and modulating device further includes a polarization control system and a diffraction beam splitting device, the polarization control system is used to adjust the polarization direction of the excitation light, and the diffraction beam splitting device is used to divide the excitation light into Multiple beams to form the structured light having a specific phase and a specific illumination pattern direction, and the structured light control device is used for controlling the diffraction beam splitting device to change the phase of the structured light and the direction of the illumination pattern, and for controlling The polarization control system makes the illumination pattern of the structured light in any phase and any direction meet the preset requirements.
  • the structured light control device is used to control the polarization control system so that the contrast of the illumination pattern of the structured light meets a preset requirement.
  • the structured light control device is used to control the linear movement of the diffraction beam splitter to change the phase of the structured light, and control the rotation of the diffraction beam splitter to change the direction of the structured light illumination pattern.
  • the super-resolution imaging system further includes an objective lens
  • the structured light generating and modulating device further includes a focusing device for focusing the structured light emitted by the diffraction beam splitting device to the objective lens
  • the objective lens is used to emit the structured light onto the biological sample in parallel light, and form interference fringes on the plane where the biological sample is located at a certain angle.
  • the diffraction beam splitting device includes a phase grating
  • the polarization control system includes a polarizer
  • phase grating and the polarizer are set together on a one-dimensional mobile platform, the one-dimensional mobile platform is set on a turntable, and the structured light control device is used to control the one-dimensional mobile platform to drive the
  • the phase grating and the polarizer move to change the phase of the structured light, and are used to control the turntable to drive the phase grating and the polarizer to rotate to change the direction of the structured light illumination pattern.
  • the focusing device includes a first lens, a second lens, and a third lens.
  • a block is provided between the first lens and the second lens, and the block is used to block and prevent part of the structure. The light enters the subsequent light path.
  • the polarizer is used to convert the excitation light into linearly polarized light
  • the phase grating is used to convert the linearly polarized light into +1, -1, and 0-order diffracted light.
  • the first-order diffracted light is blocked by the baffle, and the +1-order diffracted light and the -1st-order diffracted light are focused to the back focal plane of the objective lens after passing through the second lens and the third lens.
  • the structured light generating and modulating device further includes an adaptive optics device, and the adaptive optics device is used to shape the wavefront of the structured light.
  • the structured light control device is also used to control the adaptive optics device to optimize the contrast and/or uniformity of the structured light.
  • the super-resolution imaging system further includes a master control device for controlling the excitation light source, the structured light generating and modulating device, and the imaging system to work in coordination.
  • this application also provides a super-resolution imaging method, including:
  • the structured light is in the first phase and the illumination pattern of the structured light is in the second direction, and a fourth image of the laser light is captured.
  • the structured light is controlled to be in different phases by linearly moving the diffraction beam splitting device of the illumination system, the lighting patterns of the structured light are controlled to be in different directions by rotating the diffraction beam splitting device, and the polarization control system is controlled to move or The rotation makes the illumination pattern of the structured light in each direction and each phase meet the preset requirements.
  • making the illumination pattern of the structured light meet the preset requirement includes making the contrast of the illumination pattern of the structure light meet the preset requirement.
  • the super-resolution imaging method further includes: switching the wavelength of the excitation light and repeating the steps of switching the phase and/or direction of the structured light and shooting the laser-received image.
  • the super-resolution imaging method further includes: after each step of switching the phase and/or direction of the structured light and shooting the lasered image is completed, it further includes: switching the wavelength of the excitation light and repeating the switching and shooting. step.
  • the super-resolution imaging method further includes: switching the imaging field of view of the super-resolution imaging system and repeating the steps of switching the structured light phase and/or direction and shooting the laser-received image.
  • the super-resolution imaging method further includes: after each step of switching the structured light phase and/or direction and shooting a laser image is completed, it further includes: switching the imaging field of view of the super-resolution imaging system and repeating the Steps for switching and shooting.
  • this application also provides a biological sample identification system, including:
  • An image reconstruction and biometric identification device for receiving the laser image output by the super-resolution imaging system, and performing image reconstruction based on multiple images of the laser excited by the excitation light of the same wavelength in the same imaging field of view to obtain the reconstructed image
  • the biological characteristics of the biological sample corresponding to the laser in the imaging field of view are identified according to the reconstructed super-resolution image.
  • this application also provides a biological sample identification method, including:
  • the present application also provides a nucleic acid sequencing imaging system.
  • the nucleic acid sequencing imaging system includes the above-mentioned super-resolution imaging system.
  • the super-resolution imaging system is used to illuminate a nucleic acid sample and photograph the laser light emitted by the nucleic acid sample. Image.
  • the present application also provides a nucleic acid sequencing imaging method, which adopts the above-mentioned super-resolution imaging method to excite a nucleic acid sample to emit laser light and take an image of the laser light.
  • the present application also provides a nucleic acid identification system, the nucleic acid identification system includes the above-mentioned biological sample identification system, and the biological sample identification system is used to identify the type of base of the nucleic acid sample.
  • the present application also provides a nucleic acid identification method that uses the above-mentioned biological sample identification method to identify the type of base in a nucleic acid sample.
  • the super-resolution imaging system and method, biological sample identification system and method, nucleic acid sequencing imaging system and method, and nucleic acid identification system and method provided by the embodiments of the present invention obtain multiple laser-received images by changing the direction and phase of the structured light output illumination pattern (For example, 4) images, the multiple images are used for image reconstruction to obtain super-resolution images, the layout and category of biological samples can be identified through the super-resolution images; when applied to nucleic acid sequencing, the layout and category of bases can be identified. Since the number of lasered images that need to be taken is small, the recognition speed of biological samples is improved; when applied to nucleic acid sequencing, the speed of base recognition can be improved.
  • FIG. 1 is a schematic diagram of the structure of a super-resolution imaging system provided by an embodiment of the present invention.
  • FIG. 2 is a schematic diagram of the structure of a biological sample identification system provided by an embodiment of the present invention.
  • FIG. 3 is a schematic flowchart of a super-resolution imaging method according to an embodiment of the present invention.
  • Fig. 4 is a schematic flowchart of a biological sample identification method according to an embodiment of the present invention.
  • FIG. 5 is a schematic diagram of a specific embodiment of the super-resolution imaging system shown in FIG. 1 of the present invention.
  • 6A-6I are schematic diagrams of interference fringes generated on a sample carrier by using structured light output from the super-resolution imaging system shown in FIG. 5 in a super-resolution imaging method.
  • 7A-7D are schematic diagrams of interference fringes generated on a sample carrier using structured light output from the super-resolution imaging system shown in FIG. 5 in another super-resolution imaging method.
  • 8A-8D are schematic diagrams of interference fringes generated on the sample carrier by using the structured light output from the super-resolution imaging system shown in FIG. 5 in the third super-resolution imaging method of the present invention.
  • Fig. 9 is a schematic diagram of a nucleic acid sequencing imaging system provided by an embodiment of the present invention.
  • Fig. 10 is a schematic diagram of a nucleic acid recognition system provided by an embodiment of the present invention.
  • Imaging system B Excitation light source 10, 10a
  • Diffraction beam splitter 112 Structured light control device 113, 113a
  • Dichroic mirror 121, 143, 12a Camera 141, 141a
  • Collimating lens 115a One-dimensional mobile platform 116
  • the super-resolution imaging system 1 includes an excitation light source 10, a structured light generating and modulating device 11, an optical path guiding device 12, an objective lens 13, and Signal acquisition device 14.
  • the excitation light source 10, the structured light generation and modulation device 11, the optical path guiding device 12, and the objective lens 13 constitute the illumination system A of the super-resolution imaging system 1
  • the objective lens 13, the optical path guiding device 12 and the signal acquisition device 14 constitute the super-resolution imaging system 1.
  • the excitation light source 10 outputs excitation light, which is modulated into structured light with a specific illumination pattern direction and phase after being modulated by the structured light generating and modulating device 11, and the structured light is guided to the objective lens 13 via the optical path guiding device 12 , It is projected via the objective lens 13 onto the sample carrier 2 loaded with the processed biological sample.
  • the biological sample can be nucleic acid (DNA and RNA), protein, or cell.
  • the sample carrier 2 is placed on the sample platform 3.
  • the sample platform 3 is a movable platform for moving the sample carrier 2 so that the biological samples at different positions on the sample carrier 2 can be irradiated by excitation light in sequence. In other embodiments, the sample platform 3 may not be used.
  • the excitation light excites the marker on the biological sample to generate laser light (for example, excited fluorescence), which is collected by the objective lens 13, and then guided to the signal collection device 14 via the optical path guide device 12, and the signal collection device 14 Record and generate the laser image.
  • the objective lens 13 collects the received laser light on the one hand, and assists in generating the structured light projected on the plane of the biological sample on the other hand.
  • the objective lens 13 may be a low-magnification wide-field objective lens.
  • the excitation light source 10 is a single-color or multi-color laser light source.
  • the excitation light source may be a single-color, two-color or four-color laser to excite the biological characteristics (such as bases) of the biological sample. Fluorescent markers on.
  • the structured light generation and modulation device 11 includes a polarization control system 111, a diffraction beam splitting device 112, a structured light control device 113, and a focusing device 114.
  • the polarization control system 111 is used to adjust the polarization direction of the excitation light to make the illumination pattern of the structured light irradiated on the biological sample meet the preset requirements, for example, make the contrast of the illumination pattern of the structured light meet the requirements, or make the structure The contrast of the light illumination pattern is the highest.
  • the polarization control system 111 may include optical elements such as a polarizer, a variable phase retarder, or a wave plate.
  • the diffraction beam splitting device 112 is used to split the excitation light into multiple beams.
  • the diffraction beam splitting device 112 may include a beam splitter, a grating, a spatial light modulator, a digital micromirror device, or an optical fiber.
  • the structured light control device 113 is used to control the polarization control system 111 and the diffraction beam splitting device 112. By controlling the diffraction beam splitting device 112, the direction and phase of the illumination pattern of the structured light generated are controlled, and at the same time, by controlling The polarization control system 111 makes the pattern of structured light in each direction and each phase meet the preset requirements.
  • the structured light control device 113 controls the polarization control system 111 and the diffraction beam splitting device 112 to change the polarization direction of the structured light to follow the direction of the structured light.
  • the structured light control device 113 changes according to the received Control the polarization control system 111 to make the illumination pattern of the structured light meet the preset requirements.
  • the external command indicating that the structured light illumination pattern meets the preset contrast requirement may be a command manually input by the user, or an external device (not shown) automatically judges and outputs a command according to a preset condition.
  • the focusing device 114 is used to focus the excitation light split into the multiple beams of the diffraction beam splitter 112 to the objective lens 13, and emit the parallel light through the objective lens 13.
  • the focusing device 114 is a lens group, and the excitation light is The group focuses on the back focal plane of the objective lens 13, and emits parallel light through the objective lens 13, and forms interference fringes on the plane where the biological sample is located at a certain angle.
  • the structured light generating and modulating device 11 may also include an adaptive optics device 115 for shaping the wavefront of the structured light, and the structured light control device 113 may also be used for controlling the adaptive optics.
  • the optical device 115 is used to optimize the contrast and/or uniformity of the structured light, or to keep the contrast and/or uniformity of the structured light optimal in the imaging field of view.
  • the optical path guiding device 12 includes a dichroic mirror 121, and the dichroic mirror 121 guides the structured light output by the structured light generating and modulating device 11 to the objective lens 13 on the one hand, and the objective lens on the other hand. 13 The collected laser light is guided to the signal collection device 14.
  • the signal acquisition device 14 includes a plurality of cameras 141, a plurality of tube lenses 142, and one or more dichroic mirrors 143. Guide to a different camera 141.
  • the multiple cameras 141, multiple tube lenses 142, and one or more dichroic mirrors 143 constitute multiple detection light paths, of which only one detection light path is shown in FIG. 1.
  • the received laser light guided to the camera 141 is condensed by the corresponding tube lens 142 and then enters the camera 141, and is recorded by the camera 141 to generate a received laser image.
  • the signal collection device 14 may include four detection light paths to collect four types of laser light from a biological sample; in another embodiment, the signal collection device 14 may include two detection light paths.
  • Each detection optical path is divided into two photographs to collect two kinds of lasers of biological samples, and then the biological characteristic information of the biological samples is collected and identified by the method of chemistry and mathematical logic.
  • the signal acquisition device 14 may only include a camera 141 and a tube lens 142 to form a detection light path, and subsequently collect and identify the biological characteristic information of the biological sample through a method that cooperates with chemistry and mathematical logic.
  • the signal acquisition device 14 cooperates with the structured light generating and modulating device 11 to switch the phase of the structured light and/or the frequency of the illumination pattern direction to photograph the received laser, so as to obtain multiple different images of the same received laser.
  • the excitation light source 10 outputs excitation light of different wavelengths
  • the structured light generating and modulating device 11 and the signal collecting device 14 cooperate to switch the phase of the structured light and/or the direction of the illumination pattern and shooting, respectively.
  • the sample platform 3 moves the biological sample to switch the imaging field of view of the super-resolution imaging system 1, so as to obtain multiple different images of the biological sample in different regions.
  • the super-resolution imaging system 1 further includes an auto-focusing module 16, which is used to transmit detection light to the sample carrier 2 in real time, receive the detection light returned from the sample carrier 2, and detect light according to the return. Light real-time detection of whether the sample carrier 2 is located on the focal plane of the objective lens 13. If the sample carrier 2 is not located on the focal plane of the objective lens 13, the auto-focusing module 16 sends a signal recording the defocus direction and the defocus distance of the sample carrier 2 to A device (not shown) for controlling the relative distance between the sample carrier 2 and the objective lens 13 by which the sample carrier 2 is returned to the focal plane of the objective lens 13.
  • a device not shown
  • the detection light emitted by the autofocus module 16 is projected onto the sample carrier 2 through the optical path guiding device 12 and the objective lens 13, and the detection light returned from the sample carrier 2 is returned to the autofocus via the objective lens 13 and the optical path guiding device 12
  • the module 16 is also detected by the auto-focusing module 16.
  • the auto focus module 16 may be omitted.
  • the biological sample identification system 4 includes a super-resolution imaging system 41 and an image reconstruction and biometric identification device 42.
  • the super-resolution imaging system 41 may be the aforementioned super-resolution imaging system.
  • the super-resolution imaging system controls the phase of structured light and the direction of the illumination pattern of the structured light irradiated to the biological sample to obtain at least one field of view.
  • the image reconstruction and biometric recognition device 42 can be any computing device, and an image reconstruction program and a biometric recognition program are installed in the computing device, and the image reconstruction program receives the laser light output from the super-resolution imaging system 41.
  • the image is based on multiple images excited by the laser with the same wavelength of excitation light in the same imaging field of view.
  • Image reconstruction is performed using a specific algorithm to obtain a reconstructed super-resolution image.
  • the specific algorithm may be, for example, an open source SIM (Structured Illumination Microscopy). , Structured light illumination microscope) reconstruction algorithm, the biometric recognition program is used to recognize the biological characteristics (such as bases) of the biological sample corresponding to the laser in the corresponding field of view according to the reconstructed super-resolution image.
  • SIM Structured Illumination Microscopy
  • the biometric recognition program is used to recognize the biological characteristics (such as bases) of the biological sample corresponding to the laser in the corresponding field of view according to the reconstructed super-resolution image.
  • FIG. 3 is a schematic flowchart of a super-resolution imaging method provided in an embodiment of the present invention.
  • the method can be implemented in combination with the super-resolution imaging system 1 shown in FIG. 1 or a similar super-resolution imaging system. According to needs, some steps in the method can be omitted, and the order of some steps can be changed.
  • the super-resolution imaging method combined with the super-resolution imaging system 1 shown in FIG. 1 is taken as an example for description.
  • step S30 the excitation light source of the illumination system in the super-resolution imaging system 1 is controlled to start, so that the illumination system outputs structured light to irradiate the biological sample, so that the biological sample generates laser light.
  • Step S31 Control the structured light output by the illumination system to be in the first phase and the illumination pattern of the structured light to be in the first direction, and take a first image of the laser light;
  • the structured light output by the illumination system A is in the first phase and the illumination pattern of the structured light is in the first direction, and at the same time, the polarization control system is controlled by the structured light control device 113 111.
  • the illumination pattern of the structured light in the first direction and the first phase meet a preset requirement, for example, make the contrast of the illumination stripes of the illumination pattern meet the requirement.
  • Step S32 changing the phase of the structured light output by the lighting system to a second phase different from the first phase, controlling the structured light output by the lighting system to be in the second phase and the lighting pattern of the structured light in the first direction , Take the second image of the laser.
  • Step S33 changing the direction of the lighting pattern of the structured light output by the lighting system to a second direction different from the first direction, and controlling the structured light output by the lighting system to be in the second phase and the lighting pattern of the structured light to be in the second phase.
  • the third image of the laser light is taken.
  • Step S34 changing the direction of the lighting pattern of the structured light output by the lighting system to a third direction different from the first direction and the second direction, controlling the structured light output by the lighting system to be in the second phase and the structured light
  • the lighting pattern of the lighting system is in the third direction, or the phase of the structured light output by the lighting system is changed to the first phase, and the structured light output by the lighting system is controlled to be in the first phase and the lighting pattern of the structured light is in the second phase.
  • Direction take the fourth image of the laser.
  • steps of the present invention are not limited to the above order, they can also be adjusted as needed.
  • the "first”, “second”, and “third” used in the above expressions are only used to distinguish related phases, directions, or images. , Does not mean that the relative phases, directions, or images have a specific order. In fact, the execution order of the above steps S31-S34 is variable.
  • the structured light control device 113 controls the phase of the structured light and the direction of the illumination pattern of the structured light by controlling the diffraction spectroscopic device 112 to move linearly, rotate, etc., by synchronously moving and rotating the diffraction spectroscopic device 112 and the polarization control
  • the system 111 makes the lighting pattern of the structured light in each direction and each phase meet the preset requirements.
  • it may further include the steps of capturing the fifth image, sixth image, seventh image, eighth image, and ninth image of the laser light.
  • the steps of capturing the fifth image, sixth image, seventh image, eighth image, and ninth image of the laser light By controlling the diffraction spectroscopic device 112 and the polarization control system 111, Change the phase of the structured light and/or the direction of the structured light illumination pattern, so that each image captured by the laser is different from other images, for example, the phases of the first to ninth images and the structured light and the direction of the structured light illumination pattern.
  • the above “first” to “ninth” are only used to distinguish images, and do not mean that the images have a specific sequence. In fact, the numbers of the above images are not used to limit the sequence. In practical applications, it can be used as needed. Adjust the order of image acquisition.
  • the super-resolution imaging method may further include the step of switching the wavelength of the excitation light and repeating steps S30-S34 to obtain multiple frames of laser-induced images excited by excitation light of other wavelengths until all predetermined All the lasers excited by the excitation light of the wavelength are taken to be taken.
  • each step of switching the phase and/or direction of the structured light and shooting the lasered image in the super-resolution imaging method may further include: switching the wavelength of the excitation light and repeating the switching and shooting.
  • the step is to obtain images of the laser light excited by the excitation light of different wavelengths, until all the predetermined laser light excited by the excitation light of all wavelengths have been photographed.
  • the super-resolution imaging method may further include the step of: moving the sample carrier 2 to switch the imaging field of view of the super-resolution imaging system 1, so that the objective lens 13 can collect the biological samples at different positions of the sample carrier 2.
  • each step of switching the structured light phase and/or direction and shooting the lasered image in the super-resolution imaging method may further include: switching the imaging field of view of the super-resolution imaging system 1 and repeating it.
  • the steps of switching and shooting are to obtain laser-induced images at different positions until all predetermined positions that need to be imaged are taken.
  • FIG. 4 is a schematic flowchart of a biological sample identification method provided in an embodiment of the present invention.
  • the method can be implemented in combination with the biological sample identification system shown in FIG. 2 or a similar biological sample identification system. According to needs, some steps in the method can be omitted, and the order of some steps can be changed.
  • step S40 the excitation light source of the illumination system in the super-resolution imaging system is controlled to start, so that the illumination system outputs structured light to irradiate the biological sample, so that the biological sample generates laser light.
  • Step S41 controlling the structured light output by the illumination system to be in the first phase and the illumination pattern of the structured light to be in the first direction, and shooting the first image of the laser light.
  • Step S42 controlling the structured light output by the illumination system to be in a second phase different from the first phase and the illumination pattern of the structured light to be in the first direction, and shooting the second image of the laser light.
  • Step S43 controlling the structured light output by the illumination system to be in the second phase and the illumination pattern of the structured light to be in a second direction different from the first direction, and shooting the third image of the laser light.
  • Step S44 controlling the structured light output by the lighting system to be in the second phase and the lighting pattern of the structured light to be in a third direction different from the first direction and the second direction, or controlling the structured light output by the lighting system
  • the structured light is in the first phase and the illumination pattern of the structured light is in the second direction, and the fourth image of the laser light is captured.
  • the super-resolution imaging method in this application may be made to the description of the super-resolution imaging method in this application, which is not repeated here.
  • the fifth image, even the sixth image, and the seventh image of the laser can also be obtained.
  • the excitation light wavelength it is also possible to obtain multiple different images of the laser excited by the excitation light of other wavelengths; similarly, by changing the imaging field of view, it is also possible to obtain multiple different images of the laser in other fields of view.
  • Step S45 Perform image reconstruction to obtain a reconstructed super-resolution image based on the multiple images excited by the laser light of the same wavelength in the same field of view.
  • a SIM (Structured Illumination Microscopy) super-resolution reconstruction technology is used to reconstruct multiple images subjected to laser light.
  • the SIM super-resolution reconstruction technology can use many existing algorithms to perform reconstruction. For example, the existing open source SIM reconstruction algorithm can be used to perform the reconstruction.
  • Step S46 Perform biometric identification based on the reconstructed super-resolution image, so as to identify the biometric feature of the biological sample excited by the specific excitation light in the corresponding field of view to generate laser light.
  • the existing technology is used to identify the biological characteristics of the biological sample (such as the base of the DNA sample) based on the image, and will not be specifically introduced here.
  • the super-resolution imaging system 1a includes an excitation light source 10a, a structured light generation and modulation device 11a, a dichroic mirror 12a, an objective lens 13a, and a signal acquisition device 14a.
  • the signal acquisition device 14a includes a camera 141a and a tube Mirror 142a.
  • the structured light generating and modulating device 11a includes a polarizer 111a, a phase grating 112a, a structured light control device 113a, a focusing device 114a, and a collimator lens 115a.
  • the collimator lens 115a, the polarizer 111a, the phase grating 112a and the focusing device 114a are arranged behind the excitation light source 10a from near and far along the direction in which the excitation light is emitted, and the polarizer 111a and the phase grating 112a are arranged together.
  • the one-dimensional mobile platform 116 is set on the turntable 117 together with the polarizer 111a and the phase grating 112a.
  • the structured light control device 113a is connected to the one-dimensional mobile platform 116 and the turntable 117, and is used to control the movement of the mobile platform 116 and the rotation of the turntable 117 to adjust the phase of the output structured light and the direction of the illumination pattern of the structured light.
  • the focusing device 114a includes a first lens 1141a, a second lens 1142a, and a third lens 1143a.
  • the first lens 1141a, the second lens 1142a, and the third lens 1143a are arranged behind the phase grating 112a from near and far along the direction in which the excitation light is emitted.
  • a stopper 118 is also provided between the first lens 1141a and the second lens 1142a. The stopper 118 is used to block part of the diffracted light and prevent it from entering the subsequent light path. In this embodiment, the stopper 118 is provided on the first lens.
  • the back focal plane of 1141a is used to control the movement of the mobile platform 116 and the rotation of the turntable
  • the excitation light source 10a can emit excitation light of two wavelengths. After the excitation light is collimated by the collimator lens 115a, it enters the polarizer 111a to become linearly polarized light. The polarization direction of the linearly polarized light is related to the phase grating. The engraved line directions of 112a are parallel, and after linearly polarized light enters the phase grating 112a, +1 order, -1 order and 0 order diffracted light are generated.
  • the diffracted light is focused by the first lens 1141a
  • the 0-th order diffracted light is blocked by the baffle 118
  • the +1-th order diffracted light and the -1st-order diffracted light pass through the second lens 1142a and the third lens 1143a, and are reflected by the dichroic mirror 12a.
  • the +1 order and -1 order diffracted light overlap on the sample carrier 2a to generate interference fringes.
  • the spacing of the interference fringes is d/2M, where d is the reticle spacing of the phase grating 112a, and M is the total magnification of the combination of the first lens 1141a, the second lens 1142a, the third lens 1143a and the objective lens 13a. Therefore, the excitation light of two wavelengths produces interference fringes with the same spacing.
  • the sample carrier 2a is located on the imaging surface of the objective lens 13a, and is a conjugate surface with the reticle plane of the phase grating 112a and the sensor plane of the camera 141a.
  • the excitation light of each wavelength can excite one of the fluorescent markers to generate a fluorescent light, which is collected by the objective lens 13a, and passes through the dichroic mirror 12a and tube lens. After 142a, it is recorded by the camera 141a.
  • the following introduces three super-resolution imaging methods implemented by the super-resolution imaging system 1a.
  • the first super-resolution imaging method is as follows:
  • Step 1 Start the excitation light source 10a.
  • the excitation light source 10a first outputs the excitation light of the first wavelength.
  • the excitation light of the first wavelength passes through the super-resolution imaging system 1a and then outputs structured light to irradiate the sample carrier 2a, producing interference fringes on the sample carrier 2a.
  • the structured light excites one of the fluorescent markers on the sample carrier 2a to generate a first type of laser light, and the first type of laser light is recorded by the camera 141a.
  • the excitation light source 10a is switched to output the first type of laser light.
  • Excitation light of two wavelengths the excitation light of the second wavelength excites another fluorescent marker on the sample carrier 2a to generate a second type of laser light, and the second type of laser light is recorded by the camera 141a.
  • Step 2 The structured light control device 113a controls the one-dimensional moving platform 116 to drive the phase grating 112a to move d/6 in the grating plane, where d is the grating period, so that the phase of the interference fringes on the sample carrier 2a moves by 2 ⁇ /3, as shown in the figure Shown in 6B. Then record the first receiving laser and the second receiving laser according to the method of step 1.
  • Step 3 The structured light control device 113a continues to control the one-dimensional moving platform 116 to drive the phase grating 112a, and then continues to move d/6 forward in the grating plane, so that the phase of the interference fringes on the sample carrier 2a moves again by 2 ⁇ /3, such as Shown in Figure 6C. After that, record the first receiving laser and the second receiving laser according to the method of step 1.
  • Step 4 The structured light control device 113a controls the one-dimensional moving platform 116 to drive the phase grating 112a to move back by d/3 to return to the original position.
  • Step 5 The structured light control device 113a controls the turntable 117 to drive the phase grating 112a and the polarizer 111a to rotate 60 degrees along the grating plane. Then follow the steps 1 to 4 in sequence to record the first type of received laser light and the second type of received laser light when the interference fringes of the structured light are in different phases. The corresponding interference fringes are shown in Figure 6D-6F.
  • Step 6 the structured light control device 113a controls the turntable 117 to drive the phase grating 112a and the polarizer 111a to continue to rotate 60 degrees in the same direction along the grating plane. Then follow the steps 1 to 4 in sequence to record the first type of received laser light and the second type of received laser light when the interference fringes of the structured light are in different phases. The corresponding interference fringes are shown in Figure 6G-6I.
  • 9 images of each type of laser were obtained above. Using 9 images of each laser to perform super-resolution reconstruction, a super-resolution image of the laser can be obtained. The biological characteristics of the two super-resolution images subjected to the laser are recognized, and the biological characteristics of all biological samples on the sample carrier 2a in the field of view can be obtained.
  • the second super-resolution imaging method is as follows:
  • Step 1 Start the excitation light source 10a.
  • the excitation light source 10a first outputs the excitation light of the first wavelength.
  • the excitation light of the first wavelength passes through the super-resolution imaging system 1a and then outputs structured light to irradiate the sample carrier 2a, producing interference fringes on the sample carrier 2a.
  • the structured light excites one of the fluorescent markers on the sample carrier 2a to generate a first type of laser light, and the first type of laser light is recorded by the camera 141a.
  • the excitation light source 10a is switched to output the first type of laser light.
  • Excitation light of two wavelengths the excitation light of the second wavelength excites another fluorescent marker on the sample carrier 2a to generate a second type of laser light, and the second type of laser light is recorded by the camera 141a.
  • Step 2 The structured light control device 113a then controls the one-dimensional moving platform 116 to drive the phase grating 112a to move d/4 in the grating plane, where d is the grating period, so that the phase of the interference fringes on the sample carrier 2a moves by ⁇ /2, As shown in Figure 7B. After that, record the first type of received laser and the second type of received laser according to the method of step 1.
  • Step 3 The structured light control device 113a controls the one-dimensional moving platform 116 to drive the phase grating 112a to move back by d/4 to return to the original position.
  • Step 4 The structured light control device 113a controls the turntable 117 to drive the phase grating 112a and the polarizer 111a to rotate 60 degrees along the grating plane. Then according to the method of step one, the first type of received laser light and the second type of received laser light generated by the interference fringes of the structured light are recorded. The corresponding interference fringes are shown in Figure 7c.
  • Step 5 The structured light control device 113a controls the turntable 117 to drive the phase grating 112a and the polarizer 111a, and continue to rotate 60 degrees in the same direction along the grating plane. Then according to the method of step one, the first type of received laser light and the second type of received laser light generated by the interference fringes of the structured light are recorded. The corresponding interference fringes are shown in Figure 7d.
  • the third super-resolution imaging method is as follows:
  • Step 1 Start the excitation light source 10a.
  • the excitation light source 10a first outputs the excitation light of the first wavelength.
  • the excitation light of the first wavelength passes through the super-resolution imaging system 1a and then outputs structured light to irradiate the sample carrier 2a, producing interference fringes on the sample carrier 2a.
  • the structured light excites one of the fluorescent markers on the sample carrier 2a to generate a first type of laser light, which is recorded by the camera 141a, and then, the excitation light source 10a is switched to output the first type of laser light.
  • Excitation light of two wavelengths the excitation light of the second wavelength excites another fluorescent marker on the sample carrier 2a to generate a second type of laser light, and the second type of laser light is recorded by the camera 141a.
  • Step 2 The structured light control device 113a then controls the one-dimensional moving platform 116 to drive the phase grating 112a to move d/4 in the grating plane, where d is the grating period, so that the phase of the interference fringes on the sample carrier 2a moves by ⁇ /2, As shown in Figure 8b. After that, record the first type of received laser and the second type of received laser according to the method of step 1.
  • Step 3 The structured light control device 113a controls the one-dimensional moving platform 116 to drive the phase grating 112a to move back by d/4 and return to the original position.
  • Step 4 The structured light control device 113a controls the turntable 117 to drive the phase grating 112a and the polarizer 111a to rotate 90 degrees along the grating plane. Then sequentially record the first type of received laser light and the second type of received laser light under different phases of the interference fringes of the structured light in the manner of step 1 to step 3. The corresponding interference fringes are shown in Figure 8c.
  • Step 5 The structured light control device 113a controls the one-dimensional moving platform 116 to drive the phase grating 112a to move the d/4 grating period in the grating plane, so that the phase of the interference fringes on the sample carrier 2a moves by ⁇ /2, as shown in Fig. 8b .
  • the interference fringes of the structured light are in different phases, the first type of received laser light and the second type of received laser light are generated according to the method of step 1 to step 3.
  • Step 6 the structured light control device 113a controls the one-dimensional moving platform 116 to drive the phase grating 112a to move back by d/4 to return to the original position. In other embodiments, this step can also be omitted.
  • the four images of each type of laser are obtained above. Using 4 images of each laser to perform super-resolution reconstruction, a super-resolution image of the laser can be obtained. The biological characteristics of the two super-resolution images subjected to the laser are recognized, and the biological characteristics of all biological samples on the sample carrier 2a in the field of view can be obtained.
  • the super-resolution imaging system and method described in the above embodiments can be used in nucleic acid sequencing. Specifically, this application also provides a nucleic acid sequencing imaging system and a nucleic acid sequencing imaging method. Please refer to FIG. 9 for the nucleic acid sequencing imaging system 5 A super-resolution imaging system 51 is included.
  • the super-resolution imaging system 51 can be any of the super-resolution imaging systems introduced in the above-mentioned embodiment or a super-resolution imaging system obtained by an improvement of any of the above-mentioned super-resolution imaging systems.
  • the imaging system 51 excites the nucleic acid sample to generate laser light and takes an image of the laser light emitted from the nucleic acid sample.
  • the nucleic acid sequencing imaging method adopts a super-resolution imaging method to excite a nucleic acid sample to emit a laser and take an image of the laser.
  • the super-resolution imaging method can be any of the super-resolution imaging methods described in the above-mentioned embodiments or any of the above-mentioned methods.
  • a super-resolution imaging method improves the obtained super-resolution imaging method.
  • the biological sample identification system and method described in the above embodiments can be used in nucleic acid sequencing. Specifically, this application also provides a nucleic acid identification system and a nucleic acid identification method. Please refer to FIG. 10.
  • the nucleic acid identification system 6 includes a biological A sample identification system 61, the biological sample identification system 61 may be any of the biological sample identification systems introduced in the above embodiments or a biological sample identification system improved by any one of the above biological sample identification systems, the biological sample identification system 61
  • the nucleic acid sample is excited to generate laser light and an image of the laser light emitted by the nucleic acid sample is taken, and the base type of the nucleic acid sample is identified based on the image.
  • the nucleic acid identification method adopts a biological sample identification method to identify the base type of a nucleic acid sample
  • the biological sample identification method can be any of the biological sample identification methods described in the above embodiments or can be obtained by an improvement of any of the above biological sample identification methods. Method of identifying biological samples.
  • the super-resolution imaging system and method, biological sample identification system and method, nucleic acid sequencing imaging system and method, and nucleic acid identification system and method provided by the embodiments of the present invention are obtained by changing the direction and phase of the structured light output illumination pattern Multiple (for example, 4) images subjected to the laser are used for image reconstruction to obtain a super-resolution image.
  • the type and layout of biological samples can be identified through the super-resolution image; when applied to nucleic acid sequencing, it can identify alkali
  • the category and layout of the base Since the number of lasered images required to be taken is small, the speed of biological sample identification is increased; when applied to nucleic acid sequencing, the speed of base identification can be increased.
  • the use of super-resolution imaging technology can increase the density of the biological sample on the sample carrier, thereby solving the low imaging efficiency, the limited sample layout density, and the inefficient utilization of sample carrier caused by the use of ordinary wide-field fluorescence microscopy imaging technology in the prior art.
  • Advanced problems when applied to nucleic acid sequencing, it can solve the problems of low sequencing throughput, limited chip density, low chip utilization, and low reagent utilization.

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Abstract

一种超分辨成像系统(1, 41, 51)、超分辨成像方法、生物样品识别系统(4, 61)与方法、核酸测序成像系统(5)与方法及核酸识别系统(6)与方法，该超分辨成像系统(1, 41, 51)包括照明系统(A)与成像系统(B)，照明系统(A)输出激发光照射生物样品以生成受激光，成像系统(B)收集及记录受激光以生成受激光图像，照明系统(A)包括激发光源(10, 10a)与结构光产生及调制装置(11, 11a)，激发光源(10, 10a)输出激发光，结构光产生及调制装置(11, 11a)将激发光调制成结构光照射生物样品以产生受激光，结构光产生及调制装置(11, 11a)包括结构光控制装置(113, 113a)，结构光控制装置(113, 113a)控制结构光产生及调制装置(11, 11a)以改变其输出的结构光的相位与结构光投射于生物样品上的照明图案的方向，成像系统(B)配合结构光相位及/或照明图案的方向的改变拍摄至少一成像视场内至少一波长的激发光激发的受激光的图像，由此提升了成像效率。

Description

超分辨成像系统与方法、生物样品识别系统与方法、核酸测序成像系统与方法及核酸识别系统与方法 技术领域
本发明涉及成像领域，尤其涉及基因测序中的超分辨成像系统与方法、生物样品识别系统与方法、核酸测序成像系统与方法及核酸识别系统与方法。
背景技术
现有高通量测序仪器，主要采用普通宽场荧光显微成像技术实现信号收集。来自测序芯片表面碱基的荧光，首先由物镜进行收集，然后不同波段的荧光，通过一组或几组二向色镜分离开来，再分别经过筒镜汇聚后，最终成像在CCD/CMOS表面。普通宽场荧光显微技术受限于衍射极限，分辨率只能达到约0.61λ/NA,制约了测序芯片上DNA样品密度，因此制约测序仪的测序通量。总的来说，高通量测序仪器采用普通宽场荧光显微成像技术，会存在如下问题；
1)、受限于光学衍射极限，测序通量低；
2)、测序芯片上的DNA样品间距需大于光学分辨率，样品密度受限，芯片利用率不高；
3)、由于测序芯片上DNA样品密度受限，导致试剂利用率不高、试剂成本较高。
另外，现有超分辨成像技术通常存在成像慢等问题，制约了其在高通量测序技术方面的应用。
发明内容
有鉴于此，有必要提供一种超分辨成像系统与方法、生物样品识别 系统与方法、核酸测序成像系统与方法及核酸识别系统与方法，以解决现有技术中的至少一个存在的问题。
第一方面，本申请提供一种超分辨成像系统，所述超分辨成像系统包括照明系统与成像系统，所述照明系统用于输出激发光照射生物样品，以生成受激光，所述成像系统用于收集及记录所述受激光以生成受激光图像，所述照明系统包括激发光源与结构光产生及调制装置，所述激发光源用于输出所述激发光，所述结构光产生及调制装置用于将所述激发光调制成结构光照射所述生物样品以产生所述受激光，所述结构光产生及调制装置包括结构光控制装置，所述结构光控制装置用于控制所述结构光产生及调制装置以改变所述结构光产生及调制装置输出的结构光的相位与所述结构光投射于所述生物样品上的照明图案的方向，所述成像系统用于配合所述结构光相位及/或照明图案的方向的改变拍摄至少一成像视场内至少一波长的激发光激发的受激光的图像。
进一步地，所述结构光产生及调制装置还包括偏振控制系统与衍射分光装置，所述偏振控制系统用于调整所述激发光的偏振方向，所述衍射分光装置用于将所述激发光分成多束以形成具有特定相位及特定照明图案方向的所述结构光，所述结构光控制装置用于控制所述衍射分光装置以改变所述结构光的相位与照明图案的方向、及用于控制所述偏振控制系统以使任一相位与任一方向下的所述结构光的照明图案符合预设要求。
进一步地，所述结构光控制装置用于控制所述偏振控制系统使所述结构光的照明图案的对比度符合预设的要求。
进一步地，所述结构光控制装置用于控制所述衍射分光装置直线移动以改变所述结构光的相位，及控制所述衍射分光装置转动以改变所述结构光照明图案的方向。
进一步地，所述超分辨成像系统还包括物镜，所述结构光产生及调制装置还包括聚焦装置，所述聚焦装置用于将所述衍射分光装置出射的 所述结构光聚焦至所述物镜，所述物镜用于将所述结构光以平行光出射至所述生物样品上、并以一定夹角在所述生物样品所在平面形成干涉条纹。
进一步地，所述衍射分光装置包括相位光栅，所述偏振控制系统包括起偏器。
进一步地，所述相位光栅和所述起偏器一起设置于一维移动平台上，所述一维移动平台设置于转台上，所述结构光控制装置用于控制所述一维移动平台带动所述相位光栅和所述起偏器移动以改变所述结构光的相位、及用于控制所述转台带动所述相位光栅和所述起偏器转动以改变所述结构光照明图案的方向。
进一步地，所述聚焦装置包括第一透镜、第二透镜及第三透镜，所述第一透镜与所述第二透镜之间设置挡件，所述挡件用于遮挡并阻止部分所述结构光进入后续光路。
进一步地，所述起偏器用于将所述激发光转换成线偏振光，所述相位光栅用于将所述线偏振光转换成+1级、-1级和0级衍射光，所述0级衍射光被所述挡板遮挡，所述+1级衍射光与-1级衍射光经过所述第二透镜、第三透镜后被聚焦至所述物镜的后焦面。
进一步地，所述结构光产生及调制装置还包括自适应光学装置，所述自适应光学装置用于对所述结构光的波前进行整形。
进一步地，所述结构光控制装置还用于控制所述自适应光学装置以优化所述结构光的对比度及/或均匀度。
进一步地，所述超分辨成像系统还包括总控制装置，所述总控制装置用于控制所述激发光源、所述结构光产生及调制装置及所述成像系统协调工作。
第二方面，本申请还提供一种超分辨成像方法，包括：
控制一超分辨成像系统中的照明系统的激发光启动，使所述照明系统输出结构光照射至生物样品上，使所述生物样品产生受激光；
控制所述照明系统输出的所述结构光处于第一相位及所述结构光的照明图案处于第一方向，拍摄所述受激光的第一图像；
控制所述照明系统输出的所述结构光处于不同于所述第一相位的第二相位及所述结构光的照明图案处于所述第一方向，拍摄所述受激光的第二图像；
控制所述照明系统输出的所述结构光处于所述第二相位及所述结构光的照明图案处于不同于所述第一方向的第二方向，拍摄所述受激光的第三图像；及
控制所述照明系统输出的所述结构光处于所述第二相位及所述结构光的照明图案处于不同于所述第一方向、第二方向的第三方向，或者，控制所述照明系统输出的所述结构光处于所述第一相位及所述结构光的照明图案处于所述第二方向，拍摄所述受激光的第四图像。
进一步地，通过直线移动所述照明系统的衍射分光装置控制所述结构光处于不同相位，通过转动所述衍射分光装置控制所述结构光的照明图案处于不同方向，及通过控制偏振控制系统移动或转动使每个方向和每个相位下的所述结构光的照明图案符合预设要求。
进一步地，使所述结构光的照明图案符合预设要求包括使所述结构光的照明图案的对比度符合预设要求。
进一步地，所述超分辨成像方法还包括：切换所述激发光的波长并重复执行上述切换结构光相位及/或方向及拍摄受激光图像的步骤。
进一步地，所述超分辨成像方法还包括：在每一切换结构光相位及/或方向及拍摄受激光图像的步骤完成后还包括：切换所述激发光的波长并重复执行该切换及拍摄的步骤。
进一步地，所述超分辨成像方法还包括：切换所述超分辨成像系统的成像视场并重复执行上述切换结构光相位及/或方向及拍摄受激光图像的步骤。
进一步地，所述超分辨成像方法还包括：在每一切换结构光相位及 /或方向及拍摄受激光图像的步骤完成后还包括：切换所述超分辨成像系统的成像视场并重复执行该切换及拍摄的步骤。
第三方面，本申请还提供一种生物样品识别系统，包括：
上述的超分辨成像系统；及
图像重建与生物特征识别装置，用于接收所述超分辨成像系统输出的受激光图像，并基于同一成像视场下同一波长激发光激发的受激光的多张图像，执行图像重建以获取重建后的超分辨图像，及根据重建后的超分辨图像识别所述成像视场内所述受激光对应的生物样品的生物特征。
第四方面，本申请还提供一种生物样品识别方法，包括：
采用上述的超分辨成像方法获得至少一成像视场内同一波长激发光激发的受激光的多张图像；
基于同一成像视场下同一波长激发光激发的受激光的所述多张图像，执行图像重建以获取重建后的超分辨图像；及
根据重建后的超分辨图像识别所述成像视场内所述受激光对应的生物样品的生物特征。
第五方面，本申请还提供一种核酸测序成像系统，所述核酸测序成像系统包括上述的超分辨成像系统，所述超分辨成像系统用于照射核酸样品并拍摄所述核酸样品出射的受激光的图像。
第六方面，本申请还提供一种核酸测序成像方法，所述核酸测序成像方法采用上述的超分辨成像方法激发核酸样品出射受激光并拍摄所述受激光的图像。
第七方面，本申请还提供一种核酸识别系统，所述核酸识别系统包括上述的生物样品识别系统，所述生物样品识别系统用于识别核酸样品的碱基的类别。
第八方面，本申请还提供一种核酸识别方法，所述核酸识别方法采用上述的生物样品识别方法识别核酸样品的碱基的类别。
本发明实施方式提供的超分辨成像系统与方法、生物样品识别系统 与方法、核酸测序成像系统与方法及核酸识别系统与方法，通过改变结构光输出照明图案的方向与相位获得受激光的多张(例如4张)图像，利用该多张图像进行图像重构获得超分辨图像，通过超分辨图像即可识别生物样品的布局和类别；应用于核酸测序时，可识别碱基的布局和类别。由于所需拍摄的受激光图像数量较少，提高了生物样品的识别速度；应用于核酸测序时，可提高碱基识别的速度。而采用超分辨成像技术，可以提高样品载体上的样品密度，从而解决了现有技术中采用普通宽场荧光显微成像技术导致的成像效率低、样品布局密度受限、样品载体利用率不高等问题；应用于核酸测序时，可解决测序通量低、芯片密度受限，芯片利用率不高、试剂利用率不高等问题。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对本发明实施例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一些实施例，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，还可以根据这些附图获得其他的附图。
图1是本发明一实施方式提供的超分辨成像系统的构成示意图。
图2是本发明一实施方式提供的生物样品识别系统的构成示意图。
图3是本发明一实施方式提供的超分辨成像方法的流程示意图。
图4是本发明一实施方式提供的生物样品识别方法的流程示意图。
图5是本发明图1所示超分辨成像系统的一具体实施例的示意图。
图6A-图6I是一种超分辨成像方法中利用图5所示超分辨成像系统输出的结构光在样品载体上产生的干涉条纹的示意图。
图7A-图7D是另一种超分辨成像方法中利用图5所示超分辨成像系统输出的结构光在样品载体上产生的干涉条纹的示意图。
图8A-图8D是本发明第三种超分辨成像方法中利用图5所示超分辨成像系统输出的结构光在样品载体上产生的干涉条纹的示意图。
图9是本发明一实施方式提供的核酸测序成像系统的示意图。
图10是本发明一实施方式提供的核酸识别系统的示意图。
如下具体实施方式将结合上述附图进一步说明本发明。
主要元件符号说明
超分辨成像系   1、41、51     照明系统         A
统
成像系统       B             激发光源         10、10a
结构光产生及   11、11a       光路引导装置     12
调制装置
物镜           13、13a       信号采集装置     14、14a
样品平台       3             总控制装置       15
样品载体       2、2a         偏振控制系统     111
衍射分光装置   112           结构光控制装置   113、113a
聚焦装置       114、114a     自适应光学装置   115
二向色镜       121、143、12a 相机             141、141a
筒镜           142、142a     自动对焦模块     16
生物样品识别   4、61         图像重建与生物   42
系统                         特征识别装置
起偏器         111a          相位光栅         112a
准直透镜       115a          一维移动平台     116
转台           117           第一透镜         1141a
第二透镜       1142a         第三透镜         1143a
挡件           118           步骤             S30-S34、
                                              S40-S46
核酸测序成像   5             核酸识别系统     6
系统
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作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。
需要说明的是，当一个组件被认为是“设置于”另一个组件，它可以是直接设置在另一个组件上或者可能同时存在居中组件。本文所使用的术语“及/或”包括一个或多个相关的所列项目的所有的和任意的组合。
请参阅图1所示，为本发明一实施方式中的宽场超分辨成像系统，所述超分辨成像系统1包括激发光源10、结构光产生及调制装置11、光路引导装置12、物镜13及信号采集装置14。其中，所述激发光源10、结构光产生及调制装置11、光路引导装置12、物镜13构成超分辨成像系统1的照明系统A，所述物镜13、光路引导装置12及信号采集装置14构成超分辨成像系统1的成像系统B。
所述激发光源10输出激发光，所述激发光经结构光产生及调制装置11后被调制成具有特定的照明图案方向和相位的结构光，所述结构光经光路引导装置12引导至物镜13，经由物镜13投射至装载了经过处理的生物样品的样品载体2上。所述生物样品可以是核酸(DNA和RNA)、蛋白或者细胞等。在本实施方式中，样品载体2被置于样品平台3上。样品平台3为一可移动平台，用于移动样品载体2使样品载体2上不同位置的生物样品均能依次被激发光照射。在其它实施方式中，可以不使用样品平台3。在本实施方式中，激发光激发生物样品上的标记物产生受激光(例如受激发的荧光)，受激光经物镜13收集，再经光路引导装置12引导至信号采集装置14，由信号采集装置14记录并生成受激光图像。 在本实施方式中，物镜13一方面收集受激光，另一方面辅助产生投射于生物样品平面的所述结构光。所述物镜13可以是低倍数宽视场物镜。
在本实施方式中，激发光源10为单一色或多色激光光源，具体地，所述激发光源可以是单一色、两色或四色激光，用以激发生物样品的生物特征(如碱基)上的荧光标记物。
在本实施方式中，所述结构光产生及调制装置11包括偏振控制系统111、衍射分光装置112、结构光控制装置113及聚焦装置114。所述偏振控制系统111用于调整激发光的偏振方向，以使照射至生物样品上的结构光的照明图案符合预设要求，例如，使结构光的照明图案的对比度符合要求，或者，使结构光的照明图案的对比度最高。所述偏振控制系统111可以包括起偏器、可变相位延迟器或者波片等光学元件。所述衍射分光装置112用于将激发光分成多束，在本实施方式中，所述衍射分光装置112可以包括分束片、光栅、空间光调制器、数字微镜器件或者光纤等。所述结构光控制装置113用于控制所述偏振控制系统111与衍射分光装置112，通过控制所述衍射分光装置112，对产生的结构光的照明图案的方向和相位进行控制，同时，通过控制偏振控制系统111，使每个方向和每个相位下的结构光的图案符合预设要求。具体地，在一种实施方式中，结构光控制装置113通过控制偏振控制系统111与衍射分光装置112，使结构光的偏振方向跟随结构光的方向改变，同时，结构光控制装置113根据接收到的外部命令或者预设的参数，控制偏振控制系统111使结构光的照明图案符合预设要求，例如，在接收到一标示结构光的照明图案符合预设的对比度要求的外部命令时，所述结构光控制装置113控制所述偏振控制系统111使其保持当前设置，从而使照明系统持续输出照明图案符合预设的对比度要求的结构光，直至偏振控制系统111的偏振方向跟随结构光方向发生改变。其中，标示结构光照明图案符合预设的对比度要求的外部命令可以是用户手动输入的命令，也可以是外部装置(图未示)根据预设条件自动判断输出的命令。
所述聚焦装置114用于将衍射分光装置112分成多束的激发光聚焦至物镜13，并通过物镜13以平行光出射，在本实施方式中，聚焦装置114为一透镜组，激发光被透镜组聚焦至物镜13的后焦面上，通过物镜13以平行光出射，并以一定夹角在生物样品所在平面形成干涉条纹。
在本实施方式中，所述结构光产生及调制装置11还可包括自适应光学装置115，用于对结构光的波前进行整形，所述结构光控制装置113还可用于控制所述自适应光学装置115，以优化结构光的对比度及/或均匀度，或者，使结构光的对比度及/或均匀度在成像视场内保持最优。
在本实施方式中，所述光路引导装置12包括二向色镜121，所述二向色镜121一方面将结构光产生及调制装置11输出的结构光引导至物镜13，另一方面将物镜13收集的受激光引导至信号采集装置14。
在本实施方式中，所述信号采集装置14包括多个相机141、多个筒镜142以及一或多个二向色镜143，所述一或多个二向色镜143将不同的受激光引导至不同的相机141。所述多个相机141、多个筒镜142以及一或多个二向色镜143构成多路探测光路，其中图1中仅示出了一路探测光路。被引导至相机141的受激光经对应的筒镜142汇聚后进入相机141，被相机141记录生成受激光图像。在一种实施方式中，所述信号采集装置14可以包括四路探测光路，以采集生物样品的四种受激光；在另一种实施方式中，所述信号采集装置14可以包括两路探测光路，每路探测光路分两次拍照采集生物样品的其中两种受激光，后续通过配合化学和数学逻辑的方法采集并识别生物样品的生物特征信息。当然，在其他实施方式中，所述信号采集装置14可以只包含一个相机141与一个筒镜142构成一路探测光路，后续通过配合化学和数学逻辑的方法采集并识别生物样品的生物特征信息。
在本实施方式中，还包括总控制装置15，所述总控制装置15用于控制所述信号采集装置14、激发光源10、结构光产生及调制装置11及样品平台协同工作，例如，在总控制装置15的控制下，信号采集装置14 配合结构光产生及调制装置11切换结构光的相位及/或照明图案方向的频率对受激光进行拍摄，从而获得同一受激光的多张不同图像。例如，在总控制装置15的控制下，激发光源10输出不同波长的激发光，结构光产生及调制装置11与信号采集装置14分别配合切换结构光的相位及/或照明图案方向与拍摄，从而获得不同受激光的多张不同图像。例如，在总控制装置15的控制下，样品平台3移动生物样品，以切换超分辨成像系统1的成像视场，从而获得不同区域的生物样品的受激光的多张不同图像。
在本实施方式中，超分辨成像系统1还包括自动对焦模块16，所述自动对焦模块16用于实时发射检测光至样品载体2、接收从样品载体2返回的检测光、及根据返回的检测光实时检测样品载体2是否位于物镜13的焦平面上，若样品载体2未位于物镜13的焦平面上，则自动对焦模块16将记录样品载体2的离焦方向及离焦距离的信号发送给控制样品载体2与物镜13相对距离的装置(图未示)，通过该装置使样品载体2回到物镜13的焦平面上。在本实施方式中，自动对焦模块16发出的检测光经光路引导装置12与物镜13投射至样品载体2上，从样品载体2上返回的检测光经物镜13与光路引导装置12回到自动对焦模块16并被自动对焦模块16检测到。
在其他实施方式中，所述自动对焦模块16可以省略。
请参阅图2所示，为本发明一实施方式中的生物样品识别系统的示意图，所述生物样品识别系统4包括超分辨成像系统41及图像重建与生物特征识别装置42。所述超分辨成像系统41可以是前述介绍的超分辨成像系统，所述超分辨成像系统通过控制照射至生物样品的结构光的相位及结构光的照明图案的方向，获取至少一视场内至少一激发光激发的受激光的若干张(如四张)不同图像。所述图像重建与生物特征识别装置42可以为任一的计算装置，所述计算装置内安装有图像重建程序与生物特征识别程序，所述图像重建程序接收超分辨成像系统41输出的受激 光的图像，基于同一成像视场下同一波长激发光激发的受激光的多张图像，利用特定算法执行图像重建以获取重建后的超分辨图像，所述特定算法可以是，例如开源SIM(Structured Illumination Microscopy，结构光照明显微镜)重构算法，所述生物特征识别程序用于根据重构后的超分辨图像识别对应视场内所述受激光对应的生物样品的生物特征(如碱基)，由于本实施例中图像重建与生物特征识别均可采用现有技术，因此，在本实施例中不做具体介绍。
请参阅图3所示，为本发明一种实施方式中提供的超分辨成像方法的流程示意图，所述方法可以结合图1所示的超分辨成像系统1或者类似超分辨成像系统进行实施。根据需要，所述方法中的某些步骤可以省略，某些步骤的顺序可以改变。
在本实施方式中，以所述超分辨成像方法结合图1所示超分辨成像系统1实施为例进行说明。
步骤S30，控制超分辨成像系统1中的照明系统的激发光源启动，使照明系统输出结构光照射至生物样品上，使所述生物样品产生受激光。
步骤S31.控制所述照明系统输出的结构光处于第一相位及所述结构光的照明图案处于第一方向，拍摄所述受激光的第一图像；
在本实施方式中，通过控制衍射分光装置112，使照明系统A输出的结构光处于第一相位及所述结构光的照明图案处于第一方向，同时，通过结构光控制装置113控制偏振控制系统111，使所述第一方向与第一相位下的所述结构光的照明图案符合预设要求，例如，使照明图案的照明条纹的对比度符合要求。
步骤S32，改变所述照明系统输出的结构光的相位为不同于第一相位的第二相位，控制所述照明系统输出的结构光处于第二相位及所述结构光的照明图案处于第一方向，拍摄所述受激光的第二图像。
步骤S33，改变所述照明系统输出的结构光的照明图案的方向为不同于第一方向的第二方向，控制所述照明系统输出的结构光处于第二相 位及所述结构光的照明图案处于第二方向，拍摄所述受激光的第三图像。
步骤S34，改变所述照明系统输出的结构光的照明图案的方向为不同于第一方向与第二方向的第三方向，控制所述照明系统输出的结构光处于第二相位及所述结构光的照明图案处于第三方向，或者，改变所述照明系统输出的结构光的相位为第一相位，控制所述照明系统输出的结构光处于第一相位及所述结构光的照明图案处于第二方向，拍摄所述受激光的第四图像。
本发明的步骤不限于以上顺序，其还可以根据需要调整，此外，上述表达中所使用的“第一”、“第二”、“第三”，仅是用于区别相关相位、方向或图像，并不表示相关相位、方向或图像具有特定先后顺序，实际上，上述步骤S31-S34,其执行顺序是可变的。
在本实施方式中，结构光控制装置113通过控制衍射分光装置112进行直线移动、转动等来控制结构光的相位及结构光的照明图案的方向，通过同步移动、转动衍射分光装置112与偏振控制系统111，使每个方向和每个相位下的结构光的照明图案符合预设要求。
在另一实施方式中，还可以包括拍摄所述受激光的第五图像、第六图像、第七图像、第八图像及第九图像的步骤，通过控制衍射分光装置112与偏振控制系统111，改变结构光的相位及/或结构光的照明图案的方向，使拍摄的受激光的每一图像不同于其他图像，例如，第一至第九图像与结构光的相位及结构光照明图案的方向的对应关系可以如下表所示：
图像 相位 方向
第一图像 第一相位 第一方向
第二图像 第二相位 第一方向
第三图像 第二相位 第二方向
第四图像 第二相位 第三方向
第五图像 第三相位 第三方向
第六图像 第三相位 第二方向
第七图像 第三相位 第一方向
第八图像 第一相位 第二方向
第九图像 第一相位 第三方向
同上所述，上述“第一”至“第九”仅是用于区别图像，并不代表图像就有特定先后顺序，实际上，上述图像的编号不用于限定顺序，在实际应用中可以根据需要调整图像采集的顺序。
在其他实施方式中，所述超分辨成像方法还可包括步骤：切换激发光的波长并重复执行步骤S30-S34,以获取其他波长的激发光激发的受激光的多帧图像，直至预定的所有波长的激发光激发的受激光均完成拍照。
在其他实施方式中，所述超分辨成像方法中的每一切换结构光相位及/或方向及拍摄受激光图像的步骤完成后还可包括：切换激发光的波长并重复执行该切换及拍摄的步骤，以获取不同波长的激发光激发的受激光的图像，直至预定的所有波长的激发光激发的受激光均完成拍照。
在其他实施方式中，所述超分辨成像方法还可包括步骤：移动样品载体2以切换超分辨成像系统1的成像视场，使物镜13能收集样品载体2不同位置处的生物样品发出的受激光，并重复执行步骤S30-S34，从而获取其他位置处的受激光的多帧图像，直至预定的需要成像的所有位置均完成拍照。
在其他实施方式中，所述超分辨成像方法中的每一切换结构光相位及/或方向及拍摄受激光图像的步骤完成后还可包括：切换超分辨成像系统1的成像视场并重复执行该切换及拍摄的步骤，以获取不同位置处的受激光的图像，直至预定的需要成像的所有位置均完成拍照。
请参阅图4所示，为本发明一种实施方式中提供的生物样品识别方法的流程示意图，所述方法可以结合图2所示的生物样品识别系统或者类似的生物样品识别系统实施。根据需要，所述方法中的某些步骤可以 省略，某些步骤的顺序可以改变。
步骤S40,控制超分辨成像系统中的照明系统的激发光源启动，使照明系统输出结构光照射至生物样品上，使所述生物样品产生受激光。
步骤S41,控制所述照明系统输出的结构光处于第一相位及所述结构光的照明图案处于第一方向，拍摄所述受激光的第一图像。
步骤S42,控制所述照明系统输出的结构光处于不同于第一相位的第二相位及所述结构光的照明图案处于第一方向，拍摄所述受激光的第二图像。
步骤S43,控制所述照明系统输出的结构光处于第二相位及所述结构光的照明图案处于不同于第一方向的第二方向，拍摄所述受激光的第三图像。
步骤S44，控制所述照明系统输出的结构光处于第二相位及所述结构光的照明图案处于不同于所述第一方向、第二方向的第三方向，或者，控制所述照明系统输出的结构光处于第一相位及所述结构光的照明图案处于第二方向，拍摄所述受激光的第四图像。
本申请的上述步骤不限于以上顺序，其还可以根据需要调整。
上述步骤实施的细节可以参考本申请前面对超分辨成像方法的描述，在此不做赘述。另，同样的，通过控制照射至生物样品的结构光的相位及结构光的照明图案的方向，还可以获取受激光第五图像、甚至第六图像、第七图像……；同样的，通过改变激发光波长，还可以获取其他波长的激发光激发的受激光的多张不同图像；同样的，通过改变成像视场，还可以获取其他视场内的受激光的多张不同图像。
步骤S45，基于同一视场下同一波长激发光激发的受激光的所述多张图像，执行图像重建以获取重建后的超分辨图像。
在本实施方式中，对受激光的多张图像的重建采用SIM(Structured Illumination Microscopy，结构光照明显微镜)超分辨重构技术，所述SIM超分辨重构技术可以采用现有的诸多算法执行重构，例如，可以采 用现有的开源SIM重构算法执行重构。
步骤S46，基于重建后的超分辨图像进行生物特征识别，以识别出对应视场内被特定激发光激发产生受激光的生物样品的生物特征。
在本实施方式中，基于图像识别生物样品的生物特征(如DNA样品的碱基)采用现有技术，在此不做具体介绍。
以下给出超分辨成像系统及超分辨成像方法的具体实施例，以结合本申请前述介绍的超分辨成像系统1及超分辨成像方法进行进一步说明。
请参阅图5所示，超分辨成像系统1a包括激发光源10a、结构光产生及调制装置11a、二向色镜12a、物镜13a及信号采集装置14a，其中，信号采集装置14a包括相机141a与筒镜142a。结构光产生及调制装置11a包括起偏器111a、相位光栅112a、结构光控制装置113a、聚焦装置114a与准直透镜115a。其中，准直透镜115a、起偏器111a、相位光栅112a及聚焦装置114a沿激发光出射的方向由近及远地设置于激发光源10a的后方，起偏器111a与相位光栅112a一起设置于一维移动平台116上，一维移动平台116再连同起偏器111a、相位光栅112a一起设置于转台117上。结构光控制装置113a与一维移动平台116、转台117连接，用于控制移动平台116移动与转台117转动以调节输出的结构光的相位及结构光的照明图案的方向。聚焦装置114a包括第一透镜1141a、第二透镜1142a及第三透镜1143a，第一透镜1141a、第二透镜1142a及第三透镜1143a沿激发光出射的方向由近及远地设置于相位光栅112a后方。第一透镜1141a与第二透镜1142a之间还设置了挡件118，所述挡件118用于遮挡部分衍射光，阻止其进入后续光路，在本实施例中，挡件118设置于第一透镜1141a的后焦面上。
具体地，本实施例中，激发光源10a可以发出两种波长的激发光，激发光经准直透镜115a准直后，进入起偏器111a成为线偏振光，线偏振光的偏振方向与相位光栅112a的刻线方向平行，线偏振光进入相位光栅112a后，产生+1级、-1级和0级衍射光。衍射光经过第一透镜1141a 聚焦后，0级衍射光被挡板118挡住，+1级衍射光与-1级衍射光经过第二透镜1142a、第三透镜1143a之后，被二向色镜12a反射、聚焦至物镜13a的后焦面，再以一定夹角的平行光出射至样品载体2a上。+1级、-1级衍射光在样品载体2a上重叠，产生干涉条纹。干涉条纹的间距为d/2M，其中d为相位光栅112a的刻线间距，M为第一透镜1141a、第二透镜1142a、第三透镜1143a和物镜13a四者结合后的总放大倍率。因此，两个波长的激发光，产生的是一样间距的干涉条纹。
在本实施方式中，样品载体2a位于物镜13a的成像面上，与相位光栅112a的刻线平面、相机141a的传感器平面互为共轭面。样品载体2a上附有两种荧光标记物，每个波长的激发光可以激发其中一种荧光标记物产生荧光形式的受激光，受激光经物镜13a收集，透过二向色镜12a、筒镜142a之后，被相机141a记录。
以下介绍三种利用超分辨成像系统1a实施的超分辨成像方法。
第一种超分辨成像方法如下：
步骤一，启动激发光源10a，激发光源10a先输出第一波长的激发光，所述第一波长激发光经由超分辨成像系统1a后输出结构光照射样品载体2a，在样品载体2a上产生干涉条纹如图6A所示，所述结构光激发样品载体2a上的其中一种荧光标记物，产生第一种受激光，所述第一种受激光被相机141a记录，接着，切换激发光源10a输出第二波长的激发光，所述第二波长的激发光激发样品载体2a上的另一种荧光标记物，产生第二种受激光，所述第二种受激光被相机141a记录。
步骤二，结构光控制装置113a控制一维移动平台116带动相位光栅112a在光栅平面内移动d/6，其中d为光栅周期，使得样品载体2a上的干涉条纹的相位移动2π/3，如图6B所示。再按照步骤一的方式记录第一种受激光与第二种受激光。
步骤三，结构光控制装置113a继续控制一维移动平台116带动相位光栅112a，在光栅平面内再继续往前移动d/6，使得样品载体2a上的干涉条纹的相位再次移动2π/3，如图6C所示。之后按照步骤一的方式记录 第一种受激光与第二种受激光。
步骤四，结构光控制装置113a控制一维移动平台116带动相位光栅112a往回移动d/3，回到原点的位置。
步骤五，结构光控制装置113a控制转台117带动相位光栅112a和起偏器111a，沿着光栅平面转动60度。再依次按照步骤一至步骤四的方式记录结构光的干涉条纹在不同相位时，产生的第一种受激光与第二种受激光。相应的干涉条纹如图6D-6F所示。
步骤六，结构光控制装置113a控制转台117带动相位光栅112a和起偏器111a，沿着光栅平面继续往同一方向转动60度。再依次按照步骤一至步骤四的方式记录结构光的干涉条纹在不同相位时，产生的第一种受激光与第二种受激光。相应的干涉条纹如图6G-6I所示。
以上获得了每种受激光的9幅图像。利用每一受激光的9幅图像进行超分辨率重建，可得到一幅该受激光的超分辨率图像。对两种受激光的超分辨率图像进行生物特征识别，可以得到该视场下样品载体2a上所有生物样品的生物特征。
第二种超分辨成像方法如下：
步骤一，启动激发光源10a，激发光源10a先输出第一波长的激发光，所述第一波长激发光经由超分辨成像系统1a后输出结构光照射样品载体2a，在样品载体2a上产生干涉条纹如图7A所示，所述结构光激发样品载体2a上的其中一种荧光标记物，产生第一种受激光，所述第一种受激光被相机141a记录，接着，切换激发光源10a输出第二波长的激发光，所述第二波长的激发光激发样品载体2a上的另一种荧光标记物，产生第二种受激光，所述第二种受激光被相机141a记录。
步骤二，结构光控制装置113a接着控制一维移动平台116带动相位光栅112a，在光栅平面内移动d/4，其中d为光栅周期，使得样品载体2a上的干涉条纹的相位移动π/2，如图7B所示。之后按照步骤一的方式记录第一种受激光与第二种受激光。
步骤三，结构光控制装置113a控制一维移动平台116带动相位光栅 112a往回移动d/4，回到原点的位置。
步骤四，结构光控制装置113a控制转台117带动相位光栅112a和起偏器111a，沿着光栅平面转动60度。再按照步骤一的方式记录结构光的干涉条纹产生的第一种受激光与第二种受激光。相应的干涉条纹如图7c所示。
步骤五，结构光控制装置113a控制转台117带动相位光栅112a和起偏器111a，沿着光栅平面继续往同一方向转动60度。再按照步骤一的方式记录结构光的干涉条纹产生的第一种受激光与第二种受激光。相应的干涉条纹如图7d所示。
以上获得了每种受激光的4幅图像。利用每一受激光的4幅图进行超分辨率重建，可得到一幅该受激光的超分辨率图像。对两种受激光的超分辨率图像进行生物特征识别，可以得到该视场下样品载体2a上所有生物样品的生物特征(如DNA样品的碱基)。
第三种超分辨成像方法如下：
步骤一，启动激发光源10a，激发光源10a先输出第一波长的激发光，所述第一波长激发光经由超分辨成像系统1a后输出结构光照射样品载体2a，在样品载体2a上产生干涉条纹如图8a所示，所述结构光激发样品载体2a上的其中一种荧光标记物，产生第一种受激光，所述第一种受激光被相机141a记录，接着，切换激发光源10a输出第二波长的激发光，所述第二波长的激发光激发样品载体2a上的另一种荧光标记物，产生第二种受激光，所述第二种受激光被相机141a记录。
步骤二，结构光控制装置113a接着控制一维移动平台116带动相位光栅112a，在光栅平面内移动d/4，其中d为光栅周期，使得样品载体2a上的干涉条纹的相位移动π/2，如图8b所示。之后按照步骤一的方式记录第一种受激光与第二种受激光。
步骤三，结构光控制装置113a控制一维移动平台116带动相位光栅112a往回移动d/4，回到原来的位置。
步骤四，结构光控制装置113a控制转台117带动相位光栅112a和起偏器111a，沿着光栅平面转动90度。再依次按照步骤一至步骤三的方式记录结构光的干涉条纹在不同相位下的第一种受激光与第二种受激光。相应的干涉条纹，如图8c所示。
步骤五，结构光控制装置113a控制一维移动平台116带动相位光栅112a，在光栅平面内移动d/4光栅周期，使得样品载体2a上的干涉条纹的相位移动π/2，如图8b所示。之后按照步骤一至步骤三的方式记录结构光的干涉条纹在不同相位时，产生的第一种受激光与第二种受激光。
步骤六，结构光控制装置113a控制一维移动平台116带动相位光栅112a往回移动d/4，回到原点的位置。在其他实施例中，该步骤也可省略。
以上获得了每种受激光的4幅图。利用每一受激光的4幅图进行超分辨率重建，可得到一幅该受激光的超分辨率图像。对两种受激光的超分辨率图像进行生物特征识别，可以得到该视场下样品载体2a上所有生物样品的生物特征。
以上实施方式介绍的超分辨成像系统和方法可用于核酸测序中，具体地，本申请还提供一种核酸测序成像系统与核酸测序成像方法，请参阅图9所示，所述核酸测序成像系统5包括一超分辨成像系统51，所述超分辨成像系统51可以是上述实施方式介绍的任一个超分辨成像系统或者由上述任一个超分辨成像系统改进所获得的超分辨成像系统，所述超分辨成像系统51激发核酸样品产生受激光并拍摄核酸样品出射的受激光的图像。所述核酸测序成像方法采用一超分辨成像方法激发核酸样品出射受激光并拍摄所述受激光的图像，所述超分辨成像方法可以是上述实施方式介绍的任一个超分辨成像方法或者由上述任一个超分辨成像方法改进所获得的超分辨成像方法。
以上实施方式介绍的生物样品识别系统和方法可用于核酸测序中，具体地，本申请还提供一种核酸识别系统与核酸识别方法，请参阅图10 所示，所述核酸识别系统6包括一生物样品识别系统61，所述生物样品识别系统61可以是上述实施方式介绍的任一个生物样品识别系统或者由上述任一个生物样品识别系统改进所获得的生物样品识别系统，所述生物样品识别系统61激发核酸样品产生受激光并拍摄核酸样品出射的受激光的图像，并根据所述图像识别核酸样品的碱基类别。所述核酸识别方法采用一生物样品识别方法识别核酸样品的碱基类别，所述生物样品识别方法可以是上述实施方式介绍的任一个生物样品识别方法或者由上述任一个生物样品识别方法改进所获得的生物样品识别方法。
综上所述，本发明实施方式提供的超分辨成像系统与方法、生物样品识别系统与方法、核酸测序成像系统与方法及核酸识别系统与方法，通过改变结构光输出照明图案的方向与相位获得受激光的多张(例如4张)图像，利用该多张图像进行图像重构获得超分辨图像，通过超分辨图像即可识别生物样品的类别和布局；如应用于核酸测序时，可识别碱基的类别和布局。由于所需拍摄的受激光图像数量较少，提高了生物样品识别的速度；应用于核酸测序时，可提高碱基识别的速度。而采用超分辨成像技术，可以提高样品载体上的生物样品密度，从而解决了现有技术中采用普通宽场荧光显微成像技术导致的成像效率低、样品布局密度受限、样品载体利用率不高等问题；应用于核酸测序时，可解决测序通量低、芯片密度受限、芯片利用率不高、试剂利用率不高等问题。
最后应说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或等同替换，而不脱离本发明技术方案的精神和范围。

Claims (25)

  1. 一种超分辨成像系统，所述超分辨成像系统包括照明系统与成像系统，所述照明系统用于输出激发光照射生物样品，以生成受激光，所述成像系统用于收集及记录所述受激光以生成受激光图像，其特征在于，所述照明系统包括激发光源与结构光产生及调制装置，所述激发光源用于输出所述激发光，所述结构光产生及调制装置用于将所述激发光调制成结构光照射所述生物样品以产生所述受激光，所述结构光产生及调制装置包括结构光控制装置，所述结构光控制装置用于控制所述结构光产生及调制装置以改变所述结构光产生及调制装置输出的结构光的相位与所述结构光投射于所述生物样品上的照明图案的方向，所述成像系统用于配合所述结构光相位及/或照明图案的方向的改变拍摄至少一成像视场内至少一波长的激发光激发的受激光的图像。
  2. 如权利要求1所述的超分辨成像系统，其特征在于，所述结构光产生及调制装置还包括偏振控制系统与衍射分光装置，所述偏振控制系统用于调整所述激发光的偏振方向，所述衍射分光装置用于将所述激发光分成多束以形成具有特定相位及特定照明图案方向的所述结构光，所述结构光控制装置用于控制所述衍射分光装置以改变所述结构光的相位与照明图案的方向、及用于控制所述偏振控制系统以使任一相位与任一方向下的所述结构光的照明图案符合预设要求。
  3. 如权利要求2所述的超分辨成像方法，其特征在于，所述结构光控制装置用于控制所述偏振控制系统使所 述结构光的照明图案的对比度符合预设的要求。
  4. 如权利要求3所述的超分辨成像系统，其特征在于，所述结构光控制装置用于控制所述衍射分光装置直线移动以改变所述结构光的相位，及控制所述衍射分光装置转动以改变所述结构光照明图案的方向。
  5. 如权利要求4所述的超分辨成像系统，其特征在于，所述超分辨成像系统还包括物镜，所述结构光产生及调制装置还包括聚焦装置，所述聚焦装置用于将所述衍射分光装置出射的所述结构光聚焦至所述物镜，所述物镜用于将所述结构光以平行光出射至所述生物样品上、并以一定夹角在所述生物样品所在平面形成干涉条纹。
  6. 如权利要求5所述的超分辨成像系统，其特征在于，所述衍射分光装置包括相位光栅，所述偏振控制系统包括起偏器。
  7. 如权利要求6所述的超分辨成像系统，其特征在于，所述相位光栅和所述起偏器一起设置于一维移动平台上，所述一维移动平台设置于转台上，所述结构光控制装置用于控制所述一维移动平台带动所述相位光栅和所述起偏器移动以改变所述结构光的相位、及用于控制所述转台带动所述相位光栅和所述起偏器转动以改变所述结构光照明图案的方向。
  8. 如权利要求6所述的超分辨成像系统，其特征在于，所述聚焦装置包括第一透镜、第二透镜及第三透镜，所述第一透镜与所述第二透镜之间设置挡件，所述挡件用于遮挡并阻止部分所述结构光进入后续光路。
  9. 如权利要求8所述的超分辨成像系统，其特征在于， 所述起偏器用于将所述激发光转换成线偏振光，所述相位光栅用于将所述线偏振光转换成+1级、-1级和0级衍射光，所述0级衍射光被所述挡板遮挡，所述+1级衍射光与-1级衍射光经过所述第二透镜、第三透镜后被聚焦至所述物镜的后焦面。
  10. 如权利要求3所述的超分辨成像系统，其特征在于，所述结构光产生及调制装置还包括自适应光学装置，所述自适应光学装置用于对所述结构光的波前进行整形。
  11. 如权利要求10所述的超分辨成像系统，其特征在于，所述结构光控制装置还用于控制所述自适应光学装置以优化所述结构光的对比度及/或均匀度。
  12. 如权利要求1所述的超分辨成像系统，其特征在于，还包括总控制装置，所述总控制装置用于控制所述激发光源、所述结构光产生及调制装置及所述成像系统协调工作。
  13. 一种超分辨成像方法，其特征在于，包括：
    控制一超分辨成像系统中的照明系统的激发光源启动，使所述照明系统输出结构光照射至生物样品上，使所述生物样品产生受激光；
    控制所述照明系统输出的所述结构光处于第一相位及所述结构光的照明图案处于第一方向，拍摄所述受激光的第一图像；
    控制所述照明系统输出的所述结构光处于不同于所述第一相位的第二相位及所述结构光的照明图案处于所述第一方向，拍摄所述受激光的第二图像；
    控制所述照明系统输出的所述结构光处于所述第二相 位及所述结构光的照明图案处于不同于所述第一方向的第二方向，拍摄所述受激光的第三图像；及/或
    控制所述照明系统输出的所述结构光处于所述第二相位及所述结构光的照明图案处于不同于所述第一方向、第二方向的第三方向，或者，控制所述照明系统输出的所述结构光处于所述第一相位及所述结构光的照明图案处于所述第二方向，拍摄所述受激光的第四图像。
  14. 如权利要求12所述的超分辨成像方法，其特征在于，通过直线移动所述照明系统的衍射分光装置控制所述结构光处于不同相位，通过转动所述衍射分光装置控制所述结构光的照明图案处于不同方向，及通过控制偏振控制系统移动或转动使每个方向和每个相位下的所述结构光的照明图案符合预设要求。
  15. 如权利要求13所述的超分辨成像方法，其特征在于，使所述结构光的照明图案符合预设要求包括使所述结构光的照明图案的对比度符合预设要求。
  16. 如权利要求12所述的超分辨成像方法，其特征在于，还包括：切换所述激发光的波长并重复执行上述切换结构光相位及/或方向及拍摄受激光图像的步骤。
  17. 如权利要求12所述的超分辨成像方法，其特征在于，还包括：在每一切换结构光相位及/或方向及拍摄受激光图像的步骤完成后还包括：切换所述激发光的波长并重复执行该切换及拍摄的步骤。
  18. 如权利要求12所述的超分辨成像方法，其特征在于，还包括：切换所述超分辨成像系统的成像视场并重复执行上述切换结构光相位及/或方向及拍摄受激光图像的步骤。
  19. 如权利要求12所述的超分辨成像方法，其特征在于，还包括：在每一切换结构光相位及/或方向及拍摄受激光图像的步骤完成后还包括：切换所述超分辨成像系统的成像视场并重复执行该切换及拍摄的步骤。
  20. 一种生物样品识别系统，其特征在于，包括：
    如权利要求1-12任一项所述的超分辨成像系统；及图像重建与生物特征识别装置，用于接收所述超分辨成像系统输出的受激光图像，并基于同一成像视场下同一波长激发光激发的受激光的图像，执行图像重建以获取重建后的超分辨图像，及根据重建后的超分辨图像识别所述成像视场内所述受激光对应的生物样品的生物特征。
  21. 一种生物样品识别方法，其特征在于，包括：
    采用如权利要求13-19任一项所述的超分辨成像方法获得至少一成像视场内同一波长激发光激发的受激光的图像；
    基于同一成像视场下同一波长激发光激发的受激光的所述图像，执行图像重建以获取重建后的超分辨图像；及
    根据重建后的超分辨图像识别所述成像视场内所述受激光对应的生物样品的生物特征。
  22. 一种核酸测序成像系统，其特征在于，包括权利要求1-12任一项所述的超分辨成像系统，所述超分辨成像系统用于照射核酸样品并拍摄所述核酸样品出射的受激光的图像。
  23. 一种核酸测序成像方法，其特征在于，所述核酸测序成像方法采用如权利要求13-19任一项所述的超分辨 成像方法激发核酸样品出射受激光并拍摄所述受激光的图像。
  24. 一种核酸识别系统，其特征在于，包括权利要求20所述的生物样品识别系统，所述生物样品识别系统用于识别核酸样品的碱基的类别。
  25. 一种核酸识别方法，其特征在于，所述核酸识别方法采用权利要求21所述的生物样品识别方法识别核酸样品的碱基的类别。
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