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WO2024040871A1 - 基因测序仪及基因测序仪的使用方法 - Google Patents

基因测序仪及基因测序仪的使用方法 Download PDF

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Publication number
WO2024040871A1
WO2024040871A1 PCT/CN2023/074142 CN2023074142W WO2024040871A1 WO 2024040871 A1 WO2024040871 A1 WO 2024040871A1 CN 2023074142 W CN2023074142 W CN 2023074142W WO 2024040871 A1 WO2024040871 A1 WO 2024040871A1
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WO
WIPO (PCT)
Prior art keywords
signal
lens
laser
sequencing
dichroic mirror
Prior art date
Application number
PCT/CN2023/074142
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English (en)
French (fr)
Inventor
陈龙超
梁倩
王谷丰
赵陆洋
Original Assignee
深圳赛陆医疗科技有限公司
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Publication of WO2024040871A1 publication Critical patent/WO2024040871A1/zh

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    • 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
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B13/00—Optical objectives specially designed for the purposes specified below
    • G02B13/001—Miniaturised objectives for electronic devices, e.g. portable telephones, webcams, PDAs, small digital cameras
    • G02B13/0015—Miniaturised objectives for electronic devices, e.g. portable telephones, webcams, PDAs, small digital cameras characterised by the lens design
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B13/00—Optical objectives specially designed for the purposes specified below
    • G02B13/001—Miniaturised objectives for electronic devices, e.g. portable telephones, webcams, PDAs, small digital cameras
    • G02B13/0055—Miniaturised objectives for electronic devices, e.g. portable telephones, webcams, PDAs, small digital cameras employing a special optical element
    • G02B13/006—Miniaturised objectives for electronic devices, e.g. portable telephones, webcams, PDAs, small digital cameras employing a special optical element at least one element being a compound optical element, e.g. cemented elements
    • 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
    • G02B27/09—Beam shaping, e.g. changing the cross-sectional area, not otherwise provided for
    • G02B27/0927—Systems for changing the beam intensity distribution, e.g. Gaussian to top-hat
    • 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
    • G02B27/09—Beam shaping, e.g. changing the cross-sectional area, not otherwise provided for
    • G02B27/0938—Using specific optical elements
    • G02B27/0977—Reflective elements
    • 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
    • G02B27/09—Beam shaping, e.g. changing the cross-sectional area, not otherwise provided for
    • G02B27/0938—Using specific optical elements
    • G02B27/0988—Diaphragms, spatial filters, masks for removing or filtering a part of the beam

Definitions

  • the present invention relates to the technical field of medical equipment, and in particular to a gene sequencer and a method of using the gene sequencer.
  • Gene sequencers in related technologies cannot meet high-standard detection requirements due to low requirements for optical system design indicators.
  • the objective lenses of some gene sequencers have low sequencing throughput, and the detection time is long and inefficient.
  • the laser light used by some gene sequencers to excite fluorescent dyes conforms to a Gaussian distribution.
  • the light intensity at the edge is weak, the excitation illumination effect is poor, and the excitation efficiency is low, which affects the efficiency of scanning imaging and the accuracy of subsequent sequencing detection results, and cannot meet high detection needs.
  • the main purpose of the embodiments of the present invention is to propose a gene sequencer and a method of using the gene sequencer.
  • a microscopic objective lens with a large numerical aperture and a large imaging field of view in the gene sequencer it can satisfy high-throughput sequencing application scenarios. Improve detection efficiency.
  • the Gaussian spot can be homogenized to avoid unnecessary photobleaching, improve the efficiency of scanning imaging and the accuracy of subsequent sequencing results.
  • the first aspect of the embodiment of the present invention proposes a gene sequencer, which is used to excite the sample to be detected on the gene sequencing chip and collect the fluorescent signal emitted by the sample to be detected for fluorescence imaging, including:
  • An excitation module used to generate an excitation beam that excites the sample to be detected
  • a sequencing module configured to use the excitation beam to perform fluorescence imaging of the sample to be detected
  • a light source the light source is used to generate a laser signal
  • the aperture is placed behind the light source along the optical axis of the laser signal, and the aperture is used to spatially filter the incident laser signal to form a filtered signal;
  • Even-order aspherical reflector the even-order aspherical reflector is placed behind the aperture along the optical axis of the filtered signal, and the even-order aspherical reflector is used to form an excitation beam according to the filtered signal. ;
  • the sequencing module includes:
  • a high-throughput objective lens for receiving and focusing the excitation beam onto the sequencing unit
  • a sequencing unit configured to use the excitation beam to irradiate the sample to be detected to generate a fluorescent signal
  • At least one imaging unit for performing fluorescence imaging using the fluorescence signal
  • the high-throughput objectives include:
  • the first lens group, the second lens group and the third lens group are coaxially arranged in sequence from the object side to the image side, wherein,
  • the first lens group includes a first meniscus lens and a second meniscus lens arranged in sequence;
  • the second lens group includes a first lenticular lens, a first biconcave lens, a second lenticular lens, a third lenticular lens, a third meniscus lens and a fourth lenticular lens arranged in sequence.
  • the first lenticular lens, the third lenticular lens A biconcave lens and the second biconvex lens form a first cemented lens
  • the third meniscus lens and the fourth biconvex lens form a second cemented lens
  • the third biconvex lens has positive optical power
  • the third lens group includes a fourth meniscus lens, a fifth meniscus lens, a second biconcave lens and a fifth biconvex lens arranged in sequence.
  • the fourth meniscus lens and the fifth meniscus lens form a third cemented lens.
  • Lens, the second biconcave lens and the fifth biconvex lens form a fourth cemented lens.
  • the sequencing module further includes: a second dichroic mirror and a relay lens group;
  • the second dichroic mirror is used to transmit the fluorescence signal emitted by the sequencing unit to the relay lens group to form a first fluorescence signal.
  • the sequencing module further includes: a relay lens group, the relay lens group includes a fourth lens group and a fifth lens group;
  • the high-flux objective lens is used to receive the fluorescence signal and transmit the fluorescence signal to the second dichroic mirror;
  • the fourth lens group of the relay lens group has negative optical power, and the fourth lens group has negative optical power.
  • a lens group is arranged behind the objective lens along the optical axis of the fluorescence signal, and is used to form a first optical signal according to the fluorescence signal;
  • the fifth lens group has positive optical power, and is disposed behind the fourth lens group along the optical axis of the first optical signal for forming the first optical signal.
  • First fluorescent signal is provided.
  • the imaging unit includes: a tube lens and a camera
  • the sequencing module further includes: a third dichroic mirror and two of the imaging units;
  • the third dichroic mirror is used to reflect the first fluorescence signal to obtain a first imaging signal
  • the third dichroic mirror is also used to transmit the first fluorescence signal to obtain a second imaging signal
  • the tube lenses of the two imaging units are respectively used to receive the first imaging signal and the second imaging signal, and output optical signals to corresponding cameras to use the optical signals to perform fluorescence imaging.
  • the sequencing unit includes:
  • a sequencing chip used to carry the sample to be detected
  • a displacement stage is used to place the sequencing chip to use the excitation beam to irradiate the sample to be detected to generate a fluorescent signal.
  • the gene sequencer further includes: a first dichroic mirror and an autofocus module;
  • the first dichroic mirror is used to transmit focusing laser signals to the autofocus module
  • the autofocus module is configured to generate a relative height measurement signal according to the focusing laser signal and send it to the sequencing module;
  • the sequencing module is configured to adjust the height relative displacement between the high-throughput objective lens and the sequencing chip, the relative height measurement signal, and the relative height measurement signal according to the relative height measurement signal.
  • the autofocus module includes: a beam expander, a fourth dichroic mirror, a fifth dichroic mirror, a sixth dichroic mirror, two laser signal transmitting and filtering units and a computing unit laser signal transmitting and filter unit;
  • the fourth dichroic mirror is used to combine the collimated laser signals respectively emitted by the laser emission units of the two laser signal emission and filtering units to generate a combined laser signal;
  • the beam expander is used to expand the combined laser signal to obtain an expanded laser signal
  • the fifth color mirror is used to reflect the expanded beam laser signal to the sequencing chip through the high-flux objective lens, and transmit the light spot reflected from the sequencing chip transmitted by the high-flux objective lens;
  • the sixth dichroic mirror is used to reflect the light spot to obtain a first focusing light signal
  • the sixth dichroic mirror is also used to transmit the light spot to obtain a second focusing light signal
  • the filtering units of the two laser signal transmitting and filtering units are respectively used to receive the first focusing light signal and the second focusing light signal;
  • the calculation unit is configured to calculate and generate a relative height measurement signal based on the signal intensity ratio output by the filter unit of the two laser signal transmitting and filtering units.
  • the laser emitting unit includes: a laser diode and a collimating mirror;
  • the laser diode is used to emit laser signals to the collimating mirror;
  • the collimating mirror is used to collimate the laser signal to generate a collimated laser signal.
  • the filtering unit includes a converging mirror, a pinhole filter and a photodiode
  • the condensing mirror is used to receive the first focusing light signal or the second focusing light signal and converge it to the corresponding pinhole filter;
  • the pinhole filter is used to filter the converged first focusing light signal or the second focusing light signal to obtain a corresponding filtered signal
  • the photodiode is used to receive the corresponding filtered signal.
  • the calculation unit is also used to calculate the signal intensity ratio of the filtered signal received by the two corresponding photodiodes;
  • the calculation unit is further configured to generate the relative height measurement signal according to the corresponding relationship between the signal intensity ratio and the preset objective lens defocus amount.
  • the first dichroic mirror is used to transmit the expanded beam laser signal reflected by the fifth color mirror, and output the transmitted laser signal to the second dichroic mirror;
  • the second dichroic mirror is used to reflect the transmitted laser signal to the high-flux objective lens
  • the second dichroic mirror is also used to reflect the light spot formed by the high-flux objective lens reflecting the transmitted laser signal to the first dichroic mirror;
  • the first dichroic mirror and the fifth dichroic mirror transmit the light spot to the sixth dichroic mirror in sequence.
  • the surface shape formula of the even-order aspherical mirror satisfies the following relationship:
  • c is the curvature
  • k is the cone coefficient
  • a 1 is the second-order aspheric coefficient
  • a 2 is the fourth-order aspheric coefficient
  • a 3 is the sixth-order aspheric coefficient
  • a 4 is the eighth-order aspheric coefficient
  • x is the coordinate position of the aspheric surface.
  • the fourth lens group includes:
  • a first lens, the first lens is a biconcave lens with negative optical power
  • the second lens is cemented and connected to the first lens, and the second lens is a meniscus lens with positive optical power.
  • the fifth lens group includes:
  • a third lens is arranged behind the first lens group along the optical axis of the first optical signal, and the third lens is a biconvex lens with positive optical power;
  • the fourth lens is cemented and connected to the third lens, and the fourth lens is a meniscus lens with negative refractive power.
  • a second aspect of the embodiment of the present invention proposes a method of using a gene sequencer, which is applied to the gene sequencer described in any one of the first aspects.
  • the method includes:
  • the sample to be detected includes single-stranded DNA and four nucleotides, and the color of the fluorescence signal of each nucleotide is different;
  • the excitation module generates an excitation beam that excites the sample to be detected
  • the sequencing module adjusts the relative height displacement between the high-throughput objective lens and the sequencing chip according to the relative height measurement signal, and uses the excitation beam to scan the sample to be detected to obtain a fluorescence signal;
  • the high-throughput objective lens collects the fluorescence signal and sends it to the imaging unit;
  • the image obtained by imaging the fluorescent signal by the imaging unit is used to determine the sequence of nucleotides in the single-stranded DNA in the sample to be detected.
  • the sequencing module adjusts the height relative displacement between the high-throughput objective lens and the sequencing chip according to the relative height measurement signal, and further includes:
  • the relative height displacement between the high-throughput objective lens and the sequencing chip is adjusted in the vertical direction according to the relative height measurement signal, so that the short axis of the excitation beam moves relative to the long side of the sequencing chip, and The short axis of the excitation beam and the long side of the sequencing chip are parallel to each other;
  • the fluorescence signal of the sample to be detected is obtained.
  • adjusting the relative height displacement between the high-throughput objective lens and the sequencing chip in the vertical direction according to the relative height measurement signal includes:
  • the autofocus module reflects the laser collimation signal and converges it on the sequencing chip through a high-flux objective lens, and receives the light spot reflected by the sequencing chip to the high-flux objective lens;
  • the autofocus module uses the light spot to generate a first focusing light signal and a second focusing light signal;
  • the autofocus module generates the relative height measurement signal according to the first focusing light signal and the second focusing light signal;
  • the sequencing chip adjusts the relative height displacement according to the relative height measurement signal.
  • the imaging surface of the camera is a rectangular imaging surface, and the shape of the excitation beam is elliptical, elliptical, or rectangular; the length-to-width ratio of the rectangular imaging surface is L:W; the excitation beam The ratio of the major and minor axes of the light spot is L:W; where L and W are both positive integers.
  • the gene sequencer includes an excitation module and a sequencing module.
  • the excitation module includes: a light source, an aperture, and an even-order aspheric reflection.
  • the sequencing module includes: a high-throughput objective lens, a sequencing unit and at least one imaging unit.
  • a high-throughput microscope objective lens with a large numerical aperture and a large imaging field of view is designed in the gene sequencer to meet high-throughput sequencing application scenarios and improve detection efficiency.
  • an even-order aspherical reflector is used in combination with an aperture.
  • the Gaussian spot is homogenized with a simple optical path, effectively avoiding unnecessary photobleaching, improving the efficiency of scanning imaging and the accuracy of subsequent sequencing results.
  • Figure 1 is a schematic structural diagram of a gene sequencer provided by an embodiment of the present invention.
  • Figure 2 is a schematic structural diagram of an excitation module of a gene sequencer provided by another embodiment of the present invention.
  • Figure 3 is a light intensity distribution diagram in the long axis direction of the spot of the excitation beam of the excitation module of the gene sequencer provided by another embodiment of the present invention.
  • Figure 4 is a light intensity distribution diagram in the short-axis direction of the spot of the excitation beam of the excitation module of the gene sequencer provided by another embodiment of the present invention.
  • Figure 5 is a schematic structural diagram of a sequencing module of a gene sequencer provided by another embodiment of the present invention.
  • Figure 6 is a schematic structural diagram of a high-throughput objective lens of a gene sequencer provided by another embodiment of the present invention.
  • Figure 7 is a schematic diagram of the position of the relay lens group of the gene sequencer provided by another embodiment of the present invention.
  • Figure 8 is a schematic structural diagram of a relay lens group of a gene sequencer provided by another embodiment of the present invention.
  • Figure 9 is a schematic structural diagram of a sequencing module of a gene sequencer provided by another embodiment of the present invention.
  • Figure 10 is a schematic structural diagram of the imaging unit of a gene sequencer provided by yet another embodiment of the present invention.
  • Figure 11 is a schematic structural diagram of a sequencing module of a gene sequencer provided by yet another embodiment of the present invention.
  • Figure 12 is a schematic structural diagram of a sequencing unit of a gene sequencer provided by another embodiment of the present invention.
  • Figure 13 is a schematic structural diagram of the autofocus module of a gene sequencer provided by another embodiment of the present invention.
  • Figure 14 is a schematic structural diagram of the autofocus module of a gene sequencer provided by another embodiment of the present invention.
  • Figure 15 is a schematic structural diagram of a gene sequencer provided by yet another embodiment of the present invention.
  • Figure 16 is a flow chart of a method for using a gene sequencer provided by an embodiment of the present invention.
  • Figure 17 is an imaging schematic diagram of a method of using a gene sequencer provided by yet another embodiment of the present invention.
  • Gene sequencer 110 excitation module 200, sequencing module 300 and autofocus module 400;
  • the excitation module 200 includes: a light source 210, an aperture 220, and an even-order aspherical reflector 230;
  • the sequencing module 300 includes: a high-throughput objective lens 310, a sequencing unit 320, a sample to be detected 330, an imaging unit 340, a second dichroic mirror 350, a relay lens group 360 and a third dichroic mirror 370;
  • the high-flux objective lens 310 includes: a first lens group G1, a second lens group G2, and a third lens group G3;
  • the sequencing unit 320 includes: a sequencing chip 321 and a displacement stage 322;
  • the imaging unit 340 includes: a tube lens 341 and a camera 342;
  • the relay lens group 360 includes: the high-flux objective lens 310, the fourth lens group 362, the fifth lens group 363, the first lens 364, the second lens 365, the third lens 366 and the fourth lens 367;
  • the first dichroic mirror 410 and the autofocus module 400 includes: a fourth dichroic mirror 420, a fifth dichroic mirror 430, a sixth dichroic mirror 440, a beam expander 450, and a first laser emission Unit 461, first laser diode 4611, first collimating mirror 4612, second laser emitting unit 462, second laser diode 4621, second collimating mirror 4622, first filtering unit 464, first condensing mirror 4641, first Pinhole filter 4642, first photodiode 4643, second filter unit 463, second condensing mirror 4631, second pinhole filter 4632, second photodiode 4633 and calculation unit 470.
  • Microscopic imaging technology is widely used in sample detection.
  • fluorescence imaging of bases on biochips is required.
  • Gene sequencers are widely used in the fields of medicine and life sciences, such as detection of pathogens, genetic diseases, and tumor genes, as well as personalized drug treatment and non-invasive prenatal testing.
  • the gene sequencer works, it needs to perform fluorescence imaging of the bases on the biochip.
  • sequencing with a gene sequencer it is necessary to perform fluorescence imaging on the four bases of ATGC, namely adenine (A), thymine (T), cytosine (C) and guanine (G).
  • Multi-channel such as four-channel
  • the application of gene sequencing is becoming more and more widespread, and the detection needs are also diversified. Therefore, the detection efficiency and detection accuracy requirements of gene sequencers are also increasing.
  • some current gene sequencers cannot meet high-standard detection needs.
  • the objective lenses of some gene sequencers have low sequencing throughput, and the detection is time-consuming and inefficient.
  • the laser light used by some gene sequencers to excite fluorescent dyes conforms to a Gaussian distribution. The light intensity located in the center of the imaging field of view is strong, while the light intensity located at the edge of the imaging field of view is weak, resulting in poor excitation illumination effect and low excitation efficiency, which affects the efficiency of scanning imaging and the accuracy of subsequent sequencing detection results, and cannot meet high detection needs. .
  • embodiments of the present invention provide a gene sequencer and a method of using the gene sequencer.
  • a high-throughput microscope objective lens with a large numerical aperture and a large imaging field of view is designed in the gene sequencer to meet high-throughput sequencing application scenarios. , improve detection efficiency, and use an even-order aspherical mirror combined with an aperture to achieve homogenization of the Gaussian spot in a simple optical path, effectively avoiding unnecessary photobleaching, improving the efficiency of scanning imaging and the accuracy of subsequent sequencing results. Rate.
  • the embodiments of the present invention provide a gene sequencer and a method of using the gene sequencer, which are specifically described in the following examples. First, the gene sequencer in the embodiment of the present invention is described.
  • Figure 1 is a schematic structural diagram of a gene sequencer provided by an embodiment of the present invention.
  • the gene sequencer 100 is used to excite the sample to be detected on the gene sequencing chip, and collect the fluorescence signal emitted by the sample to be detected for fluorescence imaging, including:
  • the excitation module 200 is used to generate an excitation beam S that excites the sample to be detected.
  • the sequencing module 300 is used to use the excitation beam S to perform fluorescence imaging on the sample to be detected.
  • the excitation beam S generated by the excitation module 200 can be a laser.
  • the gene sequencer 100 can generate a laser signal through the excitation module 200 to form a corresponding illumination area and illuminate the sample to be detected.
  • the fluorescent dye in the illumination area is excited and illuminated, so that the sample to be detected generates a corresponding fluorescent signal under the excitation of the laser signal, and the fluorescent signal is imaged through the camera, so that the gene sequence can be detected.
  • the laser has the characteristics of Gaussian distribution, the light intensity in the center of the illuminated area is stronger, while the light intensity at the edge of the illuminated area is weaker, resulting in poor excitation lighting effect.
  • an even-order aspherical mirror is used in combination with an aperture to achieve homogenization of the Gaussian light spot in a simple optical path, effectively avoiding unnecessary photobleaching and improving the efficiency of scanning imaging and the accuracy of subsequent sequencing results.
  • the light source 210 is used to generate a laser signal.
  • the light source 210 is used to generate a collimated laser signal, and the spot shape of the laser signal is circular.
  • the aperture 220 is placed behind the light source 210 along the optical axis of the laser signal.
  • the aperture 220 is used to spatially filter the incident laser signal to form a filtered signal.
  • the aperture 220 refers to a device that limits the light beam in the optical system.
  • the aperture 220 can filter the beam of the laser signal, so that The edge portion with weak light intensity of the laser signal is blocked to facilitate the subsequent even-order aspherical reflector 230 to further homogenize the laser signal.
  • the even-order aspheric reflector 230 is placed behind the diaphragm 220 along the optical axis of the filtered signal.
  • the even-order aspheric reflector 230 is used to form the excitation beam S according to the filtered signal, wherein the excitation beam S is used to excite the sample to be detected to generate a fluorescent signal.
  • the laser signal is a circular spot with Gaussian distribution, that is, within the spot area (illumination area) of the laser signal, the light intensity exhibits Gaussian distribution characteristics.
  • the excitation module 200 is provided with a corresponding even-order aspherical reflector 230.
  • the even-order aspherical reflector 230 is used to perform a uniform light operation on the incident filtered signal to form an excitation waveform with a certain shape and size. The beam illuminates the spot evenly. Compared with the illumination spot that has not been subjected to the uniform light operation and is only spatially filtered by the diaphragm, the light intensity distribution in the illumination area of the excitation beam is more uniform.
  • the even-order aspherical mirror 230 is also used to reflect the excitation beam, so that the excitation beam irradiates the sample 330 to be detected.
  • the surface shape formula of the even-order aspherical mirror 230 satisfies the following relationship:
  • c is the curvature
  • k is the cone coefficient
  • a 1 is the second-order aspheric coefficient
  • a 2 is the fourth-order aspheric coefficient
  • a 3 is the sixth-order aspheric coefficient
  • a 4 is the eighth-order aspheric coefficient
  • x is the coordinate position of the aspheric surface.
  • an excitation beam with an elliptical spot can be obtained.
  • specific surface parameters curvature, cone coefficient, second-order aspheric coefficient, fourth-order aspheric coefficient, sixth-order aspheric coefficient, eighth-order aspheric coefficient, and determining the specific coordinate position of the aspheric surface, it is possible to Excitation beams of different shapes and sizes are obtained, that is, the long and short axes of the elliptical spot are different.
  • the excitation beam formed by the even-order aspherical reflector in this embodiment has a good light uniformity effect and can achieve a better excitation lighting effect.
  • FIG. 3 which is a light intensity distribution diagram in the long axis direction of the light spot of the excitation beam in the embodiment of the present application
  • FIG. 4 is a light intensity distribution diagram in the short axis direction of the light spot of the excitation beam in the embodiment of the present application.
  • the illumination uniformity is calculated using the length of the long axis of the excitation beam spot as 1.6 mm and the length of the short axis as 0.8 mm. Specifically, the minimum value of the light intensity in the lighting area is divided by the maximum value of the light intensity to obtain the lighting uniformity. Among them, the uniformity in the long axis direction is 81%, and the uniformity in the short axis direction is 81%. Sex is 85%. It can be understood that when the illumination uniformity reaches more than 75%, the efficiency of the imaging algorithm in extracting effective information about the center and edge of the illumination area corresponding to the photographing position of the camera 323 is close to the same. Therefore, the excitation module 200 of this embodiment can well meet actual usage requirements.
  • the focal length of the even-order aspherical mirror 230 satisfies the following relationship: 14.6 ⁇ f 0 ⁇ 16.1
  • f 0 is the focal length of the even-order aspherical mirror 230 .
  • the even-order aspherical reflector 230 can achieve better shaping and uniform light effects, and generate an excitation beam with a long-to-short axis ratio of 2:1.
  • the surface parameters of the even-order aspherical mirror 230 are changed, its focal length f will also be changed accordingly. This embodiment will not be described one by one here.
  • Sequencing chips are generally rectangular, so the main scanning time is spent on the long sides.
  • the illumination spot is circular, while meeting the illumination requirements of the long side of the camera, it will inevitably greatly exceed the illumination range required by the short side of the camera.
  • the excitation module 200 of this embodiment can perform a uniform light operation on the filtered signal through the even-order aspherical reflector 230 to obtain an excitation beam with uniform light intensity distribution, and illuminate the focal plane of the objective lens with an elliptical spot.
  • the elliptical spot has The long and short axis ratio is close to 2:1, which effectively avoids unnecessary photobleaching.
  • the excitation module 200 of this embodiment can achieve better excitation lighting effects with a simple structure.
  • the even-order aspherical reflector 230 of the excitation module 200 in this embodiment is not only able to perform a uniform light operation on the filtered signal, but is also used to perform a uniform illumination operation on the filtered signal. plastic surgery.
  • the even-order aspherical reflector 230 can shape the shape of the laser signal into an ellipse or a quasi-ellipse, so that the formed excitation beam can illuminate the object to be detected in the illumination area.
  • an elliptical or elliptical-like fluorescence signal is generated, in which the shape and size of the excitation beam matches the imaging surface of the camera, that is, the shape and size of the spot of the fluorescence signal matches.
  • the sample to be detected generates a fluorescence signal under the excitation illumination of the excitation beam, and the shape and size of the excitation beam are the same as the shape and size of the fluorescence signal.
  • the fluorescence signal can characterize the different detection results of the sample to be detected. For example, assume that the samples to be detected contain different gene sequences, and different gene sequences can be labeled by fluorescence of different spectra. When the excitation beam excites and illuminates the sample to be detected, different gene sequences in the sample to be detected can produce fluorescence signals of different spectra after being excited. Therefore, the detection results of the gene sequence of the sample to be detected can be obtained through different fluorescence signals.
  • FIG. 5 is a schematic structural diagram of a sequencing module according to an embodiment of the present application.
  • the sequencing module 300 includes:
  • a high-throughput objective lens 310 is used to receive and converge the excitation beam S to the sequencing unit 320;
  • the sequencing unit 320 is used to use the excitation beam S to irradiate the sample to be detected 330 to generate a fluorescent signal;
  • At least one imaging unit 340 (shown as one imaging unit in the figure) is used to perform fluorescence imaging using fluorescence signals.
  • FIG. 6 is a schematic structural diagram of a high-flux objective lens in an embodiment of the present application.
  • the imaging field of view and the numerical aperture of the objective lens are two key parameters that determine the sequencing throughput.
  • the sizes of these two parameters often trade off. That is, the field of view of a large numerical aperture objective lens is smaller, and the field of view of a large numerical aperture objective lens is smaller.
  • the numerical aperture of the objective lens of the scope is small, and it is impossible to see much at the same time.
  • the high-throughput objective lens 310 in this embodiment is a microscopic objective lens with a large numerical aperture and a large imaging field of view.
  • the high-flux objective lens 310 includes: a first lens group G1, a second lens group G2, and a third lens group G3 that are coaxially arranged in sequence from the object side to the image side, where:
  • the first lens group G1 includes: a first meniscus lens L2 and a second meniscus lens L3 arranged in sequence.
  • the second lens group G2 includes: a first biconvex lens L4, a first biconcave lens L5, a second biconvex lens L6, a third biconvex lens L7, a third meniscus lens L8 and a fourth biconvex lens L9 arranged in sequence.
  • the first biconvex lens L4, the first biconcave lens L5 and the second biconvex lens L6 combine to form a first cemented lens
  • the third meniscus lens L8 and the fourth biconvex lens L9 form a second cemented lens, so
  • the third lenticular lens has positive optical power.
  • the third lens group G3 includes: a fourth meniscus lens L10, a fifth meniscus lens L11, a second biconcave lens L12 and a fifth biconvex lens L13 arranged in sequence.
  • the fourth meniscus lens L10 and the fifth meniscus lens L10 The meniscus lens L11 forms a third cemented lens, and the second biconcave lens L12 and the fifth biconvex lens L13 form a fourth cemented lens.
  • L1 is the cover glass of the sample, or it can be the glass on the upper layer of the flow channel of the sequencing chip.
  • a lens group G1 includes L2 and L3.
  • the first lens group G1 forms a front clear surface, collects large divergence angle light signals and converts them into small angle light signals, effectively increasing the numerical aperture while reducing and avoiding overshoot. Large spherical aberration and/or coma.
  • the second lens group G2 includes lenses L4 to L9. The second lens group G2 is used to correct spherical aberration, coma aberration and/or chromatic aberration.
  • the third lens group G3 includes lenses L10 to L13, which are used to eliminate field curvature, astigmatism and/or chromatic aberration, among which L11 is a thick meniscus lens.
  • the light emitted by the sample to be detected first passes through the first lens group G1 to reduce the incident angle of the fluorescence signal, then passes through the second lens group G2 to correct one or more of spherical aberration, coma aberration or chromatic aberration, and then passes through the third lens group G2
  • the three-lens group G3 eliminates one or more of field curvature, astigmatism, or chromatic aberration, thereby simultaneously increasing the imaging field of view and numerical aperture, and improving sequencing throughput.
  • the focal length of each component in the high-throughput objective lens 310 satisfies the following relationship: 10.2 ⁇ f L23 /f ⁇ 11 6.42 ⁇ f L456 /f ⁇ 7.15 2.91 ⁇ f L7 /f ⁇ 3.32 10.6 ⁇ f L89 /f ⁇ 12.3 -6.01 ⁇ f L1011 /f ⁇ -5.66 -69.1 ⁇ f L1213 /f ⁇ -70.2
  • f represents the focal length of the high-flux objective lens 310
  • f L23 represents the focal length of the first lens group G1
  • f L456 represents the focal length of the first cemented lens
  • f L7 represents the focal length of the third lenticular lens
  • f L89 represents the second cemented lens.
  • the focal length of f L1011 represents the focal length of the third cemented lens
  • f L1213 represents the focal length of the fourth cemented lens. It can be understood that the focal length of each component is only illustrative in this embodiment and is not specifically limited.
  • the numerical aperture of the high-throughput objective lens 310 is 0.75
  • the imaging object-side field of view diameter is 1.6 mm
  • the plan apochromatic is 10 mm.
  • all lenses in the first lens group G1, the second lens group G2, and the third lens group G3 are spherical lenses. That is, all the lenses L2 to L13 in the first lens group G1 to the third lens group G3 are spherical lenses, and the spherical lenses can reduce the processing cost of the lenses.
  • this embodiment adds a relay lens group 360 between the high-flux objective lens 310 and the tube lens of the imaging unit 340 to reduce the field of view angle entering the tube lens, thereby reducing the size of the tube lens.
  • FIG. 7 is a schematic diagram of the position of the relay lens group according to the embodiment of the present application.
  • the sequencing module 300 in Figure 7 also includes: a second dichroic mirror 350 and a relay lens group 360.
  • the high-flux objective lens 310 is used to receive fluorescence signals and transmit the fluorescence signals to the second dichroic mirror 350.
  • the dichroic mirror can transmit part of the light and reflect the other part of the light.
  • the second dichroic mirror 350 350 reflects a part of the received fluorescence signal to the high-flux objective lens 310, and then receives the fluorescence signal of the sample to be detected 330 emitted by the sequencing unit 320 and transmits it to the relay lens group 360 to form a first fluorescence signal.
  • the relay lens group 360 collects, shapes and transmits the first fluorescence signal to the imaging unit 340 for subsequent fluorescence imaging.
  • the relay lens group 360 includes:
  • the relay lens group 360 includes: a fourth lens group 362 and a fifth lens group 363 .
  • the fourth lens group 362 has negative refractive power.
  • the fourth lens group 362 is arranged behind the high-flux objective lens 310 along the optical axis of the fluorescence signal, and is used to correct aberrations of the fluorescence signal and increase the light aperture. to form a first optical signal.
  • the fifth lens group 363 has positive refractive power.
  • the fifth lens group 363 is arranged behind the fourth lens group 362 along the optical axis of the first optical signal. It is used for aberration compensation of the first optical signal, increasing the light aperture and The light exit angle is reduced to form a first fluorescence signal.
  • the optical power of the fourth lens group 362 is used to characterize the convergence or divergence ability of the fourth lens group 362 for light.
  • a negative optical power of the fourth lens group 362 means that the fourth lens group 362 has a negative optical power for light. It has a divergent effect.
  • the optical power of the fifth lens group 363 is used to characterize the convergence or divergence ability of the fifth lens group 363 on light.
  • a positive optical power of the fifth lens group 363 means that the fifth lens group 363 has a convergence effect on light. .
  • the fourth lens group 362 and the fifth lens group 363 are used to jointly balance aberrations, so that the fluorescent signals incident and emitted from the relay lens group 360 are both collimated light beams. Therefore, the fourth lens group 362 and the fifth lens group 363 in this embodiment can not only form the first fluorescence signal with a smaller maximum exit angle, but also ensure good imaging performance.
  • the fourth lens group 362 includes: a first lens 364, which is a biconcave lens with negative power; a second lens 365, which is cemented with the first lens 364. Connected, the second lens 365 is a meniscus lens with positive optical power.
  • the first lens 364 and the second lens 365 are cemented and connected to form a double cemented lens (the fourth lens group 362).
  • the first lens 364 is used to receive the fluorescence signal emitted by the high-flux objective lens 310, divergence the fluorescence signal, and then transmit it to the second lens 365.
  • the second lens 365 receives the diverged fluorescence signal, converges the fluorescence signal, and corrects the field curvature of the fluorescence signal.
  • field curvature refers to field curvature.
  • the second lens 365 can correct the field curvature of the fluorescence signal and form a corresponding first optical signal.
  • the fifth lens group 363 receives the first optical signal and forms a small-angle parallel first fluorescence signal, and the tube lens 341 focuses the first fluorescence signal onto the imaging surface of the camera 342 to perform imaging detection.
  • the first lens 364 satisfies the following relationship: -15.2 ⁇ f T1 ⁇ -10.5, where f T1 is the focal length of the first lens 364; the second lens 365 satisfies the following relationship: 28.5 ⁇ f T2 ⁇ 33.1, Among them, f T2 is the focal length of the second lens 365 .
  • the first lens 364 includes a first incident surface S1 and a first exit surface (not shown in the figure), and the second lens 365 includes a second incident surface (not shown in the figure) and The second exit surface S3; wherein, the first exit surface S1 and the second incident surface are glued and connected to form the first glued surface S2; the curvature radius of the first incident surface S1 is -31.393mm, the thickness is 8mm, and the refractive index is 1.77 , the Abbe number is 49.6; the radius of curvature of the first gluing surface S2 is 15.247mm, the thickness is 8mm, the refractive index is 1.92, and the Abbe number is 20.9; the radius of curvature of the second exit surface is 25.169mm, and the thickness is 10mm.
  • the first incident surface S1 is the incident surface of the first lens 364
  • the first exit surface is the exit surface of the first lens 364 .
  • the first incident surface S1 is used for receiving fluorescent signals
  • the first exit surface is used for emitting fluorescent signals processed by the first lens 364 .
  • the first lens 364 and the second lens 365 are glued and connected, that is, the second incident surface of the second lens 365 and the first exit surface of the first lens 364 are glued and connected to form the first glued surface S2.
  • the fluorescent signal is emitted through the first exit surface and then incident on the second lens 365 through the first gluing surface. After being processed by the second lens 365, a first optical signal is formed.
  • the first optical signal is emitted from the second exit surface S3.
  • the first incident surface S1, the first gluing surface S2 and the second exit surface S3 need to meet corresponding parameter settings.
  • the parameters of the first incident surface, the first gluing surface, and the second exit surface can also be adjusted according to requirements, and this embodiment will not be described one by one here.
  • the fifth lens group 363 includes: a third lens 366.
  • the third lens 366 is disposed behind the fourth lens group 362 along the optical axis of the first optical signal.
  • the third lens 366 has a A biconvex lens with positive power; a fourth lens 367.
  • the fourth lens 367 and the third lens 366 are cemented and connected; the fourth lens 367 is a meniscus lens with negative power.
  • the third lens 366 and the fourth lens 367 are cemented and connected to form a double cemented lens (fifth lens group 363).
  • the third lens 366 is used to receive the first optical signal emitted by the fourth lens group 362, converge the first optical signal, and then transmit it to the fourth lens 367.
  • the fourth lens 367 receives the converged first optical signal and diverges the first optical signal to reduce the convergence angle of the light so that the light can be emitted in parallel.
  • the doublet lens composed of the third lens 366 and the fourth lens 367 is also used to balance the chromatic aberration of the first optical signal and compensate for the aberration generated by the fourth lens group 362.
  • the fifth lens group 363 can converge the first optical signal to form a small-angle parallel first fluorescence signal.
  • the tube lens 341 receives the first fluorescence signal and converts the A fluorescent signal is focused onto the imaging surface of the camera 342 to perform imaging detection.
  • the third lens 366 satisfies the following relationship: 19.8 ⁇ f T3 ⁇ 24.1, where f T3 is the focal length of the third lens 366; the fourth lens 367 satisfies the following relationship: -71.2 ⁇ f T4 ⁇ -66.3, Among them, f T4 is the focal length of the fourth lens 367.
  • the third lens 366 includes a third incident surface S4 and a third exit surface (not shown in the figure), and the fourth lens 367 includes a fourth incident surface (not shown in the figure) and The fourth exit surface S6; wherein, the third exit surface S4 and the fourth incident surface are glued and connected to form the second glued surface S5; the curvature radius of the third incident surface S4 is 107.462mm, the thickness is 8mm, and the refractive index is 1.59.
  • the Abbe number is 68.4; the curvature radius of the second gluing surface S5 is -14.587mm, the thickness is 7.85mm, the refractive index is 1.73, and the Abbe number is 28.4; the curvature radius of the fourth exit surface S6 is -25.279mm, and the thickness is 403.144mm.
  • the third incident surface S4 is the incident mirror surface of the third lens 366
  • the third exit surface is the exit mirror surface of the third lens 366 .
  • the third incident surface S4 is used to receive the first optical signal
  • the third exit surface is used to emit the first optical signal processed by the third lens 366 .
  • the third lens 366 and the fourth lens 367 are glued and connected, that is, the third exit surface of the third lens 366 and the fourth incident surface of the fourth lens 367 are glued and connected to form the second glued surface S5.
  • the first optical signal is emitted through the third exit surface and then incident on the fourth lens 367 through the second gluing surface. After being processed by the fourth lens 367, a first fluorescent signal is formed.
  • the first fluorescent signal is emitted from the fourth exit surface S6.
  • the third incident surface S4, the second gluing surface S5 and the fourth exit surface S6 need to meet corresponding parameter settings.
  • the parameters of the third incident surface S4, the second gluing surface S5, and the fourth exit surface S6 can also be adjusted according to requirements, and this embodiment will not be described one by one here.
  • the numerical aperture of the high-flux objective lens 310 is 0.5, the focal length is 7mm, and the imaging field diameter is 832um; the distance between the high-flux objective lens 310 and the tube lens 341 is 450mm, and the tube lens 341
  • the focal length is 200 mm, and the surface shapes of each of the first lens 364, the second lens 365, the third lens 366 and the fourth lens 367 are set according to the above parameters. It can be understood that after adding the above-mentioned relay lens group 360, the imaging quality of the sequencing module 300 is not affected, and the image quality of all fields of view is close to the diffraction limit.
  • FIG. 9 is a schematic structural diagram of a sequencing module according to an embodiment of the present application.
  • the sequencing module 300 also includes: a third dichroic mirror 370 and two imaging units 340.
  • the imaging units 340 have the same structure and include a sleeve lens 341 and a camera 342.
  • the excitation module 200 emits a two-color laser, such as a red or green excitation beam, in order to detect the four bases on the DNA of the sequencing chip 321 in the sequencing unit 320: adenine (A), Thymine (T), guanine (G) and cytosine (C) undergo fluorescence imaging respectively to determine the DNA sequence. Therefore, in order to image the four bases respectively, it is necessary to select the light color of the excitation module 200 in turn. For example, green light is first used to excite the fluorescence of base AT, which is imaged by two imaging units 340 respectively.
  • a two-color laser such as a red or green excitation beam
  • the green light of the excitation module 200 is turned off, and the red light of the excitation module 200 is turned on again to excite the fluorescence of the base GC, which is imaged by the two imaging units 340 respectively, thereby completing the identification of the four-color bases.
  • a monochromatic excitation beam can also be used to perform imaging using four imaging units. This embodiment does not limit the number of imaging units 340 and can be selected according to actual needs.
  • Figure 9 takes two imaging units as an example for illustration.
  • FIG. 10 is a schematic structural diagram of an imaging unit in an embodiment of the present application.
  • the imaging unit 340 includes a tube lens 341 and a camera 342.
  • the shape and size of the first fluorescent signal that passes through the sleeve lens 341 matches the imaging surface of the camera 342 and is imaged by the camera 342 to obtain the detection result of the gene sequence of the sample to be detected.
  • the third dichroic mirror 370 is used to reflect the first fluorescent signal to obtain a first imaging signal, and the third dichroic mirror 370 transmits the first fluorescent signal to obtain a second imaging signal, that is, the third dichroic mirror 370 in the figure reflects
  • the optical signal is the first imaging signal Y1
  • the signal transmitted by the third dichroic mirror 370 is the second imaging signal Y2.
  • the tube lenses 341 of the two imaging units 340 receive the first imaging signal Y1 and the second imaging signal Y2 respectively, and output optical signals to the corresponding cameras 342 to use the optical signals to perform fluorescence imaging of different bases.
  • the camera can be used to capture still images or video.
  • the object passes through the lens to create an optical image that is transmitted to the camera's sensor.
  • the photosensitive element can be a Charge Coupled Device (CCD) or a Complementary Metal-Oxide-Semiconductor (CMOS) phototransistor.
  • CCD Charge Coupled Device
  • CMOS Complementary Metal-Oxide-Semiconductor
  • the photosensitive element converts the optical signal into an electrical signal, and then passes the electrical signal to the IS (Image Signal processor) to convert it into a digital image signal.
  • IS Image Signal processor
  • IS Digital image signals to DS for processing.
  • DS converts digital image signals into standard RGB, YUV and other format image signals.
  • FIG. 11 is a schematic structural diagram of a sequencing module according to an embodiment of the present application.
  • the fluorescence signal formed by the sample point on the sample to be detected 330 is collected and emitted through the high-flux objective lens 310 of the relay lens group 360. After the sample point with a large field of view is emitted through the high-flux objective lens 310, it has a certain Exit angle.
  • the relay lens group 360 is placed near the exit pupil of the high-flux objective lens 310 . The relay lens group 360 receives the fluorescence signal emitted from the high-flux objective lens 310 and converges the fluorescence signal to form a first fluorescence signal output.
  • Figure 11 shows by the corresponding arrows Two sets of symmetrical light rays with the largest exit angle in the fluorescence signal are identified, and the remaining light rays with smaller exit angles are not shown in Figure 11.
  • the maximum exit angle ⁇ 1 of the high-flux objective lens 310 for emitting the fluorescence signal is greater than the maximum exit angle ⁇ 2 of the relay lens group 360 for emitting the first fluorescence signal, that is, the light beam of the first fluorescence signal is more concentrated than the light beam of the fluorescence signal.
  • the relay lens group 360 of this embodiment can converge the fluorescence signal emitted from the high-flux objective lens 310 to generate a first fluorescence signal with a smaller maximum emission angle, thereby reducing the size of the sleeve in the imaging unit 340 The size of lens 341.
  • the sequencing module 300 utilizes the relay lens group 360 and sets the corresponding relay lens group 360 between the high-flux objective lens 310 and the tube lens 341 of the imaging unit 340 to detect the fluorescence emitted from the high-flux objective lens 310.
  • the signals are converged to generate a first fluorescent signal with a maximum exit angle smaller than the maximum exit angle of the fluorescent signal emitted by the high-throughput objective lens 310, thereby reducing the size of the tube lens 341 and reducing the processing cost and cost of the sequencing module 300. volume.
  • sequencing module 300 also focuses the first fluorescent signal through the sleeve lens 341 in the imaging unit 340, so that the camera 342 in the imaging unit 340 performs imaging detection on the first fluorescent signal focused on its imaging surface.
  • sequencing The image quality of all fields of view of the module 300 is close to the diffraction limit.
  • the sequencing chip 321 is used to carry the sample 330 to be detected.
  • the sequencing chip 321 and the sample to be detected 330 may be integrated, or the sequencing chip 321 is the sample to be detected 330. In this embodiment, they are shown separately for convenience of description, and their form is not limited.
  • the displacement stage 322 is used to place the sequencing chip 321 to use the excitation beam to irradiate the sample to be detected 330 to generate a fluorescent signal.
  • the displacement stage 322 can be an electric displacement stage.
  • the gene sequencer in this embodiment also includes an autofocus module 400.
  • the feedback information adjusts the relative height of the sequencing chip 321 to ensure that each image is clear.
  • the gene sequencer 100 also includes: a first dichroic mirror 410 and an autofocus module. 400.
  • the first dichroic mirror 410 transmits the fluorescence signal to the autofocus module 400, and then the autofocus module 400 is used to generate a relative height measurement signal according to the fluorescence signal and send it to the sequencing module.
  • the sequencing module adjusts the high-throughput objective lens according to the relative height measurement signal.
  • the height relative displacement between 310 and the sequencing chip 321 avoids the imaging defocusing phenomenon caused by the height change of the sequencing chip 321 caused by the height change of the displacement stage 322.
  • the autofocus module 400 includes: a beam expander 450, a fourth dichroic mirror 420, a fifth dichroic mirror 430, a sixth dichroic mirror 440, two laser signal transmitting and filtering units, and a calculation unit 470 ,
  • the laser signal transmitting and filtering unit includes: a laser transmitting unit and a filtering unit.
  • the laser emitting unit includes: a laser diode and a collimating mirror.
  • the laser diode is used to emit the laser signal to the collimating mirror
  • the collimating mirror is used to collimate the laser signal to generate a collimated laser signal.
  • the laser emitting units are respectively: a first laser emitting unit 461 and a second laser emitting unit 462.
  • the first laser emitting unit 461 includes: a first laser diode 4611 and a first collimating mirror 4612.
  • the second laser emitting unit 462 includes: a first laser diode 4611 and a first collimating mirror 4612. Two laser diodes 4621 and a second collimating mirror 4622.
  • the filtering unit includes a converging mirror, a pinhole filter and a photodiode.
  • the condensing mirror is used to receive the first focusing light signal or the second focusing light signal, and converge it to the corresponding pinhole filter
  • the pinhole filter is used to conduct the corresponding first focusing light signal or the second focusing light signal. Filter to obtain the corresponding filtered signal
  • the photodiode is used to receive the corresponding filtered signal.
  • the filtering units are respectively: a first filtering unit 464 and a second filtering unit 463.
  • the first filtering unit 464 includes: a first converging mirror 4641, a first pinhole filter 4642 and a first photodiode 4643.
  • 463 includes: a second condensing mirror 4631, a second pinhole filter 4632 and a second photodiode 4633.
  • the autofocus process of the autofocus module 400 is described as:
  • the fourth dichroic mirror 420 combines the collimated laser signals respectively emitted by the laser emission units of the two laser signal emitting and filtering units to generate a combined laser signal.
  • the first laser signal emitted by the first laser diode 4611 of the first laser emitting unit 461 is collimated by the first collimating mirror 4612 to obtain the first collimated laser signal.
  • the second laser signal emitted by the second laser diode 4621 of the second laser emitting unit 462 is collimated by the second collimating mirror 4622 to obtain a second collimated laser signal. That is, the fourth dichroic mirror 420 combines the first collimated laser signal emitted by the first laser emitting unit 461 and the second collimated laser signal emitted by the second laser emitting unit 462 to generate a combined laser signal.
  • the beam expander 450 expands the combined laser signal generated by the fourth dichroic mirror 420 to obtain an expanded laser signal.
  • the fifth color mirror 430 is a semi-transparent mirror, which reflects the expanded laser signal emitted by the beam expander 450, passes through the high-flux objective lens 310 to the sequencing chip 321, and reflects the sequencing chip 321 transmitted from the high-flux objective lens. The reflected light spot is transmitted.
  • the sixth dichroic mirror 440 reflects the light spot transmitted by the fifth dichroic mirror 430 to obtain a first focusing light signal.
  • the sixth dichroic mirror 440 transmits the light spot transmitted by the fifth dichroic mirror 430 to obtain a second focusing light signal.
  • the filter units of the two laser signal transmitting and filtering units are respectively used to receive the first focus light signal and the second focus light signal, that is, the first filter unit 464 receives the transmitted second focus light signal, and the second filter unit 463 receives the reflected signal.
  • the first focusing light signal obtained.
  • the first converging mirror 4641 of the first filter unit 464 receives the second focus light signal, and converges the second focus light signal to the first pinhole filter 4642 of the first filter unit 464.
  • the first pinhole filter The chip 4642 filters the concentrated second focusing light signal to obtain a second filtered signal, and the first photodiode 4643 receives the second filtered signal.
  • the second converging mirror 4631 of the second filtering unit 463 receives the first focusing light signal, and converges the first focusing light signal to the second pinhole filter 4632 of the second filtering unit 463.
  • the second pinhole filter 4632 The concentrated first focusing light signal is filtered to obtain a first filtered signal, and the second photodiode 4633 receives the first filtered signal.
  • the calculation unit 470 calculates and generates a relative height measurement signal based on the signal intensity ratio output by the filter unit of the two laser signal emission and filter units.
  • the connection line between the calculation unit 470 and other components is not shown in the figure, which does not mean that there is no connection line.
  • the calculation unit 470 calculates the signal intensity ratio of the second filtered signal received by the first photodiode 4643 and the first filtered signal received by the second photodiode 4633, and then calculates the signal intensity ratio according to the signal intensity ratio and the preset objective lens defocus amount. The corresponding relationship between them generates a relative height measurement signal. It can be understood that the corresponding relationship between the signal intensity ratio and the preset objective lens defocus amount can be obtained statistically based on actual operations, and is not specifically limited in this embodiment.
  • the common idea of automatic focus tracking technology is to detect the position information of the focus tracking spot on the CCD or CMOS, that is, when the surface of the sample to be detected by the sequencing chip is out of focus from the objective lens, the focus tracking spot is the strongest point on the CCD or CMOS.
  • the pixel position will change.
  • CCD or CMOS, as multi-point detectors require a large amount of calculations and a long response time to collect the position information of a single pixel, resulting in a high delay in adjusting the height of the objective lens during the focus tracking process.
  • some other automatic focusing technologies can only be used for biochips with a single surface (such as silicon wafers) and cannot be used for biochips with multiple surfaces with flow channels (such as biochips with coverslips).
  • the reflectivity of the uppermost layer is usually more than ten times stronger than the reflectivity of the surface to be tested that is in contact with the flow channel.
  • the detected focusing spot on the surface to be tested is submerged in the cover glass. In the focus tracking spot on the surface of the chip, the focus on the surface to be measured cannot be achieved.
  • the autofocus module in the embodiment of the present application uses the photodiode of a single-point detector as a detector. Compared with a multi-point detector (CCD or CMOS), the acquisition rate is faster and the intensity information can be fed back more quickly and effectively. Reduce the delay time of changing the height of the objective lens and improve detection efficiency.
  • the autofocus module is compatible with a variety of chips (such as silicon wafers, biochips without coverslips, biochips with coverslips, etc.). And for multi-surface chips, focus tracking can be achieved on different surfaces.
  • the light source used by the autofocus module is a laser diode and the detector is a photodiode, which is small in size and low in cost.
  • Figure 15 is a schematic structural diagram of a gene sequencer according to an embodiment of the present application.
  • the excitation module 200 is composed of a light source 210, an aperture 220 and an even-order aspherical reflector 230. Its function is to shape the circular Gaussian light spot into a length and width. The elliptical uniform spot with a ratio of 2:1 outputs the excitation beam.
  • the light source 210 is a two-color laser, which can optionally output red or green laser. In the figure, dotted lines and solid lines represent excitation beams of different colors respectively.
  • the excitation beam is then reflected into the sequencing module 300 through the first dichroic mirror 410.
  • the sequencing module 300 is composed of a high-throughput objective lens 310, a sequencing unit 320, a sample to be detected 330, an imaging unit 340, a second dichroic mirror 350, a relay lens group 360 and a third dichroic mirror 370.
  • the excitation beam is reflected by the second dichroic mirror 350 into the designed high-throughput objective lens 310, and then converges on the sequencing chip 321.
  • the sequencing chip 321 is driven by the electric displacement stage 322 to complete the sample 330 to be detected on the sequencing chip 321. scan.
  • the fluorescence generated by the four ATGC bases in the sequencing chip 321 is collected again by the high-flux objective lens 310 , and is received by the relay lens group 360 through the second dichroic mirror 350 .
  • the fluorescence signal is divided into two paths through the third dichroic mirror 370, which are received by the tube lenses 341 of the two imaging units respectively, and then converged on the two cameras respectively. 342 on the imaging chip.
  • the light color of the light source 210 in order to image the four bases respectively, it is necessary to select the light color of the light source 210 in turn. For example, green light is first used to excite the fluorescence of the base AT, which is imaged by the two cameras 342 respectively, and then the light source 210 is turned off. The green light, and then the red light of the light source 210 is turned on, to excite the fluorescence of the base GC, which is imaged by the two cameras 342 respectively, thereby completing the identification of the four-color bases.
  • the autofocus module 400 needs to be used to monitor changes in the focal surface of the objective lens in real time.
  • the autofocus module consists of a first dichroic mirror 410, a fourth dichroic mirror 420, a fifth dichroic mirror 430, a sixth dichroic mirror 440, a beam expander 450, a first laser emitting unit 461, a first laser Diode 4611, first collimating mirror 4612, second laser emitting unit 462, second laser diode 4621, second collimating mirror 4622, first filter unit 464, first condensing mirror 4641, first pinhole filter 4642, It consists of a first photodiode 4643, a second filter unit 463, a second condensing mirror 4631, a second pinhole filter 4632, a second photodiode 4633 and a calculation unit 470.
  • the first laser signal emitted by the first laser diode 4611 of the first laser emitting unit 461 is collimated by the first collimating mirror 4612 to obtain the first collimated laser signal.
  • the second laser signal emitted by the second laser diode 4621 of the second laser emitting unit 462 is collimated by the second collimating mirror 4622 to obtain a second collimated laser signal.
  • the fourth dichroic mirror 420 combines the first collimated laser signal emitted by the first laser emitting unit 461 and the second collimated laser signal emitted by the second laser emitting unit 462 to generate a combined laser signal. Afterwards, the combined laser signal is expanded by the beam expander 450 to obtain the expanded laser signal.
  • the signal After the signal is reflected by the fifth color mirror 430, it is transmitted through the first dichroic mirror 410, and the transmitted laser signal is output to the second dichroic mirror 410.
  • the dichroic mirror 350 and the second dichroic mirror 350 reflect and transmit the laser signal to the high-flux objective lens 310.
  • the laser signal is reflected and transmitted by the high-flux objective lens 310 to form a light spot.
  • the second dichroic mirror 350 reflects the light spot to the first dichroic mirror 350.
  • the color mirror 410, and then the first dichroic mirror 410 and the fifth color mirror 430 sequentially transmit the light spot back along the original path to the sixth dichroic mirror 440.
  • the sixth dichroic mirror 440 reflects the above light spot to obtain the first focus.
  • the sixth dichroic mirror 440 transmits the light spot to obtain the second focusing light signal.
  • the first filter unit 464 receives the transmitted second focus light signal
  • the second filter unit 463 receives the reflected first focus light signal.
  • the first condensing mirror 4641 of the first filter unit 464 receives the second focus light signal and converges the second focus light signal to the first pinhole filter 4642 of the first filter unit 464.
  • the first pinhole filter 4642 will condense the
  • the second focusing light signal is filtered to obtain a second filtered signal
  • the first photodiode 4643 receives the second filtered signal.
  • the second converging mirror 4631 of the second filtering unit 463 receives the first focusing light signal, and converges the first focusing light signal to the second pinhole filter 4632 of the second filtering unit 463.
  • the second pinhole filter 4632 The concentrated first focusing light signal is filtered to obtain a first filtered signal, and the second photodiode 4633 receives the first filtered signal. Then the calculation unit 470 calculates the signal intensity ratio between the second filtered signal received by the first photodiode 4643 and the first filtered signal received by the second photodiode 4633, and then based on the corresponding relationship between the signal intensity ratio and the preset objective lens defocus amount Generate relative altitude measurement signals. It can be understood that the corresponding relationship between the signal intensity ratio and the preset objective lens defocus amount can be obtained statistically based on actual operations, and is not specifically limited in this embodiment.
  • the fourth dichroic mirror 420 and the fifth dichroic mirror 430 are dichroic mirrors with the same performance. Their numbers are only for convenience of description and they are interchangeable with each other.
  • the first dichroic mirror 410, the second dichroic mirror 350, the third dichroic mirror 370, the fifth dichroic mirror and the sixth dichroic mirror 440 are dichroic mirrors with different performances, and their purpose is to combine different The wavelength of light is separated, and different parameters can be selected based on actual needs or prior knowledge obtained from multiple experiments. This is not specifically limited.
  • the gene sequencer proposed in the embodiment of the present invention includes an excitation module and a sequencing module.
  • the excitation module includes: a light source, an aperture, and an even-order aspheric reflector;
  • the sequencing module includes: a high-throughput objective lens, a sequencing unit, and at least one imaging unit.
  • a high-throughput microscope objective lens with a large numerical aperture and a large imaging field of view is designed in the gene sequencer to meet high-throughput sequencing application scenarios and improve detection efficiency.
  • an even-order aspherical reflector is used in combination with an aperture.
  • the Gaussian spot is homogenized with a simple optical path, effectively avoiding unnecessary photobleaching, improving the efficiency of scanning imaging and the accuracy of subsequent sequencing results.
  • embodiments of the present invention also provide a method of using a gene sequencer, which is applied to the above gene sequencer.
  • FIG. 16 there is a flow chart of a method for using a gene sequencer provided by an embodiment of the present application.
  • the method includes steps S1610 to S1650:
  • Step S1610 Place the sample to be detected within the detection range of the sequencing chip.
  • the sample to be detected includes single-stranded DNA and four nucleotides, and the fluorescence signal of each nucleotide has a different color.
  • Step S1620 The excitation module generates an excitation beam to excite the sample to be detected.
  • Step S1630 The sequencing module adjusts the relative height displacement between the high-throughput objective lens and the sequencing chip according to the relative height measurement signal, and uses the excitation beam to scan the sample to be detected to obtain a fluorescence signal.
  • step S1630 is described as:
  • the relative height measurement signal is obtained, and then based on the relative height measurement signal, the displacement stage is controlled to adjust the height relative displacement between the high-throughput objective lens and the stage in the vertical direction, that is, the height relative displacement between the sequencing chip and the high-throughput objective lens is adjusted.
  • the short axis of the excitation beam moves relative to the long side of the sequencing chip, and the short axis of the excitation beam and the long side of the sequencing chip are parallel to each other.
  • the fluorescence signal of the sample to be detected is obtained.
  • the autofocus module generates a relative height measurement signal.
  • the specific generation process is: the autofocus module reflects the laser collimation signal and converges it on the sequencing chip through the high-flux objective lens, and receives the laser light reflected from the sequencing chip to the high-flux objective lens. light spot.
  • the autofocus module uses the light spot to generate a first focus light signal and a second focus light signal, and the autofocus module generates a first focus light signal and a second focus light signal based on the first focus light signal and the second focus light signal.
  • Relative height measurement signal The high-throughput objective lens or sequencing chip adjusts the relative height displacement based on the relative height measurement signal.
  • the camera can only image the fluorescence signal within its imaging surface A. Therefore, in order to scan and image different areas of the sequencing chip, in this embodiment, the gene sequencer is equipped with a corresponding displacement stage, and the displacement stage is used to carry The sequencing chip and the displacement stage move according to the relative height measurement signal to adjust the relative height displacement between the two, so that the excitation beam excites and illuminates different areas of the sequencing chip.
  • the sequencing chip is rectangular
  • the imaging surface A of the camera is a rectangular imaging surface
  • the length-to-width ratio of the rectangular imaging surface A is L:W
  • the long and short axis ratio of the spot D of the excitation beam is L:W.
  • the spot D of the excitation beam excites and illuminates the X1 area of the sequencing chip and generates a corresponding fluorescence signal.
  • the camera generates an image signal based on the above working principle, and generates the corresponding detection result x1 based on the image signal.
  • the displacement stage continues to move, so that the spot D of the excitation beam irradiates the X2 area of the sequencing chip, and the detection result x2 is obtained according to the above steps.
  • the displacement stage continues to move, allowing the camera to scan and image areas such as X3, X4... on the sequencing chip, thereby obtaining different detection results on the sequencing chip.
  • this embodiment excites The beam can excite and illuminate a larger area in the sequencing chip, and scan and image through the matching camera. Therefore, the scanning and detection efficiency of the gene sequencer of this embodiment is relatively high, thereby increasing the output of gene sequencing data per unit time.
  • the long axis of the spot of the excitation beam is perpendicular to the long side of the sequencing chip, and the excitation beam moves along the long side of the sequencing chip, so that the camera scans and images the X1 area, X2 area, etc. on the sequencing chip. , to obtain the detection results.
  • the displacement can be shortened.
  • the single movement distance of the stage improves the scanning efficiency of the camera.
  • the gene sequencer also uses feedback information from the autofocus module to adjust the height of the sequencing chip to ensure that each image Images are all clear.
  • the relative height measurement signal is used to control the height relative displacement between the high-flux objective lens and the displacement stage in the vertical direction.
  • the specific steps are as follows:
  • the autofocus module reflects the laser collimation signal and converges it on the sequencing chip through the high-flux objective lens, and receives the light spot reflected from the sequencing chip to the high-flux objective lens. Then the autofocus module uses the light spot to generate the first focusing light signal and the second focusing light signal. Two focusing light signals are generated, and a relative height measurement signal is generated according to the first focusing light signal and the second focusing light signal and sent to the sequencing module.
  • the sequencing module controls the displacement stage to adjust the relative height displacement between the two according to the relative height measurement signal.
  • Step S1640 During the scanning process, the high-throughput objective lens collects fluorescence signals.
  • Step S1650 Use the image obtained by imaging the fluorescence signal according to the imaging unit to determine the order of the nucleotides in the single-stranded DNA.

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Abstract

基因测序仪及基因测序仪的使用方法。其中，基因测序仪包括激发模块和测序模块，激发模块包括：光源、光阑和偶次非球面反射镜；测序模块包括：高通量物镜、测序单元和至少一个成像单元。

Description

基因测序仪及基因测序仪的使用方法
本发明要求于2022年08月22日提交中国专利局、申请号为202211004637.9，申请名称为“基因测序仪及基因测序仪的使用方法”的中国专利申请的优先权，其全部内容通过引用结合在本发明中。
技术领域
本发明涉及医疗设备技术领域，尤其涉及基因测序仪及基因测序仪的使用方法。
背景技术
目前，在基因测序中，需利用显微成像技术对生物芯片上的碱基进行荧光成像。显微成像技术在基因测序的应用越来越广泛，检测需求也随之多样化，因此对基因测序仪的检测效率和检测准确度要求也随之升高。
相关技术中的基因测序仪由于光学系统设计指标要求较低，并不能满足高标准的检测需求。例如，一些基因测序仪的物镜测序通量较低，检测耗时久效率低，另外一些基因测序仪激发荧光染料的激光符合高斯分布，其位于成像视野中心的光强较强，而位于成像视野边缘的光强较弱，激发照明效果较差，激发效率低，影响扫描成像的效率以及后续测序的检测结果准确率，不能满足高检测需求。
发明内容
本发明实施例的主要目的在于提出一种基因测序仪及基因测序仪的使用方法，通过在基因测序仪中设计大数值孔径同时兼顾大成像视野的显微物镜，满足高通量测序应用场景，提高检测效率。同时实现高斯光斑的匀化，避免不必要的光漂白，提高扫描成像的效率以及后续测序的结果准确率。
为实现上述目的，本发明实施例的第一方面提出了一种基因测序仪，用于激发基因测序芯片上的待检测样本并采集所述待检测样本发射的荧光信号进行荧光成像，包括：
激发模块，用于产生激发所述待检测样本的激发光束；
测序模块，用于利用所述激发光束对所述待检测样本进行荧光成像；
所述激发模块包括：
光源，所述光源用于产生激光信号；
光阑，所述光阑沿所述激光信号的光轴放置于所述光源的后方，所述光阑用于对入射的激光信号进行空间滤波，以形成滤波信号；
偶次非球面反射镜，所述偶次非球面反射镜沿所述滤波信号的光轴放置于所述光阑的后方，所述偶次非球面反射镜用于根据所述滤波信号形成激发光束；
所述测序模块包括：
高通量物镜，用于接收并汇聚所述激发光束到所述测序单元；
测序单元，用于利用所述激发光束照射所述待检测样本产生荧光信号；
至少一个成像单元，用于利用所述荧光信号进行荧光成像；
所述高通量物镜包括：
沿物方到像方依次同轴排列的第一透镜组、第二透镜组及第三透镜组，其中，
第一透镜组包括顺次设置的第一弯月透镜和第二弯月透镜；
第二透镜组包括顺次设置的第一双凸透镜、第一双凹透镜、第二双凸透镜、第三双凸透镜、第三弯月透镜及第四双凸透镜，所述第一双凸透镜、所述第一双凹透镜及所述第二双凸透镜组成第一胶合透镜，所述第三弯月透镜及所述第四双凸透镜组成第二胶合透镜，所述第三双凸透镜具有正光焦度；
第三透镜组包括顺次设置的第四弯月透镜、第五弯月透镜、第二双凹透镜及第五双凸透镜，所述第四弯月透镜及所述第五弯月透镜组成第三胶合透镜，所述第二双凹透镜及所述第五双凸透镜组成第四胶合透镜。
在一些实施例，所述测序模块还包括：第二二向色镜和中继透镜组；
所述第二二向色镜用于透射所述测序单元发射的荧光信号至所述中继透镜组，以形成第一荧光信号。
在一些实施例，所述测序模块还包括：中继透镜组，所述中继透镜组包括第四透镜组和第五透镜组；
所述高通量物镜用于接收所述荧光信号，并向所述第二二向色镜透射所述荧光信号；中继透镜组所述第四透镜组具有负光焦度，所述第四透镜组沿所述荧光信号的光轴设置于所述物镜的后方，用于根据所述荧光信号进行形成第一光信号；
所述第五透镜组具有正光焦度，所述第五透镜组沿所述第一光信号的光轴设置于所述第四透镜组的后方，用于根据所述第一光信号形成所述第一荧光信号。
在一些实施例，所述成像单元包括：套筒透镜和相机，所述测序模块还包括：第三二向色镜和两个所述成像单元；
所述第三二向色镜用于反射所述第一荧光信号得到第一成像信号；
所述第三二向色镜还用于透射所述第一荧光信号得到第二成像信号；
两个所述成像单元的套筒透镜分别用于接收所述第一成像信号和所述第二成像信号，并输出光信号至对应的相机，以利用光信号进行荧光成像。
在一些实施例，所述测序单元包括：
测序芯片，用于承载所述待检测样本；
位移台，用于放置所述测序芯片，以利用所述激发光束照射所述待检测样本产生荧光信号。
在一些实施例，所述基因测序仪还包括：第一二向色镜和自动对焦模块；
所述第一二向色镜用于透射对焦激光信号至所述自动对焦模块；
所述自动对焦模块用于根据所述对焦激光信号生成相对高度测量信号发送至所述测序模块；
所述测序模块用于根据所述相对高度测量信号调节所述高通量物镜与所述测序芯片的之间的高度相对位移相对高度测量信号相对高度测量信号。
在一些实施例，所述自动对焦模块包括：扩束镜、第四二向色镜、第五色镜、第六二向色镜、两个激光信号发射及滤波单元和计算单元激光信号发射及滤波单元；
所述第四二向色镜用于对两个所述激光信号发射及滤波单元的所述激光发射单元分别发射的准直激光信号进行合束，生成合束激光信号；
所述扩束镜用于对所述合束激光信号进行扩束得到扩束激光信号；
所述第五色镜用于反射所述扩束激光信号通过所述高通量物镜至所述测序芯片，并透射来自所述高通量物镜传输的所述测序芯片反射的光斑；
所述第六二向色镜用于反射所述光斑得到第一对焦光信号；
所述第六二向色镜还用于透射所述光斑得到第二对焦光信号；
两个所述激光信号发射及滤波单元的所述滤波单元分别用于接收所述第一对焦光信号和所述第二对焦光信号；
所述计算单元用于根据两个所述激光信号发射及滤波单元的所述滤波单元输出的信号强度比值计算生成相对高度测量信号。
在一些实施例，所述激光发射单元包括：激光二极管和准直镜；
所述激光二极管用于发射激光信号至所述准直镜；
所述准直镜用于对所述激光信号进行准直生成准直激光信号。
在一些实施例，所述滤波单元包括汇聚镜、针孔滤波片和光电二极管；
所述汇聚镜用于接收所述第一对焦光信号或所述第二对焦光信号，并汇聚至对应的所述针孔滤波片；
所述针孔滤波片用于将汇聚后的所述第一对焦光信号或所述第二对焦光信号进行滤波得到对应的滤波信号；
所述光电二极管用于接收对应的所述滤波信号。
在一些实施例，所述计算单元还用于计算对应的两个所述光电二极管接收到所述滤波信号的信号强度比值；
所述计算单元还用于根据所述信号强度比值和预设物镜离焦量之间的对应关系生成所述相对高度测量信号。
在一些实施例，所述第一二向色镜用于透射所述第五色镜反射的所述扩束激光信号，并输出透射激光信号至所述第二二向色镜；
所述第二二向色镜用于反射所述透射激光信号至所述高通量物镜；
所述第二二向色镜还用于反射所述高通量物镜反射所述透射激光信号形成的所述光斑至所述第一二向色镜；
所述第一二向色镜和所述第五色镜依次透射所述光斑至所述第六二向色镜。
在一些实施例，所述偶次非球面反射镜的面型公式满足以下关系：

其中，c为曲率，k为圆锥系数，a1为二阶非球面系数，a2为四阶非球面系数，a3为六阶非球面系数，a4为八阶非球面系数，x、y为非球面表面的坐标位置。
在一些实施例，所述第四透镜组包括：
第一透镜，所述第一透镜为具有负光焦度的双凹透镜；
第二透镜，所述第二透镜与所述第一透镜胶合连接，所述第二透镜为具有正光焦度的弯月透镜。
在一些实施例，所述第五透镜组包括：
第三透镜，所述第三透镜沿所述第一光信号的光轴设置于所述第一透镜组的后方，所述第三透镜为具有正光焦度的双凸透镜；
第四透镜，所述第四透镜与所述第三透镜胶合连接，所述第四透镜为具有负光焦度的弯月透镜。
本发明实施例的第二方面提出了一种基因测序仪的使用方法，应用于如第一方面任一项所述的基因测序仪，所述方法包括：
将待检测样本放入测序芯片的检测范围内，所述待检测样本包括单链DNA和四种核苷酸，每种所述核苷酸的荧光信号的颜色均不相同；
激发模块产生激发所述待检测样本的激发光束；
测序模块根据相对高度测量信号调节所述高通量物镜与所述测序芯片之间的高度相对位移，利用所述激发光束对所述待检测样本进行扫描得到荧光信号；
扫描过程中，高通量物镜采集所述荧光信号并发送至成像单元；
利用所述成像单元对所述荧光信号成像得到的图像来确定所述待检测样本中单链DNA中核苷酸的排序。
在一些实施例，所述测序模块根据相对高度测量信号调节所述高通量物镜与所述测序芯片之间的高度相对位移相对高度测量信号，还包括：
获取相对高度测量信号；
根据所述相对高度测量信号在垂直方向上调节所述高通量物镜与所述测序芯片之间的高度相对位移，以使所述激发光束的短轴相对所述测序芯片的长边移动，且所述激发光束的短轴与所述测序芯片的长边相互平行；
在移动过程中，获取所述待检测样本的荧光信号。
在一些实施例，所述根据所述相对高度测量信号在垂直方向上调节所述高通量物镜与所述测序芯片之间的高度相对位移，包括：
自动对焦模块反射激光准直信号通过高通量物镜汇聚到所述测序芯片上，并接收所述测序芯片反射至所述高通量物镜的光斑；
自动对焦模块利用所述光斑生成第一对焦光信号和第二对焦光信号；
自动对焦模块根据所述第一对焦光信号和所述第二对焦光信号生成所述相对高度测量信号；
所述测序芯片根据所述相对高度测量信号调节高度相对位移。
在一些实施例，所述相机的成像面为矩形成像面，所述激发光束的形状为椭圆形或类椭圆形或矩形；所述矩形成像面的长宽比例为L：W；所述激发光束的光斑长短轴比例为L：W；其中，L、W均为正整数。
本发明实施例提出的基因测序仪及基因测序仪的使用方法，其中，基因测序仪包括激发模块和测序模块，激发模块包括：光源、光阑和偶次非球面反射 镜；测序模块包括：高通量物镜、测序单元和至少一个成像单元。本实施例在基因测序仪中设计大数值孔径同时兼顾大成像视野的高通量显微物镜，满足高通量测序应用场景，提高检测效率，同时利用偶次非球面反射镜结合一个光阑，以简单的光路形式实现高斯光斑的匀化，有效的避免不必要的光漂白，提高扫描成像的效率以及后续测序的结果准确率。
附图说明
图1是本发明实施例提供的基因测序仪结构示意图。
图2是本发明又一实施例提供的基因测序仪的激发模块的结构示意图。
图3是本发明又一实施例提供的基因测序仪的激发模块的激发光束的光斑长轴方向上的光强分布图。
图4是本发明又一实施例提供的基因测序仪的激发模块的激发光束的光斑短轴方向上的光强分布图。
图5是本发明又一实施例提供的基因测序仪的测序模块结构示意图。
图6是本发明又一实施例提供的基因测序仪的高通量物镜的结构示意图。
图7是本发明又一实施例提供的基因测序仪的中继透镜组位置示意图。
图8是本发明又一实施例提供的基因测序仪的中继透镜组结构示意图。
图9是本发明又一实施例提供的基因测序仪的测序模块结构示意图。
图10是本发明又一实施例提供的基因测序仪的成像单元的结构示意图。
图11是本发明又一实施例提供的基因测序仪的测序模块结构示意图。
图12是本发明又一实施例提供的基因测序仪的测序单元结构示意图。
图13是本发明又一实施例提供的基因测序仪的自动对焦模块结构示意图。
图14是本发明又一实施例提供的基因测序仪的自动对焦模块结构示意图。
图15是本发明又一实施例提供的基因测序仪结构示意图。
图16是本发明一实施例提供的基因测序仪使用方法流程图。
图17是本发明又一实施例提供的基因测序仪使用方法的成像示意图。
附图标记说明：
基因测序仪110、激发模块200、测序模块300和自动对焦模块400；
激发模块200包括：光源210、光阑220和偶次非球面反射镜230；
测序模块300包括：高通量物镜310、测序单元320、待检测样本330、成像单元340、第二二向色镜350、中继透镜组360和第三二向色镜370；
高通量物镜310包括：第一透镜组G1、第二透镜组G2和第三透镜组G3；
测序单元320包括：测序芯片321和位移台322；
成像单元340包括：套筒透镜341和相机342；
中继透镜组360包括：高通量物镜310第四透镜组362、第五透镜组363、第一透镜364、第二透镜365、第三透镜366和第四透镜367；
第一二向色镜410和自动对焦模块400，自动对焦模块400包括：第四二向色镜420、第五色镜430、第六二向色镜440、扩束镜450、第一激光发射单元461、第一激光二极管4611、第一准直镜4612、第二激光发射单元462、第二激光二极管4621、第二准直镜4622、第一滤波单元464、第一汇聚镜4641、第一针孔滤波片4642、第一光电二极管4643、第二滤波单元463、第二汇聚镜4631、第二针孔滤波片4632、第二光电二极管4633和计算单元470。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中所使用的术语只是为了描述本发明实施例的目的，不是旨在限制本发明。
显微成像技术在样本检测中具有广泛应用，例如，在基因测序中，需对生物芯片上的碱基进行荧光成像。基因测序仪在医学和生命科学领域上有着广泛的应用，如病原体、遗传病、肿瘤基因的检测，以及药物个体化治疗和无创产前检测等。基因测序仪工作时需对生物芯片上的碱基进行荧光成像。基因测序仪测序时需对ATGC四种碱基，即腺嘌呤(A)、胸腺嘧啶(T)、胞嘧啶(C)与鸟嘌呤(G)，进行荧光成像，通常采用多通道(如四通道或二通道)成像，然后对每个通道得到的检测图像进行算法配准，从而使不同图像的碱基位置相匹配。
相关技术中，基因测序的应用越来越广泛，检测需求也随之多样化，因此对基因测序仪的检测效率和检测准确度要求也随之升高。但是目前一些基因测序仪并不能满足高标准的检测需求，例如一些基因测序仪的物镜测序通量较低，检测耗时久效率低，另外一些基因测序仪激发荧光染料的激光符合高斯分布，其位于成像视野中心的光强较强，而位于成像视野边缘的光强较弱，激发照明效果较差，激发效率低，影响扫描成像的效率以及后续测序的检测结果准确率，不能满足高检测需求。
基于此，本发明实施例提供一种基因测序仪及基因测序仪的使用方法，在基因测序仪中设计大数值孔径同时兼顾大成像视野的高通量显微物镜，满足高通量测序应用场景，提高检测效率，同时利用偶次非球面反射镜结合一个光阑，以简单的光路形式实现高斯光斑的匀化，有效的避免不必要的光漂白，提高扫描成像的效率以及后续测序的结果准确率。
本发明实施例提供基因测序仪及基因测序仪的使用方法，具体通过如下实施例进行说明，首先描述本发明实施例中的基因测序仪。
图1是本发明实施例提供的基因测序仪结构示意图。
该实施例中基因测序仪100用于激发基因测序芯片上的待检测样本，并采集待检测样本发射的荧光信号进行荧光成像，包括：
激发模块200，用于产生激发待检测样本的激发光束S。
测序模块300，用于利用激发光束S对待检测样本进行荧光成像。
在一实施例中，激发模块200产生的激发光束S可以是激光，基因测序仪100基于荧光染料对激光的激发特性，能够通过激发模块200产生激光信号形成相应的照明区域，照射在待检测样本上对照明区域内的荧光染料进行激发照明，使得待检测样本在激光信号的激发下产生相应的荧光信号，通过相机对荧光信号进行成像，从而能够对基因序列进行检测。但是由于激光具有高斯分布的特点，导致照明区域中心的光强较强，而照明区域边缘的光强较弱，激发照明效果较差。为了提高视野边缘的荧光亮度以达到成像所需的信噪比，就需要增加激光功率，以弥补边缘视野激发效率低的缺点。但是提高激光功率，会造成中心视野的激光强度过大，就会导致光漂白速度增加，并且过大的光强会损伤中心视野的DNA，导致后续测序的结果错误率增加。因此本实施例中利用偶次非球面反射镜结合一个光阑，以简单的光路形式实现高斯光斑的匀化，有效的避免不必要的光漂白，提高扫描成像的效率以及后续测序的结果准确率。
参照图2，是本实施例中激发模块的结构示意图。
光源210，光源210用于产生激光信号。
在一实施例中，光源210用于产生准直的激光信号，激光信号的光斑形状为圆形。
光阑220，光阑220沿激光信号的光轴放置于光源210的后方，光阑220用于对入射的激光信号进行空间滤波，以形成滤波信号。
在一实施例中，光阑220是指在光学系统中对光束起着限制作用的器件，当激光信号入射到光阑220时，光阑220能够对激光信号的光束进行过滤，从 而对激光信号光强较弱的边缘部分进行阻挡，以方便后续偶次非球面反射镜230对激光信号进行进一步匀光。
偶次非球面反射镜230，偶次非球面反射镜230沿滤波信号的光轴放置于光阑220的后方，偶次非球面反射镜230用于根据滤波信号形成激发光束S，其中，激发光束S用于激发待检测样本产生荧光信号。
在一实施例中，激光信号为高斯分布的圆形光斑，即在激光信号的光斑区域(照明区域)内，光强呈高斯分布特性。为此，本实施例中激发模块200设置了相应的偶次非球面反射镜230，偶次非球面反射镜230用于对入射的滤波信号进行匀光操作，以形成具有一定形状和大小的激发光束均匀照明光斑。相比于未经过匀光操作，仅通过光阑空间滤波的照明光斑，激发光束照明区域内的光强分布更加均匀。此外，偶次非球面反射镜230还用于对激发光束进行反射，以使激发光束照射在待检测样本330上。
在一实施例中，偶次非球面反射镜230的面型公式满足以下关系：

其中，c为曲率，k为圆锥系数，a1为二阶非球面系数，a2为四阶非球面系数，a3为六阶非球面系数，a4为八阶非球面系数，x、y为非球面表面的坐标位置。
该实施例中，当偶次非球面反射镜230满足以上公式时，滤波信号经过偶次非球面反射镜230的反射后，能够获得椭圆形光斑的激发光束。此外，通过设置具体的面型参数：曲率、圆锥系数、二阶非球面系数、四阶非球面系数、六阶非球面系数、八阶非球面系数，并确定非球面表面的具体坐标位置，能够获得不同形状大小的激发光束，即椭圆形光斑的长短轴不同。由上述内容可知，通过使得椭圆形光斑的激发光束与相机的矩形成像面相配合，能够提高相机扫描成像的效率。且本实施例中偶次非球面反射镜形成的激发光束的匀光效果好，能够实现较好的激发照明效果。
参照图3，为本申请实施例中激发光束的光斑长轴方向上的光强分布图，图4为本申请实施例中激发光束的光斑短轴方向上的光强分布图。
本实施例以激发光束光斑长轴的长度为1.6mm，短轴的长度0.8mm为例对照明均匀性进行计算。具体的，以照明区域内的光强最小值除以光强最大值，从而获得照明均匀性。其中，长轴方向上的均匀性为81％，短轴方向上的均匀 性为85％。可以理解的是，当照明均匀性达到75％以上时，成像算法对相机323拍照位置对应的照明区域的中心、边缘有效信息的提取效率接近一致。因此，本实施例的激发模块200能很好地满足实际的使用需求。
在一些实施例中，k＝-146.5，a1＝0，a2＝1.848E-4，a3＝-4.159E-6，a4＝3.216E-8。
在一实施例中，偶次非球面反射镜230的焦距满足以下关系：
14.6<f0<16.1
其中，f0为偶次非球面反射镜230的焦距。
可以理解的是，偶次非球面反射镜230的焦距满足上述关系，可以使得偶次非球面反射镜230实现较好的整形、匀光效果，并生成长短轴比例为2:1的激发光束。同理的，当偶次非球面反射镜230的面型参数进行改变时，其焦距f也要进行相应的改变，本实施例在此不进行一一说明。
由于测序时需要移动位移台配合显微镜系统完成整张测序芯片的扫描成像。测序芯片一般为长方形，因此主要的扫描时间是花费在长边上。根据以上情况，在保证相机像素数满足要求的前提下，选择长宽比为2：1的相机，使测序芯片的长边扫描方向对应相机的短边，能缩短位移台的单次移动距离，从而提高扫描效率，进而提高单位时间的测序数据输出量。根据以上情况，如果照明光斑为圆形，则在满足相机长边照明需求的情况下，必然会极大的超出相机短边所需的照明范围。这些超出的成像部分所激发的荧光信号无法被相机接收，需要在后续的扫描中被再次激发才能实现该区域的信号采集。这种多次的激发会造成该区域的光漂白，从而降低了信噪比。并且增加的光毒性也会损伤DNA，影响后续的测序错误率。本实施例的激发模块200通过偶次非球面反射镜230即可对滤波信号进行匀光操作，以获得光强分布均匀的激发光束，在物镜焦面上以椭圆光斑的照明，该椭圆光斑的长短轴比接近2：1，有效的避免了不必要的光漂白。当使用激发光束对待检测样本330进行激发照明时，各个照明区域内的光强均匀，不会出现激发效率不一致的问题。因此，本实施例的激发模块200能够以简单的结构实现较好的激发照明效果。
在一实施例中，为了提高荧光成像时，相机的扫描成像效率，本实施例中激发模块200的偶次非球面反射镜230除了能够对滤波信号进行匀光操作，还用于对滤波信号进行整形。具体的，偶次非球面反射镜230能够将激光信号的形状整形为椭圆形或者类椭圆形，以使形成的激发光束对照明区域内的待检测 样本进行激发照明后，生成椭圆形或类椭圆形的荧光信号，其中，激发光束的形状大小与相机成像面相配合，即与荧光信号的光斑的形状大小相配合。
在一实施例中，待检测样本在激发光束的激发照明下产生荧光信号，且激发光束的形状大小与荧光信号的形状大小相同。其中，荧光信号能够表征待检测样本的不同检测结果。例如，假设待检测样本含有不同的基因序列，且不同的基因序列能够被不同光谱的荧光进行标记。则当激发光束对待检测样本进行激发照明时，待检测样本中不同的基因序列被激发后能够产生不同光谱的荧光信号。因此通过不同的荧光信号能够得出待检测样本基因序列的检测结果。
参照图5，为本申请一实施例的测序模块结构示意图。
该实施例中，测序模块300包括：
高通量物镜310，用于接收并汇聚激发光束S到测序单元320；
测序单元320，用于利用激发光束S照射待检测样本330产生荧光信号；
至少一个成像单元340(图中以1个成像单元示意)，用于利用荧光信号进行荧光成像。
参照图6，为本申请一实施例中高通量物镜的结构示意图。
在一实施例中，由于物镜成像视野和数值孔径是决定测序通量的两个关键参数，但是这两个参数大小往往此消彼长，即大数值孔径物镜的视野范围较小，而大视野范围的物镜数值孔径较小，无法看的多的同时兼顾看的细。针对以上问题，本实施例中高通量物镜310是一种大数值孔径同时兼顾大成像视野的显微物镜。该实施例中，高通量物镜310包括：沿物方到像方依次同轴排列的第一透镜组G1、第二透镜组G2及第三透镜组G3，其中：
第一透镜组G1包括：顺次设置的第一弯月透镜L2和第二弯月透镜L3。
第二透镜组G2包括：顺次设置的第一双凸透镜L4、第一双凹透镜L5、第二双凸透镜L6、第三双凸透镜L7、第三弯月透镜L8及第四双凸透镜L9，所述第一双凸透镜L4、所述第一双凹透镜L5及所述第二双凸透镜L6组合第一胶合透镜，所述第三弯月透镜L8及所述第四双凸透镜L9组成第二胶合透镜，所述第三双凸透镜具有正光焦度。
第三透镜组G3包括：顺次设置的第四弯月透镜L10、第五弯月透镜L11、第二双凹透镜L12及第五双凸透镜L13，所述第四弯月透镜L10及所述第五弯月透镜L11组成第三胶合透镜，所述第二双凹透镜L12及所述第五双凸透镜L13组成第四胶合透镜。
该实施例中，L1为样品的盖玻片，也可以是测序芯片流道上层的玻璃。第 一透镜组G1包括L2和L3，第一透镜组G1形成前置齐明面，收集大发散角光信号并将其转变成小角度光信号，在有效增大数值孔径的同时，减少避免产生过大的球差和/或慧差。第二透镜组G2包括透镜L4～L9，第二透镜组G2用于矫正球差、慧差和/或色差。第三透镜组G3包括透镜L10～L13，用于消除场曲、像散和/或色差，其中，L11为厚弯月透镜。本实施例中，待检测样本发出的光首先经过第一透镜组G1减小荧光信号入射角，然后通过第二透镜组G2矫正球差、慧差或色差中的一个或多个，然后通过第三透镜组G3消除场曲、像散或色差中的一个或多个，从而实现同时增大成像视野和数值孔径，提升测序通量。
在一实施例中，高通量物镜310中各部件的焦距满足以下关系：
10.2<fL23/f<11
6.42<fL456/f<7.15
2.91<fL7/f<3.32
10.6<fL89/f<12.3
-6.01<fL1011/f<-5.66
-69.1<fL1213/f<-70.2
其中，f表示高通量物镜310的焦距,fL23表示第一透镜组G1的焦距，fL456表示第一胶合透镜的焦距，fL7表示第三双凸透镜的焦距，fL89表示第二胶合透镜的焦距，fL1011表示第三胶合透镜的焦距，fL1213表示第四胶合透镜的焦距。可以理解的是，本实施例对各部件焦距仅作示意，不做具体限定。
在一实施例中，高通量物镜310中各透镜的具体涉及参数参阅表一。参阅图6，图6中的R1-R21分别依次对应表一中的面编号1-21。
表一

在一实施例中，高通量物镜310的数值孔径为0.75，成像物方视野直径为1.6mm，平场复消色差，焦距10mm，以上参数能满足基因测序仪大部分的高通量测序应用场景。
在一实施例中，第一透镜组G1、第二透镜组G2和第三透镜组G3中的所有透镜均为球面透镜。即第一透镜组G1至第三透镜组G3中的所有透镜L2～L13均为球面透镜，球面透镜能够降低透镜的加工成本。
在一实施例中，由于进行荧光成像需要二向色镜和滤光片来分光及过滤不同波长的信号，因此物镜和套筒透镜之间难免会间隔较长的距离。而长距离会导致套筒透镜的尺寸增大，这样首先带来的是加工成本的增加，镜片面型精度难以保证。其次尺寸增加导致镜头重量增加，对镜头的固定和调节都会带来一定影响。因此该实施例在高通量物镜310和成像单元340的套筒透镜之间加入中继透镜组360，用以减小进入套筒透镜的视场角，从而减小套筒透镜的尺寸。
参照图7，为本申请实施例的中继透镜组位置示意图。
图7中测序模块300还包括：第二二向色镜350和中继透镜组360。
该实施例中，高通量物镜310用于接收荧光信号，并向第二二向色镜350透射荧光信号，二向色镜可以透过一部分光，反射另一部分光，第二二向色镜350将接收到的荧光信号的一部分反射至高通量物镜310，然后接收测序单元320发射的待检测样本330的荧光信号透射至中继透镜组360，以形成第一荧光信号。中继透镜组360对第一荧光信号收集、整形并传输至成像单元340，进行后续荧光成像。
在一实施例中，中继透镜组360包括：
高通量物镜310高通量物镜310参照图8，中继透镜组360包括：第四透镜组362和第五透镜组363。
其中，第四透镜组362具有负光焦度，第四透镜组362沿荧光信号的光轴设置于高通量物镜310的后方，用于对荧光信号进行像差矫正、增大光线孔径操作，以形成第一光信号。第五透镜组363具有正光焦度，第五透镜组363沿第一光信号的光轴设置于第四透镜组362的后方，用于对第一光信号进行像差补偿、增大光线孔径及减小光线出射角操作，以形成第一荧光信号。
在一实施例中，第四透镜组362的光焦度用于表征第四透镜组362对光线的会聚本领或发散本领，第四透镜组362为负光焦度表示第四透镜组362对光线有发散作用。同理的，第五透镜组363的光焦度用于表征第五透镜组363对光线的会聚本领或发散本领，第五透镜组363为正光焦度表示第五透镜组363对光线有会聚作用。荧光信号经过第四透镜组362和第五透镜组363后，能够形成最大出射角更小的第一荧光信号，在经过一定的传输距离后，第一荧光信号的光束直径仍然较小。
在一实施例中，第四透镜组362和第五透镜组363用于共同平衡像差，使中继透镜组360入射与出射的荧光信号均为准直光束。因此，本实施例第四透镜组362和第五透镜组363不仅能够形成最大出射角更小的第一荧光信号，同时能够保证良好的成像性能。
参照图8，该实施例中，第四透镜组362包括：第一透镜364，第一透镜364为具有负光焦度的双凹透镜；第二透镜365，第二透镜365与第一透镜364胶合连接，第二透镜365为具有正光焦度的弯月透镜。
在一实施例中，第一透镜364和第二透镜365胶合连接，从而组成双胶合透镜(第四透镜组362)。其中，第一透镜364用于接收高通量物镜310出射的荧光信号，并对荧光信号进行发散，然后传输给第二透镜365。第二透镜365接收经过发散后的荧光信号，并对荧光信号进行会聚，同时对荧光信号的场曲进行矫正。其中，场曲指的是像场弯曲，当第一透镜364或第二透镜365存在场曲时，荧光信号的交点不再与理想像点重合，整个像平面是一个曲面，使得后续的相机342对第一荧光信号进行成像时不能同时看清整个像面，给检测造成困难。因此，本实施例中第二透镜365能够对荧光信号的场曲进行矫正，并形成相应的第一光信号。第五透镜组363接收第一光信号并形成小角度平行的第一荧光信号，并由套筒透镜341将第一荧光信号聚焦至相机342的成像面，从而进行成像检测。
在一实施例中，第一透镜364满足以下关系：-15.2<fT1<-10.5，其中，fT1为第一透镜364的焦距；第二透镜365满足以下关系：28.5<fT2<33.1，其中，fT2为第二透镜365的焦距。
参照图8，在一些实施例中，第一透镜364包括第一入射面S1和第一出射面(图中未示出)，第二透镜365包括第二入射面(图中未示出)和第二出射面S3；其中，第一出射面S1与第二入射面胶合连接，以形成第一胶合面S2；第一入射面S1的曲率半径为-31.393mm，厚度为8mm，折射率为1.77，阿贝数为49.6；第一胶合面S2的曲率半径为15.247mm，厚度为8mm，折射率为1.92，阿贝数为20.9；第二出射面的曲率半径为25.169mm，厚度为10mm。
可以理解的是，第一入射面S1为第一透镜364的入射面，第一出射面为第一透镜364的出射面。第一入射面S1用于接收荧光信号，第一出射面用于出射经过第一透镜364处理后的荧光信号。此外，由上述内容可知，第一透镜364和第二透镜365胶合连接，即第二透镜365的第二入射面与第一透镜364的第一出射面胶合连接，形成第一胶合面S2。荧光信号经过第一出射面出射后通过第一胶合面入射至第二透镜365，经过第二透镜365处理后形成第一光信号，第一光信号由第二出射面S3出射。
可以理解的是，为了实现上述第一透镜364和第二透镜365的光学性能，第一入射面S1、第一胶合面S2和第二出射面S3需要满足相应的参数设置。在实际应用中，根据需求还可以对第一入射面、第一胶合面和第二出射面的参数进行调整，本实施例在此不一一说明。
参照图8，在一些实施例中，第五透镜组363包括：第三透镜366，第三透镜366沿第一光信号的光轴设置于第四透镜组362的后方，第三透镜366为具有正光焦度的双凸透镜；第四透镜367，第四透镜367与第三透镜366胶合连接，第四透镜367为具有负光焦度的弯月透镜。
可以理解的是，第三透镜366和第四透镜367胶合连接，从而组成双胶合透镜(第五透镜组363)。其中，第三透镜366用于接收第四透镜组362出射的第一光信号，并对第一光信号进行会聚，然后传输给第四透镜367。第四透镜367接收经过会聚后的第一光信号，并对第一光信号进行发散，以减小光线的会聚角度，使得光线能够平行出射。同时，第三透镜366和第四透镜367组成的双胶合透镜还用于对第一光信号的色差进行平衡，并对第四透镜组362产生的像差进行补偿。因此，本实施例中第五透镜组363能够对第一光信号进行会聚，以形成小角度平行的第一荧光信号。套筒透镜341接收第一荧光信号，并将第 一荧光信号聚焦至相机342的成像面，从而进行成像检测。
在一些实施例中，第三透镜366满足以下关系：19.8<fT3<24.1，其中，fT3为第三透镜366的焦距；第四透镜367满足以下关系：-71.2<fT4<-66.3，其中，fT4为第四透镜367的焦距。
参照图8，在一些实施例中，第三透镜366包括第三入射面S4和第三出射面(图中未示出)，第四透镜367包括第四入射面(图中未示出)和第四出射面S6；其中，第三出射面S4与第四入射面胶合连接，以形成第二胶合面S5；第三入射面S4的曲率半径为107.462mm，厚度为8mm，折射率为1.59，阿贝数为68.4；第二胶合面S5的曲率半径为-14.587mm，厚度为7.85mm，折射率为1.73，阿贝数为28.4；第四出射面S6的曲率半径为-25.279mm，厚度为403.144mm。
可以理解的是，第三入射面S4为第三透镜366的入射镜面，第三出射面为第三透镜366的出射镜面。第三入射面S4用于接收第一光信号，第三出射面用于出射经过第三透镜366处理后的第一光信号。此外，由上述内容可知，第三透镜366和第四透镜367胶合连接，即第三透镜366的第三出射面与第四透镜367的第四入射面胶合连接，以形成第二胶合面S5。第一光信号经过第三出射面出射后通过第二胶合面入射至第四透镜367，经过第四透镜367处理后形成第一荧光信号，第一荧光信号由第四出射面S6出射。
可以理解的是，为了实现上述第三透镜366和第四透镜367的光学性能，第三入射面S4、第二胶合面S5和第四出射面S6需要满足相应的参数设置。在实际应用中，根据需求还可以对第三入射面S4、第二胶合面S5和第四出射面S6的参数进行调整，本实施例在此不一一说明。
在一个具体的实施例中，高通量物镜310的数值孔径为0.5，焦距为7mm，成像视野直径为832um；高通量物镜310和套筒透镜341的距离为450mm，且套筒透镜341的焦距为200mm，第一透镜364、第二透镜365、第三透镜366和第四透镜367的各个透镜的面型按照上述参数进行设置。可以理解的是，在加入上述中继透镜组360后，测序模块300的成像质量没有受到影响，所有视场的像质均接近衍射极限。
参照图9，为本申请实施例的测序模块结构示意图。
该实施例中，测序模块300还包括：第三二向色镜370和两个成像单元340，成像单元340的结构相同，都包括套筒透镜341和相机342。
该实施例中，激发模块200发出双色激光，例如输出红色或者绿色的激发光束，为了对测序单元320中测序芯片321的DNA上四种碱基：腺嘌呤(A)、 胸腺嘧啶(T)、鸟嘌呤(G)及胞嘧啶(C)分别进行荧光成像，来实现DNA序列的测定。因此为了分别对四种碱基进行成像，需要轮流选择激发模块200的出光颜色。如先用绿光激发出碱基AT的荧光，被两个成像单元340分别成像。之后关闭激发模块200的绿光，再打开激发模块200的红光，激发出碱基GC的荧光，被两个成像单元340分别成像，以此完成对四色碱基的识别。可以理解的是，也可以采用单色的激发光束，利用四个成像单元分别成像，本实施例对成像单元340的数量不做限制，可根据实际需求进行选择。图9中以两个成像单元为例进行说明。
参照图10，为本申请实施例中成像单元的结构示意图。
该实施例中，成像单元340包括套筒透镜341和相机342。穿过套筒透镜341的第一荧光信号，其形状大小与相机342的成像面匹配，被相机342成像，得到待检测样本基因序列的检测结果。
参照图9，图9中以虚线和实线分别表示不同颜色的激发光束。第三二向色镜370用于反射第一荧光信号得到第一成像信号，第三二向色镜370透射第一荧光信号得到第二成像信号，即图中第三二向色镜370反射的光信号为第一成像信号Y1，第三二向色镜370透射的信号为第二成像信号Y2。
两个成像单元340的套筒透镜341分别接收第一成像信号Y1和第二成像信号Y2，并输出光信号至对应的相机342，以利用光信号进行不同碱基的荧光成像。
需要说明的是，相机可以用于捕获静态图像或视频。物体通过镜头生成光学图像透射到相机的感光元件。感光元件可以是电荷耦合器件(Charge Couled Device，CCD)或互补金属氧化物半导体(Comlementary Metal-Oxide-Semiconductor，CMOS)光电晶体管。感光元件把光信号转换成电信号，之后将电信号传递给IS(Image Signal rocessor，图像信号处理器)转换成数字图像信号。IS将数字图像信号输出到DS加工处理。DS将数字图像信号转换成标准的RGB，YUV等格式的图像信号。
参照图11，为本申请实施例测序模块结构示意图。
本实施例中，待检测样本330上样品点形成的荧光信号经过中继透镜组360的高通量物镜310收集并出射，大视场的样品点经过高通量物镜310出射后，具有一定的出射角度。本实施例将中继透镜组360放置于高通量物镜310的出瞳附近的位置。中继透镜组360接收高通量物镜310出射的荧光信号，并对荧光信号进行会聚，以形成第一荧光信号输出。其中，图11通过相应的箭头示出 了荧光信号中具有最大出射角度的两组对称光线，其余出射角度较小的光线在图11中未示出。其中，高通量物镜310出射荧光信号的最大出射角θ1大于中继透镜组360出射第一荧光信号的最大出射角θ2，即第一荧光信号的光束相比于荧光信号的光束更加集中。由上述内容可知，在经过相同的传输距离L1后，由于第一荧光信号的最大出射角θ2较小，因此第一荧光信号在套筒透镜341的入射镜面对应的光束半径r2比荧光信号的光束半径更小，此时套筒透镜341的直径也更小。可以理解的是，本实施例的中继透镜组360能够对高通量物镜310出射的荧光信号进行会聚，以生成最大出射角度更小的第一荧光信号，从而减小成像单元340中套筒透镜341的尺寸。
本实施例中测序模块300利用中继透镜组360，通过高通量物镜310和成像单元340的套筒透镜341之间设置相应的中继透镜组360，能够对高通量物镜310出射的荧光信号进行会聚，以生成最大出射角比高通量物镜310出射荧光信号的最大出射角更小的第一荧光信号，从而减小套筒透镜341的尺寸，并减少了测序模块300的加工成本和体积。此外测序模块300还通过成像单元340中套筒透镜341对第一荧光信号进行聚焦，以使成像单元340中相机342对聚焦在其成像面上的第一荧光信号进行成像检测，本实施例测序模块300所有视场的像质均接近衍射极限。
测序单元320进行测序时需要移动位移台或高通量物镜配合显微镜系统完成整张测序芯片的扫描成像。参照图12，为本申请实施例中测序单元结构示意图。
测序芯片321，用于承载待检测样本330。
在一实施例中，测序芯片321和待检测样本330可以是一体的，或者说测序芯片321就是待检测样本330，本实施例中为描述方便将其分开示意，不代表对其形式进行限定。
位移台322，用于放置测序芯片321，以利用激发光束照射待检测样本330产生荧光信号，位移台322可以是电动位移台。
在一实施例中，基因测序仪对测序芯片321进行扫描成像的过程中，由于测序芯片321高度的变化可能导致成像的离焦，因此该实施例的基因测序仪还包括利用自动对焦模块400的反馈信息调节测序芯片321的相对高度，来保证每一幅图像都是清晰的。
参照图13，为本申请实施例中自动对焦模块结构示意图。
该实施例中，基因测序仪100还包括：第一二向色镜410和自动对焦模块 400。
其中，第一二向色镜410透射荧光信号至自动对焦模块400，然后自动对焦模块400用于根据荧光信号生成相对高度测量信号发送至测序模块，测序模块根据相对高度测量信号调节高通量物镜310与测序芯片321之间的高度相对位移，避免由于位移台322高度变化导致的测序芯片321高度变化造成的成像离焦现象。
参照图14，为本申请实施例中自动对焦模块结构示意图。
该实施例中，自动对焦模块400包括：扩束镜450、第四二向色镜420、第五色镜430、第六二向色镜440、两个激光信号发射及滤波单元和计算单元470，激光信号发射及滤波单元包括：激光发射单元和滤波单元。
该实施例中，激光发射单元包括：激光二极管和准直镜。其中，激光二极管用于发射激光信号至准直镜，准直镜用于对激光信号进行准直生成准直激光信号。激光发射单元分别是：第一激光发射单元461和第二激光发射单元462，第一激光发射单元461包括：第一激光二极管4611和第一准直镜4612，第二激光发射单元462包括：第二激光二极管4621和第二准直镜4622。
该实施例中，滤波单元包括汇聚镜、针孔滤波片和光电二极管。其中，汇聚镜用于接收第一对焦光信号或第二对焦光信号，并汇聚至对应的针孔滤波片，针孔滤波片用于将对应的第一对焦光信号或第二对焦光信号进行滤波得到对应的滤波信号，光电二极管用于接收对应的滤波信号。滤波单元分别是：第一滤波单元464和第二滤波单元463，其中，第一滤波单元464包括：第一汇聚镜4641、第一针孔滤波片4642和第一光电二极管4643，第二滤波单元463包括：第二汇聚镜4631、第二针孔滤波片4632和第二光电二极管4633。
具体的，自动对焦模块400进行自动对焦过程描述为：
第四二向色镜420对两个激光信号发射及滤波单元的激光发射单元分别发射的准直激光信号进行合束，生成合束激光信号。其中，第一激光发射单元461的第一激光二极管4611发射的第一激光信号至第一准直镜4612进行准直，得到第一准直激光信号。第二激光发射单元462的第二激光二极管4621发射的第二激光信号至第二准直镜4622进行准直，得到第二准直激光信号。即第四二向色镜420对第一激光发射单元461发射的第一准直激光信号和第二激光发射单元462发射的第二准直激光信号进行合束，生成合束激光信号。
扩束镜450对第四二向色镜420生成的合束激光信号进行扩束，得到扩束激光信号。
第五色镜430是一种半透半反镜，其反射扩束镜450发出的扩束激光信号，通过高通量物镜310至测序芯片321，并对来自高通量物镜传输的测序芯片321反射的光斑进行透射。
第六二向色镜440反射上述第五色镜430透射得到的光斑，得到第一对焦光信号。
第六二向色镜440透射上述第五色镜430透射得到的光斑，得到第二对焦光信号。
两个激光信号发射及滤波单元的滤波单元分别用于接收第一对焦光信号和第二对焦光信号，即第一滤波单元464接收透射得到的第二对焦光信号，第二滤波单元463接收反射得到的第一对焦光信号。
该实施例中，第一滤波单元464的第一汇聚镜4641接收第二对焦光信号，并汇聚第二对焦光信号至第一滤波单元464的第一针孔滤波片4642，第一针孔滤波片4642将汇聚后的第二对焦光信号进行滤波，得到第二滤波信号，第一光电二极管4643接收第二滤波信号。同样地，第二滤波单元463的第二汇聚镜4631接收第一对焦光信号，并汇聚第一对焦光信号至第二滤波单元463的第二针孔滤波片4632，第二针孔滤波片4632将汇聚后的第一对焦光信号进行滤波，得到第一滤波信号，第二光电二极管4633接收第一滤波信号。
计算单元470根据两个激光信号发射及滤波单元的滤波单元输出的信号强度比值计算生成相对高度测量信号，图中未示出计算单元470与其他部件之间的连接线，不代表没有连接线。该实施例中，计算单元470计算第一光电二极管4643接收的第二滤波信号和第二光电二极管4633接收的第一滤波信号的信号强度比值，然后根据信号强度比值和预设物镜离焦量之间的对应关系生成相对高度测量信号。可以理解的是，信号强度比值和预设物镜离焦量之间的对应关系可以根据实际操作中统计得到，本实施例在此不做具体限定。
相关技术中，自动追焦技术的常用思路为探测追焦光斑在CCD或CMOS上的位置信息，即测序芯片待检测样本表面与物镜离焦时，追焦光斑在CCD或CMOS上的最强点的像素位置将会改变。然而，CCD或CMOS作为多点探测器，采集单个像素的位置信息需要的运算量较大，响应时间较长，导致追焦过程中，调节物镜高度的延迟较高。在一次基因测序的过程中，常需要拍摄数万至数十万张图像，即追焦数万至数十万次，因此，控制追焦响应的速度十分重要。或者，另外一些自动追焦技术只能针对单个表面的生物芯片使用(例如硅片)，无法针对具有流道的多表面的生物芯片使用(例如，具有盖玻片的生物芯片)。 对于多表面且待测表面非最上层面的生物芯片，其最上层面的反射率通常比和流道接触的待测表面反射率强十倍以上，探测到的待测表面追焦光斑淹没于盖玻片上表面的追焦光斑中，无法实现对待测表面的追焦。
因此本申请实施例的自动对焦模块通过使用单点探测器的光电二极管作为探测器，相比于多点探测器(CCD或CMOS)，其采集速率更快，能更加快速有效地反馈强度信息，减少改变物镜高度的延迟时间，提高检测效率。另外，该自动对焦模块可兼容多种芯片(例如硅片，无盖玻片的生物芯片，具有盖玻片的生物芯片等)。且对于多表面的芯片，在不同的表面均能实现追焦。并且该自动对焦模块使用的光源为激光二极管、探测器为光电二极管，其体积小、成本低廉。
参照图15，为本申请实施例基因测序仪的结构示意图。
下面结合图15，描述本申请实施例基因测序仪各部件的使用方法，首先由光源210、光阑220和偶次非球面反射镜230组成激发模块200，作用是将圆高斯光斑整形为长宽比2：1的椭圆均匀光斑，输出激发光束。其中，光源210为双色激光器，可以选择输出红色或者绿色的激光，图中用虚线和实线分别表示不同颜色的激发光束，之后激发光束通过第一二向色镜410反射入测序模块300中。测序模块300由高通量物镜310、测序单元320、待检测样本330、成像单元340、第二二向色镜350、中继透镜组360和第三二向色镜370组成。激发光束被第二二向色镜350反射入设计的高通量物镜310，之后汇聚在测序芯片321上，测序芯片321在电动的位移台322的带动下完成对测序芯片321上待检测样本330的扫描。扫描过程中，测序芯片321中ATGC四种碱基产生的荧光再次通过高通量物镜310收集，透过第二二向色镜350被中继透镜组360所接收。经过中继透镜组360缩小了视场角后，经过第三二向色镜370，将荧光信号分为两路，分别被两个成像单元的套筒透镜341接收，并分别汇聚在两个相机342的成像芯片上。本实施例中，为了分别对四种碱基进行成像，需要轮流选择光源210的出光颜色，如先用绿光激发出碱基AT的荧光，被两个相机342分别成像，之后关闭光源210的绿光，再打开光源210的红光，激发出碱基GC的荧光，被两个相机342分别成像，以此完成四色碱基的识别。
该实施例中，在电动的位移台322扫描的过程中，为了防止由于测序芯片321高度不一致导致的成像离焦，需要用自动对焦模块400实时监控物镜焦面的情况变化。其中，自动对焦模块由第一二向色镜410、第四二向色镜420、第五色镜430、第六二向色镜440、扩束镜450、第一激光发射单元461、第一激光 二极管4611、第一准直镜4612、第二激光发射单元462、第二激光二极管4621、第二准直镜4622、第一滤波单元464、第一汇聚镜4641、第一针孔滤波片4642、第一光电二极管4643、第二滤波单元463、第二汇聚镜4631、第二针孔滤波片4632、第二光电二极管4633和计算单元470构成。
该实施例中，第一激光发射单元461的第一激光二极管4611发射的第一激光信号至第一准直镜4612进行准直，得到第一准直激光信号。第二激光发射单元462的第二激光二极管4621发射的第二激光信号至第二准直镜4622进行准直，得到第二准直激光信号。第四二向色镜420对第一激光发射单元461发射的第一准直激光信号和第二激光发射单元462发射的第二准直激光信号进行合束，生成合束激光信号。之后合束激光信号通过扩束镜450扩束后，得到扩束激光信号，该信号经过第五色镜430反射后，再经过第一二向色镜410透射，输出透射激光信号至第二二向色镜350，第二二向色镜350反射透射激光信号至高通量物镜310，经过高通量物镜310反射透射激光信号形成光斑，第二二向色镜350反射该光斑至第一二向色镜410，然后第一二向色镜410和第五色镜430依次透射光斑沿原路返回到达至第六二向色镜440，第六二向色镜440反射上述光斑，得到第一对焦光信号，第六二向色镜440透射上述光斑，得到第二对焦光信号。
该实施例中，第一滤波单元464接收透射得到的第二对焦光信号，第二滤波单元463接收反射得到的第一对焦光信号。第一滤波单元464的第一汇聚镜4641接收第二对焦光信号，并汇聚第二对焦光信号至第一滤波单元464的第一针孔滤波片4642，第一针孔滤波片4642将汇聚后的第二对焦光信号进行滤波，得到第二滤波信号，第一光电二极管4643接收第二滤波信号。同样地，第二滤波单元463的第二汇聚镜4631接收第一对焦光信号，并汇聚第一对焦光信号至第二滤波单元463的第二针孔滤波片4632，第二针孔滤波片4632将汇聚后的第一对焦光信号进行滤波，得到第一滤波信号，第二光电二极管4633接收第一滤波信号。之后计算单元470计算第一光电二极管4643接收的第二滤波信号和第二光电二极管4633接收的第一滤波信号的信号强度比值，然后根据信号强度比值和预设物镜离焦量之间的对应关系生成相对高度测量信号。可以理解的是，信号强度比值和预设物镜离焦量之间的对应关系可以根据实际操作中统计得到，本实施例在此不做具体限定。
在一实施例中，可以理解的是，本申请虽并未一一指出二向色镜输出的光斑的波长不同，但是本领域相关人员知晓二向色镜对光波的透射和反射得到的 是不同波长的光斑。该实施例中，第四二向色镜420和第五色镜430是性能一致的二向色镜，对其编号仅为了描述方便，彼此之间可以互换。第一二向色镜410、第二二向色镜350、第三二向色镜370、第五色镜和第六二向色镜440是性能不同的二向色镜，其目的是将不同波长的光分开，可根据实际需求或多次实验得到的先验知识选定不同的参数，本是是对此不做具体限定。
本发明实施例提出的基因测序仪包括激发模块和测序模块，激发模块包括：光源、光阑和偶次非球面反射镜；测序模块包括：高通量物镜、测序单元和至少一个成像单元。本实施例在基因测序仪中设计大数值孔径同时兼顾大成像视野的高通量显微物镜，满足高通量测序应用场景，提高检测效率，同时利用偶次非球面反射镜结合一个光阑，以简单的光路形式实现高斯光斑的匀化，有效的避免不必要的光漂白，提高扫描成像的效率以及后续测序的结果准确率。
另外，本发明实施例还提供一种基因测序仪的使用方法，应用于上述基因测序仪。
参照图16，为本申请实施例提供的基因测序仪使用方法流程图，方法包括步骤S1610至步骤S1650：
步骤S1610，将待检测样本放入测序芯片的检测范围内。
在一实施例中，待检测样本包括单链DNA和四种核苷酸，每种核苷酸的荧光信号的颜色均不相同。
步骤S1620，激发模块产生激发待检测样本的激发光束。
步骤S1630，测序模块根据相对高度测量信号调节高通量物镜与测序芯片之间的高度相对位移，利用激发光束对待检测样本进行扫描得到荧光信号。
在一实施例中，步骤S1630具体过程描述为：
首先获取相对高度测量信号，然后根据相对高度测量信号，控制位移台在垂直方向上调节与高通量物镜之间的高度相对位移，即调节测序芯片与高通量物镜之间的高度相对位移，以使激发光束的短轴相对测序芯片的长边移动，且激发光束的短轴与测序芯片的长边相互平行，最后在移动过程中，获取待检测样本的荧光信号。
在一实施例中，自动对焦模块生成相对高度测量信号，具体的生成过程是：自动对焦模块反射激光准直信号通过高通量物镜汇聚到测序芯片上，并接收测序芯片反射至高通量物镜的光斑。自动对焦模块利用光斑生成第一对焦光信号和第二对焦光信号，自动对焦模块根据第一对焦光信号和第二对焦光信号生成 相对高度测量信号。高通量物镜或测序芯片根据相对高度测量信号调节高度相对位移。
具体的，相机只能对其成像面A内的荧光信号进行成像，因此，为了对测序芯片的不同区域进行扫描成像，本实施例中基因测序仪设置了相应的位移台，位移台用于承载测序芯片，位移台根据相对高度测量信号进行移动，以使得调节两者之间的高度相对位移，从而使得激发光束对测序芯片的不同区域进行激发照明。
该实施例中，参照图17，测序芯片为矩形，相机的成像面A为矩形成像面，且矩形成像面A的长宽比例为L：W，激发光束的光斑D长短轴比例为L：W。当位移台处于初始位置时，激发光束的光斑D对测序芯片的X1区域进行激发照明，并产生相应的荧光信号，相机根据上述工作原理生成图像信号，并根据图像信号生成相应的检测结果x1。然后位移台继续进行移动，使得激发光束的光斑D照射在测序芯片的X2区域，并按照上述步骤获得检测结果x2。位移台继续进行移动，使得相机能够对测序芯片上的X3、X4...等区域进行扫描成像，从而获得测序芯片上不同的检测结果。
可以理解的是，当相机矩形成像面A的长宽比例为2：1，激发光束的光斑D长短轴比例为2：1时，相比于相关技术中圆形的激光信号，本实施例激发光束能够对测序芯片中更大的区域进行激发照明，并通过相配合的相机进行扫描成像。因此，本实施例基因测序仪的扫描、检测效率较高，进而能够提高单位时间内基因测序数据的输出量。
图17中激发光束的光斑的长轴与测序芯片的长边相互垂直，且激发光束沿着测序芯片的长边进行移动，以使得相机对测序芯片上的X1区域、X2区域等进行扫描操作成像，从而获得检测结果。
可以理解的是，由于测序芯片的长边对应目标荧光信号的短轴，即测序芯片的长边对应相机的短边，相比于测序芯片的长边对应相机短边的扫描方式，能够缩短位移台的单次移动距离，从而提高相机的扫描效率。
进行扫描成像的过程中，由于测序芯片高度的变化可能导致成像的离焦，因此在一实施例中，基因测序仪还包括利用自动对焦模块的反馈信息调节测序芯片的高度，来保证每一幅图像都是清晰的。
本实施例中，根据相对高度测量信号，控制高通量物镜在垂直方向上调节与位移台之间的高度相对位移相对高度测量信号，具体步骤如下：
自动对焦模块反射激光准直信号通过高通量物镜汇聚到测序芯片上，并接收所述测序芯片反射至所述高通量物镜的光斑，然后自动对焦模块利用光斑生成第一对焦光信号和第二对焦光信号，并根据第一对焦光信号和第二对焦光信号生成相对高度测量信号发送给测序模块，测序模块控制位移台根据相对高度测量信号调节两者之间的高度相对位移。
步骤S1640，扫描过程中，高通量物镜采集荧光信号。
步骤S1650，利用成像单元根据荧光信号成像得到的图像来确定单链DNA中核苷酸的排序。
由此可见，上述基因测序仪实施例中的内容均适用于本实施例的基因测序仪使用方法的实施例中，本使用方法实施例所具体实现的功能与上述基因测序仪实施例相同，并且达到的有益效果与上述基因测序仪实施例所达到的有益效果也相同。
以上参照附图说明了本发明实施例的优选实施例，并非因此局限本发明实施例的权利范围。本领域技术人员不脱离本发明实施例的范围和实质内所作的任何修改、等同替换和改进，均应在本发明实施例的权利范围之内。

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  1. 一种基因测序仪，用于激发基因测序芯片上的待检测样本并采集所述待检测样本发射的荧光信号进行荧光成像，其特征在于，包括：
    激发模块，用于产生激发所述待检测样本的激发光束；
    测序模块，用于利用所述激发光束对所述待检测样本进行荧光成像；
    所述激发模块包括：
    光源，所述光源用于产生激光信号；
    光阑，所述光阑沿所述激光信号的光轴放置于所述光源的后方，所述光阑用于对入射的激光信号进行空间滤波，以形成滤波信号；
    偶次非球面反射镜，所述偶次非球面反射镜沿所述滤波信号的光轴放置于所述光阑的后方，所述偶次非球面反射镜用于根据所述滤波信号形成激发光束；
    所述测序模块包括：
    高通量物镜，用于接收并汇聚所述激发光束到测序单元；
    测序单元，用于利用所述激发光束照射所述待检测样本产生荧光信号；
    至少一个成像单元，用于利用所述荧光信号进行荧光成像；
    所述高通量物镜包括：
    沿物方到像方依次同轴排列的第一透镜组、第二透镜组及第三透镜组，其中，
    第一透镜组包括顺次设置的第一弯月透镜和第二弯月透镜；
    第二透镜组包括顺次设置的第一双凸透镜、第一双凹透镜、第二双凸透镜、第三双凸透镜、第三弯月透镜及第四双凸透镜，所述第一双凸透镜、所述第一双凹透镜及所述第二双凸透镜组成第一胶合透镜，所述第三弯月透镜及所述第四双凸透镜组成第二胶合透镜，所述第三双凸透镜具有正光焦度；
    第三透镜组包括顺次设置的第四弯月透镜、第五弯月透镜、第二双凹透镜及第五双凸透镜，所述第四弯月透镜及所述第五弯月透镜组成第三胶合透镜，所述第二双凹透镜及所述第五双凸透镜组成第四胶合透镜。
  2. 根据权利要求1所述的一种基因测序仪，其特征在于，所述测序模块还包括：第二二向色镜和中继透镜组；
    所述高通量物镜用于接收所述荧光信号，并向所述第二二向色镜透射所述荧光信号；
    所述第二二向色镜用于透射所述荧光信号至所述中继透镜组，以形成第一荧光信号。
  3. 根据权利要求2所述的一种基因测序仪，其特征在于，中继透镜组所述中继透镜组包括第四透镜组和第五透镜组；
    所述第四透镜组具有负光焦度，所述第四透镜组沿所述荧光信号的光轴设置于所述物镜的后方，用于根据所述荧光信号进行形成第一光信号；
    所述第五透镜组具有正光焦度，所述第五透镜组沿所述第一光信号的光轴设置于所述第四透镜组的后方，用于根据所述第一光信号形成所述第一荧光信号。
  4. 根据权利要求2所述的一种基因测序仪，其特征在于，所述成像单元包括：套筒透镜和相机，所述测序模块还包括：第三二向色镜和两个所述成像单元；
    所述第三二向色镜用于反射所述第一荧光信号得到第一成像信号；
    所述第三二向色镜还用于透射所述第一荧光信号得到第二成像信号；
    两个所述成像单元的套筒透镜分别用于接收所述第一成像信号和所述第二成像信号，并输出光信号至对应的相机，以利用光信号进行荧光成像。
  5. 根据权利要求2所述的一种基因测序仪，其特征在于，所述测序单元包括：
    测序芯片，用于承载所述待检测样本；
    位移台，用于放置所述测序芯片，以利用所述激发光束照射所述待检测样本产生荧光信号。
  6. 根据权利要求5所述的一种基因测序仪，其特征在于，所述基因测序仪还包括：第一二向色镜和自动对焦模块；
    所述第一二向色镜用于透射对焦激光信号至所述自动对焦模块；
    所述自动对焦模块用于根据所述对焦激光信号生成相对高度测量信号发送至所述测序模块；
    所述测序模块用于根据所述相对高度测量信号调节所述高通量物镜与所述测序芯片的之间的高度相对位移。
  7. 根据权利要求6所述的一种基因测序仪，其特征在于，所述自动对焦模块包括：扩束镜、第四二向色镜、第五色镜、第六二向色镜、激光信号发射及滤波单元和计算单元所述激光信号发射及滤波单元包括：激光发射单元和滤波单元；激光信号发射及滤波单元；
    所述第四二向色镜用于对两个所述激光信号发射及滤波单元的所述激光发射单元分别发射的准直激光信号进行合束，生成合束激光信号；
    所述扩束镜用于对所述合束激光信号进行扩束得到扩束激光信号；
    所述第五色镜用于反射所述扩束激光信号通过所述高通量物镜至所述测序芯片，并透射来自所述高通量物镜传输的所述测序芯片反射的光斑；
    所述第六二向色镜用于反射所述光斑得到第一对焦光信号；
    所述第六二向色镜还用于透射所述光斑得到第二对焦光信号；
    两个所述激光信号发射及滤波单元的所述滤波单元分别用于接收所述第一对焦光信号和所述第二对焦光信号；
    所述计算单元用于根据两个所述激光信号发射及滤波单元的所述滤波单元输出的信号强度比值计算生成相对高度测量信号。
  8. 根据权利要求7所述的一种基因测序仪，其特征在于，所述激光发射单元包括：激光二极管和准直镜；
    所述激光二极管用于发射激光信号至所述准直镜；
    所述准直镜用于对所述激光信号进行准直生成准直激光信号。
  9. 根据权利要求7所述的一种基因测序仪，其特征在于，所述滤波单元包括汇聚镜、针孔滤波片和光电二极管；
    所述汇聚镜用于接收所述第一对焦光信号或所述第二对焦光信号，并汇聚至对应的所述针孔滤波片；
    所述针孔滤波片用于将汇聚后的所述第一对焦光信号或所述第二对焦光信号进行滤波得到对应的滤波信号；
    所述光电二极管用于接收对应的所述滤波信号。
  10. 根据权利要求9所述的一种基因测序仪，其特征在于，所述计算单元还用于计算对应的两个所述光电二极管接收到所述滤波信号的信号强度比值；
    所述计算单元还用于根据所述信号强度比值和预设物镜离焦量之间的对应关系生成所述相对高度测量信号。
  11. 根据权利要求7所述的一种基因测序仪，其特征在于，所述第一二向色镜用于透射所述第五色镜反射的所述扩束激光信号，并输出透射激光信号至所述第二二向色镜；
    所述第二二向色镜用于反射所述透射激光信号至所述高通量物镜；
    所述第二二向色镜还用于反射所述高通量物镜反射所述透射激光信号形成的所述光斑至所述第一二向色镜；
    所述第一二向色镜和所述第五色镜依次透射所述光斑至所述第六二向色镜。
  12. 根据权利要求1-11任一项所述的一种基因测序仪，其特征在于，所述偶次非球面反射镜的面型公式满足以下关系：


    其中，c为曲率，k为圆锥系数，a1为二阶非球面系数，a2为四阶非球面系数，a3为六阶非球面系数，a4为八阶非球面系数，x、y为非球面表面的坐标位置。
  13. 根据权利要求3-11任一项所述的一种基因测序仪，其特征在于，所述第四透镜组包括：
    第一透镜，所述第一透镜为具有负光焦度的双凹透镜；
    第二透镜，所述第二透镜与所述第一透镜胶合连接，所述第二透镜为具有正光焦度的弯月透镜。
  14. 根据权利要求3-11任一项所述的一种基因测序仪，其特征在于，所述第五透镜组包括：
    第三透镜，所述第三透镜沿所述第一光信号的光轴设置于所述第一透镜组的后方，所述第三透镜为具有正光焦度的双凸透镜；
    第四透镜，所述第四透镜与所述第三透镜胶合连接，所述第四透镜为具有负光焦度的弯月透镜。
  15. 一种基因测序仪的使用方法，其特征在于，应用于如权利要求1至14任一项所述的基因测序仪，所述方法包括：
    将待检测样本放入测序芯片的检测范围内，所述待检测样本包括单链DNA和四种核苷酸，每种所述核苷酸的荧光信号的颜色均不相同；
    激发模块产生激发所述待检测样本的激发光束；
    测序模块根据相对高度测量信号调节所述高通量物镜与所述测序芯片之间的高度相对位移，利用所述激发光束对所述待检测样本进行扫描得到荧光信号；
    扫描过程中，高通量物镜采集所述荧光信号并发送至成像单元；
    利用所述成像单元对所述荧光信号成像得到的图像来确定所述待检测样本中单链DNA中核苷酸的排序。
  16. 根据权利要求15所述的一种基因测序仪的使用方法，其特征在于，所述测序模块根据相对高度测量信号调节所述高通量物镜与所述测序芯片之间的高度相对位移，还包括：
    获取相对高度测量信号；
    根据所述相对高度测量信号在垂直方向上调节所述高通量物镜与所述测序芯片之间的高度相对位移，以使所述激发光束的短轴相对所述测序芯片的长边移动，且所述激发光束的短轴与所述测序芯片的长边相互平行；
    在移动过程中，获取所述待检测样本的荧光信号。
  17. 根据权利要求16所述的一种基因测序仪的使用方法，其特征在于，根据所述相对高度测量信号在垂直方向上调节所述高通量物镜与所述测序芯片之间的高度相对位移，包括：
    自动对焦模块反射激光准直信号通过高通量物镜汇聚到所述测序芯片上，并接收所述测序芯片反射至所述高通量物镜的光斑；
    自动对焦模块利用所述光斑生成第一对焦光信号和第二对焦光信号；
    自动对焦模块根据所述第一对焦光信号和所述第二对焦光信号生成所述相对高度测量信号；
    所述测序芯片根据所述相对高度测量信号调节高度相对位移。
  18. 根据权利要求15-17任一项所述的一种基因测序仪的使用方法，其特征在于，相机的成像面为矩形成像面，所述激发光束的形状为椭圆形或类椭圆形或矩形；所述矩形成像面的长宽比例为L：W；所述激发光束的光斑长短轴比例为L：W；其中，L、W均为正整数。
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