WO2024040871A1

WO2024040871A1 - Gene sequencer and method for using same

Info

Publication number: WO2024040871A1
Application number: PCT/CN2023/074142
Authority: WO
Inventors: 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
Priority date: 2022-08-22
Filing date: 2023-02-01
Publication date: 2024-02-29
Also published as: CN115287168B; CN115287168A

Abstract

A gene sequencer and a method for using same. The gene sequencer comprises an excitation module and a sequencing module. The excitation module comprises a light source, a diaphragm, and an even-order aspheric reflector. The sequencing module comprises a high-throughput objective lens, a sequencing unit, and at least one imaging unit.

Description

Gene sequencers and how to use them

The present invention claims the priority of the Chinese patent application submitted to the China Patent Office on August 22, 2022, with the application number 202211004637.9, and the application name is "Gene Sequencer and Method of Using Gene Sequencer", the entire content of which is incorporated by reference. in the present invention.

Technical field

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.

Background technique

Currently, in gene sequencing, microscopic imaging technology needs to be used to perform fluorescence imaging of the bases on the biochip. The application of microscopic imaging technology in 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.

Gene sequencers in related technologies cannot meet high-standard detection requirements due to low requirements for optical system design indicators. For example, the objective lenses of some gene sequencers have low sequencing throughput, and the detection time is long and inefficient. In other cases, 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.

Contents of the invention

The main purpose of the embodiments of the present invention is to propose a gene sequencer and a method of using the gene sequencer. By designing 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. At the same time, the Gaussian spot can be homogenized to avoid unnecessary photobleaching, improve the efficiency of scanning imaging and the accuracy of subsequent sequencing results.

In order to achieve the above object, 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;

The excitation module includes:

A light source, the light source is used to generate a laser signal;

An aperture, 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, and 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, the imaging unit includes: a tube lens and a camera, and 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, the surface shape formula of the even-order aspherical mirror satisfies the following relationship:

Among them, 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, y is the coordinate position of the aspheric surface.

In some embodiments, 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.

In some embodiments, the fifth lens group includes:

A third lens, the 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:

Put the sample to be detected into the detection range of the sequencing chip, 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;

During the scanning process, 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.

In some embodiments, 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:

Obtain relative height measurement signal;

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;

During the movement, the fluorescence signal of the sample to be detected is obtained.

In some embodiments, 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.

In some embodiments, 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.

In the gene sequencer and the method of using the gene sequencer proposed by the embodiments of the present invention, 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. Mirror; the sequencing module includes: a high-throughput objective lens, a sequencing unit and at least one imaging unit. In this embodiment, 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. At the same time, 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.

Description of drawings

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.

Explanation of reference symbols:

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. 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.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention and are not intended to limit the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which the invention belongs. The terminology used herein is for the purpose of describing embodiments of the present invention only and is not intended to limit the present invention.

Microscopic imaging technology is widely used in sample detection. For example, in gene sequencing, 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. When the gene sequencer works, it needs to perform fluorescence imaging of the bases on the biochip. When 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) is usually used. or two-channel) imaging, and then algorithmically register the detection images obtained from each channel to match the base positions of different images.

Among related technologies, 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. However, some current gene sequencers cannot meet high-standard detection needs. For example, the objective lenses of some gene sequencers have low sequencing throughput, and the detection is time-consuming and inefficient. In other cases, 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. .

Based on this, 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.

In this embodiment, 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.

In one embodiment, the excitation beam S generated by the excitation module 200 can be a laser. Based on the excitation characteristics of fluorescent dyes on lasers, 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. However, because 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. In order to increase the fluorescence brightness at the edge of the field of view to achieve the signal-to-noise ratio required for imaging, it is necessary to increase the laser power to make up for the low excitation efficiency of the edge field of view. However, increasing the laser power will cause the laser intensity in the central field of view to be too high, which will increase the photobleaching rate. Excessive light intensity will damage the DNA in the central field of view, leading to an increase in the error rate of subsequent sequencing results. Therefore, in this embodiment, 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. .

Refer to Figure 2, which is a schematic structural diagram of the excitation module in this embodiment.

Light source 210. The light source 210 is used to generate a laser signal.

In one embodiment, 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.

In one embodiment, the aperture 220 refers to a device that limits the light beam in the optical system. When the laser signal is incident on the aperture 220, 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.

In one embodiment, 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. To this end, in this embodiment, 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. In addition, 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.

In one embodiment, the surface shape formula of the even-order aspherical mirror 230 satisfies the following relationship:

In this embodiment, when the even-order aspherical reflector 230 satisfies the above formula, after the filtered signal is reflected by the even-order aspherical reflector 230, an excitation beam with an elliptical spot can be obtained. In addition, by setting 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. It can be seen from the above that by matching the excitation beam of the elliptical spot with the rectangular imaging surface of the camera, the efficiency of camera scanning and imaging can be improved. In addition, 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.

Referring to 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, and 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.

In this embodiment, 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.

In some embodiments, k=-146.5, a ₁ =0, a ₂ =1.848E ^-4 , a ₃ =-4.159E ^-6 , a ₄ =3.216E ^-8 .

In one embodiment, the focal length of the even-order aspherical mirror 230 satisfies the following relationship:
14.6<f ₀ <16.1

Wherein, f ₀ is the focal length of the even-order aspherical mirror 230 .

It can be understood that if the focal length of the even-order aspherical reflector 230 satisfies the above relationship, 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. Similarly, when 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.

During sequencing, a moving stage is required to cooperate with a microscope system to complete scanning and imaging of the entire sequencing chip. Sequencing chips are generally rectangular, so the main scanning time is spent on the long sides. Based on the above situation, on the premise of ensuring that the number of camera pixels meets the requirements, choose a camera with an aspect ratio of 2:1 so that the long side scanning direction of the sequencing chip corresponds to the short side of the camera, which can shorten the single movement distance of the displacement stage. This improves scanning efficiency and thereby increases sequencing data output per unit time. Based on the above situation, if 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 fluorescence signals excited by these excess imaging parts cannot be received by the camera and need to be excited again in subsequent scans to achieve signal collection in this area. This multiple excitations can cause photobleaching of the area, thereby reducing the signal-to-noise ratio. And the increased phototoxicity will also damage DNA, affecting subsequent sequencing error rates. 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. When the excitation beam is used to excite and illuminate the sample to be detected 330, the light intensity in each illumination area is uniform, and there is no problem of inconsistent excitation efficiency. Therefore, the excitation module 200 of this embodiment can achieve better excitation lighting effects with a simple structure.

In one embodiment, in order to improve the scanning imaging efficiency of the camera during fluorescence imaging, 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. Specifically, 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. After the sample is excited and illuminated, 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.

In one embodiment, 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. Among them, 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.

Refer to Figure 5, which is a schematic structural diagram of a sequencing module according to an embodiment of the present application.

In this embodiment, 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.

Refer to FIG. 6 , which is a schematic structural diagram of a high-flux objective lens in an embodiment of the present application.

In one embodiment, the imaging field of view and the numerical aperture of the objective lens are two key parameters that determine the sequencing throughput. However, 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. To address the above problems, 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. In this embodiment, 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, and 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.

In this embodiment, 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. No. 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. In this embodiment, 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.

In one embodiment, 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

Among them, 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, and f _L89 represents the second cemented lens. The focal length of f _L1011 represents the focal length of the third cemented lens, and 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.

In one embodiment, please refer to Table 1 for specific parameters of each lens in the high-flux objective lens 310 . Referring to Figure 6, R1-R21 in Figure 6 respectively correspond to the surface numbers 1-21 in Table 1.

Table I

In one embodiment, 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, and the focal length are 10 mm. The above parameters can meet most high-throughput sequencing applications of gene sequencers. Scenes.

In one embodiment, 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.

In one embodiment, since fluorescence imaging requires dichroic mirrors and filters to split light and filter signals of different wavelengths, there is inevitably a long distance between the objective lens and the tube lens. Long distance will lead to an increase in the size of the tube lens, which will first increase the processing cost and make it difficult to guarantee the surface accuracy of the lens. Secondly, the increase in size will increase the weight of the lens, which will have a certain impact on the fixation and adjustment of the lens. Therefore, 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.

Refer to FIG. 7 , which 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.

In this embodiment, 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.

In one embodiment, the relay lens group 360 includes:

High Flux Objective Lens 310 High Flux Objective Lens 310 Referring to FIG. 8 , the relay lens group 360 includes: a fourth lens group 362 and a fifth lens group 363 .

Among them, 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.

In one embodiment, 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. Similarly, 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. . After the fluorescence signal passes through the fourth lens group 362 and the fifth lens group 363, it can form a first fluorescence signal with a smaller maximum exit angle. After a certain transmission distance, the beam diameter of the first fluorescence signal is still small.

In one embodiment, 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.

Referring to Figure 8, in this embodiment, 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.

In one embodiment, the first lens 364 and the second lens 365 are cemented and connected to form a double cemented lens (the fourth lens group 362). Among them, 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. Among them, field curvature refers to field curvature. When field curvature exists in the first lens 364 or the second lens 365, the intersection point of the fluorescence signal no longer coincides with the ideal image point, and the entire image plane is a curved surface, making the subsequent camera 342 When imaging the first fluorescent signal, the entire image plane cannot be seen clearly at the same time, which makes detection difficult. Therefore, in this embodiment, 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.

In one embodiment, 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 .

Referring to FIG. 8 , in some embodiments, 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.

It can be understood that the first incident surface S1 is the incident surface of the first lens 364 , and the first exit surface is the exit surface of the first lens 364 . The first incident surface S1 is used for receiving fluorescent signals, and the first exit surface is used for emitting fluorescent signals processed by the first lens 364 . In addition, it can be seen from the above that 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.

It can be understood that in order to achieve the above-mentioned optical performance of the first lens 364 and the second lens 365, the first incident surface S1, the first gluing surface S2 and the second exit surface S3 need to meet corresponding parameter settings. In practical applications, 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.

Referring to Figure 8, in some embodiments, 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.

It can be understood that 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. At the same time, 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. Therefore, in this embodiment, 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.

In some embodiments, 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.

Referring to FIG. 8 , in some embodiments, 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.

It can be understood that the third incident surface S4 is the incident mirror surface of the third lens 366 , and 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, and the third exit surface is used to emit the first optical signal processed by the third lens 366 . In addition, it can be seen from the above that 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.

It can be understood that in order to achieve the above-mentioned optical performance of the third lens 366 and the fourth lens 367, the third incident surface S4, the second gluing surface S5 and the fourth exit surface S6 need to meet corresponding parameter settings. In practical applications, 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.

In a specific embodiment, 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.

Refer to Figure 9, which is a schematic structural diagram of a sequencing module according to an embodiment of the present application.

In this embodiment, 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.

In this embodiment, 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. Afterwards, 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. It can be understood that 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.

Refer to FIG. 10 , which is a schematic structural diagram of an imaging unit in an embodiment of the present application.

In this embodiment, 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.

Referring to FIG. 9 , dotted lines and solid lines respectively represent excitation beams of different colors in FIG. 9 . 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, and 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.

To be clear, 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. 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 outputs digital image signals to DS for processing. DS converts digital image signals into standard RGB, YUV and other format image signals.

Refer to Figure 11, which is a schematic structural diagram of a sequencing module according to an embodiment of the present application.

In this embodiment, 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. In this embodiment, 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. Among them, 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. Among them, the maximum exit angle θ1 of the high-flux objective lens 310 for emitting the fluorescence signal is greater than the maximum exit angle θ2 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. It can be seen from the above that after passing through the same transmission distance L1, since the maximum exit angle θ2 of the first fluorescence signal is smaller, the beam radius r2 corresponding to the incident mirror surface of the tube lens 341 of the first fluorescence signal is larger than the beam radius r2 of the fluorescence signal. The radius is smaller, and the diameter of the tube lens 341 is also smaller. It can be understood that 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.

In this embodiment, 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. In addition, the 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. In this embodiment, sequencing The image quality of all fields of view of the module 300 is close to the diffraction limit.

When the sequencing unit 320 performs sequencing, a moving stage or a high-throughput objective lens is required to cooperate with a microscope system to complete scanning and imaging of the entire sequencing chip. Refer to Figure 12, which is a schematic structural diagram of the sequencing unit in the embodiment of the present application.

The sequencing chip 321 is used to carry the sample 330 to be detected.

In one embodiment, 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.

In one embodiment, during the process of scanning and imaging the sequencing chip 321 by the gene sequencer, changes in the height of the sequencing chip 321 may cause the image to be defocused. Therefore, 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.

Refer to Figure 13, which is a schematic structural diagram of the autofocus module in the embodiment of the present application.

In this embodiment, the gene sequencer 100 also includes: a first dichroic mirror 410 and an autofocus module. 400.

Among them, 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.

Refer to Figure 14, which is a schematic structural diagram of the autofocus module in the embodiment of the present application.

In this embodiment, 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.

In this embodiment, the laser emitting unit includes: a laser diode and a collimating mirror. Among them, the laser diode is used to emit the laser signal to the collimating mirror, and 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.

In this embodiment, the filtering unit includes a converging mirror, a pinhole filter and a photodiode. Wherein, 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, and 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, and 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.

Specifically, 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. Among them, 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.

In this embodiment, 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. Similarly, 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. In this embodiment, 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.

In related technologies, 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. However, 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. In a gene sequencing process, it is often necessary to take tens of thousands to hundreds of thousands of images, that is, to focus on tens of thousands to hundreds of thousands of times. Therefore, it is very important to control the speed of focus tracking response. Alternatively, 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). For biochips with multiple surfaces and the surface to be tested is not the uppermost layer, 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.

Therefore, 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. In addition, 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. Moreover, 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.

Refer to Figure 15, which is a schematic structural diagram of a gene sequencer according to an embodiment of the present application.

Next, with reference to Figure 15, the method of using each component of the gene sequencer according to the embodiment of the present application is described. First, 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. Among them, 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. During the scanning process, 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 . After the field of view is reduced by the relay lens group 360, 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. In this embodiment, 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.

In this embodiment, during the scanning process of the electric displacement stage 322, in order to prevent imaging defocus due to inconsistent heights of the sequencing chips 321, the autofocus module 400 needs to be used to monitor changes in the focal surface of the objective lens in real time. Among them, 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.

In this embodiment, 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. 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.

In this embodiment, the first filter unit 464 receives the transmitted second focus light signal, and 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, and the first photodiode 4643 receives the second filtered signal. Similarly, 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.

In an embodiment, it can be understood that although this application does not point out one by one that the wavelengths of the light spots output by the dichroic mirror are different, those relevant in the art know that the wavelengths obtained by the transmission and reflection of light waves by the dichroic mirror are They are light spots of different wavelengths. 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. In this embodiment, 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. At the same time, 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.

In addition, embodiments of the present invention also provide a method of using a gene sequencer, which is applied to the above gene sequencer.

Referring to Figure 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.

In one embodiment, 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.

In one embodiment, the specific process of step S1630 is described as:

First, 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. Finally, during the movement, the fluorescence signal of the sample to be detected is obtained.

In one embodiment, 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.

Specifically, 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.

In this embodiment, referring to Figure 17, the sequencing chip is rectangular, the imaging surface A of the camera is a rectangular imaging surface, and the length-to-width ratio of the rectangular imaging surface A is L:W, and the long and short axis ratio of the spot D of the excitation beam is L:W. . When the displacement stage is in the initial position, 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. Then 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.

It can be understood that when the length-to-width ratio of the rectangular imaging surface A of the camera is 2:1, and the long and short axis ratio of the spot D of the excitation beam is 2:1, compared with the circular laser signal in the related art, 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.

In Figure 17, 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.

It can be understood that since the long side of the sequencing chip corresponds to the short axis of the target fluorescence signal, that is, the long side of the sequencing chip corresponds to the short side of the camera, compared with the scanning method in which the long side of the sequencing chip corresponds to the short side of the camera, the displacement can be shortened. The single movement distance of the stage improves the scanning efficiency of the camera.

During the scanning and imaging process, changes in the height of the sequencing chip may cause the imaging to be out of focus. Therefore, in one embodiment, 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.

In this embodiment, 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.

It can be seen that the content in the above embodiment of the gene sequencer is applicable to the embodiment of the method of using the gene sequencer of this embodiment. The functions specifically implemented by this embodiment of the method of use are the same as those of the above embodiment of the gene sequencer, and The beneficial effects achieved are also the same as those achieved by the above-mentioned gene sequencer embodiment.

The preferred embodiments of the embodiments of the present invention have been described above with reference to the accompanying drawings, but this does not limit the scope of rights of the embodiments of the present invention. Any modifications, equivalent substitutions and improvements made by those skilled in the art without departing from the scope and essence of the embodiments of the present invention shall be within the scope of rights of the embodiments of the present invention.

Claims

A gene sequencer 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, which is characterized by 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;

The excitation module includes:

A light source, the light source is used to generate a laser signal;

An aperture, 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, and 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.
A gene sequencer according to claim 1, characterized in that the sequencing module further includes: a second dichroic mirror and a relay 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 second dichroic mirror is used to transmit the fluorescent signal to the relay lens group to form a first fluorescent signal.
A gene sequencer according to claim 2, characterized in that the relay lens group includes a fourth lens group and a fifth lens group;

The fourth lens group has negative refractive power, and is disposed behind the objective lens along the optical axis of the fluorescence signal for forming 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.
A gene sequencer according to claim 2, wherein the imaging unit includes: a sleeve lens and a camera, and 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.
A gene sequencer according to claim 2, characterized in that 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.
A gene sequencer according to claim 5, characterized in that 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 used to adjust the relative height displacement between the high-throughput objective lens and the sequencing chip according to the relative height measurement signal.
A gene sequencer according to claim 6, characterized in that the automatic focusing module includes: a beam expander, a fourth dichroic mirror, a fifth dichroic mirror, a sixth dichroic mirror, a laser signal transmitter and filtering unit and computing unit. The laser signal transmitting and filtering unit includes: a laser transmitting unit and a filtering unit; a laser signal transmitting and filtering 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.
A gene sequencer according to claim 7, characterized in that 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.
A gene sequencer according to claim 7, characterized in that 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.
A gene sequencer according to claim 9, characterized in that 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.
A gene sequencer according to claim 7, characterized in that 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 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.
A gene sequencer according to any one of claims 1-11, characterized in that the surface shape formula of the even-order aspherical mirror satisfies the following relationship:

Among them, 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, y is the coordinate position of the aspheric surface.
A gene sequencer according to any one of claims 3-11, characterized in that 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.
A gene sequencer according to any one of claims 3-11, characterized in that the fifth lens group includes:

A third lens, the 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 method of using a gene sequencer, characterized in that it is applied to the gene sequencer according to any one of claims 1 to 14, and the method includes:

Put the sample to be detected into the detection range of the sequencing chip, 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;

During the scanning process, 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 method of using a gene sequencer according to claim 15, wherein 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 further includes: :

Obtain relative height measurement signal;

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;

During the movement, the fluorescence signal of the sample to be detected is obtained.
The method of using a gene sequencer according to claim 16, characterized in that 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, include:

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 method of using a gene sequencer according to any one of claims 15 to 17, characterized in that the imaging surface of the camera is a rectangular imaging surface, and the shape of the excitation beam is elliptical, elliptical-like or rectangular; The length-to-width ratio of the rectangular imaging surface is L:W; the long and short axis ratio of the light spot of the excitation beam is L:W; where L and W are both positive integers.