CN115287168A

CN115287168A - Gene sequencer and application method thereof

Info

Publication number: CN115287168A
Application number: CN202211004637.9A
Authority: CN
Inventors: 陈龙超; 梁倩; 王谷丰
Original assignee: Shenzhen Sailu Medical Technology Co ltd
Current assignee: Shenzhen Sailu Medical Technology Co ltd
Priority date: 2022-08-22
Filing date: 2022-08-22
Publication date: 2022-11-04
Also published as: WO2024040871A1

Abstract

The embodiment of the invention provides a gene sequencer and a using method of the gene sequencer, and relates to the technical field of medical equipment. Wherein, gene sequencer includes excitation module and sequencing module, and the excitation module includes: a light source, a diaphragm and an even aspheric reflector; the sequencing module comprises: a high-throughput objective lens, a sequencing unit and at least one imaging unit. The embodiment designs the high-flux microobjective with a large numerical aperture and a large imaging field of vision simultaneously in the gene sequencer, satisfies the application scene of high-flux sequencing, improves the detection efficiency, simultaneously utilizes an even aspheric mirror to combine with a diaphragm to the homogenization of Gaussian light spots is realized in a simple light path form, the unnecessary photobleaching is effectively avoided, and the efficiency of scanning imaging and the result accuracy of subsequent sequencing are improved.

Description

Gene sequencer and application method thereof

Technical Field

The invention relates to the technical field of medical equipment, in particular to a gene sequencer and a use method of the gene sequencer.

Background

At present, in gene sequencing, a microscopic imaging technology is required to perform fluorescence imaging on bases on a biochip. The application of the microscopic imaging technology in gene sequencing is more and more extensive, and the detection requirements are diversified, so that the requirements on the detection efficiency and the detection accuracy of the gene sequencer are increased.

The gene sequencer in the related art cannot meet the high-standard detection requirement due to the low requirement of the optical system design index. For example, some gene sequencers have low objective sequencing flux and low detection efficiency for a long time, and other gene sequencers excite laser of a fluorescent dye to conform to gaussian distribution, so that the light intensity at the center of an imaging field of view is strong, the light intensity at the edge of the imaging field of view is weak, the excitation lighting effect is poor, the excitation efficiency is low, the scanning imaging efficiency and the detection result accuracy of subsequent sequencing are influenced, and the high detection requirement cannot be met.

Disclosure of Invention

The embodiment of the invention mainly aims to provide a gene sequencer and a use method of the gene sequencer, and the micro objective with large numerical aperture and large imaging visual field is designed in the gene sequencer, so that a high-throughput sequencing application scene is met, and the detection efficiency is improved. Meanwhile, homogenization of Gaussian spots is realized, unnecessary photobleaching is avoided, and the scanning imaging efficiency and the result accuracy of subsequent sequencing are improved.

In order to achieve the above object, a first aspect of the embodiments of the present invention provides a gene sequencer for exciting a sample to be detected on a gene sequencing chip and collecting a fluorescence signal emitted by the sample to be detected for fluorescence imaging, including:

the excitation module is used for generating an excitation light beam for exciting the sample to be detected;

the sequencing module is used for carrying out fluorescence imaging on the sample to be detected by utilizing the excitation light beam;

the excitation module comprises:

a light source for generating a laser signal;

the diaphragm is arranged behind the light source along the optical axis of the laser signal and is used for carrying out spatial filtering on the incident laser signal so as to form a filtering signal;

the even-order aspheric reflector is placed behind the diaphragm along the optical axis of the filtering signal and is used for forming an excitation beam according to the filtering signal;

the sequencing module comprises:

the high-flux objective lens is used for receiving and converging the excitation light beam to the sequencing unit;

the sequencing unit is used for irradiating the sample to be detected by using the excitation light beam to generate a fluorescence signal;

at least one imaging unit for fluorescence imaging using the fluorescence signal;

the high-throughput objective lens includes:

a first lens group, a second lens group and a third lens group coaxially arranged in sequence from an object side to an image side,

the first lens group comprises a first meniscus lens and a second meniscus lens which are sequentially arranged;

the second lens group comprises a first biconvex lens, a first biconcave lens, a second biconvex lens, a third meniscus lens and a fourth biconvex lens which are sequentially arranged, wherein the first biconvex lens, the first 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 focal power;

the third lens group comprises a fourth meniscus lens, a fifth meniscus lens, a second biconcave lens and a fifth biconvex lens which are arranged in sequence, the fourth meniscus lens and the fifth meniscus lens form a third cemented lens, and the second biconcave lens and the fifth biconvex lens form a fourth cemented lens.

In some embodiments, the sequencing module further comprises: a second dichroic mirror and a relay lens group;

the second dichroic mirror is used for transmitting the fluorescent signal emitted by the sequencing unit to the relay lens group so as to form a first fluorescent signal.

In some embodiments, the sequencing module further comprises: a relay lens group including a fourth lens group and a fifth lens group;

the high-flux objective lens is used for receiving the fluorescence signal and transmitting the fluorescence signal to the second dichroic mirror; the fourth lens group of the relay lens has negative focal power, is arranged behind the objective lens along the optical axis of the fluorescence signal and is used for forming a first optical signal according to the fluorescence signal;

the fifth lens group has positive focal power, is arranged behind the fourth lens group along the optical axis of the first optical signal, and is used for forming the first fluorescent signal according to the first optical signal.

In some embodiments, the imaging unit comprises: a sleeve lens and a camera, the sequencing module further comprising: a third dichroic mirror and two of the imaging units;

the third dichroic mirror is used for reflecting the first fluorescent signal to obtain a first imaging signal;

the third dichroic mirror is further used for transmitting the first fluorescent signal to obtain a second imaging signal;

the sleeve lenses of the two imaging units are respectively used for receiving the first imaging signal and the second imaging signal and outputting optical signals to corresponding cameras so as to perform fluorescence imaging by using the optical signals.

In some embodiments, the sequencing unit comprises:

the sequencing chip is used for bearing the sample to be detected;

and the displacement table is used for placing the sequencing chip so as to utilize the excitation light beam to irradiate the sample to be detected to generate a fluorescence signal.

In some embodiments, the gene sequencer further comprises: the automatic focusing device comprises a first dichroic mirror and an automatic focusing module;

the first dichroic mirror is used for transmitting a focusing laser signal to the automatic focusing module;

the automatic focusing module is used for generating a relative height measuring signal according to the focusing laser signal and sending the relative height measuring signal to the sequencing module;

the sequencing module is used for adjusting a height relative displacement relative height measurement signal between the high-throughput objective lens and the sequencing chip according to the relative height measurement signal.

In some embodiments, the auto-focus module comprises: the device comprises a beam expanding lens, a fourth dichroic mirror, a fifth dichroic mirror, a sixth dichroic mirror, two laser signal transmitting and filtering units and a calculating unit laser signal transmitting and filtering unit;

the fourth dichroic mirror is used for combining the collimated laser signals respectively emitted by the laser emission units of the two laser signal emission and filtering units to generate combined laser signals;

the beam expanding lens is used for expanding the beam of the combined laser signal to obtain a beam expanded laser signal;

the fifth chromatic mirror is used for reflecting the expanded beam laser signals to the sequencing chip through the high-flux objective lens and transmitting light spots reflected by the sequencing chip and transmitted by the high-flux objective lens;

the sixth dichroic mirror is used for reflecting the light spots to obtain a first focusing light signal;

the sixth dichroic mirror is further used for transmitting the light spots to obtain a second focused light signal;

the filter units of the two laser signal transmitting and filtering units are respectively used for receiving the first focusing light signal and the second focusing light signal;

the calculating unit is used for calculating and generating a relative height measuring signal according to the signal intensity ratio output by the filtering 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 for transmitting a laser signal to the collimating mirror;

the collimating mirror is used for collimating 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 converging mirror is used for receiving the first focusing light signal or the second focusing light signal and converging the first focusing light signal or the second focusing light signal to the corresponding pinhole filter;

the pinhole filter is used for filtering the converged first focusing light signal or the converged second focusing light signal to obtain a corresponding filtering signal;

the photodiode is used for receiving the corresponding filtering signal.

In some embodiments, the calculating unit is further configured to calculate a signal strength ratio of the filtered signals received by the corresponding two photodiodes;

the calculating unit is further used for generating the relative height measuring signal according to the corresponding relation between the signal intensity ratio and the defocus amount of the preset objective lens.

In some embodiments, the first dichroic mirror is configured to transmit the expanded beam laser signal reflected by the fifth dichroic mirror and output a transmitted laser signal to the second dichroic mirror;

the second dichroic mirror is used for reflecting the transmission laser signal to the high-flux objective lens;

the second dichroic mirror is further used for reflecting the light spot formed by the transmission laser signal reflected by the high-flux objective mirror to the first dichroic mirror;

the first dichroic mirror and the fifth dichroic mirror sequentially transmit the light spots to the sixth dichroic mirror.

In some embodiments, the even aspheric mirror has a face formula that satisfies the following relationship:

wherein c is the curvature, k is the conic coefficient, a ₁ Is a second order aspheric coefficient, a ₂ Is a fourth order aspheric coefficient, a ₃ Is a sixth order aspheric coefficient, a ₄ And x and y are coordinate positions of the aspheric surface.

In some embodiments, the fourth lens group includes:

a first lens that is a biconcave lens having a negative optical power;

the second lens is in glued joint with the first lens, and the second lens is a meniscus lens with positive focal power.

In some embodiments, the fifth lens group includes:

a third lens disposed behind the first lens group along an optical axis of the first optical signal, the third lens being a biconvex lens having a positive optical power;

and the fourth lens is in glued joint with the third lens and is a meniscus lens with negative focal power.

A second aspect of the embodiments of the present invention provides a method for using a gene sequencer, the method being applied to the gene sequencer according to any one of the first aspect, the method comprising:

putting a sample to be detected into a detection range of a sequencing chip, wherein the sample to be detected comprises single-stranded DNA and four nucleotides, and the color of a fluorescent signal of each nucleotide is different;

the excitation module generates an excitation beam for exciting the sample to be detected;

the sequencing module adjusts the height relative displacement between the high-flux objective lens and the sequencing chip according to the relative height measurement signal, and the excitation light beam is used for scanning the sample to be detected to obtain a fluorescence signal;

in the scanning process, the high-flux objective lens collects the fluorescence signal and sends the fluorescence signal to the imaging unit;

and determining the sequencing of nucleotides in the single-stranded DNA in the sample to be detected by utilizing an image obtained by imaging the fluorescence signal by the imaging unit.

In some embodiments, the sequencing module adjusts a height relative displacement relative height measurement signal between the high-throughput objective lens and the sequencing chip as a function of the relative height measurement signal, further comprising:

acquiring a relative height measurement signal;

adjusting the height relative displacement between the high-flux objective lens and the sequencing chip in the vertical direction according to the relative height measurement signal so as to enable the short axis of the excitation light beam to move relative to the long edge of the sequencing chip, wherein the short axis of the excitation light beam is parallel to the long edge of the sequencing chip;

and acquiring a fluorescence signal of the sample to be detected in the moving process.

In some embodiments, said adjusting a height relative displacement between said high-throughput objective lens and said sequencing chip in a vertical direction according to said relative height measurement signal comprises:

the automatic focusing module reflects the laser collimation signal to be converged on the sequencing chip through the high-flux objective lens and receives light spots reflected to the high-flux objective lens by the sequencing chip;

the automatic focusing module generates a first focusing light signal and a second focusing light signal by using the light spots;

the automatic focusing module generates the relative height measuring signal according to the first focusing light signal and the second focusing light signal;

and the sequencing chip adjusts the height relative displacement according to the relative height measurement signal.

In some embodiments, the imaging plane of the camera is a rectangular imaging plane, the shape of the excitation beam is elliptical or elliptical-like or rectangular; the length-width ratio of the rectangular imaging surface is L: w; the ratio of the long axis to the short axis of the light spot of the excitation light beam is L: w; wherein, L and W are positive integers.

The embodiment of the invention provides a gene sequencer and a use method of the gene sequencer, wherein the gene sequencer comprises an excitation module and a sequencing module, and the excitation module comprises: a light source, a diaphragm and an even aspheric reflector; the sequencing module comprises: a high-throughput objective lens, a sequencing unit and at least one imaging unit. The embodiment designs a high-flux microobjective with a large numerical aperture and a large imaging visual field in a gene sequencer, satisfies the application scene of high-flux sequencing, improves the detection efficiency, utilizes an even aspheric reflector to combine with a diaphragm, realizes the homogenization of Gaussian spots in a simple light path form, effectively avoids unnecessary photobleaching, and improves the efficiency of scanning imaging and the result accuracy of subsequent sequencing.

Drawings

FIG. 1 is a schematic structural diagram of a gene sequencer according to an embodiment of the present invention.

FIG. 2 is a schematic diagram showing the structure of an excitation module of a gene sequencer according to still another embodiment of the present invention.

FIG. 3 is a light intensity distribution diagram in the long axis direction of the light spot of the excitation beam of the excitation module of the gene sequencer according to still another embodiment of the present invention.

FIG. 4 is a diagram showing a distribution of light intensity in the minor axis direction of a spot of an excitation beam of an excitation module of a gene sequencer according to still another embodiment of the present invention.

FIG. 5 is a schematic diagram showing the structure of a sequencing module of a gene sequencer according to still another embodiment of the present invention.

FIG. 6 is a schematic diagram of a high-throughput objective lens of a gene sequencer according to still another embodiment of the present invention.

FIG. 7 is a schematic diagram of a relay lens set position of a gene sequencer according to still another embodiment of the present invention.

FIG. 8 is a schematic diagram of a relay lens set of a gene sequencer according to still another embodiment of the present invention.

FIG. 9 is a schematic diagram showing the structure of a sequencing module of a gene sequencer according to still another embodiment of the present invention.

FIG. 10 is a schematic diagram showing the structure of an imaging unit of a gene sequencer according to still another embodiment of the present invention.

FIG. 11 is a schematic diagram showing the structure of a sequencing module of a gene sequencer according to still another embodiment of the present invention.

FIG. 12 is a schematic diagram showing the structure of a sequencing unit of a gene sequencer according to still another embodiment of the present invention.

FIG. 13 is a schematic diagram of an autofocus module of the gene sequencer according to another embodiment of the present invention.

FIG. 14 is a schematic diagram of an autofocus module of the gene sequencer according to another embodiment of the present invention.

FIG. 15 is a schematic diagram of a gene sequencer according to still another embodiment of the present invention.

FIG. 16 is a flow chart of a method for using a gene sequencer according to an embodiment of the present invention.

FIG. 17 is a schematic diagram of an image of a method of using a gene sequencer according to yet another embodiment of the invention.

Description of reference numerals:

a gene sequencer 110, an excitation module 200, a sequencing module 300, and an auto-focus module 400;

the excitation module 200 includes: light source 210, stop 220, and even aspheric mirror 230;

the sequencing module 300 includes: the high-flux objective lens 310, the sequencing unit 320, the sample to be detected 330, the imaging unit 340, the second dichroic mirror 350, the relay lens group 360 and the 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 sleeve lens 341 and a camera 342;

the relay lens group 360 includes: high-flux objective lens 310 fourth lens group 362, fifth lens group 363, first lens 364, second lens 365, third lens 366, and fourth lens 367;

first dichroic mirror 410 and autofocus module 400, autofocus module 400 comprising: fourth dichroic mirror 420, fifth dichroic mirror 430, sixth dichroic mirror 440, beam expander 450, first laser emitting 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 converging mirror 4641, first pinhole filter 4642, first photodiode 4643, second filtering unit 463, second converging mirror 4631, second pinhole filter 4632, second photodiode 4633 and calculating unit 470.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.

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

Microscopic imaging techniques have wide application in sample detection, for example, in gene sequencing, where fluorescence imaging of bases on a biochip is required. The gene sequencer has wide application in the fields of medicine and life science, such as detection of pathogens, genetic diseases and tumor genes, medicine individualized treatment, noninvasive prenatal detection and the like. When the gene sequencer works, fluorescence imaging needs to be carried out on bases on a biochip. When a gene sequencer is used for sequencing, fluorescence imaging is carried out on four bases of ATGC, namely adenine (A), thymine (T), cytosine (C) and guanine (G), multi-channel (such as four-channel or two-channel) imaging is usually adopted, and then algorithm registration is carried out on detection images obtained by each channel, so that the base positions of different images are matched.

In the related art, the application of gene sequencing is more and more extensive, and the detection requirements are diversified, so that the requirements on the detection efficiency and the detection accuracy of the gene sequencer are increased. However, some current gene sequencers cannot meet high-standard detection requirements, for example, objective sequencing fluxes of some gene sequencers are low, detection takes a long time and the efficiency is low, and laser of some other gene sequencers for exciting fluorescent dyes conforms to gaussian distribution, the light intensity of the laser at the center of an imaging visual field is strong, the light intensity at the edge of the imaging visual field is weak, excitation illumination effect is poor, excitation efficiency is low, scanning imaging efficiency and detection result accuracy of subsequent sequencing are affected, and high detection requirements cannot be met.

Based on this, the embodiment of the invention provides a gene sequencer and a use method of the gene sequencer, wherein a high-flux microobjective with a large numerical aperture and a large imaging field of view is designed in the gene sequencer, so that a high-flux sequencing application scene is met, the detection efficiency is improved, and meanwhile, an even aspheric mirror is combined with a diaphragm to realize homogenization of Gaussian spots in a simple light path manner, so that unnecessary photobleaching is effectively avoided, and the scanning imaging efficiency and the result accuracy of subsequent sequencing are improved.

The embodiments of the present invention provide a gene sequencer and a method for using the gene sequencer, and specifically, the following embodiments are provided to describe a gene sequencer in the embodiments of the present invention.

In this embodiment, the gene sequencer 100 is used to excite a sample to be detected on a gene sequencing chip, and collect a fluorescence signal emitted by the sample to be detected to perform fluorescence imaging, and includes:

the excitation module 200 is configured to generate an excitation beam S for exciting a sample to be detected.

The sequencing module 300 is configured to perform fluorescence imaging on the sample to be detected by using the excitation beam S.

In an embodiment, the excitation light beam S generated by the excitation module 200 may be laser, and the gene sequencer 100 can generate a laser signal through the excitation module 200 to form a corresponding illumination region based on the excitation characteristic of the fluorescent dye to the laser, irradiate the sample to be detected to excite and illuminate the fluorescent dye in the illumination region, so that the sample to be detected generates a corresponding fluorescent signal under the excitation of the laser signal, and image the fluorescent signal through the camera, thereby detecting the gene sequence. However, because the laser has the characteristic of gaussian distribution, the light intensity at the center of the illumination area is strong, while the light intensity at the edge of the illumination area is weak, and the excitation illumination effect is poor. In order to increase the fluorescence brightness at the edge of the visual field to achieve the signal-to-noise ratio required by imaging, the laser power needs to be increased to compensate the defect of low excitation efficiency of the edge visual field. However, increasing the laser power causes the laser intensity in the central field to be too high, which results in an increase in the photobleaching speed, and the too high intensity damages the DNA in the central field, which results in an increase in the error rate of the subsequent sequencing. Therefore, in the embodiment, the even-order aspheric mirror is combined with a diaphragm, so that the homogenization of the Gaussian spots is realized in a simple light path mode, unnecessary photobleaching is effectively avoided, and the efficiency of scanning imaging and the accuracy of the subsequent sequencing result are improved.

Fig. 2 is a schematic structural diagram of the excitation module in this embodiment.

A light source 210, the light source 210 being for generating a laser signal.

In one embodiment, the light source 210 is used to generate a collimated laser signal having a spot shape that is circular.

And the diaphragm 220 is arranged behind the light source 210 along the optical axis of the laser signal, and the diaphragm 220 is used for spatially filtering the incident laser signal to form a filtered signal.

In an embodiment, the diaphragm 220 is a device that limits a light beam in an optical system, and when a laser signal enters the diaphragm 220, the diaphragm 220 can filter the light beam of the laser signal, so as to block an edge portion with weak light intensity of the laser signal, so as to facilitate further light homogenization of the laser signal by the even-order aspheric mirror 230 in the following.

The even aspheric mirror 230 is disposed behind the diaphragm 220 along an optical axis of the filtering signal, and the even aspheric mirror 230 is configured to form an excitation beam S according to the filtering signal, wherein the excitation beam S is configured to excite the sample to be detected to generate a fluorescence signal.

In one embodiment, the laser signal is a circular spot with a gaussian distribution, i.e. the light intensity is in a gaussian distribution in the spot area (illumination area) of the laser signal. For this purpose, the excitation module 200 of the present embodiment is provided with a corresponding even aspheric mirror 230, and the even aspheric mirror 230 is used for performing a light uniformizing operation on the incident filtered signal to form a uniform illumination spot of the excitation light beam with a certain shape and size. Compared with the illuminating light spot which is not subjected to the dodging operation and only passes through the diaphragm spatial filtering, the light intensity distribution in the illuminating area of the excitation light beam is more uniform. The even-aspheric mirror 230 is also used to reflect the excitation beam so that the excitation beam impinges on the sample 330 to be detected.

In one embodiment, the surface type formula of the even aspheric mirror 230 satisfies the following relationship:

In this embodiment, when the even-order aspheric mirror 230 satisfies the above formula, the filtered signal is reflected by the even-order aspheric mirror 230, and then the excitation beam with the elliptical light spot can be obtained. In addition, by setting specific face parameters: curvature, cone coefficient, second-order aspheric coefficient, fourth-order aspheric coefficient, sixth-order aspheric coefficient and eighth-order aspheric coefficient, and specific coordinate positions of aspheric surfaces are determined, so that excitation light beams with different shapes and sizes can be obtained, namely, the major axis and the minor axis of the elliptic light spots are different. According to the above, the efficiency of the camera scanning imaging can be improved by matching the excitation light beam of the elliptical light spot with the rectangular imaging surface of the camera. In addition, the even-order aspheric reflector in the embodiment forms an excitation light beam with a good light-homogenizing effect, so that a good excitation illumination effect can be realized.

Referring to fig. 3, a distribution diagram of the intensity of the excitation beam in the long axis direction of the spot in the present embodiment is shown, and fig. 4 is a distribution diagram of the intensity of the excitation beam in the short axis direction of the spot in the present embodiment.

In the embodiment, the length of the major axis of the excitation beam spot is 1.6mm, and the length of the minor axis is 0.8 mm. Specifically, the light intensity minimum value in the illumination area is divided by the light intensity maximum value, thereby obtaining illumination uniformity. The uniformity in the major axis direction was 81%, and the uniformity in the minor axis direction was 85%. It can be understood that when the illumination uniformity reaches more than 75%, the extraction efficiency of the imaging algorithm on the effective information of the center and the edge of the illumination area corresponding to the photographing position of the camera is nearly consistent. Therefore, the excitation module 200 of the present embodiment can well meet the actual use requirements.

In some embodiments of the present invention, the,

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 aspheric mirror 230 satisfies the following relationship:

14.6<f ₀ <16.1

wherein f is ₀ Is the focal length of the even aspheric mirror 230.

It can be understood that the focal length of the even-order aspheric mirror 230 satisfies the above relationship, so that the even-order aspheric mirror 230 can achieve better shaping and dodging effects and generate an excitation beam with a long-short axis ratio of 2. Similarly, when the surface type parameter of the even-order aspheric mirror 230 is changed, the focal length f is also changed accordingly, and this embodiment is not described herein.

During sequencing, the displacement table is required to be moved to complete scanning imaging of the whole sequencing chip in cooperation with a microscope system. Sequencing chips are typically rectangular in shape, so that the major scan time is spent on the long side. According to the above situation, on the premise of ensuring that the number of camera pixels meets the requirement, the aspect ratio is selected to be 2: the camera of 1, the long limit scanning direction that makes the sequencing chip corresponds the minor face of camera, can shorten the single displacement distance of displacement platform to improve scanning efficiency, and then improve the sequencing data output volume of unit interval. According to the above situation, if the illumination light spot is circular, the illumination range required by the short side of the camera can be greatly exceeded under the condition of meeting the illumination requirement of the long side of the camera. The fluorescence signals excited by these excess imaging portions cannot be received by the camera and need to be excited again in a subsequent scan to achieve signal acquisition in this region. This multiple excitation can cause photobleaching of the region, thereby reducing the signal-to-noise ratio. And the increased phototoxicity can also damage DNA, affecting the subsequent sequencing error rate. The excitation module 200 of this embodiment can carry out dodging operation to the filtering signal through the even aspheric mirror 230 to obtain the excitation beam with uniform light intensity distribution, and with the illumination of oval facula on the objective lens focal plane, the major-minor axis ratio of this oval facula is close to 2:1, unnecessary photobleaching is effectively avoided. When the excitation light beam is used for exciting and illuminating the sample 330 to be detected, the light intensity in each illumination area is uniform, and the problem of inconsistent excitation efficiency can be avoided. Therefore, the excitation module 200 of the present embodiment can achieve a better excitation illumination effect with a simple structure.

In an embodiment, in order to improve the scanning imaging efficiency of the camera during fluorescence imaging, the even aspheric mirror 230 of the excitation module 200 in this embodiment is used to shape the filtered signal in addition to performing the dodging operation on the filtered signal. Specifically, the even-order aspheric mirror 230 can shape the shape of the laser signal into an ellipse or an ellipse-like shape, so that the formed excitation light beam excites and illuminates the sample to be detected in the illumination area to generate an elliptical or ellipse-like fluorescence signal, wherein the shape and size of the excitation light beam are matched with the imaging surface of the camera, that is, the shape and size of the light spot of the fluorescence signal are matched.

In one embodiment, the sample to be detected generates a fluorescence signal under excitation illumination of the excitation light beam, and the shape and size of the excitation light beam are the same as those of the fluorescence signal. Wherein the fluorescent signal is capable of characterizing different detection results of the sample to be detected. For example, it is assumed that the sample to be detected contains different gene sequences, and that the different gene sequences can be labeled with different spectra of fluorescence. When the excitation light beam excites and illuminates the sample to be detected, different gene sequences in the sample to be detected can generate fluorescent signals with different spectrums after being excited. Therefore, the detection result of the gene sequence of the sample to be detected can be obtained through different fluorescent signals.

Referring to fig. 5, a schematic structural diagram of a sequencing module according to an embodiment of the present application is shown.

In this embodiment, the sequencing module 300 includes:

a high-flux objective lens 310 for receiving and converging the excitation beam S to the sequencing unit 320;

a sequencing unit 320 for irradiating the sample to be detected 330 with the excitation beam S to generate a fluorescence signal;

at least one imaging unit 340 (schematically illustrated by 1 imaging unit) for fluorescence imaging using the fluorescence signal.

Fig. 6 is a schematic structural diagram of a high-flux objective lens according to an embodiment of the present application.

In one embodiment, the objective lens imaging field and numerical aperture are two key parameters for determining the sequencing throughput, but the two parameters are usually balanced in size, that is, the field range of the objective lens with large numerical aperture is smaller, and the numerical aperture of the objective lens with large field range is smaller, so that the objective lens with large field range cannot be seen and is thin at the same time. In view of the above problems, the high-flux objective lens 310 in the present embodiment is a microscope objective lens with a large numerical aperture and a large imaging field. In this embodiment, the high-throughput objective lens 310 includes: first lens group G1, second lens group G2 and third lens group G3 along object space to image space coaxial arrangement in proper order, wherein:

the first lens group G1 includes: a first meniscus lens L2 and a second meniscus lens L3, which are sequentially provided.

The second lens group G2 includes: the optical lens comprises 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 which are arranged in sequence, wherein the first biconvex lens L4, the first biconcave lens L5 and the second biconvex lens L6 are combined with a first cemented lens, the third meniscus lens L8 and the fourth biconvex lens L9 form a second cemented lens, and the third biconvex lens has positive focal power.

The third lens group G3 includes: the fourth meniscus lens L10, the fifth meniscus lens L11, the second biconcave lens L12, and the fifth biconvex lens L13 are sequentially disposed, the fourth meniscus lens L10 and the fifth meniscus lens L11 form 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 a cover glass of the sample, and may also be glass on the upper layer of the flow channel of the sequencing chip. The first lens group G1 comprises L2 and L3, the first lens group G1 forms a front collimating surface, collects a large divergence angle optical signal and converts the large divergence angle optical signal into a small angle optical signal, and the spherical aberration and/or the coma aberration are reduced and avoided from being generated too much while the numerical aperture is effectively increased. The second lens group G2 comprises lenses L4-L9, and the second lens group G2 is used for correcting spherical aberration, coma aberration and/or chromatic aberration. The third lens group G3 includes lenses L10 to L13 for eliminating curvature of field, astigmatism and/or chromatic aberration, wherein L11 is a thick meniscus lens. In this embodiment, light emitted by a sample to be detected first passes through the first lens group G1 to reduce a fluorescence signal incident angle, 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 G3 to eliminate one or more of field curvature, astigmatism, or chromatic aberration, thereby simultaneously increasing an imaging field of view and a numerical aperture and improving sequencing flux.

In one embodiment, the focal lengths of the components in high-flux objective lens 310 satisfy 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

where f denotes the focal length of the high-flux objective lens 310, and f _L23 Denotes a focal length, f, of the first lens group G1 _L456 Denotes the focal length of the first cemented lens, f _L7 Denotes the focal length of the third biconvex lens, f _L89 Denotes the focal length of the second cemented lens, f _L1011 Denotes the focal length of the third cemented lens, f _L1213 Denotes the focal length of the fourth cemented lens. It should be understood that the focal length of each component is only illustrated in the present embodiment, and is not particularly limited.

In one embodiment, the parameters of each lens in the high-throughput objective lens 310 are referred to in table one. Referring to FIG. 6, R1-R21 in FIG. 6 correspond to the first surface numbers 1-21, respectively.

Watch 1

In one embodiment, the numerical aperture of the high-throughput objective lens 310 is 0.75, the diameter of the field of view on the imaging object side is 1.6mm, the flat field apochromatism and the focal length are 10mm, and the above parameters can meet most high-throughput sequencing application scenarios of the gene sequencer.

In an embodiment, all lenses of 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 a dichroic mirror and a filter are required for splitting and filtering signals with different wavelengths, a long distance is inevitably required between the objective lens and the sleeve lens. The long distance causes the size of the sleeve lens to increase, which firstly causes the increase of the processing cost, and the accuracy of the lens surface shape is difficult to guarantee. Secondly, the increase of the size leads to the increase of the weight of the lens, and certain influence is brought to the fixation and the adjustment of the lens. This embodiment thus incorporates a relay lens group 360 between the high flux objective lens 310 and the sleeve lens of the imaging unit 340 to reduce the field angle into the sleeve lens, thereby reducing the size of the sleeve lens.

Fig. 7 is a schematic diagram of a position of a relay lens group according to an embodiment of the present application.

The sequencing module 300 in FIG. 7 further comprises: a second dichroic mirror 350 and a relay lens group 360.

In this embodiment, the high-flux objective lens 310 is configured to receive a fluorescence signal and transmit the fluorescence signal to the second dichroic mirror 350, the dichroic mirror may transmit a portion of light and reflect another portion of light, the second dichroic mirror 350 reflects a portion of the received fluorescence signal to the high-flux objective lens 310, and then the fluorescence signal of the sample to be detected 330 emitted by the receiving and sequencing unit 320 is transmitted 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, relay lens group 360 includes:

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.

The fourth lens group 362 has negative power, and the fourth lens group 362 is disposed behind the high-throughput objective lens 310 along the optical axis of the fluorescence signal, and is used for performing aberration correction and increasing the light aperture operation on the fluorescence signal to form a first light signal. The fifth lens group 363 has positive power, and the fifth lens group 363 is disposed behind the fourth lens group 362 along the optical axis of the first optical signal, and is configured to perform operations of aberration compensation, increasing the aperture of light, and decreasing the exit angle of light on the first optical signal, so as to form a first fluorescence signal.

In one embodiment, the power of the fourth lens group 362 is used to characterize the convergence or divergence of the fourth lens group 362, and a negative power of the fourth lens group 362 means that the fourth lens group 362 has a divergence effect on light. Similarly, the focal power of the fifth lens group 363 is used to represent the convergence or divergence of the fifth lens group 363 on light, and the positive focal power of the fifth lens group 363 indicates that the fifth lens group 363 has convergence on light. After passing through the fourth lens group 362 and the fifth lens group 363, the fluorescence signal can form a first fluorescence signal with a smaller maximum emergence angle, and after a certain transmission distance, the beam diameter of the first fluorescence signal is still smaller.

In an embodiment, the fourth lens group 362 and the fifth lens group 363 are configured to balance aberrations together, so that the fluorescence signals incident and emergent from the relay lens group 360 are collimated light beams. Therefore, the fourth lens group 362 and the fifth lens group 363 of the present embodiment can not only form the first fluorescence signal with a smaller maximum exit angle, but also ensure good imaging performance.

Referring to fig. 8, in this embodiment, the fourth lens group 362 includes: a first lens 364, the first lens 364 being a biconcave lens having a negative optical power; a second lens 365, the second lens 365 being cemented with the first lens 364, the second lens 365 being a meniscus lens having a positive power.

In one embodiment, the first lens 364 and the second lens 365 are cemented together to form a double cemented lens (the fourth lens group 362). The first lens 364 is used for receiving the fluorescence signal emitted from the high-flux objective lens 310, diverging the fluorescence signal, and then transmitting the signal to the second lens 365. The second lens 365 receives the diverged fluorescence signal, converges the fluorescence signal, and corrects the curvature of field of the fluorescence signal. The field curvature refers to field curvature, when the first lens 364 or the second lens 365 has field curvature, the intersection point of the fluorescence signal is no longer overlapped with an ideal image point, and the whole image plane is a curved surface, so that the whole image plane cannot be seen clearly when the subsequent camera 342 images the first fluorescence signal, which causes difficulty in detection. Therefore, the second lens 365 of the present embodiment can correct the curvature of field of the fluorescent signal and form a corresponding first optical signal. The fifth lens group 363 receives the first optical signal and forms a parallel first fluorescence signal with a small angle, and focuses the first fluorescence signal to an imaging surface of the camera 342 by the sleeve lens 341, so as to perform imaging detection.

In one embodiment, the first lens 364 satisfies the following relationship: -15.2<f _T1 <-10.5, wherein 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 wherein 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), and the second lens 365 includes a second incident surface (not shown) and a second exit surface S3; the first emergent surface S1 is connected with the second incident surface in a gluing way to form a first gluing surface S2; the curvature radius of the first incident surface S1 is-31.393 mm, the thickness is 8mm, the refractive index is 1.77, and the Abbe number is 49.6; the curvature radius of the first adhesive 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 face is 25.169mm and the thickness is 10mm.

It is understood that the first incident surface S1 is an incident surface of the first lens 364, and the first exit surface is an exit surface of the first lens 364. The first incident surface S1 is used for receiving the fluorescent signal, and the first emitting surface is used for emitting the fluorescent signal processed by the first lens 364. As can be seen from the above, the first lens 364 and the second lens 365 are bonded together, that is, the second incident surface of the second lens 365 is bonded to the first exit surface of the first lens 364, so as to form the first bonded surface S2. The fluorescent signal is emitted through the first emitting surface and then enters the second lens 365 through the first adhesive surface, and is processed by the second lens 365 to form a first optical signal, and the first optical signal is emitted through the second emitting surface S3.

It is understood that, in order to realize the optical performance of the first lens 364 and the second lens 365, the first incident surface S1, the first adhesive surface S2 and the second exit surface S3 need to satisfy the corresponding parameter settings. In practical application, parameters of the first incident surface, the first bonding surface, and the second exit surface may also be adjusted according to requirements, which is not described in this embodiment one by one.

Referring to fig. 8, in some embodiments, fifth lens group 363 includes: a third lens 366, the third lens 366 being disposed behind the fourth lens group 362 along an optical axis of the first optical signal, the third lens 366 being a double-convex lens having positive optical power; a fourth lens 367, the fourth lens 367 being cemented to the third lens 366, the fourth lens 367 being a meniscus lens having a negative power.

It is understood that the third lens 366 and the fourth lens 367 are cemented together to constitute a double cemented lens (fifth lens group 363). The third lens 366 is configured to receive the first optical signal emitted from the fourth lens group 362, converge the first optical signal, and transmit the first optical signal 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 rays, so that the light rays can be emitted in parallel. Meanwhile, the double cemented lens of the third lens 366 and the fourth lens 367 also serves to balance chromatic aberration of the first optical signal and compensate aberration generated by the fourth lens group 362. Therefore, the fifth lens group 363 in this embodiment can converge the first optical signal to form a small-angle parallel first fluorescence signal. The sleeve lens 341 receives the first fluorescence signal and focuses the first fluorescence signal on an imaging surface of the camera 342, thereby performing imaging detection.

In some embodiments, the third lens 366 satisfies the following relationship: 19.8<f _T3 <24.1 wherein f _T3 The focal length of the third lens 366; the fourth lens 367 satisfies the following relationship: -71.2<f _T4 <-66.3, wherein f _T4 Is the focal length of fourth lens 367.

Referring to fig. 8, in some embodiments, the third lens 366 includes a third entrance surface S4 and a third exit surface (not shown), and the fourth lens 367 includes a fourth entrance surface (not shown) and a fourth exit surface S6; the third emergent surface S4 is connected with the fourth incident surface in a gluing way to form a second gluing surface S5; the curvature radius of the third incident surface S4 is 107.462mm, the thickness is 8mm, the refractive index is 1.59, and the Abbe number is 68.4; the curvature radius of the second adhesive surface S5 is-14.587 mm, the thickness is 7.85mm, the refractive index is 1.73, and the Abbe number is 28.4; the fourth exit surface S6 has a radius of curvature of-25.279 mm and a thickness of 403.144mm.

It is understood that the third incident surface S4 is an incident mirror surface of the third lens 366, and the third emergent surface is an emergent mirror surface of the third lens 366. The third incident surface S4 is configured to receive the first optical signal, and the third emergent surface is configured to emit the first optical signal processed by the third lens 366. In addition, as can be seen from the above, the third lens 366 and the fourth lens 367 are bonded, that is, the third exit surface of the third lens 366 and the fourth entrance surface of the fourth lens 367 are bonded to form the second bonding surface S5. The first optical signal is emitted through the third emitting surface and then enters the fourth lens 367 through the second bonding surface, and the first fluorescent signal is formed after the fourth lens 367 is processed, and is emitted through the fourth emitting surface S6.

It is understood that, in order to achieve the optical performance of the third lens 366 and the fourth lens 367, the third incident surface S4, the second adhesive surface S5 and the fourth exit surface S6 need to satisfy the corresponding parameter settings. In practical application, parameters of the third incident surface S4, the second bonding surface S5, and the fourth emergent surface S6 may also be adjusted according to requirements, which is not described herein.

In one specific embodiment, the high-flux objective lens 310 has a numerical aperture of 0.5, a focal length of 7mm, and an imaging field of view diameter of 832um; the distance between the high flux objective lens 310 and the sleeve lens 341 is 450mm, and the focal length of the sleeve lens 341 is 200mm, and the surface shapes of the respective lenses 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 will be appreciated that with the addition of the relay lens group 360 described above, the imaging quality of the sequencing module 300 is not affected and the image quality of all fields of view is near the diffraction limit.

Referring to fig. 9, a schematic structural diagram of a sequencing module according to an embodiment of the present application is shown.

In this embodiment, the sequencing module 300 further comprises: the third dichroic mirror 370 and the two imaging units 340, the imaging units 340 having the same structure, include a sleeve lens 341 and a camera 342.

In this embodiment, the excitation module 200 emits two-color laser light, for example, outputs a red or green excitation beam, in order to perform the following operations on four bases on the DNA of the sequencing chip 321 in the sequencing unit 320: measurement of DNA sequence was carried out by fluorescence imaging of adenine (A), thymine (T), guanine (G) and cytosine (C), respectively. Therefore, in order to image the four bases respectively, the light emitting color of the excitation module 200 needs to be selected in turn. If the fluorescence of the base AT is excited by green light, it is imaged by two imaging units 340. Then, the green light of the excitation module 200 is turned off, the red light of the excitation module 200 is turned on, and the fluorescence of the base GC is excited and imaged by the two imaging units 340, so as to complete the identification of the four-color base. It can be understood that a monochromatic excitation beam may also be used, and four imaging units are used to respectively image, and the number of the imaging units 340 is not limited in this embodiment, and may be selected according to actual requirements. Two imaging units are illustrated in fig. 9 as an example.

Fig. 10 is a schematic structural view of an imaging unit in the embodiment of the present application.

In this embodiment, the imaging unit 340 includes a sleeve lens 341 and a camera 342. The first fluorescence signal passing through the sleeve lens 341 has a shape and size matched with 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, the excitation light beams of different colors are indicated by dotted lines and solid lines, respectively, in fig. 9. The third dichroic mirror 370 is configured 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, in the figure, a light signal reflected by the third dichroic mirror 370 is the first imaging signal Y1, and a signal transmitted by the third dichroic mirror 370 is the second imaging signal Y2.

The sleeve 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 perform fluorescence imaging of different bases using the optical signals.

It should be noted that the camera may be used to capture still images or video. The object is transmitted through the lens to the light sensing element of the camera to generate an optical image. The photosensitive element may 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 transfers the electrical Signal to an IS (Image Signal processor) to be converted into a digital Image Signal. The IS outputs the digital image signal to the DS processing. The DS converts the digital image signal into an image signal in a standard RGB, YUV, or the like format.

Referring to fig. 11, a schematic structural diagram of a sequencing module in an embodiment of the present application is shown.

In this embodiment, the fluorescence signal formed by the sample point on the sample 330 to be detected is collected and emitted through the high-flux objective lens 310 of the relay lens group 360, and the sample point with a large field of view has a certain emission angle after being emitted through the high-flux objective lens 310. The present embodiment places the relay lens group 360 at a position 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. Wherein fig. 11 shows two sets of symmetrical rays with the largest exit angle in the fluorescence signal by corresponding arrows, the remaining rays with smaller exit angles are not shown in fig. 11. The maximum exit angle θ 1 of the fluorescence signal emitted by the high-flux objective lens 310 is greater than the maximum exit angle θ 2 of the first fluorescence signal emitted by the relay lens group 360, i.e. the light beam of the first fluorescence signal is more concentrated than the light beam of the fluorescence signal. As is clear from the above, after the same transmission distance L1, since the maximum emission angle θ 2 of the first fluorescent signal is small, the beam radius r2 of the first fluorescent signal corresponding to the entrance mirror surface of the sleeve lens 341 is smaller than the beam radius of the fluorescent signal, and the diameter of the sleeve lens 341 is also smaller in this case. It is understood that the relay lens group 360 of the present embodiment can converge the fluorescence signal emitted from the high flux objective lens 310 to generate the first fluorescence signal with a smaller maximum emission angle, thereby reducing the size of the sleeve lens 341 in the imaging unit 340.

In the embodiment, the sequencing module 300 utilizes the relay lens group 360, and by arranging the corresponding relay lens group 360 between the high-flux objective lens 310 and the sleeve lens 341 of the imaging unit 340, the fluorescence signal emitted from the high-flux objective lens 310 can be converged to generate the first fluorescence signal with the maximum emission angle smaller than that of the fluorescence signal emitted from the high-flux objective lens 310, so as to reduce the size of the sleeve lens 341 and reduce the processing cost and volume of the sequencing module 300. In addition, the sequencing module 300 further focuses the first fluorescence 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 fluorescence signal focused on the imaging surface thereof, and the image quality of all the fields of view of the sequencing module 300 in this embodiment is close to the diffraction limit.

When the sequencing unit 320 performs sequencing, the displacement table or the high-throughput objective lens needs to be moved to complete scanning imaging of the whole sequencing chip in cooperation with the microscope system. FIG. 12 is a schematic diagram of the structure of the sequencing unit in the example of the present application.

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

In an embodiment, the sequencing chip 321 and the sample 330 to be detected may be integrated, or the sequencing chip 321 is the sample 330 to be detected, and in this embodiment, the form is illustrated separately for convenience of description and is not meant to be limited.

And a displacement stage 322 for placing the sequencing chip 321 to irradiate the sample to be detected 330 with an excitation beam to generate a fluorescence signal, wherein the displacement stage 322 may be an electric displacement stage.

In an embodiment, during the scanning and imaging of the sequencing chip 321 by the gene sequencer, since the imaging is out of focus due to the change of the height of the sequencing chip 321, the gene sequencer of this embodiment further adjusts the relative height of the sequencing chip 321 by using the feedback information of the auto-focus module 400 to ensure that each image is clear.

Fig. 13 is a schematic structural diagram of an auto-focusing module in the embodiment of the present application.

In this embodiment, the gene sequencer 100 further comprises: first dichroic mirror 410 and autofocus module 400.

The first dichroic mirror 410 transmits a fluorescence signal to the automatic focusing module 400, the automatic focusing module 400 is used for generating a relative height measurement signal according to the fluorescence signal and sending the relative height measurement signal to the sequencing module, and the sequencing module adjusts the height relative displacement between the high-flux objective lens 310 and the sequencing chip 321 according to the relative height measurement signal, so that the phenomenon of imaging defocusing caused by the height change of the sequencing chip 321 due to the height change of the displacement table 322 is avoided.

Fig. 14 is a schematic structural diagram of an auto-focusing module in the embodiment of the present application.

In this embodiment, the auto-focusing module 400 includes: beam expanding lens 450, fourth dichroic mirror 420, fifth dichroic mirror 430, sixth dichroic mirror 440, two laser signal emission and filtering unit and calculating unit 470, laser signal emission and filtering unit includes: laser emission unit and filter unit.

In this embodiment, the laser emitting unit includes: a laser diode and a collimating mirror. The laser diode is used for emitting laser signals to the collimating mirror, and the collimating mirror is used for collimating the laser signals to generate collimated laser signals. The laser emission units are respectively: a first laser light emitting unit 461 and a second laser light emitting unit 462, the first laser light emitting unit 461 comprising: the first laser diode 4611 and the first collimating mirror 4612, and the second laser emitting unit 462 includes: a second laser diode 4621 and a second collimating mirror 4622.

In this embodiment, the filtering unit includes a converging mirror, a pinhole filter, and a photodiode. The converging lens is used for receiving the first focusing light signal or the second focusing light signal and converging the first focusing light signal or the second focusing light signal to the corresponding pinhole filter, the pinhole filter is used for filtering the corresponding first focusing light signal or the corresponding second focusing light signal to obtain a corresponding filtering signal, and the photodiode is used for receiving the corresponding filtering signal. The filtering units are respectively: a first filtering unit 464 and a second filtering unit 463, wherein the first filtering unit 464 includes: the first condensing lens 4641, the first aperture filter 4642 and the first photodiode 4643, the second filter unit 463 includes: a second converging mirror 4631, a second pinhole filter 4632 and a second photodiode 4633.

Specifically, the auto-focusing process performed by the auto-focusing module 400 is described as follows:

the fourth dichroic mirror 420 combines the collimated laser signals respectively emitted by the laser emitting units of the two laser signal emitting and filtering units to generate a combined laser signal. The first laser signal emitted by the first laser diode 4611 of the first laser emitting unit 461 is collimated by the first collimating mirror 4612, so as to obtain a 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 a beam expanded laser signal.

The fifth mirror 430 is a half-mirror, which reflects the expanded beam laser signal emitted from the expanded beam mirror 450, passes through the high-throughput objective lens 310 to the sequencing chip 321, and transmits the light spot reflected by the sequencing chip 321 transmitted by the high-throughput objective lens.

The sixth dichroic mirror 440 reflects the light spot transmitted by the fifth dichroic mirror 430 to obtain a first focused light signal.

The sixth dichroic mirror 440 transmits the light spot obtained by the transmission of the fifth dichroic mirror 430, and obtains a second focused light signal.

The filtering units of the two laser signal transmitting and filtering units are respectively configured to receive the first and second focus light signals, that is, the first filtering unit 464 receives the transmitted second focus light signal, and the second filtering unit 463 receives the reflected first focus light signal.

In this embodiment, the first converging mirror 4641 of the first filtering unit 464 receives the second focusing light signal and converges the second focusing light signal to the first pinhole filter 4642 of the first filtering unit 464, the first pinhole filter 4642 filters the converged second focusing light signal to obtain a second filtering signal, and the first photodiode 4643 receives the second filtering signal. Similarly, the second focusing lens 4631 of the second filtering unit 463 receives the first focusing optical signal and converges the first focusing optical signal to the second pinhole filter 4632 of the second filtering unit 463, the second pinhole filter 4632 filters the converged first focusing optical signal to obtain a first filtered signal, and the second photodiode 4633 receives the first filtered signal.

The calculating unit 470 generates a relative height measuring signal according to the signal intensity ratio of the outputs of the two laser signal emitting and filtering units, and the connecting lines between the calculating unit 470 and other components are not shown in the figure, and do not represent the absence of connecting lines. In this embodiment, the calculation unit 470 calculates a 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 generates a relative height measurement signal according to a correspondence between the signal intensity ratio and a preset defocus amount of the objective lens. It can be understood that the corresponding relationship between the signal intensity ratio and the defocus amount of the preset objective lens can be obtained by statistics in actual operation, and the embodiment is not specifically limited herein.

In the related art, a common idea of the automatic focus following technology is to detect position information of a focus following spot on a CCD or a CMOS, that is, when a sample surface to be detected of a sequencing chip and an objective lens are out of focus, a pixel position of a strongest point of the focus following spot on the CCD or the CMOS will be changed. However, when the CCD or CMOS is used as a multi-point detector, the amount of calculation required to acquire the position information of a single pixel is large, and the response time is long, which results in a high delay in adjusting the height of the objective lens during the focus tracking process. In the process of gene sequencing, tens of thousands to hundreds of thousands of images are often required to be shot, namely, tens of thousands to tens of thousands of times of focus tracking is carried out, so that the speed of focus tracking response is very important to control. Alternatively, other auto focus techniques can only be used for single surface biochips (e.g., silicon wafers) and not for multi-surface biochips with flow channels (e.g., biochips with coverslips). For the biochip with multiple surfaces and the surface to be detected which is not the uppermost layer, the reflectivity of the uppermost layer is usually more than ten times stronger than that of the surface to be detected which is in contact with the flow channel, and the detected focus-following light spot of the surface to be detected is submerged in the focus-following light spot on the upper surface of the cover glass, so that the focus following of the surface to be detected can not be realized.

Therefore, the automatic focusing module of the embodiment of the application uses the photodiode of the single-point detector as the detector, and compared with a multi-point detector (a CCD or a CMOS), the automatic focusing module has a faster acquisition rate, can feed back intensity information more quickly and effectively, reduces delay time for changing the height of the objective lens, and improves detection efficiency. In addition, the autofocus module is compatible with a variety of chips (e.g., silicon wafers, coverslipless biochips, biochips with coverslips, etc.). And for the multi-surface chip, the focus tracking can be realized on different surfaces. And the light source used by the automatic focusing module is a laser diode, and the detector is a photodiode, so that the automatic focusing module has small volume and low cost.

FIG. 15 is a schematic diagram of the structure of the gene sequencer according to the embodiment of the present application.

Referring to fig. 15, a method for using each component of the gene sequencer according to the embodiment of the present application is described, in which an excitation module 200 is first composed of a light source 210, a diaphragm 220, and an even aspheric mirror 230, and is used to shape a round gaussian spot into an aspect ratio of 2:1, and outputting an excitation beam. The light source 210 is a two-color laser, and can selectively output red or green laser, in the figure, the dashed lines and the solid lines respectively represent excitation light beams of different colors, and then the excitation light beams are reflected into the sequencing module 300 by the first dichroic mirror 410. The sequencing module 300 is composed of a high-flux 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 light beam is reflected by the second dichroic mirror 350 into the designed high-flux objective lens 310, and then is converged on the sequencing chip 321, and the sequencing chip 321 is driven by the electric displacement table 322 to complete scanning of the sample 330 to be detected on the sequencing chip 321. During the scanning process, the fluorescence generated by the ATGC four 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 angle is reduced by the relay lens group 360, the fluorescent signal is divided into two paths by the third dichroic mirror 370, and the two paths are received by the sleeve lenses 341 of the two imaging units and respectively collected on the imaging chips of the two cameras 342. In this embodiment, in order to image the four bases respectively, the light emitting colors of the light sources 210 need to be selected in turn, for example, green light is firstly used to excite fluorescence of the base AT and then the fluorescence is imaged by the two cameras 342 respectively, then the green light of the light source 210 is turned off, then the red light of the light source 210 is turned on, fluorescence of the base GC is excited and then the fluorescence is imaged by the two cameras 342 respectively, so as to complete the identification of the four-color base.

In this embodiment, during the scanning process of the electric displacement stage 322, in order to prevent the defocusing of the image caused by the inconsistent height of the sequencing chip 321, the autofocus module 400 needs to monitor the change of the focal plane of the objective lens in real time. The auto-focusing module is composed 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, a first collimating mirror 4612, a second laser emitting unit 462, a second laser diode 4621, a second collimating mirror 4622, a first filtering unit 464, a first converging mirror 4641, a first pinhole filter 4642, a first photodiode 4643, a second filtering unit 463, a second converging mirror 4631, a second pinhole filter 4632, a second photodiode 4633 and a calculating 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, so as to obtain a 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. And then, after the combined laser signal is expanded by the beam expanding lens 450, a beam expanded laser signal is obtained, the signal is reflected by the fifth dichroic mirror 430 and then is transmitted by the first dichroic mirror 410, a transmission laser signal is output to the second dichroic mirror 350, the second dichroic mirror 350 reflects the transmission laser signal to the high-flux objective lens 310, the transmission laser signal is reflected by the high-flux objective lens 310 to form a light spot, the light spot is reflected by the second dichroic mirror 350 to the first dichroic mirror 410, then the light spot is sequentially transmitted by the first dichroic mirror 410 and the fifth dichroic mirror 430 and returns to the sixth dichroic mirror 440 along the original path, the light spot is reflected by the sixth dichroic mirror 440 to obtain a first focusing light signal, and the light spot is transmitted by the sixth dichroic mirror 440 to obtain a second focusing light signal.

In this embodiment, the first filtering unit 464 receives the transmitted second focused light signal, and the second filtering unit 463 receives the reflected first focused light signal. The first converging mirror 4641 of the first filtering unit 464 receives the second focusing light signal and converges the second focusing light signal to the first pinhole filter 4642 of the first filtering unit 464, the first pinhole filter 4642 filters the converged second focusing light signal to obtain a second filtering signal, and the first photodiode 4643 receives the second filtering signal. Similarly, the second focusing lens 4631 of the second filtering unit 463 receives the first focusing light signal and focuses the first focusing light signal to the second pinhole filter 4632 of the second filtering unit 463, the second pinhole filter 4632 filters the focused first focusing light signal to obtain a first filtered signal, and the second photodiode 4633 receives the first filtered signal. The calculating unit 470 then 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 generates a relative height measuring signal according to the correspondence between the signal intensity ratio and the preset defocus amount of the objective lens. It can be understood that the corresponding relationship between the signal intensity ratio and the defocus amount of the preset objective lens can be obtained by statistics in actual operation, and the embodiment is not specifically limited herein.

In one embodiment, it is understood that although the application does not indicate that the dichroic mirror outputs light spots with different wavelengths, those skilled in the art will know that the dichroic mirror transmits and reflects light waves to obtain light spots with different wavelengths. In this embodiment, the fourth dichroic mirror 420 and the fifth dichroic mirror 430 are dichroic mirrors having the same performance, and the numbers thereof are merely for convenience of description and are interchangeable with each other. First dichroic mirror 410, second dichroic mirror 350, third dichroic mirror 370, fifth dichroic mirror and sixth dichroic mirror 440 are dichroic mirrors with different properties, which are intended to separate light of different wavelengths, and different parameters can be selected according to actual requirements or priori knowledge obtained through multiple experiments, which is not specifically limited in this respect.

The gene sequencer provided by the embodiment of the invention comprises an excitation module and a sequencing module, wherein the excitation module comprises: a light source, a diaphragm and an even aspheric reflector; the sequencing module comprises: a high-throughput objective lens, a sequencing unit and at least one imaging unit. The embodiment designs a high-flux microobjective with a large numerical aperture and a large imaging visual field in a gene sequencer, satisfies the application scene of high-flux sequencing, improves the detection efficiency, utilizes an even aspheric reflector to combine with a diaphragm, realizes the homogenization of Gaussian spots in a simple light path form, effectively avoids unnecessary photobleaching, and improves the efficiency of scanning imaging and the result accuracy of subsequent sequencing.

In addition, the embodiment of the invention also provides a using method of the gene sequencer, which is applied to the gene sequencer.

Referring to fig. 16, a flowchart of a method for using a gene sequencer according to an embodiment of the present disclosure is provided, the method including steps S1610 to S1650:

step S1610, the sample to be detected is placed in the detection range of the sequencing chip.

In one embodiment, the sample to be detected comprises single-stranded DNA and four nucleotides, each of which has a different color of fluorescent signal.

In step S1620, the excitation module generates an excitation beam for exciting the sample to be detected.

Step S1630, the sequencing module adjusts the height relative displacement between the high-flux objective lens and the sequencing chip according to the relative height measurement signal, and the excitation light beam is used for scanning the sample to be detected to obtain a fluorescence signal.

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

firstly, obtaining a relative height measurement signal, then controlling a displacement table to adjust the height relative displacement between the displacement table and a high-flux objective lens in the vertical direction according to the relative height measurement signal, namely adjusting the height relative displacement between a sequencing chip and the high-flux objective lens so as to enable a short shaft of an excitation light beam to move relative to a long side of the sequencing chip, enabling the short shaft of the excitation light beam to be parallel to the long side of the sequencing chip, and finally obtaining a fluorescence signal of a sample to be detected in the moving process.

In one embodiment, the auto-focusing module generates the relative height measurement signal by the following specific process: the automatic focusing module reflects the laser alignment signal to be converged on the sequencing chip through the high-flux objective lens, and receives the light spot reflected by the sequencing chip to the high-flux objective lens. The automatic focusing module generates a first focusing light signal and a second focusing light signal by using the light spot, and the automatic focusing module generates a relative height measuring signal according to the first focusing light signal and the second focusing light signal. The high-flux objective lens or the sequencing chip adjusts the height relative displacement according to the relative height measurement signal.

Specifically, the camera can only image the fluorescence signal in its imaging surface A, consequently, in order to scan the formation of image to the different regions of sequencing chip, the gene sequencer has set up corresponding displacement platform in this embodiment, and the displacement platform is used for bearing the sequencing chip, and the displacement platform removes according to relative height measuring signal to make and adjust the high relative displacement between the two, thereby make the excitation light beam to the different regions of sequencing chip arouse the illumination.

In this embodiment, referring to fig. 17, the sequencing chip is rectangular, the imaging plane a of the camera is a rectangular imaging plane, and the length-width ratio of the rectangular imaging plane a is L: w, the ratio of the long axis to the short axis of a light spot D of the excitation light beam is L: w is added. When the displacement table is at an initial position, the light spot D of the excitation light beam excites and illuminates the X1 area of the sequencing chip and generates a corresponding fluorescent signal, and the camera generates an image signal according to the working principle and generates a corresponding detection result X1 according to the image signal. And then, the displacement table continues to move, so that the light spot D of the excitation light beam irradiates on the X2 area of the sequencing chip, and the detection result X2 is obtained according to the steps. And the displacement table continues to move, so that the camera can scan and image areas such as X3, X4.

It can be understood that when the length-width ratio of the rectangular imaging plane a of the camera is 2:1, the ratio of the long axis to the short axis of a light spot D of an excitation beam is 2:1, compared with a circular laser signal in the related art, the excitation beam of the present embodiment can excite and illuminate a larger area in the sequencing chip, and scan and image through a matched camera. Therefore, the gene sequencer of the embodiment has high scanning and detection efficiency, and can improve the output of gene sequencing data in unit time.

In fig. 17, the long axis of the light spot of the excitation light beam is perpendicular to the long side of the sequencing chip, and the excitation light beam moves along the long side of the sequencing chip, so that the camera scans and images the X1 region, the X2 region, and the like on the sequencing chip, thereby obtaining the detection result.

It can be understood that, because the long side of the sequencing chip corresponds to the short side 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 mode that the long side of the sequencing chip corresponds to the short side of the camera, the single moving distance of the displacement table can be shortened, thereby improving the scanning efficiency of the camera.

In the process of scanning and imaging, because the change of the height of the sequencing chip can cause defocusing of imaging, in an embodiment, the gene sequencer further adjusts the height of the sequencing chip by using feedback information of the automatic focusing module to ensure that each image is clear.

In this embodiment, according to the relative height measurement signal, the high-throughput objective lens is controlled to adjust the height relative displacement relative height measurement signal with the displacement stage in the vertical direction, and the specific steps are as follows:

the automatic focusing module reflects laser collimation signals to be converged on a sequencing chip through a high-flux objective lens, receives light spots reflected to the high-flux objective lens by the sequencing chip, then the automatic focusing module generates a first focusing light signal and a second focusing light signal by the aid of the light spots, generates a relative height measuring signal according to the first focusing light signal and the second focusing light signal, and sends the relative height measuring signal to the sequencing module, and the sequencing module controls a displacement platform to adjust height relative displacement between the first focusing light signal and the second focusing light signal according to the relative height measuring signal.

And step S1640, in the scanning process, the high-flux objective lens collects fluorescence signals.

Step S1650, determining the order of nucleotides in the single-stranded DNA using an image obtained by imaging the fluorescent signal using the imaging unit.

Therefore, the contents in the embodiment of the gene sequencer are all applicable to the embodiment of the method for using the gene sequencer in this embodiment, the functions specifically realized in the embodiment of the method for using the gene sequencer are the same as those in the embodiment of the gene sequencer, and the beneficial effects achieved by the embodiment of the gene sequencer are also the same as those achieved by the embodiment of the gene sequencer.

The preferred embodiments of the present invention have been described above with reference to the accompanying drawings, and the scope of the embodiments of the present invention is not limited thereby. Any modifications, equivalents and improvements that may occur to those skilled in the art without departing from the scope and spirit of the embodiments of the present invention are intended to be within the scope of the claims of the embodiments of the present invention.

Claims

1. A gene sequencer is used for exciting a sample to be detected on a gene sequencing chip and collecting a fluorescence signal emitted by the sample to be detected for fluorescence imaging, and is characterized by comprising the following components:

the excitation module is used for generating an excitation beam for exciting the sample to be detected;

the excitation module comprises:

a light source for generating a laser signal;

the even-order aspheric mirror is placed behind the diaphragm along the optical axis of the filtering signal and is used for forming an excitation beam according to the filtering signal;

the sequencing module comprises:

the high-throughput objective lens includes:

a first lens group, a second lens group and a third lens group coaxially arranged in sequence from an object space to an image space,

2. The gene sequencer of claim 1, wherein the sequencing module further comprises: a second dichroic mirror and a relay lens group;

the high-flux objective lens is used for receiving the fluorescence signal and transmitting the fluorescence signal to the second dichroic mirror;

the second dichroic mirror is used for transmitting the fluorescence signal to the relay lens group to form a first fluorescence signal.

3. The gene sequencer according to claim 2, wherein the relay lens group comprises a fourth lens group and a fifth lens group;

the fourth lens group has negative focal power, is arranged behind the objective lens along the optical axis of the fluorescence signal and is used for forming a first optical signal according to the fluorescence signal;

4. The gene sequencer of claim 2, wherein the imaging unit comprises: a sleeve lens and a camera, the sequencing module further comprising: a third dichroic mirror and two of the imaging units;

the third dichroic mirror is further used for transmitting the first fluorescence signal to obtain a second imaging signal;

5. The gene sequencer of claim 2, wherein the sequencing unit comprises:

the sequencing chip is used for bearing the sample to be detected;

and the displacement table is used for placing the sequencing chip so as to irradiate the sample to be detected with the excitation light beam to generate a fluorescence signal.

6. The gene sequencer of claim 5, further comprising: the automatic focusing device comprises a first dichroic mirror and an automatic focusing module;

the sequencing module is used for adjusting the height relative displacement between the high-flux objective lens and the sequencing chip according to the relative height measurement signal.

7. The gene sequencer of claim 6, wherein the auto-focus module comprises: beam expanding lens, fourth dichroic mirror, fifth dichroic mirror, sixth dichroic mirror, laser signal transmission and filtering unit and computational element laser signal transmission and filtering unit includes: the laser emitting unit and the filtering unit; laser signal transmitting and filtering unit

the fifth chromatic mirror is used for reflecting the expanded beam laser signal to the sequencing chip through the high-flux objective lens and transmitting light spots reflected by the sequencing chip and transmitted by the high-flux objective lens;

the sixth dichroic mirror is used for reflecting the light spots to obtain a first focused light signal;

the calculation unit is used for calculating and generating a relative height measurement signal according to the signal intensity ratio output by the filter unit of the two laser signal emission and filter units.

8. The gene sequencer according to claim 7, wherein the laser emitting unit comprises: a laser diode and a collimating mirror;

9. The gene sequencer according to claim 7, wherein the filter unit comprises a converging mirror, a pinhole filter and a photodiode;

the photodiode is used for receiving the corresponding filtering signal.

10. The gene sequencer according to claim 9, wherein the computing unit is further configured to compute a ratio of signal intensities of the filtered signals received by the corresponding two photodiodes;

the calculating unit is also used for generating the relative height measuring signal according to the corresponding relation between the signal intensity ratio and the defocusing amount of the preset objective lens.

11. The gene sequencer according to claim 7, wherein the first dichroic mirror is configured to transmit the expanded beam laser signal reflected by the fifth dichroic mirror and output a transmitted laser signal to the second dichroic mirror;

12. A gene sequencer according to any one of claims 1-11, wherein the even aspheric mirror has a surface profile that satisfies the following relationship:

13. A gene sequencer according to any one of claims 1-11, wherein the fourth lens group comprises:

a first lens that is a biconcave lens having a negative optical power;

a second lens cemented with the first lens, the second lens being a meniscus lens having a positive optical power.

14. A gene sequencer according to any one of claims 1 to 11, wherein the fifth lens group comprises:

a fourth lens cemented with the third lens, the fourth lens being a meniscus lens having a negative power.

15. A method of using a gene sequencer according to any one of claims 1 to 14, the method comprising:

and determining the sequencing of the nucleotides in the single-stranded DNA in the sample to be detected by using an image obtained by imaging the fluorescent signal by using the imaging unit.

16. The method of claim 15, wherein the sequencing module adjusts the relative displacement of the high-throughput objective lens with respect to the sequencing chip according to a relative height measurement signal, and further comprising:

acquiring a relative height measurement signal;

17. The method of claim 16, wherein adjusting the relative height displacement between the high-throughput objective and the sequencing chip in a vertical direction according to the relative height measurement signal comprises:

18. The method of any one of claims 15 to 17, wherein the imaging plane of the camera is a rectangular imaging plane, and the shape of the excitation beam is elliptical or elliptical-like or rectangular; the length-width ratio of the rectangular imaging surface is L: w; the ratio of the long axis to the short axis of the light spot of the excitation light beam is L: w; wherein, L and W are both positive integers.