Genetic sequencing optical system and genetic sequencing optical system sequencing method

Abstract

This invention provides a gene sequencing optical system and a sequencing method using the same system, relating to the field of medical device technology. The gene sequencing optical system includes an excitation module, a sequencing module, and a TDI imaging component. The sequencing module uses a spectrometer to divide the fluorescence signal generated by the sample into four target fluorescence signals. The TDI imaging component integrates four TDI imaging chips that perform fluorescence imaging on the bases based on the target fluorescence signals, obtaining the gene sequencing results. Since only one imaging channel is needed, only one set of lens sleeves and filters is required for each channel, significantly reducing costs. Furthermore, because the TDI imaging component integrates four TDI imaging chips corresponding one-to-one with each base, images of all four bases can be obtained in a single imaging process, enabling rapid imaging. The integrated imaging chips also allow for circuit miniaturization, further reducing system costs.

Description

Technical Field

[0001] This invention relates to the field of medical device technology, and more particularly to gene sequencing optical systems and sequencing methods for gene sequencing optical systems. Background Technology

[0002] Gene sequencing refers to the analysis of the base sequence of a specific DNA fragment, specifically the arrangement of adenine (A), thymine (T), cytosine (C), and guanine (G). Currently, fluorescent labeling is commonly used for gene sequencing. The gene sequencing optical system uses a laser to excite fluorescent labels on a gene sequencing chip, generating fluorescence, and then collects the fluorescence signals. The four bases bind to different fluorescent labels, producing four different fluorescence bands, thus identifying the bases. A gene molecule contains multiple bases; during sequencing, one base is attached to a fluorescent label, which, when excited by a laser, produces a fluorescence signal.

[0003] In related technologies, gene sequencing optical systems require multi-channel imaging, commonly four-channel or two-channel imaging systems. For two-channel imaging systems, each camera needs to expose the same location on the sample twice. Due to camera limitations, there is a data transmission time of tens of milliseconds after each exposure. Adding the time for the two data transmissions inevitably prolongs the imaging time. In a four-channel imaging system, each camera takes a single image at the same location on the sample, obtaining images of the four bases. Although four-channel imaging systems can save at least half the imaging time compared to two-channel systems, the cost is high due to the expensive lens and filter components for each channel. Therefore, how to achieve rapid imaging while reducing system cost has become a pressing technical problem. Summary of the Invention

[0004] The main objective of this invention is to propose a gene sequencing optical system and a gene sequencing optical system sequencing method that can reduce system costs and achieve rapid imaging.

[0005] To achieve the above objectives, a first aspect of the present invention provides a gene sequencing optical system for exciting a sample to be tested and acquiring the fluorescence signal emitted by the sample for fluorescence imaging. The sample to be tested includes four bases, including:

[0006] The excitation module is used to generate an excitation beam that excites the sample to be tested;

[0007] An imaging module is used to image the sample to be detected using the excitation beam;

[0008] The imaging module includes:

[0009] The sequencing module is used to generate four target fluorescence signals by irradiating the sample to be tested with the excitation beam;

[0010] The TDI imaging component includes four integrated TDI imaging chips, each corresponding to a base. The TDI imaging component is used to perform fluorescence imaging on the bases based on the target fluorescence signal using the TDI imaging chips to obtain gene sequencing results.

[0011] The sequencing module includes:

[0012] The objective lens is used to receive and focus the excitation beam onto the sample to be tested, and also to collect the fluorescence signal generated by the excited sample to be tested;

[0013] A spectrometer is positioned behind the objective lens along the optical path of the fluorescence signal to receive the fluorescence signal and split it into four target fluorescence signals, each of which corresponds one-to-one with a base.

[0014] In one embodiment, the TDI imaging assembly further includes: a sleeve lens;

[0015] The sleeve lens is used to receive the four target fluorescence signals and converge the optical path of the target fluorescence signals to match the corresponding TDI imaging chip;

[0016] The TDI imaging chip is used to receive the target fluorescence signal through the converging optical path of the sleeve lens at a preset position, and to perform fluorescence imaging on the base.

[0017] In one embodiment, the sequencing module further includes: a first dichroic mirror;

[0018] The first dichroic mirror is used to reflect the received excitation beam to the objective lens;

[0019] The first dichroic mirror is also used to transmit the received fluorescence signal emitted from the objective lens to the beam splitter.

[0020] In one embodiment, the spectrophotometer is used to split the fluorescence signal to obtain four target fluorescence signals corresponding to the base, and the imaging surface of the TDI imaging chip is located at the focal position of the target fluorescence signal corresponding to the base.

[0021] In one embodiment, the spectrophotometer is used to spectrate the fluorescence signal to obtain four target fluorescence signals corresponding to the base.

[0022] In one embodiment, the beam-splitting component includes at least one of the following: a transmission grating, a reflection grating, or a prism.

[0023] In one embodiment, the excitation device includes: a light source and an illumination assembly;

[0024] The light source is used to generate laser signals;

[0025] The illumination component is positioned behind the light source along the optical axis of the laser signal, and is used to form an excitation beam according to the laser signal.

[0026] In one embodiment, the light source includes: a first light source, a second light source, and a beam combining assembly;

[0027] The first light source is used to emit a first laser signal;

[0028] The second light source is used to emit a second laser signal;

[0029] The beam combining component is disposed at the intersection of the first laser signal and the second laser signal. The beam combining component is used to transmit the first laser signal and reflect the second laser signal to combine the first laser signal and the second laser signal to form the laser signal.

[0030] In one embodiment, the illumination assembly includes at least one of the following: a spherical lens, an aspherical lens, a cylindrical lens, a Powell prism, a lens array, a reflector, or a filter.

[0031] In one embodiment, the device further includes a displacement stage that carries the sample to be tested. The displacement stage is used to move the sample to be tested at a preset moving speed, so that different positions of the sample to be tested can be excited to generate the fluorescence signal during the movement. The preset moving speed is determined according to the line frequency of the TDI imaging chip.

[0032] In one embodiment, the preset moving speed is expressed as:

[0033]

[0034] Among them, V s The preset moving speed is represented by f, the line frequency of the TDI imaging chip is represented by C, the pixel size of the TDI imaging chip is represented by M, and the imaging magnification of the optical system is represented by M.

[0035] In one embodiment, a filter is also included, which is disposed behind the sleeve lens along the optical axis of the target fluorescence signal. The filter is used to filter the four target fluorescence signals after the optical path is converged by the sleeve lens, and to filter out the residual excitation beam mixed in the target fluorescence signal to obtain the corresponding four filtered signals.

[0036] The TDI imaging chip is used to receive the filter signal at a preset position and perform fluorescence imaging on the bases based on the filter signal.

[0037] In one embodiment, the excitation module further includes a beam shaping module disposed in the optical path of the excitation beam emitted by the illumination component, and the beam shaping module is used to shape the excitation beam into an illumination spot in a one-dimensional direction.

[0038] To achieve the above objectives, a second aspect of the present invention provides a sequencing method for a gene sequencing optical system, characterized in that it is applied to a gene sequencing optical system as described in any one of the first aspects, the method comprising:

[0039] The excitation module generates an excitation beam to excite the sample to be tested. The sample to be tested includes four bases, each of which emits a fluorescence signal in a different wavelength band.

[0040] The sequencing module uses the excitation beam to scan the sample to be tested at a preset scanning frequency to generate four target fluorescent signals corresponding to the bases.

[0041] During the scanning process, the objective lens acquires the target fluorescence signal and transmits it to the TDI imaging module;

[0042] The TDI imaging component performs fluorescence imaging on the bases based on the target fluorescence signal to obtain gene sequencing results.

[0043] In one embodiment, the TDI imaging component performs fluorescence imaging on the bases based on the target fluorescence signal to obtain gene sequencing results, and further includes:

[0044] The TDI imaging chip is used to obtain images of the corresponding bases based on the target fluorescence signal;

[0045] The base images corresponding to the four bases are registered to obtain the gene sequencing results.

[0046] The gene sequencing optical system and sequencing method proposed in this invention include an excitation module and an imaging module. The imaging module comprises a sequencing module and a TDI imaging component. The sequencing module uses a beam splitter to divide the fluorescence signal generated by the excitation beam illuminating the sample into four target fluorescence signals. The four TDI imaging chips integrated in the TDI imaging component perform fluorescence imaging on the bases based on the target fluorescence signals to obtain the gene sequencing results. Since only one imaging channel is needed, only one set of lens sleeves and filters is required for each channel, significantly reducing costs. Furthermore, because the TDI imaging component integrates four TDI imaging chips corresponding one-to-one with the bases, images of all four bases can be obtained in a single imaging process, enabling rapid imaging. The integrated imaging chips also allow for circuit miniaturization, further reducing system costs. Attached Figure Description

[0047] Figure 1 This is a schematic diagram of the structure of the gene sequencing optical system provided in an embodiment of the present invention.

[0048] Figure 2 This is a schematic diagram of the TDI imaging chip in the TDI imaging component of a gene sequencing optical system provided in another embodiment of the present invention.

[0049] Figure 3 This is a schematic diagram of the optical path of a gene sequencing optical system provided in another embodiment of the present invention.

[0050] Figure 4 This is a schematic diagram of the optical path of a gene sequencing optical system provided in another embodiment of the present invention.

[0051] Figure 5 This is a schematic diagram of the optical path of a gene sequencing optical system provided in another embodiment of the present invention.

[0052] Figure 6 This is a schematic diagram of the optical path of a gene sequencing optical system provided in another embodiment of the present invention.

[0053] Figure 7 This is a schematic diagram of the excitation module of a gene sequencing optical system provided in another embodiment of the present invention.

[0054] Figure 8 This is a schematic diagram of the excitation module of a gene sequencing optical system provided in another embodiment of the present invention.

[0055] Figure 9 This is a schematic diagram of a DNA fragment in a sample to be tested in a gene sequencing optical system provided in another embodiment of the present invention.

[0056] Figure 10This is a schematic diagram of the structure of a gene sequencing optical system provided in another embodiment of the present invention.

[0057] Figure 11 This is an imaging schematic diagram of the method of using a gene sequencing optical system provided in an embodiment of the present invention.

[0058] Explanation of reference numerals in the attached figures:

[0059] Gene sequencing optical system 100, excitation module 200, sequencing module 400, TDI imaging component 500, sample to be tested 600, first TDI imaging chip 5101, second TDI imaging chip 5102, third TDI imaging chip 5103, fourth TDI imaging chip 5104, first dichroic mirror 410, objective lens 420, beam splitter 430, sleeve lens 520, filter 530, light source 210, illumination component 220, first light source 2101, second light source 2102 and beam combiner 2103.

[0060] Excitation beam S, fluorescence signal F, first target fluorescence signal F1, second target fluorescence signal F2, third target fluorescence signal F3, fourth target fluorescence signal F4, first filter signal F1', second filter signal F2', third filter signal F3', fourth filter signal F4'. Detailed Implementation

[0061] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0062] Unless otherwise defined, 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 pertains. The terminology used herein is for the purpose of describing embodiments of the invention only and is not intended to limit the invention.

[0063] Microscopic imaging technology has wide applications in sample testing. For example, in gene sequencing, fluorescence imaging of bases on biochips is required. Gene sequencing optical systems have extensive applications in medicine and life sciences, such as the detection of pathogens, genetic diseases, and tumor genes, as well as personalized drug therapy and non-invasive prenatal testing. Gene sequencing optical systems require fluorescence imaging of bases on biochips during operation. Sequencing with these systems requires fluorescence imaging of the four bases ATGC: adenine (A), thymine (T), cytosine (C), and guanine (G). Multi-channel (e.g., four-channel or two-channel) imaging is typically used, and then the images obtained from each channel are registered using algorithms to match the base positions in different images. Microscopic imaging systems are widely used in gene sequencing optical systems. When gene sequencing optical systems are working, fluorescence imaging of the four bases ATGC is required to determine the base sequence in DNA.

[0064] In related technologies, in order to distinguish the four bases (i.e., the four fluorescence bands), gene sequencing optical systems need to perform imaging through multiple channels. Common ones are four-channel imaging systems or dual-channel imaging systems, each of which consists of a sleeve lens, a filter and a camera.

[0065] The applicant discovered that dual-channel imaging systems require two wavelengths of excitation light to be alternately activated sequentially. For each location on the biological sample, each channel of the imaging system takes an image under different wavelengths of excitation light, thus obtaining images of the four bases ATGC. Due to the limitations of the camera itself, a data transmission time of tens of milliseconds is required after each exposure. However, each camera in the dual-channel imaging system needs to expose the same location on the sample twice. Adding the time for the two data transmissions, this inevitably prolongs the imaging time.

[0066] The applicant also discovered that, with a four-channel imaging system, two excitation wavelengths can be continuously activated simultaneously. For each location on the biological sample, the imaging system of each channel takes one image, thus obtaining images of the four bases ATGC. Compared to a two-channel imaging system, the four-channel imaging system can save at least half the imaging time. However, the sleeve lenses and filters for each channel are more expensive, inevitably increasing the cost of the four-channel system.

[0067] Based on this, this invention proposes a gene sequencing optical system and a sequencing method using the same system, addressing the issue of achieving rapid imaging while reducing system costs. The gene sequencing optical system includes an excitation module and an imaging module. The imaging module comprises a sequencing module and a TDI imaging component. The sequencing module uses a beam splitter to divide the fluorescence signal generated by the excitation beam illuminating the sample into four target fluorescence signals. The four TDI imaging chips integrated in the TDI imaging component perform fluorescence imaging on the bases based on the target fluorescence signals, obtaining the gene sequencing results. Since only one imaging channel is needed, only one set of lens sleeves and filters is required for each channel, significantly reducing costs. Furthermore, because the TDI imaging component integrates four TDI imaging chips corresponding one-to-one with each base, images of all four bases can be obtained in a single imaging process, enabling rapid imaging. The integrated imaging chips also allow for circuit miniaturization, further reducing system costs.

[0068] This invention provides a gene sequencing optical system and a gene sequencing optical system sequencing method, which are specifically described through the following embodiments. First, the gene sequencing optical system in this invention is described.

[0069] In this embodiment of the invention, a gene sequencing chip is used as the sample to be tested. Taking the four bases ATCG on the gene sequencing chip as an example, the four bases are stained with different fluorescent dyes. After being irradiated by a laser, the four bases will be excited to emit fluorescence in four different wavelengths.

[0070] Generally, gene sequencing chips contain several DNA clusters arranged in an array or randomly distributed. Each DNA cluster is a chain of bases containing a number of bases. During sequencing, the bases in each DNA cluster on the gene sequencing chip are identified one by one, and different types of bases are attached with one of four different fluorescent labels. When a laser signal of a corresponding wavelength shines on the corresponding fluorescent label, the different fluorescent labels will be excited to produce fluorescence signals of different wavelengths.

[0071] The process of gene sequencing using a gene sequencing optical system on a gene sequencing chip is described as follows: The gene sequencing chip contains four bases (A, T, G, and C) and two fluorescent dyes. The A and G bases each bind to only one fluorescent dye, the T base does not bind to any fluorescent dye, and the C base binds to both fluorescent dyes. Each fluorescent dye can only be excited by one of two lasers (red or green); the other laser cannot excite it. Two bases are excited by laser 1, and the other two bases are excited by laser 2. Both lasers are activated simultaneously, and each of the four chips simultaneously receives one image, thus obtaining images of the four bases.

[0072] Figure 1This is a schematic diagram of the structure of the gene sequencing optical system provided in an embodiment of the present invention.

[0073] In this embodiment, the gene sequencing optical system 100 is used to excite the sample to be tested and collect the fluorescence signal emitted by the sample to be tested for fluorescence imaging. The sample to be tested includes four bases.

[0074] Gene sequencing optical system 100 includes:

[0075] Excitation module 200 is used to generate an excitation beam S to excite the sample to be tested.

[0076] In one embodiment, the excitation beam S can be generated by a laser signal produced by an exciter as a light source. In traditional gene sequencing technologies, mercury lamps and argon lamps are often chosen as excitation sources. However, the lifespan of a mercury lamp is related to the number of starts and the working time; the more starts and the shorter the working time each time, the shorter the lifespan. Argon lamps require a dedicated low-voltage DC power supply box, and the lamp temperature is extremely high, making them more expensive than mercury lamps. Therefore, in this embodiment, a laser is used as the excitation source, which has advantages such as high brightness, good directionality, good monochromaticity, and good coherence.

[0077] The imaging module 300 is used to image the sample to be tested using the excitation beam S to obtain gene sequencing results.

[0078] The imaging module 300 includes:

[0079] The sequencing module 400 is used to generate four target fluorescence signals by irradiating the sample to be tested with an excitation beam S.

[0080] The TDI imaging module 500 includes four integrated TDI imaging chips, each corresponding one-to-one with a base. The TDI imaging module 500 uses these chips to perform fluorescence imaging on each base based on the target fluorescence signal, obtaining gene sequencing results. Because the TDI imaging module integrates four TDI imaging chips, each corresponding to a base, images of all four bases can be obtained in a single imaging process, enabling rapid imaging. Furthermore, the integrated imaging chips allow for smaller circuitry, further reducing system cost.

[0081] For example, in one embodiment, referencing Figure 2The TDI imaging component 500 contains four TDI imaging chips: a first TDI imaging chip 5101, a second TDI imaging chip 5102, a third TDI imaging chip 5103, and a fourth TDI imaging chip 5104. The correspondence between these chips and bases is as follows: the first TDI imaging chip 5101 is used for imaging A bases, the second TDI imaging chip 5102 is used for imaging T bases, the third TDI imaging chip 5103 is used for imaging G bases, and the fourth TDI imaging chip 5104 is used for imaging C bases. It is understood that the above correspondence between the TDI imaging chips and base imaging is for illustrative purposes only and does not constitute a limitation. Figure 2 Four TDI imaging chips can be arranged in a row, or four TDI imaging chips can be arranged in a column. Figure 2 This is just one possible arrangement of the four TDI imaging chips; this embodiment does not limit the arrangement of the four TDI imaging chips.

[0082] Compared to traditional linear array cameras, the TDI imaging method used in this application has a very fast scanning speed, typically taking less than 20 seconds to scan from one end of the sequencing chip to the other. This significantly shortens the scanning and imaging time, reducing sequencing time and improving the efficiency of the gene sequencing optical system. Furthermore, TDI imaging offers very high scanning accuracy. The TDI imaging scanning method uses confocal focusing, ensuring the focused light source is positioned on a single line. This allows for adjustment of the focusing position to scan different layers of the target object and avoids the influence of the surrounding environment.

[0083] In one embodiment, the excitation beam generated by the excitation module 200 can be a laser signal. The gene sequencing optical system 100, based on the excitation characteristics of fluorescent dyes, can generate a corresponding illumination area by the laser signal generated by the excitation module 200, which is then irradiated onto the sample to be tested. This excites and illuminates the fluorescent dye in the illumination area, causing the sample to generate a corresponding fluorescence signal under the excitation signal. The fluorescence signal is then imaged by a camera, thereby enabling the detection of gene sequences.

[0084] It should be noted that the gene sequencing optical system in this embodiment of the invention can be an imaging system. Specifically, this embodiment of the invention can obtain a data cube containing information about the sample to be tested, which includes not only the spatial information of the sample but also its spectral information. The excitation module 200, sequencing module 400, and TDI imaging component 500 in this embodiment of the invention can each be equipped with multiple elements to perform the functions required by the modules.

[0085] Reference Figure 3 The sequencing module 400 includes: a first dichroic mirror 410, an objective lens 420, and a spectrophotometer 430.

[0086] The first dichroic mirror 410 can transmit a portion of light while reflecting another portion. (See reference...) Figure 3 The optical path diagram shows that the first surface of the first dichroic mirror 410 reflects the excitation beam S to the objective lens 420 to excite the sample 600 to be tested.

[0087] Objective lens 420 is used to receive the excitation beam S reflected by the first dichroic mirror 410 and converge the excitation beam S to the sample 600 to be tested. It is also used to receive the fluorescence signal F generated by the excited sample 600. (Refer to...) Figure 3 The optical path diagram shows that the fluorescence signal F is incident on the first surface of the first dichroic mirror 410, and after transmission, it exits from the second surface of the first dichroic mirror 410 and enters the beam splitter 430.

[0088] The spectrometer assembly 430 is positioned behind the first dichroic mirror 410 and also behind the objective lens 420, along the optical path of the fluorescence signal F. It receives the fluorescence signal F emitted from the second surface of the first dichroic mirror 410 and splits it into four target fluorescence signals, each corresponding to a base. In one embodiment, the four target fluorescence signals are: a first target fluorescence signal F1, a second target fluorescence signal F2, a third target fluorescence signal F3, and a fourth target fluorescence signal F4. Specifically, the first target fluorescence signal F1 is used to detect A bases, the second target fluorescence signal F2 is used to detect T bases, the third target fluorescence signal F3 is used to detect G bases, and the fourth target fluorescence signal F4 is used to detect C bases. It is understood that the above correspondence between the target fluorescence signals and bases is for illustrative purposes only and does not constitute a limitation.

[0089] In one embodiment, reference is made to Figure 4 The optical path diagram shows that the TDI imaging assembly 500 also includes a sleeve lens 520.

[0090] The sleeve lens 520 is used to receive the four target fluorescence signals emitted by the beam splitter 430 and to converge the optical path of the target fluorescence signals to match the corresponding TDI imaging chip. The TDI imaging chip is used to receive the target fluorescence signals through the converged optical path of the sleeve lens 520 at a preset position to perform fluorescence imaging of the bases.

[0091] In one embodiment, reference is made to Figure 4Different TDI imaging chips are located in the optical path of the corresponding target fluorescence signal and are used to image the corresponding bases. The first TDI imaging chip 5101 receives the first target fluorescence signal F1 and is used to image the A base; the second TDI imaging chip 5102 receives the second target fluorescence signal F2 and is used to image the T base; the third TDI imaging chip 5103 receives the third target fluorescence signal F3 and is used to image the G base; and the fourth TDI imaging chip 5104 receives the fourth target fluorescence signal F4 and is used to image the C base. It is understood that the above correspondence between the TDI imaging chips and base imaging is for illustrative purposes only and does not represent a limitation.

[0092] In one embodiment, a filter is used to further improve imaging performance and reduce signal impurities in the target fluorescence signal. (Refer to...) Figure 5 The TDI imaging assembly 500 also includes a filter 530. The filter 530 is positioned behind the sleeve lens along the optical axis of the target fluorescence signal and is used to filter the four target fluorescence signals after they have passed through the converged optical path of the sleeve lens, thereby filtering out residual excitation beams mixed in with the target fluorescence signals and obtaining the corresponding four filtered signals.

[0093] Figure 5 In this design, filter 530 comprises four regions: a first filter region 5301, a second filter region 5302, a third filter region 5303, and a fourth filter region 5304, corresponding to four target fluorescence signals. For clarity, the four filter regions are dispersed in the diagram and do not represent their actual positions; the filter regions can be different areas divided on a single filter. In one embodiment, the first target fluorescence signal passes through the first filter region 5301, forming a first filter signal F1'; the second target fluorescence signal passes through the second filter region 5302, forming a second filter signal F2'; the third target fluorescence signal passes through the third filter region 5303, forming a third filter signal F3'; and the fourth target fluorescence signal passes through the fourth filter region 5304, forming a fourth filter signal F4'. Four TDI imaging chips 410 are respectively positioned at preset locations to receive the corresponding filter signals and perform fluorescence imaging of the bases based on the filter signals.

[0094] In this embodiment of the invention, fluorescence signals of four bases can be recorded in a single shot through a single channel. Only one spectrometer is needed to separate the four fluorescence signals according to the base type, thereby achieving efficient shooting without the need for other optical components.

[0095] In this embodiment of the invention, the target fluorescence signal with spectral dispersion is used to identify the base category of the base by recognizing the corresponding wavelength of the target fluorescence signal. Therefore, in this embodiment of the invention, the fluorescence of four bases can be recorded in a single image in a single channel, without the need for a complex optical structure, thus achieving gene sequencing and reducing system cost.

[0096] In one embodiment, the spectrophotometer 430 can be a grating or a prism, and the grating can be a transmission grating or a reflection grating, etc. Taking a grating as an example, in this embodiment of the invention, the spectrum of base fluorescence is directly obtained by grating spectrophotometry. The fluorescence signal generated by base excitation reaches the grating and is diffracted by the grating to obtain four fluorescence signals. It is understood that in this embodiment of the invention, only one grating capable of generating the target fluorescence signal is needed to achieve spatial separation of the imaging of four bases with different fluorescent groups, so that the TDI imaging component 500 can acquire the target fluorescence signal generated after grating diffraction, thus achieving separate imaging of bases in a single scan. It is understood that the choice of grating can be set according to the actual sequencing parameters, and this embodiment does not specifically limit it. This embodiment of the invention uses a low-cost grating to achieve spectrophotometry, thereby reducing the system design cost.

[0097] It should be noted that, in designing the beam splitter 430, sleeve lens 520, filter 530 and four TDI imaging chips 410 in this embodiment of the application, the focal position of the target fluorescence signal can be obtained as a preset position based on knowledge of optical parameters, and the imaging surface of the TDI imaging chip 410 can be set at the focal position of the target fluorescence signal of the corresponding base.

[0098] In one embodiment, reference is made to Figure 5 The focal positions of the four filtered signals after passing through the filter 530 are matched with the imaging surfaces of the corresponding TDI imaging chip 410, so that the fluorescence of the four bases can be recorded in a single shot in a single channel and clearly imaged separately.

[0099] In one embodiment, reference is made to Figure 6 After passing through the beam splitter 430, the fluorescence signal is split into four target fluorescence signals that enter the sleeve lens 520. The sleeve lens 520 simultaneously converges the four fluorescence signals, enabling them to be focused on the TDI imaging chip 410 at the corresponding positions, thus obtaining a clear imaging result on the imaging surface of the TDI imaging chip 410. The imaging surface in the figure is for illustration only and does not represent that the imaging surface of each TDI imaging chip 410 is located at the same vertical position.

[0100] Understandably, after calculating the focal position of the target fluorescence signal corresponding to the four bases, the installation position of the TDI imaging chip is obtained. After the components are installed and designed according to the optical parameters, there is no need to adjust the position, and simultaneous imaging of the four bases can be completed.

[0101] The gene sequencing optical system of this application requires only one imaging channel, therefore only one set of sleeve lenses and filters is needed for each channel, significantly reducing costs. Furthermore, since the TDI imaging component integrates four TDI imaging chips corresponding one-to-one with each base, images of all four bases can be obtained in a single imaging process, enabling rapid imaging. The integrated imaging chips also allow for smaller circuitry, further reducing system costs.

[0102] In one embodiment, reference is made to Figure 7 The excitation module 200 includes a light source 210 and an illumination component 220. The light source 210 can simultaneously emit dual-color laser light, such as outputting a red or green excitation beam. The illumination component 220 is positioned behind the light source 210 along the optical axis of the laser signal and generates an excitation beam S based on the laser signal. The excitation beam S is used to perform fluorescence imaging of four bases on the sample to be tested: adenine (A), thymine (T), guanine (G), and cytosine (C), thereby determining the DNA sequence. The excitation beam S simultaneously contains red and green light; for example, green light is used to excite the fluorescence of base AT, and red light is used to excite the fluorescence of base GC, thus completing the identification of the four bases.

[0103] In one embodiment, reference is made to Figure 8 The light source 210 includes a first light source 2101, a second light source 2102, and a beam combiner 2103. The first light source 2101 emits a first laser signal S1, such as red light; the second light source 2102 emits a second laser signal S2, such as green light; the beam combiner 2103 is located at the intersection of the first laser signal S1 and the second laser signal S2. The beam combiner 2103 transmits the first laser signal S1 and reflects the second laser signal S2 to combine the first laser signal S1 and the second laser signal S2 into a laser signal. An excitation beam S is then generated by the illumination component 220. The excitation beam S contains two colors of laser light simultaneously; for example, green light excites the fluorescence of the base AT, and red light excites the fluorescence of the base GC, thereby completing the identification of four-color bases. During sequencing, the two lasers can be continuously turned on without switching, effectively reducing imaging time. In one embodiment, the beam combiner 2103 is also a dichroic mirror.

[0104] As can be seen, in the above embodiments, the excitation module simultaneously excites multiple specific wavelengths of visible light, thereby improving the sequencing efficiency of the above gene sequencing optical system.

[0105] In one embodiment, the illumination assembly includes at least one of the following: a spherical lens, an aspherical lens, a cylindrical lens, a Powell prism, a lens array, a reflector, or a filter.

[0106] In one embodiment, the sample 600 to be tested contains multiple rows of DNA arrays arranged sequentially, each row of the DNA array containing several DNA fragments, such as... Figure 9 Each row of the DNA fragment array shown contains multiple DNA fragments, each DNA fragment being as follows: Figure 9 The circular pattern shown can contain multiple DNA fragments, and each DNA fragment contains multiple bases. Since the DNA fragments in the sample 600 to be tested are mostly scanned in rows, to improve the utilization efficiency of the excitation light source and the excitation effect of the excitation beam during scanning, the excitation module 200 of this embodiment further includes a beam shaping module. The beam shaping module is disposed in the optical path of the excitation beam S emitted by the illumination component 220. The beam shaping module is used to shape the excitation beam S into an illumination spot in one dimension, that is, to shape the excitation beam S in one dimension to form a linear spot so that it can precisely illuminate a certain row of DNA array in the sample 600 to improve the imaging effect of the row scan. It is understood that... Figure 9 The illustration shows DNA fragments arranged in an array, but this does not mean that DNA fragments can only be arranged in an array. This embodiment is for illustrative purposes only and does not limit the arrangement.

[0107] Specifically, the TDI imaging chip in the TDI imaging component only images the target fluorescence signal within its imaging plane. Therefore, in order to scan and image different regions of the sample 600 to be tested, the gene sequencing optical system of this application embodiment further includes a displacement stage, which is used to carry the sample 600 to be tested. The displacement stage is used to move the sample 600 to be tested row by row at a preset moving speed during row scanning, so that the position of the sample to be tested can be adjusted during the movement, thereby enabling the excitation beam to excite and illuminate different regions of the sequencing chip. Specifically, the sample 600 to be tested is excited sequentially row by row to generate fluorescence signals. The preset moving speed is determined according to the row frequency of the TDI imaging chip.

[0108] In this embodiment, the sequencing chip (sample to be tested) is rectangular, and the imaging surface of the TDI imaging chip is also rectangular with an aspect ratio of L:W. Therefore, the major and minor axes of the excitation beam S spot D are also L:W. When the stage is in its initial position, the excitation beam S spot D illuminates the X1 region of the sequencing chip, generating a corresponding fluorescence signal. The TDI imaging chip generates an image signal based on the above working principle and then generates a corresponding detection result x1 based on the image signal. The stage then continues to move, causing the excitation beam spot D to illuminate the X2 region of the sequencing chip, and the detection result x2 is obtained according to the above steps. The stage continues to move, allowing the camera to scan and image regions X3, X4, etc., on the sequencing chip, thereby obtaining different detection results on the sequencing chip.

[0109] In one embodiment, the long axis of the excitation beam S is perpendicular to the long side of the sequencing chip, and the sequencing chip moves along the long side of the excitation beam so that the camera can scan and image the X1 region, X2 region, etc. on the sequencing chip to obtain the detection result.

[0110] It is understandable that, since the long side of the sequencing chip corresponds to the short axis of the target fluorescence signal, that is, the long side of the sequencing chip corresponds to the short side of the camera, compared with the scanning method where the long side of the sequencing chip corresponds to the long side of the camera, the single movement distance of the stage can be shortened, thereby improving the scanning efficiency of the camera.

[0111] In one embodiment, the preset moving speed of the displacement stage is expressed as:

[0112]

[0113] Among them, V s The preset moving speed is indicated by f, the line frequency of the TDI imaging chip is indicated by f, the pixel size of the TDI imaging chip is indicated by C, and the imaging magnification of the TDI imaging chip is indicated by M.

[0114] It is understandable that the displacement stage can be an electrically driven stage, which moves the sample horizontally under motor control, enabling the entire optical system to scan the sample to be tested. Furthermore, since each TDI imaging chip 410 in the TDI imaging assembly 500 can perform independent imaging and computation, the preset movement speed of the displacement stage can be matched with the line scanning frequency of the TDI imaging assembly by pre-setting the aforementioned relationship. This allows for the acquisition of clear images of the four bases during rapid scanning, improving imaging efficiency.

[0115] Reference Figure 10 This is a schematic diagram of the system structure according to an embodiment of this application.

[0116] Figure 10 The following description uses a light source 210, comprising a first light source 2101, a second light source 2102, and a beam combiner 2103, as an example. The beam combiner 2103 is a dichroic mirror. The first light source 2101 emits a first laser signal S1, and the second light source 2102 emits a second laser signal S2. The first laser signal S1 is reflected by the beam combiner 2103, which is positioned at the intersection of the first laser signal S1 and the second laser signal S2. The beam combiner 2103 transmits the first laser signal S1 and reflects the second laser signal S2, thus combining the two laser signals to form a laser signal S'. The combined laser signal S' then passes through the illumination component 220 to form an excitation beam S.

[0117] Figure 10 The excitation beam S is incident on the first dichroic mirror 410. The first dichroic mirror 410 can transmit part of the light and reflect the other part. (Refer to...) Figure 11 The first surface of the first dichroic mirror 410 reflects the excitation beam S to the objective lens 420, which then reaches the sample and excites the sample 600 to generate a fluorescence signal F. The fluorescence signal F is collected by the objective lens 420, transmitted through the second surface of the first dichroic mirror 410, and enters the spectrometer 430.

[0118] Figure 10 The mid-spectrum splitter 430 is positioned behind the first dichroic mirror 410 along the optical path of the fluorescence signal F. It receives the fluorescence signal F emitted from the second surface of the first dichroic mirror 410 and splits it into four target fluorescence signals, each corresponding to a base. (Refer to...) Figure 10 The four target fluorescence signals are: the first target fluorescence signal F1, the second target fluorescence signal F2, the third target fluorescence signal F3, and the fourth target fluorescence signal F4. The spectrophotometer 430 separates the four target fluorescence signals (corresponding to the four bases of ATGC) in space.

[0119] Figure 10 The middle sleeve lens 520 receives four target fluorescence signals (F1, F2, F3, and F4) at different angles emitted by the beam splitter 430, and converges the optical path of the target fluorescence signals to match the corresponding TDI imaging chip 410, thus focusing the target fluorescence signals onto the corresponding TDI imaging chip. (Refer to...) Figure 10The filter 530 is positioned behind the sleeve lens 520 along the optical axis of the target fluorescence signal. It filters the four target fluorescence signals after they converge through the sleeve lens, removing residual excitation beams mixed in with the target fluorescence signals to obtain the corresponding four filtered signals (F1', F2', F3', and F4'). Four TDI imaging chips 410 are respectively positioned at preset locations to receive the corresponding filtered signals and perform fluorescence imaging of the bases based on the filtered signals. The gene sequencing optical system of the above embodiment requires only one imaging channel; therefore, only one set of sleeve lenses and filters is needed for each channel, significantly reducing costs.

[0120] In one embodiment, the TDI imaging component 500 uses a 4K / 256-level TDI imaging chip with an imaging pixel size of 5µm. By integrating four different TDI imaging chips, images of four different bases can be obtained in a single imaging process, achieving rapid imaging. Furthermore, the integrated imaging chip enables circuit miniaturization, further reducing system costs.

[0121] In one embodiment, the beam-splitting component is a grating. Four fluorescence signals acquired by the objective lens are split by the grating and incident at different angles according to their wavelengths into the telescopic lens. The telescopic lens then converges the target fluorescence signals at different angles onto four TDI imaging chips. In this embodiment, the grating parameters are 800 lines / mm, the focal length of the telescopic lens is 150mm, the center-to-center spacing between the different TDI imaging chips is 10mm, and the width of the TDI imaging chips is 1.28mm.

[0122] The gene sequencing optical system of this application requires only one imaging channel, therefore only one set of sleeve lenses and filters is needed for each channel, significantly reducing costs. Furthermore, since the TDI imaging component integrates four TDI imaging chips corresponding one-to-one with each base, images of all four bases can be obtained in a single imaging process, enabling rapid imaging. The integrated imaging chips also allow for smaller circuitry, further reducing system costs.

[0123] In addition, this embodiment of the invention also provides a sequencing method for a gene sequencing optical system, which is applied to the above-mentioned gene sequencing optical system.

[0124] Reference Figure 11 This is a flowchart illustrating the method of using the gene sequencing optical system provided in this application embodiment. The method is mainly executed by a controller or processor and includes steps S1210 to S1230:

[0125] Step S1110: The excitation module generates an excitation beam to excite the sample to be tested.

[0126] In one embodiment, the sample to be tested is first placed within the detection range of the gene sequencing optical system. The sample contains multiple rows of DNA arrays arranged sequentially, each row containing several DNA fragments, such as... Figure 10 Each row of the DNA fragment array shown contains multiple DNA fragments, each DNA fragment being as follows: Figure 10 The circular pattern shown can contain multiple DNA segments, and each DNA segment contains four bases, each with a different fluorescent signal color.

[0127] In step S1120, the sequencing module uses an excitation beam to scan the sample to be tested at a preset scanning frequency to generate four target fluorescent signals corresponding to the bases.

[0128] In one embodiment, the imaging module of the sequencing module uses an excitation beam to scan the sample to be tested at a preset scanning frequency to obtain fluorescence signals, and uses a spectrometer to split the fluorescence signals into four target fluorescence signals corresponding to the bases. During the scanning process, the objective lens acquires the target fluorescence signals and sends them to the TDI imaging module. It is understood that the preset scanning frequency here is matched with the preset moving speed of the stage, and the fluorescence signals obtained by scanning correspond one-to-one with the bases.

[0129] In one embodiment, the four target fluorescence signals separated by the spectrophotometer are: a first target fluorescence signal F1, a second target fluorescence signal F2, a third target fluorescence signal F3, and a fourth target fluorescence signal F4. The first target fluorescence signal F1 is used to detect A bases, the second target fluorescence signal F2 is used to detect T bases, the third target fluorescence signal F3 is used to detect G bases, and the fourth target fluorescence signal F4 is used to detect C bases. It is understood that the correspondence between the target fluorescence signals and bases described above is for illustrative purposes only and does not constitute a limitation.

[0130] In step S1130, the TDI imaging component performs fluorescence imaging on the bases based on the target fluorescence signal to obtain gene sequencing results.

[0131] Therefore, the gene sequencing optical system method of this application significantly reduces costs by utilizing a single imaging channel. Furthermore, since the TDI imaging component integrates four TDI imaging chips that correspond one-to-one with each base, images of all four bases can be obtained in a single imaging process, enabling rapid imaging.

[0132] In one embodiment, step S1230, where the TDI imaging component performs fluorescence imaging on the bases based on the target fluorescence signal to obtain gene sequencing results, specifically includes: using the TDI imaging chip to obtain base images of the corresponding bases based on the target fluorescence signal, and then registering the base images corresponding to the four bases to obtain gene sequencing results.

[0133] Understandably, by using a single imaging channel and performing a single scan, the corresponding bases can be imaged simultaneously based on the target fluorescence signals of four bases. After obtaining the base images, the four base images can be registered and further analyzed to obtain the gene sequencing results of the sample to be tested, thus significantly reducing the imaging time.

[0134] Therefore, the contents of the above-described gene sequencing optical system embodiments are all applicable to the embodiments of the gene sequencing optical system usage method of this embodiment. The specific functions implemented in this usage method embodiment are the same as those in the above-described gene sequencing optical system embodiments, and the beneficial effects achieved are also the same as those achieved in the above-described gene sequencing optical system embodiments.

[0135] It should be noted that the structure of the gene sequencing optical system described in the embodiments of the present invention is for the purpose of more clearly illustrating the technical solutions of the embodiments of the present invention, and does not constitute a limitation on the technical solutions provided in the embodiments of the present invention. As those skilled in the art will know, with the evolution of equipment architecture and the emergence of new application scenarios, the technical solutions provided in the embodiments of the present invention are also applicable to similar technical problems.

[0136] Those skilled in the art will understand that the gene sequencing optical system shown in the figures does not constitute a limitation on the embodiments of the present invention, and may include more or fewer components than shown, or combine certain components, or have different component arrangements. The above description with reference to the accompanying drawings is merely illustrative of preferred embodiments of the present invention and is not intended to limit the scope of the claims of the present invention. Any modifications, equivalent substitutions, and improvements made by those skilled in the art without departing from the scope and spirit of the embodiments of the present invention should be within the scope of the claims of the present invention.

Claims

1. A gene sequencing optical system for exciting a sample to be tested and acquiring the fluorescence signal emitted by the sample for fluorescence imaging, wherein the sample to be tested comprises four bases, characterized in that, include: The excitation module is used to generate an excitation beam that excites the sample to be tested; An imaging module is used to image the sample to be detected using the excitation beam; The imaging module includes: a sequencing module for generating four target fluorescence signals by irradiating the sample to be tested with the excitation beam; a TDI imaging component including four integrated TDI imaging chips, each TDI imaging chip corresponding to a base, the TDI imaging component being used to perform fluorescence imaging on the bases based on the target fluorescence signals using the TDI imaging chips to obtain gene sequencing results; the sequencing module includes: an objective lens for receiving and focusing the excitation beam onto the sample to be tested, and also for collecting the fluorescence signals generated by the excited sample to be tested; a spectrometer disposed behind the objective lens along the optical path of the fluorescence signal, for receiving the fluorescence signal and splitting the fluorescence signal into four target fluorescence signals, each target fluorescence signal corresponding to a base. The TDI imaging assembly further includes: a sleeve lens; the sleeve lens is used to receive four target fluorescence signals and converge the optical path of the target fluorescence signals to match the corresponding TDI imaging chip; the TDI imaging chip is used to receive the target fluorescence signals converged by the sleeve lens at a preset position and perform fluorescence imaging on the base; the beam splitter is used to split the fluorescence signals to obtain four target fluorescence signals corresponding to the base, and the imaging surface of the TDI imaging chip is located at the focal position of the target fluorescence signal of the corresponding base; It also includes a displacement stage that carries the sample to be tested. The displacement stage is used to move the sample to be tested at a preset moving speed so that different positions of the sample to be tested can be excited to generate the fluorescence signal during the movement. The preset moving speed is determined according to the line frequency of the TDI imaging chip.

2. The gene sequencing optical system according to claim 1, characterized in that, The sequencing module further includes: a first dichroic mirror; The first dichroic mirror is used to reflect the received excitation beam to the objective lens; The first dichroic mirror is also used to transmit the received fluorescence signal emitted from the objective lens to the beam splitter.

3. The gene sequencing optical system according to claim 1, characterized in that, The beam splitting component includes at least one of the following: a transmission grating, a reflection grating, or a prism.

4. The gene sequencing optical system according to claim 1, characterized in that, The excitation device includes: a light source and an illumination assembly; The light source is used to generate laser signals; The illumination component is positioned behind the light source along the optical axis of the laser signal, and is used to form an excitation beam according to the laser signal.

5. A gene sequencing optical system according to claim 4, characterized in that, The light source includes: a first light source, a second light source, and a beam combining assembly; The first light source is used to emit a first laser signal; The second light source is used to emit a second laser signal; The beam combining component is disposed at the intersection of the first laser signal and the second laser signal. The beam combining component is used to transmit the first laser signal and reflect the second laser signal to combine the first laser signal and the second laser signal to form the laser signal.

6. The gene sequencing optical system according to claim 4, characterized in that, The lighting assembly includes at least one of the following: a spherical lens, an aspherical lens, a cylindrical lens, a Powell prism, a lens array, a reflector, or a filter.

7. The gene sequencing optical system according to claim 1, characterized in that, The preset moving speed is expressed as: in, The preset moving speed is represented by f, the line frequency of the TDI imaging chip is represented by C, the pixel size of the TDI imaging chip is represented by M, and the imaging magnification of the optical system is represented by M.

8. A gene sequencing optical system according to any one of claims 1 to 7, characterized in that, It also includes a filter, which is disposed behind the sleeve lens along the optical axis of the target fluorescence signal. The filter is used to filter the four target fluorescence signals after the optical path is converged by the sleeve lens, and to filter out the residual excitation beam mixed in the target fluorescence signal to obtain the corresponding four filtered signals. The TDI imaging chip is used to receive the filter signal at a preset position and perform fluorescence imaging on the bases based on the filter signal.

9. A gene sequencing optical system according to any one of claims 1 to 7, characterized in that, The excitation module further includes a beam shaping module, which is disposed in the optical path of the excitation beam emitted by the illumination component. The beam shaping module is used to shape the excitation beam into an illumination spot in a one-dimensional direction.

10. A sequencing method using an optical system for gene sequencing, characterized in that, The method, applied to the gene sequencing optical system as described in any one of claims 1 to 9, comprises: The excitation module generates an excitation beam to excite the sample to be tested. The sample to be tested includes four bases, each of which emits a fluorescence signal in a different wavelength band. The sequencing module uses the excitation beam to scan the sample to be tested at a preset scanning frequency to generate four target fluorescent signals corresponding to the bases. During the scanning process, the objective lens acquires the target fluorescence signal and transmits it to the TDI imaging module; The TDI imaging component performs fluorescence imaging on the bases based on the target fluorescence signal to obtain gene sequencing results.

11. The sequencing method of a gene sequencing optical system according to claim 10, characterized in that, The TDI imaging component performs fluorescence imaging on the bases based on the target fluorescence signal to obtain gene sequencing results, and further includes: The TDI imaging chip is used to obtain images of the corresponding bases based on the target fluorescence signal; The base images corresponding to the four bases are registered to obtain the gene sequencing results.

Images