WO2023082179A1 - 提高测序分辨率的方法、测序装置和系统 - Google Patents

提高测序分辨率的方法、测序装置和系统 Download PDF

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Publication number
WO2023082179A1
WO2023082179A1 PCT/CN2021/130332 CN2021130332W WO2023082179A1 WO 2023082179 A1 WO2023082179 A1 WO 2023082179A1 CN 2021130332 W CN2021130332 W CN 2021130332W WO 2023082179 A1 WO2023082179 A1 WO 2023082179A1
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excitation light
dichroic mirror
sequencing
optical path
fluorescent
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PCT/CN2021/130332
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English (en)
French (fr)
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苏泽宇
梁元庆
沈梦哲
倪洁蕾
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深圳华大生命科学研究院
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Priority to CN202180099401.5A priority Critical patent/CN117501103A/zh
Priority to PCT/CN2021/130332 priority patent/WO2023082179A1/zh
Publication of WO2023082179A1 publication Critical patent/WO2023082179A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

Definitions

  • the invention relates to the field of biotechnology, in particular to a method for improving sequencing resolution, a sequencing device and a system.
  • DNA sequencing technology is one of the most commonly used technical means in molecular biology-related research, and at the same time, it also promotes the rapid development of this field.
  • the human genome project, transcriptome analysis, microbial genome resequencing, single nucleotide polymorphisms (single nucleotide polymorphisms, SNP) analysis, etc. have also greatly promoted the research and development of other biological fields.
  • the second-generation DNA sequencing technology also known as high-throughput sequencing technology (high-throughput sequencing, HTS), at a low cost and with an accuracy of over 99%, can perform hundreds of thousands to several hundreds of thousands of samples at a time. Millions of DNA molecules are subjected to rapid sequencing analysis at the same time.
  • Representative technologies include Roche's 454, Illumina's Solexa, and ABI's SOLID.
  • the second-generation DNA sequencing is the core of the epoch-making change to the first-generation sequencing technology; at present, the sequencing system based on the second-generation DNA sequencing technology uses the method of detecting optical signals to realize the four bases (A, T/U, C and G) identification and distinction, of course, the premise is that the base needs to be fluorescently labeled.
  • the central wavelength of fluorescence excited by red light is about 720nm, and the lens NA is 0.8. It can be known that its theoretical resolution is about 550nm. Because the NA of the lens is affected by the production process, it is very difficult to improve, which makes it difficult for the red-green excitation light sequencing technology to make a breakthrough in resolution, which limits the sequencing throughput and sequencing quality to a certain extent.
  • the present invention provides a method for improving the sequencing resolution, so as to solve the problem that in the existing sequencing technology, the red-green excitation light sequencing technology is actually difficult to break through in the resolution, which limits the sequencing throughput and sequencing quality to a certain extent. technical problems.
  • the present invention also provides a sequencing device implementing a method for improving sequencing resolution, and a breakthrough in sequencing resolution can be achieved through the combination of the sequencing device and the method.
  • the present invention also provides a sequencing system for application.
  • a method for improving sequencing resolution comprising the steps of:
  • the maximum wavelength is smaller than the red light wavelength
  • the generated fluorescent signal is transmitted through the optical path, it is collected and imaged.
  • the core lies in the specific selection of excitation light and wavelength.
  • the red and green light improves the resolution of detection and further increases the throughput of sequencing.
  • the invention solves the technical problem that in the existing sequencing technology, it is difficult for the red-green excitation light sequencing technology to make a breakthrough in resolution, thereby limiting the sequencing throughput and sequencing quality to a certain extent.
  • test sample containing fluorescent dye can exist in various forms in actual application scenarios; as long as the fluorescent dye is used to mark the test substance with specific physical and chemical characteristics.
  • sample to be tested containing a fluorescent dye may be a nucleic acid sequence in which the fluorescent dye is bound by some binding enzymes.
  • blue light is used as the first excitation light and green light is used as the second excitation light for the excitation light.
  • the spectrum of the second excitation light also includes yellow light, so green light is the most preferred second excitation light, but the embodiment containing yellow light is not excluded.
  • the wavelength range of the first excitation light is 440-500 nm
  • the wavelength range of the second excitation light is 500-600 nm.
  • the wavelength range of the first excitation light is 470-490nm; the wavelength range of the second excitation light is 550-580nm.
  • the two kinds of excitation light are coupled through an optical fiber, and share the same optical fiber port to output to the sample to be tested, and the type of the optical fiber port includes one of FC/PC, FC/APC or SMA.
  • the first excitation light is blue light with a wavelength range between 440-500nm, and its wavelength is smaller than the traditional red and green light, so that It will improve the detection efficiency; preferably, the FC/APC interface is used, which can reduce the return loss and further guarantee the detection result.
  • the uniformity of the light spots of the two excitation lights is greater than 85%; the length of the optical fiber is greater than 2000mm; the output power of the optical fiber is greater than or equal to 2000mw.
  • the fluorescent dye includes a first fluorescent dye excited by the first excitation light and a second fluorescent dye excited by the second excitation light;
  • the first fluorescent dye includes: one or more of Alexa Fluor 488, iF488, ATTO488, DyLight488, Hilyte488, FAM488 or FITC488;
  • the second fluorescent dye includes: one or more of CY3.5, AF568 or ROX.
  • the above-mentioned preferred specific fluorescent dyes correspond to the wavelengths of the two kinds of excitation light, so as to improve the excitation efficiency as much as possible.
  • both the above-mentioned first fluorescent dye and the second fluorescent dye can be purchased directly.
  • the dosage ratio of the first fluorescent dye to the second fluorescent dye is 3:1 ⁇ 4:1.
  • the above dosage ratio of the first fluorescent dye and the second fluorescent dye is more likely to meet the sequencing requirements at a high standard.
  • the dosage unit of the two fluorescent dyes is preferably ⁇ mol.
  • the optical path transmission it also includes the step of using two types of dichroic mirrors to guide the fluorescent signal and the non-fluorescent signal to different optical paths;
  • the transmission window of one type of dichroic mirror includes 500-550nm and/or Or 580-650nm;
  • Another type of dichroic mirror has a transmission window of at least 800nm.
  • the dichroic mirror distinguishes light of different wavelengths in the form of reflection and transmission, and for light in a specific wavelength range, its reflectivity and transmittance are both greater than 95%. It should be understood that the specific setting scenarios of the two types of dichroic mirrors are selected based on their effects in the optical path, and their setting positions are not unique.
  • an operation of passing through a filter component with a transmission window including 483nm-550nm or 580-700nm is also included.
  • the sample to be tested includes DNA and is set on a substrate, and the substrate includes one of silicon, glass or sapphire.
  • the sequencing device is provided with an excitation light source assembly capable of emitting excitation light with a maximum wavelength smaller than the wavelength of red light; at least including a dichroic mirror, an optical path assembly of an objective lens, and an imaging assembly; the dichroic mirror is arranged on On the optical path after the excitation light emitted by the excitation light source component, the excitation light and/or the fluorescence signal generated by the sample to be tested after being excited by the excitation light is reflected, transmitted or distinguished; on the optical path; the imaging component is arranged on the optical path after the fluorescent signals are distinguished, so as to realize the collection and imaging of different fluorescent signals.
  • the excitation light passes through optical path components such as dichroic mirrors and objective lenses, it excites the sample to be tested to generate a fluorescent signal.
  • the imaging component After processing or distinguishing, the imaging component finally installed at the end of the optical path collects the image, and then achieves the purpose of detection.
  • the sequencing device of the present invention uses a hardware carrier to realize the above-mentioned method for improving the sequencing resolution.
  • the excitation light source component includes two excitation light sources; one of the excitation light sources excites blue light as the first excitation light, and the other excitation light source excites green light as the second excitation light;
  • the two excitation light sources are optically coupled, and the two excitation lights share the same optical fiber port for output.
  • the excitation light source component generally needs to emit at least two excitation lights with different wavelengths, both of which are smaller than the wavelength of red light, so as to separately excite the fluorescent dyes in the sample to be tested and generate two fluorescent signals.
  • the distinguishing function of the dichroic mirror not only refers to distinguishing the fluorescent signal from other lights, but also reflects the distinguishing function of the two different fluorescent signals when there are two excitation lights. After differentiation, it is imaged by the imaging component.
  • the wavelength of the blue light is between 440-500nm, and the preferred wavelength range is between 470nm-490nm.
  • Optional components include: a semiconductor laser of 470nm, a semiconductor pumped solid-state laser of 473nm ( DPSS) laser or 488nm semiconductor laser;
  • the wavelength of another light source (green light) is between 500-600nm, the preferred wavelength range is 550-580nm, such as 556nm solid-state laser, 561nm solid-state semiconductor laser (DPSS) laser or 577nm solid-state laser.
  • the light source may also be an LED.
  • the two excitation light sources are optically coupled, and the two excitation lights share the same optical fiber port for output, which makes the device more integrated as a whole and is also easy to control.
  • the optical path assembly also includes an autofocus component; the autofocus component emits probe light with a wavelength different from that of the fluorescent signal; After collecting the feedback signal, the objective lens is driven to achieve focusing.
  • the autofocus component emits probe light with a wavelength different from that of the fluorescent signal; After collecting the feedback signal, the objective lens is driven to achieve focusing.
  • a probe light with a wavelength different from that of the fluorescent signal is emitted, generally infrared light, with a waveband between 800-900nm, and in a preferred embodiment, the waveband is 830-860nm.
  • the probe light emitted by the auto-focus component will pass through the dichroic mirror and the objective lens in sequence, and then irradiate the sample to be tested, and then return to the auto-focus component in the same way, and the auto-focus component will drive the objective lens to move to the focal plane position according to the feedback signal collected , to realize the focus function.
  • the dichroic mirror includes a second dichroic mirror, a first dichroic mirror and a third dichroic mirror arranged from top to bottom; the third dichroic mirror and the second dichroic mirror The light paths of the dichroic mirrors are communicated and arranged adjacently.
  • the dichroic mirror distinguishes excitation light, probe light or fluorescence signals with different wavelengths through reflection/transmission; due to the existence of the second dichroic mirror and the first dichroic mirror arranged from top to bottom; in this way, the excitation light first After entering the first dichroic mirror, it is reflected, and then reaches the sample to be tested after passing through the objective lens, prompting the generation of fluorescent signals.
  • the fluorescent signal passes through the objective lens, and after passing through the first dichroic mirror, it is reflected by the second dichroic mirror arranged above it. Since the third dichroic mirror communicates with the second dichroic mirror and is adjacent to the optical path, Thus, the fluorescence signal is reflected to the third dichroic mirror, differentiated, and collected and imaged by the imaging component.
  • the imaging component includes a tube lens and a camera arranged on different output light paths of the third dichroic mirror; a reflective component is also arranged between the tube lens and the camera.
  • two fluorescent signals will be generated, which will be output in different optical paths after being distinguished by the third dichroic mirror. were collected and imaged separately.
  • a collimator lens is arranged on the optical path between the excitation light source assembly and the first dichroic mirror; the free end of the collimator lens is used to access the excitation light emitted by the excitation light source assembly; the other end extends to the first dichroic Color mirror inside.
  • the collimating lens is mainly used as the optical path carrier of the excitation light, so that the excitation light enters the first dichroic mirror from the excitation light source component, and the first dichroic mirror reflects the excitation light to the objective lens and Arriving at the sample to be tested to realize the excitation of the fluorescent signal.
  • it also includes a mounting base; the dichroic mirror and the imaging component are arranged on the mounting base;
  • the optical path assembly includes an auto-focus component; the focus components are all arranged on the installation base;
  • the objective lens passes through the mounting base and is used for one end opposite to the sample to be measured, and the other end communicates with the first dichroic mirror and/or the second dichroic mirror.
  • the mounting base is used as a bearing platform for dichroic mirrors (the first dichroic mirror, the second dichroic mirror and the third dichroic mirror), imaging components, autofocus components, collimating lenses, etc. Installation and fixing are integrated.
  • the function of the installation base is to fix and install the above-mentioned various components, and at the same time, it is also a carrier for connecting and fixing the entire device and other functional parts of the sequencing system. Therefore, according to actual requirements, various forms of installation can be arranged on it. holes or mounting cavities, etc.
  • the camera is preset with a data interface for data exchange
  • the focus component is preset with a data interface for data exchange.
  • the data interface is used as a guarantee for the direct connection between the autofocus component or the camera and other functional modules (such as power supply, control system). It is easy to understand that actions that need to exchange data with other functional modules, such as autofocus components or cameras, can also be implemented through Bluetooth or other wireless connections.
  • a filter component is also included, and the filter component is arranged in the optical path before or after the tube lens; in a preferred solution, the filter component is set between the third dichroic mirror and the tube lens between mirrors.
  • the objective lens is provided with a plurality of optical lenses, and the objective lens is a dry or immersion objective lens, and its magnification is not lower than 16 times.
  • filtering components shielding the excitation light reflected by the sample
  • multiple optical lenses and magnification is to ensure the detection efficiency as much as possible from the hardware.
  • a sequencing system including a sample to be tested and the above-mentioned sequencing device
  • the sample to be tested includes DNA sequence and fluorescent dye, and is opposite to the objective lens;
  • control unit is connected with one or more components of the sequencing device to exchange, control or display data.
  • the method, sequencing device and system for improving the sequencing resolution provided by the present invention, in the specific device, after the excitation light passes through optical path components such as dichroic mirrors and objective lenses, it excites the sample to be tested to generate a fluorescent signal, and the fluorescent signal is the same
  • optical path components such as dichroic mirrors and objective lenses
  • the imaging components at the end of the optical path are finally collected and imaged, and then the detection purpose is achieved.
  • the principle is to irradiate and excite the sample to be tested containing the fluorescent dye with at least one excitation light, and generate a fluorescence signal, and in the excitation light, the maximum wavelength is smaller than the wavelength of the red light;
  • FIG. 1 is a schematic diagram of the principle of a sequencing device provided by an embodiment of the present invention
  • Figures 2-3 are views of two different perspectives of the sequencing device provided by the embodiment of the present invention.
  • Fig. 4 is the front view of Fig. 2 and Fig. 3;
  • Fig. 5 is the B-B direction sectional view of Fig. 4;
  • Fig. 6 is a view of the bottom view of the sequencing device provided by the embodiment of the present invention.
  • Fig. 7 is the transmission spectrum diagram of the dichroic mirror provided by the embodiment of the present invention.
  • Fig. 8 is two kinds of dyes and corresponding optical filter spectrogram
  • Fig. 9 is the emission intensity of different dyes in the microplate reader test in the embodiment of the present invention.
  • Figure 10 is the blue-green dual-channel sequencing scatter plot analysis results of the blue dye and the green dye in the sequencing imaging system of the two schemes in the implementation of the present invention
  • Fig. 11 is the sequencing result of SE50 based on blue light in the practice of the present invention.
  • Light source assembly 101; optical path assembly—102; imaging assembly—103; sample to be tested—104;
  • Objective lens 201; Autofocus component—202; Second dichroic mirror—203; First dichroic mirror—204; Third dichroic mirror—205; Tube mirror—206; Camera—207; Mounting frame—208 ; Reflecting component—209; Collimating lens—210; Mounting base—211; Data interface—213.
  • first and second are used for descriptive purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features. Thus, features defined as “first” and “second” may explicitly or implicitly include at least one of these features. In the description of the present invention, “plurality” means at least two, such as two, three, etc., unless otherwise specifically defined.
  • the first feature may be in direct contact with the first feature or the first and second feature may be in direct contact with the second feature through an intermediary. touch.
  • “above”, “above” and “above” the first feature on the second feature may mean that the first feature is directly above or obliquely above the second feature, or simply means that the first feature is higher in level than the second feature.
  • “Below”, “beneath” and “beneath” the first feature may mean that the first feature is directly below or obliquely below the second feature, or simply means that the first feature is less horizontally than the second feature.
  • FIG. 1 shows an embodiment of the sequencing device provided by the present invention
  • the direction of the arrow indicates the direction of light
  • the sequencing device is provided with an excitation light source assembly 101 that can emit excitation light with a maximum wavelength smaller than the wavelength of red light
  • at least The optical path assembly 102 and the imaging assembly 103 that comprise dichroic mirror, objective lens
  • the generated fluorescent signals are reflected, transmitted or distinguished
  • the objective lens is set on the optical path where the fluorescent signals enter the dichroic mirror
  • the imaging component 103 is set on the optical path after the fluorescent signals are distinguished, so as to collect and image different fluorescent signals.
  • the excitation light source assembly includes two excitation light sources; one excitation light source excites blue light as the first excitation light, and the other excitation light source excites green light as the second excitation light; the two excitation light sources are optically coupled, and the two excitation lights share the output of the same optical fiber port; an autofocus component 202 is set in the optical path assembly; the autofocus component 202 can emit detection light with a wavelength different from that of the fluorescence signal, and the detection light is transmitted through the dichroic mirror and the objective lens 201 successively, and then irradiates the sample to be tested. Then go back the same way, and the autofocus component 202 drives the objective lens 201 to focus according to the feedback signal collected after returning;
  • the dichroic mirror includes a first dichroic mirror 204, a second dichroic mirror 203 and a third dichroic mirror 205; the second dichroic mirror 203 and the first dichroic mirror 204 are arranged from top to bottom;
  • the third dichroic mirror 205 communicates with the second dichroic mirror 203 on the optical path and is adjacently arranged;
  • the imaging assembly includes a tube lens 206 and a camera 207 arranged on different output optical paths of the third dichroic mirror 205; a reflective component 209 is also arranged between the tube lens 206 and the camera 207;
  • a collimating lens 210 is arranged on the optical path between the excitation light source assembly and the first dichroic mirror 204; the free end of the collimation lens 210 is used to access the excitation light that the excitation light source assembly sends; The interior of the first dichroic mirror 204 communicates.
  • the excitation light is collimated by the collimator lens 210 and then reflected to the objective lens 201 by the first dichroic mirror 204, and then focused on the sample to be measured.
  • Dichroic mirror, imaging assembly and focusing part are all arranged on a mounting base 211; One end of objective lens 201 passes through mounting base 211 and is opposite to the sample to be measured, and the other end communicates with the first dichroic mirror 204; Camera 207 and automatic A data interface 213 for data exchange is preset on the focusing component 202 .
  • the light path before or after the tube lens 206 can also be provided with a filter component to achieve the filtering effect.
  • a filter component to achieve the filtering effect.
  • a filter component is set between the camera 207 and the tube lens 206, and in some embodiments, the filter component is set between the third dichroic mirror 205 and the tube lens 206;
  • the objective lens 201 is provided with a plurality of Optical lenses, and the objective lens 201 is a dry or immersion objective lens, and the immersion objective lens can also be a water immersion or oil immersion objective lens; the magnification of the objective lens 201 is not less than 16 times.
  • FIG. 2-FIG. 11 in a specific embodiment of the sequencing device provided by the present invention, it includes a light source, a collimating lens 210, a first dichroic mirror 204, a second dichroic mirror 203, a third dichroic Chromatic mirror 205, objective lens 201, autofocus component 202 and imaging assembly etc.; Imaging assembly comprises the tube lens 206 and the camera 207 that are arranged on the different output light paths of the third dichroic mirror 205, and between the tube lens 206 and the camera 207 There is a reflective member 209 .
  • the collimator lens 210, the first dichroic mirror 204, the second dichroic mirror 203, the third dichroic mirror 205, the autofocus component 202 and the imaging assembly are all arranged on the installation base 211, and the second dichroic mirror
  • the optical paths of the mirror 203 and the third dichroic mirror 205 are communicated, and the tube lens 206 and the camera 207 are set in pairs to collect and image two fluorescent signals.
  • a filter component may also be provided between the tube lens 206 and the reflective component 209 , and in some embodiments, the filter component directly adopts a filter.
  • the light source (not shown in the figure) includes two excitation lights, one is a 473nm solid-state semiconductor laser (DPSS); the other is a 561nm solid-state semiconductor laser (DPSS).
  • DPSS solid-state semiconductor laser
  • the type of optical fiber port uses FC/APC interface to reduce return loss.
  • parameters of the light source please refer to Table 1 below;
  • the excitation light After the excitation light is collimated by the collimating lens 210, it is guided by the first dichroic mirror 204 into the objective lens 201, and finally projected onto the surface of the sample to be measured; the focal length of the collimating lens 210 is adjusted according to the size of the required illumination range , in this embodiment, its focal length is 2.9mm.
  • the first dichroic mirror 204 can distinguish the excitation light and the fluorescence signal, and the first dichroic mirror 204 guides the excitation light into the objective lens 201 while allowing the fluorescence signal emitted from the sample to pass through.
  • Figure 7 shows the spectrum of the dichroic mirror, the abscissa in the figure represents the wavelength, and the ordinate represents the transmittance.
  • the reflectance of the first dichroic mirror 204 for the 473/561nm excitation spectrum is above 95%
  • the transmittance for the fluorescence bands 500-550nm and 580-650nm is above 95%.
  • the objective lens 201 receives the excitation light reflected from the first dichroic mirror 204 and projects it onto the sample to be measured.
  • the objective lens 201 includes a plurality of optical lenses, its magnification is 16X, and its NA is 0.8.
  • the sample to be tested is a biological sample, including DNA sequences and fluorescent dyes, which can be combined with DNA by some binding enzymes; and the DNA is arranged on a substrate, and the substrate includes a silicon base, One of glass or sapphire, preferably silicon-based.
  • the first fluorescent dye one is excited by blue light, called the first fluorescent dye, and the fluorescent signal emitted by it is called the first fluorescent signal; the other is excited by green light, called the second fluorescent dye, and the fluorescent signal emitted by it is called the first fluorescent signal. is the second fluorescent signal.
  • the selection of fluorescent dyes corresponds to the wavelength of the excitation light, so as to improve the excitation efficiency as much as possible.
  • Alexa Fluor 488 and ROX are used as the first fluorescent dye and the second fluorescent dye respectively as examples.
  • Fig. 8 shows the spectrograms of the above-mentioned two kinds of dyes and corresponding filter parts (the filter part is specifically set as a filter, and placed in front of the camera 207, specifically between the camera 207 and the tube lens 206, not shown in the figure Shows).
  • the excitation efficiency of the absorption spectrum 601 of the first fluorescent dye Alexa Fluor 488 at the first excitation wavelength 473nm is about 0.4
  • the transmission window of the first optical filter 603 is 500-550nm
  • the combining efficiency of the spectrum 602 with the first filter 603 is about 0.67.
  • the excitation efficiency of the absorption spectrum 604 of the second fluorescent dye ROX at 561nm is about 0.59
  • the transmission window of the second optical filter 606 is 580-650nm
  • the combination of the emission spectrum 605 of the second fluorescent dye and the second optical filter 606 The efficiency is about 0.59.
  • the setting of the optical filter in this embodiment can filter the excitation light to a high degree and transmit the fluorescent signal, achieving a good discrimination effect.
  • Crosstalk signal crosstalk
  • Crosstalk is actually equivalent to background noise for the camera acquisition system, which will affect the signal-to-noise ratio of the system.
  • Crosstalk is about 8%, achieving a higher signal-to-noise ratio and making the sequencing quality more accurate.
  • the two fluorescent signals emitted from the sample to be measured are collected by the objective lens 201 , transmitted through the first dichroic mirror 204 , and reach the second dichroic mirror 203 .
  • the second dichroic mirror 203 distinguishes the fluorescent signal from the detection signal of the autofocus component 202 .
  • the second dichroic mirror 203 is an 800nm long pass filter.
  • the fluorescent signal ranges from 450-750 nm.
  • the detection light emitted by the auto-focus component 202 is infrared light, which has a different wavelength from the fluorescence signal.
  • the detection signal wavelength band of the auto-focus component 202 is between 800-900 nm, preferably 820-860 nm.
  • the detection light emitted by the auto-focus component 202 will pass through the second dichroic mirror 203 and the first dichroic mirror 204 in sequence, and then reach the sample to be tested, and then return to the auto-focus component 202 from the original path of the sample to be tested.
  • the auto-focus component 202 drives the objective lens 201 to move to the position of the focal plane according to the collected feedback signal to realize the focusing function.
  • the fluorescent signal is reflected from the second dichroic mirror 203 to the third dichroic mirror 205, and the third dichroic mirror 205 will separate the first fluorescent signal and the second fluorescent signal and guide them to the two cameras 207 respectively Among them, the spectrum of the third dichroic mirror 205 is shown in FIG. 7 .
  • the first fluorescent signal and the second fluorescent signal excited by the blue light and the green light are reflected from the third dichroic mirror 205 to the tube lens 206 and finally collected by the camera 207 .
  • the dichroic mirror includes a first dichroic mirror 204 and a second dichroic mirror 203, and the second dichroic mirror 203 and the first dichroic mirror 204 are from above And under Settings. Both dichroic mirrors have fine-tuning structures to ensure that the dichroic mirrors are at the right angle.
  • the objective lens 201 is fixed on the motorized translation stage with threads, and combined with the autofocus component 202, the objective lens 201 is locked on the focal plane of the sample to be measured.
  • the electric translation stage is driven by a voice coil motor to ensure high precision requirements, specifically, its positioning accuracy is better than 100nm, preferably, its accuracy is better than 20nm.
  • DNA small balls
  • fluorescent dyes are attached to the surface of the sample to be tested.
  • the sample to be tested is placed on the two-dimensional electric translation stage, and under the cooperation of the auto-focus component 202 and the two-dimensional electric translation stage, a comprehensive scanning observation of the sample to be tested can be realized.
  • the two-dimensional electric translation stage is driven by a linear motor to meet the requirements of large stroke and high-precision positioning for scanning.
  • its stroke range is greater than 80mm*80mm, and its positioning accuracy is better than 200nm.
  • the fluorescent signal will enter the camera 207 through the tube lens 206 and reflective components.
  • the reflective component 209 is used to fold the optical path to make the structure more compact, and the reflective component 209 has a two-dimensional rotationally adjustable structure to ensure alignment of the optical paths.
  • the tube mirror 206 is installed on the base where the third dichroic mirror 205 is located, and the installation method can be threaded fixing, sleeve fixing and the like. In order to eliminate the influence of ambient light, there is a light shield (not shown in the figure) in the space between the tube lens 206 and the camera 207 .
  • the camera 207 is installed on a base with five-axis adjustment.
  • the mounting frame 208 is integrated with the mounting base 211. According to the actual incident angle and position of the fluorescence signal, the camera 207 can be adjusted so that the target surface of the camera 207 is aligned with the incident light path. More specifically, the camera 207 is a CMOS type detector, and its number of pixels is better than 5000*5000 pixels.
  • the collimating lens 210 can be a doublet lens;
  • the objective lens 201 is an immersion objective lens, which can be water immersion or oil immersion; the blue light with a wavelength of 473nm,
  • the first fluorescent dye that can be selected also includes iF488, ATTO488, DyLight488, Hilyte488, FAM488 or FITC488, etc.; for the green light with a wavelength of 561nm, the second fluorescent dye that can be selected includes CY3.5, AF568, etc.
  • the efficiency of the system can be improved by customizing the spectral lines of the optical filter, for example, the window of the first optical filter 603 can be optimized to 483-550nm, and the window of the second optical filter 606
  • the window can be optimized to 580-700nm; at the same time, by selecting different dye combinations and optimizing the filter spectrum, the Crosstalk can be lower than 5%; because the Crosstalk is very low, it has a high signal-to-noise ratio and the sequencing quality is accurate.
  • the first fluorescent signal band is 500-550nm
  • the second fluorescent signal band is 580-650nm.
  • its stroke range is greater than 100*100mm ; In a more preferred embodiment, its positioning accuracy is better than 50nm.
  • the sequencing device mentioned in any of the above schemes plus a test sample including DNA sequence and fluorescent dye, and make the test sample and the objective lens face each other, can constitute a more complex sequencing system.
  • the excitation light emitted by the excitation light source component is excited to generate a fluorescent signal, which enters the optical path of the dichroic mirror through the objective lens, thereby realizing the sequencing effect.
  • a control unit is included; the control unit is connected with one or more components of the sequencing device to exchange, control or display data.
  • Embodiments of the present invention also provide a method for improving sequencing resolution, comprising the following steps:
  • the maximum wavelength is smaller than the red light wavelength
  • the generated fluorescent signal is transmitted through the optical path, it is collected and imaged.
  • the excitation light is blue light with a wavelength range of 440-500nm as the first excitation light; and green light with a wavelength range of 500-600nm as the second excitation light; the two excitation lights are coupled through optical fibers and share the same
  • the output of the fiber port is directed to the sample to be tested, and the type of the fiber port includes one of FC/PC, FC/APC or SMA.
  • the wavelength range of the first excitation light is 470-490nm; the wavelength range of the second excitation light is 550-580nm; the uniformity of the spots of the two excitation lights is greater than 85%; the length of the optical fiber is equal to Greater than 2000mm; fiber output power is approximately equal to 2000mw.
  • the first excitation light is blue light with a wavelength of 470nm, 473nm or 488nm; the second excitation light is green light with a wavelength of 556nm, 561nm or 577nm.
  • the wavelength of blue light can be extended to green light, and the wavelength of green light can be extended to yellow and red.
  • the first excitation light is blue light, and the wavelength can simply have an upper limit, such as 470nm, 473nm or 488nm; the second excitation light is green light, and the wavelength can simply have a lower limit, such as 556nm, 561nm or 577nm .
  • another specific implementation method provided by the present invention includes: using an excitation light source component to emit two kinds of excitation light with different wavelengths and smaller than the wavelength of red light;
  • the reflection of a dichroic mirror 204 passes through the objective lens 201, and irradiates the sample to be tested (containing two kinds of fluorescent dyes), and generates two fluorescent signals;
  • the two fluorescent signals are transmitted through the first dichroic mirror 204, After being reflected by the second dichroic mirror 203 and distinguished by the third dichroic mirror 205, the images are collected and imaged by an imaging component.
  • the optical path transmission also includes the step of using two types of dichroic mirrors to guide the fluorescent signal and the non-fluorescent signal to different optical paths; one of the dichroic mirrors
  • the transmission window includes 500-550nm and/or 580-650nm; another type of dichroic mirror has a transmission window of at least 800nm.
  • the transmission of the fluorescent signal through the optical path also includes the operation of passing through a filter component with a transmission window including 483nm-550nm or 580-700nm.
  • assay optimization is performed with respect to fluorescent dyes.
  • the fluorescent dyes that match the absorption and emission wavelengths.
  • available dyes include Alexa Fluor 488, iF488, ATTO488, DyLight488, Hilyte488, FAM488, FITC488 etc., hereinafter referred to as the first dye;
  • AF568, ROX dye in DNBSEQTM technology, etc. can be selected, hereinafter referred to as the second dye.
  • the base is labeled with the first dye, and the selected first dye is used to label the four bases of A, C, G, and T respectively, and then the performance of the dye is tested using a microplate reader.
  • Figure 9 shows the emission intensities of different dyes tested on a microplate reader. The results show that due to the physical and chemical properties of the fluorescent dyes, the yields of labeled products in the labeling reaction are different, and the luminescence intensities are also different. Alexa Fluor 488 has the worst luminescent effect when labeling G and A bases. In the blue light cPAS protocol, C and T bases will be selected for labeling of the first dye, and A and G for labeling of the second dye.
  • the first dye is Alexa Fluor 488; the second dye is ROX.
  • cPAS is a well-known technical term in the art, and is an abbreviation for Combinatorial Probe Anchor Synthesis (cPAS).
  • the dye base labeling scheme can be combined according to actual needs.
  • two dyes can be marked on base C, or two dyes can be marked on base T, as shown in Table 2 below. :
  • Option One Option II A-second dye A-second dye C-Second Dye C-first dye C-first dye T-second dye T-first dye T-first dye G - unmarked G - unmarked
  • Figure 10 shows the blue-green dual-channel sequencing scatter plot analysis results of the first dye and the second dye of the two schemes on the sequencing imaging system, wherein (a) is the first dye and the second dye in scheme one in The blue-green dual-channel sequencing scattergram analysis results on the sequencing imaging system; (b) is the blue-green dual-channel sequencing scattergram analysis results of the first dye and the second dye in scheme 2 on the sequencing imaging system.
  • the horizontal coordinate is the blue light channel, and the vertical coordinate is the green light channel.
  • the total amount of the first dye and the second dye is 1 ⁇ mol, and in the combination of 1-9, the situations of different amounts of the first dye and the second dye are respectively shown; for example, in the first kind of ratio, the first The dosages of the dye and the second dye are 0.5 ⁇ mol respectively, and the specific dosages of the other combinations are determined according to the ratio of the two dyes, which will not be repeated here.
  • the SE50 test was performed on the sequencing device.
  • the present invention under the premise that the NA remains unchanged, by setting a specific optical path structure on the sequencing device, selecting a specific excitation light source (blue light and green light) and matching fluorescent dyes, it is realized without increasing In the case of low material cost, the resolution of the optical system is improved by using the blue-green light excitation method with a shorter wavelength (the first excitation light is blue light, and the second excitation light is green light), thereby improving the sequencing quality or sequencing throughput. Reduce the cost of sequencing.

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Abstract

一种提高测序分辨率的方法、测序装置和系统；方法包括，以至少一种激发光对含有荧光染料的待测样进行照射激发，产生荧光信号，且激发光中，最大的波长小于红光波长；对产生的荧光信号通过光路传输后，收集并成像。

Description

提高测序分辨率的方法、测序装置和系统 技术领域
本发明涉及生物技术领域，尤其是涉及提高测序分辨率的方法、测序装置和系统。
背景技术
DNA测序技术是分子生物学相关研究中最常用的技术手段之一，同时，也推动了该领域的快速发展。人类基因组计划、转录组分析、微生物基因组重测序、单核苷酸的多态性(single nucleotide polymorphisms，SNP)分析等方也很大程度上促进了其他生物学领域的研究和发展。
每一代测序技术的更替都标志着生物学中基因芯片、数据分析、表面化学、生物工程等技术领域有了新的突破，主要体现在往测序成本降低，测序效率提高，使测序向着高通量、低成本、高安全性和商业化的方向发展。
尽管第一代DNA测序技术以其可达1000bp的测序读长、99.99％的高准确性帮助人们完成了大量的测序工作，但其测试速度慢、成本高、通量低等方面的不足，也致使其不能得到大众化、商业化的应用。
2005年，Roche公司发布的454测序系统标志着测序技术进入第二代DNA测序技术。第二代DNA测序技术又称高通量测序技术(high-throughput sequencing，HTS)，以低成本、99％以上的准确度，1次可对几百、几千个样本的几十万至几百万条DNA分子同时进行快速测序分析，代表技术有Roche公司的454、Illumina公司的Solexa、ABI公司的SOLID。
第二代DNA测序是对第一代测序技术的划时代变革的核心；目前，以二代DNA测序技术为主的测序系统采用检测光信号的方法以实现4种碱基 (A、T/U、C和G)的鉴别和区分，当然，前提是需要对碱基进行荧光标记。
比较常见的测序技术中，一般采用4种荧光染料分别对4种碱基进行标记；同时，也有采用2种荧光染料对4种碱基的鉴别和区分的测试手段，比如，Illumina公司开发的NextSeq测序系统和Mini-Seq测序系统，使用了基于双荧光染料的测序方法。无论是采用4种荧光染料还是2种荧光染料进行染色，都不可避免需要匹配激发光源。
目前市面上的测序技术中，大多是采用红绿激发光源。根据瑞利判据，光学系统的分辨率x＝0.61λ/NA。以Illumina和MGI-DNBSEQTM的红绿激发光测序技术为例，其红光激发的荧光中心波长约720nm，镜头NA为0.8，可以知道其理论分辨率约为550nm。由于镜头的NA受生产工艺影响，非常难以提高，导致红绿激发光测序技术实际很难在分辨率上有所突破，从而对测序通量与测序质量产生一定程度的限制。
从提高测序质量，或实现提供芯片表面DNA结合位点的密度达到提高测序的通量的目标出发，如何提高测序技术的分辨率将成为本领域技术人员亟需突破的技术问题。
有鉴于此，特提出本发明。
发明内容
本发明提供了一种提高测序分辨率的方法，以解决现有的测序技术中，红绿激发光测序技术实际很难在分辨率上突破，从而对测序通量与测序质量产生一定程度的限制的技术问题。
另一方面，本发明还提供了实现提高测序分辨率的方法的测序装置，通过测序装置以及方法的结合以实现测序分辨率的突破。再一方面，本发明还提供了一种测序系统，以实现应用。
一种提高测序分辨率的方法，包括以下步骤：
以至少一种激发光对含有荧光染料的待测样进行照射激发，产生荧光信号，且激发光中，最大的波长小于红光波长；
对产生的荧光信号通过光路传输后，收集并成像。
该方法中，其核心在于对激发光以及波长进行了特定了选择，至少一种激发光照射含有荧光染料的待测样后产生荧光信号；荧光信号通过光路传输后，收集并成像，进而实现测序的目的。由于存在最大的波长小于红光波长的激发光，据瑞利判据，分辨率x＝0.61λ/NA，本发明在不增加物料成本的情况下，通过使用波长更短的激发光替代原有的红绿光，提高了检测的分辨率，并进一步提高了测序的通量。本发明解决了现有的测序技术中，红绿激发光测序技术很难在分辨率上突破，从而对测序通量与测序质量产生一定程度的限制的技术问题。
应理解，在本发明中，“含有荧光染料的待测样”，在实际的应用场景中，可以有多种存在形式；只要满足荧光染料对特定理化特性的待测物进行标记的目的即可。更为具体的，“含有荧光染料的待测样”可以是通过一些结合酶使荧光染料被结合的核酸序列。
优选的，所述激发光以蓝光作为第一激发光，以绿光作为第二激发光。
需要进一步说明的是，第二激发光的光谱在一些情况下，还会涵盖有黄光的情况，因此第二激发光以绿光作为最优选，但不排除含有黄光的实施方案。
优选的，所述第一激发光的波长范围为440-500nm，所述第二激发光的波长范围为500-600nm。
更优选的，所述第一激发光的波长范围为470-490nm；所述第二激发光的波长范围为550-580nm。
优选的，两种激发光经过光纤耦合，共用同一光纤端口输出射至待测样，且所述光纤端口类型包括FC/PC或FC/APC或SMA中的一种。
采用第一激发光和第二激发光，由此，其可以激发出至少两类荧光信号，第一激发光为波长范围在440-500nm之间的蓝光，其波长小于传统的红绿光，从而会提高检测效率；优选的，采用FC/APC接口，FC/APC接口可减少回波损耗，进一步保障检测结果。
优选的，两种激发光的光斑的均匀性均大于85％；光纤长度均大于2000mm；光纤输出功率大于等于2000mw。
上述光源的参数等，作为提高测序分辨率的优选方式，并不是唯一具体的限定。
优选的，所述荧光染料包括由第一激发光激发的第一荧光染料和第二激发光激发的第二荧光染料；
第一荧光染料包括：Alexa Fluor 488、iF488、ATTO488、DyLight488、Hilyte488、FAM488或FITC488中的一种或多种；
第二荧光染料包括：CY3.5、AF568或ROX中的一种或多种。
上述优选的特定的荧光染料，和两种激发光的波长相对应，以尽可能地提高激发效率。另外，在本发明中，上述的第一荧光染料和第二荧光染料都可以直接购买获得。
优选的，所述第一荧光染料与所述第二荧光染料的用量比为3:1～4:1。
处在上述用量比的所述第一荧光染料与所述第二荧光染料，更容易高标准达到测序要求，在实际应用中，两种荧光染料的用量单位优选为μmol。
优选的，在光路传输中，还包括利用两类二向色镜将荧光信号与非荧光信号分别引导到不同的光路的步骤；其中一类二向色镜的透过窗口包括500-550nm和/或580-650nm；另外一类二向色镜的透过窗口至少为800nm。
二向色镜对不同波长的光以反射和透过的形式实现区分，且对特定波长范围的光，其反射率和透过率均大于95％。应理解，两类二向色镜的具体设置场景以其在光路中实现的效果进行选择，其设置位置并不是唯一的。
优选的，荧光信号在光路传输过程中，还包括经过透过窗口包括483nm-550nm或者580-700nm的滤光部件的操作。
优选的，所述待测样包括DNA，且设置在基材上，基材包括硅基、玻璃或者蓝宝石中的一种。
一种实现上述方法的测序装置，测序装置设置有能发射最大波长小于红光波长的激发光的激发光源组件；至少包括二向色镜、物镜的光路组件及成像组件；二向色镜设置在激发光源组件所发激发光后的光路上，对激发光和/或待测样经激发光激发后产生的荧光信号进行反射、透过或区分；物镜设置在荧光信号进入二向色镜的光路上；成像组件设置在将荧光信号被区分后的光路上，实现对不同荧光信号收集并成像。
本发明中，激发光经过二向色镜、物镜等光路组件之后，激发待测样产生荧光信号，荧光信号同样在二向色镜、物镜等光路组件组成的光路中，经过一系列反射、透过或区分后，最终被设置在光路末端的成像组件收集成像，进而达到检测目的。
本发明的测序装置通过硬件载体，以实现上述的提高测序分辨率的方法，该装置与传统的红绿激发光相比，光源组件发出的激发光的波长相对较短，据瑞利判据(分辨率x＝0.61λ/NA)，在NA不变的前提下，本发明突破了现有技术中的理论分辨率(550nm)，提高了测序质量或测序通量，在一定程度上使测序成本低廉化。
优选的，激发光源组件包括两个激发光源；其中一个激发光源激发出作为第一激发光的蓝光，另一个激发光源激发出作为第二激发光的绿光；
两个激发光源光纤耦合，并使两种激发光共用同一光纤端口输出。
具体应用中，激发光源组件一般需要发射至少两种波长不同，且均小于红光波长的激发光，以实现对待测样中的荧光染料分别进行激发，并产生两路荧光信号。当然，在本发明中，二向色镜的区分功能不仅仅指代其将荧光信号和其他光进行区分，当有两种激发光时，其区分功能还体现在对不同的两路荧光信号进行区分后被成像组件成像。
如前文所述，蓝光(第一激发光)的波长在440-500nm之间，优选的波长范围在470nm-490nm之间，可选部件包括：470nm的半导体激光器、473nm的半导体泵浦固态激光器(DPSS)激光器或者488nm的半导体激光器；
另一个光源(绿光)波长在500-600nm之间，优选的波长范围在550-580nm，如说556nm的固体激光器、561nm的固态半导体激光器(DPSS)激光器或者577nm的固体激光器。另外，光源还可以是LED。
两个激发光源光纤耦合，并使两种激发光共用同一光纤端口输出，使得装置整体上更为集成一体化，同时也便于控制。
优选的，光路组件还包括自动对焦部件；自动对焦部件通过发射与荧光信号波长不同的探测光；且探测光经过二向色镜、物镜以及待测样传输并原路返回，自动对焦部件根据返回后收集到的反馈信号，驱动物镜实现对焦。
通过自动对焦部件，发出与荧光信号波长不同的探测光，一般选红外光，波段在800-900nm之间，在优选的实施例中，其波段为830-860nm。自动对焦部件发出的探测光会依次经过二向色镜、物镜后照射至待测样，随后再原路返回至自动对焦部件，自动对焦部件根据收集到的反馈信号，驱动物镜移动到焦面位置，实现对焦功能。
优选的，所述二向色镜包括自上而下设置的第二二向色镜、第一二向色镜以及第三二向色镜；所述第三二向色镜与所述第二二向色镜光路相通且接 邻设置。
二向色镜通过反射/透射对波长不同的激发光、探测光或荧光信号进行区分；由于存在自上而下设置的第二二向色镜和第一二向色镜；这样，激发光先进入第一二向色镜后被反射，通过物镜后到达待测样，促使产生荧光信号。荧光信号通过物镜，穿过第一二向色镜后，被设置在其上部的第二二向色镜反射，由于第三二向色镜与第二二向色镜光路相通且接邻设置，由此，荧光信号被反射至第三二向色镜，并得以区分，并被成像组件收集成像。
优选的，成像组件包括设置在第三二向色镜不同输出光路上的筒镜以及相机；筒镜以及相机之间还设置有反射部件。
如采用两种激发光，则会产生两路荧光信号，经过第三二向色镜的区分后，分别以不同的光路输出，此时设置光路上的筒镜、反射部件以及相机可实现荧光信号的分别收集和成像。
优选的，激发光源组件与第一二向色镜之间的光路上设置有一准直透镜；准直透镜的自由端用于接入激发光源组件发出的激发光；另一端延伸至第一二向色镜内部。
准直透镜通过特殊的位置关系，其主要是作为激发光的光路载体，实现激发光从激发光源组件进入至第一二向色镜，同时第一二向色镜会将激发光反射至物镜并抵达待测样，以实现荧光信号的激发。
优选的，还包括安装底座；二向色镜以及成像组件设置在安装底座上；
和/或；当光路组件包括自动对焦部件时；对焦部件均设置在安装底座上；
物镜存在穿过安装底座，且用于和待测样相对的一端，其另一端与第一二向色镜和/或第二二向色镜相通。
安装底座作为二向色镜(第一二向色镜、第二二向色镜和第三二向色镜)、成像组件、自动对焦部件、准直透镜等部件的承载平台，实现各部件的安装 和固定，一体集约。
应理解，安装底座其作用一方面是对上述多种组件的固定和安装，同时还是整个装置与测序系统其他功能件连接固定的载体，因此，根据实际要求，其上可以设置多种形式的安装孔或者安装腔等。
优选的，相机上预设有用于进行数据交换的数据接口；
和/或；当光路组件包括自动对焦部件时，对焦部件预设有用于进行数据交换的数据接口。
数据接口是作为自动对焦部件或相机与其他功能模块(如电源，控制系统)进行直接连接的保障。易理解，诸如自动对焦部件或相机等需要和其他功能模块进行数据交换的动作，还可以通过蓝牙或者其他无线连接的方式实现。
优选的，还包括滤光部件，所述滤光部件设置在所述筒镜之前或之后的光路中；在一种优选的方案中，所述滤光部件设置在第三二向色镜与筒镜之间。
优选的，物镜上设置有多个光学镜片，且物镜为干式或浸式物镜，其放大倍数不低于16倍。
通过滤光部件(屏蔽由样品反射回来的激发光)、多个光学镜片以及放大倍数等的目的均是从硬件上尽可能保障检测效率。
一种测序系统，包括待测样和上述的测序装置；
待测样包括DNA序列和荧光染料，并与物镜相对；
当荧光染料被激发光源组件发出的激发光激发，产生荧光信号，并通过物镜进入二向色镜的光路。
优选的，还包括控制部件；控制部件与测序装置的一个或者多个组件连接，进行数据的交换、控制或显示。
综上，本发明提供的提高测序分辨率的方法、测序装置和系统，在具体的装置中，激发光经过二向色镜、物镜等光路组件之后，激发待测样产生荧光信号，荧光信号同样在二向色镜、物镜等光路组件组成的光路中，经过一系列反射、透过或区分后，最终被设置在光路末端的成像组件收集成像，进而达到检测目的。
方法中，其原理在于以至少一种激发光对含有荧光染料的待测样进行照射激发，并产生荧光信号，且激发光中，最大的波长小于红光波长；由于光源组件发出的激发光的波长相对较短，据瑞利判据，分辨率x＝0.61λ/NA，在NA不变的前提下，通过优选激发光源(蓝光)和配套的荧光染料，并且在整套装置特定的光路系统下，不增加物料成本，而通过使用波长更短的蓝光，提高光学系统的分辨率，并进一步提高了测序质量或测序通量，使测序成本低廉化。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明实施例提供的测序装置的原理示意图；
图2-3分别为本发明实施例提供的测序装置两个不同视角的图；
图4为图2和图3的主视图；
图5为图4的B-B向剖视图；
图6为本发明实施例提供的测序装置的底部视角的图；
图7为本发明实施例提供的二向色镜的透射光谱图；
图8为两种染料以及对应的滤光片光谱图；
图9为本发明实施例中不同染料在酶标仪测试的发射强度；
图10为本发明实施中两种方案的蓝色染料和绿色染料在测序成像系统上的蓝绿双通道测序散点图分析结果；
图11为本发明实施中基于蓝光的SE50的测序结果。
以上附图中，各标号所代表的部件列表如下：
光源组件—101；光路组件—102；成像组件—103；待测样—104；
物镜—201；自动对焦部件—202；第二二向色镜—203；第一二向色镜—204；第三二向色镜—205；筒镜—206；相机—207；安装架—208；反射部件—209；准直透镜—210；安装底座—211；数据接口—213。
具体实施方式
为了使本发明的上述以及其他特征和优点更加清楚，下面结合附图进一步描述本发明。应当理解，本文给出的具体实施例是出于向本领域技术人员解释的目的，仅是示例性的，而非限制性的。
在本发明的描述中，需要理解的是，术语“中心”、“纵向”、“横向”、“长度”、“宽度”、“厚度”、“上”、“下”、“前”、“后”、“左”、“右”、“竖直”、“水平”、“顶”、“底”、“内”、“外”、“顺时针”、“逆时针”、“轴向”、“径向”、“周向”等指示的方位或位置关系为基于附图所示的方位或位置关系，仅是为了便于描述本发明和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本发明的限制。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第 二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
在本发明中，除非另有明确的规定和限定，术语“安装”、“相连”、“连接”、“固定”等术语应做广义理解，例如，可以是固定连接，也可以是可拆卸连接，或成一体；可以是机械连接，也可以是电连接；可以是直接相连，也可以通过中间媒介间接相连，可以是两个元件内部的连通或两个元件的相互作用关系，除非另有明确的限定。对于本领域的普通技术人员而言，可以根据具体情况理解上述术语在本发明中的具体含义。
在本发明中，除非另有明确的规定和限定，第一特征在第二特征“上”或“下”可以是第一和第二特征直接接触，或第一和第二特征通过中间媒介间接接触。而且，第一特征在第二特征“之上”、“上方”和“上面”可是第一特征在第二特征正上方或斜上方，或仅仅表示第一特征水平高度高于第二特征。第一特征在第二特征“之下”、“下方”和“下面”可以是第一特征在第二特征正下方或斜下方，或仅仅表示第一特征水平高度小于第二特征。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
请参考图1，展示了本发明提供的测序装置的一个实施例；其中，箭头方向表示光线的前进方向；测序装置设置有能发射最大波长小于红光波长的 激发光的激发光源组件101；至少包括二向色镜、物镜的光路组件102及成像组件103；二向色镜设置在激发光源组件101所发激发光后的光路上，以对激发光和/或待测样104经激发光激发后产生的荧光信号进行反射、透过或区分；物镜设置在荧光信号进入二向色镜的光路上；成像组件103设置在将荧光信号区分后的光路上，实现对不同荧光信号收集并成像。
请参考图1-图11；本发明优选的实施例可以是在上述实施例的基础之上结合以下任一项或者多项的组合；
激发光源组件包括两个激发光源；一个激发光源激发出作为第一激发光的蓝光，另一个激发光源激发出作为第二激发光的绿光；两个激发光源光纤耦合，并使两种激发光共用同一光纤端口输出；光路组件中设置自动对焦部件202；自动对焦部件202可发射与荧光信号波长不同的探测光，探测光先后在二向色镜、物镜201传输后，照射到待测样，然后再原路返回，自动对焦部件202根据返回后收集到的反馈信号，驱动物镜201对焦；
二向色镜包括第一二向色镜204、第二二向色镜203和第三二向色镜205；第二二向色镜203和第一二向色镜204自上而下设置；第三二向色镜205与第二二向色镜203光路相通且接邻设置；
成像组件包括设置在第三二向色镜205不同输出光路上的筒镜206以及相机207；筒镜206以及相机207之间还设置有反射部件209；
激发光源组件与第一二向色镜204之间的光路上设置有一准直透镜210；准直透镜210的自由端用于接入激发光源组件发出的激发光；另一端延伸至能够与所述第一二向色镜204的内部相通。激发光从准直透镜210准直后通过第一二向色镜204反射到物镜201，然后聚焦到待测样上。
二向色镜、成像组件以及对焦部件均设置在一安装底座211上；物镜201的一端穿过安装底座211和待测样相对，另一端与第一二向色镜204相通； 相机207和自动对焦部件202上预设有用于进行数据交换的数据接口213。
筒镜206之前或之后的光路中还可以设置有滤光部件，以实现滤光效果，至于滤光部件设置在筒镜206之前还是之后的光路，并无严格限定，一般根据实际操作和装置的便捷程度选取。
在一些方案中，相机207和筒镜206之间设置滤光部件，在一些实施方式中，滤光部件设置在第三二向色镜205与筒镜206之间；物镜201上设置有多个光学镜片，且物镜201为干式或浸式物镜，浸式物镜还可以是浸水式或者浸油式物镜；物镜201的放大倍数不低于16倍。
请参考图2-图11，在本发明提供的测序装置的一具体实施方式中，包括光源、准直透镜210、第一二向色镜204、第二二向色镜203、第三二向色镜205、物镜201、自动对焦部件202和成像组件等；成像组件包括设置在第三二向色镜205不同输出光路上的筒镜206以及相机207，且筒镜206以及相机207之间设置有反射部件209。准直透镜210、第一二向色镜204、第二二向色镜203、第三二向色镜205、自动对焦部件202和成像组件均设置在安装底座211上，且第二二向色镜203和第三二向色镜205的光路相通，筒镜206以及相机207均成对设置，用以对两路荧光信号进行收集和成像。
在另一些方案中，筒镜206以及反射部件209之间还可以设置滤光部件，另外，在一些实施例中，滤光部件直接采用滤光片。
更为具体的，光源(图中未示出)包含两个激发光，一个是473nm的固态半导体激光器(DPSS)；另一个是561nm的固态半导体激光器(DPSS)。两个激发光在光源内部使用光纤耦合后，在同一根光纤端口输出，光纤端口类型，使用FC/APC接口，以减少回波损耗。
光源的示例参数，请参考下表1；
表1光源的参数
Figure PCTCN2021130332-appb-000001
激发光经过准直透镜210准直后，由第一二向色镜204引导进入到物镜201，并最终投射到待测样表面；准直透镜210的焦距，根据所需照明范围的大小来调整，在本实施例中，其焦距是2.9mm。
第一二向色镜204的可区分激发光和荧光信号，第一二向色镜204引导激发光进入物镜201中，同时允许从待测样发出的荧光信号透过。
图7展示了二向色镜的光谱，图中横坐标表示波长，纵坐标表示透过率。在本实施例中，第一二向色镜204对于473/561nm激发光谱的反射率在95％以上，对于荧光波段500-550nm、580-650nm的透过率在95％以上。
物镜201接收从第一二向色镜204反射过来的激发光，使其投射到待测样上。在本实施例中，物镜201包含了多个光学镜片，其放大倍数是16X，NA是0.8。
在其中一个实施方案中，待测样是一个生物样品，包含了DNA序列和荧光染料，荧光染料可通过一些结合酶使其与DNA结合；并且DNA设置在基材上，基材包括硅基、玻璃或者蓝宝石中的一种，优选硅基。上述的荧光染料有两个，一个由蓝光激发，称第一荧光染料，其发出的荧光信号称为第 一荧光信号；另一个由绿光激发，称第二荧光染料，其发出的荧光信号称为第二荧光信号。
荧光染料的选取与激发光的波长对应，以尽可能地提高激发效率，在本实施例中，以Alexa Fluor 488和ROX为例分别作为第一荧光染料和第二荧光染料。
图8展示了上述的两种染料以及对应的滤光部件的光谱图(滤光部件具体设置为滤光片，并置于相机207前面，具体在相机207和筒镜206之间，图中未示出)。在图8中，第一荧光染料Alexa Fluor 488的吸收谱601在第一激发波长473nm的激发效率约为0.4，第一滤光片603的透过窗口为500-550nm，第一荧光染料的发射谱602与第一滤光片603的结合效率约为0.67。
第二荧光染料ROX的吸收谱604在561nm的激发效率约为0.59，第二滤光片606的透过窗口为580-650nm，第二荧光染料的发射谱605和第二滤光片606的结合效率约0.59。本实施方案滤光片的设置，高程度地过滤激发光，透过荧光信号，达到了很好的区分效果。
需要说明的是，上述的第一荧光染料的发射谱602有部分光谱和第二滤光片606的光谱重合，这部分信号称为Crosstalk(信号串扰)。Crosstalk对于相机采集系统来说其实是相当于背景噪音，会影响系统的信噪比，在荧光染料组合选取的过程中应当尽量选取Crosstalk较少的组合。在本实施例中，通过荧光染料和滤光片的组合，Crosstalk约8％，达到了较高的信噪比，使得测序质量更加精准。
从待测样发出的两路荧光信号被物镜201收集，经过第一二向色镜204透射，到达第二二向色镜203。第二二向色镜203区分荧光信号和自动对焦部件202的探测信号。在本实施例中，第二二向色镜203是一个800nm的长 通滤光片。荧光信号的范围在450-750nm内。
自动对焦部件202发出的探测光是红外光，和荧光信号的波长不一样。具体地说，自动对焦部件202的探测信号波段在800-900nm之间，在优选为820-860nm。自动对焦部件202发出的探测光会依次经过第二二向色镜203和第一二向色镜204后到达待测样，然后从待测样原路返回自动对焦部件202。自动对焦部件202根据收集到的反馈信号，驱动物镜201移动到焦面位置，实现对焦功能。
进一步的，荧光信号从第二二向色镜203反射到第三二向色镜205，第三二向色镜205会把第一荧光信号和第二荧光信号分开，分别引导至两个相机207中，第三二向色镜205的光谱如图7所示。由蓝光和绿光激发的第一荧光信号和第二荧光信号从第三二向色镜205反射到筒镜206，最终被相机207收集。
本发明的其中一个优选的实施方案中，二向色镜包括第一二向色镜204和第二二向色镜203，且第二二向色镜203和第一二向色镜204自上而下设置。两个二向色镜都有可微调的结构，保证二向色镜位于合适的角度。
物镜201用螺纹固定在电动位移台上，结合自动对焦部件202使得物镜201锁定在待测样的焦面上。电动位移台由音圈电机驱动，以保证高精度要求，具体地说，其定位精度优于100nm，优选的，其精度优于20nm。
待测样表面附着DNA(小球状)和荧光染料。待测样置于二维电动平移台上，在自动对焦部件202与二维电动平移台的配合下，可以实现对待测样全面扫描观测。可选的，二维电动平移台由直线电机驱动，满足扫描的大行程和高精度定位要求。在本实施例中，其行程范围大于80mm*80mm，其定位精度优于200nm。
荧光信号会经过筒镜206和反射部件进入相机207。反射部件209用于 折叠光路，使结构更加紧凑，且反射部件209具有二维旋转可调结构，以保证光路对齐。
筒镜206安装在第三二向色镜205所在底座上，安装方式可以是螺纹固定、套筒固定等。为了消除环境光的影响，在筒镜206至相机207之间的空间还有一个遮光罩(图中未示出)。
相机207安装在具有五轴调节的底座上，通过安装架208与安装底座211设为一体，根据荧光信号实际入射角度和位置，可以调节相机207使得相机207靶面与入射光路对齐。更为具体的，相机207是一个CMOS类型的探测器，其像素数优于5000*5000像素。
在本发明的其他一些实施例中，为了消除色差，准直透镜210可以是一个双胶合透镜；另外，物镜201为浸式物镜，可以是浸水式或者是浸油式；波长为473nm的蓝光，可以选取的第一荧光染料还包括iF488、ATTO488、DyLight488、Hilyte488、FAM488或FITC488等；针对波长为561nm的绿光，可以选取的第二荧光染料有CY3.5、AF568等。
在本发明的另一些优选的实施例中，通过定制滤光片的谱线可以提高系统的效率，比如说第一滤光片603的窗口可以优化至483-550nm，第二滤光片606的窗口可以优化至580-700nm；同时，通过选取不同的染料组合和优化滤光片光谱，Crosstalk可以低于5％；由于Crosstalk非常低，因此具有很高的信噪比，测序质量精准。
在本发明的一些优选的实施例中，第一荧光信号波段为500-550nm，第二荧光信号波段为580-650nm，另外，对于二维电动平移台，作为优选，其行程范围大于100*100mm；在更为优选的实施例中，其定位精度优于50nm。
上述的任一方案中所提及的测序装置，再外加一包括DNA序列和荧光染料的待测样，并且使得待测样和物镜相对，即可构成更为复杂的测序系统， 荧光染料被所述激发光源组件发出的激发光激发，产生荧光信号，并通过所述物镜进入二向色镜的光路，进而实现测序效果。
在优选的实施例中，包括控制部件；控制部件与测序装置的一个或者多个组件连接，进行数据的交换、控制或显示。
本发明的实施例中还提供了一种提高测序分辨率的方法，包括以下步骤：
以至少一种激发光对含有荧光染料的待测样进行照射激发，产生荧光信号，且激发光中，最大的波长小于红光波长；
对产生的荧光信号通过光路传输后，收集并成像。
在一些具体的方案中，激发光以波长范围在440-500nm的蓝光为第一激发光；以及波长范围为500-600nm的绿光为第二激发光；两种激发光经过光纤耦合，共用同一光纤端口输出射至待测样，且所述光纤端口类型包括FC/PC或FC/APC或SMA中的一种。
在更为具体的一个方案中，第一激发光的波长范围为470-490nm；第二激发光的波长范围为550-580nm；两种激发光的光斑的均匀性均大于85％；光纤长度均大于2000mm；光纤输出功率大约等于2000mw。在更为优选的方案中，第一激发光为蓝光，波长为470nm、473nm或488nm；第二激发光为绿光，波长为556nm、561nm或者577nm。
需要说明的是，根据分析需要，蓝光波长可扩展到绿光中，绿光波长可扩展到黄色和红色中。
在另一些具体的方案中，第一激发光为蓝光，波长可以简单地具有上限，例如470nm、473nm或488nm；第二激发光为绿光，波长可以简单地具有下限，例如556nm、561nm或者577nm。
基于上文中提供的测序装置，结合上述的方法，本发明提供的又一具体的实施方法包括：采用激发光源组件发射两种波长互不相同，且小于红光波 长的激发光；激发光通过第一二向色镜204的反射，经过物镜201，并照射至待测样(含有两种荧光染料)，并产生两路荧光信号；两路荧光信号在第一二向色镜204进行透过，被第二二向色镜203进行反射，被第三二向色镜205进行区分后，采用成像组件收集并成像。
和上述的装置相对应，在一些实施方案中，在光路传输中还包括利用两类二向色镜将荧光信号与非荧光信号分别引导到不同的光路的步骤；其中一类二向色镜的透过窗口包括500-550nm和/或580-650nm；另外一类二向色镜的透过窗口至少为800nm。
结合上述装置的一些实施方式中，荧光信号在光路传输过程中，还包括经过透过窗口包括483nm-550nm或者580-700nm的滤光部件的操作。
在本发明的优选实施方案中，关于荧光染料，进行了测试优化。具体的，根据光源与滤光片，筛选吸收和发射波长与之相匹配的荧光染料，针对第一激发光(蓝光)，可用染料包括Alexa Fluor 488、iF488、ATTO488、DyLight488、Hilyte488、FAM488、FITC488等，以下称第一染料；针对第二激发光(绿光)，可以选用AF568、DNBSEQTM技术中的ROX染料等，以下称第二染料。
以Alexa Fluor 488为例，对碱基进行第一染料的标记，将所选第一染料分别对A、C、G及T四种碱基进行标记然后使用酶标仪测验染料性能。
图9展示了不同染料在酶标仪测试的发射强度，结果显示，由于荧光染料本身的理化性质，在标记反应中标记产物的得率不同，发光强度也不同。Alexa Fluor 488标记G碱基和A碱基时发光效果最差。在蓝光cPAS方案中，将选择C和T碱基进行第一染料的标记，A和G标记第二染料。
作为以上的优选，第一染料为Alexa Fluor 488；第二染料为ROX。
应理解，cPAS为本领域公知的技术用语，是联合探针锚定聚合 (Combinatorial Probe Anchor Synthesis，cPAS)的简写。
在双色测序方案中，染料的碱基标记方案可以根据实际需求进行不同的组合，例如可以碱基C上标记两种染料，也可以在碱基T上标记两种染料，具体如下表2所示：
表2染料标记方案
方案一 方案二
A-第二染料 A-第二染料
C-第二染料 C-第一染料
C-第一染料 T-第二染料
T-第一染料 T-第一染料
G-无标记 G-无标记
图10展示了两种方案的第一染料和第二染料在测序成像系统上的蓝绿双通道测序散点图分析结果，其中，(a)为方案一中的第一染料和第二染料在测序成像系统上的蓝绿双通道测序散点图分析结果；(b)为方案二中的第一染料和第二染料在测序成像系统上的蓝绿双通道测序散点图分析结果。横向坐标为蓝光通道，纵向坐标为绿光通道。
通过图10可以看出两种方案的第一染料和第二染料在测序成像系统上的蓝绿双通道测序散点结果，均呈现出了4种碱基彼此之间串联少，而且区分明显的效果。
进一步的，对上述cPAS方案中双染料标记的碱基比例进行优化测试，具体的，采用上述表2中方案一的标记方式，利用两种染料标记不同的碱基，并且设定第一染料和第二染料的不同用量比例。测试的比例与起始Q30情况如下表3所示：
表3测试的比例与起始Q30情况统计表
  1 2 3 4 5 6 7 8 9
第一染料 50％ 60％ 65％ 70％ 80％ 78％ 75％ 75％ 75％
第二染料 50％ 40％ 35％ 30％ 20％ 22％ 25％ 25％ 25％
Q30(％) 57.4 59.8 62.1 68.0 70.3 73.1 80.9 81.7 84.74
在本实验中，第一染料和第二染料的总共用量为1μmol，1-9的组合中，分别示意了第一染料和第二染料不同用量的情况；比如在第1种比例中，第一染料和第二染料的用量分别为0.5μmol，其余的组合依据两种染料的比例确定具体的用量，在此不做一一赘述。
根据起始Q30的结果，第一染料/第二染料的使用量比值的范围固定在3～4之间；根据测试结果，选择第一染料/第二染料＝3的比例组合在上述优选实施例的测序装置上进行SE50测试。
结果如图11所示，当前基于蓝光的SE50的测序水平mapping率在94～95％，在15ms和20ms的曝光条件下，Q30达到测序要求。
综上所述，本发明中，在NA不变的前提下，通过对测序装置进行特定的光路结构设置，选择特定的激发光源(蓝光和绿光)和配套的荧光染料，实现了在不增加物料成本的情况下，通过使用波长更短的蓝绿光激发方式(第一激发光为蓝光，第二激发光为绿光)提高光学系统的分辨率，进而提高了测序质量或测序通量，使测序成本低廉化。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (23)

  1. 一种提高测序分辨率的方法，其特征在于，包括以下步骤：
    以至少一种激发光对含有荧光染料的待测样进行照射激发，产生荧光信号，且激发光中，最大的波长小于红光波长；
    对产生的荧光信号通过光路传输后，收集并成像。
  2. 根据权利要求1所述的提高测序分辨率的方法，其特征在于，所述激发光以蓝光作为第一激发光，以绿光作为第二激发光。
  3. 根据权利要求2所述的提高测序分辨率的方法，其特征在于，所述第一激发光的波长范围在440-500nm，所述第二激发光的波长范围为500-600nm。
  4. 根据权利要求2所述的提高测序分辨率的方法，其特征在于，所述第一激发光的波长范围为470-490nm；所述第二激发光的波长范围为550-580nm。
  5. 根据权利要求2所述的提高测序分辨率的方法，其特征在于，两种激发光的光斑的均匀性均大于85％；光纤长度均大于2000mm；光纤输出功率大于等于2000mw。
  6. 根据权利要求2所述的提高测序分辨率的方法，其特征在于，所述荧光染料包括由第一激发光激发的第一荧光染料和第二激发光激发的第二荧光染料；
    第一荧光染料包括：Alexa Fluor 488、iF488、ATTO488、DyLight488、Hilyte488、FAM488或FITC488中的一种或多种；
    第二荧光染料包括：CY3.5、AF568或ROX中的一种或多种。
  7. 根据权利要求6所述的提高测序分辨率的方法，其特征在于，所述第一荧光染料与所述第二荧光染料的用量比为3:1～4:1。
  8. 根据权利要求2所述的提高测序分辨率的方法，其特征在于，在光路传输中，还包括利用两类二向色镜将荧光信号与非荧光信号分别引导到不同的光路的步骤；其中一类二向色镜的透过窗口包括500-550nm和/或580-650nm；另外一类二向色镜的透过窗口至少为800nm。
  9. 根据权利要求2所述的提高测序分辨率的方法，其特征在于，荧光信号在光路传输过程中，还包括经过透过窗口包括483nm-550nm或者580-700nm的滤光部件的操作。
  10. 根据权利要求1-9任一项所述的提高测序分辨率的方法，其特征在于，所述待测样包括DNA，且设置在基材上，基材包括硅基、玻璃或者蓝宝石中的一种。
  11. 一种实现权利要求1-10任一项所述的方法的测序装置，其特征在于，所述测序装置设置有能发射最大波长小于红光波长的激发光的激发光源组件；至少包括二向色镜、物镜的光路组件及成像组件；
    所述二向色镜设置在所述激发光源组件所发激发光后的光路上，对激发光和/或待测样经激发光激发后产生的荧光信号进行反射、透过或区分；所述物镜设置在荧光信号进入二向色镜的光路上；
    所述成像组件设置在将荧光信号被区分后的光路上，实现对不同荧光信号收集并成像。
  12. 根据权利要求11所述测序装置，其特征在于，所述激发光源组件包括两个激发光源；其中一个激发光源激发出作为第一激发光的蓝光，另一个激发光源激发出作为第二激发光的绿光；
    两个所述激发光源光纤耦合，并使两种激发光共用同一光纤端口输出。
  13. 根据权利要求11所述的测序装置，其特征在于，所述光路组件还包括自动对焦部件；所述自动对焦部件通过发射与所述荧光信号波长不同的探测光；且所述探测光在所述二向色镜、所述物镜以及待测样传输并原路返回，所述自动对焦部件根据返回后收集到的反馈信号，驱动所述物镜实现对焦。
  14. 根据权利要求11-13任一项所述的测序装置，其特征在于，所述二向色镜包括自上而下设置的第二二向色镜、第一二向色镜；以及
    第三二向色镜，所述第三二向色镜与所述第二二向色镜光路相通且接邻设置。
  15. 根据权利要求14所述的测序装置，其特征在于，所述成像组件包括设置在所述第三二向色镜不同输出光路上的筒镜以及相机；
    所述筒镜以及所述相机之间还设置有反射部件。
  16. 根据权利要求14所述的测序装置，其特征在于，所述激发光源组件与所述第一二向色镜之间的光路上设置有一准直透镜；所述准直透镜的自由 端用于接入所述激发光源组件发出的激发光；另一端延伸至所述第一二向色镜的内部。
  17. 根据权利要求14所述的测序装置，其特征在于，还包括安装底座；所述二向色镜以及所述成像组件设置在所述安装底座上；
    和/或；当所述光路组件包括自动对焦部件时；所述自动对焦部件均设置在所述安装底座上；
    所述物镜存在穿过所述安装底座，且用于和待测样相对的一端，其另一端与所述第一二向色镜和/或所述第二二向色镜色镜相通。
  18. 根据权利要求15所述的测序装置，其特征在于，所述相机上预设有用于进行数据交换的数据接口；
    和/或；当所述光路组件包括自动对焦部件时，所述自动对焦部件预设有用于进行数据交换的数据接口。
  19. 根据权利要求15所述的测序装置，其特征在于，还包括滤光部件，所述滤光部件设置在所述筒镜之前或之后的光路中。
  20. 根据权利要求19所述的测序装置，其特征在于，所述滤光部件设置在第三二向色镜与筒镜之间。
  21. 根据权利要求14所述的测序装置，其特征在于，所述物镜上设置有多个光学镜片，且所述物镜为干式或浸式物镜，其放大倍数不低于16倍。
  22. 一种测序系统，其特征在于，包括待测样和如权利要求11-21任一项所述的测序装置；
    所述待测样包括DNA序列和荧光染料，并与所述物镜相对；
    当荧光染料被所述激发光源组件发出的激发光激发，产生荧光信号，并通过所述物镜进入二向色镜的光路。
  23. 根据权利要求22所述的测序系统，其特在于，还包括控制部件；所述控制部件与所述测序装置的一个或者多个组件连接，进行数据的交换、控制或显示。
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CN108449958A (zh) * 2015-09-01 2018-08-24 凯杰器械有限公司 高通量核酸测序系统中用于颜色探测的系统和方法
CN205368376U (zh) * 2016-02-03 2016-07-06 深圳华大基因研究院 基因测序仪光学系统及其对焦系统
CN206607236U (zh) * 2016-11-29 2017-11-03 深圳华大智造科技有限公司 一种用于基因测序仪的光学系统
JP2019012082A (ja) * 2018-09-27 2019-01-24 株式会社日立ハイテクノロジーズ 核酸分析装置、および核酸分析方法
CN111323397A (zh) * 2018-12-14 2020-06-23 深圳华大生命科学研究院 光学成像系统、成像检测系统与方法及基因测序方法
CN109504752A (zh) * 2018-12-29 2019-03-22 广州市锐博生物科技有限公司 混合波长激光系统及测序仪
CN109682784A (zh) * 2018-12-29 2019-04-26 广州市锐博生物科技有限公司 生成彩色图像的系统及高通量测序仪

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