WO2020119226A1 - 光学系统、调校光学系统的方法和测序系统 - Google Patents

光学系统、调校光学系统的方法和测序系统 Download PDF

Info

Publication number
WO2020119226A1
WO2020119226A1 PCT/CN2019/109045 CN2019109045W WO2020119226A1 WO 2020119226 A1 WO2020119226 A1 WO 2020119226A1 CN 2019109045 W CN2019109045 W CN 2019109045W WO 2020119226 A1 WO2020119226 A1 WO 2020119226A1
Authority
WO
WIPO (PCT)
Prior art keywords
lens
optical system
light beam
camera
light
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2019/109045
Other languages
English (en)
French (fr)
Inventor
齐继芳
周志良
姜泽飞
颜钦
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genemind Biosciences Co Ltd
Original Assignee
Genemind Biosciences Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN201822089550.1U external-priority patent/CN209759461U/zh
Priority claimed from CN201811516793.7A external-priority patent/CN111308726A/zh
Application filed by Genemind Biosciences Co Ltd filed Critical Genemind Biosciences Co Ltd
Priority to EP19896106.2A priority Critical patent/EP3896146A4/en
Priority to US17/311,179 priority patent/US11874453B2/en
Publication of WO2020119226A1 publication Critical patent/WO2020119226A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/361—Optical details, e.g. image relay to the camera or image sensor
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/10—Beam splitting or combining systems
    • G02B27/14—Beam splitting or combining systems operating by reflection only
    • G02B27/141—Beam splitting or combining systems operating by reflection only using dichroic mirrors
    • 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
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/02—Objectives
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/24—Base structure
    • G02B21/241—Devices for focusing
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/24—Base structure
    • G02B21/241—Devices for focusing
    • G02B21/245—Devices for focusing using auxiliary sources, detectors
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/10—Beam splitting or combining systems
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/30—Collimators
    • 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
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6421—Measuring at two or more wavelengths
    • 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
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels
    • 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
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N2021/6463—Optics
    • G01N2021/6478—Special lenses
    • 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
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence

Definitions

  • the invention relates to the technical field of optical detection, in particular to an optical system, a method for adjusting the optical system, and a sequencing system.
  • the optical imaging system can achieve sub-micron resolution and is a powerful tool for studying samples. For example, in the field of biological sample detection, some optically detectable groups/compounds/structures are used to label the biomolecules to be detected, and an optical imaging system is used to collect signals from these labels to achieve the location or location of the biomolecules to be detected. Detection.
  • a nucleotide modification with an optically detectable label such as a fluorescent molecule
  • the type of base at the corresponding position of the template is determined by exciting fluorescent molecules to emit light, thereby realizing sequencing.
  • This kind of sequencing platform hopes that the optical imaging system it contains can occupy a small space, is not easily affected by other components, and can closely cooperate with other related components to achieve stable, continuous and fast signal acquisition and output clear images. Design and build an optical imaging system that can achieve the above goals, which needs to be solved or improved.
  • the invention provides an optical system for gene sequencing, a method for adjusting the optical system, and a sequencing system.
  • An optical system includes a first light source, a first lens, and a beam splitting module.
  • the beam splitting module includes a first beam splitter, a second lens, a first camera, and a second camera.
  • the first The lens is used to receive the first light beam from the first light source and collimate the first light beam to the sample, and to receive the light beam from the sample and collimate the light beam
  • the second lens is used
  • the first beam splitter is used to divide the focused beam from the second lens into a second beam and a second beam Three beams
  • the first camera is used to receive the second beam
  • the second camera is used to receive the third beam.
  • the above-mentioned optical system is based on the above-mentioned split optical path design, which includes passing the light beam through the second lens and then passing through the beam splitter, which can shorten the total optical path length of the optical system, reduce the required space, and contribute to the miniaturization of the optical system.
  • the sample has an optically detectable label, such as one or more fluorescent molecular labels.
  • Laser is a commonly used fluorescent excitation light source.
  • a variety of fluorescent molecular markers can be excited by laser to emit fluorescence at different wavelengths.
  • the inventor also built and designed another optical system, which uses the microscope to split the light in the parallel light path (that is, between the objective lens and the barrel lens).
  • the fluorescence is split in the microscope parallel light path, that is, the fluorescence passes through the objective lens ( Lens)
  • the beam split by the beam splitter 90 forms a plurality of beam splitting light paths.
  • each beam splitting light path each light beam is focused to the camera 91 or 92 through the barrel lens 93 or 94
  • the inventor's comparison test found that compared with the latter, the optical system including the splitting optical path for splitting light in the converging optical path has a small total optical path length and a small required space, including fewer required optical components, which is beneficial to the miniaturization of the optical system , Conducive to the industrialization of the optical system.
  • a sequencing system includes a platform and the optical system of any of the above embodiments, and the platform is used to carry samples.
  • the above description of the technical characteristics and advantages of the optical system in any embodiment of the present invention is also applicable to the sequencing system of this embodiment, and will not be repeated here.
  • An embodiment of the present invention provides a method for adjusting an optical system.
  • the optical system includes a beam splitting module.
  • the beam splitting module includes a second lens, a first beam splitter, a first camera, and a second camera.
  • the second The lens, the first beam splitter and the first camera are arranged in sequence along the optical axis of the second lens, the method includes: using a collimated light pipe to emit a collimated light beam to the second lens, the collimated light
  • the tube includes a reticle, the reticle includes one or more patterns, the collimated light beam is condensed to the first beam splitter through the second lens, and is divided into second through the first beam splitter Beam and third beam, the first camera receives the second beam to obtain a first image of the pattern, the second camera receives the third beam and obtains a second image of the pattern; adjust the The angle and/or position of the first camera and/or the second camera to make the contrast of the first image and the second image consistent.
  • the method of adjusting the optical system uses the splitting module as a single module of the entire optical system to individually debug, which can reduce the limitation of the debugging space of the whole machine, can easily and conveniently realize the perpendicularity of multiple cameras and the optical axis, and realize the inclusion of splitting Adjustment of the optical system of the optical path.
  • FIG. 1 is a schematic structural diagram of an optical system according to an embodiment of the present invention.
  • FIG. 2 is a schematic structural diagram of a first light source according to an embodiment of the present invention.
  • FIG. 3 is another schematic structural diagram of an optical system according to an embodiment of the present invention.
  • FIG. 4 is a schematic structural diagram of a second light source according to an embodiment of the present invention.
  • FIG. 5 is a schematic diagram of the simulation result of the spot size of the imaging beam when the optical system of the embodiment of the present invention does not include the first beam splitter;
  • FIG. 6 is a schematic diagram of the simulation result of the size of the spot of the imaging beam including the first beam splitter of the optical system according to the embodiment of the present invention
  • FIG. 7 is a schematic diagram of the simulation result of the size of the spot of the imaging beam introduced into the compensation lens of the optical system according to the embodiment of the present invention.
  • FIG. 8 is a schematic partial perspective view of an optical system according to an embodiment of the present invention.
  • FIG. 9 is a front view of the optical system of FIG. 8.
  • FIG. 10 is a plan view of the optical system of FIG. 8;
  • FIG. 11 is a schematic structural diagram of a spectroscopic module according to an embodiment of the present invention during debugging
  • FIG. 12 is a schematic structural view of a collimator according to an embodiment of the present invention.
  • FIG. 13 is a schematic diagram of a pattern of a resolution line pair reticle according to an embodiment of the present invention.
  • optical splitting module 14 is another schematic structural diagram of the optical splitting module according to the embodiment of the present invention during debugging
  • 15 is a schematic structural diagram of an optical system of the related art
  • 16 is a perspective schematic diagram of a sequencing system according to an embodiment of the present invention.
  • first and second are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance, order, or implicitly indicating the number of technical features indicated. Thus, features defined as “first” and “second” may explicitly or implicitly include one or more of the features. In the description of the present invention, unless otherwise defined, “plurality” means two or more, unless specifically defined otherwise.
  • connection should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be The mechanical connection can also be an electrical connection or can communicate with each other; it can be directly connected or indirectly connected through an intermediary, it can be the connection between two components or the interaction between two components.
  • an optical system 10 includes a first light source 12, a first lens 16, and a beam splitting module 40.
  • the beam splitting module 40 includes a first beam splitter 14, a second lens 18, and a first Camera 20 and second camera 22.
  • the first lens 16 is used to receive the first light beam from the first light source 12 and collimate the light beam onto the sample 24, and to receive the light beam from the sample 24 and collimate the light beam.
  • the second lens 18 is used to focus the collimated light beam from the first lens 16 to the first camera 20 and the second camera 22.
  • the first beam splitter 14 is used to split the focused beam from the second lens 18 into a second beam and a third beam.
  • the first camera 20 is used to receive the second light beam.
  • the second camera 22 is used to receive the third light beam.
  • the beam splitter 14 which can reduce the use of optical elements and the length of the split optical path is small, so that the total optical path length of the optical system becomes Shortness is conducive to miniaturization of the optical system 10 and industrialization.
  • the sample 24 may be a nucleic acid sample to be tested, and the nucleic acid sample to be tested may be placed in a reaction device, such as a chip.
  • the first light source 12 may be a laser light source.
  • the chip includes a substrate, a channel is provided on the substrate, and a glass is provided on the substrate.
  • the nucleic acid, enzyme, and fluorescently labeled nucleoside to be tested Acid reagents or solutions are mixed in the channel to react, and then the first light source 12 emits laser light through the first lens 16 to enter a specific field of view of the chip, the fluorescent group of the field of view is excited to emit fluorescence, and the fluorescence passes through the first lens 16 and the second
  • the lens 18 focuses on the first beam splitter 14, and the first beam splitter 14 splits the fluorescent beam into a second beam and a third beam.
  • the first camera 20 receives the second beam, and the second camera 22 receives the third beam.
  • the first image and the second image of the field of view are mixed in the channel to react, and then the first light source 12 emits laser light through the first lens 16 to enter a specific field of view of the chip, the fluorescent group of the field of view is excited to emit fluorescence, and the fluorescence passes through the first lens 16 and the second
  • the lens 18 focuses on the first beam splitter 14, and the first beam splitter
  • the first light source 12 may include a first light emitter 13 and a third lens 15, and the first light beam is a light beam emitted by the first light emitter 13 passing through the third lens 15 After the collimated light beam, the first light beam is focused on the back focal plane of the first lens 16 through the fourth lens 17, and then collimated and incident on the sample 40 through the first lens 16.
  • the first light source 12 further includes an optical fiber coupler, such as a single-mode optical coupler.
  • the optical system 10 is a total internal reflection optical system, and the collimated light beam (parallel light beam) passing through the first lens 16 is incident on the chip surface at a angle greater than the critical angle, total internal reflection occurs, and evanescence occurs on the lower surface of the chip glass Field (evanescent wave). The fluorescence emitted by the fluorescent molecules in the evanescent field is excited and received by the first lens 16.
  • the light beam from the sample 24 received by the first lens 16 is the light beam emitted by the sample 24.
  • the image sensors of the first camera 20 and the second camera 22 may use CCD or CMOS.
  • the types of image sensors used by the first camera 20 and the second camera 22 are the same, for example, both are CCD or both are CMOS.
  • the first beam splitter 14 may be a dichroic mirror.
  • the second beam is the transmitted beam of the first beam splitter 14 and the third beam is the reflected beam of the first beam splitter 14.
  • the first camera 20 and the second camera 22 are set at 90 degrees or 270 degrees. In this way, it is convenient to configure multiple cameras of the first camera 20 and the second camera 22 into the optical system in a limited space.
  • the first beam splitter 14 has a first reflecting surface 26, and the angle between the first reflecting surface 26 and the horizontal plane is 45 degrees, and is incident on a portion of the first reflecting surface 26 in the horizontal direction The light beam is reflected and turned 90 degrees to reach the second camera 22, and another part of the light beam incident on the first reflection surface 26 in the horizontal direction passes through the first reflection surface 26 and enters the first camera 20.
  • the first camera 20 and the second camera 22 are arranged at 90 degrees clockwise and 270 degrees counterclockwise.
  • the sample is provided with two fluorescent labels, such as Cy3 and Atto647N, and the emission bands of the two fluorescent molecules are 550-620nm and 650-750nm, respectively (the peaks are approximately 564nm and 670nm, respectively);
  • the device 14 is a dichroic mirror.
  • the dichroic mirror has a high transmittance for light with a wavelength of 550-620 nm and a high reflectance for light above 650 nm.
  • the so-called fluorescent-labeled nucleotide reagents include four types of nucleotide reagents, A, T, C, and G. Different kinds of nucleotide reagents can be separately contained in different containers. In one example, the four nucleotides carry the same fluorescent label.
  • each round of sequencing reaction includes four base extension reactions, and the four base extension reactions are the sequential addition of the four nucleotides. And get the corresponding image.
  • the four types of nucleotides are respectively provided with a first fluorescent label and a second fluorescent label, and the first fluorescent label and the second fluorescent label can be excited to emit different fluorescence.
  • Two-color sequencing each round of sequencing reaction includes two base extension reactions.
  • the nucleic acid, enzyme and the first fluorescent label and the second fluorescent label are tested
  • the two nucleotide reagents or solutions are mixed in the channel to react.
  • the first light source 12 simultaneously emits the first laser light and the second laser light through the first lens 16 to enter a specific field of view of the chip.
  • the two fluorescent marks are excited by the first laser light and the second laser light, respectively, to emit the first fluorescence light and the second fluorescence light.
  • the first fluorescence light and the second fluorescence light are condensed to the first beam splitter 14 by the first lens 16 and the second lens 18 (two-way Chromatic mirror), the dichroic mirror separates the first and second fluorescent lights that converge.
  • the first fluorescent light is focused on the image plane of the first camera 20, and the second fluorescent light is focused on the image plane of the second camera 22.
  • the first image and the second image formed by the first fluorescence and the second fluorescence of the visual field. Based on the sequence of nucleotide addition and the information of the first image and the second image of different sequencing reactions, base recognition/sequencing is realized.
  • each round of sequencing reaction includes a base extension reaction, when sequencing using the sequencing system containing the optical system 10, under certain conditions, the nucleic acid to be tested, the enzyme and the above four nucleotide reagents Or a solution or the like mixed in the channel to react, the first light source 12 simultaneously emits the first laser light and the second laser light into the specific field of view of the chip through the first lens 16, and the fluorescent marks of the field of view are excited and emitted by the first laser light and the second laser light, respectively Fluorescence, which is condensed by the first lens 16 and the second lens 18 to the first beam splitter 14 (dichroic mirror), the dichroic mirror divides the fluorescence into fluorescence from the fluorescent mark a and from the fluorescent
  • the first lens 16 includes one or more lenses
  • the second lens 18 includes one or more lenses.
  • one or more lenses of the first lens 16 constitute an objective lens
  • one or more lenses of the second lens 18 constitute a tube lens.
  • the first lens 16 includes one or more lenses
  • or the second lens 18 includes one or more lenses.
  • the optical system 10 includes a second beam splitter 28 for receiving the first beam from the first light source 12 and turning the first beam to the first lens 16, so that the first A light beam is merged into the optical path (imaging optical path) where the optical axis of the first lens is located.
  • the arrangement of the second beam splitter 28 allows the first light source 12 to be located outside the optical path where the optical axis of the first lens 16 is located, enabling compact and reasonable arrangement of the components of the optical system 10, which is beneficial to the miniaturization of the optical system 10, Conducive to industrial applications.
  • the second beam splitter 28 is used to rotate the first light beam by 90 degrees.
  • the position for facilitating the configuration of the first light source 12 includes the relative position of the elements contained therein.
  • the optical system 10 includes a third beam splitter 30 and an autofocus module 32.
  • the autofocus module 32 is used to emit a fourth beam and to receive the fourth beam reflected back by the sample 40, and the third beam splitter 30 is used to receive the fourth beam and redirect the fourth beam to the first lens 16, It is also used to receive the fourth light beam reflected by the sample 40 and redirect the fourth light beam to the autofocus module 32.
  • the auto-focus module 32 can be used for focusing, and the optical system 10 can be used for image collection.
  • the autofocus module 32 includes a second light source 34 and a receiver 36.
  • the second light source 34 is used to emit a fourth light beam to the third beam splitter 30, and the receiver 36 is used to receive the collimated first lens 16 Four beams.
  • the second light source 34 may be an infrared light source.
  • the receiver 36 may be a photodiode.
  • the second light source 34 emits a fourth light beam, which is turned to the first lens 16 via the third beam splitter 30, and the fourth light beam is condensed to the sample 24 via the first lens 16.
  • the fourth light beam reflected by the sample 24 is collimated by the first lens and enters the third beam splitter 30.
  • the platform carrying the sample can be moved to move the sample 24 closer to or farther away from the first lens 16, thereby achieving focusing.
  • the receiver 36 includes a sensor, such as a two-dimensional PSD sensor, and the second light source 34 includes an LED light source and a mask in front of the LED light source; the light emitted by the LED light source is irradiated onto the pickling mold to obtain a specific pattern of light spots, the The spot of the specific pattern is transferred to the first lens 16 by the third beam splitter 30 to converge on the sample 24, and the spot reflected by the sample 24 finally reaches the sensor; the autofocus module 32 further includes a signal processing module, and the sensor is connected to the The signal processing module obtains the information of the light spot through the signal processing module. Further, the autofocus module 32 further includes a signal output module for outputting changes in the spot information, so that the platform carrying the sample moves the sample to the object plane of the imaging optical path (for example, the fluorescent optical path).
  • the second light source 34 includes an LED light source and a mask in front of the LED light source
  • the light emitted by the LED light source is irradiated onto the pickling mold to obtain a
  • the second light source 34 includes a second light emitter 35 and a fifth lens 37.
  • the fourth light beam is a collimated light beam after the light beam emitted by the second light emitter 38 passes through the fifth lens 37
  • the fourth light beam reflected by the sample 40 is condensed by the fifth lens 37 to the receiver 36.
  • the second light beam is a light beam transmitted from the second lens 18 through the first beam splitter 14, the optical system 10 includes a compensation lens 38, and the compensation lens 38 is located in the first beam splitter Between 14 and the first camera 20, a compensation lens 38 is used to compensate for astigmatism caused by the second light beam.
  • the imaging simulation results are shown in Figures 5 and 6, respectively. It can be seen that the beams (diffused spots) formed by the beams from the same field of view after passing through the second lens 18 and the first beam splitter 14 and only passing through the second The light spot (diffused spot) formed by the lens 18 has significantly increased astigmatism. For example, the spot sizes at coordinates (0, 0), (0, 3.250), and (0, 6.500) in FIG.
  • RMS radius mean square root radius
  • the root mean square radius is an important radius parameter. It is the coordinate of each point of the diffuse spot, the reference center point, the coordinate sum of the squares, divided by the number of points, and then the square root value. The radius of this value can reflect a typical dispersion.
  • the spot size reflects the actual spot size of this system quantitatively.
  • GEO RADIUS GEO radius
  • the inventor introduces the compensation lens 38 at any position between the second lens 18 and the first camera, hoping to compensate the astigmatism caused by imaging after the light beam is transmitted.
  • the compensating lens 38 please refer to FIG. 7, the spot formed by the beam from the same field of view, the spot size of FIG. 7 is significantly smaller than the spot size of FIG. 6, and from the RMS radius, at the same coordinate, the The spot size is close to or even smaller than that of FIG. 5.
  • the compensation lens 38 may use a parallel flat plate or a dichroic mirror. In the embodiment of FIGS. 8-10, the compensation lens 38 uses a dichroic mirror.
  • the angle T between the compensation lens 38 and a plane P is 45 degrees.
  • the first beam splitter 14 is perpendicular to the plane P.
  • the plane P is jointly defined by the optical axis A of the second beam and the optical axis B of the third beam.
  • Embodiments of the present invention also provide a method for adjusting an optical system.
  • the optical system includes a beam splitting module 40.
  • the beam splitting module 40 includes a second lens 18, a first beam splitter 14, a first camera 20, and a The two cameras 22, the second lens 18, the first beam splitter 14, and the first camera 20 are arranged in sequence along the optical axis of the second lens 18.
  • the method includes: using the collimator 50 to emit a collimated light beam to the second lens 18, the parallel light
  • the tube 50 includes a reticle 42 including one or more patterns.
  • the collimated light beam is condensed by the second lens 18 to the first beam splitter 14, and is divided into the second beam and the third beam by the first beam splitter 14
  • Light beam the first camera 20 receives the second light beam to obtain the first image of the pattern
  • the second camera 22 receives the third light beam to obtain the second image of the pattern; adjust the angle of the first camera 20 and/or the second camera 22 and/or Or position so that the contrast between the first image and the second image is consistent.
  • the light splitting module 40 is used as a single module of the entire optical system 10 to individually debug, which can reduce the limitation of the debugging space of the whole machine, and can easily realize the perpendicularity of multiple cameras and the optical axis, which is beneficial to Quickly realize the adjustment of the optical system including the split beam path.
  • the collimator 50 further includes a third light source 52, frosted glass 54 and objective lens 56.
  • the third light source 52, frosted glass 54, reticle 42 and objective lens 56 are arranged in this order.
  • the light beams emitted by the three light sources 52 sequentially exit through the frosted glass 54, the reticle 42 and the objective lens 56 to the second lens 18.
  • the collimated light beam emitted by the parallel light pipe 50 is a parallel light beam, and the parallel light beam is incident on the first beam splitter 14 through the second lens 18 to be divided into a second light beam and a third light beam.
  • the first image contrast of the pattern of the reticle 42 acquired by the first camera 20 and the second image contrast of the pattern of the reticle 42 acquired by the second camera 22 are the same, that is, the The plane of the image sensor is perpendicular to the optical axis of the second lens 18, and the plane of the image sensor of the second camera 22 is perpendicular to the optical axis of the spectroscopic optical path.
  • the reticle 42 is a customized resolution line pair reticle designed by the inventor.
  • the resolution line pair reticle includes five patterns A1-A5 with different distribution positions.
  • the image of the pattern A1-A5 of the reticle 42 acquired by a camera 20 and the image of the pattern A1-A5 of the reticle 42 acquired by the second camera 22, when the contrast of the corresponding image is consistent, the first camera can be determined
  • the plane of the image sensor of 20 is perpendicular to the optical axis of the second lens 18, and the plane of the image sensor of the second camera 22 is perpendicular to the optical axis of the light beam reflected by the first beam splitter 14, which meets the requirements for sequence measurement image acquisition.
  • the MTF values are Modulation, Transfer, and Function values
  • the difference between two or more MTF values is less than 10%, preferably less than 5%, it is determined that the MTF values are the same.
  • the MTF values of the images of the patterns A1-A5 are 0.80, 0.80, 0.80, 0.78, 0.80, 0.78, 0.80, 0.80, and 0.80, 0.80, thereby determining the first image
  • the adjustment of the optical system 10 is completed. The closer the MTF value is to 1, the better the performance of the optical system 10 is.
  • the reticle 40 is provided with multiple patterns. As shown in FIG. 13, the size of the first image and the second image are respectively the size of the circle formed by the images of the five patterns A1-A5. When the optical system 10 is used, the size of the first image and/or the second image is required to be not less than forty percent of the size of the image required for actual imaging using the optical system 10. In this way, when the optical system 10 is used for imaging, it is advantageous to obtain high-quality images and meet the requirements for sequence determination.
  • the size of the first image may refer to the size of the image formed by the images of multiple patterns distributed on the first camera 20.
  • the size of the second image may refer to the size of the image formed by the images of the multiple patterns distributed on the second camera 22.
  • the size of the first image and/or the second image is not less than fifty percent of the size of the image actually obtained by imaging with the optical system.
  • the spectroscopic module 40 further includes a compensation lens 38.
  • the compensation lens 38 is located between the first beam splitter 14 and the first camera 20.
  • the first camera 20 receives the first Two beams to obtain the first image. In this way, the imaging effect of the first image is good.
  • an embodiment of the present invention provides a sequencing system 100 including a platform 116 and an optical system 10.
  • the platform is used to carry a sample 40.
  • the optical system 10 is the optical system 10 in any of the foregoing embodiments.
  • the above-mentioned sequencing system 100 includes the optical system 10 having any of the above technical features and advantages, the structure is compact, which is advantageous for miniaturization and industrialization.
  • the platform 116 is movable.
  • the platform 116 can drive the sample 40 to move relative to the optical system, for example, in the direction perpendicular to the optical axis of the first lens 16, or in the direction parallel to the optical axis of the first lens 16, or in the light inclined to the first lens 16.
  • the movement in the axis direction makes the different positions of the sample 40 directly under the first lens 16, so that the sequencing system 100 including the optical system 10 can be used to realize the image acquisition of the sample 40 and the sequence determination.
  • sequencing system 100 of the embodiment of the present invention may include the optical system 10 of any of the above embodiments.

Landscapes

  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Analytical Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Multimedia (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

一种光学系统、调校光学系统的方法和测序系统，光学系统包括第一光源（12）、第一透镜（16）、第一分光器（14）、第二透镜（18）、第一相机（20）和第二相机（22），第一透镜（16）用于接收来自第一光源（12）的第一光束并使第一光束准直入射至样品（24）上，以及用于接收来自样品（24）的光束并使光束准直，第二透镜（18）用于将来自第一透镜（16）的准直光束聚焦至第一相机（20）和第二相机（22），第一分光器（14）用于将来自第二透镜（18）的聚焦光束分为第二光束和第三光束，第一相机（20）用于接收第二光束，第二相机（22）用于接收第三光束。光学系统，基于分光光路设计，包括使光束通过第二透镜后，再经过分光器，这样能够使光学系统的总光路长度变短、所需空间减小，有利于光学系统的小型化。

Description

光学系统、调校光学系统的方法和测序系统 技术领域
本发明涉及光学检测技术领域，特别涉及一种光学系统、一种调校光学系统的方法和一种测序系统。
背景技术
光学成像系统能够实现亚微米级的分辨率,是研究样本的一种有力工具。例如，在生物样品检测领域中，使用一些光学可检测基团/化合物/结构来标记待检测的生物分子，并且用光学成像系统来采集来自这些标记的信号，以实现待检测生物分子的定位或检测。
在基于光学成像进行核酸序列测定的平台，例如基于边合成边测序原理的测序平台，一般是利用加入带有光学可检测标记例如荧光分子的核苷酸修饰物，在使该核苷酸修饰物结合到模板(待测核酸序列)后，通过激发荧光分子发光，来确定模板相应位置的碱基类型，以此实现测序。该类测序平台希望其包含的光学成像系统能够占用空间小、不易受其它部件的影响且能够与其它关联组件紧密配合，实现稳定、连续且快速的信号采集，输出清晰的图像。设计搭建出能够实现上述目标的光学成像系统，有待解决或改进。
发明内容
本发明提供一种用于基因测序的光学系统、调校光学系统的方法和测序系统。
本发明实施方式的一种光学系统，包括第一光源、第一透镜和分光模组,所述分光模组包括第一分光器、第二透镜、第一相机和第二相机，所述第一透镜用于接收来自所述第一光源的第一光束并使该第一光束准直入射至样品上，以及用于接收来自所述样品的光束并使该光束准直，所述第二透镜用于将来自所述第一透镜的准直光束聚焦至所述第一相机和所述第二相机，所述第一分光器用于将来自所述第二透镜的聚焦光束分为第二光束和第三光束，所述第一相机用于接收所述第二光束，所述第二相机用于接收所述第三光束。
上述光学系统，基于上述分光光路设计，包括使光束通过第二透镜后，再经过分光器，这样能够使光学系统的总光路长度变短、所需空间减小，有利于光学系统的小型化。
通常地，样品上带有光学可检测标记,例如带有一种或多种荧光分子标记。激光是常用的荧光激发光源。多种荧光分子标记可被激光激发发出不同波长的荧光。
发明人还搭建设计了另一种光学系统，采用在显微镜平行光路中(即物镜、筒镜之间)进行分光，如图15所示，荧光在显微镜平行光路中进行分光，即荧光经物镜(透镜)后准直，经分光器90分束形成多个分光光路，在各分光光路中各光束经过筒镜93或94聚焦到相机91或92
发明人对比测试发现，相较于后者，包含了在会聚光路中进行分光的分光光路的光学系统，总光路长度小、所需空间小包括所需光学元件较少，利于光学系统的小型化，利于该光学系统的工业化。
本发明实施方式的一种测序系统，包括平台和上述任一实施方式的光学系统，所述平台用于承载样品。上述对本发明任一实施方式中的光学系统的技术特征和优点的描述，也适用于本实施方式的测序系统，在此不再赘述。
本发明实施方式提供一种调校光学系统的方法，所述光学系统包括分光模组,所述分光模组包括第二透镜、第一分光器、第一相机和第二相机，所述第二透镜、所述第一分光器和所述第一相机沿所述第二透镜的光轴依次布置，所述方法包括：利用平行光管发射准 直光束至所述第二透镜，所述平行光管包括分划板，所述分划板包括一个或多个图案，所述准直光束经过所述第二透镜会聚至所述第一分光器，并经过所述第一分光器分为第二光束和第三光束，所述第一相机接收所述第二光束，获得所述图案的第一图像，所述第二相机接收所述第三光束，获取所述图案的第二图像；调节所述第一相机和/或所述第二相机的角度和/或位置，以使所述第一图像和所述第二图像的对比度一致。该调校光学系统的方法，将分光模组作为整个光学系统的一个模组单独调试，可降低整机调试空间上的限制，能简单方便地实现多个相机与光轴的垂直，实现包含分光光路的光学系统的调校。
本发明实施例的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1是本发明实施方式的光学系统的结构示意图；
图2是本发明实施方式的第一光源的结构示意图；
图3是本发明实施方式的光学系统的另一结构示意图；
图4是本发明实施方式的第二光源的结构示意图；
图5是本发明实施方式的光学系统在不包含第一分光器时的成像光束的光斑尺寸仿真结果示意图；
图6是本发明实施方式的光学系统在包含第一分光器的成像光束的光斑的尺寸仿真结果示意图；
图7是本发明实施方式的光学系统在引入补偿镜片的成像光束的光斑的尺寸仿真结果示意图；
图8是本发明实施方式的光学系统的部分立体示意图；
图9是图8的光学系统的主视图；
图10是图8的光学系统的俯视图；
图11是本发明实施方式的分光模组在调试时的结构示意图；
图12是本发明实施方式的平行光管的结构示意图；
图13是本发明实施方式的分辨率线对分划板的图案示意图；
图14是本发明实施方式的分光模组在调试时的另一结构示意图；
图15是相关技术的光学系统的结构示意图；
图16是本发明实施方式的测序系统的立体示意图。
具体实施例
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
在本发明的描述中，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、顺序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一个或者更多个所述特征。在本发明的描述中，除非另有限定，“多个”的含义是两个或两个以上，除非另有明确具体的限定。
在本发明的描述中，除非另有限定，术语“安装”、“相连”、“连接”应做广义理解，例如，可以是固定连接，也可以是可拆卸连接，或一体地连接；可以是机械连接，也可以是电连接或可以相互通讯；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。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。
请参阅图1，本发明实施方式的一种光学系统10，包括第一光源12、第一透镜16和分光模组40，分光模组40包括第一分光器14、第二透镜18、第一相机20和第二相机22。第一透镜16用于接收来自第一光源12的第一光束并使该光束准直入射至样品24上，以及用于接收来自样品24的光束并使该光束准直。第二透镜18用于将来自第一透镜16的准直光束聚焦至第一相机20和第二相机22。第一分光器14用于将来自第二透镜18的聚焦光束分为第二光束和第三光束。第一相机20用于接收第二光束。第二相机22用于接收第三光束。
上述光学系统10，由于第二透镜18将光聚焦后，再由分光器14分成第二光束和第三光束，这样可减少光学元件的使用、分光光路长度小，使得光学系统的总光路长度变短，有利于光学系统10的小型化，利于工业化。
具体地，样品24可为待测核酸样本，待测核酸样本可放置在反应装置内，如芯片内。第一光源12可为激光光源。在一个示例中，芯片包含基底，基底上设有通道，基底上设有玻璃，在利用该光学系统的测序系统进行测序时，在一定条件下，待测核酸、酶、带荧光标记的核苷酸试剂或溶液等混合于通道中发生反应，然后第一光源12发射激光经第一透镜16入射至芯片特定视野，该视野的荧光基团被激发发出荧光，荧光经第一透镜16和第二透镜18聚焦到达第一分光器14，第一分光器14将该荧光会聚光束分成第二光束和第三光束，第一相机20接收第二光束，第二相机22接收第三光束，分别采集得该视野的第一图像和第二图像。
在一个例子中，请结合图2，第一光源12可包括第一发光器13和第三透镜15，所述第一光束为所述第一发光器13发出的光束经过所述第三透镜15后的准直光束，所述第一光束经第四透镜17聚焦到第一透镜16的后焦面、进而经过所述第一透镜16准直入射至所述样品40上。在一个示例中,第一光源12还包括光纤耦合器，例如单模光线耦合器。具体地，光学系统10为全内反射光学系统，经过所述第一透镜16的准直光束(平行光束)以大于临界角入射至芯片表面，发生全内反射，在芯片玻璃的下表面产生消逝场(消逝波)。该消逝场中的荧光分子被激发发出的荧光被第一透镜16接收。
在第一光源16发射的光束激发样品24的荧光基团发光时，第一透镜16接收的来自样品24的光束是样品24发出的光束。
第一相机20和第二相机22的图像传感器可采用CCD或CMOS。较佳地，第一相机20和第二相机22所采用的图像传感器的类型相同，例如，均为CCD或均为CMOS。第一分光器14可为二向色镜。
在图示的实施方式中，第二光束为第一分光器14的透射光束，第三光束为第一分光器14的反射光束。
在某些实施方式中，第一相机20与第二相机22呈90度或270度设置。如此，便于在有限空间内将第一相机20和第二相机22多个相机配置到该光学系统中。具体地，在图1所示的方位中，第一分光器14具有第一反射面26，第一反射面26与水平面的夹角成45度，沿水平方向入射至第一反射面26的一部分光束被反射转向90度到达第二相机22，而沿水平方向入射至第一反射面26的另一部分光束穿过第一反射面26并入射至第一相机20。在图1和图3中，第一相机20与第二相机22沿顺时针呈90度设置，沿逆时针呈270度设置。在一个示例中，样品带有两种荧光标记，例如为Cy3和Atto647N，该两种荧光分子的发射光波段分别为550-620nm和650-750nm(波峰分别大约为564nm和670nm)；第一分光器14为二向色镜，该二向色镜对波长550-620nm的光具有较高的透射率，对650nm以上的光具有较高的反射率。
所称的带荧光标记的核苷酸试剂包括A、T、C和G四种类型核苷酸试剂，不同种核苷 酸试剂可分别盛放于不同容器中。在一个示例中，四种核苷酸带有同一种荧光标记，在DNA测序时，每轮测序反应包括四次碱基延伸反应，四次碱基延伸反应分别为依次加入该四种核苷酸以及获得相应的图像。
在一个示例中，四种核苷酸两两分别带有第一荧光标记和第二荧光标记，第一荧光标记和第二荧光标记可被激发发出不同的荧光，利用该四种核苷酸进行双色测序，每轮测序反应包括两次碱基延伸反应，在利用包含该光学系统10的测序系统进行测序时，在一定条件下，待测核酸、酶以及带第一荧光标记和第二荧光标记的两种核苷酸试剂或溶液等混合于通道中发生反应，第一光源12同时发射第一激光和第二激光经第一透镜16入射至芯片特定视野，该视野的第一荧光标记和第二荧光标记分别被第一激光和第二激光激发发出第一荧光和第二荧光，该第一荧光和第二荧光经第一透镜16和第二透镜18会聚至第一分光器14(二向色镜)，该二向色镜分开会聚的第一荧光和第二荧光，第一荧光聚焦到第一相机20像面，第二荧光聚焦到第二相机22像面，由此，分别获得该视野的第一荧光和第二荧光形成的第一图像和第二图像。基于核苷酸加入顺序和不同轮测序反应的第一图像和第二图像信息，实现碱基识别/测序。
在另一个示例中，四种核苷酸分别带荧光标记a、荧光标记b、双荧光标记a-b以及不带标记，荧光标记a和荧光标记b可被激发发出不同的荧光，利用该四种核苷酸进行四色测序，每轮测序反应包括一次碱基延伸反应，在利用包含该光学系统10的测序系统进行测序时，在一定条件下，待测核酸、酶以及上述四种核苷酸试剂或溶液等混合于通道中发生反应，第一光源12同时发射第一激光和第二激光经第一透镜16入射至芯片特定视野，该视野的荧光标记分别被第一激光和第二激光激发发出荧光，该荧光经第一透镜16和第二透镜18会聚至第一分光器14(二向色镜)，该二向色镜将该荧光分为来自荧光标记a的荧光和自荧光标记b的荧光，来自荧光标记a的荧光聚焦到第一相机20像面，来自荧光标记b的荧光聚焦到第二相机22像面，由此，分别获得得该视野的第一图像和第二图像。通过不同轮测序反应的第一图像和第二图像以及合并同一轮测序反应的第一图像和第二图像的信息，实现碱基识别/测序。
在某些实施方式中，第一透镜16包含一个或多个透镜，和第二透镜18包含一个或多个透镜。具体地，在显微镜系统中，第一透镜16的一个或多个透镜构成了物镜；第二透镜18的一个或多个透镜构成了筒镜(tube lens)。在其它实施方式中，第一透镜16包含一个或多个透镜，或第二透镜18包含一个或多个透镜。
在某些实施方式中，光学系统10包括第二分光器28，第二分光器28用于接收来自第一光源12的第一光束并使该第一光束转向至第一透镜16，使得该第一光束并入到第一透镜光轴所在的光路(成像光路)中。如此，第二分光器28的设置可使得第一光源12位于第一透镜16的光轴所在光路外，能够使该光学系统10的各元件紧凑合理的设置，利于该光学系统10的小型化，利于工业应用。
具体地，第二分光器28用于使该第一光束转90度角。如此，便于配置第一光源12的位置包括其包含的元件的相对位置。
在某些实施方式中，光学系统10包括第三分光器30和自动对焦模组32。自动对焦模组32用于发出第四光束，以及用于接收被样品40反射回的第四光束，第三分光器30用于接收第四光束并使该第四光束转向至第一透镜16，还用于接收被样品40反射回的第四光束并使该第四光束转向至自动对焦模组32。如此，可利用自动对焦模组32实现对焦，实现利用该光学系统10进行图像采集。
具体地，自动对焦模组32包括第二光源34和接收器36，第二光源34用于发射第四光束至第三分光器30，接收器36用于接收第一透镜16准直后的第四光束。在一个例子中，第二光源34可以是红外光源。接收器36可为光电二极管。在进行对焦时，第二光源34发射第四光束，经第三分光器30转向至第一透镜16，第四光束经第一透镜16会聚至样品 24。样品24反射回的第四光束经过第一透镜准直入射至第三分光器30。此时，通过确定接收器36接收到的由样品24反射回的第四光束的信息的变化，可移动承载样品的平台使样品24靠近或远离第一透镜16，由此实现对焦。
在一个示例中，接收器36包括传感器，例如二维PSD传感器，第二光源34包括LED光源和位于LED光源前的掩膜；LED光源发出的光照射到腌模上得到特定图案的光斑，该特定图案的光斑经第三分光器30转至第一透镜16会聚到样品24上，经样品24反射回的该光斑最后到达传感器；该自动对焦模组32还包括信号处理模块，该传感器连接该信号处理模块，通过该信号处理模块后得到该光斑的信息。进一步地，该自动对焦模组32还包括信号输出模块，用于输出光斑信息的变化，以使承载样品的平台带动样品移动到成像光路(例如荧光光路)的物面。
在某些实施方式中，请结合图4，第二光源34包括第二发光器35和第五透镜37，第四光束为第二发光器38发出的光束经过第五透镜后37的准直光束，被样品40反射回的第四光束经第五透镜37会聚至接收器36。
在某些实施方式中，请结合图3，第二光束为来自第二透镜18的聚焦光束透射经过第一分光器14的光束，光学系统10包括补偿镜片38，补偿镜片38位于第一分光器14和第一相机20之间，补偿镜片38用于补偿第二光束带来的像散。
具体地，在一些示例中，利用软件(例如Zemax)对第二透镜18的聚焦光束进行成像仿真时发现，对比第二透镜18后不引入和引入第一分光器14即聚焦后不进行分光和进行分光，成像仿真结果分别如图5和图6所示，可以看出，来自相同视场的光束经过第二透镜18和第一分光器14后形成的光斑(弥散斑)和只经过第二透镜18形成的光斑(弥散斑)，像散明显增大。例如，图6中坐标(0，0)、(0,3.250)和(0,6.500)处的光斑尺寸均大于图5中相应坐标处的光斑尺寸。可以利用RMS半径(RMS RADIUS，均平方根半径)来衡量光斑的大小，来定量的反映某光学系统实际成像的光斑大小。均平方根半径是一个重要的半径参数，它是弥散斑各个点坐标，参考中心点，进行的坐标平方和后，除以点数量，然后开方的值，这个值的半径可以反映一个典型的弥散斑的大小，以定量的反映这个系统实际的斑点大小。另外，GEO RADIUS(GEO半径)表示弥散斑的直径。明显可看出，来自相同视场的光束聚焦形成的光斑，图6的较图5的弥散，RMS半径较大。
基于此，在一些示例中，发明人引入补偿镜片38于第二透镜18和第一相机之间的任意位置，希望能补偿光束透射后成像造成的像散。在引入补偿镜片38后，请结合图7，来自相同视场的光束形成的光斑，图7的光斑尺寸明显小于图6的光斑尺寸，并且从RMS半径来看，在相同坐标处，图7的光斑尺寸接近甚至小于图5的光斑尺寸。
补偿镜片38可采用平行平板或二向色镜。在图8-10的实施方式中，补偿镜片38采用二向色镜。补偿镜片38与一平面P的夹角T成45度，第一分光器14与平面P垂直，平面P由第二光束的光轴A和第三光束的光轴B共同限定。
本发明实施方式还提供一种调校光学系统的方法，请结合图11，光学系统包括分光模组40,分光模组40包括第二透镜18、第一分光器14、第一相机20和第二相机22，第二透镜18、第一分光器14和第一相机20沿第二透镜18的光轴依次布置，方法包括：利用平行光管50发射准直光束至第二透镜18，平行光管50包括分划板42，分划板42包括一个或多个图案，准直光束经过第二透镜18会聚至第一分光器14，并经过第一分光器14分为第二光束和第三光束，第一相机20接收第二光束，获得图案的第一图像，第二相机22接收第三光束，获取图案的第二图像；调节第一相机20和/或第二相机22的角度和/或位置，以使第一图像和第二图像的对比度一致。
该调校光学系统的方法，将分光模组40作为整个光学系统10的一个模组单独调试，可降低整机调试空间上的限制，能简单方便地实现多个相机与光轴的垂直，利于快速地实现包含分光光路的光学系统的调校。
具体地，在本实施方式中，请结图12，平行光管50还包括第三光源52、毛玻璃54和物镜56，第三光源52、毛玻璃54、分划板42和物镜56依次排列，第三光源52发射的光束依次经毛玻璃54、分划板42和物镜56出射至第二透镜18。平行光管50发射的准直光束为平行光束，平行光束经过第二透镜18入射至第一分光器14分成第二光束和第三光束。
在本实施方式中，第一相机20获取的分划板42的图案的第一图像对比度和第二相机22获取的分划板42的图案的第二图像对比度一致，即说明第一相机20的图像传感器的平面与第二透镜18的光轴垂直，第二相机22的图像传感器的平面与分光光路的的光轴垂直。
在本实施方式中，分划板42为发明人设计定制的分辨率线对分划板，请结合图13，该分辨率线对分划板包括分布位置不同的5个图案A1-A5，第一相机20获取的分划板42的图案A1-A5的图像与第二相机22获取的该分划板42的图案A1-A5的图像，相应的图像的对比度一致时，则可确定第一相机20的图像传感器的平面与第二透镜18的光轴垂直，第二相机22的图像传感器的平面与经过第一分光器14反射的光束的光轴垂直，符合用于序列测定图像采集的要求。
在某些实施方式中，图案的多个像的MTF值(MTF值是Modulation Transfer Function值，调制传递函数值)相同即判定该些图像的对比度一致。在一些示例中，两个或多个MTF值差异小于10％、较佳地小于5％，判定该些MTF值相同。具体地，在第一图像和第二图像中，图案A1-A5的像的MTF值分别为0.80、0.80，0.80、0.78，0.80、0.78,0.80、0.80以及0.80、0.80，由此判定第一图像和第二图像的对比度一致，该光学系统10调校完成。MTF值越接近1，说明光学系统10的性能越优异。
在某些实施方式中，分划板40带有多个图案，如图13所示，第一图像和第二图像的大小分别为5个图案A1-A5的像形成的圆的大小，设计该光学系统10时，要求第一图像和/或第二图像的大小不小于实际利用该光学系统10进行成像所要求的图像的大小的百分四十。如此，利用该光学系统10进行成像时，利于获得高质量的图像，符合序列测定的要求。
具体地，第一图像的大小可指多个图案的像分布在第一相机20上所形成的图像的大小。第二图像的大小可指多个图案的像分布在第二相机22上所形成的图像的大小。较佳地，第一图像和/或第二图像的大小不小于实际利用该光学系统进行成像所得的图像的大小的百分五十。
在某些实施方式中，请结合图14，分光模组40还包括补偿镜片38，补偿镜片38位于第一分光器14和第一相机20之间，第一相机20接收经过补偿镜片38的第二光束，获得第一图像。如此，第一图像的成像效果好。
请参图16，本发明实施方式提供一种测序系统100，包括平台116和光学系统10，平台用于承载样品40，光学系统10为上述任一实施方式中的光学系统10。
上述测序系统100，由于包含具有上述任一技术特征和优点的光学系统10，结构紧凑，利于小型化、工业化。
具体地，本实施方式中，平台116是可移动的。平台116可带动样品40相对于光学系统运动，例如沿垂直于第一透镜16的光轴方向运动、或沿平行于第一透镜16的光轴方向运动，或沿倾斜于第一透镜16的光轴方向运动，以使得样品40的不同位置位于第一透镜16的正下方，使得能够利用包含该光学系统10的测序系统100实现对样品40的图像采集，进而实现序列测定。
可以理解，本发明实施方式的测序系统100可包括上述任一实施方式的光学系统10。
在本说明书的描述中，参考术语“一个实施例”、“某些实施例”、“示意性实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合实施例或示例 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一定指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何的一个或多个实施例或示例中以合适的方式结合。
尽管已经示出和描述了本发明的实施例，本领域的普通技术人员可以理解：在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由权利要求及其等同物限定。

Claims (17)

  1. 一种光学系统，其特征在于，包括第一光源、第一透镜和分光模组,所述分光模组包括第一分光器、第二透镜、第一相机和第二相机，
    所述第一透镜用于接收来自所述第一光源的第一光束并使该第一光束准直入射至样品上，以及用于接收来自所述样品的光束并使该光束准直，
    所述第二透镜用于将来自所述第一透镜的准直光束聚焦至所述第一相机和所述第二相机，
    所述第一分光器用于将来自所述第二透镜的聚焦光束分为第二光束和第三光束，
    所述第一相机用于接收所述第二光束，
    所述第二相机用于接收所述第三光束。
  2. 根据权利要求1所述的光学系统，其特征在于，所述第一光源包括第一发光器和第三透镜，所述光学系统包括第四透镜,所述第一光束为所述第一发光器发出的光束经过所述第三透镜后的准直光束，所述第一光束经过所述第四透镜聚焦到所述第一透镜的后焦面、进而经过所述第一透镜准直入射至所述样品上。
  3. 根据权利要求1所述的光学系统，其特征在于，所述光学系统包括第二分光器，所述第二分光器用于接收来自所述第一光源的所述第一光束并使该第一光束转向至所述第一透镜。
  4. 根据权利要求1或3所述的光学系统，其特征在于，所述光学系统包括第三分光器和自动对焦模组，
    所述自动对焦模组用于发出第四光束，以及用于接收被所述样品反射回的第四光束，
    所述第三分光器用于接收所述第四光束并使该第四光束转向至所述第一透镜，还用于接收所述被样品反射回的第四光束并使该第四光束转向至所述自动对焦模组。
  5. 根据权利要求4所述的光学系统，其特征在于，所述自动对焦模组包括第二光源和接收器，所述第二光源用于发射所述第四光束至所述第三分光器，所述接收器用于接收所述被样品反射回的第四光束。
  6. 根据权利要求4或5所述的光学系统，其特征在于，所述第四光束经过所述第一透镜会聚到所述样品上；
    所述被样品反射回的第四光束经过所述第一透镜准直入射至所述第三分光器。
  7. 根据权利要求5所述的光学系统，其特征在于，所述第二光源包括第二发光器和第五透镜，所述第四光束为所述第二发光器发出的光束经过所述第五透镜后的准直光束，所述被样品反射回的第四光束经所述第五透镜会聚至所述接收器。
  8. 根据权利要求1-7任一项所述的光学系统，其特征在于，所述第一透镜包含一个或多个透镜，和/或所述第二透镜包含一个或多个透镜。
  9. 根据权利要求1-8任一项所述的光学系统，其特征在于，所述第一相机与所述第二相机呈90度或270度设置。
  10. 根据权利要求3所述的光学系统，其特征在于，所述第二分光器用于使该第一光束转90度角。
  11. 根据权利要求1所述的光学系统，其特征在于，所述第二光束为来自所述第二透镜的聚焦光束透射经过所述第一分光器的光束，所述光学系统包括补偿镜片，所述补偿镜片位于所述第一分光器和所述第一相机之间，所述补偿镜片用于补偿所述第二光束带来的像散。
  12. 根据权利要求11所述的光学系统，其特征在于，所述补偿镜片与一平面的夹角成45度，所述第一分光器与所述平面垂直，所述平面由所述第二光束的光轴和所述第三 光束的光轴共同限定。
  13. 一种测序系统，其特征在于，包括平台和权利要求1-12任一项所述的光学系统，所述平台用于承载样品。
  14. 一种调校光学系统的方法，其特征在于，所述光学系统包括分光模组,所述分光模组包括第二透镜、第一分光器、第一相机和第二相机，所述第二透镜、所述第一分光器和所述第一相机沿所述第二透镜的光轴依次布置，所述方法包括：
    利用平行光管发射准直光束至所述第二透镜，所述平行光管包括分划板，所述分划板包括一个或多个图案，
    所述准直光束经过所述第二透镜会聚至所述第一分光器，并经过所述第一分光器分为第二光束和第三光束，所述第一相机接收所述第二光束，获得所述图案的第一图像，
    所述第二相机接收所述第三光束，获取所述图案的第二图像；
    调节所述第一相机和/或所述第二相机的角度和/或位置，以使所述第一图像和所述第二图像的对比度一致。
  15. 根据权利要求14所述的方法，其特征在于，所述分划板带有多个图案，所述第一图像和/或所述第二图像的大小不小于利用所述光学系统进行成像所得的图像的大小的百分四十。
  16. 根据权利要求14所述的方法，其特征在于，所述分光模组还包括补偿镜片，所述补偿镜片位于所述第一分光器和所述第一相机之间，所述第一相机接收经过所述补偿镜片的第二光束，获得所述第一图像。
  17. 根据权利要求16所述的方法，其特征在于，所述补偿镜片与一平面的夹角成45度，所述第一分光器与所述平面垂直，所述平面由所述第二光束的光轴和所述第三光束的光轴共同限定。
PCT/CN2019/109045 2018-12-12 2019-09-29 光学系统、调校光学系统的方法和测序系统 Ceased WO2020119226A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP19896106.2A EP3896146A4 (en) 2018-12-12 2019-09-29 OPTICAL SYSTEM, METHOD OF CALIBRATION OF AN OPTICAL SYSTEM AND SEQUENCING SYSTEM
US17/311,179 US11874453B2 (en) 2018-12-12 2019-09-29 Optical system, method for calibrating optical system, and sequencing system

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN201822089550.1U CN209759461U (zh) 2018-12-12 2018-12-12 光学系统和测序系统
CN201822089550.1 2018-12-12
CN201811516793.7A CN111308726A (zh) 2018-12-12 2018-12-12 光学系统、调校光学系统的方法和测序系统
CN201811516793.7 2018-12-12

Publications (1)

Publication Number Publication Date
WO2020119226A1 true WO2020119226A1 (zh) 2020-06-18

Family

ID=71075255

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/109045 Ceased WO2020119226A1 (zh) 2018-12-12 2019-09-29 光学系统、调校光学系统的方法和测序系统

Country Status (3)

Country Link
US (1) US11874453B2 (zh)
EP (1) EP3896146A4 (zh)
WO (1) WO2020119226A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112646703A (zh) * 2020-12-29 2021-04-13 中国科学院长春光学精密机械与物理研究所 一种单分子荧光基因测序光学系统
US20220137425A1 (en) * 2019-03-06 2022-05-05 Konrad Gmbh Collimator

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11639846B2 (en) * 2019-09-27 2023-05-02 Honeywell International Inc. Dual-pattern optical 3D dimensioning
US11450083B2 (en) 2019-09-27 2022-09-20 Honeywell International Inc. Dual-pattern optical 3D dimensioning

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004048936A2 (en) * 2002-11-26 2004-06-10 University Of Utah Research Foundation Microporous materials, methods, and articles for localizing and quantifying analytes
CN104293648A (zh) * 2014-09-29 2015-01-21 深圳市大族激光科技股份有限公司 基因测序光路系统
CN105371757A (zh) * 2015-10-27 2016-03-02 西安交通大学 一种差动式并行光学层析显微测量装置及方法
CN106460034A (zh) * 2014-01-10 2017-02-22 格诺特斯控股股份公司 具有高准确度的单分子分析
CN108286936A (zh) * 2017-04-18 2018-07-17 北京理工大学 激光微纳加工差动共焦在线监测一体化方法与装置
CN209759461U (zh) * 2018-12-12 2019-12-10 深圳市真迈生物科技有限公司 光学系统和测序系统

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7538890B2 (en) * 2004-06-07 2009-05-26 Fujinon Corporation Wavefront-measuring interferometer apparatus, and light beam measurement apparatus and method thereof
WO2009042862A1 (en) 2007-09-28 2009-04-02 Illumina, Inc Fluorescence excitation and detection system and method
TWI406025B (zh) 2010-11-25 2013-08-21 Ind Tech Res Inst 自動聚焦裝置及方法
CN103278934B (zh) 2013-05-31 2017-03-29 中国科学院西安光学精密机械研究所 一种用于星载光学遥感器的焦面对接装置及方法
CN105068232B (zh) 2015-08-31 2017-11-24 福建师范大学 一种双通道结构光照明超分辨成像方法与装置
CN112955534B (zh) 2019-09-24 2023-01-24 深圳市真迈生物科技有限公司 减振结构、检测系统和测序系统

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004048936A2 (en) * 2002-11-26 2004-06-10 University Of Utah Research Foundation Microporous materials, methods, and articles for localizing and quantifying analytes
CN106460034A (zh) * 2014-01-10 2017-02-22 格诺特斯控股股份公司 具有高准确度的单分子分析
CN104293648A (zh) * 2014-09-29 2015-01-21 深圳市大族激光科技股份有限公司 基因测序光路系统
CN105371757A (zh) * 2015-10-27 2016-03-02 西安交通大学 一种差动式并行光学层析显微测量装置及方法
CN108286936A (zh) * 2017-04-18 2018-07-17 北京理工大学 激光微纳加工差动共焦在线监测一体化方法与装置
CN209759461U (zh) * 2018-12-12 2019-12-10 深圳市真迈生物科技有限公司 光学系统和测序系统

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3896146A4 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220137425A1 (en) * 2019-03-06 2022-05-05 Konrad Gmbh Collimator
US12066638B2 (en) * 2019-03-06 2024-08-20 Konrad Gmbh Collimator
CN112646703A (zh) * 2020-12-29 2021-04-13 中国科学院长春光学精密机械与物理研究所 一种单分子荧光基因测序光学系统

Also Published As

Publication number Publication date
EP3896146A1 (en) 2021-10-20
EP3896146A4 (en) 2022-01-19
US20210373314A1 (en) 2021-12-02
US11874453B2 (en) 2024-01-16

Similar Documents

Publication Publication Date Title
CN111308726A (zh) 光学系统、调校光学系统的方法和测序系统
US6355934B1 (en) Imaging system for an optical scanner
KR101022769B1 (ko) 바이오칩용 광검출 장치
DK2594981T3 (en) Methods and apparatus for confocal imaging
CN209759461U (zh) 光学系统和测序系统
US11874453B2 (en) Optical system, method for calibrating optical system, and sequencing system
CN101868752B (zh) 用于利用线束照射样本的光学照射装置
WO2005113832A2 (en) Wide field imager for quantitative analysis of microarrays
CN111349553A (zh) 一种基因测序仪光学系统
AU2020366521B2 (en) Virtual fiducials
WO2023077306A1 (zh) 检测设备、基因测序系统及检测方法
US8742384B2 (en) Optical illumination apparatus and method having a reflective arrangement with moveable components for adjusting incident light
WO2022094803A1 (zh) 检测系统
JP2004191252A (ja) 蛍光読み取り装置
CN113959363B (zh) 基于高色散柱透镜和柱形凹面光栅的线扫描式光谱共焦装置
HK40062282A (zh) 光学系统、调校光学系统的方法和测序系统
CN218546517U (zh) 一种接收装置及基因测序系统
JP2023530641A (ja) 生体試料画像採取装置および遺伝子シーケンサー
JP7863544B2 (ja) 多次元撮像のための方法およびシステム
JP2007212171A (ja) 蛍光検出装置
CN118671040A (zh) 一种用于快速荧光共振能量转移定量测量的光路系统
CN119959133A (zh) 一种光学成像系统及一种生物分子检测系统
HK40080170B (zh) 生物样本图像采集装置及基因测序仪

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19896106

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2019896106

Country of ref document: EP

Effective date: 20210712