WO2023103240A1

WO2023103240A1 - Sequencer fluorescent light splitting system and light splitting method

Info

Publication number: WO2023103240A1
Application number: PCT/CN2022/086206
Authority: WO
Inventors: 周藩; 陈海东; 陈鑫
Original assignee: 深圳铭毅智造科技有限公司
Priority date: 2021-12-06
Filing date: 2022-04-12
Publication date: 2023-06-15
Also published as: CN114252420B; CN114252420A

Abstract

The present invention relates to a sequencer fluorescent light splitting system, comprising a light source assembly, an optical path assembly, and an imaging device; the optical path assembly comprises an excitation filter, an excitation dichroic mirror, an objective lens, a reflection module that cannot transmit light, and an emission filter module; both the excitation filter and the excitation dichroic mirror are installed on the optical path of excitation light emitted by the light source assembly; the objective lens is installed on the optical path of the reflected excitation light; the imaging device comprises a first imaging device and a second imaging device; the first imaging device is installed on the optical path of fluorescent light transmitted by the excitation dichroic mirror; the reflection module is movably disposed on the optical path of the fluorescent light transmitted by the excitation dichroic mirror; the second imaging device is installed on the optical path of the fluorescent light reflected by the reflection module; the first imaging device and the second imaging device each is provided with an emission filter on the optical path for receiving the fluorescent light. Also disclosed is a method for splitting light using the system. The invention has low cost, high universality, low requirements for use conditions, and better compatibility.

Description

Fluorescence spectroscopic system and spectroscopic method for sequencer

technical field

The invention relates to the field of sequencers, in particular to a fluorescence spectroscopic system and a spectroscopic method for a sequencer.

Background technique

The schematic diagram of the optical path of a commonly used sequencer is shown in Figure 6. The collimated light source emits excitation light, passes through the excitation filter, is reflected by the excitation dichroic mirror into the objective lens, and the focused light is irradiated on the samples labeled with various fluorescent substances. , the generated fluorescence is collected by the objective lens, passes through the excitation dichroic mirror, passes through the fluorescence dichroic mirror, and the fluorescence in the specified wavelength range is reflected and filtered by the emission filter module (including tube lens and emission filter) After being received by the imaging device, image 1 is generated; in addition, the fluorescence in the specified wavelength range is transmitted through the fluorescent dichroic mirror, filtered by the emission filter module, and then received by the imaging device to generate image 2.

Therefore, it can be seen that the light splitting of various fluorescent dyes in commonly used sequencers uses dichroic mirrors to achieve fluorescence light splitting, as shown in the device in Figure 6. There are several defects in this method:

1. The development cost of the dichroic mirror is high. The dichroic mirror needs to achieve selective transmission and reflection for specific wavelengths, and the transmittance of the transmitted wavelength is required to be higher than 90%, and the transmittance of the non-transmitted wavelength is low. The transmittance of some non-transmissive wavelengths is even required to be lower than 10 ^-7 . These technical indicators have extremely high requirements on the coating process, and the defect rate in the production process is high ^, which leads to high development costs. expensive;

2. Dichroic mirrors rely on imports, the fluorescence signal is generally weak, and the transmittance and cut-off rate of dichroic mirrors are extremely high. Currently, high-quality dichroic mirrors are all foreign brands, basically relying on imports;

3. The versatility of the dichroic mirror is not strong. The dichroic mirror is a customized product and needs to be customized and developed according to the wavelength of the fluorescent dye used. The development cost of the coating process in the early stage is high, and once the fluorescent dye is replaced, the dichroic mirror needs to be re-customized. The customization cycle is long, time-consuming and expensive;

4. Dichroic mirrors are used under harsh conditions. Dichroic mirrors have high requirements for the collimation and angle of incident light. They have requirements for the range of incident angles and the size of the light cone. Once the requirements are exceeded, blue light will occur in the wavelength range that can be transmitted. Shift or red shift, affect the final image quality.

technical problem

Aiming at the existing deficiencies, the invention provides a fluorescence spectroscopic system and a spectroscopic method of a sequencer.

technical solution

The technical solution adopted by the present invention to solve the technical problem is: a fluorescence spectroscopic system of a sequencer, including a light source assembly, an optical path assembly and an imaging device, the light source assembly is a light source assembly for emitting excitation light; the optical path assembly includes An excitation filter, an excitation dichroic mirror, an objective lens, a light-impermeable reflection module, and an emission filter module; the excitation filter and the excitation dichroic mirror are all installed on the optical path of the excitation light emitted by the light source assembly , and the excitation filter is between the excitation dichroic mirror and the light source assembly; the objective lens is installed on the optical path of the excitation light reflected by the excitation dichroic mirror; the imaging device includes a first imaging device and a second imaging device device, the first imaging device is installed on the optical path of the fluorescence transmitted by the excitation dichroic mirror; the reflection module is movable between the first imaging device and the excitation dichroic mirror on the optical path; the second imaging device is installed on the optical path of the fluorescent light reflected by the reflective module; a first emission filter module is installed on the optical path of the fluorescent light received between the first imaging device and the reflective module, so A second emission filter module is installed on the optical path for receiving fluorescence between the second imaging device and the reflection module.

Preferably, the reflection module includes a light-tight reflection mirror and a mounting base for installing the reflection mirror. The mirror is set on the mount at an angle of 45° corresponding to the incident direction of the fluorescence.

Preferably, the mount is movable horizontally on the optical path that excites the fluorescence transmitted by the dichroic mirror, and the mount is mounted on a translation stage that is close to or far away from the second imaging device in a one-dimensional direction, The displacement platform is electrically connected with a control terminal.

Preferably, the mounting seat is rotatably arranged on the optical path of the fluorescent light transmitted by the excitation dichroic mirror, and the mounting seat is set on the optical path of the fluorescent light transmitted by the excitation dichroic mirror with any one end as the rotation center limit rotation. On the optical path, and the axis line of the rotation center of the mounting seat is perpendicular to the optical path of the fluorescent light transmitted by the excitation dichroic mirror and the optical path of the fluorescent light reflected by the reflector.

As a preference, the mounting seat is rotatably arranged on the optical path of the fluorescent light transmitted by the excitation dichroic mirror, and the mounting seat can be clamped clockwise at 45° with the optical path of the fluorescent light transmitted by the excitation dichroic mirror. The angular direction is a turntable for limiting rotation in the axial direction, and a plurality of through holes in the direction of an angle of 45° in the clockwise direction with the axial direction and the light path of the fluorescent light transmitted by the excitation dichroic mirror are arranged in parallel on the turntable, and the reflector There are also a plurality of reflecting mirrors spaced apart in the through holes.

Preferably, the mounting base is connected with a motor through transmission, and the motor is electrically connected with a control terminal.

Preferably, the reflector is any one of a metal layer reflector and a dielectric film reflector.

Preferably, multiple second imaging devices are arranged side by side, the number of the reflection module and the second emission filter module is the same as the number of the second imaging device, and the reflection module, the second emission filter module The module and the second imaging device are arranged correspondingly one by one.

Preferably, the reflective module is movably arranged at an angle of 45 degrees on the optical path that excites the fluorescent light transmitted by the dichroic mirror.

A method for fluorescence spectroscopy of a sequencer, using the fluorescence spectroscopy system of a sequencer as described in any one of the preceding items to perform fluorescence spectroscopy.

Beneficial effect

The beneficial effect of the present invention is that: in the present invention, the reflective module is movably arranged on the transmitted fluorescent light path, and when the reflective module is on the fluorescent light path, the fluorescent light is reflected, and then the light filtered by the emission filter module is absorbed by the imaging device to generate For the fluorescence of one dye, when the reflection module is removed from the fluorescence light path, the fluorescence will directly shine on another emission filter module, and the fluorescence of another dye can be obtained after filtering the light. Low cost, the components contained in the reflection module are common components, mature processing, low technical threshold, relatively lower individual price, and domestic products can basically meet the requirements, do not need to rely on imports; and the versatility of the reflection module Relatively strong, the reflective module is not limited by fluorescent dyes, different fluorescent dyes can be used, the reflector can reflect ultraviolet light, visible light, and near-infrared light with high reflectivity; The collimation and angle requirements of the incident light are not high, the reflectivity is only related to the wavelength of the reflected light, and the compatibility is better.

Description of drawings

Fig. 1 is a schematic diagram of the principle structure of the horizontal movement of the reflection module according to the embodiment of the present invention;

Fig. 2 is a schematic structural diagram of the first rotation of the reflective module according to the embodiment of the present invention;

Fig. 3 is a schematic structural diagram of the second rotation of the reflection module according to the embodiment of the present invention;

Fig. 4 is a schematic structural diagram of the second rotating mounting base of the reflective module according to the embodiment of the present invention;

Fig. 5 is a schematic structural diagram of the principle of arranging multiple reflection modules in parallel according to an embodiment of the present invention;

Fig. 6 is a schematic structural diagram of the principle structure of an existing sequencer spectroscopic system;

Part names and serial numbers in the figure: 1-light source assembly 2-excitation filter 3-excitation dichroic mirror 4-objective lens 5-reflection module 6-imaging device 60-first imaging device 61-second imaging device 7- First emission filter module 70 - second emission filter module 8 - fluorescent sample.

Embodiments of the present invention

In order to more clearly illustrate the purpose, technical solutions and advantages of the embodiments of the present invention, the present invention will be further described below in conjunction with the embodiments, and a clear and complete description will be made. Obviously, the described embodiments are part of the embodiments of the present invention. rather than all examples. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

In the present invention, as shown in Figures 1 to 5, a fluorescence spectroscopic system for a sequencer includes a light source assembly 1, an optical path assembly and an imaging device 6, the light source assembly 1 is a light source assembly for emitting excitation light, Contains a light source and a collimation system; the optical path assembly includes an excitation filter 2, an excitation dichroic mirror 3, an objective lens 4, a reflective module 5 that cannot transmit light, and an emission filter module, and the excitation filter 2 makes it pass through Among the light emitted by the light source assembly 1, the light of the wavelength that can excite fluorescence passes through, and the light of this wavelength passes through the excitation dichroic mirror 3 and is reflected into the objective lens 4, and the light focused by the objective lens 4 is irradiated to the sample 8 marked with various fluorescent substances Above, the generated fluorescence is collected by the objective lens 4 and transmitted through the excitation dichroic mirror 3, and the passing fluorescence is split by the reflection module 5; both the excitation filter 2 and the excitation dichroic mirror 3 are installed on the The optical path of the light, and the excitation filter 2 is between the excitation dichroic mirror 3 and the light source assembly 1, so that the wavelength that can excite the fluorescence is filtered out and passed through the excitation filter 2, and then passed through the excitation The reflection of the dichroic mirror 3 goes to irradiate the fluorescence of the excitation fluorescent sample 8; at this moment, the objective lens 4 is installed on the optical path of the excitation light reflected by the excitation dichroic mirror 3, and the objective lens 4 will excite the reflected light of the dichroic mirror 3 irradiate on the sample to excite the sample to generate fluorescence; the imaging device 6 includes a first imaging device 60 and a second imaging device 61 to receive the fluorescent signal to generate a corresponding image, and the first imaging device 60 is installed in the excitation two-way The optical path of the fluorescent light transmitted by the color mirror 3; the reflective module 5 is movable between the first imaging device 60 and the excitation dichroic mirror 3 and is arranged on the optical path of the fluorescent light transmitted by the excitation dichroic mirror 3, and the reflective module 5 is movable Being arranged on the optical path of the fluorescent light transmitted by the excitation dichroic mirror 3 means that the reflective module 5 can be on the optical path or removed from the optical path. When it is on the optical path, it will reflect the fluorescent light and the reflected light Just shoot to the second imaging device 61. At this time, the reflective module 5 is movably arranged at an angle of 45 degrees on the optical path that excites the fluorescent light transmitted by the dichroic mirror 3, which can better reflect the fluorescent light. When it is removed from the optical path, If there is no obstacle on the optical path, it directly shoots to the first imaging device 60, and the two different states of the reflective module 5 relative to the optical path allow the fluorescence to be captured and imaged in different routes; the second imaging device 61 is installed on the reflective The optical path of the fluorescent light reflected by the module 5; the first emission filter module 7 is installed between the first imaging device 60 and the reflective module 5 on the optical path that receives the fluorescent light, and the second imaging device 61 and the reflective module 5, a second emission filter module 70 is installed on the optical path for receiving fluorescence, and the first emission filter module 7 filters the fluorescence to obtain the fluorescence of a dye in the sample, which is then captured by the first imaging device 60 receives and generates an image, and the second emission filter module 70 also filters the fluorescence to obtain the fluorescence of another dye in the sample, which is then received by the second imaging device 61 to generate an image. Both emission filter modules include Tube lens and emission filter, two different emission filter modules get different fluorescence. The operation process of the whole system is that the light source component 1 emits the collimated excitation light, passes through the excitation filter 2, is reflected by the excitation dichroic mirror 3 into the objective lens 4, and the light focused by the objective lens 4 irradiates various fluorescent substance marks On the sample 8, the generated fluorescence is collected by the objective lens 4, passes through the excitation dichroic mirror 3, and the transmitted fluorescence is split by the reflection module 5. When the reflection module 5 is in the optical path, as shown by the dotted line, the fluorescence is reflected, The fluorescence of one of the dyes is obtained after being filtered by the second emission filter module 70, and is received by the second imaging device 61 to generate image one; The fluorescence of another dye is obtained after being filtered by the first emission filter module 7 and received by the first imaging device 60 to generate image two. Light splitting is simple, easy to use, and the development cost of the reflective module 5 is low. The components contained in the reflective module 5 are commonly used components. The processing is mature, the technical threshold is low, and the individual price is relatively lower. Domestic products can basically meet the requirements. No need to rely on imports; and the versatility of the reflective module 5 is relatively strong, the reflective module 5 is not limited by fluorescent dyes, and different fluorescent dyes can be used, and the reflector of the reflective module 5 can be used for ultraviolet light, visible light, and near-infrared light. High reflectivity reflection; the use conditions of the reflective module 5 are not high, and the reflective module 5 does not have high requirements on the collimation and angle of the incident light. The reflectivity is only related to the wavelength of the reflected light, and the compatibility is better.

As a further improvement, the reflective module 5 includes a light-tight reflector and a mount for mounting the reflector, and the mount is movable horizontally or rotated on the optical path that excites the fluorescent light transmitted by the dichroic mirror 3 , and the reflector is set on the mounting base at an angle of 45° corresponding to the incident direction of the fluorescence, the reflector moves synchronously with the mounting base, and the movable setting of the mounting base on the optical path is the movement relative to the optical path in the horizontal direction Or it can be realized by rotating relative to the optical path. At the same time, when the reflector moves horizontally or rotates to the optical path, its reflective surface can always face the incident direction of the fluorescence. At this time, the reflector is any one of the metal layer reflector and the dielectric film reflector. The reflective layer on the metal layer reflector is gold, silver, aluminum, copper, tin, nano and other specular reflection gold. The shape of the reflector is Then it is any existing reflector shape such as plane reflector and reflector prism. For the movable setting of the mount on the optical path, as shown in Figure 1, the mount is movable horizontally on the optical path that excites the fluorescent light transmitted by the dichroic mirror 3, and the mount is installed on a On the translation stage that is close to or far away from the second imaging device in the one-dimensional direction, the translation platform is electrically connected to the control terminal, and the horizontal movement of the mounting base is realized through the translation platform, and the mounting base is fixedly installed on the translation platform. The translation stage is a one-dimensional movement, and any existing translation stage can be used, only to make it move in any one-dimensional direction on the XYZ axis according to the direction of the optical path of the system, such as a screw, in the second imaging device 61 When it is at the left and right positions of the optical path, the translation stage pushes the mounting seat to move left and right; when the second imaging device 61 is at the front and rear positions of the optical path, the translation stage pushes the mounting seat to move back and forth, and the movement of the translation stage is controlled by the control terminal. The terminal uses a computer, and installs the corresponding control program on the computer for control. The control terminal issues control instructions according to different needs, and the instructions are transmitted to the control circuit board of the translation platform, and the control circuit board controls the relevant components of the translation platform according to the instructions. The work makes the translation stage move accordingly, and then drives the mount to move horizontally, so that when the reflector enters the optical path, the fluorescence is reflected, and when it moves out of the optical path, the fluorescence is transmitted, thereby realizing the spectroscopic function of fluorescence. The mounting seat is movably arranged on the optical path that excites the fluorescent light transmitted by the dichroic mirror 3 in a rotating manner. There are two different ways to realize the rotating manner, as shown in FIG. 2 , one is that the mounting seat With any end as the rotation center, the limited rotation is set on the optical path of the fluorescent light transmitted by the excitation dichroic mirror 3, and the axis line of the rotation center of the mounting base is perpendicular to the optical path of the fluorescent light transmitted by the excitation dichroic mirror 3 and the reflector The optical path of the reflected fluorescent light, that is to say, the axis of rotation of the mount is perpendicular to the plane formed by the transmission and reflection of the fluorescent light, so that the mount is movable on the optical path of the fluorescent light transmitted by the dichroic mirror 3 , and the limit of rotation makes it better reflect the fluorescence when it rotates to the corresponding angle, maintain the consistency of imaging, and avoid systematic errors. Any existing limit can be used for the limit According to different needs, make corresponding selections, such as rotating to a fixed angle position at one time, or rotating to any desired angular position through a motor drive, and the rotation center is set accordingly according to the position of the second imaging device 61 , when the mounting base and the second imaging device 61 are on the same side of the transmitted fluorescence optical path, the center of rotation is set at the upper end of the mounting base, and the mounting base rotates in a direction relatively away from the second imaging device 61, between the mounting base and the second When the imaging device 61 is on opposite sides of the transmitted fluorescence optical path, the center of rotation is set at the lower end of the mount, and the mount rotates in a direction relatively close to the second imaging device 61 to realize light splitting of the fluorescence. As shown in Figures 3 and 4, the other is that the mounting seat is a turntable that can be rotated in a clockwise direction at an angle of 45° to the optical path that excites the fluorescent light transmitted by the dichroic mirror, The turntable is provided with a plurality of through holes whose axial direction forms a clockwise angle of 45° with the optical path of the fluorescent light transmitted by the excitation dichroic mirror 3, and the reflectors are also provided with a plurality of reflectors spaced apart from each other. In the through hole, that is, the turntable is provided with a plurality of through holes around its circumference, and the installation of the reflector in the through hole is installed in a plurality of through holes at intervals, that is, a reflector is installed in a through hole, and the corresponding mirror is installed in the through hole. The reflector is not installed in the two adjacent through holes, and the installation of the reflector in the through hole is realized by analogy. At this time, the reflector is installed in the through hole with its reflective surface facing the incident direction of the fluorescent light. During the rotation of the turntable, when the through hole not equipped with a reflector is on the fluorescent light path, the fluorescent light is transmitted from the through hole and shoots to the first imaging device 60; when the through hole equipped with a reflective mirror is on the fluorescent light path , the reflector reflects the fluorescent light, and the fluorescent light shoots to the second imaging device 61 to realize light splitting. For the rotation of the mounting base, the power to drive its action is that a motor is connected to the mounting base, and the motor is electrically connected to a control terminal. The control terminal also uses a computer to drive the motor through the control program on the computer. The operation realizes the automatic adjustment, which makes the adjustment more precise and more convenient to use.

As a further improvement, as shown in FIG. 5 , multiple second imaging devices 61 are arranged side by side, and the number of reflective modules 5 and second emission filter modules 70 is equal to the number of second imaging devices 61 Identical, and reflection module 5, the second emission filter module 50, the second imaging device 61 are arranged correspondingly one by one, that is, a reflection module 5 corresponds to a second emission filter module 50, and the second emission filter module The module 70 corresponds to a second imaging device 61, which forms reflection imaging of fluorescence, and a plurality of them arranged side by side forms multi-channel light splitting. Different second emission filter modules 70 can be used to perform multiple kinds of fluorescent light splitting. It is more convenient to use and lower in cost.

A fluorescent spectroscopic method for a sequencer, which uses the fluorescent spectroscopic system of a sequencer as described in any one of the preceding items to perform fluorescence spectrometry, and uses the system to irradiate the light focused by the objective lens onto samples marked with various fluorescent substances, which is simple and convenient, Good light splitting can be achieved without high requirements for use.

It should be understood that those skilled in the art can make improvements or changes based on the above description, and all these improvements and changes should fall within the protection scope of the appended claims of the present invention.

Claims

A sequencer fluorescence spectroscopic system, characterized in that: comprise a light source assembly, an optical path assembly and an imaging device, the light source assembly is a light source assembly for emitting excitation light; the optical path assembly includes an excitation filter, an excitation dichroic mirror, objective lens, light-impermeable reflection module, and emission filter module; the excitation filter and the excitation dichroic mirror are all installed on the optical path of the excitation light emitted by the light source assembly, and the excitation filter is in the excitation Between the dichroic mirror and the light source assembly; the objective lens is installed on the optical path of the excitation light reflected by the excitation dichroic mirror; the imaging device includes a first imaging device and a second imaging device, and the first imaging device is installed On the optical path of the fluorescent light transmitted by the excitation dichroic mirror; the reflection module is movable between the first imaging device and the excitation dichroic mirror and arranged on the optical path of the fluorescent light transmitted by the excitation dichroic mirror; the second imaging The device is installed on the optical path of the fluorescent light reflected by the reflective module; a first emission filter module is installed between the first imaging device and the reflective module on the optical path for receiving the fluorescent light, and between the second imaging device and the reflective module A second emission filter module is installed on the optical path for receiving fluorescence.
According to the described sequencer fluorescence spectroscopic system of claim 1, it is characterized in that: the reflective module comprises a light-tight reflector, a mount for mounting the reflector, and the mount is movable horizontally or rotated to be arranged on the excitation The optical path of the fluorescent light transmitted by the dichroic mirror, and the reflective mirror is arranged on the mounting base at an included angle of 45° corresponding to the incident direction of the fluorescent light.
According to the described sequencer fluorescence spectroscopic system of claim 2, it is characterized in that: the mounting seat is movable and arranged on the optical path that excites the transmitted fluorescence of the dichroic mirror in a horizontal movement mode, and the mounting seat is installed in a one-dimensional direction On the translation platform that is close to or far from the second imaging device, the translation platform is electrically connected with a control terminal.
According to the described sequencer fluorescence spectroscopic system of claim 2, it is characterized in that: the mounting seat is movable and arranged on the optical path that excites the fluorescence transmitted by the dichroic mirror in a rotating manner, and the mounting seat is limited by any end as the center of rotation. The position rotation is set on the optical path of the fluorescent light transmitted by the excitation dichroic mirror, and the axis line of the rotation center of the mounting seat is perpendicular to the optical path of the fluorescent light transmitted by the excitation dichroic mirror and the optical path of the fluorescent light reflected by the reflector.
According to the described sequencer fluorescence spectroscopic system of claim 2, it is characterized in that: the mounting base is movable and arranged on the optical path of the fluorescent light transmitted by the excitation dichroic mirror in a rotating manner, and the mounting base can be dichroic with the excitation The optical path of the fluorescent light transmitted by the color mirror is clockwise with a 45° included angle, and the direction is a turntable for limited rotation. On the turntable, a plurality of axial directions are arranged in parallel with the optical path of the fluorescent light transmitted by the excitation dichroic mirror in a clockwise direction. There are also multiple through holes in the direction of an included angle of 45°, and the reflectors are arranged in the through holes at intervals.
According to claim 4 or 5, the sequencer fluorescence spectroscopic system is characterized in that: the mounting base is connected with a motor, and the motor is electrically connected with a control terminal.
The fluorescence spectroscopic system for a sequencer according to claim 2, wherein the reflector is any one of a metal layer reflector and a dielectric film reflector.
According to the described sequencer fluorescence spectroscopic system of claim 1, it is characterized in that: described second imaging device is provided with a plurality of side by side, and the quantity that described reflection module and second emission filter module are set is the same as that of second imaging device The numbers are the same, and the reflective module, the second emission filter module, and the second imaging device are arranged correspondingly.
The fluorescence spectroscopic system of the sequencer according to claim 1, characterized in that: the reflection module is movably arranged at an angle of 45 degrees on the optical path that excites the fluorescence transmitted by the dichroic mirror.
A method for fluorescence spectroscopy of a sequencer, characterized in that: the fluorescence spectroscopy of a sequencer as described in any one of claims 1 to 9 is used for fluorescence spectroscopy.