CN117368174A

CN117368174A - Imaging system and imaging method

Info

Publication number: CN117368174A
Application number: CN202311669454.3A
Authority: CN
Inventors: 陈龙超; 梁倩; 王谷丰; 赵陆洋
Original assignee: Shenzhen Sailu Medical Technology Co ltd
Current assignee: Shenzhen Sailu Medical Technology Co ltd
Priority date: 2023-12-07
Filing date: 2023-12-07
Publication date: 2024-01-09

Abstract

The invention discloses an imaging system and an imaging method, which are used for a gene sequencer. The light source assembly can emit various excitation lights, the objective table is arranged on the light path of the light source assembly, the objective table carries a sample to be detected, each excitation light irradiates the sample to be detected to excite a strip-shaped fluorescent light extending along a first direction, the imaging assembly is used for receiving the strip-shaped fluorescent light of a plurality of positions, each position comprises a plurality of strip-shaped fluorescent lights, the strip-shaped fluorescent light of the plurality of positions generates a strip fluorescent image of a surface area, the strip fluorescent lights form strip fluorescent areas on the strip fluorescent image, the extending directions of the strip fluorescent areas of each strip fluorescent image are different, and the strip fluorescent images are reconstructed into super-resolution images.

Description

Imaging system and imaging method

Technical Field

The present application relates to the field of gene sequencing technology, and more particularly, to an imaging system and an imaging method.

Background

The gene sequencer detects four bases of ATCG in the chip in the process of gene sequencing. In general, the higher the data volume density on the chip, the lower the detection cost, and the higher the detection of the high-density chip, the higher the resolution of the gene sequencer is needed, so that the gene sequencer needs to be realized by adopting a super-resolution optical means.

The imaging system of the gene sequencer can adopt a structured light illumination super-resolution microscope to realize higher resolution imaging, and at present, the widely adopted structured light illumination super-resolution microscope imaging scheme is mainly based on area array imaging, but because in an area imaging mode, each time a view field is switched, a sample object stage needs to be started and stopped once, and the time consumption is long. And image stitching is also needed to be considered in scanning imaging, and the imaging boundary may need to be scanned in an overlapping manner to maintain a good imaging effect, so that the scanning time is further prolonged.

In the related art, the above problems can be solved by a line scanning structured light illumination super-resolution microscopic imaging method, but the resolution improvement in all directions on a two-dimensional plane is difficult to satisfy in the existing line scanning super-resolution microscopic illumination implementation.

Disclosure of Invention

The embodiment of the application provides an imaging system and an imaging method capable of meeting resolution improvement in all directions on a two-dimensional plane.

An imaging system of an embodiment of the present application for a gene sequencer, comprising:

a light source assembly capable of emitting a plurality of excitation lights;

the objective table is arranged on the light path of the light source assembly, and is used for carrying a sample to be tested, and each excitation light irradiates the sample to be tested to excite a stripe-shaped fluorescence extending along a first direction;

an imaging assembly for receiving said striped fluorescence at a plurality of locations, each location comprising a plurality of said striped fluorescence, the same one of said striped fluorescence at a plurality of locations generating a striped fluorescence image of a planar area, said striped fluorescence forming striped fluorescence regions on said striped fluorescence image, each of said striped fluorescence images having a different direction of extension of said striped fluorescence regions, a plurality of said striped fluorescence images being reconstructed into a super-resolution image.

In some embodiments, the three stripe fluorescent images have three stripe fluorescent regions extending in directions of 0 °, 120 ° and 240 ° from the first direction.

In certain embodiments, the light source assembly includes a laser emitter for emitting laser light and a spatial light modulator for diffracting the laser light, and a plurality of converging mirrors for conditioning and sorting the laser light.

In some embodiments, the light source assembly further comprises a diaphragm, the diaphragm is located on the optical path, the diaphragm is provided with a plurality of light through holes, and the diaphragm allows the partially diffracted laser light to pass through from the light through holes.

In certain embodiments, the light passing holes include a first light passing hole, a second light passing hole, a third light passing hole and a fourth light passing hole, the first light passing hole, the second light passing hole, the third light passing hole and the fourth light passing hole are sequentially arranged along a straight line, the distance from the first light passing hole to the second light passing hole is equal to the distance from the third light passing hole to the fourth light passing hole, and the distance from the first light passing hole to the fourth light passing hole is twice the distance from the second light passing hole to the third light passing hole.

In some embodiments, the second light-passing hole and the third light-passing hole are symmetrical about an optical axis of the excitation light, and the first light-passing hole and the fourth light-passing hole are symmetrical about the optical axis of the excitation light.

In some embodiments, the stage is coupled to a drive assembly that drives the stage in the first direction, and the imaging system further includes a control assembly that controls the drive assembly to drive the stage in the first direction and controls the imaging assembly to receive the fringe fluorescent images such that a plurality of fringe fluorescent regions of the fringe fluorescent images have a plurality of different directions of extension.

In certain embodiments, the imaging system further comprises a first dichroic mirror for collecting the fluorescence and transmitting the fluorescence to the first dichroic mirror, and an objective for reflecting the excitation light to the sample under test and for transmitting the striped fluorescence to the imaging assembly.

In certain embodiments, the imaging assembly includes a plurality of second dichroic mirrors that distribute the fluorescence light to a plurality of imaging channels, the second dichroic mirrors being located between the imaging channels and the first dichroic mirrors, each of the imaging channels including a barrel mirror for collecting the fluorescence light to the line camera and a line camera for receiving the fluorescence light.

The embodiment of the application provides an imaging method of an imaging system, which is used for a gene sequencer, wherein the imaging system comprises a light source assembly, an objective table and an imaging assembly, a sample to be detected is carried on the objective table, and the imaging method comprises the following steps:

emitting excitation light through the light source assembly to irradiate the sample to be detected so as to excite stripe-shaped fluorescence extending along a first direction;

receiving, by the imaging assembly, imaging of the plurality of locations of the striped fluorescence to generate a striped fluorescence image of the area, the striped fluorescence forming a striped fluorescence region on the striped fluorescence image;

changing the parameters of the spatial light modulator, and changing excitation light emitted by the light source assembly, and receiving and generating at least three fringe fluorescent images through the imaging assembly, wherein the extending directions of a plurality of fringe fluorescent areas of the fringe fluorescent images are different;

and reconstructing a super-resolution image according to a plurality of fringe fluorescent images with different extending directions of a plurality of fringe fluorescent regions by the imaging component.

In some embodiments, the method for generating a fringe fluorescence image comprises:

the objective table is driven by the driving component to move the sample to be measured along the first direction, meanwhile, the light source component is controlled to enable the excitation light to irradiate the sample to be measured and excite the striped fluorescence, and imaging of the striped fluorescence at a plurality of positions is received by the imaging component to generate a striped fluorescence image of the area.

In some embodiments, the method of replacing excitation light emitted by the light source assembly comprises:

the light source assembly is internally provided with a diaphragm, the diaphragm is positioned on the excitation light path, a plurality of light passing holes are formed in the diaphragm, and the excitation light is changed by changing the service condition of the light passing holes in the diaphragm.

In the imaging system of the embodiment of the application, the light source component emits excitation light to excite stripe fluorescence on the sample to be detected, and the imaging component generates stripe fluorescence images with different extending directions of a plurality of stripe fluorescence areas to reconstruct into a final super-resolution image, so that the imaging system can equivalently replace structured light illumination in a plurality of directions, further meet the resolution improvement of each direction on a two-dimensional plane, and effectively improve the imaging quality of the super-resolution image.

Additional aspects and advantages of embodiments of the application will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of embodiments of the application.

Drawings

The foregoing and/or additional aspects and advantages of the present application will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings, in which:

FIG. 1 is a schematic diagram of the main structure of an imaging system of an embodiment of the present application;

FIG. 2 is a block schematic diagram of an imaging system of an embodiment of the present application;

FIG. 3 is a frequency domain diagram of SIM imaging in different fringe directions in the related art;

FIG. 4 is a SIM imaging frequency domain plot of excitation light according to an embodiment of the present application;

FIG. 5 is a schematic structural view of an imaging system according to an embodiment of the present application;

FIG. 6 is a schematic diagram of a first fringe fluorescent image generation mode of an embodiment of the present application;

FIG. 7 is a schematic view of illumination directions of first stripe fluorescent image excitation light according to an embodiment of the present application;

FIG. 8 is a schematic representation of a first fringe fluorescence image imaging of an embodiment of the present application;

FIG. 9 is a schematic representation of actual fringes of excitation light of a first fringe fluorescence image of an embodiment of the present application;

FIG. 10 is a lateral intensity distribution plot of excitation light for a first fringe fluorescent image of an embodiment of the present application;

FIG. 11 is a schematic diagram of a second fringe fluorescent image generating mode of an embodiment of the present application;

FIG. 12 is a schematic view of the illumination direction of the excitation light of the second fringe fluorescent image in accordance with the embodiments of the present application;

FIG. 13 is a schematic view of a diaphragm structure according to an embodiment of the present application;

FIG. 14 is a schematic representation of a second fringe fluorescence image imaging of an embodiment of the present application;

FIG. 15 is a schematic representation of actual fringes of excitation light of a second fringe fluorescence image of an embodiment of the present application;

FIG. 16 is a lateral intensity distribution plot of excitation light for a second fringe fluorescent image of an embodiment of the present application;

FIG. 17 is a schematic diagram of a third fringe fluorescent image generating mode of an embodiment of the present application;

FIG. 18 is a schematic view of illumination directions of third fringe fluorescent image excitation light in accordance with an embodiment of the present application;

FIG. 19 is a third fringe fluorescence imaging schematic of an embodiment of the present application;

FIG. 20 is a schematic representation of actual fringes of excitation light of a third fringe fluorescence image of an embodiment of the present application;

FIG. 21 is a lateral intensity distribution of excitation light of a third fringe fluorescent image of the embodiments of the present application;

fig. 22 is a flow chart of an imaging method of an embodiment of the present application.

Description of main reference numerals: the imaging system 100, the light source assembly 10, the laser emitter 11, the spatial light modulator (or micro-mirror array) 12, the multimode optical fiber 13, the first converging mirror 14, the second converging mirror 15, the third converging mirror 16, the fourth converging mirror 17, the diaphragm 18, the first light passing hole 181, the second light passing hole 182, the third light passing hole 183, the fourth light passing hole 184, the stage 20, the imaging assembly 30, the second dichroic mirror 31, the imaging channel 32, the barrel lens 321, the line camera 322, the optical filter 323, the sample under test 40, the striped fluorescence 41, the striped fluorescence region 42, the imaging region 43, the driving assembly 50, the control assembly 60, the objective lens 71, and the first dichroic mirror 72.

Detailed Description

Embodiments of the present application are further described below with reference to the accompanying drawings. The same or similar reference numbers in the drawings refer to the same or similar elements or elements having the same or similar functions throughout.

In addition, the embodiments of the present application described below in conjunction with the drawings are exemplary only and are not to be construed as limiting the present application.

In this application, unless expressly stated or limited otherwise, a first feature "up" or "down" a second feature may be the first and second features in direct contact, or the first and second features in indirect contact via an intervening medium. Moreover, a first feature being "above," "over" and "on" a second feature may be a first feature being directly above or obliquely above the second feature, or simply indicating that the first feature is level higher than the second feature. The first feature being "under", "below" and "beneath" the second feature may be the first feature being directly under or obliquely below the second feature, or simply indicating that the first feature is less level than the second feature.

In the sequencing process of the gene sequencer, fluorescent imaging is needed to be carried out on four bases of ATCG respectively, and as each target point to be detected is in a submicron scale range, the gene sequencer can inevitably adopt a microscopic imaging mode to realize the detection purpose. An important index of a gene sequencer is the dot density on the chip, which determines the amount of data on a chip, and also determines the cost of production of unit data. Since the difference between the dot density and the amount of the reagent used per unit area is not large, the higher the dot density is, the lower the detection cost is.

High density chips require coordination of higher resolution microscopy systems. However, conventional wide field microscope systems have diffraction limits and resolution is typically up to around 200 nm. In order to achieve higher resolution and thus higher chip dot density, it is necessary to implement this by means of "super-resolution" optics.

The term "super resolution" is used to refer to exceeding the diffraction limit of an optical system. The most probable super-resolution technique for the gene sequencer is an optical illumination super-resolution microscope (Structured Illumination Microscopy, SIM), and the method utilizes the principle of molar fringes, namely that the superposition of two pieces of high-frequency information is low-frequency information, so that the high-frequency information which cannot pass through an objective lens originally can be received by an optical system, the spectrum range of the original wide-field microscopic imaging (shown as a in fig. 3) is expanded by 2 times in the corresponding fringe modulation direction (shown as b in fig. 3), and the improvement of twice resolution is finally realized.

The imaging system of the gene sequencer can adopt a structured light illumination super-resolution microscope to realize higher resolution imaging, and at present, the widely adopted structured light illumination super-resolution microscope imaging scheme is mainly based on area array imaging, but because in the area array imaging mode, each time a view field is switched, a sample object stage is started and stopped once, and the time consumption is long. And image stitching is also needed to be considered in scanning imaging, and the imaging boundary may need to be scanned in an overlapping manner to maintain a good imaging effect, so that the scanning time is further prolonged. In the field of gene sequencing, imaging speed is an important indicator that determines the time to output a sequencing report. In order to further increase the imaging detection speed, a scheme of adopting line scanning imaging is a trend in the future.

In the related art, the above problems can be solved by a line scanning super-resolution microscopic imaging method, and the existing illumination implementation of the line scanning super-resolution microscope is usually structured light illumination, that is, the sample to be measured is illuminated by structured light and fluorescence is excited, and then the sample to be measured is acquired by an imaging component and reconstructed into a super-resolution image.

In view of this, referring to fig. 1-22, an imaging system 100 for a gene sequencer includes a light source assembly 10, a stage 20, and an imaging assembly 30.

The light source assembly 10 can emit various excitation lights, the objective table 20 is arranged on the light path of the light source assembly 10, the objective table 20 carries a sample 40 to be measured, each excitation light irradiates the sample 40 to be measured to excite a strip-shaped fluorescent light 41 extending along a first direction, the imaging assembly 30 is used for receiving the strip-shaped fluorescent light 41 at a plurality of positions, each position comprises the strip-shaped fluorescent light 41, the strip-shaped fluorescent light 41 at the same one of the plurality of positions generates a strip-shaped fluorescent image of a surface area, the strip-shaped fluorescent light 41 forms a strip-shaped fluorescent area 42 on the strip-shaped fluorescent image, the strip-shaped fluorescent light area 42 of each strip-shaped fluorescent image has different extending directions, and the strip-shaped fluorescent images are reconstructed into super-resolution images.

In the imaging system 100 of the embodiment of the present application, the light source assembly 10 emits excitation light to excite the striped fluorescence 41 on the sample 40 to be measured, and the imaging assembly 30 generates a plurality of striped fluorescence images with different extending directions of the striped fluorescence regions 42 to reconstruct into a final super-resolution image, which equivalently replaces the structured light illumination in a plurality of directions, thereby meeting the resolution improvement in each direction on the two-dimensional plane and effectively improving the imaging quality of the super-resolution image.

Specifically, the sample 40 to be measured is a biological sample with fluorescent dye, the biological sample is arranged on a gene sequencing chip, the gene sequencing chip is placed on the objective table 20, excitation light emitted by the light source assembly 10 irradiates the sample 40 to be measured to form a first light spot, the fluorescent dye on the biological sample is excited by the light spot to form striped fluorescence 41, and the light spot and the striped fluorescence 41 extend towards the first direction X.

In some embodiments, the three stripe fluorescent images have three stripe fluorescent images, and the included angles between the extending directions of the three stripe fluorescent regions 42 of the three stripe fluorescent images and the first direction are respectively 0 °, 120 ° and 240 °, specifically, the included angles between the extending directions of the three stripe fluorescent regions 42 of the three stripe fluorescent images and the first direction are respectively 0 °, 120 ° and 240 °, which can be equivalent to the structured light illumination in the three directions of 0 °, 120 ° and 240 °, as shown in fig. 4, and the structured light illumination in the three directions can satisfy the resolution improvement in each direction on the two-dimensional plane.

Referring to fig. 5, in some embodiments, the light source assembly 10 includes a laser emitter 11 and a spatial light modulator 12, where the laser emitter 11 is configured to emit laser light, and the spatial light modulator 12 is configured to diffract the laser light, and in particular, the laser emitter 11 may emit laser light with a specific wavelength to excite the sample 40 to be measured to generate fluorescence; the light source assembly 10 may further include a multimode optical fiber 13, the multimode optical fiber 13 being positioned between the laser transmitter 11 and the spatial light modulator 12, the laser transmitter 11 transmitting laser light to the spatial light modulator 12 through the multimode optical fiber 13.

In some embodiments, the light source assembly 10 further includes a plurality of converging mirrors for adjusting and sorting the laser light, and in particular, the converging mirrors include a first converging mirror 14, the first converging mirror 14 is located between the multimode optical fiber 13 and the spatial light modulator 12, and the first converging mirror 14 is used for collimating the laser light emitted from the laser emitter 11 through the multimode optical fiber 13 into parallel light.

In some embodiments, the light source assembly 10 further includes a diaphragm 18, the diaphragm 18 is located on an optical path, the diaphragm 18 has a plurality of light holes, the diaphragm 18 makes part of diffracted laser light pass through from the different light holes, specifically, the converging mirror further includes a second converging mirror 15, a third converging mirror 16 and a fourth converging mirror 17, the first converging mirror 14 is located between the spatial light modulator 12 and the diaphragm 18, the spatial light modulator 12 reflects and diffracts parallel light collimated by the first converging mirror 14, and then focuses the parallel light onto the diaphragm 18 through the second converging mirror 15, the diaphragm 18 only allows ±1-order light to pass through, and light of 0 order and higher orders in the diffracted laser light is blocked from passing through the diaphragm 18 sequentially through the third converging mirror 16 and the fourth converging mirror 17, and after the light passing through the diaphragm 18 is collimated by the third converging mirror 16, the light is focused by the fourth converging mirror 17.

Referring to fig. 5 and 13, in some embodiments, the light passing holes include a first light passing hole 181, a second light passing hole 182, a third light passing hole 183, and a fourth light passing hole 184, the first light passing hole 181, the second light passing hole 182, the third light passing hole 183, and the fourth light passing hole 184 are sequentially disposed along a straight line, a distance from the first light passing hole 181 to the second light passing hole 182 is equal to a distance from the third light passing hole 183 to the fourth light passing hole 184, a distance from the first light passing hole 181 to the fourth light passing hole 184 is twice a distance from the second light passing hole 182 to the third light passing hole 183, further, the second light passing hole 182 is symmetrical with the third light passing hole 183 with respect to an optical axis of the excitation light, the first light passing hole 181 is symmetrical with the fourth light passing hole 184 with respect to the optical axis of the excitation light, specifically, the diaphragm 18 is circular, the first light passing hole 181, the second light passing hole 182, the third light passing hole 183 and the fourth light passing hole 184 are arranged along a diameter direction of the diaphragm 18, the center of the optical cable is located at a midpoint of a connection line between the second light passing hole 182 and the third light passing hole 183, wherein when the laser diffracted by the spatial light modulator 12 passes through the first light passing hole 181 and the fourth light passing hole 184, first excitation light is formed on the sample 40 to be tested, when the laser diffracted by the spatial light modulator 12 passes through the second light passing hole 182 and the third light passing hole 183, second excitation light is formed on the sample 40 to be tested, and a period of the second excitation light is twice that of the first excitation light.

Referring to fig. 2, in some embodiments, the stage 20 is connected to a driving assembly 50, and the driving assembly 50 drives the stage 20 to move along a first direction, and in particular, the driving assembly 50 is an electric displacement stage.

In certain embodiments, imaging system 100 further comprises a control assembly 60, control assembly 60 controlling drive assembly 50 to drive stage 20 in a first direction and imaging assembly 30 to receive the fringe fluorescent images such that fringe fluorescent regions 42 of the plurality of fringe fluorescent images have a plurality of different directions of extension.

Referring to fig. 5, in some embodiments, the imaging system 100 further includes a first dichroic mirror 72 and an objective 71, the objective 71 is configured to collect fluorescence and transmit the fluorescence to the first dichroic mirror 72, the first dichroic mirror 72 is configured to reflect excitation light to the sample 40 to be measured and transmit striped fluorescence 41 to the imaging assembly 30, and in particular, the fourth converging mirror 17 converges two lights passing through the aperture 18 at two points on a back focal plane of the objective 71, thereby generating excitation light on the sample 40 to be measured, wherein the excitation light and the striped fluorescence 41 are both sinusoidal stripes.

In certain embodiments, imaging assembly 30 comprises a plurality of second dichroic mirrors 31, plurality of second dichroic mirrors 31 distributing fluorescence light to a plurality of imaging channels 32, second dichroic mirrors 31 being located between imaging channels 32 and first dichroic mirrors 72, each imaging channel 32 comprising a barrel mirror 321 and a line camera 322, barrel mirror 321 for collecting fluorescence light to line camera 322, line camera 322 for receiving fluorescence light.

In certain embodiments, imaging assembly 30 further comprises a filter 323, filter 323 being positioned between barrel 321 and line camera 322, filter 323 being configured to filter fluorescence.

In some embodiments, the number of the second dichroic mirrors 31 is three, in particular, the ATCG four bases form four kinds of fluorescence, at least three second dichroic mirrors 31 are required for separating the four kinds of fluorescence one by one, the three second dichroic mirrors 31 are different kinds of dichroic mirrors, and further, the three second dichroic mirrors 31 are different in coating film for separating the four kinds of fluorescence formed by the ATCG four bases.

The embodiment of the application provides an imaging method of an imaging system 100, the imaging system 100 includes a light source assembly 10, an objective table 20 and an imaging assembly 30, the objective table 20 carries a sample 40 to be measured, and the imaging method includes:

step 01: emitting excitation light through the light source assembly 10 to be irradiated onto the sample 40 to be measured to excite the striped fluorescence 41 extending in the first direction;

step 02: receiving imaging of multiple locations of the striped fluorescence 41 by the imaging assembly 30 to generate a striped fluorescence image of the area, the striped fluorescence 41 forming a striped fluorescence region 42 on the striped fluorescence image;

step 03: changing the excitation light emitted by the light source assembly 10, receiving and generating at least three stripe fluorescent images through the imaging assembly 30, wherein the extending directions of a plurality of stripe fluorescent areas 42 of the plurality of stripe fluorescent images are different;

step 04: a super-resolution image is reconstructed by the imaging assembly 30 from a plurality of fringe fluorescent images having different directions of extension of the plurality of fringe fluorescent regions 42.

Specifically, the imaging system 100 further includes a control component 60, where the control component 60 emits excitation light through the light source component 10 to irradiate the sample 40 to be measured to excite the striped fluorescent light 41 extending along the first direction, and then the control component 60 controls the imaging component 30 to receive imaging of a plurality of positions of the striped fluorescent light 41 to generate a striped fluorescent image of the area, the striped fluorescent light 41 forms a striped fluorescent region 42 on the striped fluorescent image, then the excitation light emitted by the light source component 10 is replaced, at least three striped fluorescent images are received and generated through the imaging component 30, the extending directions of the plurality of striped fluorescent regions 42 of the plurality of striped fluorescent images are different, and a super-resolution image is reconstructed through the imaging component 30 according to the plurality of striped fluorescent images with the extending directions of the plurality of striped fluorescent regions 42 being different.

Further, the method for generating the streak fluorescent image includes:

the stage 20 is driven by the driving assembly 50 to move the sample 40 to be measured in the first direction, and simultaneously the light source assembly 10 is controlled to irradiate the sample 40 to be measured and excite the striped fluorescence 41, and the imaging assembly 30 receives the imaging of the striped fluorescence 41 at a plurality of positions to generate a striped fluorescence image of the area.

In some embodiments, the method of replacing excitation light emitted by the light source assembly 10 includes:

the light source assembly 10 is internally provided with a diaphragm 18, the diaphragm 18 is located on the light path, a plurality of light-passing holes are formed in the diaphragm 18, excitation light is changed by changing the service condition of the light-passing holes in the diaphragm 18, specifically, the light-passing holes in the diaphragm 18 comprise a first light-passing hole 181, a second light-passing hole 182, a third light-passing hole 183 and a fourth light-passing hole 184, and excitation light periods formed by passing through the light-passing holes from different light-passing holes are different.

In some embodiments, imaging using the line camera 322 is accomplished in conjunction with moving the motorized displacement stage to scan the sample 40 under test. Thus, this imaging approach results in the excitation light being fixed relative to the camera. SIM imaging, however, requires that the excitation light be fixed relative to the sample to achieve a structured light illumination effect. Therefore, in the scanning process of the electric displacement table, the excitation light needs to be changed correspondingly according to the scanning position to meet the actual illumination requirement, and in particular, the excitation light can be controlled by the spatial light modulator 12.

Since three stripe fluorescent images are required, the included angles between the extending directions of the three stripe fluorescent regions 42 of the three stripe fluorescent images and the first direction are respectively 0 °, 120 ° and 240 °, so as to be used for equivalent three-phase line scanning structure light, and the binary stripe with a period of 6 pixels is used as a simplified model to introduce a method for generating the extending directions of the three stripe fluorescent regions 42.

Referring to fig. 6, first, a first fringe fluorescent image is generated by forming an angle of 0 ° between the extending direction of the fringe fluorescent region 42 and the first direction. Taking a in fig. 6 as an example, each square represents each pixel of the final image. The thick and long frame in the figure indicates that the line camera 322 photographs the imaging region 43 once, and the imaging region 43 extends in a second direction, which is perpendicular to the first direction. Therefore, to complete 1-24 lines of imaging, the line camera 322 is required to be implemented in combination with the electric displacement stage scanning. The grey squares in the figure represent pixels with signals, i.e. illuminated areas on the side of the sample 40 to be measured. The illumination mode is stripe light illumination, and the period is 6 pixels. B in fig. 6 is the position of the stripe when the camera captures the second line image, c in fig. 6 is the position of the stripe when the 24 th line image is captured, after a group of 24 line images are completed, the phase of the excitation light is adjusted, and imaging is performed, as shown by d in fig. 6 to f in fig. 6 and h in fig. 6 to i in fig. 6, the excitation light of a in fig. 6 to c in fig. 6 is 0 ° phase, the excitation light of d in fig. 6 to f in fig. 6 is 120 ° phase, and the excitation light of h in fig. 6 to i in fig. 6 is 240 ° phase.

Referring to fig. 7, in order to achieve the above-mentioned illumination effect, when the line camera 322 shoots a certain line once, the actual excitation light on the whole surface of the sample 40 to be measured is shown. The fringes are produced by the spatial light modulator 12. I in fig. 7 a to 7 and i in fig. 6 a to 6 correspond to each other, and it is ensured that the position of the excitation light is the result shown in fig. 6 when the line camera 322 scans the corresponding number of lines. After the line camera 322 finishes scanning 1 to 24 lines of the sample, the final fringe fluorescent image is shown in fig. 8, specifically, the fringe fluorescent images generated corresponding to a to c in fig. 6 are a in fig. 8, the fringe fluorescent images generated corresponding to d to f in fig. 6 are b in fig. 8, and the fringe fluorescent images generated corresponding to h to i in fig. 6 are c in fig. 8.

Note that the illumination intensity of the actual excitation light emitted through the objective lens 71 is sinusoidal, so the final actual excitation light is shown in fig. 9 and 10, where fig. 9 is an actual stripe schematic, and fig. 10 is a lateral intensity distribution of the actual stripe in line 12. Although its intensity is changed from a binary stripe to a sinusoidal stripe, its period is still 6 pixels.

Referring to fig. 11, the second fringe fluorescent image with an angle of 120 ° between the extending direction of the fringe fluorescent region 42 and the first direction is generated by the method shown in fig. 11, and the projection of the excitation light with a period of 6 pixels in the second direction is abs (6/cos (120 °) =12 pixels in the second direction after the excitation light is tilted by 120 ° as shown in a in fig. 11. The method can be extended to any periodic stripe, i.e. the horizontal projection of the oblique stripe is 2 times the period of the vertical stripe. Since the stripes are oblique, when the line camera 322 is shifted, the excitation light needs to be shifted correspondingly to the right by tan (60 °) = 1.7321 pixels. Since a single pixel cannot be further split in a minimum unit, the actual offset pixel number of the excitation light with respect to the imaging first line at the time of imaging is:

X=ceil(i*tan(60°))

where ceil is the round up and i is the number of rows currently scanned. A in fig. 11 to c in fig. 11 are schematic line scanning procedures when excitation light in the 120 ° direction is in the 0 ° phase. Since the projection period of the stripe in the horizontal direction is 12 pixels, when the phase is changed from 0 ° to 120 °, the excitation light of each line needs to be shifted by 4 pixels in the phase change direction, which shift is controlled by the spatial light modulator 12, as indicated by d in fig. 11 to f in fig. 11. Similarly, when the phase is changed from 120 ° to 240 °, the excitation light of each line needs to be shifted by 4 pixels in the phase change direction, as shown by g in fig. 11 to i in fig. 11.

To achieve the above-described illumination effect, the actual excitation light on the entire sample surface is as shown in fig. 12 when the line camera 322 photographs a certain line at a time. The fringes are produced by the spatial light modulator 12. I in fig. 11 to 11 and i in fig. 12 to 12 correspond to each other, and the position of the excitation light is ensured as shown in fig. 12 when the line camera 322 scans the corresponding number of lines.

Since the period of the actual illumination of the excitation light inclined in the 120 ° direction on the sample 40 to be measured is twice as large as that of the excitation light in the 0 ° direction, the diaphragm 18 is designed as shown in fig. 13. Wherein the first light passing hole 181 and the fourth light passing hole 184 correspond to the conjugate of 0 ° direction excitation light in the frequency domain ±1 stage, and the second light passing hole 182 and the third light passing hole 183 correspond to the conjugate of 120 ° direction stripes in the frequency domain ±1 stage. The first light passing hole 181 and the fourth light passing hole 184 are equidistant from the center of the diaphragm 18, and the second light passing hole 182 and the third light passing hole 183 are equidistant from the center of the diaphragm 18. And the first and fourth light passing holes 181 and 184 are twice as far from the center of the diaphragm 18 as the second and third light passing holes 182 and 183 are from the center of the diaphragm 18.

After the line camera 322 finishes scanning 1 to 24 lines of the sample, the final second stripe fluorescent image is shown in fig. 14. The actual excitation light is shown in fig. 15 and 16. Fig. 15 is a schematic view showing the sinusoidal intensity distribution of the actual stripe, and fig. 16 is a lateral intensity distribution of the actual stripe in line 12. Although the intensity thereof is changed from the binary stripe to the sinusoidal stripe, the period thereof in the lateral direction is still 12 pixels.

Similar to the generation of the second stripe fluorescent image with an angle of 120 ° between the extending direction of the stripe fluorescent region 42 and the first direction, the generation of the third stripe fluorescent image with an angle of 240 ° between the extending direction of the stripe fluorescent region 42 and the first direction is shown in fig. 17. When the line camera 322 is shifted, the excitation light needs to be shifted leftward by tan (60 °) = 1.7321 pixels accordingly. Since a single pixel cannot be further split in a minimum unit, the actual offset pixel number of the excitation light with respect to the imaging first line at the time of imaging is:

X=ceil(i*tan(60°))

where ceil is the round up and i is the number of rows currently scanned. A in fig. 17 to c in fig. 17 are schematic line scanning procedures when excitation light in the 120 ° direction is in the 0 ° phase. Since the projection period of the excitation light in the horizontal direction is 12 pixels, when the phase is changed from 0 ° to 120 °, the excitation light of each line needs to be shifted by 4 pixels in the phase change direction, as shown by d in fig. 17 to f in fig. 17. Similarly, when the phase is changed from 120 ° to 240 °, the excitation light of each line needs to be shifted by 4 pixels in the phase change direction, as shown by g in fig. 17 to i in fig. 17.

To achieve the above-described illumination effect, the actual excitation light on the entire sample surface is as shown in fig. 18 when the line camera 322 photographs a certain line at a time. The fringes are produced by the spatial light modulator 12. I in fig. 17 a to 17 and i in fig. 18 a to 18 correspond to each other, and when the line camera 322 scans the corresponding number of lines, the position of the excitation light is ensured to be the result shown in fig. 18. After the line camera 322 finishes scanning 1 to 24 lines of the sample, the final third fringe fluorescent image is shown in fig. 19. The actual excitation light is shown in fig. 20 and 21. Fig. 20 is a schematic view of the sinusoidal intensity distribution of the actual stripe, and fig. 21 is a lateral intensity distribution of the actual stripe in line 12. Although the intensity thereof is changed from the binary stripe to the sinusoidal stripe, the period thereof in the lateral direction is still 12 pixels.

In summary, in the imaging system and the imaging method according to the embodiments of the present application, through the arrangement of the laser emitter 11, the spatial light modulator 12, the first converging mirror 14, the second converging mirror 15, and the diaphragm 18 of the light source assembly, the laser emitted by the laser emitter 11 can be diffracted by the spatial light modulator 12 and irradiated onto the diaphragm 18, the diaphragm 18 can enable the laser of ±1 level to pass, and the laser of 0 level and higher level in the diffracted laser is blocked, so that the laser after passing through the diaphragm 18 is irradiated onto the sample to be measured by the excitation light formed by the collimation of the third converging mirror 16 and the fourth converging mirror 17 and forms the excitation stripe fluorescence, the high-frequency information which cannot be obtained originally can be shifted to the passband to be represented by the low-frequency intensity, when the subsequent algorithm is shifted back to the correct position, the higher resolution can be obtained, and simultaneously, the spatial light modulator 12 can adjust the phase of the laser, so that the phase of the stripe fluorescence is changed, and the imaging assembly 30 can obtain images of different phases according to the stripe fluorescence of different phases. The laser is controlled to pass out of the light passing hole of the diaphragm 18, so that periodic change can be realized, and further, the imaging difficulty of the imaging assembly in different directions can be simplified, so that the imaging system is simple to operate and easy to implement. The collimated laser light is reflected by the first dichroic mirror 72, passes through the objective lens 71, irradiates the sample, and excites stripe-shaped fluorescence, which is transmitted to the imaging assembly 30 through the objective lens 71. After entering the imaging assembly 30, the striped fluorescence is distributed by a plurality of second dichroic mirrors 31 into four different imaging channels 32 according to four colors to correspond to four different bases; the imaging channel 32 is provided with an optical filter 323 for filtering stray light to avoid interference with imaging, the end of the imaging channel 32 is provided with a line camera 322, the line camera 322 is used for forming stripe fluorescent images with stripe fluorescent regions of 0 DEG, 120 DEG and 240 DEG respectively from stripes of different phases, and then reconstructing a plurality of stripe fluorescent images into a super-resolution image.

In some embodiments of the present invention, the module involved may be a single-chip microcomputer chip, integrated with a processor, a memory, a communication module, etc. The processor may be a central processing unit (Central Processing Unit, CPU), but may also be other general purpose processors, digital signal processors (Digital Signal Processor, DSP), application specific integrated circuits (Application Specific Integrated Circuit, ASIC), off-the-shelf programmable gate arrays (Field Programmable Gate Array, FPGA) or other programmable logic devices, discrete gate or transistor logic devices, discrete hardware components, or the like.

Any process or method descriptions in flow charts or otherwise described herein may be understood as representing modules, segments, or portions of code which include one or more executable instructions for implementing specific logical functions or steps of the process, and further implementations are included within the scope of the preferred embodiment of the present invention in which functions may be executed out of order from that shown or discussed, including substantially concurrently or in reverse order, depending on the functionality involved, as would be understood by those reasonably skilled in the art of the present invention.

Logic and/or steps represented in the flowcharts or otherwise described herein, e.g., a ordered listing of executable instructions for implementing logical functions, can be embodied in any computer-readable medium for use by or in connection with an instruction execution system, apparatus, or device, such as a computer-based system, system that includes a processing module, or other system that can fetch the instructions from the instruction execution system, apparatus, or device and execute the instructions.

In the description of the present specification, reference to the terms "certain embodiments," "one embodiment," "some embodiments," "an exemplary embodiment," "an example," "a particular example," or "some examples" means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present application, the meaning of "plurality" is at least two, for example two, three, unless explicitly defined otherwise.

Although embodiments of the present application have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the application, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the application, which is defined by the claims and their equivalents.

Claims

1. An imaging system for a gene sequencer, comprising:

a light source assembly capable of emitting a plurality of excitation lights;

2. The imaging system of claim 1, wherein there are three of said fringe fluorescent images, and wherein the directions of extension of three of said fringe fluorescent regions of three of said fringe fluorescent images are at 0 °, 120 ° and 240 ° with respect to the first direction, respectively.

3. The imaging system of claim 2, wherein the light source assembly comprises a laser emitter for emitting laser light and a spatial light modulator for diffracting the laser light, the light source assembly further comprising a plurality of converging mirrors for conditioning and sorting the laser light or the excitation light.

4. The imaging system of claim 3, wherein the light source assembly further comprises a diaphragm positioned in the optical path, the diaphragm having a plurality of light passing apertures therein, the diaphragm passing the partially diffracted laser light therethrough.

5. The imaging system of claim 4, wherein the light passing holes comprise a first light passing hole, a second light passing hole, a third light passing hole and a fourth light passing hole, the first light passing hole, the second light passing hole, the third light passing hole and the fourth light passing hole are sequentially arranged along a straight line, a distance from the first light passing hole to the second light passing hole is equal to a distance from the third light passing hole to the fourth light passing hole, and a distance from the first light passing hole to the fourth light passing hole is twice a distance from the second light passing hole to the third light passing hole.

6. The imaging system of claim 5, wherein the second light passing aperture and the third light passing aperture are symmetrical about an optical axis of the excitation light, and the first light passing aperture and the fourth light passing aperture are symmetrical about the optical axis of the excitation light.

7. The imaging system of claim 1, wherein the stage has a drive assembly coupled thereto, the drive assembly driving the stage in the first direction, the imaging system further comprising a control assembly controlling the drive assembly to drive the stage in the first direction and controlling the imaging assembly to receive the fringe fluorescent images such that a plurality of the fringe fluorescent regions of the fringe fluorescent images have a plurality of different directions of extension.

8. The imaging system of claim 1, further comprising a first dichroic mirror for collecting the fluorescence and transmitting the fluorescence to the first dichroic mirror, and an objective for reflecting the excitation light to the sample under test and for transmitting the striped fluorescence to the imaging assembly.

9. The imaging system of claim 8, wherein the imaging assembly comprises a plurality of second dichroic mirrors that distribute the fluorescence light to a plurality of imaging channels, the second dichroic mirrors being located between the imaging channels and the first dichroic mirrors, each of the imaging channels comprising a barrel mirror for pooling the fluorescence light to the line camera and a line camera for receiving the fluorescence light.

10. An imaging method for an imaging system for a gene sequencer, the imaging system comprising a light source assembly, a stage, and an imaging assembly, the stage carrying a sample to be tested thereon, the imaging method comprising:

changing excitation light emitted by the light source assembly, and receiving and generating at least three stripe fluorescent images through the imaging assembly, wherein the extending directions of a plurality of stripe fluorescent areas of the stripe fluorescent images are different;

11. The imaging method of an imaging system of claim 10, wherein the method of generating the fringe fluorescence image comprises:

12. The imaging method of an imaging system of claim 10, wherein the method of replacing excitation light emitted by the light source assembly comprises: