Imaging system, imaging method and gene sequencer

Abstract

Disclosed are an imaging system, an imaging method and a gene sequencer, the imaging system comprising a light source assembly, a stage and an imaging assembly. The light source component can emit multiple types of excitation light; the stage is disposed on an optical path of the light source component; the stage carries a sample to be measured; each type of excitation light is irradiated to the sample to be tested, to excite a stripe-shaped fluorescence extending along a first direction; the imaging component is used to receive stripe-shaped fluorescence at multiple positions, each position comprising multiple stripe-shaped fluorescences, and the same stripe-shaped fluorescence at multiple positions generating a stripe fluorescence image of a surface area; the stripe-shaped fluorescence forms a stripe fluorescence area in the stripe fluorescence image, the extension direction of the stripe fluorescence area of ​​each stripe fluorescence image being different; and multiple stripe fluorescence images are reconstructed into a super-resolution image.

Description

Imaging system, imaging method and gene sequencer

[0001] The present invention claims priority to Chinese patent application number 202311669454.3, filed with the Patent Office of China on December 7, 2023, entitled “IMAGING SYSTEM AND IMAGING METHOD,” the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention relates to the technical field of gene sequencing, and in particular to an imaging system, an imaging method and a gene sequencer. Background Art

[0003] During the gene sequencing process, a gene sequencer detects the four bases ATCG on the chip. Generally, higher data density on the chip leads to lower detection costs. However, detection on high-density chips requires a higher-resolution gene sequencer, which requires super-resolution optical methods.

[0004] The imaging system of a gene sequencer can use structured light super-resolution microscopy to achieve higher-resolution imaging. Currently, the most widely used structured light super-resolution microscopy imaging solution is based on area array imaging. However, in area imaging mode, the sample stage must be started and stopped every time a field of view is switched, which is time-consuming. Furthermore, image stitching must be considered during scanning, and image boundaries may require overlapping scans to maintain good imaging quality, further extending scanning time.

[0005] In related technologies, the above problems can be solved by line scanning structured light illumination super-resolution microscopy imaging methods, but existing line scanning super-resolution microscope illumination implementation schemes are difficult to meet the resolution improvement in all directions on a two-dimensional plane.

[0006] Summary of the Invention

[0007] In order to solve the existing technical problems, the embodiments of the present invention provide an imaging system, an imaging method and a gene sequencer that can meet the requirements of improving the resolution in all directions on a two-dimensional plane.

[0008] An imaging system according to an embodiment of the present application is used in a gene sequencer, comprising:

[0009] A light source component capable of emitting a variety of excitation lights;

[0010] A stage is provided in the optical path of the light source assembly, the stage carries a sample to be tested, and each of the excitation lights is irradiated on the sample to be tested to excite a stripe-shaped fluorescence extending along a first direction;

[0011] An imaging component is provided, wherein the imaging component is used to receive the stripe fluorescence at multiple positions, each position including multiple stripe fluorescences, the same stripe fluorescence at multiple positions generating a stripe fluorescence image of a surface area, the stripe fluorescence forming a stripe fluorescence area on the stripe fluorescence image, the stripe fluorescence area of ​​each stripe fluorescence image extending in a different direction, and the multiple stripe fluorescence images are reconstructed into a super-resolution image.

[0012] In some embodiments, there are three types of stripe fluorescence images, and the angles between the extension directions of the three stripe fluorescence areas of the three stripe fluorescence images and the first direction are 0°, 120°, and 240°, respectively.

[0013] In some embodiments, the light source assembly includes a laser emitter and a spatial light modulator, wherein the laser emitter is used to emit laser light, and the spatial light modulator is used to diffract the laser light, and the diffracted laser light forms the excitation light on the sample to be tested; the light source assembly also includes a plurality of converging mirrors, and the converging mirrors are used to adjust and organize the laser light.

[0014] In some embodiments, the light source assembly further includes a multimode optical fiber, wherein the multimode optical fiber is located between the laser emitter and the spatial light modulator, and the laser emitter emits laser light to the spatial light modulator through the multimode optical fiber.

[0015] In some embodiments, the light source assembly further includes an aperture, which is located on the optical path and has a plurality of light-through holes. The aperture allows the partially diffracted laser light to pass through different light-through holes.

[0016] In some embodiments, the converging mirror includes a first converging mirror located between the laser emitter and the spatial light modulator, a second converging mirror located between the spatial light modulator and the aperture, and a third converging mirror and a fourth converging mirror located behind the aperture;

[0017] The first converging mirror is used to collimate the laser emitted by the laser emitter; the second converging mirror is used to focus the parallel light reflected and diffracted by the spatial light modulator and then pass it through the light hole; the third converging mirror is used to collimate the light passing through the aperture; and the fourth converging mirror is used to focus the light collimated by the third converging mirror on two points on the back focal plane of the target objective lens.

[0018] In some embodiments, the light through hole includes a first light through hole, a second light through hole, a third light through hole and a fourth light through hole, and the first light through hole, the second light through hole, the third light through hole and the fourth light through hole are arranged in sequence along a straight line, the distance from the first light through hole to the second light through hole is equal to the distance from the third light through hole to the fourth light through hole, and the distance from the first light through hole to the fourth light through hole is twice the distance from the second light through hole to the third light through hole.

[0019] In some embodiments, the second light hole and the third light hole are symmetrical about the optical axis of the excitation light, and the first light hole and the fourth light hole are symmetrical about the optical axis of the excitation light.

[0020] In some embodiments, the aperture is circular, and the center of the aperture is located at the midpoint of the second light-through hole and the line connecting the second light-through hole.

[0021] In some embodiments, the laser light diffracted by the spatial light modulator forms a first excitation light on the sample to be tested after passing through the first light hole and the fourth light hole; the laser light diffracted by the spatial light modulator forms a second excitation light on the sample to be tested after passing through the second light hole and the third light hole; the period of the second excitation light is twice the period of the first excitation light.

[0022] In some embodiments, the stage is connected to a driving component, which drives the stage to move along the first direction. The imaging system also includes a control component, which controls the driving component to drive the stage to move along the first direction and controls the imaging component to receive the stripe fluorescence image, so that the stripe fluorescence areas of the multiple stripe fluorescence images have multiple different extension directions.

[0023] In some embodiments, the imaging system further includes a first dichroic mirror and an objective lens, wherein the objective lens is used to collect fluorescence and transmit the fluorescence to the first dichroic mirror, and the first dichroic mirror is used to reflect the excitation light emitted from the light source assembly to the sample to be measured, and to transmit the stripe-shaped fluorescence to the imaging assembly.

[0024] In some embodiments, the imaging assembly includes a plurality of second dichroic mirrors, which distribute the fluorescence to a plurality of imaging channels. The second dichroic mirrors are located between the imaging channels and the first dichroic mirrors. Each of the imaging channels includes a tube lens and a line array camera. The tube lens is used to collect the fluorescence to the line array camera, and the line array camera is used to receive the fluorescence.

[0025] In some embodiments, the number of the second dichroic mirrors is three, and each second dichroic mirror has a different coating, so as to separate four types of fluorescence corresponding to the four base types of ATCG.

[0026] The present application provides an imaging method of an imaging system for a gene sequencer. The imaging system includes a light source assembly, a stage, and an imaging assembly. The stage carries a sample to be tested. The imaging method includes:

[0027] The light source assembly emits excitation light to illuminate the sample to be tested and excite stripe-shaped fluorescence extending along a first direction;

[0028] receiving images of a plurality of positions of the stripe-shaped fluorescence through the imaging component to generate a stripe fluorescence image of a surface area, wherein the stripe-shaped fluorescence forms a stripe fluorescence area on the stripe fluorescence image;

[0029] By changing the parameters of the spatial light modulator, the excitation light emitted by the light source assembly is replaced, and at least three stripe fluorescence images are received and generated by the imaging assembly, wherein the extension directions of the multiple stripe fluorescence areas of the multiple stripe fluorescence images are different;

[0030] The imaging component reconstructs a super-resolution image based on a plurality of stripe fluorescence images with different extension directions of the stripe fluorescence areas.

[0031] In some embodiments, the method for generating the stripe fluorescence image includes:

[0032] The stage is driven by a driving component to move the sample to be tested along the first direction, and the light source component is controlled to make the excitation light illuminate the sample to be tested and excite the stripe fluorescence. The imaging component receives the imaging of the stripe fluorescence at multiple positions to generate a stripe fluorescence image of the surface area.

[0033] In some embodiments, the method of replacing the excitation light emitted by the light source assembly includes:

[0034] An aperture is provided inside the light source assembly, and the aperture is located on the optical path of the excitation light. The aperture is provided with a plurality of light-through holes, and the excitation light is changed by changing the usage of the light-through holes on the aperture.

[0035] An embodiment of the present application provides a gene sequencer, comprising the imaging system described in any embodiment of the present application.

[0036] In the imaging system of the embodiment of the present application, the light source component emits excitation light to excite stripe fluorescence on the sample to be tested, and the imaging component generates multiple stripe fluorescence images with different extension directions of the stripe fluorescence areas to reconstruct the final super-resolution image, which is equivalent to replacing the structured light illumination in multiple directions, thereby meeting the resolution improvement in all directions on the two-dimensional plane and effectively improving the imaging quality of the super-resolution image.

[0037] Additional aspects and advantages of the embodiments of the present application will be given in part in the description below, and in part will become obvious from the description below, or will be learned through practice of the embodiments of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0039] FIG1 is a schematic diagram of the main structure of an imaging system according to an embodiment of the present application;

[0040] FIG2 is a schematic diagram of a module of an imaging system according to an embodiment of the present application;

[0041] FIG3 is a frequency domain diagram of SIM imaging of different fringe directions in the related art;

[0042] FIG4 is a frequency domain diagram of SIM imaging of excitation light according to an embodiment of the present application;

[0043] FIG5 is a schematic structural diagram of an imaging system according to an embodiment of the present application;

[0044] FIG6 is a schematic diagram of a method for generating a first stripe fluorescent image according to an embodiment of the present application;

[0045] FIG7 is a schematic diagram of the illumination direction of the excitation light for the first stripe fluorescence image according to an embodiment of the present application;

[0046] FIG8 is a schematic diagram of a first stripe fluorescence image according to an embodiment of the present application;

[0047] FIG9 is a schematic diagram of actual stripes of excitation light in a first stripe fluorescence image according to an embodiment of the present application;

[0048] FIG10 is a diagram showing the lateral intensity distribution of the excitation light of the first fringe fluorescence image according to an embodiment of the present application;

[0049] FIG11 is a schematic diagram of a second stripe fluorescent image generation method according to an embodiment of the present application;

[0050] FIG12 is a schematic diagram of the illumination direction of the second stripe fluorescence image excitation light according to an embodiment of the present application;

[0051] FIG13 is a schematic diagram of the aperture structure according to an embodiment of the present application;

[0052] FIG14 is a schematic diagram of a second stripe fluorescence image imaging according to an embodiment of the present application;

[0053] FIG15 is a schematic diagram of actual stripes of excitation light in a second stripe fluorescence image according to an embodiment of the present application;

[0054] FIG16 is a diagram showing the lateral intensity distribution of the excitation light of the second fringe fluorescence image according to an embodiment of the present application;

[0055] FIG17 is a schematic diagram of a third stripe fluorescent image generation method according to an embodiment of the present application;

[0056] FIG18 is a schematic diagram of the illumination direction of the third fringe fluorescence image excitation light according to an embodiment of the present application;

[0057] FIG19 is a schematic diagram of a third stripe fluorescence image according to an embodiment of the present application;

[0058] FIG20 is a schematic diagram of actual stripes of excitation light in a third stripe fluorescence image according to an embodiment of the present application;

[0059] FIG21 is a diagram showing the lateral intensity distribution of the excitation light of the third fringe fluorescence image according to an embodiment of the present application;

[0060] FIG22 is a flow chart of an imaging method according to an embodiment of the present application.

[0061] Component symbol explanation: imaging system 100, light source assembly 10, laser emitter 11, spatial light modulator (or micro-mirror array) 12, multimode optical fiber 13, first converging mirror 14, second converging mirror 15, third converging mirror 16, fourth converging mirror 17, aperture 18, first light hole 181, second light hole 182, third light hole 183, fourth light hole 184, stage 20, imaging assembly 30, second dichroic mirror 31, imaging channel 32, tube lens 321, linear array camera 322, filter 323, sample to be measured 40, stripe fluorescence 41, stripe fluorescence area 42, imaging area 43, drive assembly 50, control assembly 60, objective lens 71, first dichroic mirror 72. DETAILED DESCRIPTION

[0062] The following further describes the embodiments of the present application in conjunction with the accompanying drawings. The same or similar reference numerals in the accompanying drawings represent the same or similar elements or elements with the same or similar functions.

[0063] In addition, the embodiments of the present application described below in conjunction with the accompanying drawings are exemplary and are only used to explain the embodiments of the present application and should not be understood as limiting the present application.

[0064] In this application, unless otherwise expressly specified or limited, when a first feature is "above" or "below" a second feature, it may mean that the first and second features are in direct contact, or the first and second features are in indirect contact through an intermediate medium. Furthermore, when a first feature is "above," "above," or "above" a second feature, it may mean that the first feature is directly above or diagonally above the second feature, or simply means that the first feature is at a higher level than the second feature. When a first feature is "below," "below," or "below" a second feature, it may mean that the first feature is directly below or diagonally below the second feature, or simply means that the first feature is at a lower level than the second feature.

[0065] During the sequencing process, gene sequencers require fluorescence imaging of each of the four bases (ATCG). Because each target point is located at the submicron scale, gene sequencers inevitably employ microscopic imaging to achieve detection. A key metric for gene sequencers is the density of the dots on the chip, which determines the amount of data on a chip and, consequently, the cost per unit of data. Because the amount of reagents used per unit area is minimally affected by dot density, higher dot density results in lower detection costs.

[0066] High-density chips require higher-resolution microscopes. However, traditional wide-field microscopes are diffraction-limited, with resolutions typically capped at around 200nm. To achieve higher resolution and thus higher chip point density, "super-resolution" optical techniques are required.

[0067] Super-resolution, or "super-resolution," exceeds the diffraction limit of an optical system. The super-resolution technology most likely to be used in gene sequencers is Structured Illumination Microscopy (SIM). This method exploits the principle of moiré fringes, whereby two high-frequency signals are superimposed to form low-frequency signals. This allows high-frequency signals, previously unable to pass through the objective lens, to be received by the optical system. This allows the spectral range of the original wide-field microscopic imaging (as shown in Figure 3, a) to be doubled in the corresponding fringe modulation direction (as shown in Figure 3, b), ultimately doubling the resolution.

[0068] The imaging system of a gene sequencer can use structured light illumination super-resolution microscopy to achieve higher-resolution imaging. Currently, the widely used structured light illumination super-resolution microscopy imaging solution is mainly based on area array imaging. However, in area array imaging mode, the sample stage starts and stops every time a field of view is switched, which takes a long time. In addition, image stitching must be considered during scanning imaging. The imaging boundaries may require overlapping scans to maintain good imaging effects, further extending the scanning time. In the field of gene sequencing, imaging speed is a key indicator, which determines the time it takes to output the sequencing report. To further improve imaging detection speed, the use of line scanning imaging solutions is the future trend.

[0069] In the related art, the above-mentioned problems can be solved by line scanning super-resolution microscopy imaging methods. The existing line scanning super-resolution microscope illumination implementation scheme is usually structured light illumination, that is, the sample to be tested is illuminated by structured light and fluorescence is excited, and then the image is acquired and reconstructed into a super-resolution image through the imaging component. The resolution improvement effect of the above-mentioned illumination method and imaging method is shown in b in Figure 3. It is not difficult to see that the resolution improvement effect is relatively limited and cannot achieve the resolution improvement that satisfies all directions on the two-dimensional plane.

[0070] In view of this, referring to FIG. 1 to FIG. 22 , an imaging system 100 for a gene sequencer includes a light source assembly 10 , a stage 20 , and an imaging assembly 30 .

[0071] The light source assembly 10 is capable of emitting multiple excitation lights. The stage 20 is arranged on the optical path of the light source assembly 10. The stage 20 carries a sample 40 to be tested. Each excitation light is irradiated onto the sample 40 to be tested to excite a stripe-shaped fluorescence 41 extending along a first direction. The imaging assembly 30 is used to receive the stripe-shaped fluorescence 41 at multiple positions. Each position includes multiple stripe-shaped fluorescence 41. The same stripe-shaped fluorescence 41 at multiple positions generates a stripe fluorescence image of a surface area. The stripe fluorescence 41 forms a stripe fluorescence area 42 on the stripe fluorescence image. The extension direction of the stripe fluorescence area 42 of each stripe fluorescence image is different. The multiple stripe fluorescence images are reconstructed into a super-resolution image.

[0072] In the imaging system 100 of the embodiment of the present application, the light source component 10 emits excitation light to excite stripe fluorescence 41 on the sample to be tested 40, and the imaging component 30 generates multiple stripe fluorescence images with different extension directions of the stripe fluorescence area 42 to reconstruct the final super-resolution image, which is equivalent to replacing the structured light illumination in multiple directions, thereby meeting the resolution improvement in various directions on the two-dimensional plane and effectively improving the imaging quality of the super-resolution image.

[0073] Specifically, the sample to be tested 40 is a biological sample with a fluorescent dye, which is set on a gene sequencing chip. The gene sequencing chip is placed on the stage 20. The excitation light emitted by the light source assembly 10 irradiates the sample to be tested 40 to form a first light spot. The light spot excites the fluorescent dye on the biological sample to form a stripe-shaped fluorescence 41. Both the light spot and the stripe-shaped fluorescence 41 extend in the first direction X.

[0074] In some embodiments, there are three types of stripe fluorescent images, and the angles between the extension directions of the three stripe fluorescent areas 42 of the three stripe fluorescent images and the first direction are 0°, 120°, and 240°, respectively. Specifically, the angles between the extension directions of the three stripe fluorescent areas 42 of the three stripe fluorescent images and the first direction are 0°, 120°, and 240°, respectively, which can be equivalent to structured light illumination in three directions of 0°, 120°, and 240°. As shown in FIG. 4 , the structured light illumination in these three directions can meet the resolution improvement in various directions on the two-dimensional plane.

[0075] Please refer to Figure 5. In some embodiments, the light source assembly 10 includes a laser emitter 11 and a spatial light modulator 12. The laser emitter 11 is used to emit laser light, and the spatial light modulator 12 is used to diffract laser light. Specifically, the laser emitter 11 can emit laser light of a specific wavelength to excite the sample 40 to produce fluorescence. The light source assembly 10 can also include a multimode optical fiber 13. The multimode optical fiber 13 is located between the laser emitter 11 and the spatial light modulator 12. The laser emitter 11 emits laser light to the spatial light modulator 12 through the multimode optical fiber 13.

[0076] In some embodiments, the light source assembly 10 also includes a plurality of converging mirrors, which are used to adjust and organize the laser. Specifically, the converging mirrors include a first converging mirror 14, which is located between the multimode optical fiber 13 and the spatial light modulator 12. The first converging mirror 14 is used to collimate the laser emitted by the laser emitter 11 through the multimode optical fiber 13 into parallel light.

[0077] In some embodiments, the light source assembly 10 also includes an aperture 18, which is located on the optical path and has multiple light holes. The aperture 18 allows part of the diffracted laser to pass through different light holes. Specifically, the converging mirror also 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 laser emitter 11 and the spatial light modulator 12. The spatial light modulator 12 reflects and diffracts the parallel light collimated by the first converging mirror 14, and then focuses it onto the aperture 18 through the second converging mirror 15. The function of the aperture 18 is to allow only ±1 order light to pass through and block the 0th order and higher order light in the diffracted laser. The light passing through the aperture 18 passes through the third converging mirror 16 and the fourth converging mirror 17 in sequence. The light passing through the aperture 18 is collimated by the third converging mirror 16 and then focused by the fourth converging mirror 17.

[0078] Please refer to Figures 5 and 13. In some embodiments, the light holes include a first light hole 181, a second light hole 182, a third light hole 183 and a fourth light hole 184. The first light hole 181, the second light hole 182, the third light hole 183 and the fourth light hole 184 are arranged in sequence along a straight line. The distance from the first light hole 181 to the second light hole 182 is equal to the distance from the third light hole 183 to the fourth light hole 184. The distance from the first light hole 181 to the fourth light hole 184 is twice the distance from the second light hole 182 to the third light hole 183. Furthermore, the second light hole 182 and the third light hole 183 are symmetrical about the optical axis of the excitation light. The first light hole 181 and the fourth light hole 184 are symmetrical about the optical axis of the excitation light. The light hole 184 is symmetrical about the optical axis of the excitation light. Specifically, the aperture 18 is circular, and the first light hole 181, the second light hole 182, the third light hole 183 and the fourth light hole 184 are arranged along a diameter direction of the aperture 18. The center of the aperture 18 is located at the midpoint of the line connecting the second light hole 182 and the third light hole 183. The laser light diffracted by the spatial light modulator 12 forms a first excitation light on the sample to be tested 40 when passing through the first light hole 181 and the fourth light hole 184. The laser light diffracted by the spatial light modulator 12 forms a second excitation light on the sample to be tested 40 when passing through the second light hole 182 and the third light hole 183. The period of the second excitation light is twice that of the first excitation light.

[0079] Please refer to FIG. 2 . In some embodiments, the stage 20 is connected to a driving assembly 50 . The driving assembly 50 drives the stage 20 to move along a first direction. Specifically, the driving assembly 50 is an electric translation stage.

[0080] In some embodiments, the imaging system 100 further includes a control component 60, which controls the driving component 50 to drive the stage 20 to move along a first direction and controls the imaging component 30 to receive the stripe fluorescence image, so that the stripe fluorescence areas 42 of the multiple stripe fluorescence images have multiple different extension directions.

[0081] Please refer to Figure 5. In some embodiments, the imaging system 100 also includes a first dichroic mirror 72 and an objective lens 71. The objective lens 71 is used to collect fluorescence and transmit the fluorescence to the first dichroic mirror 72. The first dichroic mirror 72 is used to reflect the excitation light to the sample to be tested 40, and to transmit the stripe-shaped fluorescence 41 to the imaging component 30. Specifically, the fourth converging mirror 17 converges the two lights passing through the aperture 18 at two points on the rear focal plane of the objective lens 71, thereby generating excitation light on the sample to be tested 40, wherein the excitation light and the stripe-shaped fluorescence 41 are both sinusoidal stripes.

[0082] In some embodiments, the imaging assembly 30 includes multiple second dichroic mirrors 31, which distribute fluorescence to multiple imaging channels 32. The second dichroic mirrors 31 are located between the imaging channels 32 and the first dichroic mirror 72. Each imaging channel 32 includes a tube lens 321 and a line array camera 322. The tube lens 321 is used to collect fluorescence to the line array camera 322, and the line array camera 322 is used to receive fluorescence.

[0083] In some embodiments, the imaging assembly 30 further includes a filter 323 . The filter 323 is located between the tube lens 321 and the linear array camera 322 . The filter 323 is used to filter fluorescence.

[0084] In some embodiments, the number of second dichroic mirrors 31 is three. Specifically, the four bases of ATCG form four types of fluorescence. At least three second dichroic mirrors 31 are required to separate the four fluorescences one by one. The three second dichroic mirrors 31 are different types of dichroic mirrors. Furthermore, the three second dichroic mirrors 31 have different coatings and are used to separate the four fluorescences formed by the four bases of ATCG.

[0085] The present application provides an imaging method of an imaging system 100. The imaging system 100 includes a light source assembly 10, a stage 20, and an imaging assembly 30. The stage 20 carries a sample 40 to be measured. The imaging method includes:

[0086] Step 01: emitting excitation light from a light source assembly to illuminate a sample to be tested, thereby exciting stripe-shaped fluorescence extending along a first direction;

[0087] Step 02: Receive images of multiple positions of the stripe-shaped fluorescence through an imaging component to generate a stripe fluorescence image of the surface area, wherein the stripe-shaped fluorescence forms a stripe fluorescence area on the stripe fluorescence image;

[0088] Step 03: Replace the excitation light emitted by the light source assembly, receive and generate at least three stripe fluorescence images through the imaging assembly, and the extension directions of the multiple stripe fluorescence areas of the multiple stripe fluorescence images are different;

[0089] Step 04: Reconstruct a super-resolution image based on the multiple stripe fluorescence images with different extension directions of the multiple stripe fluorescence areas through an imaging component.

[0090] Specifically, the imaging system 100 also includes a control component 60, which emits excitation light through the light source component 10 to irradiate the sample to be tested 40 and excite the stripe fluorescence 41 extending along the first direction. Then, the control component 60 controls the imaging component 30 to receive imaging of multiple positions of the stripe fluorescence 41 to generate a stripe fluorescence image of the surface area. The stripe fluorescence 41 forms a stripe fluorescence area 42 on the stripe fluorescence image. Then, the excitation light emitted by the light source component 10 is replaced, and at least three stripe fluorescence images are received and generated through the imaging component 30. The extension directions of the multiple stripe fluorescence areas 42 of the multiple stripe fluorescence images are different. The imaging component 30 reconstructs the multiple stripe fluorescence images with different extension directions of the multiple stripe fluorescence areas 42 into a super-resolution image.

[0091] Furthermore, the method for generating the stripe fluorescence image includes:

[0092] The driving component 50 drives the stage 20 to move the sample to be tested 40 along the first direction, and at the same time controls the light source component 10 to make the excitation light irradiate the sample to be tested 40 and excite the stripe fluorescence 41. The imaging component 30 receives the imaging of the stripe fluorescence 41 at multiple positions to generate a stripe fluorescence image of the surface area.

[0093] In some embodiments, the method of replacing the excitation light emitted by the light source assembly 10 includes:

[0094] An aperture 18 is provided inside the light source assembly 10. The aperture 18 is located on the light path and is provided with multiple light holes. The excitation light is changed by changing the usage of the light holes on the aperture 18. Specifically, the light holes on the aperture 18 include a first light hole 181, a second light hole 182, a third light hole 183 and a fourth light hole 184. The excitation light period formed by passing through different light holes is different.

[0095] In certain embodiments, imaging using a linear array camera 322 requires the coordination of a motorized stage to move and scan the sample 40. Therefore, this imaging method results in the excitation light being fixed relative to the camera. However, SIM imaging requires the excitation light to be fixed relative to the sample to achieve structured light illumination. Therefore, during the motorized stage scanning process, the excitation light must vary according to the scanning position to meet actual lighting requirements. Specifically, the excitation light can be controlled by a spatial light modulator 12.

[0096] Since three types of stripe fluorescence images are required, the extension directions of the three stripe fluorescence areas 42 of the three stripe fluorescence images are at angles of 0°, 120°, and 240° with the first direction, respectively, to be equivalent to three-phase line scanning structured light. The following uses a 6-pixel periodic binary stripe as a simplified model to introduce a method for generating the extension directions of the three stripe fluorescence areas 42.

[0097] Please refer to Figure 6. First, we will introduce the method of generating the first stripe fluorescent image in which the angle between the extension direction of the stripe fluorescent area 42 and the first direction is 0°. Take a in Figure 6 as an example, where each square represents each pixel of the final image. The bold long box in the figure indicates that the linear array camera 322 takes a single shot of the imaging area 43, and the imaging area 43 extends along the second direction, and the second direction is perpendicular to the first direction. Therefore, to complete the imaging of 1 to 24 rows, the linear array camera 322 needs to be scanned in conjunction with the electric translation stage. The gray squares in the figure represent pixels with signals, that is, the illuminated areas on the surface of the sample 40 to be tested. The lighting method is stripe light illumination with a period of 6 pixels. b in Figure 6 is the position of the stripes when the camera captures the second row of images, and c in Figure 6 is the position of the stripes when the 24th row is captured. After completing a set of 24 rows of shooting, the phase of the excitation light is adjusted, and then imaging is performed separately, as shown in d to f in Figure 6 and h to i in Figure 6. The excitation light of a to c in Figure 6 is in a phase of 0°, the excitation light of d to f in Figure 6 is in a phase of 120°, and the excitation light of h to i in Figure 6 is in a phase of 240°.

[0098] Please refer to Figure 7. To achieve the aforementioned illumination effect, when the line array camera 322 takes a single shot of a row, the actual excitation light across the entire surface of the sample 40 is shown in the figure. These stripes are generated by the spatial light modulator 12. Figures a through i in Figure 7 correspond to Figures a through i in Figure 6, ensuring that when the line array camera 322 scans the corresponding rows, the position of the excitation light is as shown in Figure 6. After the line array camera 322 completes scanning of rows 1 through 24 on the sample, the final stripe fluorescence image is shown in Figure 8. Specifically, Figures a through c in Figure 6 correspond to the stripe fluorescence image generated in Figure 8, Figure 8b in Figure 8, and Figures h through i in Figure 6 correspond to the stripe fluorescence image generated in Figure 8.

[0099] It should be noted that the actual excitation light emitted through objective lens 71 has a sinusoidal intensity distribution, so the final actual excitation light is shown in Figures 9 and 10, where Figure 9 is a schematic diagram of the actual stripes, and Figure 10 is the transverse intensity distribution of the actual stripes in row 12. Although the intensity changes from binary stripes to sinusoidal stripes, the period remains 6 pixels.

[0100] Please refer to Figure 11. The method for generating a second stripe fluorescence image in which the angle between the extension direction of the stripe fluorescence region 42 and the first direction is 120° is shown in the figure. After the excitation light of 6 pixel period is tilted by 120 degrees, its projection in the second direction is abs(6 / cos(120°)) = 12 pixel periods, as shown in a in Figure 11. This method can be extended to stripes of any period, that is, the horizontal projection of the tilted stripes is twice the vertical stripe period. Because the stripes are tilted, when the line array camera 322 changes lines, the excitation light needs to be shifted to the right by tan(60°) = 1.7321 pixels. Since a single pixel is the smallest unit and cannot be further divided, the actual number of pixels offset by the excitation light relative to the first line of imaging during imaging is:

[0101] X=ceil(i*tan(60°))

[0102] Where ceil is rounded up, and i is the number of rows currently scanned. Figures 11a to 11c are schematic diagrams of the line scanning process of the excitation light in the 120° direction at a phase of 0°. Since the projection period of the stripes in the horizontal direction is 12 pixels, when the phase changes from 0° to 120°, the excitation light of each row needs to be moved 4 pixels along the phase change direction. The movement is controlled by the spatial light modulator 12, as shown in Figure 11d to Figure 11f. Similarly, when the phase changes from 120° to 240°, the excitation light of each row also needs to be moved 4 pixels along the phase change direction, as shown in Figure 11g to Figure 11i.

[0103] To achieve the aforementioned illumination effect, when line scan camera 322 captures a single line, the actual excitation light across the entire sample surface is shown in Figure 12. This stripe is generated by spatial light modulator 12. Figures a through i in Figure 11 correspond to Figures a through i in Figure 12, ensuring that the excitation light is positioned as shown in Figure 12 when line scan camera 322 scans the corresponding line.

[0104] Because the actual illumination period of the excitation light at an angle of 120° on the sample 40 is twice that of the excitation light at an angle of 0°, the aperture 18 is designed as shown in Figure 13. The first and fourth light holes 181, 184 correspond to the ±1st order conjugates of the 0° excitation light in the frequency domain, while the second and third light holes 182, 183 correspond to the ±1st order conjugates of the 120° stripes in the frequency domain. The distances from the first and fourth light holes 181, 184 to the center of the aperture 18 are equal, as are the distances from the second and third light holes 182, 183 to the center of the aperture 18. Furthermore, the distances from the first and fourth light holes 181, 184 to the center of the aperture 18 are twice the distances from the second and third light holes 182, 183 to the center of the aperture 18.

[0105] After line scan camera 322 completes scanning of lines 1 through 24 on the sample, the final second fringe fluorescence image is shown in Figure 14 . The actual excitation light is shown in Figures 15 and 16 . Figure 15 illustrates the sinusoidal intensity distribution of the actual fringe, while Figure 16 shows the lateral intensity distribution of the actual fringe at line 12. Although the intensity changes from binary to sinusoidal, the lateral period remains at 12 pixels.

[0106] Similar to the method for generating the second fringe fluorescence image, where the angle between the extension direction of the fringe fluorescence region 42 and the first direction is 120°, the method for generating the third fringe fluorescence image, where the angle between the extension direction of the fringe fluorescence region 42 and the first direction is 240°, is shown in Figure 17 . When the line array camera 322 changes lines, the excitation light needs to be shifted to the left by tan(60°) = 1.7321 pixels. Since a single pixel is the smallest unit and cannot be further divided, the actual number of pixels offset by the excitation light relative to the first imaging line during imaging is: X = ceil(i * tan(60°))

[0107] Where ceil is rounded up, and i is the number of rows currently scanned. Figures 17a to 17c are schematic diagrams of the line scanning process of the excitation light in the 120° direction at a phase of 0°. Since the projection period of the excitation light in the horizontal direction is 12 pixels, when the phase changes from 0° to 120°, the excitation light of each row needs to be moved 4 pixels along the phase change direction, as shown in Figure 17d to Figure 17f. Similarly, when the phase changes from 120° to 240°, the excitation light of each row also needs to be moved 4 pixels along the phase change direction, as shown in Figure 17g to Figure 17i.

[0108] To achieve the aforementioned lighting effect, when the line array camera 322 takes a single shot of a row, the actual excitation light on the entire sample surface is shown in FIG18 . This stripe is generated by the spatial light modulator 12 . The lines a through i in FIG17 and a through i in FIG18 correspond to each other, ensuring that when the line array camera 322 scans the corresponding number of rows, the position of the excitation light is as shown in FIG18 . After the line array camera 322 completes scanning rows 1 to 24 on the sample, the final fluorescence image of the third stripe is shown in FIG19 . The actual excitation light is shown in FIG20 and FIG21 . FIG20 illustrates the sinusoidal intensity distribution of the actual stripe, and FIG21 illustrates the lateral intensity distribution of the actual stripe at row 12. Although its intensity changes from binary stripes to sinusoidal stripes, its lateral period remains 12 pixels.

[0109] In summary, in the imaging system and imaging method of the embodiment 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 aperture 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 aperture 18. The aperture 18 can allow the ±1 order lasers to pass through, while blocking the 0th order and higher order lasers in the diffracted lasers. Therefore, the laser light after passing through the aperture 18 is collimated by the third converging mirror 16 and the fourth converging mirror 17 to form an excitation light that is irradiated onto the sample to be measured and forms an excited stripe fluorescence. The high-frequency information that was originally unavailable can be shifted to the passband and represented by low-frequency intensity. When the frequency is subsequently shifted back to the correct position through an algorithm, a higher resolution can be obtained. At the same time, the spatial light modulator 12 can adjust the phase of the laser, thereby changing the phase of the stripe fluorescence. Then, the imaging assembly 30 can obtain images of different phases according to the stripe fluorescence of different phases. By controlling the laser light to pass through the aperture 18, periodic changes can be achieved, thereby simplifying the imaging difficulty of the imaging component in different directions, making the imaging system simple to operate and easy to implement. The collimated laser light is reflected by the first dichroic mirror 72 and then passes through the objective lens 71 to illuminate the sample, stimulating streak fluorescence. The streak fluorescence is then transmitted through the objective lens 71 to the imaging component 30. After entering the imaging component 30, the streak fluorescence is distributed by multiple second dichroic mirrors 31 into four different imaging channels 32 according to four colors, corresponding to the four different bases. The imaging channels 32 are equipped with filters 323 to filter stray light and prevent interference with imaging. At the end of the imaging channel 32, a line array camera 322 is provided. The line array camera 322 is used to convert the streaks of different phases into streak fluorescence images with 0°, 120°, and 240° streak fluorescence areas, and then reconstruct the multiple streak fluorescence images into a super-resolution image.

[0110] In certain embodiments of the present invention, the module involved may be a single-chip microcomputer chip that integrates a processor, memory, communication module, etc. The processor may be a central processing unit (CPU), or other general-purpose processors, digital signal processors (DSP), application-specific integrated circuits (ASIC), field programmable gate arrays (FPGA), or other programmable logic devices, discrete gate or transistor logic devices, discrete hardware components, etc.

[0111] Any process or method description in a flowchart or otherwise described herein may be understood to represent a module, segment or portion of code comprising one or more executable instructions for implementing the steps of a specific logical function or process, and the scope of the preferred embodiments of the present invention includes alternative implementations in which functions may be performed out of the order shown or discussed, including performing functions in a substantially simultaneous manner or in the reverse order depending on the functions involved, which should be understood by those skilled in the art to which the embodiments of the present invention pertain.

[0112] The logic and / or steps represented in the flowchart or otherwise described herein, for example, can be considered as an ordered list of executable instructions for implementing logical functions, and can be embodied in any computer-readable medium for use by an instruction execution system, apparatus or device (such as a computer-based system, a system including a processing module, or other system that can fetch instructions from an instruction execution system, apparatus or device and execute instructions), or used in conjunction with such instruction execution systems, apparatuses or devices.

[0113] Throughout this specification, reference to the terms "certain embodiments," "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples" means that a specific feature, structure, material, or characteristic described in conjunction with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0114] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of technical features indicated. Thus, a feature specified as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of this application, "plurality" means at least two, for example, two or three, unless otherwise specifically defined.

[0115] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are illustrative and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application. The scope of the present application is defined by the claims and their equivalents.

Claims

1. An imaging system, characterized in that: For gene sequencers, including: A light source component capable of emitting a variety of excitation lights; A stage is arranged on the optical path of the light source assembly, the stage carries a sample to be tested, and each of the excitation lights is irradiated to the sample to be tested to excite a stripe-shaped fluorescence extending along a first direction; An imaging component is provided, wherein the imaging component is used to receive the stripe fluorescence at multiple positions, each position includes multiple stripe fluorescences, the same stripe fluorescence at multiple positions generates a stripe fluorescence image of a surface area, the stripe fluorescence forms a stripe fluorescence area on the stripe fluorescence image, the extension direction of the stripe fluorescence area of ​​each stripe fluorescence image is different, and multiple stripe fluorescence images are reconstructed into a super-resolution image.

2. The imaging system according to claim 1, characterized in that There are three types of stripe fluorescent images, and the included angles between the extension directions of the three stripe fluorescent areas of the three stripe fluorescent images and the first direction are 0°, 120° and 240° respectively.

3. The imaging system according to claim 2, characterized in that The light source assembly includes a laser emitter and a spatial light modulator, wherein the laser emitter is used to emit laser light, and the spatial light modulator is used to diffract the laser light, and the diffracted laser light forms the excitation light on the sample to be tested; The light source assembly further includes a plurality of converging mirrors, which are used to adjust and organize the laser or the excitation light.

4. The imaging system according to claim 3, characterized in that The light source assembly further comprises a multimode optical fiber, wherein the multimode optical fiber is located between the laser transmitter and the spatial light modulator, and the laser transmitter transmits laser light to the spatial light modulator through the multimode optical fiber.

5. The imaging system according to claim 3, characterized in that: The light source assembly further includes an aperture, which is located on the light path and has a plurality of light-through holes. The aperture allows the partially diffracted laser light to pass through the light-through holes.

6. The imaging system according to claim 5, characterized in that: The converging mirror comprises a first converging mirror located between the laser emitter and the spatial light modulator, a second converging mirror located between the spatial light modulator and the aperture, and a third converging mirror and a fourth converging mirror located behind the aperture; The first converging mirror is used to collimate the laser emitted by the laser transmitter; the second converging mirror is used to focus the parallel light reflected and diffracted by the spatial light modulator and then pass it through the light hole; the third converging mirror is used to collimate the light passing through the aperture; and the fourth converging mirror is used to focus the light collimated by the third converging mirror on two points of the rear focal plane of the target objective lens.

7. The imaging system according to claim 5, characterized in that: The light through holes include a first light through hole, a second light through hole, a third light through hole and a fourth light through hole, wherein the first light through hole, the second light through hole, the third light through hole and the fourth light through hole are arranged in sequence along a straight line, the distance from the first light through hole to the second light through hole is equal to the distance from the third light through hole to the fourth light through hole, and the distance from the first light through hole to the fourth light through hole is twice the distance from the second light through hole to the third light through hole.

8. The imaging system according to claim 7, characterized in that: The second light through hole and the third light through hole are symmetrical about the optical axis of the excitation light, and the first light through hole and the fourth light through hole are symmetrical about the optical axis of the excitation light.

9. The imaging system according to claim 7, characterized in that: The aperture is circular, and the center of the aperture is located at the midpoint of the second light-through hole and the line connecting the second light-through hole.

10. The imaging system according to claim 7, characterized in that: The laser light diffracted by the spatial light modulator passes through the first light through hole and the fourth light through hole to form a first excitation light on the sample to be tested; The laser light diffracted by the spatial light modulator passes through the second light through hole and the third light through hole to form a second excitation light on the sample to be tested; The period of the second excitation light is twice the period of the first excitation light.

11. The imaging system according to claim 1, characterized in that: The stage is connected to a driving component, which drives the stage to move along the first direction. The imaging system also includes a control component, which controls the driving component to drive the stage to move along the first direction and controls the imaging component to receive the stripe fluorescence image, so that the stripe fluorescence areas of a plurality of the stripe fluorescence images have a plurality of different extension directions.

12. The imaging system according to claim 1, characterized in that The imaging system also includes a first dichroic mirror and an objective lens, wherein the objective lens is used to collect fluorescence and transmit the fluorescence to the first dichroic mirror, and the first dichroic mirror is used to reflect the excitation light emitted from the light source assembly to the sample to be tested, and to transmit the striped fluorescence to the imaging assembly.

13. The imaging system according to claim 12, characterized in that: The imaging component includes a plurality of second dichroic mirrors, which distribute the fluorescence to a plurality of imaging channels. The second dichroic mirrors are located between the imaging channels and the first dichroic mirrors. Each of the imaging channels includes a tube lens and a line array camera. The tube lens is used to collect the fluorescence to the line array camera, and the line array camera is used to receive the fluorescence.

14. The imaging system according to claim 13, characterized in that There are three second dichroic mirrors, each of which has a different coating, and is used to separate four types of fluorescence corresponding to the four base types of ATCG.

15. An imaging method of an imaging system, characterized in that: For a gene sequencer, the imaging system comprises a light source component, a stage and an imaging component, the stage carries a sample to be tested, and the imaging method comprises: The light source assembly emits excitation light to illuminate the sample to be tested and excite stripe-shaped fluorescence extending along a first direction; Receiving images of multiple positions of the stripe-shaped fluorescence through the imaging component to generate a stripe fluorescence image of a surface area, wherein the stripe-shaped fluorescence forms a stripe fluorescence area on the stripe fluorescence image; The excitation light emitted by the light source assembly is replaced, and at least three stripe fluorescence images are received and generated by the imaging assembly, wherein the extension directions of the stripe fluorescence areas of the stripe fluorescence images are different; The imaging component reconstructs a super-resolution image based on a plurality of stripe fluorescence images with different extension directions of the stripe fluorescence areas.

16. The imaging method of the imaging system according to claim 15, characterized in that: The method for generating the stripe fluorescence image comprises: The stage is driven by a driving component to move the sample to be tested along the first direction, and the light source component is controlled to make the excitation light illuminate the sample to be tested and excite the stripe fluorescence. The imaging component receives the imaging of the stripe fluorescence at multiple positions to generate a stripe fluorescence image of the surface area.

17. The imaging method of the imaging system according to claim 15, characterized in that: The method for replacing the excitation light emitted by the light source assembly comprises: An aperture is arranged inside the light source assembly, and the aperture is located on the optical path of the excitation light. The aperture is provided with a plurality of light-through holes, and the excitation light is changed by changing the usage of the light-through holes on the aperture.

18. A gene sequencer, characterized in that: Comprising the imaging system of any one of claims 1 to 14.

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