Imaging systems and methods, identification devices and methods, sequencers and sequencing methods

Abstract

An imaging system and method, an identification device and method, a sequencer, and a sequencing method are disclosed. The imaging system includes: a laser source and a structured light modulation element, wherein the structured light modulation element is used to modulate excitation light to generate and emit a first light and a second light with separated optical paths; a light guide element, which guides the first light and the second light to interfere on the surface of the sample under test to form fringe structured light in a first angle state, and guides the first light or the second light to form a wide field light on the surface of the sample under test in a second angle state; the fringe structured light excites and generates a structured light fluorescence signal; when the light guide element emits the first light or the second light at the second angle, the first light or the second light forms a wide field light spot on the surface of the sample under test to excite and generate a wide field fluorescence signal; and an imaging module, which is used to acquire the structured light fluorescence signal to generate a structured light image and acquire the wide field fluorescence signal to generate a wide field image.

Description

Imaging system and method, identification device and method, sequencer and sequencing method Technical Field

[0001] The present application relates to super-resolution imaging technology, and in particular to a super-resolution imaging system, a super-resolution imaging method, a biological sample identification device based on super-resolution imaging, a biological sample identification method applied to the biological sample identification device, a gene sequencer based on super-resolution imaging, and a gene sequencing method applied to the gene sequencer. Background Art

[0002] Super-resolution imaging technology has broad application prospects in the medical device field, especially in gene sequencing. In super-resolution imaging, structured illumination microscopy (SIM) generates striped light (the light spot pattern on the sample is a stripe structure of alternating light and dark) instead of traditional uniform light beam scanning.

[0003] The Time Delay Integration (TDI) camera is a new type of imaging element. Along the scanning direction of the light scanning sample, the photosensitive unit of the TDI camera can realize multi-level sequential light accumulation and accumulate it into the charge register, and finally generate an image signal through the electronic circuit module. Compared with general area array cameras, TDI cameras increase exposure time through multi-level accumulation, reduce scanning time, and improve system throughput. When the arrangement direction of the stripes in the stripe structured light is perpendicular to the scanning direction (that is, the extension direction of the stripes is parallel to the scanning direction), the TDI camera can form an image normally. However, when the arrangement direction of the stripes in the stripe structured light is parallel to the scanning direction, due to the integration effect of the TDI camera, the electrical signal generated by the stripe structured light will be homogenized and unusable.

[0004] Traditional super-resolution imaging techniques often require two stripe structured light beams, each arranged perpendicularly to the sample, to illuminate the sample. However, one of the stripe structured light beams cannot be integrated and imaged by a TDI camera. Therefore, TDI cameras cannot be used in systems that illuminate the sample with stripe structured light, limiting the system's throughput. Some existing systems have modified TDI cameras to accommodate dual stripe structured light illumination, but this approach is costly, complex, and challenging to implement.

[0005] Summary of the Invention

[0006] The first aspect of the present application provides a super-resolution imaging system, comprising:

[0007] A light source module includes a laser light source and a structured light modulation element, wherein the laser light source has a light-emitting end face for emitting excitation light, and the structured light generating element is located on one side of the light-emitting end face of the laser light source and is used to modulate the phase of the excitation light to generate and emit first light and second light with separate optical paths;

[0008] a light guide module, comprising a light guide element located on the optical path of the first light and the second light, wherein the light guide element guides the first light and the second light to interfere with each other on the surface of the sample to be tested to form stripe structured light at a first angle, and guides the first light or the second light to form wide-field light on the surface of the sample to be tested at a second angle; the stripe structured light excites and generates a structured light fluorescence signal, and when the light guide element emits the first light and the second light at the second angle, the first light or the second light forms a wide-field light spot on the surface of the sample to be tested to excite and generate a wide-field fluorescence signal; and

[0009] An imaging module is used to collect the structured light fluorescence signal generated by the stripe structured light excitation to generate a structured light image, and to collect the wide-field fluorescence signal generated by the wide-field light excitation to generate a wide-field image. The structured light image and the wide-field image are used to obtain a super-resolution image of the sample to be tested.

[0010] A second aspect of the present application provides a super-resolution imaging method, comprising:

[0011] Setting the light guide element to a first angle state;

[0012] driving a laser light source to emit excitation light, modulating the excitation light to separate the first light and the second light into an output light path; controlling the light guide element to emit the first light and the second light onto the surface of the sample to be measured to interfere with each other to form a stripe structured light, thereby exciting and generating a structured light fluorescence signal, and collecting the structured light fluorescence signal to generate a structured light image; and

[0013] Switch the light-guiding element to a second angle state, control the light-guiding element to emit the first light or the second light to the surface of the sample to be tested as wide-field light, so as to excite and generate a wide-field fluorescence signal, and collect the wide-field fluorescence signal to generate a wide-field image; the structured light image and the wide-field image are used to obtain a super-resolution image of the sample to be tested.

[0014] A third aspect of the present application provides a biological sample identification device, comprising:

[0015] As in the above-mentioned super-resolution imaging system, the sample to be tested is a biological sample; and

[0016] A control system is electrically connected to the light source module, the light guide module and the imaging module, respectively, and is used to control the laser light source to emit the excitation light, switch the light guide element to the first angle state or the second angle state, control the imaging module to collect the structured light fluorescence signal and the wide-field fluorescence signal, and obtain a super-resolution image of the sample to be tested based on the structured light image and the wide-field image, so as to identify the characteristic information of the sample to be tested according to the super-resolution image.

[0017] A fourth aspect of the present application provides a biological sample identification method, comprising:

[0018] Setting the light guide element to a first angle state;

[0019] driving a laser light source to emit excitation light, modulating the excitation light to separate a first light and a second light into an output optical path; controlling the light guide element to emit the first light and the second light onto the surface of a sample to be tested to interfere with each other to form a stripe structured light, thereby exciting and generating a structured light fluorescence signal; collecting the structured light fluorescence signal to generate a structured light image, wherein the sample to be tested is a biological sample; and

[0020] Switching the light guide element to a second angle state, controlling the light guide element to emit the first light or the second light to the surface of the sample to be measured as wide-field light to excite and generate a wide-field fluorescence signal, and collecting the wide-field fluorescence signal to generate a wide-field image;

[0021] The structured light image and the wide-field image are super-resolved and reconstructed based on deep learning to obtain a super-resolved image of the sample to be tested, so as to obtain feature information of the sample to be tested according to the super-resolved image.

[0022] A fifth aspect of the present application provides a gene sequencer, comprising:

[0023] In the super-resolution imaging system described above, the sample to be tested is a nucleic acid sample; and

[0024] A control system is electrically connected to the light source module, the light guide module and the imaging module, respectively, and is used to control the laser light source to emit the excitation light, switch the light guide element to the first angle state or the second angle state, control the imaging module to collect the structured light fluorescence signal and the wide-field fluorescence signal, and perform super-resolution reconstruction of the structured light image and the wide-field image based on deep learning to obtain a super-resolution image of the sample to be tested, so as to obtain base sequence information of the sample to be tested based on the super-resolution image.

[0025] A sixth aspect of the present application provides a gene sequencing method, comprising:

[0026] Setting the light guide element to a first angle state;

[0027] driving a laser light source to emit excitation light, modulating the excitation light to separate the first light and the second light into an output light path; controlling the light-guiding element to emit the first light and the second light onto the surface of a sample to be tested to interfere with each other to form a stripe structured light, thereby exciting and generating a structured light fluorescence signal; collecting the structured light fluorescence signal to generate a structured light image, wherein the sample to be tested is a nucleic acid sample; and

[0028] Switching the light guide element to a second angle state, controlling the light guide element to emit the first light or the second light to the surface of the sample to be measured as wide-field light to excite and generate a wide-field fluorescence signal, and collecting the wide-field fluorescence signal to generate a wide-field image;

[0029] The structured light image and the wide-field image are super-resolved reconstructed based on deep learning to obtain a super-resolved image of the sample to be tested, so as to obtain base sequence information of the sample to be tested according to the super-resolved image.

[0030] In the above-mentioned super-resolution imaging system, super-resolution imaging method, biological sample identification device, biological sample identification method, gene sequencer and gene sequencing method, the light-guiding element is used to guide the first light and the second light emitted by the light source module to the surface of the sample to be tested; the light-guiding element has a first angle state and a second angle state; when the light-guiding element is in the first angle state, the first light and the second light are simultaneously guided to the surface of the sample to be tested, so that the first light and the second light interfere with each other on the surface of the sample to be tested to generate stripe structured light; when the light-guiding element is in the second angle state, only one of the first light and the second light is guided to the surface of the sample to be tested, so that the first light or the second light alone irradiates the sample to be tested, then no interference occurs at this time, and the first light or the second light alone serves as wide-field light to irradiate the sample to be tested; in a pair of proportions, two stripe structured lights with different stripe arrangement directions are used to irradiate the sample to be tested respectively, and only one of them One type of stripe structured light can be used in conjunction with a TDI camera for integral imaging, while another type of stripe structured light cannot be used in conjunction with a TDI camera for integral imaging when irradiated; the above-mentioned light-guiding element switches between a first angle state and a second angle state, and can use stripe structured light and wide-field light to illuminate the sample to be tested, respectively. That is, wide-field light is used to replace one of the stripe structured lights in the comparative example, which can effectively avoid the problem that one of the stripe structured lights cannot be used in conjunction with a TDI camera for integral imaging, and based on a deep learning network and a super-resolution reconstruction algorithm, a super-resolution image of the sample to be tested can also be obtained; therefore, the super-resolution imaging system of an embodiment of the present application switches stripe structured light and wide-field light through a light-guiding element to respectively irradiate the sample to be tested, so that the stripe structured light can be combined with a TDI camera for imaging, which is beneficial to simultaneously improve the resolution and throughput of the super-resolution imaging system, and the optical path structure of the super-resolution imaging system is simple, which is beneficial to reducing the system cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG1 is a schematic diagram of the module structure of a biological sample identification device according to an embodiment of the present application.

[0032] FIG. 2 is a schematic diagram of the optical path structure of the biological sample identification device in FIG. 1 .

[0033] FIG. 3 is a schematic diagram showing the propagation process of the excitation light by the collimating assembly in FIG. 2 on the first plane.

[0034] FIG. 4 is a schematic diagram showing the propagation process of the excitation light by the collimating assembly in FIG. 2 on the second plane.

[0035] FIG. 5 is a schematic diagram of the light spot structure on the light receiving surface of the structured light modulation element in FIG. 2 .

[0036] FIG6 is a schematic diagram of the planar structure of the spatial filter in FIG2 .

[0037] FIG7 is a schematic diagram showing the structure of a light spot formed by stripe structured light on the surface of a sample to be measured.

[0038] FIG8 is a schematic diagram showing the structure of a light spot formed by wide-field light on the surface of a sample to be measured.

[0039] FIG9 is a schematic flow chart of the steps of the biological sample identification method according to an embodiment of the present application.

[0040] : Description of the main component symbols Biological sample identification device 1 Super-resolution imaging system 100 Light source module 10 Laser light source 11 Structured light modulation element 12 Light receiving surface 121 Long side a Short side b Spatial filter 13 Channels 131, 132 Collimation assembly 14 Circular lens 141 Cylindrical lens 142 First lens 151 Second lens 152 Light guide module 20 Light guide element 21 Reflection surface 211 Third lens 22 Imaging module 30 First imaging channel 31 First camera 311 First tube lens 312 Second imaging channel 32 Second camera 321 Second tube lens 322 Beam splitter 33 Focusing module 40 Light guiding module 50 Objective lens 51 First dichroic mirror 52 Second dichroic mirror 53 Control system 200 Sample stage 300 Sample to be measured 2 First lightL1 Second light L2 Light spots S0, S1, S2 Steps S11, S12, S13, S14, S15

[0041] The following specific implementation methods will further illustrate the present application in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0042] In order to more clearly understand the above-mentioned objectives, features and advantages of the present application, the present application is described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the embodiments of the present application and the features therein can be combined with each other in the absence of conflict.

[0043] The following description sets forth many specific details to facilitate a full understanding of the present application. The embodiments described are only a portion of the embodiments of the present application, not all of the embodiments. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present application without creative effort are intended to fall within the scope of protection of the present application.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0045] The present application provides a super-resolution imaging system that can be applied to both structured light super-resolution imaging scenarios and wide-field illumination imaging scenarios. The super-resolution imaging system is used to respectively obtain structured light images and wide-field images of the sample to be tested. Based on a deep learning network and a super-resolution reconstruction algorithm, a super-resolution image of the sample to be tested can be generated based on the structured light image and the wide-field image. Feature information of the sample to be tested can be obtained from the super-resolution image. When the super-resolution imaging system is applied to a biological sample identification device, the sample to be tested is a biological sample, such as a nucleic acid sample (DNA or RNA), protein or cell, etc., and the biological sample identification device is used to identify the biological feature information of the sample to be tested.

[0046] In the embodiment of the present application, an example is given in which the sample to be tested is a nucleic acid sample and the biological sample identification device is a gene sequencer, and the method for obtaining the base sequence information of the sample to be tested is described.

[0047] Referring to Figure 1 , the biological sample identification device 1 of this embodiment includes a super-resolution imaging system 100, a control system 200, and a sample carrier 300. The control system 200 is electrically connected to the super-resolution imaging system 100 and the sample carrier 300. The surface of the sample carrier 300, facing the super-resolution imaging system 100, is used to support a sample 2 to be tested. Different bases (e.g., including bases A, T, G, and C) on the sample 2 to be tested are bound to different types of fluorescent dyes.

[0048] The control system 200 is used to control the super-resolution imaging system 100 to emit laser light toward the sample 2 to excite different types of fluorescent dyes, generating multiple fluorescent signals with different wavelengths. The control system 200 is also used to control the super-resolution imaging system 100 to receive these multiple fluorescent signals to generate a structured light image and a widefield image. The control system 200 is further used to generate a super-resolution image of the sample 2 based on the structured light image and widefield image, thereby obtaining base sequence information of the sample 2.

[0049] Since the field of view of the laser is limited, it is difficult for the laser to completely cover the surface of the sample to be tested 2, and therefore it is difficult to simultaneously excite the fluorescent dyes in all areas of the sample to be tested 2. In this embodiment, the control system 200 is used to drive the sample carrier 300 to synchronously drive the sample to be tested 2 to move in a plane perpendicular to the optical axis of the objective lens (see the objective lens 51 in FIG2 ), so that different areas of the surface of the sample to be tested 2 are sequentially located within the field of view of the laser and irradiated by the laser. The above process can be referred to as a laser scanning process of the sample to be tested 2. Through the above scanning process, the fluorescent dyes in various areas of the sample to be tested 2 can be excited, and a complete structured light image and wide-field image of the sample to be tested 2 can be obtained, thereby obtaining the base sequence information of all areas of the sample to be tested 2.

[0050] 2 , the super-resolution imaging system 100 of this embodiment includes a light source module 10, a light guide module 20, an imaging module 30, a focus module 40, and a light guiding module 50. The light source module 10, the light guide module 20, the imaging module 30, and the focus module 40 are each electrically connected to a control system (not shown).

[0051] The light source module 10 includes a laser light source 11, a structured light modulation element 12, a spatial filter 13, a collimator 14, a first lens 151, and a second lens 152. In this embodiment, the laser light source 11, the collimator 14, the structured light modulation element 12, the first lens 151, the spatial filter 13, and the second lens 152 are arranged in sequence.

[0052] The laser light source 11 includes a laser (not shown) and an optical fiber (not shown) connected to the laser emission end. The laser outputs excitation light through the optical fiber. The excitation light includes a first laser and a second laser with different wavelengths. The laser can emit the first laser and the second laser simultaneously, or it can emit the first laser and the second laser in sequence. The optical fiber has a light-emitting end face for emitting excitation light. The light-emitting end face is rectangular, so that the excitation light can form a rectangular spot. The collimating component 14, the structured light modulation element 12, the first lens 151, the spatial filter 13, and the second lens 152 are located on one side of the light-emitting end face of the laser light source 11, and are sequentially located on the optical path of the excitation light. In this embodiment, the first laser is a green laser with a wavelength of 532 nm, and the second laser is a red laser with a wavelength of 640 nm. The aspect ratio of the light-emitting end face is 3:1. In this embodiment, the long side dimension of the light-emitting end face is 450 μm, and the short side dimension is 150 μm.

[0053] The collimating component 14 is used to collimate the received excitation light into a parallel beam so that the excitation light is vertically incident on the structured light modulation element 12. The collimating component 14 may include one or more lenses.

[0054] Please refer to Figure 3. In the embodiment, the collimating component 14 includes a circular lens 141 and a cylindrical lens 142. In the first plane direction, the excitation light emitted by the laser light source 11 is collimated by the circular lens 141 and focused by the cylindrical lens 142 in sequence, and then is incident on the structured light modulation element 12, forming the short side of a rectangular light spot. Please refer to Figure 4. In the second plane direction perpendicular to the first plane direction, the cylindrical lens 142 does not have a focusing function. After the excitation light is collimated by the circular lens 141, it is transmitted from the cylindrical lens 142 and vertically incident on the structured light modulation element 12, forming the long side of the rectangular light spot. That is, because the cylindrical lens 142 has different light shaping effects in different directions, the light emitted from the cylindrical lens 142 can form a rectangular light spot.

[0055] The structured light modulation element 12 has a light-receiving surface 121 facing the collimator assembly 14. The structure of the light spot S0 formed by the excitation light on the light-receiving surface 121 is shown in Figure 5 . The light spot is a rectangular light spot with a long side a and a short side b. In this embodiment, because the long side a of the light spot is much larger than the short side b, it can also be called a stripe light spot or a linear light spot.

[0056] Referring again to FIG. 2 , in this embodiment, the structured light modulation element 12 is a phase modulation grating used to modulate the excitation light to generate diffracted light. The first lens 151 is used to receive the diffracted light and converge the diffracted light onto the spatial filter 13. The spatial filter 13 is used to filter out all diffracted light except for the ±1st diffraction order. That is, after the diffracted light enters the spatial filter 13, the diffracted light in the ±1st diffraction order can be transmitted through the spatial filter 13 to propagate to the subsequent optical path, while the diffracted light in the remaining diffraction orders is filtered by the spatial filter 13.

[0057] In this embodiment, the planar structure of the spatial filter 13 is shown in FIG6 , which includes two spaced-apart light output channels: a first light output channel 131 and a second light output channel 132 . Therefore, the spatial filter 13 is used to output two light beams separated by optical paths: the first light L1 and the second light L2 . Referring again to FIG2 , the second lens 152 is used to focus the first light L1 and the second light L2 from the spatial filter 13 onto the light guide element 21 .

[0058] The first lens 151 and the second lens 152 form a 4f system. The structured light modulation element 12 is located at the front focal plane of the 4f system. The light guide element 21 is located at the back focal plane of the 4f system. The spatial filter 13 is located at the spectrum plane of the 4f system.

[0059] The light guide module 20 includes a light guide element 21 and a third lens 22. In this embodiment, the light guide element 21 is a galvanometer. The light guide element 21 has a reflective surface 211. When the first light L1 and the second light L2 are incident on the reflective surface 211, they are reflected by the reflective surface 211.

[0060] The light guide element 21 of this embodiment can be switched between a first angular state (the state indicated by the solid line in FIG. 2 ) and a second angular state (the state indicated by the dashed line in FIG. 2 ) by rotation under the control of a control system. When the light guide element 21 is in the first angular state and the second angular state, the first light L1 and the second light L2 have different incident angles, resulting in different exit angles of the first light L1 and the second light L2 when reflected from the reflective surface 211.

[0061] When the light guide element 21 is in a first angle state, the first light L1 and the second light L2 are emitted at a first angle, so that both the first light L1 and the second light L2 are received by the third lens 22, focused, and transmitted to the subsequent optical path (see the optical path shown by the solid line in FIG2 ). When the light guide element 21 is in a second angle state, the first light L1 and the second light L2 are emitted at a second angle, so that one of the first light L1 and the second light L2 can be received by the third lens 22, focused, and transmitted to the subsequent optical path, while the other cannot be received by the third lens 22 and is dispersed in space (see the optical path shown by the dotted line in FIG2 ). That is, when the light guide element 21 is in the first angle state, it is used to guide the first light L1 and the second light L2 to the subsequent optical path to illuminate the sample 2 to be measured. When the light guide element 21 is in the second angle state, it is used to guide one of the first light L1 and the second light L2 to the subsequent optical path to illuminate the sample 2 to be measured, and cut the other of the first light L1 and the second light L2 out of the optical path of the super-resolution imaging system 100. In this embodiment, when the light guide element 21 is in the second angle state, the first light L1 is received and focused by the third lens 22 and transmitted to the subsequent optical path, while the second light L2 cannot be received by the third lens 22 and is diverged in space.

[0062] In other embodiments of the present application, the light-guiding element 21 may be other optical elements that enable light to be emitted in different directions by switching states, such as a reflective plate that is driven to rotate by a motor. In other embodiments of the present application, the light-guiding element 21 may also switch states by other means besides rotation, such as by switching states by translation. In other embodiments of the present application, the light-guiding module 20 may enable light to be emitted in different directions by a combination of multiple optical elements, for example, the light-guiding module 20 includes two independent optical paths, which guide the first light L1 and the second light L2 to different directions respectively. In this embodiment, the light-guiding element 21 adopts a galvanometer, which is conducive to simplifying the optical path structure and saving costs. In addition, the setting time of the galvanometer is less than 20ms, or even less than 10ms, which is conducive to realizing the high-speed rotation process of the light-guiding element 21, and is suitable for the super-resolution imaging system 100 for high-speed imaging.

[0063] When the first light L1 and the second light L2 simultaneously illuminate the sample 2, they interfere with each other to produce striped structured light. This striped structured light can form a striped light spot S1 on the surface of the sample 2, consisting of alternating parallel arrangements of light and dark stripes, as shown in Figure 7. When only one of the first light L1 and the second light L2 illuminates the sample 2, no interference occurs. The first light L1 or the second light L2 directed toward the sample 2 acts solely as wide-field light to illuminate the sample 2, forming a wide-field light spot S2 on the surface of the sample 2, as shown in Figure 8.

[0064] 2 , when the sample 2 is illuminated by the stripe structured light, the fluorescent dye is excited to generate a structured light fluorescence signal. When the sample 2 is illuminated by the wide-field light, the fluorescent dye is excited to generate a wide-field fluorescence signal.

[0065] In this embodiment, the imaging module 30 includes a first imaging channel 31 and a second imaging channel 32 that are independent of each other. The structured light fluorescence signal includes the first fluorescence and the second fluorescence, and the wide-field fluorescence signal also includes the first fluorescence and the second fluorescence. The first fluorescence and the second fluorescence have different wavelengths. The imaging module 30 also includes a spectrometer 33. In this embodiment, the spectrometer 33 is a dichroic mirror for reflecting the first fluorescence to the first imaging channel 31 and transmitting the second fluorescence to the second imaging channel 32. The first imaging channel 31 is used to collect the first fluorescence to generate a structured light image and a wide-field image, respectively. The second imaging channel 32 is used to collect the second fluorescence to generate a structured light image and a wide-field image, respectively. In this embodiment, the wavelength of the first fluorescence is between 550 and 600 nm (including the endpoint values), and the wavelength of the second fluorescence is between 660 and 700 nm (including the endpoint values).

[0066] The first imaging channel 31 includes a first camera 311 and a first tube lens 312, and the second imaging channel 32 includes a second camera 321 and a second tube lens 322. The first imaging channel 31 and the second imaging channel 32 have substantially the same structure and function. The following description will take the first imaging channel 31 as an example. The first camera 311 is a TDI camera having a photosensitive surface facing the first tube lens 312. The first tube lens 312 is used to focus the first fluorescence onto the photosensitive surface of the first camera 311. When the photosensitive surface of the first camera 311 receives the first fluorescence, it can generate an electrical signal. During the process of continuous laser scanning of the sample 2 to be tested, the first camera 311 is used to continuously integrate the electrical signal until the scan is completed and the first camera 311 stops integrating.

[0067] In this embodiment, the super-resolution imaging system 100 contains two fluorescence signals of different wavelengths: a first fluorescence and a second fluorescence. The imaging module 30 includes two imaging channels: a first imaging channel 31 and a second imaging channel 32. In this embodiment, the laser light source 11 simultaneously emits the first laser and the second laser, and the first camera 311 and the second camera 321 simultaneously initiate exposure, enabling simultaneous imaging of the first imaging channel 31 and the second imaging channel 32.

[0068] In other embodiments of the present application, other numbers of fluorescence signals may be present within the super-resolution imaging system 100, for example, fluorescence signals of four different wavelengths. The number of imaging channels in the imaging module 30 may vary, for example, the imaging module 30 may include one or four imaging channels. When the number of imaging channels in the imaging module 30 is less than the number of fluorescence signals, the laser light source 11 needs to emit the first laser and the second laser in a time-sharing manner, so that the sample 2 to be tested is stimulated in a time-sharing manner to generate fluorescence of different wavelengths, and the imaging channels collect fluorescence images of the different wavelengths in a time-sharing manner.

[0069] The focus module 40 is used to emit infrared light in real time and receive infrared light reflected by the sample 2 to be tested. The focus module 40 calculates the defocus value based on the infrared light reflected by the sample 2. The control system 200 is used to adjust the position of the objective lens in the system in real time based on the defocus value to ensure the accuracy and stability of the optical path structure. In this embodiment, the wavelength of the infrared light emitted by the focus module 40 is 850nm. In other embodiments of the present application, the biological sample identification device 100 may not include the focus module 40.

[0070] The biological sample identification device 1 also includes a plurality of optical elements for guiding the transmission direction of light signals (such as the first light, the second light, the first fluorescence, the second fluorescence, infrared light, etc.). In this embodiment, the above-mentioned optical elements include an objective lens 51, a first dichroic mirror 52 and a second dichroic mirror 53. The first dichroic mirror 52 is used to reflect the first light and / or the second light to the objective lens 51, transmit the structured light fluorescence signal and the wide-field fluorescence signal from the sample to be tested 2, and transmit infrared light. The objective lens 51 is used to focus the first light and / or the second light to the sample to be tested 2, and to guide the structured light fluorescence signal and the wide-field fluorescence signal from the sample to be tested 2 to the first dichroic mirror 52. The second dichroic mirror 53 is located between the focusing module 40 and the first dichroic mirror 52, and is used to transmit infrared light and reflect the structured light fluorescence signal and the wide-field fluorescence signal.

[0071] In other embodiments of the present application, the biological sample identification device 1 may further include other optical elements, such as reflectors and lenses, for guiding light signals to corresponding spatial locations, so that the space occupied by the biological sample identification device 1 meets the required requirements. In other embodiments of the present application, the biological sample identification device 1 may also not include optical elements for guiding light signals, such as the first dichroic mirror 52 and the second dichroic mirror 53.

[0072] In this embodiment, to accommodate the different wavelengths of the first and second laser beams emitted by the laser light source 11, lenses 141, 142, 151, 152, and 22 are achromatic lenses. In at least one alternative embodiment of the present application, lenses 141, 142, 151, 152, and 22 can be the same type of lens to reduce hardware costs. In this embodiment, the first dichroic mirror 52, the second dichroic mirror 53, and the beam splitter 33 have high reflectivity for light of specific wavelengths and high transmittance for light of other wavelengths, thereby achieving highly efficient spectroscopic function. For example, the first dichroic mirror 52 has high reflectivity for light near the wavelengths (532 nm and 640 nm) of the first and second laser beams emitted by the laser light source 11, while having high transmittance for light near the wavelengths (550-600 nm and 660-700 nm) of the first and second fluorescent light emitted by the sample 2. In this embodiment, the objective lens 51 has a numerical aperture of 0.8 and a magnification of 20X. The first camera 311 and the second camera 321 are 256-order TDI cameras with a resolution of 256×9072 pixels.

[0073] The embodiment of the present application further provides a biological sample identification method, which is applied to the biological sample identification device 1 described above.

[0074] Referring to FIG. 9 , the biological sample identification method includes:

[0075] Step S11, setting the light guide element to a first angle state;

[0076] Step S12: driving a light source to emit excitation light, modulating the excitation light to separate the first light and the second light into an output light path; controlling a light guide element to emit the received first light and the second light to a sample to be tested, so that the first light and the second light interfere with each other on the surface of the sample to be tested to form a stripe structured light, thereby exciting and generating a structured light fluorescence signal; and collecting the structured light fluorescence signal to generate a structured light image.

[0077] Step S13, turning off the driving light source and switching the light guide element to a second angle state;

[0078] Step S14: driving the light source to emit excitation light, modulating the excitation light to separate the first light and the second light into an output light path; controlling the light guide element to output one of the received first light and the second light to the sample to be tested, so that the first light or the second light forms a wide-field light on the surface of the sample to be tested to excite and generate a wide-field fluorescence signal; collecting the wide-field fluorescence signal to generate a wide-field image; and

[0079] Step S15 , performing super-resolution reconstruction based on the structured light image and the wide-field image to obtain a super-resolution image of the sample to be tested, so as to identify feature information of the sample to be tested according to the super-resolution image.

[0080] 2 , in step S11 , the light guide element 21 is set at a 45° angle, that is, the reflective surface 211 of the light guide element 21 and the optical axis of the excitation light have an included angle of 45°. This is referred to as the light guide element 21 being in a first angle state.

[0081] In step S12, the laser light source 11 is driven to turn on and simultaneously emit the combined light of the first laser and the second laser as the excitation light. The excitation light is modulated by the structured light modulation element 12 and then filtered by the spatial filter 13. The light guide element 21 receives the first light and the second light and reflects the first light and the second light to the third lens 22 at the same time. The third lens 22, the first dichroic mirror 52, and the objective lens 51 sequentially guide the first light and the second light to the surface of the sample 2 to be tested, and interference generates stripe structured light. The stripe structured light excites the fluorescent dye to generate a structured light fluorescence signal. The structured light fluorescence signal is sequentially guided to the imaging module 30 through the objective lens 51, the first dichroic mirror 52, the second dichroic mirror 53, and the beam splitter 33. Among them, the first fluorescence in the first wavelength band of the structured light fluorescence signal is reflected to the first imaging channel 31, and the second fluorescence in the second wavelength band is transmitted to the second imaging channel 31. When the laser light source 11 is turned on, the first camera 311 and the second camera 321 are turned on at the same time. The first tube lens 312 in the first imaging channel 31 focuses the first fluorescence to the first camera 311, so that the first camera 311 generates a first structured light image; at the same time, the second tube lens 322 in the second imaging channel 32 focuses the second fluorescence to the second camera 321, so that the second camera 321 generates a second structured light image.

[0082] In this embodiment, the arrangement direction of the stripes in the stripe structured light is perpendicular to the scanning direction of the stripe structured light on the sample 2 to be measured, and the arrangement direction of the stripes in the stripe structured light is perpendicular to the integration direction of the first camera 311 and the second camera 321 .

[0083] After the stripe structured light scans the sample 2, in step S13, the laser light source 11 is turned off, the excitation light emission is stopped, and the first camera 311 and the second camera 321 are turned off. The control system 200 rotates the light guide element 21, causing the light guide element 21 to be at a 40° angle. This means that the reflective surface 211 of the light guide element 21 forms a 50° angle with the optical axis of the excitation light. This is referred to as the light guide element 21 being at the second angle.

[0084] The angle of the light-guiding element 21 in the first angle state and the second angle state differs by at least 5°, which is 5° in this embodiment (45° and 40° in the two angle states respectively); in other embodiments of the present application, for example, the angle of the light-guiding element 21 in the first angle state and the second angle state may differ by 10°.

[0085] After the angle of the light guide element 21 is switched, in step S14, the laser light source 11 is turned on again, emitting excitation light including the first laser and the second laser. The first camera 311 and the second camera 321 are also turned on simultaneously. The excitation light is modulated by the structured light modulator 12 and then filtered by the spatial filter 13. The light guide element 21 receives the first and second light beams, reflecting one of the first and second beams to the third lens 22 and the other into space for dispersion. In other words, the third lens 22 receives only the first or second beam. The third lens 22, the first dichroic mirror 52, and the objective lens 51 sequentially guide the first or second beam to the surface of the sample 2 to be tested. The first or second beam, acting alone as widefield light, illuminates the sample 2 without generating interference fringes. The widefield light excites the fluorescent dye to produce a widefield fluorescence signal. The widefield fluorescence signal is sequentially guided through the objective lens 51, the first dichroic mirror 52, the second dichroic mirror 53, and the beam splitter 33 to the imaging module 30. The first fluorescence in the first wavelength band of the wide-field fluorescence signal is reflected to the first imaging channel 31, and the second fluorescence in the second wavelength band is transmitted to the second imaging channel 31. When the laser light source 11 is turned on, the first camera 311 and the second camera 321 are turned on simultaneously. The first tube lens 312 in the first imaging channel 31 focuses the first fluorescence onto the camera 311, causing the camera to generate a first wide-field image. Simultaneously, the second tube lens 322 in the second imaging channel 32 focuses the second fluorescence onto the camera 311, causing the camera to generate a second wide-field image.

[0086] After the wide-field light completes scanning the sample 2 to be measured, the laser light source 11 , the camera 311 and the second camera 321 are turned off simultaneously.

[0087] During steps S12 and S14, sample stage 300 continuously translates at a constant speed in a plane perpendicular to objective lens 51. Specifically, sample stage 300 begins translation simultaneously when laser light source 11 is turned on and first camera 311 and second camera 321 begin exposure. Translation ceases when laser light source 11 is turned off and first camera 311 and second camera 321 cease exposure. In this embodiment, the numbers used for the above method steps do not limit the order in which the steps should be performed.

[0088] The biological sample identification method of this embodiment is applied to a gene sequencer as a gene sequencing method, and the test sample 2 is a nucleic acid sample. In step S15, the control system 200 performs super-resolution reconstruction on the first structured light image, the second structured light image, the first widefield image, and the second widefield image based on a super-resolution reconstruction algorithm and a deep learning network, obtaining a super-resolution image of the test sample 2. The base sequence information of the test sample 2 is then obtained based on the super-resolution image.

[0089] In other embodiments of the present application, the sample 2 to be tested may be other biological samples, and the biological sample identification method may be used to obtain other characteristic information of the sample 2 to be tested. The above steps S11 to S14 may be used as a super-resolution imaging method and applied to other optical devices that require super-resolution imaging to obtain super-resolution images.

[0090] In the super-resolution imaging system 100 of the embodiment of the present application, the light guide module 20 includes a light guide element 21, which is used to guide the first light and the second light emitted by the light source module 10 to the surface of the sample to be tested 2. The light guide element 21 has a first angle state and a second angle state. When the light guide element 21 is in the first angle state, the first light and the second light are simultaneously guided to the surface of the sample to be tested 2, so that the first light and the second light interfere with each other on the surface of the sample to be tested 2, generating stripe structured light; when the light guide element 21 is in the second angle state, only one of the first light and the second light is guided to the surface of the sample to be tested 2, so that the first light or the second light alone illuminates the sample to be tested 2, then no interference occurs at this time, and the first light or the second light alone serves as wide-field light to illuminate the sample to be tested 2. When the stripe structured light illuminates the sample to be tested, it excites and generates a structured light fluorescence signal, and when the wide-field light illuminates the sample to be tested 2, it excites and generates a wide-field fluorescence signal. The imaging module 30 collects structured light fluorescence signals to generate structured light images, collects wide-field fluorescence signals to generate wide-field images, and generates a super-resolution image of the sample 2 to be tested based on the structured light image and the wide-field image.

[0091] In a comparative example, two stripe structured lights with different stripe arrangement directions were used to illuminate the sample under test. Only one of the stripe structured lights could be used with a TDI camera for integral imaging, while the other stripe structured light could not. In the present application, by switching the light-guiding element 21 between a first angle state and a second angle state, the sample under test 2 can be illuminated with both stripe structured light and widefield light. This effectively avoids the problem of one of the stripe structured lights being unable to be used with a TDI camera for integral imaging. Furthermore, based on a deep learning network and a super-resolution reconstruction algorithm, a super-resolution image of the sample under test can be obtained.

[0092] Therefore, the super-resolution imaging system 100 of the embodiment of the present application switches the stripe structured light and the wide-field light through the light-guiding element 21 to respectively illuminate the sample 2 to be tested, so that the stripe structured light can be combined with the TDI camera for imaging, which is beneficial to simultaneously improve the resolution and throughput of the super-resolution imaging system 100. In addition, the optical path structure of the super-resolution imaging system 100 is simple, which is beneficial to reducing the system cost.

[0093] In this embodiment, the super-resolution imaging method, the biological sample identification device 1 , the biological sample identification method, the gene sequencer, and the gene sequencing method can achieve all the beneficial effects of the super-resolution imaging system 100 described above.

[0094] Those skilled in the art should recognize that the above embodiments are merely intended to illustrate the present application and are not intended to limit the present application. As long as they are within the spirit of the present application, appropriate changes and modifications to the above embodiments are within the scope of protection claimed in the present application.

Claims

1. A super-resolution imaging system, characterized in that, include: A light source module, comprising a laser light source and a structured light modulation element, wherein the laser light source has a light-emitting end face for emitting excitation light, and the structured light generating element is located on one side of the light-emitting end face of the laser light source, and is used to modulate the phase of the excitation light to generate and emit a first light and a second light with separated optical paths; A light guide module, comprising a light guide element located on the optical path of the first light and the second light, wherein the light guide element guides the first light and the second light to interfere on the surface of the sample to be tested to form stripe structured light in a first angle state, and guides the first light or the second light to form wide-field light on the surface of the sample to be tested in a second angle state; the stripe structured light excites and generates a structured light fluorescence signal, and when the light guide element emits the first light or the second light in the second angle state, the first light or the second light forms a wide-field light spot on the surface of the sample to be tested to excite and generate a wide-field fluorescence signal; as well as The imaging module is used to collect the structured light fluorescence signal generated by the stripe structured light excitation to generate a structured light image, and to collect the wide-field fluorescence signal generated by the wide-field light excitation to generate a wide-field image. The structured light image and the wide-field image are used to obtain a super-resolution image of the sample to be tested.

2. The super-resolution imaging system according to claim 1, characterized in that, The imaging module includes a time-delay integration camera, which is used to sense the structured light fluorescence to generate the structured light image and to sense the wide-field fluorescence to generate the wide-field image.

3. The super-resolution imaging system according to claim 2, wherein The stripe structured light continuously scans the sample to be tested, and during the period when the stripe structured light scans the sample to be tested, the time-delay integration camera continuously senses the structured light fluorescence signal to continuously integrate and generate the structured light image; The arrangement direction of the stripes in the stripe structured light is perpendicular to the scanning direction of the stripe structured light scanning the sample to be tested, and the arrangement direction of the stripes in the stripe structured light is perpendicular to the integration direction of the time delay integration camera.

4. The super-resolution imaging system according to claim 2, characterized in that, The light emitting end surface is rectangular, and when the first light or the second light irradiates the sample to be tested alone, a stripe-shaped light spot is formed on the surface of the sample to be tested.

5. The super-resolution imaging system according to claim 2, characterized in that, The light guide element switches between the first angle state and the second angle state by rotating; The incident angles of the first light and the second light of the light-guiding element are different when the light-guiding element is in the first angle state and the second angle state, so that the light-guiding element reflects the first light and the second light at the first angle when in the first angle state, and reflects the first light and the second light at the second angle when in the second angle state.

6. The super-resolution imaging system according to claim 5, characterized in that, The angle of the light guide element in the first angle state and the second angle state differs by at least 5°.

7. The super-resolution imaging system according to claim 5, wherein The light guiding element is a galvanometer.

8. The super-resolution imaging system according to claim 2, characterized in that, The first light and the second light are ±1st order diffracted light of the excitation light.

9. The super-resolution imaging system according to claim 2, wherein The excitation light includes a first laser and a second laser having different wavelengths, and the laser light source is used to emit the first laser and the second laser simultaneously or in a time-sharing manner.

10. The super-resolution imaging system according to claim 2, wherein The structured light modulation element is a phase modulation grating.

11. A super-resolution imaging method, characterized in that, include: Setting the light guide element to a first angle state; Drive a laser light source to emit excitation light, and modulate the excitation light to emit a first light and a second light separated in the optical path; Control the light guiding element to emit the first light and the second light to the surface of the sample to be measured, and interfere to form fringe structured light, so as to excite and generate a structured light fluorescence signal, and collect the structured light fluorescence signal to generate a structured light image; And Switch the light guiding element to a second angle state, control the light guiding element to emit the first light or the second light to the surface of the sample to be measured as wide-field light, so as to excite and generate a wide-field fluorescence signal, and collect the wide-field fluorescence signal to generate a wide-field image; the structured light image and the wide-field image are used to obtain a super-resolution image of the sample to be measured.

12. The super-resolution imaging method according to claim 11, wherein The step of switching the light guiding element to the second angle state includes: Rotate the light guiding element so that the light guiding element is switched from the first angle state to the second angle state.

13. The super-resolution imaging method according to claim 10 or 11, characterized in that, The step of collecting the structured light fluorescence to generate a structured light image includes: Collect the structured light fluorescence signal through a time-delay integration camera to generate a structured light image; The step of collecting the wide-field fluorescence to generate a wide-field image includes: Collect the wide-field fluorescence signal through a time-delay integration camera to generate a wide-field image.

14. The super-resolution imaging method according to claim 13, wherein The step of switching the light guiding element to the second angle state includes: Turn off the laser light source and turn off the time-delay integration camera; and Switch the light guiding element to the second angle state.

15. The super-resolution imaging method according to claim 11, wherein The step of driving the light source to emit excitation light includes: Drive the laser light source to simultaneously emit a first laser and a second laser with different wavelengths; or Drive the light source to emit a first laser and a second laser with different wavelengths in a time-sharing manner.

16. A biological sample identification device, characterized in that, Includes: The super-resolution imaging system according to any one of claims 1-10, wherein the sample to be measured is a biological sample; And A control system, electrically connected to the light source module, the light guiding module and the imaging module respectively, for controlling the laser light source to emit the excitation light, switching the light guiding element to be in the first angle state or the second angle state, controlling the imaging module to collect the structured light fluorescence signal and the wide-field fluorescence signal, and obtaining a super-resolution image of the sample to be measured based on the structured light image and the wide-field image, so as to identify the characteristic information of the sample to be measured according to the super-resolution image.

17. The biological sample identification device according to claim 16, wherein, It further includes a sample stage, and the super-resolution imaging system further includes an objective lens, and the objective lens is used to guide the first light and / or the second light to the sample to be measured; The sample stage is used to carry the sample to be measured, and synchronously drive the sample to be measured to displace in a plane perpendicular to the optical axis of the objective lens, and the direction in which the sample stage drives the sample to be measured to displace is perpendicular to the arrangement direction of the fringes in the fringe structured light.

18. A biological sample recognition method, characterized in that, Includes: Set the light guiding element to be in the first angle state; Drive the laser light source to emit excitation light, and modulate the excitation light to emit a first light and a second light separated in the optical path; Control the light guiding element to emit the first light and the second light to the surface of the sample to be measured, and interfere to form fringe structured light, so as to excite and generate a structured light fluorescence signal, and collect the structured light fluorescence signal to generate a structured light image, and the sample to be measured is a biological sample; and Switch the light guiding element to the second angular state, control the light guiding element to emit the first light or the second light to the surface of the sample to be measured as wide-field light, so as to excite the generation of wide-field fluorescence signals, and collect the wide-field fluorescence signals to generate a wide-field image; Based on deep learning, perform super-resolution reconstruction on the structured light image and the wide-field image to obtain the super-resolution image of the sample to be measured, so as to obtain the characteristic information of the sample to be measured according to the super-resolution image.

19. A gene sequencer, characterized in that, Comprising: The super-resolution imaging system according to any one of claims 1-10, wherein the sample to be measured is a nucleic acid sample; And A control system, electrically connected to the light source module, the light guiding module and the imaging module respectively, for controlling the laser light source to emit the excitation light, switching the light guiding element to be in the first angular state or the second angular state, controlling the imaging module to collect the structured light fluorescence signals and the wide-field fluorescence signals, and performing super-resolution reconstruction on the structured light image and the wide-field image based on deep learning to obtain the super-resolution image of the sample to be measured, so as to obtain the base sequence information of the sample to be measured according to the super-resolution image.

20. A gene sequencing method, characterized in that, Comprising: Set the light guiding element to be in the first angular state; Drive the laser light source to emit the excitation light, and modulate the excitation light to emit the first light and the second light with separated optical paths; Control the light guiding element to emit the first light and the second light to the surface of the sample to be measured to interfere and form a fringe structured light, so as to excite the generation of structured light fluorescence signals, collect the structured light fluorescence signals to generate a structured light image, wherein the sample to be measured is a nucleic acid sample; and Switch the light guiding element to the second angular state, control the light guiding element to emit the first light or the second light to the surface of the sample to be measured as wide-field light, so as to excite the generation of wide-field fluorescence signals, and collect the wide-field fluorescence signals to generate a wide-field image; Based on deep learning, perform super-resolution reconstruction on the structured light image and the wide-field image to obtain the super-resolution image of the sample to be measured, so as to obtain the base sequence information of the sample to be measured according to the super-resolution image.

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