Microscopic imaging apparatus, sequencing device, and microscopic imaging method

Abstract

A microscopic imaging apparatus, a sequencing device, and a microscopic imaging method. The microscopic imaging apparatus comprises a light source (10), an illumination light path assembly, an imaging light path assembly, and an imaging processing apparatus (14); the illumination light path assembly and the imaging light path assembly are arranged opposite to each other, and an illumination chip (12) is arranged between the illumination light path assembly and the imaging light path assembly; the light source (10) irradiates, by means of the illumination light path assembly, light having different irradiation angles to the illumination chip (12), so that the illumination chip (12) generates plasma structured light, wherein a substrate of the illumination chip (12) is made of a transparent material; and the imaging processing apparatus (14) captures, by means of the imaging light path assembly, an original image of a fluorescence signal of the illumination chip (12) under the irradiation of light having different irradiation angles.

Description

Microscopic imaging device, sequencing equipment and microscopic imaging method Technical Field

[0001] The present disclosure relates to the field of optical imaging technology, for example, to a microscopic imaging device, a sequencing device, and a microscopic imaging method. Background Art

[0002] The resolution of traditional optical microscopy systems is limited by the optical diffraction limit, hindering the advancement of optical microscopy technology. This theory, proposed by German scientist E. Abbe in 1873, states that the resolution of an optical system cannot exceed λ / (2*NA), where λ is the wavelength of the incident light and NA is the numerical aperture of the optical system. This theory led scientists to believe for much of the 20th century that optical microscopy would never be able to observe objects at smaller scales, such as the interactions of individual molecules within cells.

[0003] However, the diffraction limit theory was broken at the end of the 20th century. Currently, there are many mature super-resolution microscopy methods in the world: structured illumination microscopy (SIM), stimulated emission depletion microscopy (STED), and stochastic optical reconstruction microscopy (STORM). Among them, the structured illumination microscopy method was proposed by Professor Gustafsson in 2000. It is based on wide-field microscopy and uses structured light with periodic intensity modulation to illuminate the sample. High-frequency information is extracted through a specific algorithm to reconstruct a super-resolution image, achieving a breakthrough in the diffraction limit. Compared with other super-resolution methods, the structured illumination microscopy method has the advantages of fast imaging speed, low dye requirements, simple optical path structure, low light damage, and can be used for real-time dynamic three-dimensional imaging of living cells. It is widely used in the biomedical field.

[0004] Traditional SIM is based on frequency domain solutions, and reconstructing a super-resolution image requires at least nine consecutive images. According to the reconstruction method proposed by Professor Gustafsson, three different phase values ​​need to be obtained in each illumination direction, and high-frequency information is obtained by solving a system of linear equations, so three images need to be taken; in addition, in order to obtain super-resolution in each direction, the illumination direction needs to be rotated by three angles (generally 0°, 60°, and 120°), which means that a total of nine images are required. Sometimes, in order to obtain more precise reconstructed data, more images need to be collected. In addition, reconstruction algorithms based on frequency domain solutions inevitably introduce artifacts, which confuse the judgment of the true morphology of the observed sample structure.

[0005] In summary, microscopic imaging methods generally have the following defects:

[0006] 1) Reconstruction through frequency domain algorithms will inevitably introduce artifacts, affecting the imaging results;

[0007] 2) Traditional structured light illumination super-resolution imaging uses multiple imaging in a wide field, and repeated laser irradiation sequencing will cause additional damage to the sample.

[0008] Summary of the Invention

[0009] The present disclosure provides a microscopic imaging device, a sequencing device, and a microscopic imaging method.

[0010] In a first aspect, a microscopic imaging device is provided, comprising a light source, an illumination optical path component, an imaging optical path component, and an imaging processing device; the illumination optical path component and the imaging optical path component are arranged opposite to each other, with an illumination chip disposed therebetween;

[0011] The light source irradiates light at different irradiation angles to the illumination chip through the illumination optical path component, so that the illumination chip generates plasma structured light, wherein the substrate of the illumination chip is made of a transparent material;

[0012] The imaging processing device captures original images of the fluorescence signal of the illumination chip under the illumination of light at different illumination angles through the imaging optical path component.

[0013] In a second aspect, a sequencing device is provided, comprising the microscopic imaging device as described in the first aspect.

[0014] In a third aspect, a microscopic imaging method is provided, which is applied to the microscopic imaging device as described in the first aspect or the sequencing device as described in the second aspect, the method comprising:

[0015] Adjusting the irradiation angle of the light in the first direction at least twice, and adjusting the irradiation angle of the light in the second direction at least twice after each adjustment of the irradiation angle of the light in the first direction; wherein the sum of the changes in the irradiation angle of the light in the first direction is 0, and the sum of the changes in the irradiation angle of the light in the second direction is 0;

[0016] A corresponding original image is captured for each adjustment of the illumination angle of the light, and the original image is used to synthesize a corresponding microscopic image.

[0017] On the basis of conforming to the common sense in this field, the above-mentioned optional conditions can be arbitrarily combined to obtain the embodiments of the present disclosure.

[0018] Based on this disclosure, those skilled in the art can understand other aspects of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The features and advantages of the present disclosure will be better understood after reading the detailed description of the embodiments of the present disclosure in conjunction with the following drawings. In the drawings, components are not necessarily drawn to scale, and components with similar related properties or characteristics may have the same or similar reference numerals.

[0020] FIG1 is a schematic structural diagram of a microscopic imaging device provided by an exemplary embodiment of the present disclosure;

[0021] FIG2 is a front view of a microscopic imaging device provided by an exemplary embodiment of the present disclosure;

[0022] FIG3 is an imaging effect diagram of a first objective lens with an imaging aperture of 0.95 provided by an exemplary embodiment of the present disclosure;

[0023] FIG4 is an imaging effect diagram of a first objective lens with an imaging aperture of 0.75 provided by an exemplary embodiment of the present disclosure;

[0024] FIG5 is a schematic diagram of the movement of a TDI camera provided by an exemplary embodiment of the present disclosure;

[0025] FIG6 is a schematic structural diagram of a sequencing device provided by an exemplary embodiment of the present disclosure;

[0026] FIG7 is a schematic diagram of the distribution of illumination columns provided by an exemplary embodiment of the present disclosure;

[0027] FIG8 is a heat map showing the height distribution of an illumination column according to an exemplary embodiment of the present disclosure;

[0028] FIG9 is a diagram showing a resonance simulation effect of a lighting unit according to an exemplary embodiment of the present disclosure;

[0029] FIG10 is a schematic diagram of a first structure of an illumination chip provided by an exemplary embodiment of the present disclosure;

[0030] FIG11 is a side view of an illumination chip provided by an exemplary embodiment of the present disclosure;

[0031] FIG12 is a top view of an illumination chip provided by an exemplary embodiment of the present disclosure;

[0032] FIG13 is a second structural diagram of an illumination chip provided by an exemplary embodiment of the present disclosure;

[0033] FIG14 is a diagram showing the local electric field distribution of a sapphire substrate and an oxidation unit of silicon oxide provided by an exemplary embodiment of the present disclosure;

[0034] FIG15 is a diagram showing the local electric field distribution of a quartz substrate and an oxidation unit of silicon oxide provided by an exemplary embodiment of the present disclosure;

[0035] FIG16 is a schematic structural diagram of a bearing assembly provided by an exemplary embodiment of the present disclosure;

[0036] FIG17 is an enlarged view of a sequencing device provided by an exemplary embodiment of the present disclosure;

[0037] FIG18 is a flow chart of a microscopic imaging method provided by an exemplary embodiment of the present disclosure;

[0038] FIG19 is a schematic diagram showing changes in an illumination spot according to an exemplary embodiment of the present disclosure;

[0039] FIG20 is a first comparison effect diagram of an original image and a microscopic image provided by an exemplary embodiment of the present disclosure;

[0040] FIG21 is a second comparison effect diagram of the original image and the microscopic image provided by an exemplary embodiment of the present disclosure.

[0041] Explanation of Reference Numerals: Light source 10; illumination chip 12; imaging processing device 14; first stage 21; second stage 22; vertical support member 23; shock-absorbing foot member 24; carrying assembly 25; light angle control device 111; lens group 112; polarization adjustment member 113; first reflector 114; first objective lens 115; first galvanometer mirror 1111; second galvanometer mirror 1112; back focal plane of first objective lens 1151; front focal plane of first objective lens 1152; substrate 121; illumination unit 122; seed unit 123; placement unit 124; side wall 125; oxidation unit 126; sample 127; second objective lens 131; filter 132; tube lens 133; first dichroic mirror 134; second dichroic mirror 135; third reflector 136; fourth reflector 137; first imaging processing device 141; Second imaging processing device 142; first cylindrical mirror 1331; second cylindrical mirror 1332; positioning pin 251; adsorption platform 252; moving platform 26; bracket 27; horizontal moving assembly 28; fine adjustment moving assembly 281; coarse adjustment moving assembly 282;Focusing assembly 29; first reflecting mirror 291.; DETAILED DESCRIPTION

[0042] The present disclosure is further illustrated below by way of examples, but the present disclosure is not limited to the scope of the examples.

[0043] It should be noted that references in the specification to "one embodiment," "an alternative embodiment," "another embodiment," etc. indicate that the described embodiment may include a particular feature, structure, or characteristic, but every embodiment may not necessarily include that particular feature, structure, or characteristic. Moreover, such phrases do not necessarily refer to the same embodiment. In addition, when a particular feature, structure, or characteristic is described in conjunction with an embodiment, whether or not explicitly described, it is within the knowledge of those skilled in the relevant art to implement such feature, structure, or characteristic in conjunction with other embodiments.

[0044] In the description of the present disclosure, it should be understood that the terms "center", "lateral", "up", "down", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present disclosure and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present disclosure. In addition, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present disclosure, unless otherwise specified, "multiple" means two or more. In addition, the term "including" and any variations thereof are intended to cover non-exclusive inclusions.

[0045] In the description of the present disclosure, it should be noted that, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood in a broad sense. For example, they can refer to fixed connections, detachable connections, or integral connections; mechanical connections or electrical connections; direct connections or indirect connections through an intermediate medium; and internal connections between two components. Those skilled in the art will understand the specific meanings of the above terms in the present disclosure based on the specific circumstances.

[0046] The terms used herein are intended only to describe specific embodiments and are not intended to limit exemplary embodiments. Unless the context clearly indicates otherwise, the singular forms "a", "an", "an item" used herein are also intended to include the plural. It should also be understood that the terms "comprise" and / or "include" used herein specify the presence of stated features, integers, steps, operations, units and / or components, and do not preclude the presence or addition of one or more other features, integers, steps, operations, units, components and / or combinations thereof.

[0047] In order to overcome the above-mentioned defects that currently exist, as shown in Figure 1, an exemplary embodiment of the present disclosure provides a microscopic imaging device, which includes a light source 10, an illumination optical path component, an imaging optical path component and an imaging processing device 14; the illumination optical path component and the imaging optical path component are arranged opposite to each other, and an illumination chip 12 is arranged in the middle.

[0048] The light source 10 irradiates light at different angles to the illumination chip 12 through the illumination optical path component, causing the illumination chip 12 to generate plasma structured light. The imaging processing device 14 captures original images of the fluorescence signal of the illumination chip 12 under light at different angles through the imaging optical path component.

[0049] The light emitted by the light source 10 may be a laser to excite the illumination chip to generate plasma structured light, which is also referred to as excitation light in this embodiment. The light is not limited to one type of laser and can be selected according to actual conditions.

[0050] The substrate of illumination chip 12 can be made of a transparent material, including but not limited to sapphire, quartz, or any other material. The material of choice can be tailored to the specific application and is not particularly limited in this embodiment. A transparent substrate allows light from the light source to pass through illumination chip 12 and excite the sample on the illumination chip, generating a fluorescent signal that is received by the imaging optical path assembly.

[0051] In this embodiment, changing the angle of light illumination alters the location of the enhanced region within the light, thereby changing the phase of the structured light. Due to the localized nature of the plasma structured light, a regularly varying illumination distribution can be formed during imaging. This produces the spectral shift required for structured light super-resolution, eliminating the need for frequency-domain reconstruction algorithms. This reduces artifacts, does not affect the observation of oriented samples, and effectively reduces the difficulty and cost of constructing the microscopic imaging apparatus. Furthermore, the near-field effect of the plasma structured light effectively enhances the fluorescence signal within the illuminated chip, reducing the required exposure time and light dose, thereby reducing phototoxicity to the sample within the illuminated chip.

[0052] In one embodiment, as shown in FIG. 1 and FIG. 2 , the illumination optical path assembly includes: a light angle control device 111 , and the light angle control device 111 includes a first galvanometer 1111 and a second galvanometer 1112 (not shown in FIG. 1 ).

[0053] The first galvanometer 1111 is used to adjust the irradiation angle of the light emitted by the light source 10 toward the illumination chip 12 in a first direction, and the second galvanometer 1112 is used to adjust the irradiation angle of the light emitted by the light source 10 toward the illumination chip 12 in a second direction.

[0054] Specifically, the irradiation angle in the first direction may be the irradiation angle of the light on the x-axis, and the irradiation angle in the second direction may be the irradiation angle of the light on the y-axis. The specific selection can be made according to actual conditions and is not particularly limited in this embodiment.

[0055] Generally speaking, the irradiation angle and / or phase of the light is adjusted by the light angle control device 111 to generate plasma structured light of corresponding phase and frequency. The specific method for controlling the irradiation angle will be discussed in detail in the microscopic imaging method.

[0056] Similarly, the light angle control device 111 can also change the orientation of the light in the first direction and the second direction through the first galvanometer 1111 and the second galvanometer 1112. The control method is the same as the control method of the illumination angle, which will not be repeated here.

[0057] In this embodiment, the purpose of changing the irradiation angle of the light is to change the projection of the light wave vector incident on the chip on the horizontal plane, thereby changing the phase of the plasma structured light. The purpose of changing the orientation of the light is to ensure an isotropic resolution improvement effect on the horizontal plane.

[0058] As an embodiment, as shown in FIG. 1 and FIG. 2 , the illumination optical path assembly further includes a lens group 112 , a polarization adjustment member 113 , a first reflector 114 and a first objective lens 115 .

[0059] The lens group 112 is used to receive the light adjusted by the light angle control device 111, and converge the light to irradiate it to the polarization adjustment member 113; the polarization adjustment member 113 is used to keep the light of different irradiation angles in the same polarization state, and the first reflector 114 is used to reflect the light to the first objective lens 115; the first objective lens 115 converges the light to the illumination chip 12.

[0060] Among them, the lens assembly 112 is used to compensate for the optical path difference generated when light passes through the first galvanometer mirror 1111 and the second galvanometer mirror 1112. When the light angle control device 111 changes the illumination angle of the light incident on the illumination chip 12, it is generally required that the illumination spots formed by light at all illumination angles overlap on the illumination chip. Therefore, the optical path of light at different illumination angles must be consistent. In this embodiment, the lens assembly 112 is used to compensate for the optical path difference at different illumination angles. The lens assembly 112 can be a cylindrical lens assembly or a specially designed lens assembly, which is not particularly limited in this embodiment and can be selected according to actual conditions.

[0061] As an implementation, the imaging aperture of the first objective lens 115 can be adjusted according to imaging requirements.

[0062] Different imaging apertures of the first objective lens 115 result in different entrance pupil ranges for the first objective lens 115. As shown in FIG3 or FIG4 , when light is focused onto the surface of the illumination chip 12 by the lens assembly 112, light rays at different illumination angles sequentially pass through the polarization adjustment element 113, the back focal plane 1151, and the front focal plane 1152 of the first objective lens 115. Finally, the light spots of the light rays at different illumination angles overlap at the front focal plane 1152 of the first objective lens 115.

[0063] As an example provided in this embodiment, the imaging aperture of the first objective lens 115 in Figure 3 is set to 0.95, and the imaging aperture of the first objective lens 115 in Figure 4 is set to 0.75. It can be seen from the imaging diagrams of the first objective lens in Figures 3 and 4 that the entrance pupil range of the first objective lens 115 with an imaging aperture of 0.95 is smaller than that of the first objective lens 115 with an imaging aperture of 0.75.

[0064] It should be noted that, since the objective lens with a larger imaging aperture usually has a smaller entrance pupil, the light spot may have part of its energy blocked during the entrance pupil process of the first objective lens 115, forming a vignetting effect, thereby affecting the formation of plasma structure light on the illuminated chip. For example, when the light spot radius is 0.17 mm, if the first objective lens 115 with an imaging aperture of 0.95 is used as shown in FIG3 , the light at its edge cannot pass through the first objective lens 115 to enter the pupil without obstruction. At this time, a first objective lens 115 with a smaller imaging aperture can be used, such as the first objective lens 115 with an imaging aperture of 0.75 as shown in FIG4 , to reduce the vignetting effect. In addition, from the perspectives of light collection efficiency, difficulty of assembly and adjustment, and hardware cost, the use of a first objective lens 115 with a smaller imaging aperture has greater advantages.

[0065] As an embodiment, the polarization adjusting member 113 includes a plurality of polarization units, each polarization unit corresponds to an irradiation angle of the light, and the polarization units are distributed in a ring shape on the polarization adjusting member.

[0066] The polarization adjusting element 113 is used to ensure that light with different irradiation angles can maintain the same polarization state, and to balance the polarization of different light to ensure that the intensity of the plasma structure light is the same.

[0067] The polarization adjustment element 113 selectively modulates the polarization according to different illumination angles. For example, if the light angle control element 111 controls the light to produce nine different illumination angles, the polarization adjustment element is also equipped with nine polarization units. As light propagating at each different illumination angle passes through the corresponding polarization unit, the polarization unit passively adjusts the light. Because the distribution of the light beam in the optical path is generally axisymmetric, the polarization units are arranged in a circular pattern on the polarization adjustment element. It should be noted that the arrangement of the polarization units in this embodiment is not limited to a circular distribution and can be selected according to actual conditions.

[0068] In one embodiment, the imaging optical path components include a second objective lens 131 , a tube lens 133 , and a filter 132 .

[0069] The second objective lens 131 is used to transmit the fluorescent signal on the illumination chip 12 and filter the excitation light in the fluorescent signal through the filter 132 ; the tube lens 133 focuses the fluorescent signal after the excitation light is filtered by the filter 132 to the imaging processing device 14 .

[0070] In one embodiment, the illumination chip 12 includes a plurality of illumination units distributed on a substrate of the illumination chip; the illumination units are configured to generate surface plasmon structured light when illuminated by a light source.

[0071] The illumination unit may be made of solid metal, and the solid metal may be silver, copper, aluminum, gold, or the like.

[0072] As an embodiment, the illumination chip 12 further includes a plurality of seed units. The seed units are disposed between corresponding illumination units and the substrate to increase the adhesion of the illumination units.

[0073] In one embodiment, the imaging processing device accumulates the fluorescence signals of each row of illumination chips during their movement. Generally speaking, the imaging processing device 14 can be a camera, including but not limited to staring cameras and time delay integration (TDI) cameras. TDI is a technology that images objects while they are in motion. TDI cameras can synchronously transfer the collected photoelectric signals row by row during movement to accumulate fluorescence signals.

[0074] As shown in Figure 5, when a row of illumination chip 12 is at position 1, it is imaged at position 1' in the TDI camera, and the corresponding pixel elements in the TDI camera generate photoelectric signals. When a row of illumination chip 12 moves to position 2, it is imaged at position 2' in the TDI camera, and the corresponding pixel elements in the TDI camera generate photoelectric signals. When a row of illumination chip 12 moves from position 1 to position 2, the photoelectric signals generated at position 1' in the TDI camera are synchronously transferred to position 2' for accumulation. The TDI imaging provided in this embodiment ensures the effective exposure time of illumination chip 12 while maintaining microscopic imaging speed, thus preventing image quality from being affected by insufficient exposure time.

[0075] During the imaging process, illumination chip 12 maintains a constant motion, with its speed highly correlated with the speed at which the imaging processing device transfers the photoelectric signals. The plasma structured light generated by illumination chip 12 is parallel light. As long as the angle of incidence on illumination chip 12 remains constant, the plasma structured light generated by illumination chip 12 remains constant during its motion. Therefore, imaging processing device 14 can simply and effectively superimpose the photoelectric signals transferred line by line.

[0076] Furthermore, during the imaging process of a row on the illumination chip 12, in order to further achieve super-resolution microscopic imaging, the illumination angle and / or orientation of the light incident on the illumination chip 12 needs to be changed multiple times. A raw image of a row on the illumination chip 12 is captured each time the illumination angle and orientation are changed. After all changes in the illumination angle and / or orientation of the light for that row are complete, the next row's raw image is captured.

[0077] In this embodiment, changes in illumination angle and orientation are primarily achieved with the aid of a light angle control device 111. A lens assembly 112 and a polarization adjustment element 113 are added to the optical path following the light angle control device 111 to control and balance the spot position, propagation quality, and polarization state of light at different illumination angles. After the aforementioned microscopic imaging device completes imaging of the entire illumination chip 12, combined with an image reconstruction algorithm, a large-scale, uniform, high-resolution image can be obtained. Furthermore, the imaging device utilizing plasma structured light imaging offers significant advantages in imaging speed.

[0078] In addition, microscopic imaging devices can be applied to various fields such as gene sequencing and medical sampling. Reconstructing images from raw images requires only simple spatial domain processing, rather than complex frequency domain algorithm processing. This reduces artifacts and produces the most realistic image results. This is very important for quantitative analysis applications and can effectively improve the accuracy of analysis results. For example, in second-generation gene sequencing, the DNA beads on the chip are regularly arranged, and the accuracy of recognition directly affects the reliability of the final sequencing results. The frequency domain reconstruction algorithm in traditional structured illumination microscopy inevitably introduces artifacts, thereby affecting recognition accuracy.

[0079] In order to overcome the above-mentioned existing defects, as shown in FIG6 , an exemplary embodiment of the present disclosure further provides a sequencing device, which includes the microscopic imaging device as described in the above embodiment.

[0080] The sequencing device further includes a support assembly; the support assembly includes a first stage 21 , a second stage 22 and a vertical support member 23 .

[0081] The first stage 21 is positioned above the second stage 22. The second stage 22 is equipped with an illumination optical path assembly, while the first stage 21 is equipped with an imaging optical path assembly. A vertical support 23 is mounted between the first stage 21 and the second stage 22 to support the first stage 21.

[0082] Among them, in addition to providing structural support for the first stage 21, the vertical support member 23 can also reduce the impact of internal and external low-frequency vibrations on the displacement of the illumination chip 12, affecting the shooting of the illumination chip 12, and thus affecting the sequencing results of the sequencing equipment.

[0083] Since this embodiment provides a sequencing device, the sample placed in the illumination chip 12 can be a sample for sequencing, such as DNA (deoxyribonucleic acid) beads, quantum dots, fluorescent nanospheres, etc. Of course, this embodiment does not specifically limit the type of sample, and the sample can be selected according to actual conditions.

[0084] In one embodiment, a sample is placed on the illumination chip 12 . When the illumination chip is illuminated by a light source, surface plasmon structured light is generated to excite fluorescent dyes in the sample and generate fluorescent signals.

[0085] When the illumination chip 12 includes a plurality of illumination units 122 distributed on the substrate 121, the illumination units 122 may include illumination columns. This embodiment does not limit the shape and material of the illumination units 122 to columns; any material can be selected based on actual circumstances as long as it can achieve the corresponding function. As shown in Figures 7 and 8, Figure 7 is a schematic diagram of the distribution of illumination columns, and Figure 8 is a heat map of the distribution of the heights of the illumination columns. It can be seen that the height of the illumination columns affects the ratio of the intensity of the near-field excited plasma structure light to the intensity of the surrounding background light.

[0086] Different materials and dimensional parameters for illumination unit 122 are selected based on the laser wavelength and resolution requirements, combined with finite element analysis and simulation. As shown in Figure 9, when the illumination unit is a cylindrical illumination unit with a pitch (the distance between the centers of two adjacent illumination units 122) of 350nm, simulation shows that when the illumination unit is irradiated with 532nm excitation light, the surface plasmon has good resonance energy and meets the resolution improvement requirements when the height of the illumination unit is 60nm and the diameter is 80nm.

[0087] Furthermore, a plurality of illumination units 122 are distributed in an array on substrate 121. When a beam of light strikes chip 12 at a certain angle, it induces electron oscillations within the metal-structured illumination units 122. These oscillations are concentrated at the interface between the metal and dielectric, and their distribution depends on the specific structure. If illumination units 122 are arranged in a regular array, they can generate any desired spatial frequency of plasma structured light.

[0088] The following provides a specific schematic structure of the illumination chip 12. As shown in Figures 10-12, the illumination chip 12 includes a plurality of illumination units 122 distributed on a substrate 121. A seed unit 123 is disposed between the illumination units 122 and the substrate 121. Furthermore, a plurality of placement units 124 for placing samples are defined on an upper surface extending from the illumination units 122. Each placement unit 124 is distributed on a corresponding illumination unit 122. Sidewalls 125 are provided between adjacent placement units.

[0089] The distance between two adjacent illumination units 122 must match the sample-carrying area of ​​the placement unit 124. A larger sample-carrying area allows for more samples to be accommodated, resulting in a stronger signal. However, excessively large sample-carrying areas can easily cause signal crosstalk between adjacent placement units 124. For example, if the distance between the centers of two adjacent illumination units 122 is 195 nm, a sample diameter of 140-160 nm is acceptable.

[0090] The following provides a specific schematic structure of the illumination chip 12. As shown in FIG13 , the illumination chip 12 also includes a plurality of oxidation units 126, each of which is distributed over a corresponding illumination unit 122. The oxidation units 126 are used to prevent the illumination units 122 from being oxidized. Furthermore, the oxidation units 126 may also have modified regions 127 for adsorbing samples. These modified regions can generally be modified electrically or chemically to enable sample adsorption.

[0091] The oxidation unit 126 may include, but is not limited to, silicon oxide or titanium oxide, and may be selected based on actual conditions. By combining substrates 121 of different materials, the oxidation unit 126 generates different local electric field distributions on the illumination unit 122, as shown in Figures 14 and 15. Figure 14 shows the local electric field distribution of a sapphire substrate 121 and an oxidation unit 126 of silicon oxide under 532nm excitation light; Figure 15 shows the local electric field distribution of a quartz substrate 121 and an oxidation unit 126 of silicon oxide under 532nm excitation light.

[0092] In one embodiment, the support assembly further includes a shock-absorbing foot 24 mounted on the bottom of the second stage 22. The shock-absorbing foot 24 is used to reduce the impact of external vibration on the displacement of the illumination chip 12, thereby affecting the imaging of the illumination chip 12 and thus affecting the sequencing results.

[0093] In order to allow light to pass through the first stage 21 and be received by the imaging optical path component mounted on the first stage 21, a through hole is provided on the first stage 21 in this embodiment, and the imaging optical path component and the illumination optical path component are arranged relative to each other through the through hole.

[0094] In one embodiment, the sequencing device further includes a carrying assembly 25 , which is installed between the imaging optical path assembly and the illumination optical path assembly. The carrying assembly 25 is used to carry the illumination chip 12 .

[0095] As one embodiment, as shown in FIG16 , the carrier assembly 25 is provided with positioning pins 251 and an adsorption platform 252. The positioning pins 251 are used to locate the position of the illumination chip 12. The adsorption platform 252 adsorbs and fixes the illumination chip 12 to the carrier assembly 25 via a connected vacuum generator.

[0096] As one embodiment, specifically as shown in FIG17 , the sequencing device further includes a moving platform 26, which is used to support the carrier assembly 25 and drive the carrier assembly 25 to move at a uniform speed under the imaging optical path assembly; the moving platform 26 is used to control the carrier assembly 25 to move down one row after the imaging processing device completes one row of imaging.

[0097] Specifically, the movable platform 26 is generally used to adjust the carrier assembly 25 on a plane. The movable platform 26 includes adjustment in at least two directions, such as adjustment in the X direction or the Y direction. In this embodiment, the movable platform 26 is used to adjust the carrier assembly 25 on a plane, thereby adjusting the movement of the illumination chip 12 on the plane.

[0098] In one embodiment, as shown in FIG17 , the sequencing device further includes a bracket 27 mounted on the second stage 22. The upper portion of the bracket 27 is used to support the carrier assembly 25, and the lower portion of the bracket 27 has a receiving space for accommodating the illumination optical path assembly.

[0099] In one embodiment, as specifically shown in FIG2 , the sequencing device further includes a horizontal moving assembly 28 , which is disposed at the bottom of the illumination optical path assembly and is configured to drive the illumination optical path assembly to move. To ensure that the illumination spot of the light can accurately illuminate the sample position on the illumination chip 12 , the horizontal moving assembly 28 includes a fine adjustment moving assembly 281 and a coarse adjustment moving assembly 282 .

[0100] The coarse adjustment moving assembly 281 is used to adjust the position of the illumination light path component, and the fine adjustment moving assembly 281 is used to adjust the position of the illumination light path component after the coarse adjustment moving assembly 282 is adjusted.

[0101] In one embodiment, the imaging processing device 14 includes a line scan camera or a linear array camera. One advantage of plasma structured light illumination is that the illumination does not change with the relative motion of the second objective lens 131 and the movable platform 26, but is only dependent on the incident angle and / or phase of the light. Imaging using a line scan camera or a linear array camera can achieve rapid imaging while ensuring imaging accuracy.

[0102] In one embodiment, as specifically shown in FIG6 , when the imaging optical path component includes the second objective lens 131 , the sequencing device further includes a focusing component 29 , which is configured to emit an outgoing laser, which is emitted to the sample on the illumination chip 12 through the second objective lens 131 ; the sample on the illumination chip 12 reflects the outgoing laser to obtain a reflected laser, which is emitted to the focusing component 29 through the second objective lens 131 .

[0103] Focusing assembly 29 determines the offset based on the outgoing and reflected laser light to drive the second objective lens 131 to focus. Focusing assembly 29 has an extremely sensitive and accurate focus feedback system. Based on the returned reflected laser light, it determines whether the second objective lens 131 is in the optimal imaging position. Ultimately, by driving the second objective lens 131 to move along the Z axis, it adjusts the distance between the second objective lens 131 and the illumination chip 12, thereby achieving autofocus.

[0104] As one embodiment, specifically as shown in FIG18 , the sequencing device further includes a second reflector 291 , which is disposed between the focusing assembly 29 and the second objective lens 131 . The second reflector 291 is configured to reflect the outgoing laser emitted by the focusing assembly 29 to the second objective lens 131 , and to reflect the outgoing laser reflected by the second objective lens to the focusing assembly 29 .

[0105] As one embodiment, the imaging optical path component also includes a first dichroic mirror 134, which is arranged between the second objective lens 131 and the focusing component 29. Since the light reflected from the illumination chip 12 through the second objective lens 131 includes light such as the outgoing laser mentioned in the above embodiment, in order to enable the imaging processing device 14 to capture a relatively pure original image and reduce the interference of the outgoing laser, the first dichroic mirror 134 is used to transmit the outgoing laser and reflect the fluorescent signal to reflect the fluorescent signal to the imaging processing device 14.

[0106] In one embodiment, the light source 10 in the sequencing device is often a fluorescence excitation light source. Each excitation light source simultaneously excites two wavelengths of fluorescence signals on the illuminated chip 12, thus requiring spectroscopic processing of the two fluorescence signals. Specifically, as shown in FIG18 , when the imaging optical path assembly includes a second objective lens 131 , the imaging optical path assembly also includes a first dichroic mirror 134 and a second dichroic mirror 135 , and the imaging processing device 14 includes a first imaging processing device 141 and a second imaging processing device 142 .

[0107] It should be noted that, since the light emitted from the illumination chip 12 through the second objective lens 131 contains a large amount of other excitation light, such as the emitted laser mentioned in the above embodiment, the excitation light generated by the light source 10, etc., in order to enable the imaging processing device 14 to capture a relatively pure original image and reduce the interference of other light, the first dichroic mirror 134 is used to receive the fluorescent signal transmitted by the second objective lens 131 and reflect the fluorescent signal after filtering the excitation light to the second dichroic mirror 135.

[0108] The second dichroic mirror 135 is arranged at a predetermined angle relative to the first dichroic mirror 134. This predetermined angle can be selected based on the position of the imaging processing device 14 or the positions of the first dichroic mirror 134 and the second dichroic mirror 135, and is not particularly limited in this embodiment. The second dichroic mirror 135 is configured to receive the fluorescent signal reflected by the first dichroic mirror 134, transmit the fluorescent signal within the first wavelength range to the first imaging processing device 141, and reflect the fluorescent signal within the second wavelength range to the second imaging processing device 142.

[0109] As an embodiment, the imaging optical path assembly further includes a third reflector 136 and a fourth reflector 137. The cylindrical lens 133 further includes a first cylindrical lens 1331 and a second cylindrical lens 1332. The third reflector 136 and the first cylindrical lens 1331 are disposed in front of the first imaging processing device 141 and are configured to reflect the fluorescence signal onto the target surface of the first imaging device 141. The fourth reflector 137 and the second cylindrical lens 1332 are disposed in front of the second imaging processing device 142 and are configured to reflect the fluorescence signal onto the target surface of the second imaging device 142.

[0110] In this embodiment, the excitation light and other light are filtered by the first dichroic mirror 134, and the fluorescence signals of different wavelengths are split by the second dichroic mirror 135 to generate different imaging channels according to different fluorescence signals, thereby reducing costs while ensuring imaging quality.

[0111] It should be noted that the sequencing device in this embodiment is not limited to aligning the imaging positions of the two imaging processing devices. When the sequencing device adds more imaging channels, the sequencing device needs to add corresponding dichroic mirrors and imaging processing devices respectively.

[0112] In this embodiment, the phenomenon of plasma structured light formed in the near field of the illumination chip 12 by the light emitted by the light source 10 is utilized to excite the sample on the illumination chip 12 to produce a fluorescence signal, thereby achieving the illumination conditions required for structured light super-resolution technology. Furthermore, the distribution of the plasma structured light illumination is not affected by the relative position of the illumination chip 12 and the imaging processing device. By switching the illumination angle and / or phase of the light, the imaging processing device can be used to achieve imaging while in motion, thereby meeting the requirements of rapid super-resolution gene sequencing. Furthermore, due to the high efficiency of plasma structured light excited in the near field, the illumination chip 12 can be illuminated with a smaller amount of light, significantly reducing light damage to the sample, thereby facilitating the realization of more sequencing rounds and improving the performance of the sequencing equipment.

[0113] To overcome the above-mentioned existing defects, as shown in FIG18 , an exemplary embodiment of the present disclosure provides a microscopic imaging method, which is applied to a microscopic imaging device or sequencing device as described in the above-mentioned embodiments. The method includes:

[0114] S10, adjusting the irradiation angle of the light in the first direction at least twice, and adjusting the irradiation angle of the light in the second direction at least twice after each adjustment of the irradiation angle of the light in the first direction.

[0115] The sum of changes in the irradiation angle of the light in the first direction is 0, and the sum of changes in the irradiation angle of the light in the second direction is 0. The first direction may be the X axis, and the second direction may be the Y axis.

[0116] S11 . Capturing a corresponding original image for each adjustment of the light irradiation angle. The original image is used to synthesize a corresponding microscopic image.

[0117] In one embodiment, synthesizing the corresponding microscopic image in step S11 may specifically include: constructing a residual between the original image, sample information, and the original image obtained by the imaging processing device, and converging the image by gradient descent to obtain a super-resolution microscopic image.

[0118] In one embodiment, capturing the corresponding original image for each adjustment of the light irradiation angle in step S11 includes:

[0119] Each time the irradiation angle of the light is adjusted, the imaging processing device is controlled to shoot the illumination chip line by line to obtain a corresponding original image.

[0120] As an embodiment, controlling the imaging processing device to capture the illumination chip line by line to obtain the corresponding original image specifically includes:

[0121] After completing the adjustment of the irradiation angle of the light on each row of the illumination chips and the photographing, the illumination chips are controlled to move downward one row until the illumination chips complete the photographing.

[0122] As an embodiment, the imaging processing device accumulates the fluorescence signals of each row of illumination chips during the movement of the illumination chips.

[0123] Therefore, in the process of controlling the imaging processing device to shoot the illumination chip row by row to obtain the corresponding original image, the illumination chip is controlled to move at a uniform speed, and during the movement of the illumination chip, the imaging processing device is controlled to accumulate the light on each row of the illumination chip to obtain the corresponding original image.

[0124] The microscopic imaging method in the above embodiment is described by a specific implementation method:

[0125] The light angle control device 111 adjusts the irradiation angle and phase of the light and then incidents on the illumination chip 12. Specifically, the irradiation angle of the light on the Y axis is kept at 0°, and the irradiation angle of the X axis is adjusted to -60°, 0° and +60° respectively. The irradiation angle of the light on the Y axis is adjusted to -120°, and the irradiation angle of the light on the x axis is repeatedly adjusted to -60°, 0° and +60° respectively. Finally, the irradiation angle of the light on the Y axis is adjusted to +120°, and the irradiation angle of the light on the x axis is repeatedly adjusted to -60°, 0° and +60° respectively. The imaging processing device 14 is controlled to take pictures of each adjustment of the irradiation angle. Figure 19 is a schematic diagram of the change of the illumination spot when the irradiation angle of the light on the Y axis is 0° and the irradiation angle of the X axis is -60°, 0° and +60° respectively. A total of 9 different illumination spot distributions are formed by the arrangement and combination of the irradiation angles on the X axis and the Y axis, and the fluorescence signal after excitation is imaged on the imaging processing device 14.

[0126] The carrier assembly drives the illumination chip 12 to continuously and uniformly move beneath the second objective lens 13, eliminating the need to pause for adjustments to the original image. After the original images for all illumination angles for a row on the illumination chip 12 are captured, the chip moves to the next row, continuing to image while moving. This imaging process repeats until all rows on the chip have completed capturing original images.

[0127] By constructing the residual between the original image, sample information and imaging processing device to obtain the original image, the image is converged by gradient descent to obtain a super-resolution microscopic image. Specifically, as shown in Figures 20 and 21, Figure 20 is a comparison of a set of original images and microscopic images, with the original image on the left and the microscopic image on the right. The intensity distribution measured along the tangent direction of the figure shows a significant improvement in resolution. The half-height width is reduced from 29 pixels to 16.4 pixels, an increase of nearly 2 times. The size of a pixel is about 15nm, so the corresponding diameter of the sample is about 240nm. Figure 21 is another comparison of the original image and microscopic images, with the original image on the left and the microscopic image on the right. The intensity distribution measured along the tangent direction of the figure shows a significant improvement in resolution. The half-height width is reduced from 29 pixels to 10.2 pixels, an increase of nearly 3 times. The corresponding diameter of the nucleic acid molecule nanosphere is about 150nm.

[0128] Although the specific embodiments of the present disclosure have been described above, those skilled in the art will appreciate that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present disclosure. Therefore, the scope of protection of the present disclosure is defined by the appended claims.

Claims

1. A microscopic imaging device, comprising a light source, an illumination optical path component, an imaging optical path component and an imaging processing device; the illumination optical path component and the imaging optical path component are arranged opposite to each other, with an illumination chip arranged in between; The light source irradiates light at different irradiation angles to the illumination chip through the illumination optical path component, so that the illumination chip generates plasma structure light, wherein: The substrate of the illumination chip is made of transparent material; The imaging processing device captures original images of the fluorescence signal of the illumination chip under the illumination of light at different illumination angles through the imaging optical path component.

2. The microscopic imaging device according to claim 1, wherein: The illumination optical path assembly comprises: a light angle control device, wherein the light angle control device comprises a first galvanometer and a second galvanometer; The first galvanometer is used to adjust the irradiation angle of the light irradiated by the light source to the illumination chip in a first direction, and the second galvanometer is used to adjust the irradiation angle of the light irradiated by the light source to the illumination chip in a second direction.

3. The microscopic imaging device according to claim 2, wherein: The illumination optical path assembly further includes a lens group, a polarization adjustment member, a first reflector and a first objective lens; The lens group is used to receive the light adjusted by the light angle controller and converge the light to irradiate the polarization adjustment member; The polarization adjusting element is used to keep light rays at different irradiation angles in the same polarization state; The first reflector is used to reflect light to the first objective lens; The first objective lens converges the light to the illumination chip.

4. The microscopic imaging device according to claim 3, wherein: The polarization adjusting member includes a plurality of polarization units, each of which corresponds to an irradiation angle of the light, and the polarization units are distributed in a ring shape on the polarization adjusting member.

5. The microscopic imaging device according to claim 1, wherein: The imaging optical path assembly includes a second objective lens, a tube lens and a filter; The second objective lens is used to transmit the fluorescent signal on the illumination chip and filter the excitation light in the fluorescent signal through the filter; The tube lens converges the fluorescence signal after the excitation light is filtered by the filter to the imaging processing device.

6. The microscopic imaging device according to claim 1, wherein: The illumination chip comprises a plurality of illumination units distributed on the substrate of the illumination chip; the illumination units are used to generate surface plasma structure light when illuminated by a light source.

7. The microscopic imaging device according to claim 6, wherein: The illumination chip further includes a plurality of seed units; The seed unit is disposed between the corresponding illumination unit and the substrate to increase the adhesion of the illumination unit.

8. A sequencing device, comprising the microscopic imaging device according to any one of claims 1 to 7.

9. The sequencing device according to claim 8, further comprising a support assembly; the support assembly comprises a first stage, a second stage and a vertical support member; The first loading platform is arranged above the second loading platform; The second stage is equipped with an illumination optical path component, and the first stage is equipped with an imaging optical path component; The vertical support member is installed between the first loading platform and the second loading platform, and the vertical support member is used to support the first loading platform.

10. The sequencing device according to claim 9, wherein: The support assembly also includes a shock-absorbing foot piece, and the shock-absorbing foot piece is installed at the bottom of the second loading platform.

11. The sequencing device according to claim 9, wherein: The first stage is provided with a through hole, and the imaging optical path component and the illumination optical path component are arranged relative to each other through the through hole.

12. The sequencing device according to claim 9, further comprising a supporting component, wherein the supporting component is installed between the imaging optical path component and the illumination optical path component, and the supporting component is used to support an illumination chip.

13. The sequencing device according to claim 12, further comprising a bracket, wherein the bracket is mounted on the second stage; The upper part of the bracket is used to support the bearing assembly, and the lower part of the bracket has a receiving space, and the receiving space is used to receive the lighting optical path assembly.

14. The sequencing device according to claim 12, wherein: The carrier assembly is provided with a positioning pin, and the positioning pin is used to locate the position of the illumination chip; And / or, an adsorption platform is provided on the carrier component, and the adsorption platform fixes the illumination chip to the carrier component through a connected vacuum generator.

15. The sequencing device of claim 12, further comprising a mobile platform; The mobile platform is used to support the bearing assembly and drive the bearing assembly to move at a constant speed under the imaging optical path assembly; the mobile platform is used to control the bearing assembly to move down one row after the imaging processing device completes one row of imaging.

16. The sequencing device according to claim 8, further comprising a horizontal moving assembly, the horizontal moving assembly being disposed at the bottom of the illumination light path assembly, the horizontal moving assembly being used to drive the illumination light path assembly to move; the horizontal moving assembly comprising a fine adjustment moving assembly and a coarse adjustment moving assembly; The coarse adjustment movable assembly is used to adjust the position of the illumination optical path assembly; the fine adjustment movable assembly is used to adjust the position of the illumination optical path assembly after the coarse adjustment movable assembly has been adjusted.

17. The sequencing device according to claim 8, wherein: The imaging processing device includes a line scan camera or a line array camera.

18. The sequencing device according to claim 8, wherein: A sample is placed on the illumination chip. When the illumination chip is illuminated by a light source, surface plasma structure light is generated to excite the fluorescent dye in the sample and generate a fluorescent signal.

19. The sequencing device according to claim 8, wherein: When the illumination chip includes a plurality of illumination units distributed on the substrate, a plurality of placement units for placing samples are divided on an upper surface extending from the illumination unit, and each of the placement units is distributed on a corresponding illumination unit.

20. The sequencing device according to claim 8, wherein: When the illumination chip includes a plurality of illumination units distributed on the substrate, the illumination chip further includes a plurality of oxidation units, and the oxidation units are correspondingly distributed on the corresponding illumination units; The oxidation unit is used to prevent the illumination unit from being oxidized, and / or the oxidation unit has a modified area for adsorbing samples.

21. The sequencing device according to claim 8, wherein: When the illumination chip includes a plurality of illumination units distributed on the substrate, the illumination unit includes an illumination column, and / or the plurality of illumination units are distributed on the substrate in an array manner.

22. The sequencing device according to claim 8, wherein: When the imaging optical path component includes a second objective lens, the sequencing device further includes a focusing component; The focusing assembly is used to emit outgoing excitation light, and the outgoing excitation light is emitted to the sample on the illumination chip through the second objective lens; The sample on the illumination chip reflects the outgoing excitation light to obtain reflected excitation light, and the reflected excitation light is emitted to the focusing component through the second objective lens; The focusing component determines an offset according to the outgoing excitation light and the reflected excitation light to drive the second objective lens to focus.

23. The sequencing device of claim 22, wherein: The imaging optical path component also includes a first dichroic mirror, which is arranged between the second objective lens and the focusing component. The first dichroic mirror is used to transmit the outgoing excitation light and reflect the fluorescent signal, and reflect the fluorescent signal to the imaging processing device.

24. The sequencing device according to claim 8, wherein: When the imaging optical path component includes a second objective lens, the imaging optical path component further includes a first dichroic mirror and a second dichroic mirror, and the imaging processing device includes a first imaging processing device and a second imaging processing device; The first dichroic mirror is used to receive the fluorescent signal transmitted by the second objective lens, and reflect the fluorescent signal after filtering the excitation light to the second dichroic mirror; The second dichroic mirror is arranged at a preset angle with the first dichroic mirror. The second dichroic mirror is used to receive the fluorescent signal reflected by the first dichroic mirror, transmit the fluorescent signal with a wavelength within the first wavelength range to the first imaging processing device, and reflect the fluorescent signal with a wavelength within the second wavelength range to the second imaging processing device.

25. A microscopic imaging method, applied to the microscopic imaging device according to any one of claims 1 to 7 or the sequencing device according to any one of claims 8 to 24, the method comprising: Adjusting the irradiation angle of the light in the first direction at least twice, and adjusting the irradiation angle of the light in the second direction at least twice after each adjustment of the irradiation angle of the light in the first direction; wherein the sum of changes in the irradiation angle of the light in the first direction is 0, and the sum of changes in the light in the second direction is 0; A corresponding original image is captured for each adjustment of the illumination angle of the light, and the original image is used to synthesize a corresponding microscopic image.

26. The microscopic imaging method of claim 25, wherein: For each adjustment of the light angle, the corresponding original image is captured, including: For each adjustment of the irradiation angle of the light, the imaging processing device is controlled to shoot the illumination chip line by line to obtain a corresponding original image.

27. The microscopic imaging method of claim 26, wherein: Controlling the imaging processing device to photograph the illumination chip line by line to obtain a corresponding original image includes: After the irradiation angle adjustment and shooting of the light on each row of the illumination chips are completed, the illumination chips are controlled to move downward by one row until the illumination chips complete shooting.

28. The microscopic imaging method of claim 27, wherein: The imaging processing device accumulates the fluorescence signals of each row of the illumination chips during the movement of the illumination chips.

29. The microscopic imaging method of claim 27, wherein: Controlling the imaging processing device to photograph the illumination chip line by line to obtain a corresponding original image includes: Controlling the illumination chip to move at a uniform speed; During the movement of the illumination chip, the imaging processing device is controlled to accumulate the light on each row of the illumination chip to obtain a corresponding original image.

Images