Optical microscopic imaging device and method for determining nucleotide sequence of biological sample

Abstract

The invention provides an optical microscopic imaging device and a method for determining a nucleotide sequence of a biological sample. The optical microscopic imaging device comprises a light source assembly used for emitting exciting light; the light beam transmission assembly at least comprises an optical fiber structure and is used for guiding the exciting light to a preset view field of a to-be-detected sample; the fluorescence acquisition assembly comprises a super-structure lens, and the super-structure lens is used for receiving a fluorescence signal generated when the to-be-detected sample is excited by the exciting light in a preset view field; the light beam scanning reset assembly at least comprises a scanning galvanometer, the scanning galvanometer corresponds to the super-structure lens, the angle of the scanning galvanometer can be adjusted, and the scanning galvanometer is used for correcting the transmission direction of the fluorescence signal; the fluorescence imaging assembly is located on one side of the scanning galvanometer and used for receiving the fluorescence signals corrected by the scanning galvanometer, and the transmission direction of the corrected fluorescence signals in the fluorescence imaging assembly is parallel to the optical axis of the fluorescence imaging assembly. According to the invention, the problem that a large numerical aperture and a large field of view of an optical microscopic imaging device in the prior art are difficult to consider at the same time is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of optical detection equipment, and in particular to an optical microscopic imaging device and a method for determining the nucleotide sequence of a biological sample. Background Art

[0002] Gene sequencing technology has played a huge role in promoting the exploration of the mysteries of life, the treatment of diseases, and the development of the entire biological science and even medicine, and has broad application prospects. The mainstream high-throughput gene sequencing technology (second-generation gene sequencing technology) in the world is based on the traditional optical microscopic imaging system. Through biochemical methods, the four bases of the gene sequence on the sequencing chip carry corresponding fluorescent groups, and the specific bases are identified by collecting the fluorescent signals of the fluorescent groups. Due to the limitation of the optical diffraction limit, the sequencing throughput of the traditional gene sequencing system is also limited by the resolution and field of view of the optical system. Among them, the optical resolution determines the density of the sequencing points in the sequencing chip, and the field of view determines the size of the image that the camera can collect at a time, that is, the number of points collected at a time.

[0003] In existing optical microscopy imaging devices, the objective lens, as the most important optical component, determines the imaging quality and various optical parameters of the system, including magnification, resolution and imaging field of view on the object side. The resolution of the optical system is mainly determined by the numerical aperture (NA) of the objective lens. As one of the most important parameters of the objective lens, the numerical aperture determines the spatial resolution capability of the optical system. An objective lens with a higher numerical aperture has a higher ability to resolve spatial details than one with a lower numerical aperture. However, increasing the numerical aperture means that the light cone angle of the objective lens becomes larger, and the ability of light to be focused is greater, thereby reducing the imaging field of view on the object side of the imaging system. From formula (1), it can be seen that the limit d of the resolution of the optical system is inversely proportional to the numerical aperture NA and the pupil diameter (D). Therefore, in a sequencing system, it is very difficult to improve the sequencing throughput by simultaneously improving the resolution and field of view of the optical system.

[0004]

[0005] Therefore, in existing high-throughput sequencing systems, under the premise of ensuring the resolution of the imaging system, a dual-axis motion control platform is usually used to move the sequencing chip, and images of multiple fields of view are collected at different positions of the sequencing chip, and the photographed area is spliced, regional positioning and base recognition are performed through algorithms. The use of the transport control platform to scan the chip in multiple fields of view requires coupling the motion control system in the imaging system, and the frequent movement and stopping of the motion platform greatly deepens the complexity of the sequencing system. Among them, the movement and setting time of the platform increase the time cost of taking pictures; affect the stability of the imaging system; and the size of the sequencing system is also limited by the size of the transport control platform.

[0006] That is to say, the optical microscopic imaging device in the prior art has the problem that it is difficult to take into account both a large numerical aperture and a large field of view at the same time. Summary of the invention

[0007] The main purpose of the present invention is to provide an optical microscopic imaging device and a method for determining the nucleotide sequence of a biological sample to solve the problem that the optical microscopic imaging device in the prior art has a large numerical aperture and a large field of view that are difficult to take into account at the same time.

[0008] In order to achieve the above-mentioned purpose, according to one aspect of the present invention, there is provided an optical microscopy imaging device, comprising: a light source assembly, the light source assembly is used to emit excitation light; a light beam transmission assembly, the light beam transmission assembly at least comprises an optical fiber structure, the optical fiber structure is connected to the light source assembly, and is used to guide the excitation light to a predetermined field of view of a sample to be tested; a fluorescence collection assembly, the fluorescence collection assembly comprises a meta-lens, and the meta-lens is used to receive a fluorescence signal generated by the sample to be tested in the predetermined field of view when the sample is excited by the excitation light; a light beam scanning and resetting assembly, the light beam scanning and resetting assembly at least comprises a scanning galvanometer, the scanning galvanometer corresponds to the meta-lens and the angle of the scanning galvanometer is adjustable, and is used to correct the transmission direction of the fluorescence signal; a fluorescence imaging assembly, the fluorescence imaging assembly is located on one side of the scanning galvanometer, and is used to receive the fluorescence signal corrected by the scanning galvanometer, and the transmission direction of the corrected fluorescence signal in the fluorescence imaging assembly is parallel to the optical axis direction of the fluorescence imaging assembly.

[0009] Furthermore, the excitation light includes a first light and a second light of different wavelengths, and the fluorescence imaging component includes a fluorescence dichroic mirror, a first fluorescence detection channel and a second fluorescence detection channel. The fluorescence dichroic mirror is used to separate the fluorescence signal according to the wavelength, the first fluorescence detection channel is used to receive the fluorescence signal corresponding to the first light, and the second fluorescence detection channel is used to receive the fluorescence signal corresponding to the second light.

[0010] Furthermore, the meta-lens has a nano-array structure.

[0011] Furthermore, the beam scanning and resetting assembly also includes a 4f optical system composed of a first scanning lens and a second scanning lens, and the first scanning lens and the second scanning lens are sequentially arranged between the meta-lens and the scanning galvanometer in a direction away from the meta-lens.

[0012] Furthermore, the light source assembly is located on the side of the sample to be tested away from the meta-lens and is used to project excitation light toward the side of the sample to be tested away from the meta-lens. The optical fiber structure includes an optical fiber array corresponding to the object plane where the sample to be tested is located; the optical fiber array includes a plurality of optical fibers corresponding one by one to a predetermined field of view; the fluorescence collection assembly also includes a notch filter located between the sample to be tested and the fluorescence imaging assembly, and the notch filter is used to transmit the fluorescence signal and block the excitation light transmitted through the sample to be tested.

[0013] Furthermore, the excitation light emitted by the light source assembly is projected onto the sample to be tested via the scanning galvanometer, and the light beam transmission assembly further includes a first collimating lens and a second collimating lens located between the optical fiber structure and the scanning galvanometer.

[0014] Further, the predetermined field of view is arranged along a first direction and a second direction which are perpendicular to each other, and the scanning galvanometer includes a first galvanometer which drives the excitation light to move along the first direction and a second galvanometer which drives the excitation light to move along the second direction.

[0015] Furthermore, the optical microscopic imaging device also includes a first dichroic mirror located between the light beam transmission component and the scanning galvanometer, and the first dichroic mirror has a transmission band for transmitting the excitation light and a reflection band for reflecting the fluorescence signal.

[0016] According to another aspect of the present invention, there is also included a method for determining the nucleotide sequence of a biological sample, which is implemented using the above-mentioned optical microscopy imaging device, and the method includes: starting a light source component to sequentially project excitation light toward multiple fields of view of an object plane where a sample to be tested is located, to excite the sample to be tested to generate a fluorescence signal in the field of view, and using a meta-lens to respectively collect the fluorescence signals corresponding to different fields of view according to the emission order of the excitation light; adjusting the deflection angle of the scanning galvanometer according to the propagation direction of the fluorescence signals of different fields of view to correct the fluorescence signals of different fields of view to propagate along the optical axis direction parallel to the optical path where the fluorescence imaging component is located; the corrected fluorescence signal is collected by the fluorescence imaging component according to the emission order and a fluorescence image of the corresponding fluorescence signal is generated; the fluorescence images obtained under different fields of view are spliced ​​to obtain a spliced ​​image of the field of view corresponding to the object plane, and the nucleotide sequence of the sample to be tested is identified based on the spliced ​​image.

[0017] Furthermore, the excitation light scans the corresponding field of view, the scanning galvanometer corrects the fluorescence signal, and the fluorescence imaging component exposes and collects the fluorescence signal simultaneously.

[0018] Furthermore, in the process of collecting the corrected fluorescence signal by the fluorescence imaging component according to the emission sequence and generating a fluorescence image corresponding to the fluorescence signal, it includes: collecting fluorescence signals of different wavelengths in at least two different fluorescence collection channels respectively.

[0019] Furthermore, the light source assembly includes an optical fiber array corresponding to the object plane, and the optical fiber array includes multiple optical fibers corresponding one by one to the fields of view; when the light source assembly is started to sequentially project excitation light toward the multiple fields of view on the object plane where the sample to be tested is located, and the process of exciting the sample to be tested to generate a fluorescent signal in the field of view includes: sequentially controlling the optical fiber to irradiate the corresponding field of view according to a pre-set excitation sequence, and the excitation light is emitted from the side of the sample to be tested away from the meta-lens.

[0020] Furthermore, the optical microscopic imaging device also includes a notch filter located between the sample to be tested and the fluorescent imaging component, and the method also includes: the notch filter transmits the fluorescent signal and blocks the excitation light transmitted through the sample to be tested.

[0021] Furthermore, in the process of starting the light source component to sequentially project excitation light toward multiple fields of view on the object plane where the sample to be tested is located, thereby exciting the sample to be tested to generate a fluorescence signal in the field of view, the light source component is also included in the process of projecting the excitation light onto the scanning galvanometer, and the scanning galvanometer deflects and projects the excitation light to different fields of view in sequence according to a pre-set excitation sequence.

[0022] Furthermore, the multiple fields of view are arranged along a first direction and a second direction that are perpendicular to each other, and the scanning galvanometer includes a first galvanometer that drives the excitation light to deflect along the first direction and a second galvanometer that drives the excitation light to deflect along the second direction.

[0023] According to the technical solution of the present invention, the optical microscopic imaging device includes a light source assembly, which is used to emit excitation light; a light beam transmission assembly, which includes at least an optical fiber structure, which is connected to the light source assembly and is used to guide the excitation light to a predetermined field of view of a sample to be tested; a fluorescence collection assembly, which includes a meta-lens, which is used to receive a fluorescence signal generated by the sample to be tested in the predetermined field of view when the sample is excited by the excitation light; a light beam scanning and resetting assembly, which includes at least a scanning galvanometer, which corresponds to the meta-lens and has an adjustable angle, so as to correct the transmission direction of the fluorescence signal; and a fluorescence imaging assembly, which is located on one side of the scanning galvanometer and is used to receive the fluorescence signal corrected by the scanning galvanometer, and the transmission direction of the corrected fluorescence signal in the fluorescence imaging assembly is parallel to the optical axis direction of the fluorescence imaging assembly.

[0024] The present application adopts a meta-lens to receive the fluorescence signal generated by the sample to be tested in a predetermined field of view when stimulated by the excitation light, and cooperates with a scanning galvanometer and subsequent structures to realize the collection and detection of the fluorescence signal. Compared with traditional optical lenses, meta-lenses have the advantages of thinner volume, lighter weight, lower cost, better imaging, and easier integration. Setting the meta-lens in the optical microscopic imaging device of the present application is conducive to ensuring that the numerical aperture and field of view of the optical microscopic imaging device are increased without increasing the size of the optical elements in the device, achieving high numerical aperture and large field of view, and then achieving high resolution. And by adjusting the deflection angle of the scanning galvanometer, the illumination beam can scan the full field of view of the meta-lens, reducing the size of the optical elements in the system, and effectively avoiding the disadvantages of the operation and control platform scanning the full field of view, and the structure is simple, easy to implement, and suitable for mass production. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0026] Figure 1 A schematic diagram of a light path of an optical microscopic imaging device according to a first embodiment of the present invention is shown;

[0027] Figure 2 Shows Figure 1 A local light path diagram of an optical microscopic imaging device in FIG.

[0028] Figure 3 A schematic diagram of a light path of an optical microscopic imaging device according to a second embodiment of the present invention is shown;

[0029] Figure 4 Shows Figure 3 A local light path diagram of an optical microscopic imaging device in FIG.

[0030] Figure 5 A scanning process diagram of an optical microscopic imaging device according to an optional embodiment of the present invention is shown;

[0031] Figure 6 Shows Figure 5 The scanning process of the full field of view of the optical microscopy imaging device in

[0032] The above drawings include the following reference numerals:

[0033] 10. Light source assembly; 21. Fiber array; 22. Multimode fiber; 23. First collimating lens; 24. Second collimating lens; 30. Meta-lens; 40. Sample to be tested; 50. Notch filter; 61. Scanning galvanometer; 611. First galvanometer; 612. Second galvanometer; 62. First scanning lens; 63. Second scanning lens; 71. Fluorescence spectroscopic dichroic mirror; 72. First dichroic mirror; 74. First filter; 75. First tube lens; 76. First camera; 77. Second filter; 78. Second tube lens; 79. Second camera. DETAILED DESCRIPTION

[0034] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0035] It should be noted that, unless otherwise specified, all technical and scientific terms used in this application have the same meanings as commonly understood by ordinary technicians in the technical field to which this application belongs.

[0036] In the present invention, unless otherwise specified, the directional words used, such as "up, down, top, bottom", usually refer to the directions shown in the drawings, or to the components themselves in the vertical, perpendicular or gravity directions; similarly, for ease of understanding and description, "inside and outside" refer to the inside and outside relative to the outline of each component itself, but the above-mentioned directional words are not used to limit the present invention.

[0037] In order to solve the problem that it is difficult to simultaneously take into account a large numerical aperture and a large field of view in optical microscopic imaging devices in the prior art, the present invention provides an optical microscopic imaging device and a method for determining the nucleotide sequence of a biological sample.

[0038] like Figures 1 to 6 As shown, the optical microscopy imaging device includes a light source assembly, which is used to emit excitation light; a light beam transmission assembly, which includes at least an optical fiber structure, which is connected to the light source assembly and is used to guide the excitation light to a predetermined field of view of a sample to be tested; a fluorescence collection assembly, which includes a meta-lens, which is used to receive a fluorescence signal generated by the sample to be tested in a predetermined field of view when the sample is excited by the excitation light; a light beam scanning and resetting assembly, which includes at least a scanning galvanometer, which corresponds to the meta-lens and has an adjustable angle, so as to correct the transmission direction of the fluorescence signal; and a fluorescence imaging assembly, which is located on one side of the scanning galvanometer and is used to receive the fluorescence signal corrected by the scanning galvanometer, and the transmission direction of the corrected fluorescence signal in the fluorescence imaging assembly is parallel to the optical axis direction of the fluorescence imaging assembly.

[0039] The present application adopts a meta-lens to receive the fluorescence signal generated by the sample to be tested in a predetermined field of view when stimulated by the excitation light, and cooperates with a scanning galvanometer and subsequent structures to realize the collection and detection of the fluorescence signal. Compared with traditional optical lenses, meta-lenses have the advantages of thinner volume, lighter weight, lower cost, better imaging, and easier integration. Setting the meta-lens in the optical microscopic imaging device of the present application is conducive to ensuring that the numerical aperture and field of view of the optical microscopic imaging device are increased without increasing the size of the optical elements in the device, achieving high numerical aperture and large field of view, and then achieving high resolution. And by adjusting the deflection angle of the scanning galvanometer, the illumination beam can scan the full field of view of the meta-lens, reducing the size of the optical elements in the system, and effectively avoiding the disadvantages of the operation and control platform scanning the full field of view, and the structure is simple, easy to implement, and suitable for mass production.

[0040] Specifically, the above-mentioned meta-lens 30 is a two-dimensional plane lens, the meta-lens 30 has a nano-array structure, and the surface of one side of the meta-lens 30 having the nano-array structure is a meta-surface. The nano-array structure is composed of a two-dimensional metamaterial with sub-wavelength thickness and size, so as to achieve focused imaging of the incident light. By adjusting the shape, rotation direction, height and other parameters of the nano-array structure, the properties of the light such as polarization, phase and amplitude can be regulated. The design of the meta-lens 30 can meet the numerical aperture (NA>0.75) of the optical system while increasing the size and improving the field of view of the imaging system, thereby meeting the demand for a large field of view. Therefore, the present application adopts a super-groove lens instead of an objective lens, which effectively reduces the cost of the system, reduces the complexity of the system, and also enables the optical system to be better integrated into the detection part.

[0041] In an optional embodiment of the present application, the numerical aperture of the above-mentioned meta-lens 30 is 0.8, and the field of view size is 10mm*10mm.

[0042] Specifically, the light source assembly 10 includes one of an LED and a laser light source. The light source assembly 10 can be an LED or laser light source of any wavelength and type.

[0043] Specifically, the optical fiber structure is an optical fiber array 21 or a multimode optical fiber 22. The optical fiber array 21 is composed of a plurality of optical fibers, and the plurality of optical fibers can be arrayed into arrays of different shapes as required.

[0044] Specifically, the excitation light includes a first light and a second light of different wavelengths, and the fluorescence imaging component includes a fluorescence dichroic mirror 71, a first fluorescence detection channel and a second fluorescence detection channel. The fluorescence dichroic mirror 71 is used to separate the fluorescence signal according to the wavelength, the first fluorescence detection channel is used to receive the fluorescence signal corresponding to the first light, and the second fluorescence detection channel is used to receive the fluorescence signal corresponding to the second light.

[0045] Specifically, the beam scanning and resetting assembly further includes a 4f optical system consisting of a first scanning lens 62 and a second scanning lens 63, which are sequentially arranged between the meta-lens 30 and the scanning galvanometer 61 in a direction away from the meta-lens 30. The first scanning lens 62 and the second scanning lens 63 are both double-cemented lenses.

[0046] like Figure 5 and Figure 6 As shown, there are multiple predetermined fields of view, and the multiple predetermined fields of view are arranged along a first direction and a second direction perpendicular to each other. The scanning galvanometer 61 includes a first galvanometer 611 that drives the excitation light to move along the first direction, and a second galvanometer 612 that drives the excitation light to move along the second direction. The first direction is the X-axis direction in the figure, and the second direction is the Y-axis direction in the figure. Figure 5A schematic diagram is described of a light beam illuminating a single field of view after passing through the first galvanometer 611 and the second galvanometer 612 (for simplicity, two scanning lenses are omitted). Figure 6 A schematic diagram of the full field of view is shown, which describes the scanning logic of the optical microscopy imaging device: the first galvanometer 611 first performs a transverse scan of each single field of view along the X-axis at a fixed Y-axis position (the black arrow indicates the scanning direction). After completing the scanning of the full field of view in the X-axis direction, the second galvanometer 612 switches the coordinates along the Y-axis and continues to perform a transverse scan in the opposite direction of the X-axis, and cooperates with the second galvanometer 612 to achieve the Y-axis switching of the longitudinal position until the center field of view and the edge field of view are completely scanned. Among them, the area illuminated each time is defined as a single field of view, and the illumination time of each single field of view is consistent with the exposure time of the camera. After the current single field of view is illuminated, the illumination beam is deflected to the next single field of view for data collection. The principle of the imaging optical path is similar to that of Example 1 and will not be repeated here.

[0047] The optical microscopic imaging device of the present application is described below in conjunction with specific embodiments.

[0048] Embodiment 1

[0049] like Figure 1 and Figure 2 As shown, the optical microscopic imaging device of embodiment 1 is described.

[0050] In embodiment 1, the optical microscopy imaging device includes a light source assembly 10, an optical fiber structure, a meta-lens 30, a sample to be tested 40, a notch filter 50, a scanning galvanometer 61, a first scanning lens 62, a second scanning lens 63, a fluorescence dichroic mirror 71, a first fluorescence detection channel, and a second fluorescence detection channel.

[0051] In the first embodiment, the optical fiber structure is an optical fiber array 21, which is composed of a plurality of optical fibers. The first fluorescence detection channel includes a first filter 74, a first tube lens 75 and a first camera 76; the second fluorescence detection channel includes a second filter 77, a second tube lens 78 and a second camera 79.

[0052] like Figure 1As shown, the light source assembly 10 is located on the side of the sample to be tested 40 away from the metalens 30 and is used to project excitation light toward the side of the sample to be tested 40 away from the metalens 30. The optical fiber structure is an optical fiber array 21 corresponding to the object plane where the sample to be tested 40 is located; the optical fiber array 21 includes a plurality of optical fibers corresponding to a predetermined field of view; the light emitted by the light source assembly 10 is coupled into the optical fiber array 21, and the end faces of the plurality of optical fibers are arranged in a certain order into a required geometric shape (here in a rectangular array). The optical fiber is a single-mode optical fiber, which is used to form the optical fiber array 21 and the transmission of the light beam, so as to ensure that a single optical fiber can illuminate the preset field of view during imaging; the notch filter 50 is located between the sample to be tested and the fluorescence imaging assembly, specifically between the metalens 30 and the first scanning lens 62, and the notch filter is used to transmit the fluorescence signal and block the excitation light transmitted through the sample to be tested 40.

[0053] Specifically, the scanning galvanometer 61 is a dual-axis scanning galvanometer. Through the conjugate relationship between the rear focal plane of the meta-lens 30 and the surface of the scanning galvanometer 61, the scanning galvanometer 61 transmits the fluorescent signals emitted from different fields of view and diverging at different field angles in the same direction that coincides with the main optical axis, and finally their spatial positions coincide on the camera image plane.

[0054] Specifically, in this embodiment, the fluorescence imaging component is a dual fluorescence collection channel, the excitation light includes a first light and a second light of different wavelengths, and the fluorescence imaging component includes a fluorescence dichroic mirror 71, a first fluorescence detection channel and a second fluorescence detection channel. The fluorescence dichroic mirror 71 is used to separate the fluorescence signal according to the wavelength, the first fluorescence detection channel is used to receive the fluorescence signal corresponding to the first light, and the second fluorescence detection channel is used to receive the fluorescence signal corresponding to the second light; the first filter 74, the first tube lens 75 and the first camera 76 of the first fluorescence detection channel are sequentially arranged on the reflection path of the fluorescence dichroic mirror 71 in the direction away from the fluorescence dichroic mirror 71, and the second filter 77, the second tube lens 78 and the second camera 79 of the second fluorescence detection channel are sequentially arranged on the transmission path of the fluorescence dichroic mirror 71 in the direction away from the fluorescence dichroic mirror 71. The fluorescence signals of different wavelengths enter the camera after passing through the corresponding filters and tube lenses, so as to realize the collection of fluorescence signals and the detection of bases.

[0055] Specifically, the optical microscopic imaging device also includes a control component, which includes a signal transmitter, an image acquisition card and a computer. The computer sends a TTL trigger signal to the light source component 10, the scanning galvanometer 61 and the camera at a fixed frequency and duty cycle through the signal transmitter to achieve the setting and synchronous triggering of the imaging field of view, the deflection angle of the scanning galvanometer 61 and the camera exposure, and ensure that the spatial position of the fluorescence in different fields of view can overlap on the image plane and be synchronously collected by the camera. The single optical fiber in the optical fiber array 21 is controlled by the control component, turned on in turn, collimated into a parallel beam through the built-in collimating lens, and illuminates different positions of the sample to be tested 40 in turn due to the distribution of the spatial position on the end face of the optical fiber. Each optical fiber corresponds to the spatial position of a part of the field of view in the imaging field of view of the meta-lens 30. Each time the laser is turned on, only one optical fiber is turned on to illuminate the preset field of view of the sample. Fluorescent groups at different positions on the object surface of the sample 40 to be tested are excited, and the emitted fluorescence signals are collected by the meta-lens 30, and then collected by the camera after passing through the subsequent devices. The excitation light transmitted through the sample 40 to be tested is filtered by the notch filter 50 and does not pass through the subsequent imaging system.

[0056] refer to Figure 1 The first scanning lens 62 and the second scanning lens 63 are both double-glued lenses, the light incident surface of the first scanning lens 62 is a convex surface, the gluing surface of the first scanning lens 62 is convexly arranged toward the light emitting surface, and the light emitting surface of the first scanning lens 62 is a plane; the light incident surface of the second scanning lens 63 is a plane, the gluing surface of the second scanning lens 63 is convexly arranged toward the light incident surface, and the light emitting surface of the first scanning lens 62 is a convex surface.

[0057] like Figure 2 As shown, it is a schematic diagram of the optical path of the beam scanning reset component and the fluorescent imaging component. In the figure, X0, X1 and X2 correspond to the fluorescence emitted from different fields of view of the object plane, where X0 represents the central field of view, and X1 and X2 represent the edge fields of view in the X direction, respectively. Since the X-axis and Y-axis of the optical path and the scanning galvanometer 61 are symmetrical, only the X-axis is taken as an example here. Among them, the central field of view X0 propagates along the main optical axis of the optical path, passes through two scanning lenses in turn, and is reflected to the image plane of the tube mirror and the camera through the scanning galvanometer 61. Its fluorescence signal is collected by the camera. At this time, relative to the main optical axis, the deflection angle (X0) of the scanning galvanometer 61 is 0°.

[0058] like Figure 2As shown, for the fluorescence signals (X1 and X2) excited by the edge field of view, due to different spatial positions, they coincide with the rear focal plane (spectral plane) of the metalens 30 after passing through the metalens 30, and are projected onto the scanning galvanometer 61 through a group of 4f scanning lenses at different field angles. Among them, the positions of the two scanning lenses are in a 4f relationship, so that the rear focal plane of the metalens 30 and the surface of the scanning galvanometer 61 are mutually conjugate, that is, the light beams of different fields of view are incident on the surface of the scanning galvanometer 61 at different angles after passing through the 4f scanning lens, but their spatial positions can all coincide on the surface of the scanning galvanometer 61. At this time, the angle difference between the incident angle of the edge field of view to the scanning galvanometer 61 and the main optical axis is corrected by the deflection angle of the scanning galvanometer 61, that is, the deflection angles X1 and X2 respectively correct the deflection angles of the two edge fields of view, and by deflecting the scanning galvanometer 61, the fluorescent light beam emitted from the scanning galvanometer 61 is propagated along the main optical axis of the optical path (the angle with the main optical axis is 0°). Since the light beam corrected by the scanning galvanometer 61 has no deflection angle, the light beams in different fields of view coincide with the main optical axis during propagation and are imaged at the same spatial position on the image plane of the camera.

[0059] Specifically, the meta-lens 30, the first scanning lens 62, the second scanning lens 63 and the tube lens are mutually 4f systems, so that the object plane and the image plane are mutually conjugate, which is used to ensure the magnification and project the object plane information onto the image plane.

[0060] The sample 40 to be tested in this embodiment may be a transmission type slide, specifically glass.

[0061] Embodiment 2

[0062] like Figure 3 and Figure 4 As shown, the optical microscopic imaging device of embodiment 2 is described.

[0063] In the second embodiment, the optical microscopic imaging device includes a light source assembly 10, an optical fiber structure, a first collimating lens 23, a second collimating lens 24, a meta-lens 30, a sample to be tested 40, a scanning galvanometer 61, a first scanning lens 62, a second scanning lens 63, a fluorescence beam splitting dichroic mirror 71, a first dichroic mirror 72, a first fluorescence detection channel and a second fluorescence detection channel. The optical fiber structure is an optical fiber array 21, and the optical fiber array 21 is composed of a plurality of optical fibers. The first fluorescence detection channel includes a first filter 74, a first tube lens 75 and a first camera 76; the second fluorescence detection channel includes a second filter 77, a second tube lens 78 and a second camera 79.

[0064] In this embodiment, the optical fiber structure is a multimode optical fiber 22 .

[0065] like Figure 3As shown, the first collimating lens 23 and the second collimating lens 24 are sequentially arranged in a direction away from the optical fiber structure, and the first collimating lens 23 and the second collimating lens 24 are used to realize the coaxial collimation, beam reduction and transmission of the light beam. The meta-lens 30 and the sample to be tested 40 are sequentially arranged on the reflection path of the scanning galvanometer 61 in a direction away from the scanning galvanometer 61. The scanning galvanometer 61 reflects the excitation light, and then incidents on the sample to be tested 40 through the meta-lens 30. The sample to be tested 40 generates a fluorescence signal and reflects it to the meta-lens 30, and then is reflected by the scanning galvanometer 61 through the meta-lens 30 to the fluorescence imaging component. By constructing a conjugate relationship between the surface of the scanning galvanometer 61 and the back focal plane of the meta-lens 30, the excitation light is deflected so that the excitation light can sequentially illuminate different fields of view of the meta-lens 30; at the same time, the scanning galvanometer 61 propagates the fluorescence signals emitted from different fields of view at different deflection angles (relative to the main optical axis), and finally their spatial positions coincide on the image plane of the camera.

[0066] Specifically, the first collimating lens 23 and the second collimating lens 24 are both double-glued lenses, the light incident surface of the first collimating lens 23 is a convex surface, the gluing surface is protruding in the direction away from the light incident surface, and the light emitting surface is a plane; the light incident surface of the second collimating lens 24 is a plane, the gluing surface is protruding in the direction close to the light incident surface, and the light emitting surface is a convex surface.

[0067] Specifically, the optical microscope imaging device further includes a first dichroic mirror 72 located between the beam transmission component and the scanning galvanometer 61, and the first dichroic mirror 72 has a transmission band for transmitting the excitation light and a reflection band for reflecting the fluorescence signal. Specifically, the first dichroic mirror 72 is located between the second collimating lens 24 and the scanning galvanometer 61, and the fluorescence beam splitting dichroic mirror 71 is located on the reflection path of the first dichroic mirror 72. Figure 3 It can be seen that the optical path from the first dichroic mirror 72 to the second fluorescence detection channel is parallel to the optical path from the scanning galvanometer 61 to the meta-lens 30. The fluorescence signal reflected by the sample 40 to be tested is sequentially transmitted through the meta-lens 30, the second scanning lens 63, the first scanning lens 62, and the projection of the scanning galvanometer 61 to the scanning galvanometer 61, and the scanning galvanometer 61 reflects the fluorescence signal to the first dichroic mirror 72, and further the first dichroic mirror 72 reflects the fluorescence signal to the fluorescence splitting dichroic mirror 71, and the fluorescence splitting dichroic mirror 71 reflects the fluorescence signal of one wavelength to the first fluorescence detection channel, and enters the first camera 76 through the first filter 74 and the first tube lens 75; the fluorescence splitting dichroic mirror 71 transmits the fluorescence signal of another wavelength to the second fluorescence detection channel, and enters the second camera 79 through the second filter 77 and the second tube lens 78, completing the collection of the fluorescence signal and the detection of the base by the two cameras.

[0068] The main difference between the optical path system of this embodiment and the first embodiment lies in the scanning method. The first embodiment uses the optical fiber array 21 to sequentially illuminate the partial field of view to achieve the illumination of the entire field of view; the reflective system of this embodiment uses the scanning galvanometer 61 to adopt a scanning method, and scans the illumination beam to cover the entire field of view by changing the deflection angle of the scanning galvanometer 61. In this case, the illumination optical path and the imaging optical path share the beam scanning reset component (such as Figure 4 As shown), that is, under the principle of reversible optical path, the scanning galvanometer 61 realizes the functions of deflection illumination to change the illumination field and fluorescence angle correction at the same deflection angle.

[0069] The sample 40 to be tested in this embodiment may be a reflective wafer, specifically a silicon wafer.

[0070] The present invention also provides a method for determining the nucleotide sequence of a biological sample, which is implemented using the above-mentioned optical microscopy imaging device, and the method includes: starting the light source component 10 to project excitation light in turn toward multiple fields of view on the object plane where the sample to be tested 40 is located, exciting the sample to be tested to generate a fluorescence signal in the field of view, and using the meta-lens 30 to collect the fluorescence signals corresponding to the different fields of view according to the emission order of the excitation light; adjusting the deflection angle of the scanning galvanometer 61 according to the propagation direction of the fluorescence signals in the different fields of view to correct the fluorescence signals in the different fields of view to propagate along the optical axis direction parallel to the optical path where the fluorescence imaging component is located; the corrected fluorescence signal is collected by the fluorescence imaging component according to the emission order and a fluorescence image of the corresponding fluorescence signal is generated; the fluorescence images obtained under different fields of view are spliced ​​to obtain a spliced ​​image of the field of view corresponding to the object plane, and the nucleotide sequence of the sample to be tested 40 is identified based on the spliced ​​image.

[0071] Specifically, the excitation light scans the corresponding field of view, the scanning galvanometer 61 corrects the fluorescence signal, and the fluorescence imaging component exposes and collects the fluorescence signal.

[0072] In addition, the process of collecting the corrected fluorescence signal by the fluorescence imaging component according to the emission sequence and generating a fluorescence image corresponding to the fluorescence signal includes: collecting fluorescence signals of different wavelengths in at least two different fluorescence collection channels respectively.

[0073] Specifically, the light source assembly includes an optical fiber array corresponding to the object plane, and the optical fiber array includes multiple optical fibers corresponding to the fields of view one by one; when the light source assembly 10 is started, the excitation light is sequentially projected toward the multiple fields of view on the object plane where the sample to be tested 40 is located, and the process of exciting the sample to be tested to generate a fluorescent signal in the field of view includes: sequentially controlling the optical fiber to irradiate the corresponding field of view according to a pre-set excitation sequence, and the excitation light is emitted from the side of the sample to be tested 40 away from the meta-lens.

[0074] Optionally, the optical microscopic imaging device further includes a notch filter 50 located between the sample to be tested 40 and the fluorescence imaging component, and the method further includes: the notch filter 50 transmits the fluorescence signal and blocks the excitation light transmitted through the sample to be tested 40.

[0075] Specifically, when starting the light source assembly 10 to sequentially project excitation light toward multiple fields of view on the object plane where the sample to be tested 40 is located, and exciting the sample to be tested to generate a fluorescence signal in the field of view, the process also includes: the light source assembly 10 projects the excitation light to the scanning galvanometer 61, and the scanning galvanometer 61 deflects and projects the excitation light to different fields of view in sequence according to a pre-set excitation sequence.

[0076] Specifically, the multiple fields of view are arranged along a first direction and a second direction perpendicular to each other, and the scanning galvanometer includes a first galvanometer 611 driving the excitation light to deflect along the first direction, and a second galvanometer 612 driving the excitation light to deflect along the second direction.

[0077] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects:

[0078] 1. The present invention combines the meta-lens 30, uses the optical fiber structure to illuminate part of the field of view separately, and uses the two-axis scanning galvanometer to offset the imaging field of view, so that the spatial positions of different imaging fields of view overlap on the image plane of the camera, which can effectively solve the problem of excessive size of components in the existing system, and can replace the operation and control platform by controlling the optical fiber structure without expanding the size of the imaging system, and has the advantages of simple structure, easy implementation, high stability, low cost, etc.

[0079] 2. The optical microscopic imaging device of the present invention can achieve the effects of large field of view and high resolution. Taking the meta-lens 30 with parameters of 0.8NA and 10mm*10mm field of view as an example, the resolution is about 500nm (wavelength 630nm). At this time, the density of the sequencing chip is calculated based on a 500nm pitch (sequencing site spacing 500nm), and the points covered in the chip are approximately: (10x103 / 0.5μm)2≈4x108→400M. Compared with the traditional 20X, 0.8NA objective lens, the maximum field of view is about 1.5mm*1.5mm. Under the same numerical aperture and resolution, the solution using the meta-lens 30 can cover nearly 40 times its field of view. In other words, the present application uses the meta-lens 30 in combination with the scanning galvanometer 61 for scanning, which can cover nearly 40 times the field of view under the same numerical aperture and resolution.

[0080] 3. Scanning the sample 40 to be tested without a control platform: The whole field of view is scanned by the scanning galvanometer 61, and there is no need to couple the motion control system in the imaging system, thus avoiding frequent movement and stopping of the motion platform, reducing the time cost of taking pictures, increasing the stability of the imaging system, and greatly simplifying the complexity of the sequencing system. At the same time, the sequencing system is not limited by the size of the control platform.

[0081] 4. No need to enlarge the size of all original components in the optical system to adapt to the large field of view: by scanning the scanning galvanometer 61, the fluorescent signal except the central field of view is deflected and reset, so that the light spots of the fields of view at different spatial positions can overlap at the same position on the image plane, avoiding the enlargement of the size of the optical components (camera, tube mirror, lens and filter), effectively reducing the size of the system, reducing the cost and making the system easy to mass produce;

[0082] 5. The optical microscopic imaging device of the present application provides two systems, namely the transmission system of the first embodiment and the reflection system of the second embodiment, which can be applied to reflective slides (silicon slides) and transmissive slides (glass), covering most sample types, and has the advantages of wide application range, flexibility, and easy implementation;

[0083] 6. The light source assembly 10, the scanning galvanometer 61 and the camera are synchronously controlled by the control system. Compared with the movement of the traditional operation and control platform system, the operation of the scanning galvanometer 61 takes less time (milliseconds), and the entire field of view can be quickly scanned and imaged, which improves the scanning speed and time resolution of the sequencing system and also increases the stability of the system.

[0084] Obviously, the above-described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0085] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0086] It should be noted that the terms "first", "second", etc. in the specification and claims of the present application and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the numbers used in this way can be interchanged where appropriate, so that the embodiments of the present application described herein can be implemented in an order other than those illustrated or described herein.

[0087] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An optical microscopic imaging device, characterized in that: include: A light source assembly (10), the light source assembly (10) being used to emit excitation light; A light beam transmission component, the light beam transmission component at least comprising an optical fiber structure, the optical fiber structure being connected to the light source component (10) and being used to guide the excitation light to a predetermined field of view of a sample to be tested (40); A fluorescence collection component, the fluorescence collection component comprising a meta-lens (30), the meta-lens (30) being used to receive a fluorescence signal generated by a sample to be tested (40) in the predetermined field of view when excited by the excitation light; A beam scanning resetting component, the beam scanning resetting component at least comprising a scanning galvanometer (61), the scanning galvanometer (61) corresponding to the meta-lens (30) and the angle of the scanning galvanometer (61) being adjustable, and used to correct the transmission direction of the fluorescent signal; A fluorescence imaging component is located on one side of the scanning galvanometer (61) and is used to receive the fluorescence signal corrected by the scanning galvanometer (61), wherein the transmission direction of the corrected fluorescence signal in the fluorescence imaging component is parallel to the optical axis direction of the fluorescence imaging component.

2. The optical microscopic imaging device according to claim 1, characterized in that: The excitation light comprises a first light and a second light of different wavelengths, and the fluorescence imaging component comprises a fluorescence dichroic mirror (71), a first fluorescence detection channel and a second fluorescence detection channel, the fluorescence dichroic mirror (71) is used to separate the fluorescence signal according to wavelength, the first fluorescence detection channel is used to receive the fluorescence signal corresponding to the first light, and the second fluorescence detection channel is used to receive the fluorescence signal corresponding to the second light.

3. The optical microscopic imaging device according to any one of claims 1 to 2, characterized in that: The meta-lens (30) has a nano-array structure.

4. The optical microscopic imaging device according to claim 1, characterized in that: The beam scanning and resetting assembly also includes a 4f optical system composed of a first scanning lens (62) and a second scanning lens (63), wherein the first scanning lens (62) and the second scanning lens (63) are sequentially arranged between the meta-lens (30) and the scanning galvanometer (61) in a direction away from the meta-lens (30).

5. The optical microscopic imaging device according to claim 1, characterized in that: The light source assembly (10) is located on a side of the sample to be tested (40) away from the metalens (30) and is used to project the excitation light toward the side of the sample to be tested (40) away from the metalens (30); the optical fiber structure comprises an optical fiber array (21) corresponding to the object plane where the sample to be tested is located; the optical fiber array (21) comprises a plurality of optical fibers corresponding one by one to the predetermined field of view; the fluorescence collection assembly further comprises a notch filter (50) located between the sample to be tested (40) and the fluorescence imaging assembly, the notch filter (50) being used to transmit the fluorescence signal and block the excitation light transmitted through the sample to be tested (40).

6. The optical microscopic imaging device according to claim 1, characterized in that: The excitation light emitted by the light source assembly (10) is projected onto the sample to be tested (40) via the scanning galvanometer (61), and the light beam transmission assembly further comprises a first collimating lens (23) and a second collimating lens (24) located between the optical fiber structure and the scanning galvanometer (61).

7. The optical microscopic imaging device according to claim 6, characterized in that: The predetermined field of view is arranged along a first direction and a second direction perpendicular to each other, and the scanning galvanometer (61) comprises a first galvanometer (611) driving the excitation light to move along the first direction and a second galvanometer (612) driving the excitation light to move along the second direction.

8. The optical microscopic imaging device according to claim 7, characterized in that: The optical microscopic imaging device further comprises a first dichroic mirror (72) located between the light beam transmission component and the scanning galvanometer (61), wherein the first dichroic mirror (72) has a transmission band for transmitting the excitation light and a reflection band for reflecting the fluorescence signal.

9. A method for determining the nucleotide sequence of a biological sample, characterized in that: The method is implemented by using the optical microscopic imaging device according to any one of claims 1 to 8, and the method comprises: Starting the light source assembly (10) to sequentially project excitation light toward a plurality of viewing fields on the object plane where the sample to be tested (40) is located, thereby exciting the sample to be tested to generate a fluorescence signal in the viewing field, and using a meta-lens (30) to respectively collect the fluorescence signals corresponding to the different viewing fields according to the emission sequence of the excitation light; Adjusting the deflection angle of the scanning galvanometer (61) according to the propagation direction of the fluorescent signals in different fields of view to correct the fluorescent signals in different fields of view to propagate in the direction of the optical axis parallel to the optical path where the fluorescent imaging component is located; The corrected fluorescence signal is collected by the fluorescence imaging component according to the emission sequence and a fluorescence image corresponding to the fluorescence signal is generated; The fluorescent images obtained under different fields of view are stitched together to obtain a stitched image of the field of view corresponding to the object plane, and the nucleotide sequence of the sample to be tested (40) is identified based on the stitched image.

10. The method for determining the nucleotide sequence of a biological sample according to claim 9, characterized in that: The excitation light scans the corresponding field of view, the scanning galvanometer (61) corrects the fluorescence signal, and the fluorescence imaging component exposes and collects the fluorescence signal.

11. The method for determining the nucleotide sequence of a biological sample according to claim 9, characterized in that: The process in which the corrected fluorescence signal is collected by the fluorescence imaging component according to the emission sequence and a fluorescence image corresponding to the fluorescence signal is generated includes: The fluorescence signals of different wavelengths are collected respectively in at least two different fluorescence collection channels.

12. The method for determining the nucleotide sequence of a biological sample according to any one of claims 9 to 11, characterized in that: The light source assembly includes an optical fiber array corresponding to the object plane, and the optical fiber array includes a plurality of optical fibers corresponding to the fields of view one by one; when the light source assembly (10) is started to sequentially project excitation light toward the plurality of fields of view on the object plane where the sample to be tested (40) is located, and the process of exciting the sample to be tested to generate a fluorescent signal in the field of view includes: The optical fiber is controlled to illuminate the corresponding field of view in sequence according to a preset excitation sequence, and the excitation light is emitted from a side of the sample to be tested (40) away from the meta-lens.

13. The method for determining the nucleotide sequence of a biological sample according to claim 12, characterized in that: The optical microscopic imaging device further comprises a notch filter (50) located between the sample to be tested (40) and the fluorescent imaging component, and the method further comprises: The notch filter (50) transmits the fluorescent signal and blocks the excitation light transmitted through the sample to be tested (40).

14. The method for determining the nucleotide sequence of a biological sample according to any one of claims 9 to 11, characterized in that: In the process of starting the light source assembly (10) to sequentially project excitation light toward a plurality of viewing fields on the object plane where the sample to be tested (40) is located, and exciting the sample to be tested to generate a fluorescent signal in the viewing field, the process further includes: The light source assembly (10) projects the excitation light onto the scanning galvanometer (61), and the scanning galvanometer (61) sequentially deflects and projects the excitation light onto different viewing fields according to a pre-set excitation sequence.

15. The method for determining the nucleotide sequence of a biological sample according to any one of claims 9 to 11, characterized in that: The multiple fields of view are arranged along a first direction and a second direction that are perpendicular to each other, and the scanning galvanometer comprises a first galvanometer (611) that drives the excitation light to deflect along the first direction and a second galvanometer (612) that drives the excitation light to deflect along the second direction.

Images