Microscopic imaging apparatus, microscopic imaging method, and storage medium

Abstract

The embodiment of the application discloses a microscopic imaging device, a microscopic imaging method and a storage medium, and relates to the technical field of microscopic imaging. When performing fluorescence imaging on a microscopic sequencing chip, first, an excitation light beam generated by an excitation module excites the sequencing chip to generate a fluorescence signal, the fluorescence signal passes through an objective lens to reach an imaging unit for imaging, wherein a sleeve lens in the imaging unit converges the fluorescence signal to form an optical signal which is received by a detector for imaging. At this time, only the relative distance between the detector and the sleeve lens in the imaging unit needs to be adjusted, the objective lens is controlled to move by an automatic focusing device so that the sample is kept on the focal point of the objective lens, and thus clear imaging of the sample on different measurement surfaces of the chip can be performed without the need of designing a special zoom lens with high cost, the operation is simple and convenient, the cost is low, and the fluid reagent can be maximally utilized.

Description

Technical Field

[0001] This invention relates to the field of microscopic imaging technology, and particularly to microscopic imaging equipment, microscopic imaging methods, and storage media. Background Technology

[0002] Microscopic imaging technology has wide applications in sample testing. For example, in gene sequencing, fluorescence imaging of bases on biochips is required. As the application of microscopic imaging technology becomes more widespread, testing needs are also diversifying.

[0003] Due to their short depth of field, microscope objectives in related technologies can generally only provide clear imaging of one surface of a biochip. However, biochips can contain multiple surfaces, each carrying a sample to be tested. To achieve clear imaging of samples on multiple surfaces, the gene sequencer's imaging system needs to be refocused multiple times, or a dedicated zoom lens needs to be designed. However, the process of multiple refocusings is extremely cumbersome, and zoom lenses are expensive to manufacture and difficult to design, making it difficult to achieve clear imaging of samples on multiple surfaces. Summary of the Invention

[0004] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a microscopic imaging device, a microscopic imaging method, and a storage medium, which can easily and quickly perform clear microscopic imaging of multi-surface samples.

[0005] A microscopic imaging apparatus according to a first aspect of the present invention is used for imaging fluorescence signals of a sequencing chip, the sequencing chip including a first measurement surface and a second measurement surface, comprising:

[0006] An excitation module is used to generate an excitation beam so that the sequencing chip is excited by the excitation beam to produce a fluorescence signal;

[0007] An objective lens, positioned on the optical axis of the fluorescence signal, is used to acquire the fluorescence signal;

[0008] At least one imaging unit is configured to perform fluorescence imaging on the sequencing chip based on the fluorescence signal. The fluorescence imaging includes: a first imaging or a second imaging, wherein the first imaging is configured to image a first measurement surface of the sequencing chip to obtain a first imaging image, and the second imaging is configured to image a second measurement surface of the sequencing chip to obtain a second imaging image.

[0009] The imaging unit includes:

[0010] A sleeve lens is used to converge the fluorescence signal emitted from the objective lens to form the target light signal;

[0011] The detector is positioned behind the sleeve lens along the optical axis of the target optical signal;

[0012] When the detector is used to perform a first imaging based on the target light signal, the distance between the detector and the sleeve lens is the first imaging distance;

[0013] When the detector is used to perform a second imaging based on the target light signal, the distance between the detector and the sleeve lens is the second imaging distance, and the first imaging distance is different from the second imaging distance.

[0014] According to some embodiments of the present invention, the imaging unit further includes: a first displacement stage, the first displacement stage being connected to the detector or the sleeve lens, the first displacement stage being used to control the detector to move along the optical axis of the target light signal, or to control the sleeve lens to move along the optical axis of the target light signal, such that the distance between the sleeve lens and the detector is the first imaging distance or the second imaging distance.

[0015] According to some embodiments of the present invention, the device further includes: a second displacement stage and an autofocus device, the autofocus device being connected to the objective lens or the second displacement stage, for moving the objective lens or the second displacement stage along the optical axis of the fluorescence signal during the first imaging of the detector, so that the first measuring surface is located at the focal position of the objective lens; or for moving the objective lens or the second displacement stage along the optical axis of the fluorescence signal during the second imaging of the detector, so that the second measuring surface is located at the focal position of the objective lens.

[0016] According to some embodiments of the present invention, the autofocus device is used to obtain a first focusing distance between the first measuring surface and the objective lens or a second focusing distance between the second measuring surface and the objective lens, and is also used to adjust the position of the objective lens or the second displacement stage in real time along the optical axis of the fluorescence signal according to the first focusing distance when the detector performs a first imaging, and is also used to adjust the position of the objective lens or the second displacement stage in real time along the optical axis of the fluorescence signal according to the second focusing distance when the detector performs a second imaging.

[0017] According to some embodiments of the present invention, it further includes: an optical element located between the objective lens and the sleeve lens, the optical element comprising one or more of a dichroic mirror, a filter, or a reflector.

[0018] A microscopic imaging method according to a second aspect of the present invention includes:

[0019] Acquire imaging state, which is used to characterize whether a first imaging is performed on a first measurement surface of the sequencing chip or a second imaging is performed on a second measurement surface of the sequencing chip;

[0020] The distance between the detector and the sleeve lens is adjusted to either a first imaging distance or a second imaging distance based on the imaging state.

[0021] The detector is used to perform the first imaging to obtain the first imaging image, or to perform the second imaging to obtain the second imaging image.

[0022] According to some embodiments of the present invention, acquiring the imaging state further includes:

[0023] If the imaging state indicates that the detector is performing the first imaging, the objective lens is moved along the optical axis of the fluorescence signal so that the first measuring surface is located at the focal position of the objective lens;

[0024] If the imaging state indicates that the detector is performing a second imaging, the objective lens is moved along the optical axis of the fluorescence signal so that the first measuring surface is located at the focal point of the objective lens.

[0025] According to some embodiments of the present invention, it further includes:

[0026] Obtain the first focusing distance between the first measuring surface and the objective lens or the second focusing distance between the second measuring surface and the objective lens;

[0027] If the imaging state indicates that the detector is performing the first imaging, the position of the objective lens is adjusted in real time along the optical axis of the fluorescence signal according to the first focusing distance;

[0028] If the imaging state indicates that the detector is performing a second imaging, the position of the objective lens is adjusted in real time along the optical axis of the fluorescence signal according to the second focusing distance.

[0029] According to some embodiments of the present invention, adjusting the distance between the detector and the sleeve lens to a first imaging distance or a second imaging distance based on the imaging state further includes: controlling the detector to move along the optical axis of the target light signal, or controlling the sleeve lens to move along the optical axis of the target light signal, so that the distance between the detector and the sleeve lens is the first imaging distance or the second imaging distance.

[0030] According to a third aspect of the present invention, a computer-readable storage medium stores computer-executable instructions for performing the above-described microscopic imaging method.

[0031] The microscopic imaging device, microscopic imaging method, and storage medium proposed in this invention have at least the following beneficial effects: When performing fluorescence imaging on a microscopic sequencing chip, the chip is first excited by the excitation beam generated by the excitation module to generate a fluorescence signal. The fluorescence signal passes through the objective lens to reach the imaging unit for imaging. The sleeve lens in the imaging unit converges the fluorescence signal to form a light signal, which is then received and imaged by the detector. At this time, it is only necessary to adjust the relative distance between the detector and the sleeve lens in the imaging unit, and control the movement of the objective lens through the autofocus device to keep the sample at the focal point of the objective lens. This allows for clear imaging of different measurement surfaces of the sample on the chip without the need to design a high-cost dedicated zoom lens, making the operation simple and convenient.

[0032] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0033] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0034] Figure 1 This is a schematic diagram of the sequencing chip structure according to an embodiment of the present invention;

[0035] Figure 2 This is the first measurement surface imaging image of its quality in the prior art;

[0036] Figure 3 The image quality of the second measurement surface in the prior art;

[0037] Figure 4 This is a schematic diagram of the structure of a microscopic imaging device according to an embodiment of the present invention;

[0038] Figure 5 for Figure 4 The diagram shows the imaging process of different measurement surfaces in a microscopic imaging device.

[0039] Figure 6 This is the first measurement surface imaging image quality image according to an embodiment of the present invention;

[0040] Figure 7 This is the second measurement surface imaging image quality image according to an embodiment of the present invention;

[0041] Figure 8 This is a schematic diagram of the microscopic imaging method implemented in this invention;

[0042] Figure 9 This is a schematic diagram of the structure of a multi-channel microscopic imaging device according to an embodiment of the present invention.

[0043] Reference numerals: excitation module 100, objective lens 200, imaging unit 300, sleeve lens 310, detector 320, first displacement stage 330, autofocus device 400, optical element 500, sequencing chip 600, flow channel 610, biological sample 620, coverslip 630, slide 640, first measurement surface 650, second measurement surface 660, second displacement stage 700. Detailed Implementation

[0044] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0045] In the description of this invention, it should be understood that the orientation descriptions, such as up, down, front, back, left, right, etc., are based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting this invention.

[0046] In the description of this invention, "several" means one or more, "more than" means two or more, "greater than," "less than," and "exceeding" are understood to exclude the stated number, while "above," "below," and "within" are understood to include the stated number. The use of "first" and "second" in the description is merely for distinguishing technical features and should not be construed as indicating or implying relative importance, or implicitly indicating the number of indicated technical features, or implicitly indicating the order of the indicated technical features.

[0047] In the description of this invention, unless otherwise explicitly defined, terms such as "set up," "install," and "connect" should be interpreted broadly, and those skilled in the art can reasonably determine the specific meaning of the above terms in this invention in conjunction with the specific content of the technical solution.

[0048] To better understand the technical solution provided by this invention, the terms appearing in this document are explained accordingly:

[0049] Depth of field refers to the range of distances within the object side (in front of and behind the focal point) where objects appear as blurry images on the image side within this range, all within the limits of the circle of confusion (the acceptable range of sharpness). In other words, it's the acceptable range of sharpness in front of and behind an object along the optical axis from the object side to the image side.

[0050] The diffraction limit refers to the limitation that when an ideal object point is imaged by an optical system, an ideal image point cannot be obtained due to diffraction; instead, a Fraunhofer diffraction image is obtained. Since the aperture of a typical optical system is circular, Fraunhofer diffraction is known as the Airy disk. Thus, the image of each object point is a diffuse spot, and two diffuse spots are difficult to distinguish when close together, thereby limiting the system's resolution. The larger the spot, the lower the resolution. This limitation is a limitation of physical optics, caused by the diffraction of light.

[0051] NA: refers to the aperture ratio, which is the amount of light that can be captured by the objective lens frame. It is the numerical aperture of an optical microscope, abbreviated as NA (numerical aperture) or A. It is a key parameter of the objective lens and condenser and is directly proportional to the resolution of the microscope.

[0052] Point plot: In geometrical optics imaging, many rays emanating from a single point, after being imaged by an optical system, no longer converge at a fixed point due to aberrations, forming a diffuse spot distributed within a certain range. The method of measuring the imaging quality of an optical system using the density of these points in the point plot is called the point plot method. When evaluating the imaging quality of photographic lenses, etc., the point plot method typically uses the area formed by points or rays converging at more than 30% as its actual effective diffuse spot. The reciprocal of the diameter of the diffuse spot is the system's resolution. This resolution is limited by geometrical aberrations and does not consider diffraction effects.

[0053] Airy disk: A spot of light formed at the focal point by diffraction when a point light source is imaged under diffraction-limited conditions. It has a bright circular spot in the center, surrounded by a set of concentric rings of weaker light and dark. The central bright spot, bounded by the first dark ring, is called the Airy disk.

[0054] Microscopic imaging technology has wide applications in sample detection. In gene sequencing, fluorescence imaging of bases on the sequencing chip is required. When the gene sequencer is working, the excitation beam excites the biological sample on the sequencing chip to generate a fluorescence signal. The generated fluorescence signal is collected and shaped by a series of optical elements and finally imaged onto the detector. In one test cycle, sequencing reagents can interact with biological samples on multiple surfaces simultaneously, and the excitation beam can excite biological samples on multiple surfaces of the sequencing chip at the same time. However, due to their short depth of field, the microscope objectives in related technologies can generally only provide a clear image of one surface of the sequencing chip. Figure 2 and Figure 3These are imaging images of different measurement surfaces of a sequencing chip in related technologies. It can be seen that due to aberrations caused by the liquid layer in the flow channel, multiple surfaces cannot achieve optimal imaging results simultaneously. Therefore, if clear imaging of multiple surfaces is desired, it is necessary to perform multiple focusing adjustments on the imaging system of the gene sequencer or design expensive dedicated zoom lenses.

[0055] This invention provides a method and apparatus for microscopic imaging. When imaging different measurement surfaces, clear multi-surface microscopic imaging can be achieved simply by moving the working distance between the objective lens and the sequencing chip, and the working distance between the tube lens and the detector. This method is low-cost, easy to operate, and can maximize the utilization of fluid reagents during gene sequencing.

[0056] Reference Figure 1 The sequencing chip 600 structure shown has a biological sample 620 loaded between the lower surface of a coverslip 630 and the upper surface of a slide 640. The coverslip 630 covers the slide 640, forming a flow channel 610 between them. Thus, the lower surface of the coverslip 630 is the upper surface of the flow channel, i.e., the first measurement surface 650, and the upper surface of the slide 640 is the lower surface of the flow channel, i.e., the second measurement surface 660.

[0057] In gene sequencing, biological sample 620 is a multi-surfaced biological DNA cluster with varying heights and depths. In existing gene sequencers, sequencing reagents can interact with biological sample 620 simultaneously, and excitation beams can simultaneously excite biological sample 620 on different measurement surfaces of sequencing chip 600 to generate fluorescence signals. However, due to the short depth of field, the microscope objectives of existing microscopic imaging devices can generally only clearly image one surface of sequencing chip 600. During the imaging process, the presence of liquid in channel 610 introduces aberrations into the imaging results of the other surface, resulting in blurred imaging of the other surface. This makes it impossible to achieve the best imaging effect for multiple surfaces simultaneously.

[0058] For example, refer to Figure 2 and Figure 3These are schematic diagrams of image quality of the first measurement surface 650 and the second measurement surface 660 of the sequencing chip 600 in the prior art. In the example, the microscopic imaging device images the sequencing chip 600 with a cover glass thickness of 170 μm and a channel thickness of 80 μm. The objective lens parameters are as follows: NA 0.7, field of view 1.2 mm, and magnification 20x. As can be seen from the images, the Ally spot radius (i.e., optical diffraction limit) of the microscopic imaging device at a wavelength of 587.563 nm is 0.4779 μm. If the GEO spot radius of each field of view obtained from the dot plot is greater than the diffraction limit, then for an infinitesimally small point, its spot radius after passing through the optical system is the GEO radius; if it is less than the diffraction limit, then the spot radius is the diffraction limit. For example, in the image quality of the first measurement surface 650, such as... Figure 2 As shown, the GEO radius of the third field of view is 0.309 μm, which is smaller than the radius of the Airy disk. Therefore, its spot radius is the Airy disk radius, i.e., 0.4779 μm. In the imaging quality of the second measurement surface 660, as... Figure 3 As shown, the GEO radius of the third field of view is 4.835 μm, which is much larger than the radius of the Airy disk. Therefore, its spot radius is its GEO radius of 4.835 μm. It is understood that the wavelength of light, cover glass thickness, channel thickness, objective lens parameters, and Airy disk radius mentioned above are all examples and do not represent a limitation on the above parameters in this embodiment. The above parameters can be set according to actual needs.

[0059] Generally, a GEO radius less than or close to the diffraction limit in each field of view indicates a clearer image; if it exceeds the diffraction limit, the image quality is considered poor. When using a sequencing chip 600 with a coverslip thickness of 170µm and a channel thickness of 80µm for gene sequencing, such as... Figure 2 As shown, the GEO radii of each field of view are close to the Airy disk radius, thus the imaging of the first measurement surface 650 is close to the diffraction limit, resulting in clear and good imaging. However, due to aberrations caused by the liquid in the 80µm thick flow channel, the GEO radii of each field of view are much larger than the Airy disk radius, leading to poorer image quality for the second measurement surface 660. Figure 3 As shown, it falls far short of the diffraction limit, resulting in poor image blurring.

[0060] Based on this, embodiments of the present invention provide a microscopic imaging device and its usage method that can effectively solve the above problems.

[0061] Reference Figure 4The microscopic imaging device shown mainly includes an excitation module 100, which generates an excitation beam to excite the sequencing chip 600 to produce a fluorescence signal; an objective lens 200, which is positioned on the optical axis of the fluorescence signal to acquire the fluorescence signal; and an imaging unit 300, which performs fluorescence imaging on the sequencing chip 600 based on the fluorescence signal. The imaging unit 300 mainly includes a sleeve lens 310 and a detector 320. The sleeve lens 310 is used to converge the fluorescence signal emitted from the objective lens 200 to form a target light signal, and the detector 320 is positioned on the sleeve lens 310 along the optical axis of the target light signal. Behind the lens 310, the imaging unit 300 also includes a first displacement stage 330, which is connected to the detector 320 and used to control the detector 320 to move along the optical axis of the target light signal; an autofocus device 400, connected to the objective lens 200, used to adjust the position of the objective lens 200 in real time along the optical axis of the fluorescence signal; one or more optical elements 500, located between the objective lens 200 and the sleeve lens 310, used to collect and process excitation signals or fluorescence signals; and a sequencing chip 600, containing multiple surface samples to be tested, positioned below the objective lens 200 and connected to the second displacement stage 700.

[0062] Fluorescence imaging for gene sequencing includes either a first imaging process or a second imaging process. The first imaging process images the first measurement surface 650 of the sequencing chip 600 to obtain a first image. The second imaging process images the second measurement surface 660 of the sequencing chip 600 to obtain a second image. When the detector 320 performs the first imaging based on the target light signal, the autofocus device 400 moves the objective lens 200 along the optical axis of the fluorescence signal according to a first focusing distance, so that the first measurement surface 650 is located at the focal point of the objective lens 200. The distance between the detector 320 and the sleeve lens 310 is controlled by the first displacement stage 330 to be the first imaging distance. When the detector 320 performs the second imaging based on the target light signal, the autofocus device 400 moves the objective lens 200 along the optical axis of the fluorescence signal according to a second focusing distance, so that the second measurement surface 660 is located at the focal point of the objective lens 200. The distance between the detector 320 and the sleeve lens 310 is controlled by the first displacement stage 330 to be the second imaging distance. This allows for clear imaging of multiple surfaces of the sequencing chip 600.

[0063] In the above embodiment, during the microscopic imaging process, an excitation beam is first emitted by the excitation module 100. After being reflected by the optical element 500, the excitation beam passes through the objective lens 200 and reaches the sequencing chip 600 below, exciting the sequencing chip 600 to generate a fluorescence signal. The fluorescence is collected by the objective lens 200 and, after being transmitted and reflected by the optical element 500, reaches the imaging unit 300. First, the target light signal is acquired and converged by the sleeve lens 310 in the imaging unit 300, and then collected and imaged by the detector 320 located behind the sleeve lens 310 in the imaging unit 300. The sequencing chip 600 is placed on the second displacement stage 700, and the detector 320 is placed on the first displacement stage 330. The autofocus device 400 detects the height of the moving sample in real time and then controls and adjusts the height of the objective lens 200 to keep the distance between the objective lens 200 and the sequencing chip 600 constant, so as to obtain a clear imaging image.

[0064] It is understood that the autofocus device 400 is connected to the objective lens 200 and controls the movement of the objective lens 200 to achieve the distance between the objective lens 200 and the sequencing chip 600, so that the first measurement surface 650 or the second measurement surface 660 is located outside the focal position of the objective lens 200. In some embodiments, the autofocus device 400 can also be connected to the second displacement stage 700, and the same effect can be achieved by moving the second displacement stage 700.

[0065] Furthermore, referring to Figure 5 The imaging process shown allows for preset adjustment of the relative position between the sleeve lens 310 and the detector 320 on different measurement surfaces via the first displacement stage 330, so that the device can clearly image both the first measurement surface 650 and the second measurement surface 660 of the sequencing chip 600. Specifically, when imaging the first measurement surface 650 of the sequencing chip 600, the objective lens 200 collects the fluorescence signal generated by the biological sample 620. Subsequently, the fluorescence signal is focused onto the detector 320 by the sleeve lens 310 for imaging. At this time, the distance between the detector 320 and the sleeve lens is the first imaging distance. When imaging the second measurement surface 660 of the sequencing chip 600, the objective lens 200 acquires the fluorescence signal generated by the biological sample 620. This fluorescence signal passes through the flow channel 610 and the coverslip 630, and is subsequently focused onto the detector 320 by the sleeve lens 310 for imaging. At this time, the distance between the detector 320 and the sleeve lens is the second imaging distance. Due to the aberrations caused by the flow channel 610, the first imaging distance and the second imaging distance are different. The relative positional relationship between the objective lens 200, the sleeve lens 310, and the detector 320 is adjusted according to the imaging distance to achieve clear imaging of different measurement surfaces.

[0066] Understandably, the positions of the objective lens 200, the telescopic lens 310, and the detector 320 are all adjusted by moving back and forth along the optical axis of the signal. The height adjustment of the objective lens 200 is controlled by the autofocus device 400. When performing the first imaging of the first measuring surface 650, the autofocus device 400 acquires the first focusing distance between the first measuring surface 650 and the objective lens 200. Based on this first focusing distance, it adjusts the position of the objective lens 200 in real time along the optical axis of the fluorescence signal so that the first measuring surface 650 is located at the focal point of the objective lens 200. When performing the second imaging of the second measuring surface 660, the autofocus device 400 acquires the second focusing distance between the second measuring surface 660 and the objective lens 200. Based on this second focusing distance, it adjusts the position of the objective lens 200 in real time along the optical axis of the fluorescence signal so that the second measuring surface 660 is located at the focal point of the objective lens 200. This ensures that imaging of different measuring surfaces is based on a clear surface at the focal point.

[0067] It is understandable that in the actual production process, imperfections in the flatness of the coverslip 630 and slide 640 are inevitable, resulting in unevenness in the biological sample 620. When the sequencing chip 600 moves horizontally, the distance between the objective lens 200 and the sequencing chip 600 will change, leading to unclear imaging. In one embodiment, the objective lens 200 is electrically connected to the autofocus device 400. During the sequencing of the sequencing chip 600, since the glass height varies at different positions on the sequencing chip 600, the autofocus device 400 continuously monitors the height of the moving biological sample 620 in real time during imaging of different measurement surfaces to ensure that the surface of the biological sample 620 at different heights remains at the focal point of the objective lens. When there are unevenness on the surface of the biological sample 620, the objective lens 200 is moved up and down for fine-tuning, keeping the distance between the objective lens 200 and the biological sample 620 constant, ensuring that the biological sample 620 is always at the focal point of the objective lens 200. This automated operation is simple and produces clear and effective imaging.

[0068] Furthermore, the relative position of the sleeve lens 310 and the detector 320 is adjusted and controlled by the movement of the displacement 330. Specifically, the sleeve lens 310 is fixed, and the detector 320 is connected to the first displacement stage 330. After the equipment is debugged in the early stage with a cover glass 630 and a flow channel 610 of preset thickness, the imaging distance between the sleeve lens 310 and the detector 320 is designed. When the first displacement stage 330 controls the position of the two at the first imaging distance, the autofocus device 400 controls the objective lens 200 to find the imaging focal plane of the sample as the first measurement surface 650. At this time, the distance coordinate value between the first displacement stage 330 and the sleeve lens 310 is set to T1. When the first displacement stage 330 controls the position of the two at the second imaging distance, the autofocus device 400 controls the objective lens 200 to find the imaging focal plane of the sample as the second measurement surface 660. At this time, the distance coordinate value between the first displacement stage 330 and the sleeve lens 310 is set to T2. Then, the coordinate values ​​of the first imaging distance T1 of the first measuring surface 650 and the second imaging distance T2 of the second measuring surface 660 are pre-written into the control system of the first displacement stage 330.

[0069] During gene sequencing, if it is necessary to test the first measurement surface 650, the first stage 330 is moved to position T1, and then the objective lens 200 is controlled by the autofocus device 400 to find the imaging focus of the sample. At this time, the imaging focal plane of the sample is the first measurement surface 650. If it is necessary to test the second measurement surface 660, the first stage 330 is moved to position T2, and the objective lens 200 is controlled by the autofocus device 400 to find the imaging focus of the sample. At this time, the imaging focal plane of the sample is the second measurement surface 660.

[0070] It is understandable that the relative distance between the detector 320 and the sleeve lens 310 can be changed by fixing the detector 320 and connecting the sleeve lens 310 to the first displacement stage 330. Alternatively, the detector 320 and the sleeve lens 310 can be connected to different first displacement stages 330.

[0071] Understandably, depending on the actual needs, coverslips 630 and flow channels 610 of different thicknesses can be used. By obtaining T1 and T2 through a single preset adjustment, multiple multi-surface imaging can be performed on the sequencing chip using coverslips 630 and flow channels 610 of the same thickness.

[0072] Reference Figure 6 and Figure 7As shown, exemplarily, the cover glass thickness is 170 μm, the flow channel thickness is 80 μm, the focal length of the sleeve lens 310 is 200 mm, and when the objective lens 200 images the first measurement surface 650, the working distance of the objective lens 200, i.e., the distance from the cover glass 630 to the surface of the first lens of the objective lens 200, is 1 mm. Relevant optical elements are provided between the objective lens 200 and the sleeve lens 310 as needed. The distance from the sleeve lens 310 to the detector 320 is 148.655 mm. At this time, the sleeve lens 310 is fixed, the position of the detector 320 is denoted as T1, and the image quality is as follows... Figure 6 As shown, the GEO radii of each field of view in the dot plot are 9.758 μm, 8.578 μm, and 6.918 μm, respectively, all smaller than or close to the Airy disk radius of 9.55 μm at a wavelength of 588 nm. Therefore, the image quality within a 1.2 mm field of view is close to the diffraction limit, resulting in clear imaging. In the example, the flow channel 610 is a liquid layer with a height of 80 μm. When the objective lens 200 images the second measurement surface 660, the working distance of the objective lens 200 is 0.977 mm, and the distance from the sleeve lens 310 to the detector 320 is 134.169 mm. The position of the detector 320 at this time is denoted as T2, and the image quality at this time is as follows: Figure 7 As shown in the dot plot, the GEO radii of each field of view are 11.977 μm, 8.626 μm, and 8.918 μm, respectively, all smaller than or close to the Airy disk radius of 9.103 μm at a wavelength of 588 nm. Therefore, the image quality within a 1.2 mm field of view is also close to the diffraction limit, resulting in clear imaging. It is understood that the wavelength of light, cover glass thickness, channel thickness, objective lens parameters, and Airy disk radius mentioned above are merely examples and do not represent a limitation on these parameters in this embodiment. These parameters can be set according to actual needs.

[0073] It is understood that optical elements 500 include, but are not limited to, one or more of dichroic mirrors, filters, and mirrors, which can be added according to the actual needs of the sequencing work. Among them, dichroic mirrors are used to achieve spectral separation based on the wavelength of light transmitted or reflected, enabling the classification and sequencing of different bases on a dual-channel or four-channel imaging system; filters are used to filter out excitation light, allowing only fluorescence of specific wavelengths to pass through; and mirrors are used to improve the integration of the system.

[0074] In the embodiments of the present invention, the microscopic imaging device does not require the addition of other new optical elements to correct the aberrations caused by the liquid layer in the flow channel. It only needs to control the working distance of the objective lens 200 by moving it up and down through the autofocus device 400, and adjust the relative distance between the sleeve lens 310 and the detector 320 through the first displacement stage 330 to achieve multi-surface microscopic imaging. There is no need to design a high-cost special zoom lens. Therefore, the present invention has the advantages of low cost, convenient operation, and maximizing the utilization of fluid reagents in the gene sequencing process.

[0075] based on Figure 5 The aforementioned microscopic imaging device, with reference to Figure 8 The present invention provides a microscopic imaging method applied to a microscopic imaging device, which includes, but is not limited to, the following steps S100 to S300.

[0076] Step S100: Obtain the imaging state, which is used to characterize whether the first imaging is performed on the first measurement surface 650 or the second imaging is performed on the second measurement surface 660 of the sequencing chip 600.

[0077] In one embodiment, step S100 further includes the following steps;

[0078] In step S110, during the first imaging, the autofocus device 400 controls the movement of the objective lens 200 along the optical axis of the fluorescence signal according to the first focusing distance, so that the first measuring surface 650 is located at the focal position of the objective lens 200.

[0079] In step S120, during the second imaging process, the autofocus device 400 controls the movement of the objective lens 200 along the optical axis of the fluorescence signal according to the second focusing distance, so that the second measurement surface 660 is located at the focal position of the objective lens 200.

[0080] Step S200: Adjust the distance between the detector 320 and the sleeve lens 310 to a first imaging distance or a second imaging distance according to the imaging state.

[0081] In some embodiments, step S200 specifically involves controlling the detector 320 or the sleeve lens 310 to move along the optical axis of the target light signal via a first displacement stage 330, such that the distance between the detector 320 and the sleeve lens 310 is a first imaging distance or a second imaging distance. In step S210, when the imaging state is characterized as a first imaging of the first measuring surface 650, the first displacement stage 330 adjusts the distance between the detector 320 and the sleeve lens 310 to the first imaging distance; in step S220, when the imaging state is characterized as a second imaging of the second measuring surface 660, the first displacement stage 330 adjusts the distance between the detector 320 and the sleeve lens 310 to the second imaging distance.

[0082] Step S300: The detector 320 performs a first imaging to obtain a first image or performs a second imaging to obtain a second image.

[0083] In some embodiments, the above steps further include obtaining a first focusing distance between the first measuring surface 650 and the objective lens 200 or a second focusing distance between the second measuring surface 660 and the objective lens 200. If the imaging state characterization detector 320 is performing a first imaging, the position of the objective lens 200 is adjusted in real time along the optical axis of the fluorescence signal based on the first focusing distance; if the imaging state characterization detector 320 is performing a second imaging, the position of the objective lens 200 is adjusted in real time along the optical axis of the fluorescence signal based on the second focusing distance.

[0084] The microscopic imaging method provided in this application embodiment can image different measurement surfaces of the sequencing chip 600 by simply acquiring the imaging state to characterize the imaging of different surfaces, thereby controlling and adjusting the imaging distance between the detector 320 and the sleeve lens 310, and then adjusting the focusing distance between the objective lens 200 and different measurement surfaces to clearly image multi-surface samples. The method is simple, fast, and produces clear images.

[0085] It is understandable that the above example only shows single-channel imaging. In specific microscopic imaging processes, dual-channel or four-channel and other multi-channel imaging methods are commonly used, referring to... Figure 9 As shown, each channel can be used for multi-surface imaging using the microscopic imaging equipment and method of this invention.

[0086] This invention also provides a storage medium, which is a computer-readable storage medium storing a computer program that, when executed by a processor, implements the aforementioned microscopic imaging method. The memory, as a non-transitory computer-readable storage medium, can be used to store non-transitory software programs and non-transitory computer-executable programs. Furthermore, the memory may include high-speed random access memory and non-transitory memory, such as at least one disk storage device, flash memory device, or other non-transitory solid-state storage device. In some embodiments, the memory may optionally include memory remotely located relative to the processor, and these remote memories can be connected to the processor via a network. Examples of such networks include, but are not limited to, the Internet, corporate intranets, local area networks, mobile communication networks, and combinations thereof.

[0087] The microscopic imaging device, method, and storage medium provided in this invention achieve multi-surface microscopic imaging. Through an autofocus device 400, different preset focusing distances are determined based on the thickness of the coverslip 630 and the flow channel 610 during pre-tuning. This controls the working distance of the up-and-down moving objective lens 200 to switch between different measurement surfaces of the sequencing chip 600. Real-time fine-tuning maintains a constant focal height between the objective lens 200 and biological samples 620 with different heights. Then, the first displacement stage 330 adjusts the different imaging distances between the sleeve lens 310 and the detector 320. This method enables clear imaging of both the upper and lower surfaces of the biochip's flow channel without requiring the design of high-cost dedicated zoom lenses, the addition of new optical elements to the microscopic imaging device to correct aberrations caused by the liquid layer in the flow channel, or multiple focusing and imaging operations. Therefore, this invention is cost-effective, easy to operate, and maximizes the utilization of fluid reagents during gene sequencing without waste.

[0088] The device embodiments described above are merely illustrative. The units described as separate components may or may not be physically separate; that is, they may be located in one place or distributed across multiple network units. Some or all of the modules can be selected to achieve the purpose of this embodiment according to actual needs.

[0089] Those skilled in the art will understand that all or some of the steps and systems in the methods disclosed above can be implemented as software, firmware, hardware, and suitable combinations thereof. Some or all of the physical components can be implemented as software executed by a processor, such as a central processing unit, digital signal processor, or microprocessor, or as hardware, or as an integrated circuit, such as an application-specific integrated circuit. Such software can be distributed on a computer-readable medium, which can include computer storage media (or non-transitory media) and communication media (or transient media). As is known to those skilled in the art, the term computer storage media includes volatile and non-volatile, removable and non-removable media implemented in any method or technology for storing information (such as computer-readable instructions, data structures, program modules, or other data). Computer storage media includes, but is not limited to, RAM, ROM, EEPROM, flash memory or other memory technologies, CD-ROM, digital versatile disc (DVD) or other optical disc storage, magnetic cartridges, magnetic tape, storage device storage or other magnetic storage devices, or any other medium that can be used to store desired information and is accessible to a computer. Furthermore, as is known to those skilled in the art, communication media typically include computer-readable instructions, data structures, program modules, or other data in modulated data signals such as carrier waves or other transmission mechanisms, and may include any information delivery medium.

[0090] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. It should also be understood that the various implementation methods provided by the embodiments of the present invention can be arbitrarily combined to achieve different technical effects.

Claims

1. A microscopic imaging device for imaging fluorescent signals of a sequencing chip, the sequencing chip comprising a first measurement surface and a second measurement surface, characterized in that, include: An excitation module is used to generate an excitation beam so that the sequencing chip is excited by the excitation beam to produce a fluorescence signal; An objective lens, positioned on the optical axis of the fluorescence signal, is used to acquire the fluorescence signal; At least one imaging unit is configured to perform fluorescence imaging on the sequencing chip based on the fluorescence signal. The fluorescence imaging includes: a first imaging or a second imaging, wherein the first imaging is configured to image a first measurement surface of the sequencing chip to obtain a first imaging image, and the second imaging is configured to image a second measurement surface of the sequencing chip to obtain a second imaging image. The imaging unit includes: A sleeve lens is used to converge the fluorescence signal emitted from the objective lens to form the target light signal; The detector is positioned behind the sleeve lens along the optical axis of the target optical signal; When the detector is used to perform a first imaging based on the target light signal, the distance between the detector and the sleeve lens is the first imaging distance; When the detector is used to perform a second imaging based on the target light signal, the distance between the detector and the sleeve lens is the second imaging distance, and the first imaging distance is different from the second imaging distance. The imaging unit further includes: a first displacement stage, which is connected to the detector or the sleeve lens. The first displacement stage is used to control the detector to move along the optical axis of the target light signal, or to control the sleeve lens to move along the optical axis of the target light signal, so that the distance between the sleeve lens and the detector is the first imaging distance or the second imaging distance. The first imaging distance and the second imaging distance are determined by adjusting the cover glass and the flow channel with a preset thickness; the coordinate values ​​of the first imaging distance and the second imaging distance are pre-written into the control system, and the first displacement stage is used to control the detector or the sleeve lens to move to the first imaging distance or the second imaging distance according to the coordinate values, so as to correct the aberration caused by the liquid layer in the flow channel, so that the imaging quality of the first measurement surface and the second measurement surface is close to the diffraction limit. It also includes: a second displacement stage and an autofocus device, the autofocus device being connected to the objective lens or the second displacement stage, used to move the objective lens or the second displacement stage along the optical axis of the fluorescence signal during the first imaging of the detector, so that the first measuring surface is located at the focal position of the objective lens; or used to move the objective lens or the second displacement stage along the optical axis of the fluorescence signal during the second imaging of the detector, so that the second measuring surface is located at the focal position of the objective lens; the autofocus device is used to position the measuring surface at the focal position of the objective lens, and the first displacement stage is used to adjust the relative distance between the sleeve lens and the detector to correct the aberration.

2. The microimaging device of claim 1, wherein, The autofocus device is used to obtain the first focusing distance between the first measuring surface and the objective lens or the second focusing distance between the second measuring surface and the objective lens. It is also used to adjust the position of the objective lens or the second displacement stage in real time along the optical axis of the fluorescence signal according to the first focusing distance when the detector performs the first imaging. It is also used to adjust the position of the objective lens or the second displacement stage in real time along the optical axis of the fluorescence signal according to the second focusing distance when the detector performs the second imaging.

3. The microscopic imaging device according to claim 1, characterized in that, Also includes: An optical element located between the objective lens and the sleeve lens, the optical element comprising one or more of a dichroic mirror, a filter, or a reflector.

4. A microscopic imaging method, applied to the microscopic imaging device as described in claim 1, characterized in that, include: Acquire imaging state, which is used to characterize whether a first imaging is performed on a first measurement surface of the sequencing chip or a second imaging is performed on a second measurement surface of the sequencing chip; The distance between the detector and the sleeve lens is adjusted to either a first imaging distance or a second imaging distance based on the imaging state. The detector is used to perform the first imaging to obtain the first imaging image, or to perform the second imaging to obtain the second imaging image.

5. The microscopic imaging method according to claim 4, characterized in that, The acquisition of the imaging state also includes: If the imaging state indicates that the detector is performing the first imaging, the objective lens is moved along the optical axis of the fluorescence signal so that the first measuring surface is located at the focal position of the objective lens; If the imaging state indicates that the detector is performing a second imaging, the objective lens is moved along the optical axis of the fluorescence signal so that the second measuring surface is located at the focal point of the objective lens.

6. The microscopic imaging method according to claim 5, characterized in that, Also includes: Obtain the first focusing distance between the first measuring surface and the objective lens or the second focusing distance between the second measuring surface and the objective lens; If the imaging state indicates that the detector is performing the first imaging, the position of the objective lens is adjusted in real time along the optical axis of the fluorescence signal according to the first focusing distance; If the imaging state indicates that the detector is performing a second imaging, the position of the objective lens is adjusted in real time along the optical axis of the fluorescence signal according to the second focusing distance.

7. The microscopic imaging method according to claim 4, characterized in that, The step of adjusting the distance between the detector and the sleeve lens to a first imaging distance or a second imaging distance based on the imaging state further includes: The detector is controlled to move along the optical axis of the target light signal, or the sleeve lens is controlled to move along the optical axis of the target light signal, so that the distance between the detector and the sleeve lens is the first imaging distance or the second imaging distance.

8. A computer-readable storage medium storing computer-executable instructions for performing the microscopic imaging method according to any one of claims 4 to 7.

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