Focusing method, microscopic system and computer readable storage medium

Abstract

The invention discloses a focusing method, a microscopic system and a computer readable storage medium. The microscope system comprises an objective lens and an imaging assembly for imaging a target surface, and the focusing method comprises the steps that the microscope system is controlled to execute coarse focusing operation, so that a focusing position is located at a coarse focusing position, and the focusing position is a relative position between the objective lens and the target surface; the objective lens and / or the target surface are / is controlled to move multiple times at the position near the coarse focusing position, and after each time of movement, a new focusing position and a definition score of an image shot by the imaging assembly for the target surface corresponding to the new focusing position are obtained; a definition mapping curve is determined according to the multiple focusing positions and the multiple definition scores, then the objective lens and / or the target surface are / is moved so that the focusing positions can be located at the fine focusing positions, then the imaging assembly obtains a clear image of the target surface, and the definition scores at the fine focusing positions are determined according to the wave crests of the definition mapping curve. The focusing method is beneficial to improving the observation efficiency.

Description

Technical Field

[0001] The present invention relates to the field of focusing technology, and in particular to a focusing method, a microscope system and a computer-readable storage medium. Background Art

[0002] In the related art, the focusing method of the microscope is mainly optical focusing. In the actual observation process, there may be a situation where the focal plane of the objective lens needs to be adjusted multiple times according to the observation requirements. Since the focusing time of optical focusing is long and there is a problem of inaccurate focusing, it may be necessary to focus again, which will greatly affect the observation efficiency. Summary of the invention

[0003] The present invention provides a focusing method, a microscope system and a computer-readable storage medium to solve at least one of the above-mentioned technical problems.

[0004] A focusing method of the present invention is used in a microscope system, wherein the microscope system includes an objective lens and an imaging component for imaging a target surface, and the focusing method includes:

[0005] Controlling the microscope system to perform a coarse focusing operation so that the focusing position is at a coarse focusing position, wherein the focusing position is a relative position between the objective lens and the target surface;

[0006] Controlling the microscope system to perform a fine focusing operation, including:

[0007] Controlling the objective lens and / or the target surface to move multiple times near the coarse focus position, and obtaining a new focus position and a clarity score of an image taken by the imaging component for the target surface corresponding to the new focus position after each movement;

[0008] A clarity mapping curve is determined based on the multiple focus positions and the multiple clarity scores, and then the objective lens and / or the target surface is moved so that the focus position is at a fine focus position, thereby enabling the imaging component to obtain a clear image of the target surface, and the clarity score at the fine focus position is determined based on the peak of the clarity mapping curve.

[0009] The above-mentioned focusing method obtains a new focusing position and a corresponding clarity score by moving the objective lens and / or the target surface, and then obtains multiple focusing positions and multiple clarity scores. The multiple clarity scores can be used to determine a clarity mapping curve. The focusing position corresponding to the peak of the clarity mapping curve can enable the imaging component to obtain the clearest image. The focusing operation of the microscope system is controlled by the change of the clarity score, thereby achieving an automatic focusing effect, making the focusing more accurate and faster, which is conducive to improving the observation efficiency.

[0010] In an optional technical solution of the present invention, the step of controlling the microscope system to perform a coarse focus operation so that the focus position is at a coarse focus position comprises:

[0011] A first focusing light is output to the objective lens along the optical axis of the objective lens, the first focusing light can be focused into a point on the focal plane of the objective lens, in the process of the target surface moving from between the objective lens and the focal plane to the focal plane along the optical axis, the first focusing light forms an image on a first area on the target surface and the image size gradually decreases, in the process of the target surface moving from the focal plane to a direction away from the objective lens along the optical axis, the first focusing light forms an image on a second area on the target surface and the image size gradually increases, and the first area and the second area are axially symmetrical on the target surface;

[0012] According to the imaging change of the first focusing light on the target surface during the movement of the target surface, the position of the target surface when the imaging of the first focusing light is a point is determined as the position of the focal plane, and the objective lens and / or the target surface are moved so that the target surface is at the position of the focal plane.

[0013] In an optional technical solution of the present invention, the step of controlling the microscope system to perform a coarse focus operation so that the focus position is at a coarse focus position comprises:

[0014] The microscope system is controlled to image the target surface, and when the image of the target surface is not obtained, the objective lens and / or the target surface is moved with a first set step length, and then the target surface is imaged again until the image of the target surface can be obtained.

[0015] In an optional technical solution of the present invention, the step of controlling the objective lens and / or the target surface to move multiple times near the coarse focus position, and obtaining a new focus position and a clarity score of an image taken by the imaging component for the target surface corresponding to the new focus position after each movement comprises:

[0016] The objective lens and / or the target surface are controlled to move from the coarse focus position to a first set distance, and then the objective lens and / or the target surface are controlled to move multiple times in the opposite direction according to a second set step length.

[0017] In an optional technical solution of the present invention, the second setting step length is greater than the depth of field of the objective lens.

[0018] In an optional technical solution of the present invention, the microscope system includes an automatic focusing component, and the focusing method includes:

[0019] Controlling the auto-focusing assembly to send a second focusing light to the target surface along the optical axis of the objective lens, and receiving the second focusing light reflected from the target surface, and then outputting an electrical signal according to the received second focusing light, wherein the electrical signal is related to the focusing position;

[0020] The step of controlling the objective lens and / or the target surface to move multiple times near the coarse focus position and obtaining a new focus position and a clarity score of an image taken by the imaging component for the target surface corresponding to the new focus position after each movement comprises:

[0021] The objective lens and / or the target surface are controlled to move according to the electrical signal.

[0022] In an optional technical solution of the present invention, the step of determining a clarity mapping curve according to the plurality of focus positions and the plurality of clarity scores, and then moving the objective lens and / or the target surface so that the focus position is at a fine focus position comprises:

[0023] Performing fitting processing on the plurality of focus positions and the plurality of clarity scores to obtain the clarity mapping curve;

[0024] The objective lens and / or the target surface are controlled to move so that the focus position is at a fine focus position.

[0025] In an optional technical solution of the present invention, the step of fitting the change trends of the plurality of focus positions and the plurality of clarity scores to obtain the clarity mapping curve comprises:

[0026] When the clarity mapping curve meets a set condition, it is determined to control the microscope system to re-execute the coarse focus operation and / or the fine focus operation.

[0027] In an optional technical solution of the present invention, the setting condition includes at least one of the following:

[0028] When the number of the plurality of clarity scores obtained is greater than a set number, a peak of the clarity mapping curve is not fitted;

[0029] When the objective lens and / or the target surface has moved to the maximum scanning distance, the peak of the clarity mapping curve is not fitted;

[0030] The plurality of clarity scores obtained in sequence have a tendency to gradually decrease;

[0031] The fluctuation amplitude of the clarity mapping curve is smaller than the set amplitude value, and the maximum value of the clarity score is smaller than the set score value;

[0032] The clarity mapping curve has a plurality of peaks.

[0033] In an optional technical solution of the present invention, the step of performing fitting processing on the plurality of focus positions and the plurality of clarity scores to obtain the clarity mapping curve comprises:

[0034] A first fitting straight line is obtained by performing a first-order straight line fitting on a portion of the multiple clarity scores showing an upward trend, and a second fitting straight line is obtained by performing a first-order straight line fitting on a portion of the multiple clarity scores showing a downward trend, and the intersection of the first fitting straight line and the second fitting straight line is determined as the peak of the clarity mapping curve.

[0035] In an optional technical solution of the present invention, the step of performing fitting processing on the plurality of focus positions and the plurality of clarity scores to obtain the clarity mapping curve comprises:

[0036] A second-order parabola fitting is performed on the multiple clarity scores to obtain a first fitting parabola, and the vertex of the first fitting parabola is determined as the peak of the clarity mapping curve.

[0037] In an optional technical solution of the present invention, the step of performing fitting processing on the plurality of focus positions and the plurality of clarity scores to obtain the clarity mapping curve comprises:

[0038] Acquire a plurality of fine focus combinations, wherein the fine focus combinations include a plurality of clarity scores obtained by moving the objective lens and / or the target surface by corresponding step lengths, and each of the fine focus combinations has a corresponding coarse focus position and a same imaging position;

[0039] Taking the real best focal plane of the objective lens as the objective function, performing machine learning according to the multiple fine focus combinations to determine the weights of the multiple clarity scores in the clarity mapping curve, the position of the real best focal plane of the objective lens corresponding to the peak of the clarity mapping curve;

[0040] After the machine learning is completed, the plurality of clarity scores are used as input to output the peaks of the clarity mapping curve.

[0041] In an optional technical solution of the present invention, the focusing method includes:

[0042] The sharpness value of the image obtained by imaging is used as the clarity score.

[0043] In an optional technical solution of the present invention, the focusing method includes:

[0044] The bright spots in the image obtained by imaging are evaluated to determine the clarity score.

[0045] In an optional technical solution of the present invention, the evaluation process of the bright spot in the image obtained by imaging can be implemented by the following formula:

[0046] Score=[(k1*k2-1)*CV-EV] / [(CV+EV) / (k1*k2)];

[0047] Among them, Score represents the clarity score, k1*k2 represents the part of the matrix composed of odd rows and odd columns in the pixel array of the image within the range of the bright spot, CV represents the central pixel value of the matrix corresponding to the bright spot, and EV represents the sum of the non-central pixel values ​​of the matrix corresponding to the bright spot.

[0048] In an optional technical solution of the present invention, the focusing method is applied in a sequencing-by-synthesis process, and the focusing method includes:

[0049] During the first round of base extension reaction, controlling the microscope system to sequentially perform the coarse focusing operation once and the fine focusing operation once;

[0050] During each round of base extension reaction after the first round of base extension reaction, the microscope system is controlled to perform the fine focusing operation once.

[0051] In an optional technical solution of the present invention, the step of controlling the microscope system to perform the fine focusing operation once during each round of base extension reaction after the first round of base extension reaction comprises:

[0052] Controlling the objective lens and / or the target surface to move so that the focus position is at a fine focus position in a previous round of base extension reaction, and after the movement, determining a current clarity score as a first clarity score;

[0053] After controlling the objective lens and / or the target surface to move in the reverse direction at a third set step length from the focus position corresponding to the first clarity score, performing the fine focus operation to determine a new fine focus position, and determining the clarity score corresponding to the new fine focus position as a second clarity score;

[0054] When the first clarity score is less than the second clarity score, determining to use the current focus position as the fine focus position of the current round of base extension reaction;

[0055] In the case where the first sharpness score is greater than the second sharpness score, the objective lens and / or the target surface is moved back to a fine focus position corresponding to the first sharpness score.

[0056] In an optional technical solution of the present invention, the focusing method includes:

[0057] In the case that the focus position is not at the fine focus position in the previous round of base extension reaction, the microscope system is controlled to perform the coarse focus operation once, and after completion, the objective lens and / or the target surface is controlled to move to determine the second clarity score.

[0058] In an optional technical solution of the present invention, the microscope device includes a driving assembly, and the driving assembly is used to drive the objective lens and / or the target surface to move along the optical axis of the objective lens;

[0059] The focusing method comprises:

[0060] After determining the first clarity score, detecting whether a difference between a first voltage value and a second voltage value is less than or equal to a first set value, the first voltage value being a voltage value output when the drive component controls the objective lens and / or the target surface to move to a focus position corresponding to the first clarity score in the previous round of base extension reaction, and the second voltage value being a voltage value output when the drive component controls the objective lens and / or the target surface to move to a focus position corresponding to the first clarity score in the current round of base extension reaction;

[0061] When the difference is less than or equal to the first set value, controlling the objective lens and / or the target surface to move to determine the second clarity score;

[0062] In the case where the difference is greater than the first set value, the microscope system is controlled to perform the coarse focusing operation once, and after completion, the objective lens and / or the target surface is controlled to move to determine the second clarity score.

[0063] In an optional technical solution of the present invention, the step of controlling the objective lens and / or the target surface to move in the reverse direction at a third set step length from the focus position corresponding to the first clarity score, performing the fine focus operation to determine a new fine focus position, and determining the clarity score corresponding to the new fine focus position as the second clarity score comprises:

[0064] Acquiring a third voltage value, wherein the third voltage value is a voltage value output when the driving component controls the objective lens and / or the target surface to move to a position corresponding to the new fine focus position in the current round of base extension reaction;

[0065] When the third voltage value is less than the second set value, determining the clarity score corresponding to the new fine focus position as the second clarity score;

[0066] When the third voltage value is greater than or equal to the second set value, the drive component is subjected to voltage zeroing processing, and then the objective lens and / or the target surface is controlled to move in the reverse direction from the focusing position corresponding to the first clarity score at the third set step size, and then the fine focusing operation is re-executed until the third voltage value obtained is less than the second set value.

[0067] A microscopic system of the present invention comprises an objective lens, an imaging component for imaging a target surface, and a control module, wherein the control module is used to:

[0068] Controlling the microscope system to perform a coarse focusing operation so that the focusing position is at a coarse focusing position, wherein the focusing position is a relative position between the objective lens and the target surface;

[0069] Controlling the microscope system to perform a fine focusing operation, including:

[0070] Controlling the objective lens and / or the target surface to move multiple times near the coarse focus position, and obtaining a new focus position and a clarity score of an image taken by the imaging component for the target surface corresponding to the new focus position after each movement;

[0071] A clarity mapping curve is determined based on the multiple focus positions and the multiple clarity scores, and then the objective lens and / or the target surface is moved so that the focus position is at a fine focus position, thereby enabling the imaging component to obtain a clear image of the target surface, and the clarity score at the fine focus position is determined based on the peak of the clarity mapping curve.

[0072] The above-mentioned microscope system obtains a new focus position and a corresponding clarity score by moving the objective lens and / or the target surface, and then obtains multiple focus positions and multiple clarity scores. The multiple clarity scores can be used to determine a clarity mapping curve. The focus position corresponding to the peak of the clarity mapping curve can enable the imaging component to obtain the clearest image. The focus operation of the microscope system is controlled by the change of the clarity score, thereby achieving an automatic focus effect, making the focus more accurate and faster, which is conducive to improving the observation efficiency.

[0073] In an optional technical solution of the present invention, the control module is used to:

[0074] A first focusing light is output to the objective lens along the optical axis of the objective lens, the first focusing light can be focused into a point on the focal plane of the objective lens, in the process of the target surface moving from between the objective lens and the focal plane to the focal plane along the optical axis, the first focusing light forms an image on a first area on the target surface and the image size gradually decreases, in the process of the target surface moving from the focal plane to a direction away from the objective lens along the optical axis, the first focusing light forms an image on a second area on the target surface and the image size gradually increases, and the first area and the second area are axially symmetrical on the target surface;

[0075] According to the imaging change of the first focusing light on the target surface during the movement of the target surface, the position of the target surface when the imaging of the first focusing light is a point is determined as the position of the focal plane, and the objective lens and / or the target surface are moved so that the target surface is at the position of the focal plane.

[0076] In an optional technical solution of the present invention, the control module is used to:

[0077] The microscope system is controlled to image the target surface, and when the image of the target surface is not obtained, the objective lens and / or the target surface is moved with a first set step length, and then the target surface is imaged again until the image of the target surface can be obtained.

[0078] In an optional technical solution of the present invention, the control module is used to:

[0079] The objective lens and / or the target surface are controlled to move from the coarse focus position to a first set distance, and then the objective lens and / or the target surface are controlled to move multiple times in the opposite direction according to a second set step length.

[0080] In an optional technical solution of the present invention, the second setting step length is greater than the depth of field of the objective lens.

[0081] In an optional technical solution of the present invention, the microscope system includes an autofocus component, and the control module is used to:

[0082] Controlling the auto-focusing assembly to send a second focusing light to the target surface along the optical axis of the objective lens, and receiving the second focusing light reflected from the target surface, and then outputting an electrical signal according to the received second focusing light, wherein the electrical signal is related to the focusing position;

[0083] The objective lens and / or the target surface are controlled to move according to the electrical signal.

[0084] In an optional technical solution of the present invention, the control module is used to:

[0085] Performing fitting processing on the plurality of focus positions and the plurality of clarity scores to obtain the clarity mapping curve;

[0086] The objective lens and / or the target surface are controlled to move so that the focus position is at a fine focus position.

[0087] In an optional technical solution of the present invention, the control module is used to:

[0088] When the clarity mapping curve meets a set condition, it is determined to control the microscope system to re-execute the coarse focus operation and / or the fine focus operation.

[0089] In an optional technical solution of the present invention, the setting condition includes at least one of the following:

[0090] When the number of the plurality of clarity scores obtained is greater than a set number, a peak of the clarity mapping curve is not fitted;

[0091] When the objective lens and / or the target surface has moved to the maximum scanning distance, the peak of the clarity mapping curve is not fitted;

[0092] The plurality of clarity scores obtained in sequence have a tendency to gradually decrease;

[0093] The fluctuation amplitude of the clarity mapping curve is smaller than the set amplitude value, and the maximum value of the clarity score is smaller than the set score value;

[0094] The clarity mapping curve has a plurality of peaks.

[0095] In an optional technical solution of the present invention, the control module is used to:

[0096] A first fitting straight line is obtained by performing a first-order straight line fitting on a portion of the multiple clarity scores showing an upward trend, and a second fitting straight line is obtained by performing a first-order straight line fitting on a portion of the multiple clarity scores showing a downward trend, and the intersection of the first fitting straight line and the second fitting straight line is determined as the peak of the clarity mapping curve.

[0097] In an optional technical solution of the present invention, the control module is used to:

[0098] A second-order parabola fitting is performed on the multiple clarity scores to obtain a first fitting parabola, and the vertex of the first fitting parabola is determined as the peak of the clarity mapping curve.

[0099] In an optional technical solution of the present invention, the control module is used to:

[0100] Acquire a plurality of fine focus combinations, wherein the fine focus combinations include a plurality of clarity scores obtained by moving the objective lens and / or the target surface by corresponding step lengths, and each of the fine focus combinations has a corresponding coarse focus position and a same imaging position;

[0101] Taking the real best focal plane of the objective lens as the objective function, performing machine learning according to the multiple fine focus combinations to determine the weights of the multiple clarity scores in the clarity mapping curve, the position of the real best focal plane of the objective lens corresponding to the peak of the clarity mapping curve;

[0102] After the machine learning is completed, the plurality of clarity scores are used as input to output the peaks of the clarity mapping curve.

[0103] In an optional technical solution of the present invention, the control module is used to:

[0104] The sharpness value of the image obtained by imaging is used as the clarity score.

[0105] In an optional technical solution of the present invention, the control module is used to:

[0106] The bright spots in the image obtained by imaging are evaluated to determine the clarity score.

[0107] In an optional technical solution of the present invention, the evaluation process of the bright spot in the image obtained by imaging can be implemented by the following formula:

[0108] Score=[(k1*k2-1)*CV-EV] / [(CV+EV) / (k1*k2)];

[0109] Among them, Score represents the clarity score, k1*k2 represents the part of the matrix composed of odd rows and odd columns in the pixel array of the image within the range of the bright spot, CV represents the central pixel value of the matrix corresponding to the bright spot, and EV represents the sum of the non-central pixel values ​​of the matrix corresponding to the bright spot.

[0110] In an optional technical solution of the present invention, the microscopic system is applied in a sequencing-by-synthesis process, and the control module is used to:

[0111] During the first round of base extension reaction, controlling the microscope system to sequentially perform the coarse focusing operation once and the fine focusing operation once;

[0112] During each round of base extension reaction after the first round of base extension reaction, the microscope system is controlled to perform the fine focusing operation once.

[0113] In an optional technical solution of the present invention, the control module is used to:

[0114] Controlling the objective lens and / or the target surface to move so that the focus position is at a fine focus position in a previous round of base extension reaction, and after the movement, determining a current clarity score as a first clarity score;

[0115] After controlling the objective lens and / or the target surface to move in the reverse direction at a third set step length from the focus position corresponding to the first clarity score, performing the fine focus operation to determine a new fine focus position, and determining the clarity score corresponding to the new fine focus position as a second clarity score;

[0116] When the first clarity score is less than the second clarity score, determining to use the current focus position as the fine focus position of the current round of base extension reaction;

[0117] In the case where the first sharpness score is greater than the second sharpness score, the objective lens and / or the target surface is moved back to a fine focus position corresponding to the first sharpness score.

[0118] In an optional technical solution of the present invention, the control module is used to:

[0119] In the case that the focus position is not at the fine focus position in the previous round of base extension reaction, the microscope system is controlled to perform the coarse focus operation once, and after completion, the objective lens and / or the target surface is controlled to move to determine the second clarity score.

[0120] In an optional technical solution of the present invention, the microscope device includes a driving assembly, and the driving assembly is used to drive the objective lens and / or the target surface to move along the optical axis of the objective lens;

[0121] The control module is used for:

[0122] After determining the first clarity score, detecting whether a difference between a first voltage value and a second voltage value is less than or equal to a first set value, the first voltage value being a voltage value output when the drive component controls the objective lens and / or the target surface to move to a focus position corresponding to the first clarity score in the previous round of base extension reaction, and the second voltage value being a voltage value output when the drive component controls the objective lens and / or the target surface to move to a focus position corresponding to the first clarity score in the current round of base extension reaction;

[0123] When the difference is less than or equal to the first set value, controlling the objective lens and / or the target surface to move to determine the second clarity score;

[0124] In the case where the difference is greater than the first set value, the microscope system is controlled to perform the coarse focusing operation once, and after completion, the objective lens and / or the target surface is controlled to move to determine the second clarity score.

[0125] In an optional technical solution of the present invention, the control module is used to:

[0126] Acquiring a third voltage value, wherein the third voltage value is a voltage value output when the driving component controls the objective lens and / or the target surface to move to a position corresponding to the new fine focus position in the current round of base extension reaction;

[0127] When the third voltage value is less than the second set value, determining the clarity score corresponding to the new fine focus position as the second clarity score;

[0128] When the third voltage value is greater than or equal to the second set value, the drive component is subjected to voltage zeroing processing, and then the objective lens and / or the target surface is controlled to move in the reverse direction from the focusing position corresponding to the first clarity score at the third set step size, and then the fine focusing operation is re-executed until the third voltage value obtained is less than the second set value.

[0129] A microscope system of the present invention comprises a memory and a processor, wherein the memory stores a computer program, and when the processor executes the computer program, the steps of the focusing method of the present invention are implemented.

[0130] The above-mentioned microscope system obtains a new focus position and a corresponding clarity score by moving the objective lens and / or the target surface, and then obtains multiple focus positions and multiple clarity scores. The multiple clarity scores can be used to determine a clarity mapping curve. The focus position corresponding to the peak of the clarity mapping curve can enable the imaging component to obtain the clearest image. The focus operation of the microscope system is controlled by the change of the clarity score, thereby achieving an automatic focus effect, making the focus more accurate and faster, which is conducive to improving the observation efficiency.

[0131] A computer-readable storage medium of the present invention stores a computer program, which, when executed by a processor, implements the steps of the focusing method of the present invention.

[0132] The above-mentioned computer-readable storage medium obtains a new focus position and a corresponding clarity score by moving the objective lens and / or the target surface, and then obtains multiple focus positions and multiple clarity scores. The multiple clarity scores can be used to determine a clarity mapping curve. The focus position corresponding to the peak of the clarity mapping curve can enable the imaging component to obtain the clearest image. The focus operation of the microscope system is controlled by the change of the clarity score, thereby achieving an automatic focusing effect, making the focus more accurate and faster, which is conducive to improving the observation efficiency.

[0133] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0134] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0135] Figure 1 Schematic diagram of the module structure of the microscope system according to the embodiment of the present invention;

[0136] Figure 2 is a schematic flow chart of a focusing method according to an embodiment of the present invention;

[0137] Figure 3 is another schematic diagram of the module structure of the microscope system according to an embodiment of the present invention;

[0138] Figure 4 is a schematic diagram of a clarity mapping curve according to an embodiment of the present invention;

[0139] Figure 5 is a schematic diagram of a coarse focusing operation according to an embodiment of the present invention;

[0140] Figure 6 is another schematic flow chart of a focusing method according to an embodiment of the present invention;

[0141] Figure 7 is another schematic flow chart of a focusing method according to an embodiment of the present invention;

[0142] Figure 8 is a schematic diagram of performing first-order straight line fitting on a clarity mapping curve according to an embodiment of the present invention;

[0143] Fig. 9 is a schematic diagram of performing second-order parabola fitting on a clarity mapping curve according to an embodiment of the present invention;

[0144] Fig.10 is another schematic flow chart of the focusing method according to an embodiment of the present invention;

[0145] Fig.11 is another schematic flow chart of the focusing method according to an embodiment of the present invention;

[0146] Fig.12 is a schematic diagram of the structure of a sequencing chip according to an embodiment of the present invention;

[0147] Fig.13 is another schematic flow chart of the focusing method according to an embodiment of the present invention;

[0148] Fig.14 It is a schematic diagram of the process of the focusing method in the process of sequencing by synthesis according to an embodiment of the present invention;

[0149] Fig.15 It is another schematic diagram of the module structure of the microscope system according to the embodiment of the present invention.

[0150] Description of main component symbols:

[0151] Microscope system 1000;

[0152] Optical imaging system 100, objective lens 110, imaging assembly 120, first dichroic lens 121, first camera 122, second camera 123, second dichroic lens 124, first reflective lens 125, tube lens 126, first filter 127, second filter 128;

[0153] Target surface 101, sequencing chip 102, first glass plate 103, second glass plate 104;

[0154] Auto focus assembly 200, focus sensor 201, third reflector 202, control module 210;

[0155] A drive assembly 300;

[0156] Laser lighting assembly 400, laser 410, collimator 420, converging lens 430, second reflective lens 440, first lens 450, third dichroic lens 460;

[0157] Memory 510 and processor 520 . DETAILED DESCRIPTION

[0158] In the description of the present invention, some of the disclosed contents have been shown in the accompanying drawings accordingly, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The contents described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0159] In the description of the present invention, many different contents or examples are disclosed to realize different structures of the present invention. In order to simplify the disclosure of the present invention, the parts and settings of specific examples are described below. Of course, they are only examples, and the purpose is not to limit the present invention.

[0160] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0161] Specifically, please refer to Figure 1 In the present invention, the microscope system 1000 may include an optical imaging system 100. The optical imaging system 100 may include an objective lens 110 and an imaging assembly 120. The imaging assembly 120 may include a first dichroic lens 121, a first camera 122, a second camera 123, a second dichroic lens 124, a first reflective lens 125, and a tube lens 126. When the light is reflected from the target surface 101, the light may be first transmitted and reflected on the second dichroic lens 124, wherein the light transmitted from the second dichroic lens 124 may first be reflected on the first reflective lens 125, and then irradiate the first dichroic lens 121 via the tube lens 126. Similarly, the light irradiated on the first dichroic lens 121 may be transmitted and reflected on the first dichroic lens 121, so that the light with the first wavelength in the light is transmitted on the first dichroic lens 121 and enters the first camera 122 to form an image, and the light with the second wavelength in the light is reflected on the first dichroic lens 121 and enters the second camera 123 to form an image. exist Figure 1 In the embodiment, the microscope system 1000 may further include a first filter 127 and a second filter 128. The first filter 127 is disposed between the first dichroic lens 121 and the first camera 122, so as to filter the light transmitted through the first dichroic lens 121. The second filter 128 is disposed between the first dichroic lens 121 and the second camera 123, so as to filter the light reflected from the first dichroic lens 121.

[0162] In addition, Figure 1In the embodiment, the microscope system 1000 may include a laser illumination assembly 400. The laser illumination assembly 400 may include a laser 410, a collimator 420, a converging lens 430, a second reflective lens 440, a first lens 450, and a third dichroic lens 460. The laser 410 may emit a laser, which is collimated and expanded by the collimator 420, and then converged by the converging lens 430, and then reflected by the second reflective lens 440 and irradiated into the first lens 450. After the laser is emitted from the first lens 450, it is reflected by the third dichroic lens 460 and the second dichroic lens 124 in sequence, and finally irradiated on the target surface 101 through the objective lens 110. In some cases, the first lens 450 may be an EX lens.

[0163] Please refer to Figure 1 A focusing method of the present invention can be used in a microscope system 1000. The microscope system 1000 may include an objective lens 110 and an imaging component 120 for imaging a target surface 101. Figure 2 , focusing methods can include:

[0164] 01: Control the microscope system 1000 to perform a coarse focusing operation, so that the focus position is at a coarse focus position, where the focus position is a relative position between the objective lens 110 and the target surface 101;

[0165] 02: Control the microscope system 1000 to perform fine focusing operations, including:

[0166] 021: Control the objective lens 110 and / or the target surface 101 to move multiple times near the rough focus position, and after each movement, obtain a new focus position and a clarity score of an image taken by the imaging component 120 for the target surface 101 corresponding to the new focus position;

[0167] 022: Determine a clarity mapping curve based on multiple focus positions and multiple clarity scores, then move the objective lens 110 and / or the target surface 101 so that the focus position is at a fine focus position, so that the imaging component 120 obtains a clear image of the target surface 101, and the clarity score at the fine focus position is determined according to the peak of the clarity mapping curve.

[0168] It should be noted that, in some scenarios in which the present invention is applied, after the microscopic system 1000 is subjected to a fine focus operation so that the focus position is at the fine focus position, the fine focus position may be further offset by a certain distance. It is understood that, in practical applications, the hardware in the microscopic system 1000 may have mechanical structural limitations. In the process of performing the fine focus operation, such limitations may affect the movement of the objective lens 110 and / or the target surface 101, so that a deviation is formed between the focus position and the actual fine focus position. This deviation is the mechanical deviation, such as the mechanical return difference.

[0169] Based on the above, in some cases, step 022 may include:

[0170] After moving the objective lens 110 and / or the target surface 101 so that the clarity score at the focus position corresponds to the peak of the clarity mapping curve, the objective lens 110 and / or the target surface 101 are moved again by a distance, and the moved distance is related to the mechanical deviation of the microscope system 1000.

[0171] Based on the above, in some cases, step 022 may include:

[0172] The objective lens 110 and / or the target surface 101 are moved so that the focus position is at a fine focus position, and the sharpness score at the fine focus position is determined according to the peak of the sharpness mapping curve and the mechanical deviation of the microscope system 1000 .

[0173] The above situation can also be understood as, after performing the fine focus operation to make the focus position at the conceptual fine focus position, the focus position is further offset by a certain distance according to the mechanical deviation of the microscope system 1000, or when performing the fine focus operation to move the objective lens 110 and / or the target surface 101, the mechanical deviation is considered, and the movement is combined with the peak of the clarity mapping curve and the mechanical deviation, so that the focus position can reach the actual fine focus position. At present, in some cases, since the mechanical deviation has only a very small value, it can be considered that the influence caused by the mechanical deviation can be ignored.

[0174] The focusing method of the present invention can be implemented by the microscope system 1000 of the present invention. Figure 1 and Figure 3, the microscope system 1000 may further include a control module 210. The control module 210 may be used to: control the microscope system 1000 to perform a coarse focus operation so that the focus position is at a coarse focus position, which is a relative position between the objective lens 110 and the target surface 101; control the microscope system 1000 to perform a fine focus operation, including: controlling the objective lens 110 and / or the target surface 101 to move multiple times near the coarse focus position, and after each movement, obtaining a new focus position and a clarity score of an image taken by the imaging component 120 for the target surface 101 corresponding to the new focus position; determining a clarity mapping curve according to multiple focus positions and multiple clarity scores, and then moving the objective lens 110 and / or the target surface 101 so that the focus position is at a fine focus position, thereby allowing the imaging component 120 to obtain a clear image of the target surface 101, and the clarity score at the fine focus position is determined according to the peak of the clarity mapping curve.

[0175] The above-mentioned focusing method and microscope system 1000 obtain a new focusing position and a corresponding clarity score by moving the objective lens 110 and / or the target surface 101, and then obtain multiple focusing positions and multiple clarity scores. The multiple clarity scores can be used to determine a clarity mapping curve. The focusing position corresponding to the peak of the clarity mapping curve can enable the imaging component 120 to obtain the clearest image, so that the focusing operation of the microscope system 1000 can be controlled by the change of the clarity score, thereby achieving an automatic focusing effect, making the focusing more accurate and faster, which is conducive to improving the observation efficiency.

[0176] In the present invention, in some cases, the coarse focusing operation can be understood as adjusting at least one of the objective lens 110 and the target surface 101 so that the target surface 101 is quite close to the position where the focal plane of the objective lens 110 is located. At this time, the imaging component 120 can image the target surface 101 to obtain a relatively clear image; the fine focusing operation can be understood as further adjusting at least one of the objective lens 110 and the target surface 101 on the basis of the coarse focusing operation so that the target surface 101 is basically located at the position where the focal plane of the objective lens 110 is located. At this time, the imaging component 120 can image the target surface 101 to obtain a relatively clear image.

[0177] The objective lens 110 may have an optical axis. Figure 1 ,exist Figure 1In the figure, the optical axis of the objective lens 110 is represented as L. When the objective lens 110 and the target surface 101 are in a certain or determinable relative position, a distance of corresponding size is formed between the objective lens 110 and the target surface 101 along the optical axis. In this case, although the objective lens 110 or the target surface 101 can be moved to any position on the optical axis, the relative position remains unchanged, so that the distance formed between the objective lens 110 and the target surface 101 along the optical axis remains constant or substantially unchanged, so that the image obtained by the objective lens 110 imaging the target surface 101 also has a certain or substantially unchanged clarity.

[0178] In the present invention, in some cases, when the relative position between the objective lens 110 and the target surface 101 is in a coarse focus position, the distance formed between the objective lens 110 and the target surface 101 along the optical axis direction of the objective lens 110 will be smaller than the first distance; when the relative position between the objective lens 110 and the target surface 101 is in a fine focus position, the distance formed between the objective lens 110 and the target surface 101 along the optical axis direction of the objective lens 110 will be smaller than the second distance, and the second distance is smaller than the first distance.

[0179] Please combine Figure 1 and Figure 4 ,exist Figure 4 In some cases, when the microscope system 1000 is controlled to perform a coarse focus operation, at least one of the objective lens 110 and the target surface 101 may be moved along the optical axis L, so that the target surface 101 is located within the Z0 interval on the optical axis L, that is, the focus position is at the coarse focus position. Then, when the microscope system 1000 is controlled to perform a fine focus operation, at least one of the objective lens 110 and the target surface 101 may be controlled to move, so that the target surface 101 takes the position within the Z0 interval as the starting position and moves multiple times along the optical axis L relative to the objective lens 110, and then the target surface 101 moves to the coarse focus position. Figure 4 In , the positions where the target surface 101 can stay after multiple movements can be represented as Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, and Z9, respectively. In other words, Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, and Z9 can all be used as new focus positions. It can be understood that different images can be captured at these new focus positions, and because the distance between the objective lens 110 and the target surface 101 has changed, the different images captured will also have corresponding different clarity scores. By obtaining multiple focus positions and multiple clarity scores, the clarity mapping curve can be determined through processing. Figure 4 In , the clarity mapping curve is represented as X.

[0180] Since the smaller the distance between the target surface 101 and the focal plane of the objective lens 110 is, the clearer the captured image will be. Figure 4 It is easy to know that after determining the clarity mapping curve, the peak of the clarity mapping curve can be determined as the position of the focal plane of the objective lens 110, so that the position corresponding to the peak of the clarity mapping curve on the optical axis L can be used as the fine focus position, and at least one of the objective lens 110 or the target surface 101 can be controlled to move, so that the target surface 101 can eventually be in the fine focus position, thereby completing the focusing of the target surface 101 through the fine focus operation.

[0181] In the present invention, step 01 (controlling the microscope system 1000 to perform a coarse focus operation so that the focus position is at a coarse focus position) may include:

[0182] A first focusing light is outputted from the objective lens 110 along the optical axis of the objective lens 110, and the first focusing light can be focused into a point on the focal plane of the objective lens 110. When the target surface 101 moves from between the objective lens 110 and the focal plane to the focal plane along the optical axis, the first focusing light forms an image on a first area on the target surface 101, and the image size gradually decreases. When the target surface 101 moves from the focal plane to a direction away from the objective lens 110 along the optical axis, the first focusing light forms an image on a second area on the target surface 101, and the image size gradually increases. The first area and the second area are axially symmetrical on the target surface 101.

[0183] According to the imaging change of the first focusing light on the target surface 101 during the movement of the target surface 101, the position of the target surface 101 when the imaging of the first focusing light is a point is determined as the focal plane position, and the objective lens 110 and / or the target surface 101 is moved so that the target surface 101 is at the focal plane position.

[0184] The focusing method of the present invention can be implemented by the microscope system 1000 of the present invention. Figure 3The control module 210 can be used for: outputting a first focusing light to the objective lens 110 along the optical axis of the objective lens 110, the first focusing light can be focused into a point on the focal plane of the objective lens 110, when the target surface 101 moves along the optical axis from between the objective lens 110 and the focal plane to the focal plane, the first focusing light forms an image in a first area on the target surface 101 and the image size gradually decreases, when the target surface 101 moves along the optical axis from the focal plane to a direction away from the objective lens 110, the first focusing light forms an image in a second area on the target surface 101 and the image size gradually increases, and the first area and the second area are axially symmetrical on the target surface 101; according to the imaging change of the first focusing light on the target surface 101 during the movement of the target surface 101, the position of the target surface 101 when the imaging of the first focusing light is a point is determined as the position of the focal plane, and the objective lens 110 and / or the target surface 101 are moved so that the target surface 101 is at the position of the focal plane.

[0185] In this way, the coarse focusing efficiency of the microscope system 1000 can be improved.

[0186] Specifically, please refer to Figure 1 , the microscope system 1000 may include an autofocus assembly 200. The autofocus assembly 200 may include a first light source (not shown) and a focus sensor 201. The first light source is used to provide a first focus light, such as an infrared light of 850 nm, and the objective lens 110 is used to project the first focus light to the target surface 101 and collect the first focus light reflected from the target surface 101. The focus sensor 201 may receive the first focus light reflected from the target surface 101 collected by the objective lens 110 to obtain the first focus light information. In some cases, the focus sensor 201 may include a CCD (Charge-coupled Device) array, and the focus sensor 201 may detect the shape and size of the light spot formed by the first focus light through the CCD array. According to the shape and size of the light spot, it can be determined whether the target surface 101 is located on the focal plane of the objective lens 110, thereby achieving a coarse focus operation.

[0187] The focus sensor 201 and the first light source can be in different positions. In some cases, the focus sensor 201 and the first light source can be respectively located at different positions on the same horizontal plane. At this time, the target surface 101, the focus sensor 201 and the first light source can form a triangle. According to the principle of triangulation, when the relative distance between the target surface 101 and the focus sensor 201 (usually the distance in the vertical direction) remains unchanged, the first focus light reflected from the target surface 101 will be imaged at a fixed position on the CCD array, and when the relative distance between the target surface 101 and the focus sensor 201 changes, the position of the image on the CCD array will also change. Therefore, the relative distance between the target surface 101 and the focus sensor 201 can be determined based on the change of the imaging position on the CCD array.

[0188] In some cases, the CCD array can output a corresponding voltage after performing photoelectric conversion on the received first focusing light. At this time, the reference voltage of the reference position on the CCD array can be set so that the voltage output by the photoelectric conversion of the CCD array corresponds to the relative distance between the target surface 101 and the focusing sensor 201, that is, when the relative distance between the target surface 101 and the focusing sensor 201 is fixed, the voltage output by the photoelectric conversion of the CCD array is also fixed. The first light source can be a built-in light source of the focusing sensor 201. The autofocus assembly 200 can also include a third reflector 202. The third reflector 202 can be used to reflect the first focusing light, thereby changing the propagation optical path of the first focusing light, and further enabling the focusing sensor 201 to adjust the installation position in the autofocus assembly 200 according to specific needs.

[0189] The first light source can emit first focusing light in multiple different directions, and all the emitted first focusing light can be focused into one point on the focal plane. Figure 5 , the first focused light may include light l1 and light l2. Light l1 and light l2 both pass through focal point d0. All first focused light rays may be emitted toward focal point d0, so that all first focused light rays gradually converge in the process of approaching focal point d0, then converge at focal point d0, and then gradually diffuse in the process of moving away from focal point d0. For the first light source, light l1 may be emitted from one end of the first light source in a direction perpendicular to the focal plane, and light l2 may be emitted from the other end of the first light source at a corresponding inclined angle. The first focused light may be a semicircular light beam when emitted from the first light source.

[0190] exist Figure 5 In the case where the plane where the focal plane is located coincides with plane d2, the focus d0 is also located on plane d2, and the image obtained by imaging plane d2 will be a light point, that is, Figure 5When imaging the plane d1 between the auto-focusing assembly 200 and the plane d2, since the light beam formed by the first focusing light between the plane d1 and the plane d2 is gradually converged, the imaging of the first focusing light on the plane d1 can be Figure 5 When imaging the plane d3 located on the other side of the plane d2, since the light beam formed by the first focusing light between the plane d2 and the plane d3 gradually diffuses, the imaging of the first focusing light on the plane d3 can be Figure 5 D3 shown in .

[0191] Wherein, when the first focusing light is a semicircular light beam when emitted from the first light source, Figure 5 In the figure, D1 and D3 are both semicircular, and the semicircular straight edges of D1 and D3 can be located on the straight line M, and D2 is also located on the straight line M. It can be understood that according to the characteristics of the first focusing light emitted by the first light source, the image of the first focusing light on the plane d1 and the image of the first focusing light on the plane d3 are respectively located on both sides of the straight line M, and the image of the first focusing light on the plane d2 converges to a point, that is, according to the shape of the image of the first focusing light, the positional relationship between the focal plane currently being imaged and the plane where the focus d0 is located can be determined, and then the direction and distance of the focal plane to be moved can be determined, so that when performing a coarse focusing operation, the coarse focusing efficiency can be improved.

[0192] In addition, in some cases, the positional relationship between the focal plane and the plane where the focus d0 is located can be determined according to the range and intensity of light sensitivity.

[0193] In the present invention, step 01 (controlling the microscope system 1000 to perform a coarse focus operation so that the focus position is at a coarse focus position) may include:

[0194] The microscope system 1000 is controlled to image the target surface 101, and when the image of the target surface 101 is not obtained, the objective lens 110 and / or the target surface 101 is moved with a first set step length, and then the target surface 101 is imaged again until the image of the target surface 101 can be obtained.

[0195] The focusing method of the present invention can be implemented by the microscope system 1000 of the present invention. Figure 3 The control module 210 can be used to: control the microscope system 1000 to image the target surface 101, and when the image of the target surface 101 is not obtained, move the objective lens 110 and / or the target surface 101 with a first set step size, and then image the target surface 101 until the image of the target surface 101 can be obtained.

[0196] In this way, the whole image focusing effect of the microscope system 1000 can be achieved.

[0197] Specifically, the entire target surface 101 can be imaged. After imaging is performed sequentially with a first set step size, the target surface 101 can form a relatively clear outline in one or several images, thereby determining that the position of the target surface 101 is close to the focal plane of the objective lens 110, that is, the focus position is at a coarse focus position.

[0198] In addition, in some cases, if the optical focus fails to achieve coarse focus, you can use the full-image focus method to achieve coarse focus. If both the optical focus and the full-image focus cannot achieve coarse focus, it means that the focus has failed.

[0199] Please refer to Figure 6 In the present invention, step 021 (controlling the objective lens 110 and / or the target surface 101 to move multiple times near the coarse focus position, and obtaining a new focus position and a clarity score of the image taken by the imaging component 120 for the target surface 101 corresponding to the new focus position after each movement) may include:

[0200] 0211: Control the objective lens 110 and / or the target surface 101 to move a first set distance from the coarse focus position, and then control the objective lens 110 and / or the target surface 101 to move multiple times in the opposite direction according to a second set step length.

[0201] The focusing method of the present invention can be implemented by the microscope system 1000 of the present invention. Figure 3 The control module 210 may be used to control the objective lens 110 and / or the target surface 101 to move a first set distance from a coarse focus position, and then control the objective lens 110 and / or the target surface 101 to move multiple times in the opposite direction according to a second set step length.

[0202] In some cases, step 0211 may be to control the objective lens 110 and / or the target surface 101 to move a first set distance in the positive direction of the Z axis, and then to move multiple times according to the second set step length starting from the negative direction of the Z axis. In other cases, step 0211 may be to control the objective lens 110 and / or the target surface 101 to move a first set distance in the negative direction of the Z axis, and then to move multiple times according to the second set step length starting from the positive direction of the Z axis. The specific movement mode may be selected according to the actual application scenario.

[0203] In this way, the focusing effect can be guaranteed.

[0204] Specifically, please combine Figure 4, the target surface 101 is basically within the range of Z0 on the optical axis L. When performing a fine focusing operation, the target surface 101 can move from its current position along the negative direction of the optical axis L to Z1, so that the target surface 101 moves a first set distance relative to the optical axis L, and then moves along the positive direction of the optical axis L with a second set step length, so that the target surface 101 stays at Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, and Z9 in sequence, that is, the distance between adjacent Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, and Z9 is the second set step length.

[0205] It can be understood that by executing step 0211, the change of image clarity in a larger range around Z0 can be obtained, so that the clarity mapping curve can characterize the corresponding relationship between the imaging clarity of the target surface 101 and the position of the target surface 101 along the optical axis, and thus the position of the peak of the clarity mapping curve on the optical axis is as close as possible to the focal plane of the objective lens 110. After the focus position is at the fine focus position through the fine focus operation, the imaging of the target surface 101 is as clear as possible, thereby ensuring the focusing effect.

[0206] In the present invention, the second set step size may be greater than the depth of field of the objective lens 110. In some cases, the depth of field may be 0.2 mm, and the second set step size may be between 0.3 mm and 0.5 mm, such as 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5 (mm).

[0207] In this way, the focusing accuracy can be improved.

[0208] Specifically, after moving back and forth twice with the second set step size, the imaging clarity obtained by the objective lens 110 can have a more obvious change and be easy to distinguish, so that the clarity mapping curve can be closer to the actual correspondence between the clarity score and the position of the target surface 101 along the optical axis, thereby improving the focusing accuracy when performing fine focusing operations through the clarity mapping curve.

[0209] In addition, it should be noted that the second set step size should be slightly larger than the depth of field of the objective lens 110. If the second set step size is too large relative to the depth of field of the objective lens 110, otherwise the number of clarity scores obtained may be too small, which may easily cause the point to be far away from the focal plane and cause the relevant information near the focal plane to be lost, thereby failing to accurately reflect the basic trend of the actual clarity mapping curve, which will in turn greatly affect the focusing accuracy.

[0210] Please refer to Figure 1In the present invention, the microscope system 1000 may include an automatic focusing assembly 200. The focusing method may include:

[0211] Control the auto-focusing assembly 200 to send a second focusing light along the optical axis of the objective lens 110 to the target surface 101, receive the second focusing light reflected from the target surface 101, and then output an electrical signal according to the received second focusing light, wherein the electrical signal is related to the focusing position;

[0212] Step 021 (controlling the objective lens 110 and / or the target surface 101 to move multiple times near the coarse focus position, and obtaining a new focus position and a clarity score of an image taken by the imaging component 120 for the target surface 101 corresponding to the new focus position after each movement) may include:

[0213] The objective lens 110 and / or the target surface 101 are controlled to move according to the electrical signal.

[0214] The focusing method of the present invention can be implemented by the microscope system 1000 of the present invention. Figure 3 The control module 210 can be used to: control the autofocus component 200 to send a second focusing light along the optical axis of the objective lens 110 to the target surface 101, and receive the second focusing light reflected from the target surface 101, and then output an electrical signal according to the received second focusing light, the electrical signal being related to the focusing position; and control the movement of the objective lens 110 and / or the target surface 101 according to the electrical signal.

[0215] In this way, the problem of reduced focusing accuracy due to optical axis offset when focusing according to the optical axis can be avoided.

[0216] Specifically, in general, if focusing is performed according to the optical axis (particularly, the optical axis is moved in a positive direction and then moved in a negative direction so that the target surface 101 reaches the focal plane), the focusing will be inaccurate.

[0217] exist Figure 1In the embodiment, the second focusing light is emitted by the auto-focusing component 200. Since there is a corresponding relationship between the position of the target surface 101 along the optical axis and the clarity score of the imaging of the target surface 101, if the position of the target surface 101 along the optical axis changes, the second focusing light received by the auto-focusing component 200 will also change (such as the time between sending the second focusing light and receiving the second focusing light is different). According to this change, the auto-focusing component 200 can output a corresponding electrical signal according to the received second focusing light, and then establish a corresponding relationship between the electrical signal and the focusing position. According to this corresponding relationship and the electrical signal, the focusing position, or the position of the target surface 101 along the optical axis, can be determined. Compared with the method of directly adjusting and determining the focusing position through the optical axis, the situation of reducing the focusing accuracy due to the offset of the optical axis can be avoided. The electrical signal may include voltage, current, and power. When the current focusing position changes, the electrical signal output by the auto-focusing component 200 will also change accordingly in value, that is, the electrical signal is related to the focusing position.

[0218] It should be noted that the electrical signal feedback is the relative height formed between the objective lens 110 and the target surface 101 along the optical axis, and the position of the objective lens 110 on the optical axis is the absolute height formed by the objective lens 110 along the optical axis.

[0219] On the basis of the above, in some cases, during fine focus operation, the microscope system 1000 may fluctuate, for example, the target surface 101 may fluctuate, resulting in low scanning accuracy when imaging at the peak position of the clarity mapping curve, resulting in the need for multiple supplementary photos, or, for some reason, the photos cannot be taken in sequence (corresponding to step 0211 of the present invention).

[0220] In the above situation, since the value of the electrical signal is still accurate when the target surface 101 is undulating, compared with the method of taking images in a sequence of multiple moves according to the second set step length, the present invention can also take multiple images randomly at a position close to the focal plane of the objective lens 110 through the automatic focusing component 200, and then use the association between the electrical signal and the focus position to sort the taken images according to the value of the electrical signal, thereby obtaining an effect similar to taking images in a sequence of multiple moves according to the second set step length. In this way, the shooting order will not affect the accuracy of the determined clarity mapping curve.

[0221] Please refer to Figure 7 In the present invention, step 022 (determining a clarity mapping curve according to a plurality of focus positions and a plurality of clarity scores, and then moving the objective lens 110 and / or the target surface 101 so that the focus position is at a fine focus position) may include:

[0222] 0221: Perform fitting processing on multiple focus positions and multiple clarity scores to obtain a clarity mapping curve;

[0223] 0222: Control the objective lens 110 and / or the target surface 101 to move so that the focus position is at a fine focus position.

[0224] The focusing method of the present invention can be implemented by the microscope system 1000 of the present invention. Figure 3 The control module 210 can be used to: perform fitting processing on multiple focus positions and multiple clarity scores to obtain a clarity mapping curve; and control the movement of the objective lens 110 and / or the target surface 101 so that the focus position is at a fine focus position.

[0225] This is beneficial to the focusing effect of fine focusing operation.

[0226] Specifically, please combine Figure 4 After determining each focus position and the corresponding clarity score, a coordinate system can be established according to the focus position and the clarity score. Each focus position and the corresponding clarity score can determine a corresponding coordinate point in the coordinate system, thereby obtaining multiple coordinate points. According to these coordinate points, a fitting process can be performed to obtain a clarity mapping curve. The clarity mapping curve can reflect the change of the clarity score with the change of the focus position. According to the peak of the clarity mapping curve, the position of the focal plane of the objective lens 110 can be determined. The objective lens 110 and / or the target surface 101 are controlled to move so that the target surface 101 is located at the position corresponding to the peak on the optical axis, that is, the target surface 101 can be located at the position of the focal plane of the objective lens 110, thereby achieving the focusing effect of the fine focusing operation.

[0227] In the present invention, step 0221 (fitting the change trends of multiple focus positions and multiple clarity scores to obtain a clarity mapping curve) may include:

[0228] When the definition mapping curve satisfies the set condition, it is determined to control the microscope system 1000 to re-execute the coarse focus operation and / or the fine focus operation.

[0229] The focusing method of the present invention can be implemented by the microscope system 1000 of the present invention. Figure 3 The control module 210 may be used to: when the clarity mapping curve satisfies a set condition, determine to control the microscope system 1000 to re-execute a coarse focus operation and / or a fine focus operation.

[0230] In this way, it is possible to avoid the final focusing effect being affected by some factors.

[0231] It is understandable that when the focusing process is actually carried out, the influence of some relevant factors may be encountered, so that the obtained clarity mapping curve is different from the expected situation (such as the peak of the clarity mapping curve cannot be found), that is, the clarity mapping curve meets the set conditions. In this case, at least one of the coarse focus operation and the fine focus operation can be re-executed to eliminate the influence of relevant factors as much as possible, so as to ensure that the clarity mapping curve is consistent with the actual situation as much as possible.

[0232] In the present invention, the setting conditions may include at least one of the following:

[0233] When the number of the obtained multiple clarity scores is greater than the set number, the peak of the clarity mapping curve is not fitted;

[0234] When the objective lens 110 and / or the target surface 101 has moved to the maximum scanning distance, the peak of the clarity mapping curve is not fitted;

[0235] The multiple clarity scores obtained in sequence have a tendency to gradually decrease;

[0236] The fluctuation amplitude of the clarity mapping curve is less than the set amplitude value, and the maximum value of the clarity score is less than the set score;

[0237] The clarity map curve has multiple peaks.

[0238] In this way, the influence of the failure of the previous coarse focusing operation on the fitting process can be reduced.

[0239] Specifically, in some cases, if the number of multiple clarity scores obtained is greater than the set number, but the peak of the clarity mapping curve is not fitted, it can be determined that the focal plane of the objective lens 110 exceeds the adjustment range that the focus position can currently reach, making the focus position unable to reach the fine focus position, and thus it is necessary to re-acquire the clarity score.

[0240] In some cases, if the peak of the clarity mapping curve is not fitted when the objective lens 110 and / or the target surface 101 have moved to the maximum scanning distance, it can be determined that the focal plane of the objective lens 110 exceeds the distance range that the objective lens 110 and / or the target surface 101 can currently reach, making it impossible for the focus position to reach the fine focus position, thereby requiring the clarity score to be reacquired.

[0241] In some cases, if the multiple clarity scores obtained in sequence have a trend of gradually decreasing, it may mean that the multiple clarity scores obtained are all obtained by unidirectional sampling along the optical axis at the focal plane of the objective lens 110, and the position of the focal plane of the objective lens 110 along the optical axis is not within the sampling interval formed by the multiple clarity scores obtained, or the second set step size is too large compared to the depth of field of the objective lens 110, and only one or two clarity scores are obtained in one direction of the focal plane of the objective lens 110 along the optical axis, and the other clarity scores are obtained in another direction of the focal plane of the objective lens 110 along the optical axis, so that the change trend of the multiple clarity scores obtained does not accurately reflect the basic trend of the actual clarity mapping curve, and it is necessary to re-acquire the clarity scores.

[0242] In some cases, if the fluctuation amplitude of the clarity mapping curve is less than the set amplitude value, and the maximum value of the clarity score is less than the set score, it may mean that the currently acquired clarity scores correspond to points on the clarity mapping curve that are far away from the position of the focal plane of the objective lens 110 along the optical axis, and are basically at the two ends of the clarity mapping curve. This is because the actual clarity mapping curve will have a larger change trend at the peak (corresponding to the position of the focal plane of the objective lens 110 along the optical axis), and the clarity scores corresponding to the points around the peak are relatively high. Therefore, it can be determined that the current sampling points have not taken points close to the peak, and the clarity mapping curve obtained based on these clarity scores cannot reflect the actual change trend of the clarity mapping curve at the peak, so it is necessary to re-acquire the clarity score.

[0243] In some cases, if the clarity mapping curve has multiple peaks, it may mean that there are deviations in the acquisition of some clarity scores, making some clarity scores relatively too large or too small. In this way, the obtained clarity mapping curve cannot reflect the actual changing trend of the clarity mapping curve at the peaks, and it is necessary to re-acquire the clarity scores.

[0244] It can be understood that, on the basis of satisfying at least one of the above-mentioned setting conditions, it can be determined that the focal plane of the objective lens 110 cannot be specifically located according to the peak on the currently obtained clarity mapping curve, that is, the previous coarse focusing operation did not achieve the expected coarse focusing effect, or the coarse focusing failed, and it is necessary to interrupt the current fine focusing operation, restart focusing from the coarse focusing operation, or re-execute the fine focusing operation, thereby reducing the impact of the larger distance between the current target surface 101 and the focal plane of the objective lens 110, and ensuring the fitting accuracy of the subsequent clarity mapping curve.

[0245] In the present invention, step 0221 (performing fitting processing on multiple focus positions and multiple clarity scores to obtain a clarity mapping curve) may include:

[0246] A first fitting straight line is obtained by performing a first-order straight line fitting on a part of the multiple clarity scores showing an upward trend, and a second fitting straight line is obtained by performing a first-order straight line fitting on a part of the multiple clarity scores showing a downward trend, and the intersection of the first fitting straight line and the second fitting straight line is determined as the peak of the clarity mapping curve.

[0247] The focusing method of the present invention can be implemented by the microscope system 1000 of the present invention. Figure 3 The control module 210 can be used to: perform a first-order straight line fitting on a portion of the multiple clarity scores that shows an upward trend to obtain a first fitting straight line, perform a first-order straight line fitting on a portion of the multiple clarity scores that shows a downward trend to obtain a second fitting straight line, and determine the intersection of the first fitting straight line and the second fitting straight line as the peak of the clarity mapping curve.

[0248] In this way, the fitting effect can be easily achieved.

[0249] In this embodiment, the first fitting straight line and the second fitting straight line are obtained by a first-order straight line fitting method. The first-order straight line fitting is also often called linear regression fitting, and its purpose is to find a straight line so that a given set of data points is as close to the straight line as possible to some extent. The equation of this straight line is usually expressed in the form of y=ax+b, where a is the slope of the straight line, which determines the degree of inclination of the straight line, and b is the intercept of the straight line on the y-axis. Common fitting methods include least squares method, gradient descent method, etc.

[0250] Specifically, please combine Figure 8 ,exist Figure 8 In the embodiment, after controlling the objective lens 110 and / or the target surface 101 to move multiple times near the coarse focus position, multiple points located in the coordinate system can be determined by each focus position and the corresponding clarity score, that is, Figure 8 p 11 、p 12 、p 13 、p 14 、p 15 、p 16 、p 17 、p 18 、p 19 . As the focus position changes in the positive direction, p 11 、p 12 、p 13 、p 14 、p 15 There is a trend that the clarity scores increase successively, p 16 、p 17 、p 18 、p 19 The clarity scores tend to decrease successively, so p 11 、p12 、p 13 、p 14 、p 15 Perform a first-order straight line fitting to obtain the first fitting straight line X1, and you can 16 、p 17 、p 18 、p 19 A first-order straight line fitting is performed to obtain a second fitting straight line X2. Then, the intersection point p between the first fitting straight line X1 and the second fitting straight line X2 can be 10 Determined as the peak of the clarity map curve.

[0251] It can be understood that, through the above-mentioned first-order straight line fitting process, on the basis of ensuring a certain fitting accuracy, the process of fitting process can be simplified and the efficiency of fitting process can be improved.

[0252] Step 0221 (performing fitting processing on multiple focus positions and multiple clarity scores to obtain a clarity mapping curve) may include:

[0253] A second-order parabola fitting is performed on the plurality of clarity scores to obtain a first fitting parabola, and the vertex of the first fitting parabola is determined as a peak of the clarity mapping curve.

[0254] The focusing method of the present invention can be implemented by the microscope system 1000 of the present invention. Figure 3 The control module 210 may be configured to: perform a second-order parabola fitting on the plurality of clarity scores to obtain a first fitting parabola, and determine the vertex of the first fitting parabola as the peak of the clarity mapping curve.

[0255] This is helpful to improve the fitting accuracy of the actual situation.

[0256] In this embodiment, a second-order parabola fitting method is used to obtain a first fitting parabola. The second-order parabola fitting method is to find a parabola equation to approximate a given set of data points. The general equation form of the parabola is y=ax 2 +bx+c, where a, b, and c are the parameters that need to be determined. a≠0, which determines the opening direction of the parabola (a>0 opens upward, a<0 ​​opens downward) and the degree of curvature, b affects the position of the symmetry axis of the parabola, and c is the ordinate of the intersection of the parabola and the y\ axis. Commonly used fitting methods include the least squares method.

[0257] Specifically, please combine Fig. 9 ,exist Fig. 9 In the embodiment, after controlling the objective lens 110 and / or the target surface 101 to move multiple times near the coarse focus position, multiple points located in the coordinate system can be determined by each focus position and the corresponding clarity score, that is, Fig. 9 p21 、p 22 、p 23 、p 24 、p 25 、p 26 、p 27 、p 28 、p 29 . 21 、p 22 、p 23 、p 24 、p 25 、p 26 、p 27 、p 28 、p 29 A second-order parabola fitting is performed to obtain a first fitting parabola X3. Then, according to the vertex p of the first fitting parabola X3, 20 Determined as the peak of the clarity map curve.

[0258] It can be understood that, through the above-mentioned second-order parabola processing, the changing trend of image clarity in actual situations can be matched, thereby improving the fitting accuracy to the actual situation.

[0259] Please refer to Fig.10 Step 0221 (fitting multiple focus positions and multiple clarity scores to obtain a clarity mapping curve) may include:

[0260] 02211: Acquire a plurality of fine focus combinations, the fine focus combinations comprising a plurality of clarity scores obtained by moving the objective lens 110 and / or the target surface 101 by corresponding step lengths, each fine focus combination having a corresponding coarse focus position and a same imaging position;

[0261] 02212: Taking the real best focal plane of the objective lens 110 as the objective function, machine learning is performed according to multiple fine focus combinations to determine the weights of multiple clarity scores in the clarity mapping curve, and the position of the real best focal plane of the objective lens 110 corresponds to the peak of the clarity mapping curve;

[0262] 02213: After completing machine learning, take multiple clarity scores as input to output the peaks of a clarity map curve.

[0263] The focusing method of the present invention can be implemented by the microscope system 1000 of the present invention. Figure 3The control module 210 can be used to: obtain multiple fine focus combinations, the fine focus combinations include multiple clarity scores obtained by moving the objective lens 110 and / or the target surface 101 by corresponding step sizes, each fine focus combination has a corresponding coarse focus position and the same imaging position; take the real best focal plane of the objective lens 110 as the objective function, perform machine learning based on the multiple fine focus combinations to determine the weights of the multiple clarity scores in the clarity mapping curve, and the position of the real best focal plane of the objective lens 110 is determined according to the peak of the clarity mapping curve; after completing the machine learning, use the multiple clarity scores as input to output the peak of the clarity mapping curve.

[0264] In this way, the fitting results can be made as close to the actual situation as possible.

[0265] In actual application scenarios, although the clarity mapping curves obtained by fitting vary depending on the specific circumstances, the overall change trend can be determined, that is, the closer the position is to the focal plane of the objective lens 110, the larger the corresponding clarity score is, and the clarity score corresponding to the position of the focal plane of the objective lens 110 is the maximum value, that is, the part close to the peak on the actual clarity mapping curve can more clearly reflect the change trend at the peak.

[0266] Specifically, in some cases, since the clarity scores obtained by imaging at positions close to the focal plane on the optical axis are also close to the clarity scores of the target surface 101 at the position where the focal plane is located, or in other words, the fluctuation amplitude of the clarity scores within the range close to the focal plane on the optical axis is small, while the clarity scores at positions far from the focal plane on the optical axis have larger fluctuations, when the clarity scores at positions far from the focal plane are introduced for fitting processing, the fluctuations at positions far from the focal plane will also be introduced, thereby affecting the position of the peak of the clarity mapping curve.

[0267] On the basis of the above, the present invention can use the clarity scores in multiple fine focus combinations as the input of the initial learning model, use the actual focal plane of the objective lens 110 as the target function, and then compare the output of the initial learning model with the target function. According to the comparison result, the learning model parameters can be adjusted, and then the clarity scores in multiple fine focus combinations are input, and then the output is compared with the target function until the error formed by the comparison result can meet the expectation, so that the current learning model can be used as a machine model for outputting the clarity mapping curve. In some cases, adjusting the parameters of the learning model can be used to adjust the weights of each clarity score in the learning model.

[0268] It can be understood that by performing machine learning, the degree of influence of each fine focus combination's own fluctuation on the wave peak in the fitting process can be determined, so that weights can be assigned to these fine focus combinations, so that fine focus combinations that cause greater fluctuation influence on the wave peak are assigned smaller weights, and fine focus combinations that cause less fluctuation influence on the wave peak are assigned larger weights, thereby minimizing the fluctuation influence on the wave peak in the fine focus combinations introduced into the fitting process, so that the fitting results are as close to the actual situation as possible.

[0269] In addition, in some cases, for fine focus combinations with greater fluctuation effects, their weights may be adjusted to 0, that is, these fine focus combinations are removed, so as to further improve the degree of fitting to the actual situation.

[0270] In the present invention, the focusing method may include:

[0271] The sharpness value of the image obtained by imaging is used as the clarity score.

[0272] The focusing method of the present invention can be implemented by the microscope system 1000 of the present invention. Figure 3 The control module 210 may be configured to: use the sharpness value of the image obtained by imaging as the clarity score.

[0273] This is beneficial to improving the data processing efficiency in fine focusing operations.

[0274] It can be understood that for images, there is a certain relationship between the sharpness value and the image clarity. By determining the sharpness of the image obtained by imaging to obtain the corresponding sharpness value, and then judging the clarity score of the image based on the sharpness value, the process of determining the clarity score of the image can be simplified, thereby improving the efficiency of data processing.

[0275] In the present invention, the focusing method may include:

[0276] The bright spots in the image obtained by imaging are evaluated and processed to determine the clarity score.

[0277] The focusing method of the present invention can be implemented by the microscope system 1000 of the present invention. Figure 3 The control module 210 can be used to: evaluate the bright spots in the image obtained by imaging to determine the clarity score.

[0278] This is helpful to improve the accuracy of the obtained clarity score.

[0279] Specifically, after imaging the target surface 101, the light will form one or more bright spots in the image obtained by imaging. In some cases, the bright spots in the image can be determined by detecting the pixel values ​​of the image. In one case, the pixel values ​​of all pixels in the area where the bright spots are located are basically the same, and the pixel values ​​of the area where the bright spots are located are greatly different from the pixel values ​​of the area around the bright spots (which can also be the entire image) (generally speaking, the pixel values ​​in the bright spots are greater than the pixel values ​​in other areas of the image), so the bright spots in the image can be determined by comparing the pixel values.

[0280] It can be understood that the bright spot in the image has a higher degree of recognition than the image area around the bright spot (or the entire image). When determining the clarity score of the image, determining by the bright spot can improve a certain degree of accuracy. In some cases, the bright spot can be formed when the image is formed by setting a corresponding light source, or the bright spot can be formed by performing a certain degree of local overexposure when imaging.

[0281] In addition, it should be pointed out that in some cases, there may be a problem that bright spots cannot be observed on the image. For example, when the image is a high-density array image, the image itself has a very high resolution, so that the bright spot part of the image is displayed in a very small proportion and is difficult to observe. Also, when the image is an image collected during a single-target sequencing process, the image itself has no obvious point signal and only a small amount of noise.

[0282] In the case of the above-mentioned image, the present invention can determine the clarity score of the image by other means. Specifically, in one case, the grayscale image standard deviation of the image can be obtained or determined and used as the evaluation value of the image sharpness, and then the corresponding area in the image is evaluated and processed according to the grayscale image standard deviation to determine the clarity score. And, in another case, the pixel standard deviation or pixel mean of all pixels in the image can be obtained or determined, and then the sharpening degree of multiple images is evaluated according to the pixel standard deviation or pixel mean to determine the clarity score, which can eliminate brightness interference.

[0283] It is understandable that those skilled in the art can select the method of evaluating the image to determine the clarity score in the present invention according to the specific situation of the image.

[0284] In the present invention, the evaluation process of the bright spots in the image obtained by imaging can be achieved by the following formula:

[0285] Score=[(k1*k2-1)*CV-EV] / [(CV+EV) / (k1*k2)];

[0286] Among them, Score represents the clarity score, k1*k2 represents the part of the matrix composed of odd rows and odd columns in the pixel array of the image within the range of the bright spot, CV represents the central pixel value of the matrix corresponding to the bright spot, and EV represents the sum of the non-central pixel values ​​of the matrix corresponding to the bright spot.

[0287] In this way, the clarity score of the image can be easily determined by the bright spots in the image.

[0288] In the present invention, the focusing method can be applied in the sequencing-by-synthesis process.

[0289] It is understood that, generally speaking, nucleic acid sequencing includes the process of binding nucleotides (including nucleotide analogs) to templates and collecting corresponding extended base fluorescence signals. In some platforms, the binding of nucleotides to templates and the collection of corresponding fluorescence signals are performed asynchronously or in real time, and multiple rounds of sequencing reactions are usually required to achieve the determination of the nucleotide sequence or base sequence of the template nucleic acid molecule, wherein the process of extending a base and determining the type of the extended base can be referred to as a round of sequencing (cycle). In other words, in one round of sequencing, the process of determining the base type at a given position on the template nucleic acid molecule is completed, and this method is also referred to as sequencing by synthesis (SBS).

[0290] In the related art, multiple rounds of sequencing by synthesis are performed to obtain sequencing sequences or reads. For example, the nucleic acid molecule to be tested is contacted with a polymerase and a modified nucleotide and placed under conditions suitable for a polymerization reaction, and the modified nucleotide is controllably incorporated into the nucleic acid molecule to be tested, or a single base extension is controllably achieved, and the corresponding reaction signal is detected, and the type of nucleotide incorporated into the nucleic acid molecule to be tested in this reaction is determined based on the signal, and multiple controlled single base extensions and corresponding signal detections are performed in this way, so that the type of nucleotides or bases incorporated into the nucleic acid molecule to be tested in multiple or multiple rounds of reactions can be detected according to the reaction signal information, so as to read out a portion of the sequence of the nucleic acid molecule to be tested.

[0291] In the sequencing process, in order to match the fluorescent signal with the target nucleic acid molecule, the site where the target nucleic acid is fixed will be positioned at different coordinates. Specifically, in the process of sequencing by synthesis, in each extension reaction, the extended base carries an optically detectable label such as a fluorescent label. Therefore, by exciting fluorescence, at least a portion of the nucleic acid molecules or clone clusters can appear as bright spots on the image. These "bright spots" are also called "bright spots" (spots or peaks), which will appear as luminous points on the image, and one luminous point occupies at least one pixel. Thus, for a "given position" (also called a "template position"), it will match a specific target nucleic acid molecule or fragment. Thus, the obtained series of image sets can be classified by position so as to obtain the sequencing reads corresponding to each target nucleic acid molecule.

[0292] On the basis of the above, after each round of extension reaction, the excited fluorescent signal is imaged and recorded, so that an image formed by the fluorescent signal can be obtained. These images will reflect the type and intensity of the fluorescent signal at each position. It should be noted that, according to the number of base types added in the reaction mixture of an extension reaction, the image data obtained by each round of extension reaction will be different. Generally speaking, different images can be obtained for fluorescent signals of different colors. In other words, it can be understood by those skilled in the art that after each extension reaction, multiple photographs can be taken, and different detection channels can be used for photographic detection for fluorescence of different colors, so that when the image difference between different detection channels is huge, for example, in single signal sequencing, only one detection channel in multiple detection channels has a signal, which can maximize the degree of fit between the obtained focal plane and the real focal plane of the objective lens 110. As a result, in order to determine the type of extended bases, each sequencing round needs to collect at least one image, or even multiple images. For example, a base extension reaction may include one image acquisition, or may include multiple image acquisitions, and the collection of these images is used as an image set for subsequent sequencing analysis.

[0293] In addition, in some cases, after each extension reaction, the same color of fluorescence can be photographed and detected using the same detection channel, and the fine focus position can be determined according to different positions in the image.

[0294] Specifically, in some practical applications, when capturing an image, since the captured image has a large size, or there may be an angle between the objective lens 110 and the target surface 101 (this angle may be the angle formed by the non-parallelism between the optical axis direction of the objective lens 110 and the normal direction of the target surface 101), the central area and the edge area of ​​the image correspond to different fine focus positions, or there is a deviation between the best focal plane obtained through the central area of ​​the image and the best focal plane obtained through the edge area of ​​the image.

[0295] In view of the above problem, in order to ensure the optimal signal of the whole image, the corresponding fine focus position can be obtained for different positions of the image, and then the weighted average of the multiple fine focus positions obtained can be taken, and finally the average is used as the final fine focus position. In one case, the central area of ​​the image and the edge area between the central area and each of the four edges of the image can be selected, and a total of five image areas can be finely focused to obtain five fine focus positions, and then the final fine focus position is determined according to the weighted average of the five fine focus positions.

[0296] In addition, the weighted average of the five fine focus positions can be the weighted average of the heights formed by the five fine focus positions along the optical axis, or the weighted average of the numerical values ​​of the electrical signals corresponding to the five fine focus positions obtained by the autofocus component. When performing weighted average processing on the five fine focus positions, the weight of each image area can be determined based on the actual situation of the image (such as a specific part of the image is not clear enough due to equipment problems), or it can be calibrated through actual testing. In other cases, the specific position and number of the selected image areas can also be adjusted and determined as appropriate.

[0297] Please refer to Fig.11 , focusing methods can include:

[0298] 03: During the first round of base extension reaction, the microscope system 1000 is controlled to perform a coarse focus operation and a fine focus operation in sequence;

[0299] 04: During each round of base extension reaction after the first round of base extension reaction, control the microscope system 1000 to perform a fine focusing operation.

[0300] The focusing method of the present invention can be implemented by the microscope system 1000 of the present invention. Figure 3 The control module 210 can be used to: during the first round of base extension reaction, control the microscope system 1000 to perform a coarse focus operation and a fine focus operation in sequence; during each round of base extension reaction after the first round of base extension reaction, control the microscope system 1000 to perform a fine focus operation.

[0301] In this way, rapid focusing can be achieved during the sequencing-by-synthesis process.

[0302] It can be understood that in the process of sequencing by synthesis, since multiple base extension reactions need to be performed and the sequencing sample needs to be imaged after each base extension reaction to determine whether the base extension reaction proceeds as expected, a focusing operation is required after each base extension reaction to ensure that the test sample is photographed clearly enough.

[0303] The sequencing sample may be located on a sequencing chip, and the target surface 101 may be a surface on the sequencing chip where a sequencing reaction occurs. Fig.12 , Fig.12 What is shown is a sequencing chip 102 used in the process of sequencing by synthesis. The sequencing chip 102 may include a first glass plate 103 and a second glass plate 104. Fig.12In the figure, the upper surface of the first glass plate 103 can be represented as S1, the lower surface of the first glass plate 103 can be represented as S2, and the upper surface of the second glass plate 104 can be represented as S3. A gap is formed between the lower surface of the first glass plate 103 and the upper surface of the second glass plate 104, and this gap is the fluid channel of the sequencing chip 102.

[0304] In some cases, during the sequencing reaction process, the lower surface S2 of the first glass plate 103 can be specially chemically modified, and a segment of oligonucleotide sequence can be connected to its surface through hydrogen bonds, and the oligonucleotide sequence can be complementary hybridized with the linker sequence of single-stranded DNA or RNA, that is, the sample to be tested is confined on the lower surface S2 of the first glass plate 103, so that the lower surface S2 of the first glass plate 103 can be used as the target surface 101 of the present invention. Similarly, in other cases, the upper surface S3 of the second glass plate 104 can also be specially chemically modified, so that the upper surface S3 of the second glass plate 104 can also be used as the target surface 101 of the present invention.

[0305] In addition, during the first round of base extension reaction, controlling the microscope system 1000 to perform a coarse focus operation and a fine focus operation in sequence can make the target surface 101 sufficiently close to the focal plane of the objective lens 110. During each round of base extension reaction after the first round of base extension reaction, there is no need to perform a coarse focus operation, and only a fine focus operation can be performed, which is beneficial to improving the processing speed of the overall process of sequencing by synthesis, thereby improving the sequencing efficiency.

[0306] Please refer to Fig.13 In the present invention, step 04 (controlling the microscope system 1000 to perform a fine focusing operation during each round of base extension reaction after the first round of base extension reaction) may include:

[0307] 041: Control the objective lens 110 and / or the target surface 101 to move so that the focus position is at the fine focus position in the previous round of base extension reaction, and after the movement, determine the current clarity score as the first clarity score;

[0308] 042: after controlling the objective lens 110 and / or the target surface 101 to move in the reverse direction from the focus position corresponding to the first clarity score by a third set step length, a fine focus operation is performed to determine a new fine focus position, and the clarity score corresponding to the new fine focus position is determined as a second clarity score;

[0309] 043: when the first clarity score is less than the second clarity score, determine to use the current focus position as the fine focus position of the current round of base extension reaction;

[0310] 044: When the first clarity score is greater than the second clarity score, the objective lens 110 and / or the target surface 101 is moved back to the fine focus position corresponding to the first clarity score.

[0311] The focusing method of the present invention can be implemented by the microscope system 1000 of the present invention. Figure 3 The control module 210 can be used to: control the objective lens 110 and / or the target surface 101 to move so that the focus position is at the fine focus position in the previous round of base extension reaction, and after the movement, determine the current clarity score as the first clarity score; control the objective lens 110 and / or the target surface 101 to move in the opposite direction from the focus position corresponding to the first clarity score by a third set step length, perform a fine focus operation to determine a new fine focus position, and determine the clarity score corresponding to the new fine focus position as the second clarity score; when the first clarity score is less than the second clarity score, determine the current focus position as the fine focus position of the current round of base extension reaction; when the first clarity score is greater than the second clarity score, move the objective lens 110 and / or the target surface 101 back to the fine focus position corresponding to the first clarity score.

[0312] In this way, it can be ensured that the imaging of the target surface 101 is as clear as possible.

[0313] It can be understood that in the process of sequencing by synthesis, since the target surface 101 needs to be imaged once after each round of base extension reaction, it is necessary to focus during each imaging so that the focus position is at the fine focus position under the current round of base extension reaction, thereby ensuring that the imaging of the target surface 101 in each round of base extension reaction is as clear as possible.

[0314] Specifically, when performing at least two rounds of base extension reactions, since the focal plane of the objective lens 110 has been focused in the previous round of base extension reactions, when performing a new round of base extension reactions, the fine focus position in the previous round of base extension reactions can be used as the starting focus position, and then a fine focus operation is performed from the starting focus position with a third set step length to obtain a new fine focus position, and then the clarity scores corresponding to the two previous and next fine focus positions are compared. If the clarity score of the starting focus position is greater, the fine focus position in the previous round of base extension reactions is used as the fine focus position in the current round of base extension reactions. If the clarity score of the new focus position is greater, the new fine focus position is used as the fine focus position in the current round of base extension reactions.

[0315] In addition, in some cases, step 041 and step 042 may be to control the objective lens 110 and / or the target surface 101 to move in the positive direction of the Z axis so that the focus position is at the fine focus position in the previous round of base extension reaction, and then start to move in the negative direction of the Z axis according to the third set step length. In other cases, step 041 and step 042 may be to control the objective lens 110 and / or the target surface 101 to move in the negative direction of the Z axis so that the focus position is at the fine focus position in the previous round of base extension reaction, and then start to move in the positive direction of the Z axis according to the third set step length. Which specific movement method to adopt can be selected according to the actual application scenario.

[0316] In the present invention, the focusing method may include:

[0317] When the focus position is not at the fine focus position in the previous round of base extension reaction, the microscope system 1000 is controlled to perform a coarse focus operation, and after completion, the objective lens 110 and / or the target surface 101 are controlled to move to determine a second clarity score.

[0318] The focusing method of the present invention can be implemented by the microscope system 1000 of the present invention. Figure 3 The control module 210 can be used to: when the focus position in the previous round of base extension reaction is not at the fine focus position, control the microscope system 1000 to perform a coarse focus operation, and after completion, control the objective lens 110 and / or the target surface 101 to move to determine a second clarity score.

[0319] In this way, the focusing accuracy of the current round of base extension reaction can be ensured.

[0320] It can be understood that if the focus position in the previous round of base extension reaction is not at the fine focus position, it means that the focus operation performed in the previous round of base extension reaction was unsuccessful and is not suitable as a reference for the fine focus position in the current round of base extension reaction.

[0321] Specifically, if the previous round of fine focusing operation is unsuccessful, the first clarity score may not be obtained. In this case, a coarse focusing operation can be performed to ensure that the focus position can reach near the focal plane, and then a fine focusing operation can be performed to ensure that the focus position corresponding to the second clarity score obtained is as close to the focal plane as possible, thereby ensuring the focusing accuracy of the current round of base extension reaction.

[0322] Please refer to Figure 3 In the present invention, the microscope system 1000 may include a driving assembly 300. The driving assembly 300 may be used to drive the objective lens 110 and / or the target surface 101 to move along the optical axis of the objective lens 110.

[0323] In the present invention, the focusing method may include:

[0324] After determining the first clarity score, detecting whether the difference between the first voltage value and the second voltage value is less than or equal to a first set value, the first voltage value being the voltage value output when the driving component 300 controls the objective lens 110 and / or the target surface 101 to move to a focus position corresponding to the first clarity score in the previous round of base extension reaction, and the second voltage value being the voltage value output when the driving component 300 controls the objective lens 110 and / or the target surface 101 to move to a focus position corresponding to the first clarity score in the current round of base extension reaction;

[0325] When the difference is less than or equal to the first set value, controlling the objective lens 110 and / or the target surface 101 to move to determine a second clarity score;

[0326] When the difference is greater than the first set value, the microscope system 1000 is controlled to perform a coarse focusing operation, and after the coarse focusing operation is completed, the objective lens 110 and / or the target surface 101 are controlled to move to determine a second clarity score.

[0327] The focusing method of the present invention can be implemented by the microscope system 1000 of the present invention. Figure 3 The control module 210 can be used to: after determining the first clarity score, detect whether the difference between the first voltage value and the second voltage value is less than or equal to the first set value, the first voltage value is the voltage value output when the driving component 300 controls the objective lens 110 and / or the target surface 101 to move to the focusing position corresponding to the first clarity score in the previous round of base extension reaction, and the second voltage value is the voltage value output when the driving component 300 controls the objective lens 110 and / or the target surface 101 to move to the focusing position corresponding to the first clarity score in the current round of base extension reaction; when the difference is less than or equal to the first set value, control the objective lens 110 and / or the target surface 101 to move to determine the second clarity score; when the difference is greater than the first set value, control the microscope system 1000 to perform a coarse focusing operation, and after completion, control the objective lens 110 and / or the target surface 101 to move to determine the second clarity score.

[0328] In this way, it can be determined whether the obtained clarity score is affected by fluctuations.

[0329] Specifically, the voltage value output by the driving component 300 can correspond to the distance between the target surface 101 and the focal plane in the focusing position. That is, for the two previous and subsequent fine focusing operations, if the focusing position is the same, the driving component 300 will also output the same voltage value.

[0330] On the basis of the above, when a fine focus operation is performed in combination with the fine focus position in the previous round of base extension reaction, the deviation between the new fine focus position in the current round of base extension reaction and the fine focus position in the previous round of base extension reaction can be determined according to the difference between the first voltage value and the second voltage value. If the difference is less than or equal to the first set value, it means that the deviation is within an acceptable range and can basically maintain the same clarity as the image obtained by imaging in the previous round of base extension reaction. If the difference is greater than the first set value, it means that the obtained second voltage value has a fluctuation abnormality, so that the microscope system 1000 can be controlled to perform a coarse focus operation to eliminate the fluctuation abnormality in the second voltage value.

[0331] In addition, in some cases, the first set value can be determined based on actual conditions or calibrated through testing.

[0332] In the present invention, step 042 (controlling the objective lens 110 and / or the target surface 101 to move in the reverse direction at a third set step length from the focus position corresponding to the first clarity score, performing a fine focus operation to determine a new fine focus position, and determining the clarity score corresponding to the new fine focus position as the second clarity score) may include:

[0333] Acquire a third voltage value, where the third voltage value is a voltage value output when the driving component 300 controls the objective lens 110 and / or the target surface 101 to move to a corresponding new fine focus position in the current round of base extension reaction;

[0334] When the third voltage value is less than the second set value, the clarity score corresponding to the new fine focus position is determined as the second clarity score;

[0335] When the third voltage value is greater than or equal to the second set value, the drive component 300 is subjected to voltage zeroing processing, and then the objective lens 110 and / or the target surface 101 are controlled to move in the reverse direction again at a third set step size starting from the focusing position corresponding to the first clarity score, and then the fine focusing operation is re-executed until the obtained third voltage value is less than the second set value.

[0336] The focusing method of the present invention can be implemented by the microscope system 1000 of the present invention. Figure 3The control module 210 can be used to: obtain a third voltage value, where the third voltage value is a voltage value output when the driving component 300 controls the objective lens 110 and / or the target surface 101 to move to a corresponding new fine focus position in the current round of base extension reaction; when the third voltage value is less than the second set value, the clarity score corresponding to the new fine focus position is determined as the second clarity score; when the third voltage value is greater than or equal to the second set value, the driving component 300 is voltage zeroed, and then the objective lens 110 and / or the target surface 101 are controlled to move in the reverse direction from the focus position corresponding to the first clarity score at a third set step size, and then the fine focus operation is re-executed until the third voltage value obtained is less than the second set value.

[0337] In this way, the increase in voltage value can be prevented from affecting the accuracy of the fine focusing operation.

[0338] Specifically, in some cases, the voltage value output by the driving component 300 is positively correlated with the distance between the target surface 101 and the focal plane in the focus position. When the third voltage value is greater than or equal to the second set value, the distance between the target surface 101 and the focal plane of the objective lens 110 in the focus position is too large, and a small distance movement is not easy to detect, so that the output third voltage value has a certain deviation from the actual voltage value that should be output, which will lead to inaccurate focus when performing fine focusing operation later.

[0339] On the basis of the above, the driving component 300 can be subjected to voltage zeroing processing, and the voltage value output by the driving component 300 can be reset. In this way, when a fine focusing operation is subsequently performed, the third voltage value is basically at a size close to a reference value (for example, 0). At this time, the distance change formed by the focusing position can be reflected by the third voltage value, thereby ensuring the accuracy of focusing during subsequent fine focusing operations.

[0340] In addition, in some cases, the second set value can be determined based on actual conditions or calibrated through testing.

[0341] Please refer to Fig.14In an application scenario of the present invention, when a new round of base extension reaction is performed, it can be determined whether the previous round of base extension reaction is successfully focused. If successful, the focus is locked according to the voltage value when the focus is successfully locked in the previous round of base extension reaction, and then the current voltage value and the clarity score score1 are obtained. If unsuccessful, optical focusing is performed with a coarse focus operation. After obtaining score1, it can be detected whether the absolute value of the difference between the voltage value in the previous round of base extension reaction and the current voltage value is less than 2. If so, the focal plane of the objective lens 110 starts to move in the opposite direction with a step size of 3dR and starts shooting and focusing. If not, optical focusing is also performed with a coarse focus operation. After multiple images are captured and focused, one of the voltage values ​​corresponding to all the images is obtained as the optimal voltage value, and it is determined whether the optimal voltage value is less than 5. If so, the focus is locked with the optimal voltage value, and the clarity score score2 after the focus is locked is obtained. If not, make0 (clear) processing is performed, and then a fine focus operation is performed again from the focus position before the optical focal plane moves in the opposite direction, and a new optimal voltage value is obtained, and then it is determined whether the optimal voltage value is less than 5. After determining score2, the size between score1 and score2 is determined. If score1 is greater than score2, the focus is locked according to the voltage value when the focus is successfully locked in the previous round of base extension reaction. If score1 is less than score2, the focus is locked according to the optimal voltage value obtained in the current round of base extension reaction.

[0342] Please refer to Fig.15 A microscope system 1000 of the present invention may include a memory 510 and a processor 520. The memory 510 may store a computer program. When the processor 520 executes the computer program, the steps of the focusing method of the present invention may be implemented.

[0343] For example, when the computer program is executed by a processor, the focusing method that can be implemented may include:

[0344] 01: Control the microscope system 1000 to perform a coarse focusing operation so that the focus position is at a coarse focus position, where the focus position is a relative position between the objective lens 110 and the target surface 101;

[0345] 02: Control the microscope system 1000 to perform fine focusing operations, including:

[0346] 021: Control the objective lens 110 and / or the target surface 101 to move multiple times near the rough focus position, and after each movement, obtain a new focus position and a clarity score of an image taken by the imaging component 120 for the target surface 101 corresponding to the new focus position;

[0347] 022: Determine a clarity mapping curve based on multiple focus positions and multiple clarity scores, then move the objective lens 110 and / or the target surface 101 so that the focus position is at a fine focus position, so that the imaging component 120 obtains a clear image of the target surface 101, and the clarity score at the fine focus position is determined according to the peak of the clarity mapping curve.

[0348] The above-mentioned microscope system 1000 obtains a new focus position and a corresponding clarity score by moving the objective lens 110 and / or the target surface 101, and further obtains multiple focus positions and multiple clarity scores. The multiple clarity scores can be used to determine a clarity mapping curve. The focus position corresponding to the peak of the clarity mapping curve can enable the imaging component 120 to obtain the clearest image, so that the focus operation of the microscope system 1000 can be controlled by the change of the clarity score, thereby achieving an automatic focus effect, making the focus more accurate and faster, which is conducive to improving the observation efficiency.

[0349] Please refer to Fig.15 , a computer readable storage medium of the present invention may store a computer program. When the computer program is executed by the processor 520, the steps of the focusing method of the present invention may be implemented.

[0350] For example, when the computer program is executed by a processor, the focusing method that can be implemented may include:

[0351] 01: Control the microscope system 1000 to perform a coarse focusing operation, so that the focus position is at a coarse focus position, where the focus position is a relative position between the objective lens 110 and the target surface 101;

[0352] 02: Control the microscope system 1000 to perform fine focusing operations, including:

[0353] 021: Control the objective lens 110 and / or the target surface 101 to move multiple times near the rough focus position, and after each movement, obtain a new focus position and a clarity score of an image taken by the imaging component 120 for the target surface 101 corresponding to the new focus position;

[0354] 022: Determine a clarity mapping curve based on multiple focus positions and multiple clarity scores, then move the objective lens 110 and / or the target surface 101 so that the focus position is at a fine focus position, so that the imaging component 120 obtains a clear image of the target surface 101, and the clarity score at the fine focus position is determined according to the peak of the clarity mapping curve.

[0355] The above-mentioned microscope system 1000 obtains a new focus position and a corresponding clarity score by moving the objective lens 110 and / or the target surface 101, and further obtains multiple focus positions and multiple clarity scores. The multiple clarity scores can be used to determine a clarity mapping curve. The focus position corresponding to the peak of the clarity mapping curve can enable the imaging component 120 to obtain the clearest image, so that the focus operation of the microscope system 1000 can be controlled by the change of the clarity score, thereby achieving an automatic focus effect, making the focus more accurate and faster, which is conducive to improving the observation efficiency.

[0356] The computer-readable storage medium may be provided in the microscope system 1000 or in other terminals. The microscope system 1000 may communicate with other terminals to obtain corresponding programs.

[0357] It is understood that computer-readable storage media may include: any entity or device capable of carrying a computer program, recording media, USB flash drives, mobile hard disks, magnetic disks, optical disks, computer memories, read-only memories (ROMs), random access memories (RAMs), and software distribution media, etc. A computer program may include computer program code. The computer program code may be in source code form, object code form, executable files, or some intermediate form, etc.

[0358] In certain embodiments of the present invention, the control module 210 may be a single-chip microcomputer chip that integrates a processor, a memory, a communication module, etc. The processor may be a central processing unit (CPU), a graphics processing unit (GPU), or other general-purpose processors, digital signal processors (DSP), application-specific integrated circuits (ASIC), field-programmable gate arrays (FPGA), or other programmable logic devices, discrete gates or transistor logic devices, discrete hardware components, etc.

[0359] Any process or method description in a flowchart or otherwise described herein may be understood to represent a module, segment or portion of code that includes one or more executable instructions for implementing the steps of a specific logical function or process, and the scope of the preferred embodiments of the present invention includes alternative implementations in which functions may not be performed in the order shown or discussed, including performing functions in a substantially simultaneous manner or in the reverse order depending on the functions involved, which should be understood by those skilled in the art to which the embodiments of the present invention belong.

[0360] The logic and / or steps represented in the flowchart or otherwise described herein, for example, can be considered as an ordered list of executable instructions for implementing logical functions, and can be embodied in any computer-readable medium for use by an instruction execution system, apparatus or device (such as a computer-based system, a system including a processing module, or other system that can fetch instructions from an instruction execution system, apparatus or device and execute instructions), or used in combination with these instruction execution systems, apparatuses or devices.

[0361] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments of the present invention without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A focusing method for a microscope system, characterized in that: The microscope system includes an objective lens and an imaging component for imaging a target surface, and the focusing method includes: Controlling the microscope system to perform a coarse focusing operation so that the focusing position is at a coarse focusing position, wherein the focusing position is a relative position between the objective lens and the target surface; Controlling the microscope system to perform a fine focusing operation, including: Controlling the objective lens and / or the target surface to move multiple times near the coarse focus position, and obtaining a new focus position and a clarity score of an image taken by the imaging component for the target surface corresponding to the new focus position after each movement; A clarity mapping curve is determined based on the multiple focus positions and the multiple clarity scores, and then the objective lens and / or the target surface is moved so that the focus position is at a fine focus position, thereby enabling the imaging component to obtain a clear image of the target surface, and the clarity score at the fine focus position is determined based on the peak of the clarity mapping curve.

2. The focusing method according to claim 1, characterized in that: The step of controlling the microscope system to perform a coarse focusing operation so that the focus position is at a coarse focusing position comprises: A first focusing light is output to the objective lens along the optical axis of the objective lens, the first focusing light can be focused into a point on the focal plane of the objective lens, in the process of the target surface moving from between the objective lens and the focal plane to the focal plane along the optical axis, the first focusing light forms an image on a first area on the target surface and the image size gradually decreases, in the process of the target surface moving from the focal plane to a direction away from the objective lens along the optical axis, the first focusing light forms an image on a second area on the target surface and the image size gradually increases, and the first area and the second area are axially symmetrical on the target surface; According to the change of the imaging of the first focusing light on the target surface during the movement of the target surface, the position of the target surface when the imaging of the first focusing light is a point is determined as the position of the focal plane, and the objective lens and / or the target surface is moved so that the target surface is at the position of the focal plane; Optionally, the step of controlling the microscope system to perform a coarse focus operation so that the focus position is at a coarse focus position comprises: Controlling the microscope system to image the target surface, and when the image of the target surface is not obtained, moving the objective lens and / or the target surface by a first set step length, and then imaging the target surface again, until the image of the target surface can be obtained; Optionally, the step of controlling the objective lens and / or the target surface to move multiple times near the coarse focus position, and obtaining a new focus position and a clarity score of an image taken by the imaging component for the target surface corresponding to the new focus position after each movement comprises: Controlling the objective lens and / or the target surface to move from the coarse focus position to a first set distance, and then controlling the objective lens and / or the target surface to move multiple times in the opposite direction according to a second set step length; Optionally, the second setting step length is greater than the depth of field of the objective lens; Optionally, the microscope system includes an automatic focusing assembly, and the focusing method includes: Controlling the auto-focusing assembly to send a second focusing light to the target surface along the optical axis of the objective lens, and receiving the second focusing light reflected from the target surface, and then outputting an electrical signal according to the received second focusing light, wherein the electrical signal is related to the focusing position; The step of controlling the objective lens and / or the target surface to move multiple times near the coarse focus position and obtaining a new focus position and a clarity score of an image taken by the imaging component for the target surface corresponding to the new focus position after each movement comprises: The objective lens and / or the target surface are controlled to move according to the electrical signal.

3. The focusing method according to claim 1, characterized in that: The step of determining a clarity mapping curve according to the plurality of focus positions and the plurality of clarity scores, and then moving the objective lens and / or the target surface so that the focus position is at a fine focus position comprises: Performing fitting processing on the plurality of focus positions and the plurality of clarity scores to obtain the clarity mapping curve; Controlling the objective lens and / or the target surface to move so that the focus position is at a fine focus position; Optionally, the step of fitting the change trends of the plurality of focus positions and the plurality of clarity scores to obtain the clarity mapping curve comprises: When the clarity mapping curve satisfies a set condition, determining to control the microscope system to re-execute the coarse focus operation and / or the fine focus operation; Optionally, the setting conditions include at least one of the following: When the number of the plurality of clarity scores obtained is greater than a set number, a peak of the clarity mapping curve is not fitted; When the objective lens and / or the target surface has moved to the maximum scanning distance, the peak of the clarity mapping curve is not fitted; The plurality of clarity scores obtained in sequence have a tendency to gradually decrease; The fluctuation amplitude of the clarity mapping curve is smaller than the set amplitude value, and the maximum value of the clarity score is smaller than the set score value; The clarity mapping curve has a plurality of peaks; Optionally, the step of performing fitting processing on the plurality of focus positions and the plurality of clarity scores to obtain the clarity mapping curve comprises: Performing first-order straight line fitting on a portion of the plurality of clarity scores showing an upward trend to obtain a first fitting straight line, performing first-order straight line fitting on a portion of the plurality of clarity scores showing a downward trend to obtain a second fitting straight line, and determining an intersection of the first fitting straight line and the second fitting straight line as a peak of the clarity mapping curve; Optionally, the step of performing fitting processing on the plurality of focus positions and the plurality of clarity scores to obtain the clarity mapping curve comprises: Performing a second-order parabola fitting on the multiple clarity scores to obtain a first fitting parabola, and determining the vertex of the first fitting parabola as the peak of the clarity mapping curve; Optionally, the step of performing fitting processing on the plurality of focus positions and the plurality of clarity scores to obtain the clarity mapping curve comprises: Acquire a plurality of fine focus combinations, wherein the fine focus combinations include a plurality of clarity scores obtained by moving the objective lens and / or the target surface by corresponding step lengths, and each of the fine focus combinations has a corresponding coarse focus position and a same imaging position; Taking the real best focal plane of the objective lens as the objective function, performing machine learning according to the multiple fine focus combinations to determine the weights of the multiple clarity scores in the clarity mapping curve, the position of the real best focal plane of the objective lens corresponding to the peak of the clarity mapping curve; After completing the machine learning, taking the plurality of clarity scores as input to output the peaks of the clarity mapping curve; Optionally, the focusing method comprises: The sharpness value of the image obtained by imaging is used as the clarity score; Optionally, the focusing method comprises: Performing evaluation processing on bright spots in the image obtained by imaging to determine the clarity score; Optionally, the evaluation process of the bright spots in the image obtained by imaging can be implemented by the following formula: Score=[(k1*k2-1)*CV-EV] / [(CV+EV) / (k1*k2)]; Among them, Score represents the clarity score, k1*k2 represents the part of the matrix composed of odd rows and odd columns in the pixel array of the image within the range of the bright spot, CV represents the central pixel value of the matrix corresponding to the bright spot, and EV represents the sum of the non-central pixel values ​​of the matrix corresponding to the bright spot.

4. The focusing method according to claim 1, characterized in that: The focusing method is applied in a sequencing-by-synthesis process, and the focusing method comprises: During the first round of base extension reaction, controlling the microscope system to sequentially perform the coarse focusing operation once and the fine focusing operation once; During each round of base extension reaction after the first round of base extension reaction, controlling the microscope system to perform the fine focusing operation once; Optionally, during each round of base extension reaction after the first round of base extension reaction, the step of controlling the microscope system to perform the fine focus operation once comprises: Controlling the objective lens and / or the target surface to move so that the focus position is at a fine focus position in a previous round of base extension reaction, and after the movement, determining a current clarity score as a first clarity score; After controlling the objective lens and / or the target surface to move in the reverse direction at a third set step length from the focus position corresponding to the first clarity score, performing the fine focus operation to determine a new fine focus position, and determining the clarity score corresponding to the new fine focus position as a second clarity score; When the first clarity score is less than the second clarity score, determining to use the current focus position as the fine focus position of the current round of base extension reaction; When the first clarity score is greater than the second clarity score, moving the objective lens and / or the target surface back to a fine focus position corresponding to the first clarity score; Optionally, the focusing method comprises: In the case that the focus position is not at the fine focus position in the previous round of base extension reaction, controlling the microscope system to perform the coarse focus operation once, and after completion, controlling the objective lens and / or the target surface to move to determine the second clarity score; Optionally, the microscopic device comprises a driving assembly for driving the objective lens and / or the target surface to move along the optical axis of the objective lens; The focusing method comprises: After determining the first clarity score, detecting whether a difference between a first voltage value and a second voltage value is less than or equal to a first set value, the first voltage value being a voltage value output when the drive component controls the objective lens and / or the target surface to move to a focus position corresponding to the first clarity score in the previous round of base extension reaction, and the second voltage value being a voltage value output when the drive component controls the objective lens and / or the target surface to move to a focus position corresponding to the first clarity score in the current round of base extension reaction; When the difference is less than or equal to the first set value, controlling the objective lens and / or the target surface to move to determine the second clarity score; In the case where the difference is greater than the first set value, controlling the microscope system to perform the coarse focusing operation once, and after the operation is completed, controlling the objective lens and / or the target surface to move to determine the second clarity score; Optionally, the step of controlling the objective lens and / or the target surface to move in the reverse direction from the focus position corresponding to the first clarity score by a third set step length, performing the fine focus operation to determine a new fine focus position, and determining the clarity score corresponding to the new fine focus position as the second clarity score comprises: Acquiring a third voltage value, wherein the third voltage value is a voltage value output when the driving component controls the objective lens and / or the target surface to move to a position corresponding to the new fine focus position in the current round of base extension reaction; When the third voltage value is less than the second set value, determining the clarity score corresponding to the new fine focus position as the second clarity score; When the third voltage value is greater than or equal to the second set value, the drive component is subjected to voltage zeroing processing, and then the objective lens and / or the target surface is controlled to move in the reverse direction from the focusing position corresponding to the first clarity score at the third set step size, and then the fine focusing operation is re-executed until the third voltage value obtained is less than the second set value.

5. A microscope system, characterized in that: The microscopic system includes an objective lens, an imaging component for imaging a target surface, and a control module, wherein the control module is used to: Controlling the microscope system to perform a coarse focusing operation so that the focusing position is at a coarse focusing position, wherein the focusing position is a relative position between the objective lens and the target surface; Controlling the microscope system to perform a fine focusing operation, including: Controlling the objective lens and / or the target surface to move multiple times near the coarse focus position, and obtaining a new focus position and a clarity score of an image taken by the imaging component for the target surface corresponding to the new focus position after each movement; A clarity mapping curve is determined based on the multiple focus positions and the multiple clarity scores, and then the objective lens and / or the target surface is moved so that the focus position is at a fine focus position, thereby enabling the imaging component to obtain a clear image of the target surface, and the clarity score at the fine focus position is determined based on the peak of the clarity mapping curve.

6. The microscope system according to claim 5, characterized in that: The control module is used for: A first focusing light is output to the objective lens along the optical axis of the objective lens, the first focusing light can be focused into a point on the focal plane of the objective lens, in the process of the target surface moving from between the objective lens and the focal plane to the focal plane along the optical axis, the first focusing light forms an image on a first area on the target surface and the image size gradually decreases, in the process of the target surface moving from the focal plane to a direction away from the objective lens along the optical axis, the first focusing light forms an image on a second area on the target surface and the image size gradually increases, and the first area and the second area are axially symmetrical on the target surface; According to the change of the imaging of the first focusing light on the target surface during the movement of the target surface, the position of the target surface when the imaging of the first focusing light is a point is determined as the position of the focal plane, and the objective lens and / or the target surface is moved so that the target surface is at the position of the focal plane; Optionally, the control module is used to: Controlling the microscope system to image the target surface, and when the image of the target surface is not obtained, moving the objective lens and / or the target surface by a first set step length, and then imaging the target surface again, until the image of the target surface can be obtained; Optionally, the control module is used to: Controlling the objective lens and / or the target surface to move from the coarse focus position to a first set distance, and then controlling the objective lens and / or the target surface to move multiple times in the opposite direction according to a second set step length; Optionally, the second setting step length is greater than the depth of field of the objective lens; Optionally, the microscope system includes an autofocus component, and the control module is used to: Controlling the auto-focusing assembly to send a second focusing light to the target surface along the optical axis of the objective lens, and receiving the second focusing light reflected from the target surface, and then outputting an electrical signal according to the received second focusing light, wherein the electrical signal is related to the focusing position; The objective lens and / or the target surface are controlled to move according to the electrical signal.

7. The microscope system according to claim 5, characterized in that: The control module is used for: Performing fitting processing on the plurality of focus positions and the plurality of clarity scores to obtain the clarity mapping curve; Controlling the objective lens and / or the target surface to move so that the focus position is at a fine focus position; Optionally, the control module is used to: When the clarity mapping curve satisfies a set condition, determining to control the microscope system to re-execute the coarse focus operation and / or the fine focus operation; Optionally, the setting conditions include at least one of the following: When the number of the plurality of clarity scores obtained is greater than a set number, a peak of the clarity mapping curve is not fitted; When the objective lens and / or the target surface has moved to the maximum scanning distance, the peak of the clarity mapping curve is not fitted; The plurality of clarity scores obtained in sequence have a tendency to gradually decrease; The fluctuation amplitude of the clarity mapping curve is smaller than the set amplitude value, and the maximum value of the clarity score is smaller than the set score value; The clarity mapping curve has a plurality of peaks; Optionally, the control module is used to: Performing first-order straight line fitting on a portion of the plurality of clarity scores showing an upward trend to obtain a first fitting straight line, performing first-order straight line fitting on a portion of the plurality of clarity scores showing a downward trend to obtain a second fitting straight line, and determining an intersection of the first fitting straight line and the second fitting straight line as a peak of the clarity mapping curve; Optionally, the control module is used to: Performing a second-order parabola fitting on the multiple clarity scores to obtain a first fitting parabola, and determining the vertex of the first fitting parabola as the peak of the clarity mapping curve; Optionally, the control module is used to: Acquire a plurality of fine focus combinations, wherein the fine focus combinations include a plurality of clarity scores obtained by moving the objective lens and / or the target surface by corresponding step lengths, and each of the fine focus combinations has a corresponding coarse focus position and a same imaging position; Taking the real best focal plane of the objective lens as the objective function, performing machine learning according to the multiple fine focus combinations to determine the weights of the multiple clarity scores in the clarity mapping curve, the position of the real best focal plane of the objective lens corresponding to the peak of the clarity mapping curve; After completing the machine learning, taking the plurality of clarity scores as input to output the peaks of the clarity mapping curve; Optionally, the control module is used to: The sharpness value of the image obtained by imaging is used as the clarity score; Optionally, the control module is used to: Performing evaluation processing on bright spots in the image obtained by imaging to determine the clarity score; Optionally, the evaluation process of the bright spots in the image obtained by imaging can be implemented by the following formula: Score=[(k1*k2-1)*CV-EV] / [(CV+EV) / (k1*k2)]; Among them, Score represents the clarity score, k1*k2 represents the part of the matrix composed of odd rows and odd columns in the pixel array of the image within the range of the bright spot, CV represents the central pixel value of the matrix corresponding to the bright spot, and EV represents the sum of the non-central pixel values ​​of the matrix corresponding to the bright spot.

8. The microscope system according to claim 5, characterized in that: The microscopic system is applied in the process of sequencing by synthesis, and the control module is used for: During the first round of base extension reaction, controlling the microscope system to sequentially perform the coarse focusing operation once and the fine focusing operation once; During each round of base extension reaction after the first round of base extension reaction, controlling the microscope system to perform the fine focusing operation once; Optionally, the control module is used to: Controlling the objective lens and / or the target surface to move so that the focus position is at a fine focus position in a previous round of base extension reaction, and after the movement, determining a current clarity score as a first clarity score; After controlling the objective lens and / or the target surface to move in the reverse direction at a third set step length from the focus position corresponding to the first clarity score, performing the fine focus operation to determine a new fine focus position, and determining the clarity score corresponding to the new fine focus position as a second clarity score; When the first clarity score is less than the second clarity score, determining to use the current focus position as the fine focus position of the current round of base extension reaction; When the first clarity score is greater than the second clarity score, moving the objective lens and / or the target surface back to a fine focus position corresponding to the first clarity score; Optionally, the control module is used to: In the case that the focus position is not at the fine focus position in the previous round of base extension reaction, controlling the microscope system to perform the coarse focus operation once, and after completion, controlling the objective lens and / or the target surface to move to determine the second clarity score; Optionally, the microscopic device comprises a driving assembly for driving the objective lens and / or the target surface to move along the optical axis of the objective lens; The control module is used for: After determining the first clarity score, detecting whether a difference between a first voltage value and a second voltage value is less than or equal to a first set value, the first voltage value being a voltage value output when the drive component controls the objective lens and / or the target surface to move to a focus position corresponding to the first clarity score in the previous round of base extension reaction, and the second voltage value being a voltage value output when the drive component controls the objective lens and / or the target surface to move to a focus position corresponding to the first clarity score in the current round of base extension reaction; When the difference is less than or equal to the first set value, controlling the objective lens and / or the target surface to move to determine the second clarity score; In the case where the difference is greater than the first set value, controlling the microscope system to perform the coarse focusing operation once, and after the operation is completed, controlling the objective lens and / or the target surface to move to determine the second clarity score; Optionally, the control module is used to: Acquiring a third voltage value, wherein the third voltage value is a voltage value output when the driving component controls the objective lens and / or the target surface to move to a position corresponding to the new fine focus position in the current round of base extension reaction; When the third voltage value is less than the second set value, determining the clarity score corresponding to the new fine focus position as the second clarity score; When the third voltage value is greater than or equal to the second set value, the drive component is subjected to voltage zeroing processing, and then the objective lens and / or the target surface is controlled to move in the reverse direction from the focusing position corresponding to the first clarity score at the third set step size, and then the fine focusing operation is re-executed until the third voltage value obtained is less than the second set value.

9. A microscopic system, characterized in that: The microscope system includes a memory and a processor, the memory stores a computer program, and when the processor executes the computer program, the steps of the focusing method according to any one of claims 1 to 4 are implemented.

10. A computer-readable storage medium having a computer program stored thereon, characterized in that: When the computer program is executed by a processor, the steps of the focusing method according to any one of claims 1 to 4 are implemented.

Images