Method and equipment for testing repetitive positioning precision of chip platform

Abstract

The invention provides a method and equipment for testing repeatability positioning precision of a chip platform, and the method comprises the steps: controlling the chip platform and imaging equipment to carry out reciprocating relative movement for multiple times in the horizontal direction, and controlling the imaging equipment to photograph a calibration plate after the chip platform and the imaging equipment move to the same target position each time, obtaining multiple frames of target images; selecting a region of interest from the multiple frames of target images; the region of interest comprises a target marking region and a background region; for a region of interest in the multi-frame target image, identifying a boundary position formed between a target mark region and a background region in the region of interest according to the intensity values of at least part of pixel points in the region of interest; and according to the boundary position identified from the region of interest in the multiple frames of target images, determining the repetitive positioning precision during relative movement between the chip platform and the imaging equipment.

Description

Technical Field

[0001] The present disclosure relates to the technical field of gene sequencing, and more particularly, to a method and device for testing the repeatable positioning accuracy of a chip platform. Background Art

[0002] In the technical field of gene sequencing, the sequencing method based on optical microscopy imaging is the current mainstream sequencing method. In the sequencing method based on optical microscopy imaging, a specific fluorescent substance is attached to the gene fragment to be measured (for example, a DNA fragment) through a chemical reaction, and the fluorescent substance is excited by visible light (for example, generally red light, green light) to generate a fluorescent signal that can reflect the base characteristics of the gene fragment to be measured. Then, an imaging device (for example, generally an industrial camera) is used to collect the fluorescent signal and generate a fluorescent image. Subsequently, the image analysis software obtains the fluorescent image, extracts the gray value of the light spot in the fluorescent image, and identifies the base type corresponding to the light spot according to the gray value of the light spot, so as to determine the base sequence corresponding to the gene fragment to be measured.

[0003] The imaging device is integrated in the gene sequencing system. During the process of collecting the fluorescent image, the chip platform in the gene sequencing system and the imaging device move relative to each other along the length direction of the chip flow channel, so that the imaging device can collect the fluorescent image from each area of the flow channel. Therefore, in the second-generation gene sequencer, the accuracy requirements for the repeatable positioning of the chip platform and / or the imaging device in the length direction of the chip flow channel are extremely high.

[0004] The repeatable positioning error of the chip platform and / or the imaging device should be at the micron (μm) level, and this error cannot be distinguished by the naked eye. Therefore, a high-precision detection method is needed to distinguish it to determine whether the instrument meets the requirements of the sequencing experiment. Summary of the Invention

[0005] The embodiments of the present disclosure at least provide a method and device for testing the repeatable positioning accuracy of a chip platform.

[0006] In a first aspect, the embodiments of the present disclosure provide a method for testing repeatable positioning accuracy, including:

[0007] Controlling the relative movement between the chip platform and the imaging device along the horizontal direction for multiple reciprocations, and after each relative movement to the same target position, controlling the imaging device to take a picture of the calibration plate located on the chip platform to obtain multiple frames of target images; wherein, a target mark is set on the calibration plate; the multiple frames of target images are the images of the target mark;

[0008] Selecting a region of interest from the multiple frames of target images; wherein, the region of interest includes a target mark region and a background region;

[0009] For the region of interest in the multi-frame target images, based on the intensity values of at least some of the pixel points in the region of interest, identify the position of the boundary line formed between the target marker region and the background region in the region of interest;

[0010] Based on the position of the boundary line identified from the region of interest in the multi-frame target images, determine the repeatable positioning accuracy when the chip platform and the imaging device move relative to each other.

[0011] Optionally, the method further includes:

[0012] Before controlling the imaging device to take pictures of the calibration board placed on the chip platform to obtain multi-frame target images, aiming to maximize the clarity of the target images, perform focusing processing on the imaging device so that the calibration board is located on the focal plane of the imaging device.

[0013] Optionally, the performing focusing processing on the imaging device so that the calibration board is located on the focal plane of the imaging device includes:

[0014] Control the relative movement between the chip platform and the imaging device in the vertical direction, and after each relative movement to a new position, control the imaging device to take pictures of the calibration board to obtain multi-frame focusing images at different positions;

[0015] Determine the clarity corresponding to each of the multi-frame focusing images;

[0016] Based on the clarity corresponding to each of the multi-frame focusing images, determine the target shooting position of the imaging device when the calibration board is located on the focal plane of the imaging device.

[0017] Optionally, the determining the clarity corresponding to each of the multi-frame focusing images includes:

[0018] For the multi-frame focusing images, determine the sum of the squares of the intensity differences between at least some of the pixel points in the focusing image and their horizontally adjacent pixel points; and

[0019] Determine the mean value of the sum of the squares of the intensity differences between at least some of the pixel points in the focusing image and their horizontally adjacent pixel points;

[0020] Determine the mean value of the sum of the squares of the intensity differences as the clarity of the focusing image.

[0021] Optionally, the based on the clarity corresponding to each of the multi-frame focusing images, determining the target shooting position of the imaging device when the calibration board is located on the focal plane of the imaging device includes:

[0022] Performing fitting processing on the sharpness corresponding to the multiple-frame focus images respectively to obtain the corresponding relationship between the photographing position of the imaging device and the sharpness of the focus image;

[0023] Taking maximizing the sharpness as the goal, based on the corresponding relationship, determining the photographing position corresponding to the maximum value of the sharpness. The photographing position corresponding to the maximum value of the sharpness is the target photographing position of the imaging device when the calibration plate is located on the focal plane of the imaging device.

[0024] Optionally, the identifying the position of the boundary line formed between the target marking area and the background area in the region of interest according to the intensity values of at least some pixel points in the region of interest includes:

[0025] Determining a plurality of detection windows in the region of interest along the length direction of the region of interest at a preset step size; wherein, the length and width of each detection window are equal to or less than the width of the region of interest;

[0026] For the plurality of detection windows, determining the average pixel intensity of at least some pixel points in the detection window;

[0027] Determining the position of the boundary line according to the average pixel intensity corresponding to each adjacent two detection windows respectively.

[0028] Optionally, the determining the position of the boundary line according to the average pixel intensity corresponding to each adjacent two detection windows respectively includes:

[0029] Determining target adjacent detection windows from each adjacent two detection windows; the difference between the average pixel intensities corresponding to the target adjacent detection windows is greater than a preset difference threshold; and / or, the average pixel intensity of one detection window in the target adjacent detection windows is greater than a first preset threshold, and the average pixel intensity of the other detection window is less than a second preset threshold; the first preset threshold is greater than or equal to the second preset threshold;

[0030] Determining the position of the boundary line according to the position of the previous detection window in the region of interest among the target adjacent detection windows and the size of the detection window.

[0031] Optionally, it further includes:

[0032] Performing boundary sharpening processing on the region of interest by using Laplacian operator convolution to obtain the sharpened region of interest;

[0033] Determining a plurality of detection windows in the region of interest along the length direction of the region of interest at a preset step size includes:

[0034] Determine a plurality of the detection windows in the sharpened region of interest along the length direction of the region of interest according to the preset step size.

[0035] Optionally, the position of the demarcation line includes: the coordinate value of the pixel point on the demarcation line in the horizontal direction of relative movement;

[0036] Determining the repeatable positioning accuracy when the chip platform and the imaging device move relative to each other according to the position of the demarcation line recognized from the region of interest in multiple frames of target images includes:

[0037] Determine at least one of the standard deviation and the maximum difference of the coordinate values according to the coordinate values of the pixel points on the demarcation line recognized from the region of interest in multiple frames of target images in the horizontal direction of relative movement;

[0038] Use at least one of the standard deviation and the maximum difference as an index to measure the repeatable positioning accuracy.

[0039] In a second aspect, an alternative implementation of the present disclosure further provides a computer device, a processor, and a memory. The memory stores machine-readable instructions executable by the processor. The processor is configured to execute the machine-readable instructions stored in the memory. When the machine-readable instructions are executed by the processor, the machine-readable instructions execute the steps in the first aspect or any possible implementation manner in the first aspect when the machine-readable instructions are executed by the processor.

[0040] In a third aspect, an alternative implementation of the present disclosure further provides a computer-readable storage medium. A computer program is stored on the computer-readable storage medium. When the computer program is run, it executes the steps in the first aspect or any possible implementation manner in the first aspect.

[0041] In the embodiment of the present disclosure, by controlling the reciprocating relative movement between the chip platform and the imaging device in the horizontal direction, and after each relative movement to the same target position, controlling the imaging device to take a picture of the calibration board on the chip platform to obtain multiple frames of target images; a target mark is set on the calibration board, and the multiple frames of target images are images of the target mark. Then, a region of interest is selected from the multiple frames of target images, and for the region of interest in the multiple frames of target images, according to the intensity values of at least some pixel points in the region of interest, the position of the demarcation line formed between the target mark region and the background region in the region of interest is recognized, and then the repeatable positioning accuracy when the chip platform and the imaging device move relative to each other is determined by using the position of the demarcation line recognized from the region of interest in the multiple frames of target images. Thus, the repeatable positioning accuracy when the chip platform and the imaging device move relative to each other can be accurately recognized by using the calibration board and the target mark set on the calibration board, so as to determine whether the instrument meets the requirements of the sequencing experiment.

[0042] To make the above objects, features, and advantages of the present disclosure more obvious and understandable, the following provides preferred embodiments in conjunction with the accompanying drawings and makes a detailed description as follows. Description of the Drawings

[0043] To more clearly illustrate the technical solutions of the embodiments of the present disclosure, the following briefly introduces the drawings required for use in the embodiments. The drawings herein are incorporated into the specification and constitute a part of this specification. These drawings show embodiments that conform to the present disclosure and are used together with the specification to illustrate the technical solutions of the present disclosure. It should be understood that the following drawings only show some embodiments of the present disclosure and should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other relevant drawings can also be obtained based on these drawings.

[0044] Figure 1 Shows a schematic diagram of a sequencing system provided by some embodiments of the present disclosure;

[0045] Figure 2 Shows a schematic diagram of a sequencing chip provided by some embodiments of the present disclosure;

[0046] Figure 3 Shows a schematic diagram of an optical detection system provided by some embodiments of the present disclosure;

[0047] Figure 4 Shows a flowchart of a test method for the repeat positioning accuracy of a chip platform provided by some embodiments of the present disclosure;

[0048] Figure 5 Shows one of the examples of setting a calibration plate on a sequencing chip provided by some embodiments of the present disclosure;

[0049] Figure 6 Shows a flowchart of a specific method for a target camera to perform focusing processing and determine the target photographing position of the camera photographing image provided by some embodiments of the present disclosure;

[0050] Figure 7 Shows a second specific example of a pixel point and its horizontally adjacent pixel points provided by some embodiments of the present disclosure;

[0051] Figure 8 Shows a third specific example of a parabola representing the relationship between the photographing position and clarity provided by some embodiments of the present disclosure;

[0052] Figure 9 Shows a fourth example of selecting a region of interest in an image provided by some embodiments of the present disclosure;

[0053] Figure 10Shows Example 5 of determining multiple detection windows in the region of interest provided by some embodiments of the present disclosure;

[0054] Figure 11 Shows a schematic diagram of a computer device provided by some embodiments of the present disclosure. Detailed implementation manners

[0055] To make the objectives, technical solutions, and advantages of the embodiments of the present disclosure clearer, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present disclosure. Apparently, the described embodiments are only some of the embodiments of the present disclosure, rather than all of the embodiments. Usually, the components of the embodiments of the present disclosure described and illustrated here can be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present disclosure is not intended to limit the scope of the present disclosure to be protected, but merely represents the selected embodiments of the present disclosure. All other embodiments obtained by those skilled in the art based on the embodiments of the present disclosure without creative efforts belong to the scope of protection of the present disclosure.

[0056] To facilitate the understanding of the technical solutions of the present disclosure, the technical terms in the embodiments of the present disclosure will be explained first:

[0057] Library construction:

[0058] The genomic DNA or RNA molecules to be sequenced are fragmented by physical or chemical means. For example, they are fragmented by ultrasonic waves. After fragmentation, DNA or RNA fragments are formed. First, the ends of the DNA or RNA fragments are filled in with enzymes, and then specific enzymes are used to ligate specific DNA or RNA sequences (usually, this specific DNA sequence or RNA is also called an adapter) to both ends of the fragments, forming a mixture of DNA or RNA. This mixture of DNA or RNA is also called a library in the industry.

[0059] To save the sequencing cost, generally, multiple samples are sequenced simultaneously in a sequencer. To distinguish the sequencing results of different samples, when preparing the libraries of different samples, a DNA or RNA sequence (usually containing 6-8 bases) that can identify the source of the sample is included in the adapter. This DNA or RNA sequence that can identify the source of the sample can also be called a sample tag (or Index, Barcode). It can be understood that each sample library adapter contains its exclusive sample tag.

[0060] Usually, library construction is completed outside the sequencer. For example, libraries are obtained through experimental operations or automated equipment in a laboratory.

[0061] Amplification reaction:

[0062] Taking the amplification of a DNA library by bridge PCR (Polymerase Chain Reaction) as an example, after the library is constructed, the library can be inoculated onto a sequencing chip and amplified on the sequencing chip. Among them, the adapters at both ends of the library are complementary to the first amplification primer on the sequencing chip. Therefore, the library can be inoculated onto the sequencing chip through complementary hybridization.

[0063] After the library is inoculated onto the sequencing chip, the amplification reaction can be carried out using this library as the template strand. For example, the process of the amplification reaction can be to first add dNTPs and polymerase into the sequencing chip. The polymerase will start from the first amplification primer and synthesize a new DNA strand along the template strand. The new DNA strand is completely complementary to the template strand, so it is also called the complementary strand of the template strand. The complementary strand is covalently linked to the sequencing chip. Next, add NaOH alkaline solution to the sequencing chip for rinsing. In the presence of the NaOH alkaline solution, the template strand and the complementary strand are separated from each other, and the template strand is washed away with the alkaline solution, while the complementary strand covalently linked to the sequencing chip is retained. Then add a neutral liquid to the sequencing chip to neutralize the NaOH alkaline solution. The entire environment inside the sequencing chip becomes neutral, and the other end on the complementary strand will continue to undergo complementary hybridization with the second amplification primer on the sequencing chip. Add dNTPs and polymerase, and the polymerase will start from the second amplification primer and synthesize a brand-new DNA strand along the complementary strand. At this time, the new DNA strand is completely complementary to the complementary strand and identical to the template strand. Then add NaOH alkaline solution to separate the two strands from each other. In this way, two strands that are covalently linked to the sequencing chip and complementary to each other can be obtained. Repeat this process, and the number of DNA strands will increase exponentially.

[0064] After amplification, both DNA double strands identical to the template strand and the complementary strand will be retained on the sequencing chip. Then add specific reaction reagents to the sequencing chip to cut the DNA strand synthesized from one of the amplification primers. For example, cut the DNA strand identical to the complementary strand and retain the DNA strand identical to the template strand. Then add NaOH alkaline solution to the sequencing chip for rinsing. The alkaline solution separates the DNA double strands from each other, and the cut DNA strand will also be washed away with the alkaline solution. Eventually, only single-stranded DNA is retained on the sequencing chip, and the number of single-stranded DNA retained on the sequencing chip at this time is an exponential multiple of that at the beginning of the amplification, thus forming a DNA cluster, and all the single-stranded DNAs in a DNA cluster are the same. Then add a neutral solution, and the sequencing of all the single-stranded DNAs in this DNA cluster can be carried out in the neutral solution environment.

[0065] It should be noted that the above amplification reaction can be completed outside the sequencer, such as amplifying the library through experimental operations in the laboratory, or it can be completed inside the sequencer. When the amplification reaction is completed inside the sequencer, the library and various reaction reagents (for example, dNTP, polymerase, NaOH alkaline solution, neutral solution, etc.) participating in the amplification reaction can be added to the sequencing chip through the liquid path system of the sequencer or the liquid path system. In addition, the above amplification reaction is only exemplary. The present application is not limited to using the bridge PCR amplification method, and other amplification methods can also be used for amplification, for example, loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), multiplex probe amplification (MPA), etc.

[0066] Sequencing reaction:

[0067] Taking the principle of sequencing by synthesis as an example, during sequencing, 4 types of dNTPs with fluorescent groups are added to the sequencing chip through the liquid path system. Each type of dNTP can only be synthesized with one of the four bases ATCG, and the 3'-end of the dNTP has been blocked by a blocking group (the blocking group includes but is not limited to azide group). Then, polymerase is added to the sequencing chip through the liquid path system. Through the action of the polymerase, one of the 4 types of dNTPs will be synthesized with the complementary base on the single-strand to be sequenced. Since the 3'-end of the dNTP is blocked by a blocking group, only one dNTP can be extended on the single-strand to be sequenced each time. After synthesis, a specific chemical reagent is added to the sequencing chip through the liquid path system to flush away the excess dNTP and polymerase. Next, the fluorescent group of the dNTP synthesized onto the single-strand can be excited by the optical detection system, causing the fluorescent group to emit a fluorescent signal. Since the fluorescent groups of the dNTPs on each single-strand in a cluster will emit the same fluorescent signal, which plays a role in amplifying the fluorescent signal, the optical detection system can collect the fluorescent signal and generate a fluorescent image.

[0068] The computer system processes and analyzes the fluorescent image to determine which type of dNTP has been synthesized onto the single-strand to be sequenced. Then, based on the complementary principle, it can be deduced which base on the single-strand to be sequenced has been synthesized with the dNTP. Thus, one sequencing cycle is completed.

[0069] Next, specific chemical reagents are added to the sequencing chip through the liquid path system to cut off the blocking group and the fluorescent group, and then the hydroxyl group at the 3' end of the dNTP is exposed.

[0070] Next, enter the next sequencing cycle and repeat the above process.

[0071] It can be understood that one sequencing cycle can detect one base. After multiple sequencing cycles, multiple bases in the single-stranded DNA to be sequenced can be detected. Specifically, the number of sequencing cycles can be determined according to the set sequencing read length. For example, 150 or 300 sequencing cycles.

[0072] Of course, it should also be noted that the above sequencing reaction is only exemplary. The present application is not limited to using the principle of sequencing by synthesis, and other sequencing principles can also be used.

[0073] Sequencing system:

[0074] Refer to Figure 1 As shown, the sequencing system includes: a sequencing chip 10, a chip platform 20, a reagent storage container 30, a liquid path system (or diversion system) 40, an optical detection system 50, a computer system 60, and a waste liquid storage container 70. Among them:

[0075] The sequencing chip 10 is configured to provide a reaction area for the amplification reaction and the sequencing reaction;

[0076] The chip platform 20 is configured to fix and support the sequencing chip 10;

[0077] The reagent storage container 30 is configured to store one or more mixed sample libraries and one or more reagents;

[0078] The liquid path system 40 is configured to controllably transport one or more mixed sample libraries and one or more reagents from the reagent storage container 30 to the sequencing chip 10 for the amplification reaction and the sequencing reaction in the sequencing chip 10, and controllably transport the waste liquid after the reaction from the sequencing chip 10 to the waste liquid storage container 70;

[0079] The optical detection system 50 is configured to excite and collect fluorescence signals during the sequencing reaction and generate a fluorescence image according to the fluorescence signals;

[0080] The computer system 60 is configured to obtain the fluorescence image from the optical detection system 50 and identify the base sequence of the sample library according to the fluorescence image;

[0081] The waste liquid storage container 70 is configured to store the waste liquid generated after the reaction.

[0082] Sequencing chip:

[0083] As a carrier for amplification reaction and sequencing reaction, the sequencing chip can provide a reaction area for these reactions, and this area is the channel. Generally, one sequencing chip 10 may contain one or more channels 11 (for example, 2, 4, 6, 8), and the channels are isolated from each other. Refer to Figure 2 As shown, taking 4 channels as an example, there is a small hole 12 at each end of each channel 11 for the inflow and outflow of fluid (such as biological samples, reaction reagents). The upper and lower surfaces inside each channel are chemically modified respectively, and two kinds of amplification primers are inoculated in a covalent bond manner, and these two kinds of amplification primers are respectively complementary to the adapters at both ends of the library to achieve the amplification of the library.

[0084] Optical detection system:

[0085] Refer to Figure 3 As shown, taking the dual-color channel as an example, the optical detection system 50 at least includes a light source component and an imaging component. Among them, the light source component at least includes light sources (5101, 5102), field stop sheets (5201, 5202), dichroic mirror 5301 and filter element 540.

[0086] Here, in one implementation, the light sources (5101, 5102) can be light-emitting diodes (LEDs). The LED is an aspherical mirror and can diverge the LED point light source into parallel light. Of course, in addition to LEDs, other forms of point or surface light sources can also be used. In another implementation, a collimating element ( Figure 3 not shown in the figure) can also be provided behind the light sources (5101, 5102) to collimate the light emitted by the light sources (5101, 5102) into a parallel light beam. The collimating element can include one or more lenses, including but not limited to any one or any combination of single lenses, cemented lenses, spherical lenses, and aspherical lenses.

[0087] Parallel light beams are emitted through the field stop sheets (5201, 5202). The field stop sheets (5201, 5202) define the field of view range of the light emitted from the light sources (5101, 5102), and thus define the field of view range of the excitation light irradiated onto the sequencing chip 10. The light passing through the field stop sheets (5101, 5102) reaches the dichroic mirror 5301. The dichroic mirror 5301 can transmit the light emitted by one of the light sources (5101, 5102) and reflect the light emitted by the other light source. The light passing through the dichroic mirror 5301 further passes through the filter element 540. The filter element 540 allows the light of a certain wavelength band in the light emitted by the light sources (5101, 5102) to pass through and serves as the excitation light, while blocking the light of other wavelength bands. For example, it blocks the light of the same wavelength band as the fluorescence emitted by the fluorescent group, ensuring that the fluorescence emitted by the fluorescent group does not contain stray light introduced by the light sources (5101, 5102), which helps to improve the optical imaging effect.

[0088] In one embodiment, the dichroic mirror 5301 can be fixed at a certain angle in an inclined manner. Exemplarily, the dichroic mirror 5301 is fixed by dispensing glue. In another implementation, the field stop sheets (5201, 5202) can be, but are not limited to, rectangular diaphragms or circular diaphragms; the field stop sheets (5201, 5202) can be single-hole or multi-hole diaphragm sheets; the field stop sheets (5201, 5202) can be made of light-impermeable materials. Exemplarily, they can be metal sheets.

[0089] Here, it should be noted that in the optical detection system 50, the light sources (5101, 5102) emit light alternately, not simultaneously. By operating the light sources (5101, 5102) in a time-sharing manner (that is, the light emitted by the light sources 5101 and 5102 has different wavelengths and is turned on alternately during use), only one light source is turned on at a time, which will reduce the optical power of the excitation light irradiated onto the chip and is beneficial to protecting the fluorescence lifetime of the fluorescent group.

[0090] In addition, in order to obtain the best fluorescence image through the imaging component, the imaging component also needs to be focused. The light source component further includes a light source 5103, a field stop sheet 5203, an attenuation sheet 550, and a dichroic mirror 5302. In one embodiment, considering that using LED light as the excitation light for focusing will cause damage to the DNA strands to be sequenced on the sequencing chip 10 and affect the subsequent sequencing quality, the light source 5103 is, for example, a semiconductor laser (Laser Diode, LD) and emits laser light for focusing the imaging component.

[0091] When focusing on the imaging component, the laser emitted by the semiconductor laser passes through the field stop 5203. The field stop 5203 defines the field of view range of the laser emitted by the semiconductor laser, and thus defines the field of view range of the excitation light irradiated on the sequencing chip 10. The field stop 5203 can be a single-hole or multi-hole aperture stop. The field stop 5203 can be made of light-impermeable material. Exemplarily, it can be a metal sheet. The laser can only pass through the light-passing hole part of the corresponding field stop, and for the non-light-passing hole part of the field stop, the laser will be blocked. The laser passing through the field stop 5203 reaches the attenuation sheet 550. After being attenuated by the attenuation sheet 550, the laser reaches the dichroic mirror 5302. The dichroic mirror 5302 can transmit the laser and reflect the LED light. The dichroic mirror 5302 can be fixed at a certain angle, for example, by dispensing. The laser transmitted by the dichroic mirror 5302 or the LED light reflected by it reaches the convex lens 560, and the convex lens 560 collimates the laser and the LED light into parallel light beams.

[0092] The imaging component at least includes a dichroic mirror 5303, an objective lens 570, a tube lens 580, and an image sensor 590. The laser and the LED light collimated into parallel light beams pass through the dichroic mirror 5303. The dichroic mirror 5303 reflects the laser and the LED light in the direction of the sequencing chip 10. The reflected laser and LED light irradiate the sequencing chip 10 through the objective lens 570 and excite the fluorophore to emit a fluorescence signal. The objective lens 570 collects the fluorescence signal. The fluorescence signal reaches the dichroic mirror 5303 through the objective lens 570. The dichroic mirror 5303 transmits the fluorescence signal to the tube lens 580. The tube lens 580 projects the fluorescence signal onto the image sensor 590. The image sensor 590 collects the fluorescence signal and generates a fluorescence image. The computer system 60 identifies the base sequence based on the fluorescence image. In one embodiment, a filter element ( Figure 3 not shown in the figure) can be provided between the tube lens 580 and the dichroic mirror 5303, and a filter element ( Figure 3 not shown in the figure) can be provided between the tube lens 580 and the image sensor 590.

[0093] In one embodiment, the image sensor 590 can be an industrial camera, for example. In another embodiment, the collimating element can include one or more lenses, including but not limited to any one or any combination of single lenses, cemented lenses, spherical lenses, and aspherical lenses.

[0094] To achieve focusing on the imaging component, a motor 600 is arranged on one side of the objective lens 570. In one implementation, the motor can be, for example, a voice coil motor or other linear motor. Based on the image quality of the fluorescence image recognized by the computer system 60, the voice coil motor can be controlled by a driver to drive the objective lens 570 to move up and down, so that the objective lens 570 reaches the optimal focal plane and finally obtains the optimal fluorescence image.

[0095] In a next-generation sequencing gene sequencer, it is necessary to identify the base types in the gene sequence through the collected fluorescence images. During the process of collecting fluorescence images, the chip platform and the imaging device (i.e., the imaging component in the optical detection system 50) move relative to each other along the length direction of the chip flow channel, so that the imaging device can collect fluorescence images from various regions of the flow channel. Therefore, in a next-generation sequencing gene sequencer, extremely high requirements are imposed on the repetitive positioning accuracy of the chip platform and / or the imaging device along the length direction of the chip flow channel.

[0096] The repetitive positioning error of the chip platform and / or the imaging device along the length direction of the chip flow channel should be at the um level, which cannot be distinguished by the naked eye. Therefore, a high-precision detection method is needed to distinguish the repetitive positioning accuracy of the chip platform to determine whether the instrument meets the requirements of the sequencing experiment.

[0097] Based on the above research, the present disclosure provides a method for testing the repetitive positioning accuracy between a chip platform and an imaging device, which can utilize a calibration plate and target marks arranged on the calibration plate, and through image recognition and processing, repeatedly locate the position information of the target marks in the image multiple times, and use the position information of the target marks repeatedly located in the image multiple times to accurately identify the repetitive positioning accuracy of the relative movement between the chip platform and the imaging device.

[0098] All the defects existing in the above solutions are the results obtained by the inventors through practice and careful research. Therefore, the process of discovering the above problems and the solutions proposed by the present disclosure for the above problems in the following text should be the contributions made by the inventors to the present disclosure during the process of the present disclosure.

[0099] It should be noted that similar reference numerals and letters indicate similar items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0100] For ease of understanding this embodiment, first, a test method for repeat positioning accuracy disclosed in the embodiments of the present disclosure will be introduced in detail. The execution subject of the test method for repeat positioning accuracy provided in the embodiments of the present disclosure is generally a computer device with certain computing capabilities. Such a computer device includes, for example, a computer system 60. In some possible implementation manners, the test method for repeat positioning accuracy can be implemented by a processor calling computer-readable instructions stored in a memory.

[0101] The following describes the test method for repeat positioning accuracy provided in the embodiments of the present disclosure.

[0102] See Figure 4 As shown, it is a flowchart of the test method for repeat positioning accuracy provided in the embodiments of the present disclosure. The method includes steps S401 to S404, where:

[0103] S401: Control the reciprocating relative movement between the chip platform and the imaging device in the horizontal direction for multiple times. After each relative movement to the same target position, control the imaging device to take pictures of the calibration plate located on the chip platform to obtain multiple frames of target images. Among them, target marks are set on the calibration plate, and the multiple frames of target images are images of the target marks.

[0104] During the sequencing process, the imaging device can be kept stationary, and only the chip platform is controlled to move relative to the imaging device along the length direction of the chip flow channel (for example, the X direction in Figure 2 ), or the chip platform can be kept stationary, and only the imaging device is controlled to move relative to the chip platform along the length direction of the chip flow channel, or both the chip platform and the imaging device are controlled to move relative to each other along the length direction of the chip flow channel, so that the imaging device can take pictures of different regions of the chip flow channel. Similarly, when detecting the repeat positioning accuracy between the chip platform and the imaging device, in specific implementation, the imaging device can also be kept stationary, and the chip platform is controlled to move relative to the imaging device along the length direction of the chip flow channel for multiple times, or the chip platform can be kept stationary, and the imaging device is controlled to move relative to the chip platform along the length direction of the chip flow channel for multiple times, or both the imaging device and the chip platform can be controlled to move relative to each other along the length direction of the chip flow channel for multiple times.

[0105] The calibration plate is a reference object used to calibrate the repeatable positioning accuracy of the chip platform. On the calibration plate, there are target marks that can emit fluorescence signals and reflect external light under the excitation of excitation light. Other areas on the calibration plate except the target marks cannot emit fluorescence signals. Therefore, when obtaining the target image of the calibration plate placed on the chip platform through an imaging device, for example, under the irradiation condition of excitation light, the target image of the calibration plate can be obtained, so that there is a more obvious intensity difference between the area where the target mark is located (i.e., the target mark area) and the background area in the target image of the calibration plate, so as to improve the calibration accuracy when calibrating the repeatable positioning accuracy of the relative movement between the chip platform and the imaging device using the calibration plate.

[0106] The shape and size of the calibration plate can be, for example, the same as those of the sequencing chip, so that the calibration plate can be directly assembled on the chip platform. The assembly method of the calibration plate on the chip platform is usually the same as that of the sequencing chip on the chip platform.

[0107] Similar to the sequencing chip, one or more flow channels (for example, four flow channels) are provided on the calibration plate, and a target mark can be provided in one of the flow channel areas. The target mark can be imaged, for example, through excitation light, and the imaging is a specific pattern (such as a rectangle), and is used to calibrate the repeatable positioning accuracy of the relative movement between the chip platform and the imaging device.

[0108] In multiple reciprocating relative movements, each time when moving to the same position relative to each other, this position is recorded as the target position, and the target mark on the calibration plate is imaged through the imaging device to obtain the target image.

[0109] The shape of the target mark is, for example, a rectangle, such as a square or a rectangle; when the calibration plate is fixed on the chip platform, one side of the target mark is perpendicular to the length direction of the chip flow channel, and at this time, the other side adjacent to this side is parallel to the length direction of the chip flow channel.

[0110] As Figure 5 shown, the embodiments of the present disclosure show a specific example of the calibration plate. In this example, the size and shape of the calibration plate 51 are the same as or approximate to those of the Figure 2 shown sequencing chip; and a target mark s1 is provided in the second flow channel area on the left side of the calibration plate. As shown in the enlarged view corresponding to a partial area of the second flow channel, the target mark can be, for example, a rectangle, and one side of the rectangle is perpendicular to the extending direction of the flow channel.

[0111] The following takes the example of controlling the chip platform to move reciprocally relative to the imaging device for illustration. For example, controlling the chip platform to perform multiple reciprocating movements along the length direction of the flow channel. As Figure 5As shown, the control chip platform makes multiple reciprocating movements along the x direction. In the following embodiments, Figure 5 the x direction in Figure 5 is the first direction;

[0112] The control chip platform moves reciprocally multiple times along the first direction relative to the imaging device, and after each movement to the same target position, the imaging device is used to obtain the target image of the calibration plate placed on the chip platform. In this case, theoretically speaking, the target mark should be located at a certain fixed position in the target image. However, due to certain errors in the relative movement between the chip platform and the imaging device, there will be a difference between the actual position of the target mark in the target image and its theoretically fixed position.

[0113] When using the imaging device to take pictures of the calibration plate to obtain the target image, if the position of the imaging device relative to the calibration plate is too close or too far, it will cause the problem of decreased clarity of the target image. Therefore, before controlling the imaging device to take pictures of the calibration plate placed on the chip platform and obtaining multiple frames of target images, the imaging device can be pre-focused to ensure the best clarity of the target image. In another alternative embodiment of the present disclosure, the imaging device can be focused with the goal of maximizing the clarity of the target image, so that the calibration plate is located on the focal plane of the imaging device.

[0114] Specifically, referring to Figure 6 shown, the present disclosure provides a specific method for focusing the imaging device, including:

[0115] S601: Control the relative movement between the chip platform and the imaging device along the vertical direction, and after each relative movement to a new position, control the imaging device to take pictures of the calibration plate to obtain multiple frames of focus images at different positions.

[0116] Here, after placing the calibration plate on the chip platform, the relative movement between the imaging device and the chip platform in the z direction (perpendicular to the xy plane) can be controlled, and after each movement by a certain displacement (i.e., relative movement to a new position), the imaging device is controlled to take pictures of the calibration plate to obtain the focus image. By taking pictures multiple times, multiple frames of focus images are obtained.

[0117] Among them, the content in multiple frames of focus images is usually the same, but the clarity is different.

[0118] In specific implementation, it can be to keep the imaging device stationary and control the chip platform to move relative to the imaging device along the z direction, or, alternatively, keep the chip platform stationary and control the imaging device to move relative to the chip platform along the z direction, or, further, control both the imaging device and the chip platform to move relative to each other along the z direction.

[0119] S602: Determine the sharpness corresponding to each of the multiple-frame focus images;

[0120] Specifically, for example, the sharpness corresponding to any one of the focus images in the next-frame focus images can be adopted:

[0121] Determine the sum of the squares of the intensity differences between some or all of the pixel points in the focus image and their horizontally adjacent pixel points; and determine the average value of the sum of the squares of the intensity differences between some or all of the pixel points in the focus image and their horizontally adjacent pixel points;

[0122] Determine the average value of the sum of the squares of the intensity differences as the sharpness of the focus image.

[0123] In a specific implementation, the horizontally adjacent pixel points of a certain pixel point here are, for example, multiple pixel points in the x direction shown above and within a certain distance range from this pixel point. Figure 5 As shown in the example of

[0124] As Figure 7 shown, a specific example of a pixel point and its horizontally adjacent pixel points is shown. In this example, the maximum distance between pixel point A and its corresponding horizontally adjacent pixel points is 2, and the pixel point can have 4 adjacent pixel points, which are respectively Figure 7 a1 to a4 in

[0125] After that, calculate the intensity difference between pixel point A and horizontally adjacent pixel point a1, denoted as a11;

[0126] Calculate the intensity difference between pixel point A and horizontally adjacent pixel point a2, denoted as a12;

[0127] Calculate the intensity difference between pixel point A and horizontally adjacent pixel point a3, denoted as a13;

[0128] Calculate the intensity difference between pixel point A and horizontally adjacent pixel point a4, denoted as a14;

[0129] After that, calculate the sum of the squares b of a11, a12, a13, and a14, where b satisfies:

[0130] b = a11 2 + a12 2 + a13 2 + a14 2 .

[0131] Assume that there are k pixel points in the focus image, then the sums of the squares of the intensity differences between the k pixel points and their horizontally adjacent pixel points are respectively expressed as: b1, b2,..., bk.

[0132] The sharpness M of the focus image satisfies:

[0133] M = (b1 + b2 + …… + bk) / k.

[0134] S603: Determine the target photographing position of the imaging device when the calibration plate is located on the focal plane of the imaging device based on the sharpness corresponding to each of the multiple-frame focused images.

[0135] Specifically, when determining the target photographing position of the imaging device when the calibration plate is located on the focal plane of the imaging device based on the sharpness corresponding to the focused images respectively corresponding to multiple photographing positions, for example, the following method can be adopted:

[0136] Perform a fitting process on the sharpness corresponding to each of the multiple-frame focused images to obtain the correspondence between the photographing position of the imaging device and the sharpness of the focused image;

[0137] With the goal of maximizing the sharpness of the focused image, based on this correspondence, determine the photographing position corresponding to the maximum sharpness value. The photographing position corresponding to the maximum sharpness value is the target photographing position of the imaging device when the calibration plate is located on the focal plane of the imaging device.

[0138] It can be understood that the relationship between the photographing position of the imaging device and the sharpness of the focused image presents a parabola. Therefore, the obtained relationship information can usually be described by a parabola equation. Among them, the independent variable of this parabola equation is the photographing position (taking the control of the imaging device to move relative to the chip platform in the z direction as an example, that is, the position value of the imaging device in the z direction); the dependent variable of the parabola equation is the sharpness of the focused image.

[0139] After obtaining the above parabola equation, the vertex coordinates of the parabola equation can be obtained, and based on these vertex coordinates, the target photographing position can be determined.

[0140] As Figure 8 shown, the embodiments of the present disclosure provide a specific example of obtaining a parabola that can characterize the relationship between the photographing position and the sharpness by performing a fitting process on the sharpness corresponding to the focused images respectively corresponding to multiple photographing positions. The photographing position corresponding to the vertex of this parabola is the target photographing position.

[0141] After obtaining the target photographing position (that is, the optimal photographing position), when obtaining the target image on the calibration plate placed on the chip platform through the imaging device, the imaging device takes a picture of the chip platform at this target photographing position to obtain the target image on the calibration plate placed on the chip platform.

[0142] Continuing from the above S401, the method for detecting the repeatable positioning accuracy of the relative movement between the chip platform and the imaging device provided by the embodiments of the present disclosure further includes:

[0143] S402: Select a region of interest from multiple frames of target images; wherein, the region of interest includes a target marker region and a background region;

[0144] In a specific implementation, the region of interest, for example, can be an intermediate region intercepted from the target image, such as a region where n pixels above and below the vertical center position are located (it is found through practice that the boundary between the target marker region and the background region is more obvious in the intermediate region of the target image). Or, it can also be the entire target image.

[0145] Such as Figure 9 In the example shown, a specific example of selecting a region of interest in the target image is shown. In this example, s6 represents the target image; s7 represents the region of interest selected in the target image.

[0146] In the region of interest, both the target marker region and the background region are included, and a boundary line is formed between the target marker region and the background region.

[0147] S403: For the region of interest in each frame of the target image, identify the position of the boundary line formed between the target marker region and the background region according to the intensity values of some or all of the pixel points in the region of interest.

[0148] In a specific implementation, when identifying the position of the boundary line formed between the target marker region and the background region in the region of interest, for example, the following method can be adopted:

[0149] Determine multiple detection windows in the region of interest along the length direction of the region of interest according to a preset step size; wherein, the length and width of each detection window are equal to or less than the width of the region of interest;

[0150] For multiple detection windows, determine the average pixel intensity of at least some pixel points in the detection window;

[0151] Determine the position of the boundary line according to the average pixel intensities respectively corresponding to every two adjacent detection windows.

[0152] In a specific implementation, the preset step size is, for example, 1 pixel, and multiple detection windows are determined.

[0153] Such as Figure 10 As shown, a specific example of determining multiple detection windows in the region of interest is shown. The step size between two adjacent detection windows in position is 1 pixel.

[0154] Assume that the size of the region of interest is 16 pixels * 4 pixels, where 16 pixels is the length and 4 pixels is the width, and the preset step size is 1 pixel. Then the number of determined detection windows is: 16 - 4 + 1, that is, 13 detection windows.

[0155] For each detection window, determine the average pixel intensity of some or all of the pixel points in each detection window. Generally, since the intensity values of the pixel points in the target marking area are much higher than those of the pixel points in the background area. If all the pixel values in the detection window belong to the background, the average pixel intensity of this detection window will be relatively low; if some of the pixel points in the detection window belong to the background and the other part belongs to the target marking, the average pixel intensity of the pixel values of this detection window will be in the middle part between the lowest intensity of the background and the highest intensity corresponding to the target marking; if all the pixel values in the detection window belong to the target marking, the average pixel intensity corresponding to this detection window will be relatively high. Since the luminous effect of the target marking itself will cause the luminous effect to affect the intensity values of the pixel points in the background area near the target marking, therefore, in the embodiments of the present disclosure, when determining the position of the demarcation line according to the difference between the average pixel intensities respectively corresponding to every two adjacent detection windows, the following method can be adopted:

[0156] From every two adjacent detection windows, determine the target adjacent detection windows; the difference between the average pixel intensities respectively corresponding to the target adjacent detection windows is greater than the preset difference threshold; and / or, the average pixel intensity of one detection window in the target adjacent detection windows is greater than the first preset threshold, and the average pixel intensity of the other detection window is less than the second preset threshold; the first preset threshold is greater than or equal to the second preset threshold;

[0157] According to the position of the previous detection window in the region of interest in the target adjacent detection windows and the size of the detection window, determine the position of the demarcation line.

[0158] Specifically, the difference between the average pixel intensities respectively corresponding to every two adjacent detection windows can be compared with the preset difference threshold; if the difference is greater than the preset difference threshold, it indicates that a large jump occurs in the average pixel intensity between the two detection windows, and the position where the jump occurs is the demarcation line position between the target marking area and the background area. Or, the average pixel intensities respectively corresponding to every two adjacent detection windows are respectively compared with the preset threshold; if one is greater than the first preset threshold and the other is less than the second preset threshold, it indicates that a large jump occurs in the average pixel intensity between the two detection windows, and the position where the jump occurs is the demarcation line position between the target marking area and the background area. Among them, the first preset threshold is greater than or equal to the second preset threshold.

[0159] Specifically, assume that the width of the detection window is f. If the difference between the average pixel intensity corresponding to the i-th detection window and the average pixel intensity corresponding to the (i - 1)-th detection window is greater than a preset difference threshold, then the position of the dividing line is: the x-coordinate value x of the upper-left pixel point of the i-th detection window i + f.

[0160] In another alternative embodiment, before determining the detection window, the Laplace operator convolution can also be used to perform edge sharpening processing on the region of interest to obtain a sharpened region of interest.

[0161] In this case, when determining a plurality of detection windows in the region of interest along the length direction of the region of interest at a preset step size, it is possible to:

[0162] Determine a plurality of the detection windows in the sharpened region of interest along the length direction of the region of interest at the preset step size.

[0163] After that, according to the intensity values of each detection window in the sharpened region of interest, determine the average pixel intensity corresponding to each detection window.

[0164] Specifically, the Laplace operator, also known as the Laplace filter or Laplace mask, is a convolution kernel used for image processing. It performs a second-order derivative operation on an image, which helps to detect the rate of change in the image, especially edges. By applying the Laplace operator to the region of interest, the edges in the region of interest can be enhanced, making the dividing line between the target marked region and the background region more prominent.

[0165] Continuing from the above S403, the test method for the repeatable positioning accuracy of the relative movement between the chip platform and the imaging device provided by the embodiments of the present disclosure further includes:

[0166] S404: Determine the repeatable positioning accuracy when the chip platform and the imaging device move relative to each other according to the position of the dividing line identified from the regions of interest in multiple target images.

[0167] In a specific implementation, the position of the dividing line includes: the coordinate value of the pixel point on the dividing line in the horizontal direction of the relative movement (for example, the x direction).

[0168] In a specific implementation, when determining the repeatable positioning accuracy when the chip platform and the imaging device move relative to each other, for example, the following method can be adopted:

[0169] According to the coordinate value of the pixel point on the dividing line identified from the regions of interest in multiple target images in the horizontal direction of the relative movement (for example, the x direction), determine at least one of the standard deviation and the maximum difference of the coordinate values;

[0170] Use at least one of the standard deviation and the maximum difference as an index for measuring the repeatable positioning accuracy.

[0171] Here, the coordinate standard deviation satisfies, for example:

[0172]

[0173] where μ represents the mean value of the coordinate values of the pixel points on the demarcation line corresponding to multiple frames of target images in the first direction; x i represents the coordinate value of the pixel point on the demarcation line corresponding to the i-th frame of target image in multiple frames of target images in the first direction; n represents the number of target images.

[0174] The maximum difference is, for example, determined by finding the maximum coordinate value and the minimum coordinate value from the coordinate values of the pixel points on the demarcation lines corresponding to multiple frames of target images in the horizontal direction of relative movement, and taking the difference between the maximum coordinate value and the minimum coordinate value as the maximum difference.

[0175] The above-mentioned coordinate standard deviation and / or maximum difference are used as an index for measuring the repeatable positioning accuracy.

[0176] After that, the following method can be used to determine whether the repeatable positioning accuracy of the relative movement between the chip platform and the imaging device meets the requirements of the sequencing experiment based on the above index:

[0177] Compare the standard deviation with a preset standard deviation threshold to obtain a first comparison result; and / or,

[0178] Compare the maximum difference with a preset maximum difference threshold to obtain a second comparison result;

[0179] Determine whether the repeatable positioning accuracy of the relative movement between the chip platform and the imaging device meets the requirements of the sequencing experiment according to the first comparison result and / or the second comparison result.

[0180] Specifically, if the first comparison result indicates that the standard deviation is less than the standard deviation threshold, and / or, if the second comparison result indicates that the maximum difference is less than the preset maximum difference threshold, it is determined that the repeatable positioning accuracy of the relative movement between the chip platform and the imaging device meets the requirements of the sequencing experiment.

[0181] If the first comparison result indicates that the standard deviation is greater than or equal to the standard deviation threshold, and / or, if the second comparison result indicates that the maximum difference is greater than or equal to the preset maximum difference threshold, it is determined that the repeatable positioning accuracy of the relative movement between the chip platform and the imaging device does not meet the requirements of the sequencing experiment, and the motion error existing in the relative movement between the chip platform and the imaging device is the index value determined by the above method.

[0182] In the embodiments of the present disclosure, by controlling the reciprocating relative movement between the chip platform and the imaging device in the horizontal direction, and after each relative movement to the same target position, controlling the imaging device to take pictures of the calibration plate on the chip platform to obtain multiple frames of target images; a target mark is set on the calibration plate, and the multiple frames of target images are images of the target mark. Then, an area of interest is selected from the multiple frames of target images, and for the area of interest in the multiple frames of target images, according to the intensity values of at least some pixel points in the area of interest, the position of the boundary line formed between the target mark area and the background area in the area of interest is identified, and then by using the position of the boundary line identified from the area of interest in the multiple frames of target images, the repeatable positioning accuracy during the relative movement between the chip platform and the imaging device is determined, so that the repeatable positioning accuracy during the relative movement between the chip platform and the imaging device can be accurately identified by using the calibration plate and the target mark set on the calibration plate, to determine whether the instrument meets the requirements of the sequencing experiment.

[0183] Those skilled in the art can understand that in the above method of the specific embodiment, the writing order of each step does not mean a strict execution order that constitutes any limitation on the implementation process, and the specific execution order of each step should be determined according to its function and possible internal logic.

[0184] The embodiments of the present disclosure also provide a computer device, as Figure 11 shown, which is a schematic structural diagram of the computer device provided by the embodiments of the present disclosure, including:

[0185] a processor 111 and a memory 112; the memory 112 stores machine-readable instructions executable by the processor 111, and the processor 111 is used to execute the machine-readable instructions stored in the memory 112. When the machine-readable instructions are executed by the processor 111, the processor 111 executes the following steps:

[0186] Control the reciprocating relative movement between the chip platform and the imaging device in the horizontal direction, and after each relative movement to the same target position, control the imaging device to take pictures of the calibration plate located on the chip platform to obtain multiple frames of target images; wherein, a target mark is set on the calibration plate; the multiple frames of target images are images of the target mark;

[0187] Select an area of interest from the multiple frames of target images; wherein, the area of interest includes a target mark area and a background area;

[0188] For the area of interest in the multiple frames of target images, according to the intensity values of at least some pixel points in the area of interest, identify the position of the boundary line formed between the target mark area and the background area in the area of interest;

[0189] Determine the repeatable positioning accuracy when the chip platform and the imaging device move relative to each other according to the position of the demarcation line recognized from the region of interest in multiple frames of target images.

[0190] The above-mentioned memory 112 includes a main memory 1121 and an external memory 1122; the main memory 1121 here is also called internal memory, which is used to temporarily store the operation data in the processor 111 and the data exchanged with the external memory 1122 such as a hard disk. The processor 111 exchanges data with the external memory 1122 through the main memory 1121.

[0191] The specific execution process of the above instructions can refer to the steps of the test method for repeatable positioning accuracy described in the embodiments of the present disclosure, which will not be elaborated here.

[0192] The embodiments of the present disclosure also provide a computer-readable storage medium, on which a computer program is stored. When the computer program is run by a processor, it executes the steps of the test method for repeatable positioning accuracy described in the above method embodiments. Among them, the storage medium can be a volatile or non-volatile computer-readable storage medium.

[0193] The embodiments of the present disclosure also provide a computer program product, which carries program codes. The instructions included in the program codes can be used to execute the steps of the test method for repeatable positioning accuracy described in the above method embodiments. Specifically, reference can be made to the above method embodiments, which will not be elaborated here.

[0194] Among them, the above computer program product can be specifically implemented in a manner of hardware, software or a combination thereof. In an alternative embodiment, the computer program product is specifically embodied as a computer storage medium. In another alternative embodiment, the computer program product is specifically embodied as a software product, such as a Software Development Kit (SDK), etc.

[0195] Finally, it should be noted that the above embodiments are only specific implementation manners of the present disclosure, which are used to illustrate the technical solutions of the present disclosure, rather than to limit them. The protection scope of the present disclosure is not limited thereto. Although the present disclosure has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: any person skilled in the art within the technical scope disclosed by the present disclosure can still modify the technical solutions recorded in the foregoing embodiments, or can easily think of changes, or perform equivalent replacements on some of the technical features; and these modifications, changes or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present disclosure, and should all be covered by the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be subject to the protection scope of the claims.

Claims

1. A test method for repeat positioning accuracy, characterized in that, Including: Controlling the reciprocating relative movement between the control chip platform and the imaging device in the horizontal direction, and after each relative movement to the same target position, controlling the imaging device to take pictures of the calibration plate located on the chip platform to obtain multiple frames of target images; wherein, target marks are provided on the calibration plate; the multiple frames of target images are images of the target marks; Selecting a region of interest from the multiple frames of target images; wherein, the region of interest includes a target mark region and a background region; For the region of interest in the multiple frames of target images, according to the intensity values of at least some pixel points in the region of interest, identifying the position of the boundary line formed between the target mark region and the background region in the region of interest; According to the boundary line positions identified from the regions of interest in the multiple frames of target images, determining the repeatable positioning accuracy when the chip platform and the imaging device move relatively.

2. The method according to claim 1, wherein The method further includes: Before controlling the imaging device to take pictures of the calibration plate placed on the chip platform to obtain multiple frames of target images, aiming at maximizing the clarity of the target images, performing focusing processing on the imaging device so that the calibration plate is located on the focal plane of the imaging device.

3. The method according to claim 2, wherein The performing focusing processing on the imaging device so that the calibration plate is located on the focal plane of the imaging device includes: Controlling the relative movement between the chip platform and the imaging device in the vertical direction, and after each relative movement to a new position, controlling the imaging device to take pictures of the calibration plate to obtain multiple frames of focusing images at different positions; Determining the clarity corresponding to each of the multiple frames of focusing images; Based on the clarity corresponding to each of the multiple frames of focusing images, determining the target photographing position of the imaging device when the calibration plate is located on the focal plane of the imaging device.

4. The method according to claim 3, wherein The determining the clarity corresponding to each of the multiple frames of focusing images includes: For the multiple frames of focusing images, determining the sum of the squares of the intensity differences between at least some pixel points in the focusing image and their horizontally adjacent pixel points; and Determining the average value of the sum of the squares of the intensity differences between at least some pixel points in the focusing image and their horizontally adjacent pixel points; Determining the average value of the sum of the squares of the intensity differences as the clarity of the focusing image.

5. The method according to claim 3, characterized in that, The based on the clarity corresponding to each of the multiple frames of focusing images, determining the target photographing position of the imaging device when the calibration plate is located on the focal plane of the imaging device includes: Performing fitting processing on the clarity corresponding to each of the multiple frames of focusing images to obtain the correspondence between the photographing position of the imaging device and the clarity of the focusing image; Aiming at maximizing the clarity, based on the correspondence, determining the photographing position corresponding to the maximum clarity value, and the photographing position corresponding to the maximum clarity value is the target photographing position of the imaging device when the calibration plate is located on the focal plane of the imaging device.

6. The method according to claim 1, characterized in that, The according to the intensity values of at least some pixel points in the region of interest, identifying the position of the boundary line formed between the target mark region and the background region in the region of interest includes: Determine a plurality of detection windows in the region of interest along the length direction of the region of interest according to a preset step size; wherein, the length and width of each of the detection windows are equal to or less than the width of the region of interest; For the plurality of detection windows, determine the average pixel intensity of at least some pixel points in the detection windows; Determine the position of the dividing line according to the average pixel intensities respectively corresponding to every two adjacent detection windows.

7. The method according to claim 6, characterized in that, The determining the position of the dividing line according to the average pixel intensities respectively corresponding to every two adjacent detection windows includes: Determine target adjacent detection windows from every two adjacent detection windows; the difference between the average pixel intensities respectively corresponding to the target adjacent detection windows is greater than a preset difference threshold; and / or, the average pixel intensity of one detection window in the target adjacent detection windows is greater than a first preset threshold, and the average pixel intensity of the other detection window is less than a second preset threshold; the first preset threshold is greater than or equal to the second preset threshold; Determine the position of the dividing line according to the position of the previous detection window in the region of interest in the target adjacent detection windows and the size of the detection window.

8. The method according to claim 6, wherein Further includes: Perform boundary sharpening processing on the region of interest by using Laplacian operator convolution to obtain a sharpened region of interest; Determine a plurality of detection windows in the region of interest along the length direction of the region of interest according to a preset step size, including: Determine a plurality of the detection windows in the sharpened region of interest along the length direction of the region of interest according to the preset step size.

9. A computer device, characterized in that, Includes: A processor and a memory, the memory stores machine-readable instructions executable by the processor, the processor is configured to execute the machine-readable instructions stored in the memory, and when the machine-readable instructions are executed by the processor, the processor executes the steps of the test method for repeatable positioning accuracy according to any one of claims 1 to 8.

10. A computer-readable storage medium, characterized in that, A computer program is stored on the computer-readable storage medium, and when the computer program is run by a computer device, the computer device executes the steps of the test method for repeatable positioning accuracy according to any one of claims 1 to 8.

Images