Microscopy method and applications

Abstract

The present application relates to the field of imaging technology, in particular to a microscopic imaging method and application. The present application discloses a self-adaptive optical adjusting method, which estimates sequencing brightness by imaging a small area on a chip, adjusts laser power or exposure time, so that the base brightness is in the interval meeting the base recognition requirement, thereby helping the sequencer to stabilize the sequencing signal in the case that the performance of the chip, laser, reagent and optical system fluctuates.

Description

Technical Field

[0001] This invention relates to the field of imaging technology, and more specifically to a microscopic imaging method and its application. Background Technology

[0002] Fluorescence microscopy refers to the use of fluorescent molecules to label specific structures or components in a sample. These fluorescent molecules are excited to a high-energy state with short-wavelength light, and the emitted longer-wavelength fluorescence during de-excitation is then imaged under a microscope. It is also known as fluorescence microscopy. This technique enables "functional imaging" by labeling specific structures, molecules, or ions in biological samples, thereby acquiring sample information. During fluorescence microscopy, the brightness of the fluorescence signal acquired by the optical equipment determines the quality of the raw data-image, thus affecting the accuracy of the sample information. For example, when the signal brightness is too low, the signal generated by the sample is difficult to distinguish from noise (background signal); when the signal brightness is too high, energy spillover (optical scattering) from neighboring signal points causes false positives. Furthermore, the instrument is affected by multiple factors during fluorescence microscopy, and performance fluctuations may occur. This also makes the fluorescence signal generated during sample testing unstable, leading to deviations in sample information. For example, when sequencing nucleic acid molecules using microscopy, the sequencer is affected by performance fluctuations in the chip, laser, reagents, and optical system, resulting in unstable sequencing signals and deviations in the sequencing results.

[0003] Therefore, there is an urgent need to develop a method that can reduce the impact of signal fluctuations on the presentation of microscopic imaging results and, consequently, on the judgment of imaging data. Summary of the Invention

[0004] The present invention aims to at least partially solve one of the technical problems in the related art.

[0005] To this end, the first aspect of the present invention provides a microscopic imaging method, wherein the method is based on an imaging device to perform microscopic imaging processing on a biological sample on the surface of a solid carrier, wherein the imaging device includes a laser, and the microscopic imaging processing uses the laser to perform microscopic optical imaging on the fluorescence signal generated by the biological sample based on a first optical parameter, wherein the first optical parameter is a laser power with sufficient brightness and / or a laser irradiation time.

[0006] According to some specific embodiments of the present invention, the method for determining the first optical parameter includes:

[0007] (i) Based on initial optical parameters, a first reaction is performed on the biological sample, and a first fluorescence signal is collected on the surface of the solid support after the first reaction, and a first brightness of the first fluorescence signal in at least a part of the solid support is determined;

[0008] (ii) Compare the first brightness with the preset standard brightness to determine whether the first brightness meets the standard;

[0009] If so, then the initial optical parameter is determined to be the first optical parameter;

[0010] If not, adjust the initial optical parameters based on a preset function to determine the second optical parameter that meets the preset standard brightness in the preset function; repeat steps (i) and (ii) with the second optical parameter instead of the initial optical parameter until the brightness meets the standard, and determine the first optical parameter.

[0011] The preset function is a function established based on the second brightness and third optical parameters of the second fluorescence signal in at least a part of the solid carrier when the biological sample undergoes a second reaction and the second fluorescence signal is collected on the surface of the solid carrier after the second reaction. The second optical parameters are the second laser power and / or the second laser irradiation time.

[0012] The method of this invention can reduce the impact of external factors causing signal brightness fluctuations during microscopic imaging on the accuracy of the acquired data. This allows the microscopic imaging method to obtain stable fluorescence signals regardless of performance fluctuations caused by instruments, equipment, consumables, reagents, systems, or methods, thereby improving the accuracy of microscopic imaging data. Taking nucleic acid molecule sequencing technology using microscopic imaging as an example, this method can obtain stable sequencing signals even when there are performance fluctuations in the sequencer's chip, laser, reagents, and optical system, reducing sequencing result deviations and thus improving sequencing accuracy.

[0013] According to some specific embodiments of the present invention, the initial optical parameter is calculated as follows: initial optical parameter = A × b, where A represents the initial laser power or initial laser irradiation time of the laser, which is the initial laser parameter determined based on the initial laser power and the initial laser irradiation time, and b represents the ratio of the quality control optical parameter and the average optical parameter of the solid-phase carrier, wherein the quality control optical parameter is the quality control laser power, the average optical parameter is the average laser power, or the quality control optical parameter is the quality control laser irradiation time, the average optical parameter is the average irradiation time, or the quality control optical parameter is the quality control initial laser parameter, and the average optical parameter is the average laser power.

[0014] According to some specific embodiments of the present invention, the steps of performing a first base extension reaction on the biological sample, acquiring a first fluorescence signal on the surface of the solid support after the first reaction, and determining a first brightness of at least a portion of the solid support include: performing a first reaction on the biological sample, acquiring an image of at least one field of view (FOV) on the surface of the solid support where the first reaction occurs, estimating an average brightness based on the brightness of each bright spot in the image, and outputting the average brightness as a first brightness.

[0015] According to some specific embodiments of the present invention, the image is an image of a field of view (FOV).

[0016] According to some specific embodiments of the present invention, the first reaction is one of the first N reactions performed on the biological sample.

[0017] According to some specific embodiments of the present invention, the value of N ranges from 1 to 5.

[0018] According to some specific embodiments of the present invention, a method for estimating the average brightness based on the brightness of each bright spot in the image includes:

[0019] Based on images from multiple detection channels within a single field of view (FOV), identify signal peaks and background peaks.

[0020] Based on the signal peak and the background peak, the gray values ​​of all pixels in the image are fitted respectively to determine the signal peak distribution curve and the background peak distribution curve;

[0021] The average brightness is determined based on the signal peak distribution curve and the background peak distribution curve.

[0022] According to some specific embodiments of the present invention, determining the average brightness based on the signal peak distribution curve and the background peak distribution curve includes:

[0023] Based on the signal peak distribution curve, determine the median value of the signal peak;

[0024] Based on the background peak distribution curve, determine the median value of the background peak;

[0025] Based on the median value of the signal peak and the median value of the background peak, the difference between the two is determined, and the difference is used as the average brightness.

[0026] According to some specific embodiments of the present invention, the preset standard brightness is a preset standard brightness range.

[0027] According to some specific embodiments of the present invention, comparing the first brightness with a preset standard brightness to determine whether the first brightness meets the standard includes:

[0028] Compare the first brightness with the preset standard brightness. If the first brightness is within the preset standard brightness range, then the first brightness is determined to meet the standard.

[0029] If the first brightness is not within the preset standard brightness range, then the first brightness is determined to be substandard.

[0030] According to some specific embodiments of the present invention, the preset function includes at least one selected from the brightness-laser power function, the brightness-laser irradiation time function, and the brightness-laser power-laser irradiation time function.

[0031] According to some specific embodiments of the present invention, the method for establishing the preset function is as follows:

[0032] Multiple different third optical parameters are set, and the reaction is carried out based on different third optical parameters. The third fluorescence signal on the surface of the solid support after the third reaction is collected, and the second brightness of the third fluorescence signal in each round is determined. The third optical parameters are preset laser power, preset laser irradiation time, or preset laser power-laser irradiation time array.

[0033] Based on the third optical parameter and the second brightness, a preset function is obtained by fitting.

[0034] According to some specific embodiments of the present invention, the step of adjusting the initial optical parameters based on a preset function to determine the second optical parameters includes:

[0035] Based on the preset function and preset standard brightness, the fourth optical parameter is determined;

[0036] The initial optical parameters are adjusted to the fourth optical parameter, and the fourth optical parameter is determined as the second optical parameter.

[0037] According to some specific embodiments of the present invention, in repeating steps (i) and (ii), in each step (i), the first brightness is respectively derived from the first brightness of different FOVs in the image generated by the same first reaction, or

[0038] The first brightness is derived from the first brightness of the same FOV in different first reactions, or

[0039] The first brightness is derived from the first brightness of different FOVs of different first reactions.

[0040] According to some specific embodiments of the present invention, steps (i) and (ii) are repeated less than or equal to 3 times.

[0041] According to some specific embodiments of the present invention, after the step of determining the first optical parameter for sequencing, the method further includes: recording and storing the first optical parameter.

[0042] According to some specific embodiments of the present invention, the CV value of the first brightness in different regions of the solid carrier is less than or equal to 5%.

[0043] According to some specific embodiments of the present invention, the biological sample is a nucleic acid molecule, and the first reaction and the second reaction are base extension reactions performed on the biological sample on the surface of the solid support.

[0044] A second aspect of the present invention provides a microscopic imaging apparatus, the apparatus comprising a laser and a first optical parameter determination system, the first optical parameter determination system being used to determine first optical parameters for microscopic imaging processing of biological samples on the surface of a solid carrier, wherein the first optical parameters are laser power and / or laser time that meet the brightness requirements.

[0045] According to some specific embodiments of the present invention, the first optical parameter determination system includes:

[0046] The first brightness determination unit is used to perform a first reaction on the biological sample based on initial optical parameters, collect the first fluorescence signal on the surface of the solid support after the first reaction, and determine the first brightness of the first fluorescence signal in at least a part of the solid support.

[0047] The first brightness judgment unit is used to compare the first brightness with the preset standard brightness to determine whether the first brightness meets the standard.

[0048] If so, then the initial optical parameter is determined to be the first optical parameter;

[0049] If not, the initial optical parameters are adjusted based on a preset function to determine the second optical parameter that meets the preset standard brightness. The first brightness determination unit and the first brightness judgment unit are repeated with the second optical parameter instead of the initial optical parameter until the brightness meets the standard, and the first optical parameter is determined.

[0050] The preset function is a function established based on the second brightness and third optical parameters of the second fluorescence signal in at least a part of the solid carrier when the biological sample undergoes a second reaction and the second fluorescence signal is collected on the surface of the solid carrier after the second reaction. The second optical parameters are the second laser power and / or the second laser irradiation time.

[0051] According to some specific embodiments of the present invention, the initial optical parameters are calculated using the following formula: initial optical parameters = A × b, where A represents the initial laser power or initial laser irradiation time of the laser, or the initial laser parameters determined based on the initial laser power and the initial laser irradiation time, and b represents the ratio of the quality control optical parameters to the average optical parameters of the solid-phase carrier.

[0052] The quality control optical parameter is the quality control laser power, and the average optical parameter is the average laser power, or

[0053] The quality control optical parameter is the quality control laser irradiation time, and the average optical parameter is the average irradiation time, or

[0054] The quality control optical parameters are the initial laser parameters for quality control, and the average optical parameters are the average initial laser parameters.

[0055] According to some specific embodiments of the present invention, the step of performing a first reaction on the biological sample, collecting a first fluorescence signal on the surface of the solid support after the first reaction, and determining a first brightness of at least a portion of the solid support includes:

[0056] The biological sample undergoes a first reaction, and images of at least one field of view (FOV) on the surface of the solid support where the first reaction occurs are acquired. The average brightness is estimated based on the brightness of each bright spot in the image, and the average brightness is output as the first brightness.

[0057] According to some specific embodiments of the present invention, the image is an image of a field of view (FOV).

[0058] According to some specific embodiments of the present invention, the first reaction is one of the first N reactions performed on the biological sample.

[0059] According to some specific embodiments of the present invention, the value of N ranges from 1 to 5.

[0060] According to some specific embodiments of the present invention, a method for estimating the average brightness based on the brightness of each bright spot in the image includes:

[0061] Based on images from multiple detection channels within a single field of view (FOV), identify signal peaks and background peaks.

[0062] Based on the signal peak and the background peak, the gray values ​​of all pixels in the image are fitted respectively to determine the signal peak distribution curve and the background peak distribution curve;

[0063] The average brightness is determined based on the signal peak distribution curve and the background peak distribution curve.

[0064] According to some specific embodiments of the present invention, determining the average brightness based on the signal peak distribution curve and the background peak distribution curve includes:

[0065] Based on the signal peak distribution curve, determine the median value of the signal peak;

[0066] Based on the background peak distribution curve, determine the median value of the background peak;

[0067] Based on the median value of the signal peak and the median value of the background peak, the difference between the two is determined, and the difference is used as the average brightness.

[0068] According to some specific embodiments of the present invention, the preset standard brightness is a preset standard brightness range.

[0069] According to some specific embodiments of the present invention, comparing the first brightness with a preset standard brightness to determine whether the first brightness meets the standard includes:

[0070] Compare the first brightness with the preset standard brightness. If the first brightness is within the preset standard brightness range, then the first brightness is determined to meet the standard.

[0071] If the first brightness is not within the preset standard brightness range, then the first brightness is determined to be substandard.

[0072] According to some specific embodiments of the present invention, the preset function includes at least one selected from the brightness-laser power function, the brightness-laser irradiation time function, and the brightness-laser power-laser irradiation time function.

[0073] According to some specific embodiments of the present invention, the method for establishing the preset function is as follows:

[0074] Multiple third optical parameters are set, and reactions are carried out based on different third optical parameters. The third fluorescence signal on the surface of the solid support after the third reaction is collected, and the second brightness of the third fluorescence signal in each round is determined. The third optical parameters are preset laser power, preset laser irradiation time, or preset laser power-laser irradiation time array.

[0075] Based on the third optical parameter and the second brightness, a preset function is obtained by fitting.

[0076] According to some specific embodiments of the present invention, the step of adjusting the initial optical parameters based on a preset function to determine the second optical parameters includes:

[0077] Based on the preset function and preset standard brightness, the fourth optical parameter is determined;

[0078] The initial optical parameters are adjusted to the fourth optical parameter, and the fourth optical parameter is determined as the second optical parameter.

[0079] According to some specific embodiments of the present invention, when repeating the first brightness determination unit and the first brightness judgment unit, in each first brightness determination unit, the first brightness is respectively derived from the first brightness of different FOVs in the same cyclic base extension image, or

[0080] The first brightness is derived from the first brightness of the same FOV in different cyclic base extension images, or

[0081] The first brightness is derived from the first brightness of different FOVs of different round base extension images.

[0082] According to some specific embodiments of the present invention, the number of times the first brightness determination unit and the first brightness judgment unit are repeated is less than or equal to 3.

[0083] According to some specific embodiments of the present invention, the microscopic imaging device further includes a storage unit connected to the first brightness determination unit, the storage unit being used to record and store the first optical parameters.

[0084] According to some specific embodiments of the present invention, the CV value of the first brightness in different regions of the solid carrier is less than or equal to 5%.

[0085] According to some specific embodiments of the present invention, the biological sample is a nucleic acid molecule, and the first reaction and the second reaction are base extension reactions performed on the biological sample on the surface of the solid support.

[0086] The third aspect of the present invention provides the use of the microscopic imaging apparatus described in the second aspect in sequencing.

[0087] The fourth aspect of the present invention provides a method for obtaining preset optical parameters of a sequencing chip, the method comprising obtaining a first optical parameter by the method for determining a first optical parameter in the microscopic imaging method described in the first aspect, and using the value of the first optical parameter as the preset optical parameter of the sequencing chip.

[0088] The optical parameters provided by this invention can be recorded in the quality control of sequencing chips and reagents, and these "recommended optical parameters" can be saved in the product information of the reagent kit in the form of QR codes. When users read the QR code in the sequencing process, they can immediately select the optical parameters for this batch when passing the quality control based on this information, thereby reducing the frequency of use of the adaptive laser power adjustment process and saving sequencing time.

[0089] The fifth aspect of the present invention provides a sequencing method, the sequencing method comprising performing microscopic optical imaging on the fluorescence signal generated by the biological sample on the sequencing chip using the microscopic imaging method described in the first aspect, or performing microscopic optical imaging on the fluorescence signal generated by the biological sample on the sequencing chip using the microscopic imaging device described in the second aspect, and analyzing the imaging results to obtain the specific sequence of the biological sample on the sequencing chip.

[0090] A sixth aspect of the present invention provides an electronic device for determining optical parameters during sequencing, comprising a memory and a processor;

[0091] The processor reads executable program code stored in the memory to run a program corresponding to the executable program code, so as to implement the microscopic imaging method described in the first aspect.

[0092] A seventh aspect of the present invention provides a computer-readable storage medium storing a computer program that, when executed by a processor, implements the microscopic imaging method described in the first aspect.

[0093] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0094] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0095] Figure 1 This diagram illustrates the steps of a method for adjusting a first optical parameter during sequencing, according to one embodiment of the present invention.

[0096] Figure 2 This invention illustrates a flowchart of a method for adjusting a first optical parameter during sequencing, according to one embodiment of the present invention.

[0097] Figure 3 The image shows the signal peak and background peak when estimating brightness in one embodiment of the present invention;

[0098] Figure 4 The graph shows the linear relationship between laser power and sequencing brightness in one embodiment of the present invention. The left graph represents the sequencing brightness variation curve of chip 1 on different sequencers, and the right graph represents the sequencing brightness variation curve of chip 2 on different sequencers.

[0099] Figure 5 This invention illustrates sequencing brightness curves generated under different optical parameters in one embodiment of the invention.

[0100] Figure 6A graph showing the brightness response percentage versus power adjustment percentage in one embodiment of the present invention is displayed. Detailed Implementation

[0101] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0102] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of this invention, unless otherwise stated, "a plurality of" means two or more. Without departing from the scope of the embodiments of this application, a first reaction may also be referred to as a second reaction, and correspondingly, a second reaction may be referred to as a first reaction. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include one or more of that feature.

[0103] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0104] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0105] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0106] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0107] In this article, the term "FOV" stands for Field of View. If the width (W) of the imaging plane is fixed, the size of the FOV is directly determined by the Focal Length. The larger the Focal Length, the farther you can see, but the smaller the FOV. The smaller the Focal Length, the closer you can see, but the larger the FOV.

[0108] In this paper, the term "initial optical parameters" refers to the initial laser power and laser irradiation time.

[0109] In this paper, the term "CV" refers to the coefficient of variation, which is the ratio of the standard deviation to the mean, expressed as a percentage (%).

[0110] In this article, the term "sequencing" is also referred to as "nucleic acid sequencing" or "gene sequencing," and these three terms are interchangeable in meaning, all referring to the determination of the type and sequence of bases or nucleotides (including nucleotide analogs) in nucleic acid molecules. Sequencing includes the process of binding nucleotides to a template and collecting the corresponding signals emitted by the nucleotides (including analogs). Sequencing includes sequencing-by-synthesis (SBS) and / or sequencing-by-ligation (SBL), including DNA sequencing and / or RNA sequencing, including long-fragment sequencing and / or short-fragment sequencing. The terms "long-fragment" and "short-fragment" are relative; for example, nucleic acid molecules longer than 1Kb, 2Kb, 5Kb, or 10Kb can be called long fragments, while those shorter than 1Kb or 800bp can be called short fragments.

[0111] In this paper, the term "base extension reaction" refers to the reaction process in which bases or nucleotides (including nucleotide analogs) in a nucleic acid molecule are extended from the 5' end to the 3' end. In this field, any nucleic acid molecule amplification reaction involves base extension reaction, including PCR, sequencing, etc.

[0112] Sequencing generally involves multiple rounds to determine the type and sequence of multiple bases or nucleotides on a nucleic acid template. In this application, each round of "determining the type and sequence of multiple bases or nucleotides on a nucleic acid template" is referred to as "one round of sequencing." A "sequencing round" (cycle) can be defined as one base extension of four nucleotides / bases; in other words, "one round of sequencing" can be defined as determining the type of a base or nucleotide at any specified position on the template. For sequencing platforms based on polymerization or ligation reactions, one round of sequencing includes the process of binding four nucleotides (including nucleotide analogs) to the nucleic acid template via base complementarity and collecting the corresponding signals. Specifically, for platforms based on polymerization reactions, the reaction system includes reaction substrate nucleotides, polymerase, and a nucleic acid template. The nucleic acid template has a sequence (sequencing primer) bound to it. Based on the base pairing principle and the polymerization reaction principle, the added reaction substrate nucleotides, catalyzed by the polymerase, are ligated to the sequencing primers, achieving the binding of the nucleotide to a specific position on the nucleic acid template. Typically, a single sequencing run may include one or more base extensions (repeat). For example, four nucleotides are added to the reaction system sequentially, and base extensions and corresponding reaction signals are collected separately, resulting in four base extensions in one sequencing run. Alternatively, four nucleotides may be added to the reaction system in any combination, such as in pairs or in a one-to-three combination, with each combination undergoing base extension and corresponding reaction signal collection separately, resulting in two base extensions in one sequencing run. Yet another example is that all four nucleotides are added to the reaction system simultaneously for base extension and reaction signal collection, resulting in one base extension in one sequencing run.

[0113] The term "nucleic acid molecule" refers to a polymeric form of nucleotides of any length, and may include ribonucleotides or their analogues, deoxyribonucleotides or their analogues, and mixtures of the above nucleotides or their analogues. A nucleic acid molecule can refer to a single-stranded polynucleotide or a double-stranded polynucleotide. Nucleotides in a nucleic acid molecule may include naturally occurring nucleotides and their functionally substitutable analogues.

[0114] In this article, the term "phase decoupling" can also be referred to as "phase imbalance," "phase decoupling," or "phase difference," referring to the phenomenon in a chemical reaction where the reactions of nucleic acid molecules in a group, such as a cluster of nucleic acid molecules, are not synchronized, including phasing or sequence lag and prephasing or sequence lead.

[0115] In fluorescence microscopy, the brightness of the fluorescence signal acquired by the optical equipment determines the quality of the raw data-image, thus affecting the accuracy of sample information. Taking sequencing technology as an example, when sequencing is performed based on reversible terminators containing cleavable fluorescent groups, the brightness of the fluorescence signal acquired by the optical equipment determines the quality of the raw data-image, thus affecting the accuracy of base identification. For example, when the signal brightness is too low, the signal generated by the sample is difficult to distinguish from noise (background signal), resulting in base deletion errors; when the signal brightness is too high, energy overflow (optical scattering) from adjacent signal points causes excessive signal recognition, i.e., insertion errors. During the sequencing process, the sequencer is affected by multiple factors such as the chip, laser, reagents, and optical system, resulting in performance fluctuations. This also makes the sequencing signal generated during the sequencing process unstable, leading to deviations in the sequencing results and affecting the accuracy of the sequencing results.

[0116] In view of this, the present invention provides a microscopic imaging method capable of performing microscopic imaging in scenarios with insufficient signal stability, and ensuring that the obtained microscopic imaging data is unaffected by signal stability. The microscopic imaging method of the present invention is based on microscopic imaging processing of biological samples on the surface of a solid support using an imaging device. The imaging device includes a laser, which generates excitation light to excite fluorescent molecules in the biological sample to emit light, thereby facilitating microscopic optical imaging.

[0117] In this embodiment of the invention, a solid-phase support is understood as a carrier or support for biological samples. In some application examples, a solid-phase support may also be referred to as a solid-phase substrate. The biological sample referred to in this application embodiment is a biological sample that can be excited to produce a fluorescence signal under certain conditions or reactions by specific excitation light. As an example, a biological sample can be various types of cells containing fluorescent substances, or cells that can undergo changes such as generating, increasing, decreasing, or eliminating fluorescence signals through treatment or placement under certain conditions. As another example, a biological sample can be a biomolecule with a fluorescent group, or a biomolecule capable of binding a fluorescent group or a compound with a fluorescent group. Exemplarily, the biomolecule can be a nucleic acid molecule, but is not limited thereto.

[0118] This microscopic imaging method aims to obtain a fluorescence signal brightness that meets the requirements. Under this premise, a first optical parameter is determined, and a laser is used to perform microscopic optical imaging of the fluorescence signal generated by biomolecules on the surface of a solid support based on this first optical parameter. In this embodiment of the invention, the first optical parameter is the operating parameter of the laser, which can be the laser power, the laser irradiation time, or both the laser power and laser irradiation time that meet the brightness requirements. In this embodiment, "meeting the brightness requirements" refers to meeting the brightness conditions of a preset standard brightness.

[0119] Microscopic imaging methods

[0120] According to some specific embodiments of the present invention, the present invention proposes a method for determining a first optical parameter, such as... Figure 1 As shown, it includes:

[0121] S110. Based on the initial optical parameters, perform a first reaction on the biological sample, collect the first fluorescence signal on the surface of the solid support after the first reaction, and determine the first brightness of the first fluorescence signal in at least a part of the solid support.

[0122] S120. Compare the first brightness with the preset standard brightness to determine whether the first brightness meets the standard.

[0123] If so, then the initial optical parameter is determined to be the first optical parameter;

[0124] If not, adjust the initial optical parameters based on a preset function to determine the second optical parameter that meets the preset standard brightness in the preset function; repeat steps S110 and S120 with the second optical parameter instead of the initial optical parameter until the brightness meets the standard, and determine the first optical parameter.

[0125] The preset function is a function established based on the second brightness and third optical parameters of the second fluorescence signal in at least a part of the solid carrier when the biological sample undergoes a second reaction and the second fluorescence signal is collected on the surface of the solid carrier after the second reaction. The second optical parameters are the second laser power and / or the second laser irradiation time.

[0126] In step S110, after a biological sample undergoes a certain reaction, situations such as generating, enhancing, or reducing fluorescence signals may occur. This embodiment of the application collects the fluorescence signal after the reaction based on the initial optical parameters of the laser that excites the biological sample to generate a fluorescence signal, and obtains the brightness of the fluorescence signal of the biological sample under the initial optical parameters. It should be understood that this method is applicable to situations where the biological sample undergoes multiple reactions, and the fluorescence signal of the solid-phase support is collected during each reaction. Specifically, by using the fluorescence signal obtained during the first or several previous reactions and the corresponding parameters, the optical parameters used for subsequent reactions (i.e., the first optical parameters) are determined, so that in subsequent reactions, the laser directly selects these optical parameters to perform microscopic optical imaging of the fluorescence signal generated by the biological sample, and obtains an image with acceptable brightness. This reduces the impact of various factors affecting the stability of the fluorescence signal on the judgment of data obtained from the image during microscopic imaging, improving the accuracy of the image reaction data.

[0127] This application does not strictly limit the type of reaction in its embodiments. In some embodiments, the first reaction is one of the first N reactions performed on the biological sample. That is, the first reaction is the first or one of the first few reactions when the biomolecules undergo multiple reactions and the fluorescence signal of the solid-phase support is collected during each reaction. Thus, by determining the first brightness corresponding to the first fluorescence signal obtained under the initial optical parameter conditions through the first few reactions performed on the biological sample, the time for determining the first optical parameters can be saved. In some embodiments, the value of N ranges from 1 to 5, and for example, N is 1, 2, 3, 4, 5, etc. For example, the biological sample is a nucleic acid molecule, and this microscopic imaging method is used to collect fluorescence signals when sequencing nucleic acid molecules. In this case, the first reaction corresponds to the sequencing reaction, specifically the first round or the first few rounds of sequencing reaction.

[0128] In this embodiment, the initial optical parameter is the initial value of the optical parameter at the start of the reaction. Correspondingly, in this embodiment, the initial optical parameter is the optical parameter during the first reaction or the first few reactions when the biological sample undergoes multiple reactions. Taking the biological sample as a nucleic acid molecule and the reaction as a base extension reaction as an example, the initial optical parameter is the optical parameter during the first round of base extension reaction or the first few rounds of base extension reaction on the nucleic acid molecule. In some embodiments, the initial optical parameter is calculated as: initial optical parameter = A × b, where A represents the initial laser power or initial laser irradiation time of the laser, or the initial laser parameter determined based on the initial laser power and initial laser irradiation time, and b represents the ratio of the quality control optical parameter and the average optical parameter of the solid-phase support. Here, A is determined based on the optical settings of the instrument; depending on the type of A, the quality control optical parameter of the solid-phase support can be the quality control laser power, the quality control laser irradiation time, or the quality control laser parameter determined based on the quality control laser power and quality control laser irradiation time. In step b, there is a correspondence between the quality control optical parameters and the average optical parameters. For example, the quality control optical parameter is the quality control laser power, and the average optical parameter is the average laser power; the quality control optical parameter is the quality control laser irradiation time, and the average optical parameter is the average irradiation time; the quality control optical parameter is the quality control initial laser parameter, and the average optical parameter is the average initial laser parameter. Determining the initial optical parameters in this way saves the time spent figuring out the laser's operating parameters during the first reaction, thus saving microscopic imaging time.

[0129] It should be understood that b is determined by the properties of the solid-phase support and the instrument settings, and is generally within a certain range. For example, if the average laser power of the solid-phase support is 500mW and the quality control laser power is 650mW, then b is 650 / 500 = 1.3. For example, taking a sequencer as an example, the initial laser power of a sequencer is set to 550mW at the factory (i.e., A is 550mW), and b is 1.3. Therefore, the initial optical parameters are 1.3 * 550 = 715mW.

[0130] Taking biological samples as nucleic acid molecules and the reaction as sequencing of nucleic acid molecules as an example, the setting of average laser power and quality control laser power can be as follows: Based on a preset brightness range, adjust the optical parameters so that the brightness range of the fluorescence signal in the imaging image is achieved in most sequencing instruments, solid-phase carriers, and batches of reagents used in the reaction, and determine the average value of the optical parameters; set the quality control laser power based on the average value of the optical parameters, and use it as a basis to debug other sequencers and other batches of solid-phase carriers; through multiple and amplified tests on the sequencers and solid-phase carriers, adjust to obtain the final average laser power.

[0131] In some embodiments, the steps of performing a first reaction on a biological sample based on initial optical parameters, acquiring a first fluorescence signal on the surface of a solid support after the first reaction, and determining a first brightness of the first fluorescence signal in at least a portion of the solid support include: performing a first reaction on biomolecules to generate a first fluorescence signal, acquiring an image of at least one field of view (FOV) on the solid support surface where the first reaction occurs, estimating an average brightness based on the brightness of each bright spot in the image, and outputting the average brightness as the first brightness. In this embodiment, the estimation of the average brightness can be based on an image of at least one FOV on the solid support surface where the first reaction occurs, including the case based on one FOV on the solid support surface where the first reaction occurs (in this case, the image used to estimate the average brightness is an image acquired from one FOV), the case based on multiple FOVs on the solid support surface where the first reaction occurs (in this case, the image used to estimate the average brightness is an image acquired from multiple FOVs), and the case based on all FOVs on the solid support surface where the first reaction occurs (in this case, the image used to estimate the average brightness is an image acquired from all FOVs).

[0132] According to embodiments of this application, the image used to estimate the average brightness can be derived from a single first reaction or from the first few reactions in a series of reactions. The range of the selected fluorescence image is not strictly limited; a small region on the solid support can be selected for analysis. Theoretically, multiple regions on the solid support surface where the first reaction occurs, or even the entire solid support, can be selected for analysis. However, it should be understood that the larger the selected region, the longer the corresponding data processing time. The range of the small region is not strictly limited, as long as the small region contains a statistically sufficient number of data points, such as more than 10,000 points. For example, taking a base extension reaction as an example, the first brightness can be derived from the first brightness of different FOVs in the fluorescence image obtained from the same round (e.g., the first round) of base extension reaction; the first brightness can also be derived from the first brightness of the same FOV in the fluorescence images obtained from different rounds of base extension reaction in previous rounds; the first brightness can also be derived from the first brightness of different FOVs in the fluorescence images obtained from different rounds of base extension reaction in previous rounds. In some embodiments, the CV value of the first brightness of different regions of the solid support is less than or equal to 5% to obtain randomly selected regions and good data.

[0133] In some embodiments, the biomolecule is a nucleic acid molecule, and the first reaction is a base extension reaction. In this case, the average brightness is estimated based on the brightness of each bright spot in the image using the normal sequencing process, which is completed by reading data from a small region on the sequencing chip in the first or early rounds of sequencing. When estimating the brightness, fluorescence brightness and background values ​​from each camera can be read separately, simplifying the algorithm and saving time and computing resources; alternatively, the sequencer's standard image processing-basecall software can be used to calculate a more accurate sequencing brightness (patent application CN 112289377A).

[0134] In some embodiments, the biomolecule is a nucleic acid molecule, and the first reaction is a base extension reaction. In this case, the method for estimating the average brightness based on the brightness of each bright spot in the image includes: identifying signal peaks and background peaks based on an image with multiple base channels in a field of view (FOV); fitting the gray values ​​of all pixels in the image to determine the signal peak distribution curve and the background peak distribution curve based on the signal peaks and the background peaks; and then, based on the signal peak distribution curve and the background peak distribution curve... Figure 3 To determine the average brightness.

[0135] In one embodiment, determining the average brightness based on the signal peak distribution curve and the background peak distribution curve includes: determining the median value of the signal peak based on the signal peak distribution curve; determining the median value of the background peak based on the background peak distribution curve; determining the difference between the median value of the signal peak and the median value of the background peak, and using the difference as the average brightness.

[0136] In some embodiments, signal brightness during sequencing can be affected by factors such as laser power, exposure time, optical system transmittance, filter quality, purity of fluorescent bases in chemical reagents, phasing and photodamage during sequencing, chip fluorescence background, and amplification factor of chip amplification clusters. The first two, laser power and exposure time, generally have significant adjustment potential and can be adjusted through sequencing experiments to establish brightness-power and brightness-exposure time functions, serving as the basis for optical adjustment.

[0137] In step S120, the preset standard brightness is a preset standard brightness range, which can be understood as the preset brightness that can effectively identify each signal site in the image when performing microscopic imaging processing on biological samples on the surface of a solid carrier.

[0138] This application embodiment determines whether the first brightness meets the standard by comparing a first brightness with a preset standard brightness. The judgment criterion is whether the first brightness is within the preset standard brightness range. Based on the judgment structure, there are two scenarios:

[0139] In the first scenario, the initial brightness is compared with the preset standard brightness. If the initial brightness is within the preset standard brightness range, it is determined that the initial brightness meets the standard. In this case, the corresponding judgment result is "yes," and the initial optical parameter is determined to be the first optical parameter. This first optical parameter is used as the laser's operating parameter in subsequent reactions on biological samples.

[0140] In this scenario, after determining the first optical parameters for sequencing, the process further includes recording and storing these first optical parameters. When performing the same reaction on a biological sample subsequently, these first optical parameters are directly used as the laser's operating parameters. This ensures that even with numerous factors affecting performance, microscopic imaging can still proceed with the reaction based on the determined first optical parameters and achieve the required brightness in the acquired fluorescence image.

[0141] In the second scenario, the initial brightness is compared with the preset standard brightness. If the initial brightness is not within the preset standard brightness range, then the initial brightness is determined to be substandard. The corresponding judgment structure is "No," and in this case, the following processing is required:

[0142] (i) Adjust the initial optical parameters based on the preset function to determine the second optical parameter that meets the preset standard brightness in the preset function.

[0143] In this step, the preset function is a function established based on the second brightness and a third optical parameter of the second fluorescence signal in at least a portion of the solid-phase carrier when a second reaction is performed on the biological sample to generate a second fluorescence signal. It should be understood that the type of the third optical parameter corresponds to that of the first optical parameter. For example, when the first optical parameter is laser power, the third optical parameter is also laser power; when the first optical parameter is laser irradiation time, the third optical parameter is also laser irradiation time; when the first optical parameter is a correlation parameter formed by laser power and laser irradiation time (i.e., a preset laser power-laser irradiation time array), the third optical parameter is also a correlation parameter formed by laser power and laser irradiation time. Correspondingly, the preset function includes at least one selected from the brightness-laser power function, the brightness-laser irradiation time function, and the brightness-laser power-laser irradiation time function. Specifically, the brightness-laser power function is a functional relationship established based on brightness and laser power; the brightness-laser irradiation time function is a functional relationship established based on brightness and laser irradiation time; and the brightness-laser power-laser irradiation time function is a functional relationship established based on brightness, laser power, and laser irradiation time.

[0144] In some embodiments, the method for establishing the preset function is as follows: setting multiple different third optical parameters, performing a third reaction based on the different third optical parameters, collecting the third fluorescence signal on the surface of the solid support after the third reaction, determining the second brightness of each second fluorescence signal, where the third optical parameters are a preset laser power, a preset laser irradiation time, or a preset laser power-laser irradiation time array; and fitting the preset function based on the third optical parameters and the second brightness. The number of third optical parameters is not strictly limited, and their values ​​can be the same as or different from the values ​​of the first optical parameters. The third reaction is of the same type as the first reaction, but the number of rounds differs. For example, the biological sample is a nucleic acid molecule, and the reaction is a base extension reaction. The first reaction can be the first round of base extension reaction on the nucleic acid molecule, and the third reaction is the base extension reaction on the nucleic acid molecule after the first round of base extension reaction. Depending on the number of third optical parameters, the number of rounds of base extension reaction after the third base extension reaction varies. After each third reaction, collecting the third fluorescence signal on the surface of the solid support yields a fluorescence image. A preset function is obtained by fitting multiple sets of third optical parameters and the second brightness corresponding to the third fluorescence signal obtained based on the third optical parameters and the third reaction.

[0145] According to embodiments of this application, after determining a preset function, a second optical parameter can be obtained by adjusting the initial optical parameters based on the expected brightness (i.e., the preset standard brightness) when reacting with the biological sample. In some embodiments, the step of adjusting the initial optical parameters based on the preset function to determine the second optical parameter includes: determining a fourth optical parameter based on the preset function and the preset standard brightness; adjusting the initial optical parameter to the fourth optical parameter; and determining the fourth optical parameter as the second optical parameter. For example, the biological sample is a nucleic acid molecule, the reaction is a base extension reaction, and the preset standard brightness is a preset brightness range of bases that meets the requirements for base recognition.

[0146] (ii) Repeat steps S110 and S120 with the second optical parameter instead of the initial optical parameter until the brightness meets the standard, and determine the first optical parameter.

[0147] In this step, after confirming the second optical parameter, the second optical parameter is used as the initial optical parameter, and steps S110 and S120 are repeated to adjust the optical parameter until the first optical parameter with the required brightness is obtained.

[0148] In repeating steps (i) and (ii), in each step (i), the image binary of the first brightness can be derived from various cases. In some embodiments, the first brightness originates from the first brightness of different FOVs in the image generated by the same first reaction; in some embodiments, the first brightness originates from the first brightness of the same FOV in different first reactions; in some embodiments, the first brightness originates from the first brightness of different FOVs in different first reactions.

[0149] In some embodiments, the biological sample is a nucleic acid molecule, the first reaction is a base extension reaction, and steps (i) and (ii) are repeated less than or equal to 3 times. This can prevent some solid-phase carriers from experiencing faults such as contamination, leakage, or "screen bursting" during sequencing, which would result in abnormal brightness data that cannot be used normally.

[0150] According to some more specific embodiments of the present invention, the present invention proposes a method for adjusting a first optical parameter during sequencing, such as... Figure 2 As shown, it includes:

[0151] S110. Perform the first round of base extension reaction on the nucleic acid molecules to be tested on the surface of the sequencing chip, collect the fluorescence signal on the surface of the sequencing chip after the first round of base extension reaction to obtain fluorescence imaging, and estimate the sequencing brightness.

[0152] S1201: The control software determines whether the sequencing brightness meets the standard. The brightness standard is generally a range. If the quality of the sequencing image within this range meets the base recognition requirements, the sequencing brightness is considered to meet the standard, and the sequencing step is initiated; otherwise, the process proceeds to step S1202.

[0153] S1202. Calculate the laser power or exposure time value or the brightness-exposure time dual adjustment value that can make the brightness enter the target range according to the preset function.

[0154] S1203: Adjust laser power or exposure time, or dual adjustment of brightness and exposure time;

[0155] S1204. Re-image the chip using the new parameters and estimate the sequencing brightness. Re-image can be taken at a different location on the sequencing chip to prevent fluorescence quenching from the first imaging.

[0156] S1205, repeat steps S1202-S1204 until the sequencing brightness reaches the target. A maximum of 3 cycles can be set to prevent other faults from occurring on some chips, such as reagent leakage causing multiple imaging failures.

[0157] Sequencing is performed according to the compliant sequencing optical parameters. If the sequencing is a quality control test and the sequencing is successful, the first optical parameters used are output, and the optical parameter information is written into the chip QR code.

[0158] According to some more specific embodiments of the present invention, during the sequencing process, the optical parameter information carried by the chip and reagent kit when the brightness meets the standard is read in, and the optical parameter information is multiplied by the initial optical parameters on the sequencer to obtain the initial optical parameters at the start of sequencing. For example, if the factory power of the sequencer is 550mW and the optical parameter information is 1.3, the resulting initial optical parameter is 715mW. Starting sequencing with the initial optical parameter can save sequencing adaptive time. In the embodiments of this application, the sequencing adaptive time refers to the time taken for the instrument to reach the specific sequencing brightness that meets the requirements.

[0159] According to some specific embodiments of the present invention, the calculation of brightness estimation during sequencing involves signal localization, statistical median value of the signal region, background region setting, statistical median value of the signal region, signal minus background, and taking the median value of sequencing brightness within the FOV. Sequencing brightness estimation has no requirements on the sequencing process; it involves reading data from a small region, such as an FOV, on the sequencing chip during the first or early sequencing rounds, and then estimating the brightness of subsequent sequencing rounds based on the brightness values ​​from the first or early rounds.

[0160] According to some more specific embodiments of the present invention, in steps S1201 and S1205 of the above method, it is determined whether the brightness of each base is within a preset range. For example, the preset brightness range for the A- base is 60-100. The base recognition requirement refers to the brightness requirement for the four bases: such as 60-100 for the A- base and 60-100 for the T base. According to historical data, within such brightness ranges, the statistical probability that the base recognition error rate in subsequent sequencing rounds is less than 0.5% is higher than 99.9%.

[0161] According to some embodiments of the present invention, the brightness CV of the sequencing chip used in the present invention is less than 5%, so as to improve the brightness uniformity of different regions on the surface of the sequencing chip and the uniformity of optical devices.

[0162] Typically, the optical performance of sequencing chips and reagents in each batch is similar; however, there may be fluctuations in sequencing brightness between different batches. Therefore, the optical parameters used in the quality control of sequencing chips and reagents can be recorded and stored in the product information of the reagent kit in the form of QR codes. In this way, using the method provided in the embodiments of this application, users can immediately select the first optical parameters for this batch when passing quality control based on the information when reading the QR code during the sequencing process. This method reduces the process of repeatedly adjusting to obtain appropriate laser power and / or laser irradiation time, thus saving sequencing time.

[0163] Microscopic imaging device

[0164] According to some embodiments of the present invention, a microscopic imaging device is proposed, including a laser and a first optical parameter determination system, wherein the first optical parameter determination system is used to determine the first optical parameters when performing microscopic imaging processing on a biological sample on the surface of a solid carrier, and the first optical parameters are the laser power and / or laser time that meet the brightness standard.

[0165] According to some embodiments of the present invention, a first optical parameter determination system is proposed, the system comprising:

[0166] The first brightness determination unit is used to perform a first reaction on the biological sample based on initial optical parameters, collect the first fluorescence signal on the surface of the solid support after the first reaction, and determine the first brightness of the first fluorescence signal in at least a part of the solid support.

[0167] The first brightness judgment unit is used to compare the first brightness with the preset standard brightness to determine whether the first brightness meets the standard.

[0168] If so, then the initial optical parameter is determined to be the first optical parameter;

[0169] If not, the initial optical parameters are adjusted based on a preset function to determine the second optical parameter that meets the preset standard brightness in the preset function; the first brightness determination unit and the first brightness judgment unit are repeated with the second optical parameter instead of the initial optical parameter until the brightness reaches the standard, and the first optical parameter is determined.

[0170] The preset function is a function established based on the second brightness and third optical parameters of the second fluorescence signal in at least a part of the solid carrier when the biological sample undergoes a second reaction and the second fluorescence signal is collected on the surface of the solid carrier after the second reaction. The second optical parameters are the second laser power and / or the second laser irradiation time.

[0171] In the first brightness determination unit, after a biological sample undergoes a certain reaction, situations such as generating, enhancing, or reducing fluorescence signals may occur. This embodiment of the application collects the fluorescence signal after the reaction based on the initial optical parameters of the laser that excites the biological sample to generate a fluorescence signal, and obtains the brightness of the fluorescence signal of the biological sample under the initial optical parameters. It should be understood that this device is suitable for situations where biological samples undergo multiple reactions and the fluorescence signal of the solid-phase support is collected during each reaction. Specifically, by using the fluorescence signal obtained during the first or several previous reactions and the corresponding parameters, the optical parameters used for subsequent reactions (i.e., the first optical parameters) are determined, so that in subsequent reactions, the laser directly selects these optical parameters to perform microscopic optical imaging of the fluorescence signal generated by the biological sample and obtains an image with acceptable brightness. This reduces the impact of various factors affecting the stability of the fluorescence signal on the judgment of data obtained from the image during microscopic imaging, improving the accuracy of the image reaction data.

[0172] This application does not strictly limit the type of reaction in its embodiments. In some embodiments, the first reaction is one of the first N reactions performed on the biological sample. That is, the first reaction is the first or one of the first few reactions when the biomolecules undergo multiple reactions and the fluorescence signal of the solid-phase support is collected during each reaction. Thus, by determining the first brightness corresponding to the first fluorescence signal obtained under the initial optical parameter conditions through the first few reactions performed on the biological sample, the time for determining the first optical parameters can be saved. In some embodiments, the value of N ranges from 1 to 5, and for example, N is 1, 2, 3, 4, 5, etc. For example, the biological sample is a nucleic acid molecule, and this microscopic imaging method is used to collect fluorescence signals when sequencing nucleic acid molecules. In this case, the first reaction corresponds to the sequencing reaction, specifically the first round or the first few rounds of sequencing reaction.

[0173] In this embodiment, the initial optical parameter is the initial value of the optical parameter at the start of the reaction. Correspondingly, in this embodiment, the initial optical parameter is the optical parameter during the first or several previous reactions when the biological sample undergoes multiple reactions. Taking nucleic acid molecules as the biological sample and sequencing reactions as an example, the initial optical parameter is the optical parameter during the first or several previous sequencing reactions of the nucleic acid molecules. In some embodiments, the initial optical parameter is calculated as: Initial optical parameter = A × b, where A represents the initial laser power, initial laser irradiation time, or initial laser parameter determined based on the initial laser power and initial laser irradiation time, and b represents the ratio of the quality control optical parameter to the average optical parameter of the solid-phase carrier. Here, A is determined based on the instrument's optical settings; depending on the type of A, the quality control optical parameter of the solid-phase carrier can be the quality control laser power, quality control laser irradiation time, or quality control laser parameter determined based on the quality control laser power and quality control laser irradiation time. In step b, there is a correspondence between the quality control optical parameters and the average optical parameters. For example, the quality control optical parameter is the quality control laser power, and the average optical parameter is the average laser power; the quality control optical parameter is the quality control laser irradiation time, and the average optical parameter is the average irradiation time; the quality control optical parameter is the quality control initial laser parameter, and the average optical parameter is the average initial laser parameter. Determining the initial optical parameters in this way saves the time spent figuring out the laser's operating parameters during the first reaction, thus saving microscopic imaging time.

[0174] It should be understood that b is determined by the properties of the solid-phase support and the instrument settings, and is generally within a certain range. For example, if the average laser power of the solid-phase support is 500mW and the quality control laser power is 650mW, then b is 650 / 500 = 1.3. For example, taking a sequencer as an example, the initial laser power of a sequencer is set to 550mW at the factory (i.e., A is 550mW), and b is 1.3. Therefore, the initial optical parameters are 1.3 * 550 = 715mW.

[0175] Taking biological samples as nucleic acid molecules and the reaction as sequencing of nucleic acid molecules as an example, the setting of average laser power and quality control laser power can be as follows: Based on a preset brightness range, adjust the optical parameters so that the brightness range of the fluorescence signal in the imaging image is achieved in most sequencing instruments, solid-phase carriers, and batches of reagents used in the reaction, and determine the average value of the optical parameters; set the quality control laser power based on the average value of the optical parameters, and use it as a basis to debug other sequencers and other batches of solid-phase carriers; through multiple and amplified tests on the sequencers and solid-phase carriers, adjust to obtain the final average laser power.

[0176] In some embodiments, the steps of performing a first reaction on a biological sample based on initial optical parameters, acquiring a first fluorescence signal on the surface of a solid support after the first reaction, and determining a first brightness of the first fluorescence signal in at least a portion of the solid support include: performing a first reaction on biomolecules to generate a first fluorescence signal, acquiring an image of at least one field of view (FOV) on the solid support surface where the first reaction occurs, estimating an average brightness based on the brightness of each bright spot in the image, and outputting the average brightness as the first brightness. In this embodiment, the estimation of the average brightness can be based on an image of at least one FOV on the solid support surface where the first reaction occurs, including the case based on one FOV on the solid support surface where the first reaction occurs (in this case, the image used to estimate the average brightness is an image acquired from one FOV), the case based on multiple FOVs on the solid support surface where the first reaction occurs (in this case, the image used to estimate the average brightness is an image acquired from multiple FOVs), and the case based on all FOVs on the solid support surface where the first reaction occurs (in this case, the image used to estimate the average brightness is an image acquired from all FOVs).

[0177] According to embodiments of this application, the image used to estimate the average brightness can be derived from a single first reaction or from the first few reactions in multiple reactions. The range of the selected fluorescence image is not strictly limited; a small region on the solid support can be selected for analysis. Theoretically, multiple regions on the solid support surface where the first reaction occurs, or even the entire solid support, can be selected for analysis. However, it should be understood that the larger the selected region, the longer the corresponding data processing time. The range of the small region is not strictly limited, as long as the small region contains a statistically sufficient number of data points, such as more than 10,000 points. For example, taking a sequencing reaction as an example, the first brightness can be derived from the first brightness of different FOVs in the fluorescence images obtained from the same round (e.g., the first round) of sequencing reaction; the first brightness can also be derived from the first brightness of the same FOV in fluorescence images obtained from different rounds of sequencing reaction in previous rounds; the first brightness can also be derived from the first brightness of different FOVs in fluorescence images obtained from different rounds of sequencing reaction in previous rounds. In some embodiments, the CV value of the first brightness of different regions of the solid support is less than or equal to 5% to obtain randomly selected regions and good data.

[0178] In some embodiments, the biomolecule is a nucleic acid molecule, and the first reaction is a base extension reaction. In this case, the average brightness is estimated based on the brightness of each bright spot in the image using the normal sequencing process, which is completed by reading data from a small region on the sequencing chip in the first or early rounds of sequencing. When estimating the brightness, fluorescence brightness and background values ​​from each camera can be read separately, simplifying the algorithm and saving time and computing resources; alternatively, the sequencer's standard image processing-basecall software can be used to calculate a more accurate sequencing brightness (patent application CN 112289377A).

[0179] In some embodiments, the biomolecule is a nucleic acid molecule, and the first reaction is a sequencing reaction. In this case, the method for estimating the average brightness based on the brightness of each bright spot in the image includes: identifying signal peaks and background peaks based on an image with multiple base channels in a field of view (FOV); fitting the gray values ​​of all pixels in the image to determine the signal peak distribution curve and the background peak distribution curve based on the signal peaks and the background peaks; and then, based on the signal peak distribution curve and the background peak distribution curve... Figure 3 To determine the average brightness.

[0180] In one embodiment, determining the average brightness based on the signal peak distribution curve and the background peak distribution curve includes: determining the median value of the signal peak based on the signal peak distribution curve; determining the median value of the background peak based on the background peak distribution curve; determining the difference between the median value of the signal peak and the median value of the background peak, and using the difference as the average brightness.

[0181] In some embodiments, signal brightness during sequencing can be affected by factors such as laser power, exposure time, optical system transmittance, filter quality, purity of fluorescent bases in chemical reagents, phasing and photodamage during sequencing, chip fluorescence background, and amplification factor of chip amplification clusters. The first two, laser power and exposure time, generally have significant adjustment potential and can be adjusted through sequencing experiments to establish brightness-power and brightness-exposure time functions, serving as the basis for optical adjustment.

[0182] In the first brightness judgment unit, the preset standard brightness is a preset standard brightness range, which can be understood as the preset brightness that can effectively identify each signal point in the image when performing microscopic imaging processing on biological samples on the surface of a solid carrier.

[0183] This application embodiment determines whether the first brightness meets the standard by comparing a first brightness with a preset standard brightness. The judgment criterion is whether the first brightness is within the preset standard brightness range. Based on the judgment structure, there are two scenarios:

[0184] In the first scenario, the initial brightness is compared with the preset standard brightness. If the initial brightness is within the preset standard brightness range, it is determined that the initial brightness meets the standard. In this case, the corresponding judgment result is "yes," and the initial optical parameter is determined to be the first optical parameter. This first optical parameter is used as the laser's operating parameter in subsequent reactions on biological samples.

[0185] In this scenario, after determining the first optical parameters for sequencing, the process further includes recording and storing these first optical parameters. When performing the same reaction on a biological sample subsequently, these first optical parameters are directly used as the laser's operating parameters. This ensures that even with numerous factors affecting performance, microscopic imaging can still proceed with the reaction based on the determined first optical parameters and achieve the required brightness in the acquired fluorescence image.

[0186] In the second scenario, the initial brightness is compared with the preset standard brightness. If the initial brightness is not within the preset standard brightness range, then the initial brightness is determined to be substandard. The corresponding judgment structure is "No," and in this case, the following processing is required:

[0187] (i) Adjust the initial optical parameters based on the preset function to determine the second optical parameter that meets the preset standard brightness in the preset function.

[0188] In this step, the preset function is a function established based on the second brightness and a third optical parameter of the second fluorescence signal in at least a portion of the solid-phase support when the biomolecule undergoes a second reaction to generate a second fluorescence signal. It should be understood that the type of the third optical parameter corresponds to that of the first optical parameter. For example, when the first optical parameter is laser power, the third optical parameter is also laser power; when the first optical parameter is laser irradiation time, the third optical parameter is also laser irradiation time; when the first optical parameter is a correlation parameter formed by laser power and laser irradiation time (i.e., a preset laser power-laser irradiation time array), the third optical parameter is also a correlation parameter formed by laser power and laser irradiation time. Correspondingly, the preset function includes at least one selected from the brightness-laser power function, the brightness-laser irradiation time function, and the brightness-laser power-laser irradiation time function. Specifically, the brightness-laser power function is a functional relationship established based on brightness and laser power; the brightness-laser irradiation time function is a functional relationship established based on brightness and laser irradiation time; and the brightness-laser power-laser irradiation time function is a functional relationship established based on brightness, laser power, and laser irradiation time.

[0189] In some embodiments, the method for establishing the preset function is as follows: setting multiple different third optical parameters, performing a third reaction based on the different third optical parameters, collecting the third fluorescence signal on the surface of the solid support after the third reaction, determining the second brightness of each second fluorescence signal, where the third optical parameters are a preset laser power, a preset laser irradiation time, or a preset laser power-laser irradiation time array; and fitting the preset function based on the third optical parameters and the second brightness. The number of third optical parameters is not strictly limited, and their values ​​can be the same as or different from the values ​​of the first optical parameters. The third reaction is of the same type as the first reaction, but the number of rounds differs. For example, the biological sample is a nucleic acid molecule, and the reaction is a sequencing reaction. The first reaction can be the first round of sequencing of the nucleic acid molecule, and the third reaction is the sequencing reaction performed on the nucleic acid molecule after the first round of sequencing. Depending on the number of third optical parameters, the number of rounds of sequencing reactions performed after the third sequencing reaction varies. After each third reaction, collecting the third fluorescence signal on the surface of the solid support yields a fluorescence image. The preset function is obtained by fitting multiple sets of third optical parameters and the second brightness corresponding to the third fluorescence signal obtained from the third reaction based on the third optical parameters.

[0190] According to embodiments of this application, after determining a preset function, a second optical parameter can be obtained by adjusting the initial optical parameters based on the expected brightness (i.e., the preset standard brightness) when reacting with the biological sample. In some embodiments, the step of adjusting the initial optical parameters based on the preset function to determine the second optical parameter includes: determining a fourth optical parameter based on the preset function and the preset standard brightness; adjusting the initial optical parameters to the fourth optical parameter; and determining the fourth optical parameter as the second optical parameter. For example, the biological sample is a nucleic acid molecule, the reaction is a sequencing reaction, and the preset standard brightness is a preset brightness range of bases that meets the requirements for base recognition.

[0191] (ii) The first brightness determination unit and the first brightness judgment unit are repeated with the second optical parameter instead of the initial optical parameter until the brightness reaches the standard, and the first optical parameter is determined.

[0192] In this step, after confirming the second optical parameter, the second optical parameter is used as the initial optical parameter, and the first brightness determination unit and the first brightness judgment unit are re-performed to adjust the optical parameter until the first optical parameter that meets the brightness standard is obtained.

[0193] In repeating steps (i) and (ii), in each step (i), the image binary of the first brightness can be derived from various cases. In some embodiments, the first brightness originates from the first brightness of different FOVs in the image generated by the same first reaction; in some embodiments, the first brightness originates from the first brightness of the same FOV in different first reactions; in some embodiments, the first brightness originates from the first brightness of different FOVs in different first reactions.

[0194] In some embodiments, the biological sample is a nucleic acid molecule, the first reaction is a sequencing reaction, and the number of times the first brightness determination unit and the first brightness judgment unit are repeated is less than or equal to 3. This can prevent some solid-phase carriers from experiencing faults such as contamination, leakage, or "screen bursting" during the sequencing process, which would result in abnormal brightness data that cannot be used normally.

[0195] According to some specific embodiments of the present invention, the calculation of brightness estimation during sequencing involves signal localization, statistical median value of the signal region, background region setting, statistical median value of the signal region, signal minus background, and taking the median value of sequencing brightness within the FOV. Sequencing brightness estimation has no requirements on the sequencing process; it involves reading data from a small region, such as an FOV, on the sequencing chip during the first or early sequencing rounds, and then estimating the brightness of subsequent sequencing rounds based on the brightness values ​​from the first or early rounds.

[0196] According to some more specific embodiments of the present invention, it is determined whether the brightness of each base is within a preset range. For example, the preset brightness range for the A- base is 60-100. The base identification requirement refers to the brightness requirement for the four bases: such as 60-100 for the A- base and 60-100 for the T base. Based on historical data, within such brightness ranges, the statistical probability that the base identification error rate in subsequent sequencing rounds is less than 0.5% is higher than 99.9%.

[0197] According to some embodiments of the present invention, the brightness CV of the sequencing chip used in the present invention is less than 5%, so as to improve the brightness uniformity of different regions on the surface of the sequencing chip and the uniformity of optical devices.

[0198] The present disclosure will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the disclosure. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0199] Example 1: Brightness Control Function

[0200] 1. Changing the sequencing brightness by altering the laser power used for sequencing.

[0201] The experiment used three GenoLab sequencers and two sequencing chips (labeled Chip 1 and Chip 2, respectively). Different laser powers (including standard power and power obtained by increasing or decreasing the standard power by a certain percentage) were used for one round of sequencing. Table 1 below shows the laser power used for sequencing of the two chips.

[0202] Table 1

[0203]

[0204] Sequencing brightness was collected and plotted using standard image processing-base recognition software (see patent application CN 112289377 A). Figure 4 As shown, the horizontal axis represents the sequencing laser power, and the vertical axis represents the sequencing signal brightness. The left figure is a fitting graph of the brightness change of chip 1 under different power conditions in three sequencers, and the right figure is a fitting graph of the brightness change of chip 1 under different power conditions in three sequencers. The three lines correspond to the three sequencers respectively. Figure 4 The laser power and sequencing brightness show a linear relationship. Therefore, the method for determining the first optical parameter according to the embodiments of this application is feasible.

[0205] 2. Sequencing brightness is changed by continuously adjusting the optical parameters of each cycle during the sequencing process.

[0206] Figure 5The graph shows the sequencing brightness curves generated under different laser power conditions: the horizontal axis represents the number of sequencing cycles, and the vertical axis represents the sequencing signal brightness (Total: the sum of the brightness of the four bases). L01 is the brightness-cycle curve generated without any laser power adjustment. The laser power corresponding to L01 is a constant (515mW for green laser, 440mW for red laser). As shown in the figure, the sequencing brightness decreases with increasing sequencing cycle number, reflecting sequencing signal loss caused by factors such as phasing and photochemical damage during sequencing. L02 is the brightness-cycle curve after laser power adjustment based on L01, according to the function power = 515 + 1.033*n (n is the number of cycles), increasing the laser power by 1.033mW per cycle. As shown in the figure, under this condition, the brightness-cycle curve is basically flattened, and the stability of the sequencing brightness is improved, not fluctuating with the number of cycles. The brightness decreases with increasing rotation number, resulting in more stable brightness that improves sequencing quality. The brightness curve for L03, based on the laser power of L01, shows an increase of approximately 13% (580mW for green laser and 493mW for red laser), resulting in an overall brightness increase of about 20% compared to L01. The brightness curve for L04, based on the laser power of the green line L03, is adjusted according to the function power = 580 + 1.033*n (n being the rotation number), increasing the power by 1.033mW per rotation. As shown in the figure, under these conditions, the brightness-rotation number curve is essentially flattened, indicating improved sequencing brightness stability that does not decrease with increasing rotation number. This more stable brightness is beneficial for sequencing quality. This experiment demonstrates that adjusting the laser power according to the brightness control function can effectively control sequencing brightness.

[0207] Example 2: Adaptive adjustment of laser power

[0208] A 250M sequencing chip was used on a GenoLab sequencer to sequence E. coli standards, generating a brightness control function based on laser power. Specifically, the green laser (532nm laser) power was adjusted in each sequencing round: starting from a standard power of 515mW, it was initially reduced by 40%, then gradually increased to 824mW; the red laser power remained unchanged. After sequencing, the following data processing was performed:

[0209] (1) The A-base signal brightness of each round was calculated and extracted using standard image processing-basecall software;

[0210] (2) Take the average brightness of the sequencing under different laser power parameters, calculate the corresponding percentage of brightness compared with 515nm, and the results are shown in Table 2.

[0211] Table 2

[0212] Green laser power Power adjustment percentage A-Base Brightness Luminance response percentage 309 -40％ 23.06 -44％ 412 -20％ 30.18 -26％ 515 0％ 40.87 0％ 618 20％ 52.84 29％ 721 40％ 65.83 61％ 824 60％ 81.22 99％

[0213] (3) Plot the corresponding brightness percentage against the power adjustment percentage, such as... Figure 6 As shown, the linear function is determined to be y = 1.4761x, where y represents the brightness response percentage and x represents the power adjustment percentage;

[0214] (4) Use this function in subsequent sequencing: that is, if the A-base brightness is not within the threshold (60-100) range after the first round of sequencing, determine the laser power corresponding to the expected brightness in the function according to the function and the expected brightness, until the laser power that meets the sequencing brightness standard is determined. This process is also called adaptive laser process.

[0215] Sequencing was performed using a separate 250M sequencing chip with E. coli standards as samples. The first round of sequencing was performed at standard power (515mW), followed by calculation of the A-base signal intensity. The calculated signal intensity was outside the threshold range (60-100) and had a center difference of -73%. Based on the function, the laser power was calculated to be increased by 50%.

[0216] (5) Change the green laser power of the sequencer to 773mW (increase by 50%) and perform a new round of sequencing. Use standard image processing - basecall software again to calculate and extract the A-base signal brightness for each round. The brightness has met the threshold range (60-100) requirement, as shown in Table 3.

[0217] Table 3

[0218] Green laser power Power adjustment percentage A-Base Brightness 515 0％ 46.19 773 50％ 93.81

[0219] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0220] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A microscopic imaging method, characterized in that, Microscopic imaging processing of biological samples on the surface of a solid carrier is performed based on an imaging device, wherein the imaging device includes a laser, and the microscopic imaging processing uses the laser to perform microscopic optical imaging of the fluorescence signal generated by the biological sample based on a first optical parameter, wherein the first optical parameter is a laser power with sufficient brightness and / or laser irradiation time. The methods for determining the first optical parameter include: (i) Based on the initial optical parameters, a first reaction is performed on the biological sample, and a first fluorescence signal is collected on the surface of the solid support after the first reaction, and the first brightness of the first fluorescence signal in at least a part of the solid support is determined; (ii) Compare the first brightness with the preset standard brightness to determine whether the first brightness meets the standard; If so, then the initial optical parameter is determined to be the first optical parameter; If not, adjust the initial optical parameters until the brightness meets the standard, and then determine the first optical parameters.

2. The method according to claim 1, characterized in that, The method for determining the first optical parameter further includes: If the first brightness does not meet the standard, the initial optical parameters are adjusted based on a preset function to determine the second optical parameters that meet the preset standard brightness in the preset function; steps (i) and (ii) are repeated with the second optical parameters instead of the initial optical parameters until the brightness meets the standard, and the first optical parameters are determined. The preset function is a function established based on the second brightness and third optical parameters of the second fluorescence signal in at least a part of the solid carrier when the biological sample undergoes a second reaction and the second fluorescence signal is collected on the surface of the solid carrier after the second reaction. The second optical parameters are the second laser power and / or the second laser irradiation time.

3. The method according to claim 2, characterized in that, The initial optical parameters are calculated using the formula: Initial optical parameters = A × b, where A represents the initial laser power or initial laser irradiation time of the laser, or the initial laser parameters determined based on the initial laser power and the initial laser irradiation time, and b represents the ratio of the quality control optical parameters to the average optical parameters of the solid-phase carrier. The quality control optical parameter is the quality control laser power, and the average optical parameter is the average laser power, or The quality control optical parameter is the quality control laser irradiation time, and the average optical parameter is the average irradiation time, or The quality control optical parameters are the initial laser parameters for quality control, and the average optical parameters are the average initial laser parameters.

4. The method according to claim 2, wherein the step of performing a first reaction on the biological sample, collecting a first fluorescence signal on the surface of the solid support after the first reaction, and determining a first brightness of the first fluorescence signal in at least a portion of the solid support comprises: The biological sample undergoes a first reaction, and images of at least one field of view (FOV) on the surface of the solid support where the first reaction occurs are acquired. The average brightness is estimated based on the brightness of each bright spot in the image, and the average brightness is output as the first brightness.

5. The method according to claim 4, characterized in that, The image was captured from a field of view (FOV).

6. The method according to claim 4, characterized in that, The first reaction is one of the first N reactions performed on the biological sample.

7. The method according to claim 6, characterized in that, The value of N ranges from 1 to 5.

8. The method according to claim 4, characterized in that, A method for estimating the average brightness based on the brightness of each bright spot in the image includes: Based on images from multiple detection channels within a single field of view (FOV), identify signal peaks and background peaks. Based on the signal peak and the background peak, the gray values ​​of all pixels in the image are fitted respectively to determine the signal peak distribution curve and the background peak distribution curve; The average brightness is determined based on the signal peak distribution curve and the background peak distribution curve.

9. The method according to claim 8, characterized in that, Determining the average brightness based on the signal peak distribution curve and the background peak distribution curve includes: Based on the signal peak distribution curve, determine the median value of the signal peak; Based on the background peak distribution curve, determine the median value of the background peak; Based on the median value of the signal peak and the median value of the background peak, the difference between the two is determined, and the difference is used as the average brightness.

10. The method according to claim 2, characterized in that, The preset standard brightness is a preset standard brightness range.

11. The method according to claim 10, characterized in that, By comparing the first brightness with a preset standard brightness, it is determined whether the first brightness meets the standard, including: Compare the first brightness with the preset standard brightness. If the first brightness is within the preset standard brightness range, then the first brightness is determined to meet the standard. If the first brightness is not within the preset standard brightness range, then the first brightness is determined to be substandard.

12. The method according to any one of claims 1-11, characterized in that, The preset function includes at least one selected from the brightness-laser power function, brightness-laser irradiation time function, and brightness-laser power-laser irradiation time function.

13. The method according to claim 2, characterized in that, The method for establishing the preset function is as follows: Multiple different third optical parameters are set, and the reaction is carried out based on different third optical parameters. The third fluorescence signal on the surface of the solid support after the third reaction is collected, and the second brightness of the third fluorescence signal in each round is determined. The third optical parameters are preset laser power, preset laser irradiation time, or preset laser power-laser irradiation time array. Based on the third optical parameter and the second brightness, a preset function is obtained by fitting.

14. The method according to claim 2, characterized in that, The step of adjusting the initial optical parameters based on a preset function to determine the second optical parameters includes: Based on the preset function and preset standard brightness, the fourth optical parameter is determined; The initial optical parameters are adjusted to the fourth optical parameter, and the fourth optical parameter is determined as the second optical parameter.

15. The method according to claim 4, characterized in that, Repeat steps (i) and (ii), in each step (i), the first brightness is derived from the first brightness of different FOVs in the image generated by the same first reaction, or The first brightness is derived from the first brightness of the same FOV in different first reactions, or The first brightness is derived from the first brightness of different FOVs of different first reactions.

16. The method according to claim 15, characterized in that, The number of times steps (i) and (ii) are repeated is less than or equal to 3.

17. The method according to any one of claims 1-11, characterized in that, After the step of determining the first optical parameter, the method further includes: recording and storing the first optical parameter.

18. The method according to any one of claims 1-11, characterized in that, The CV value of the first brightness in different regions of the solid support is less than or equal to 5%.

19. The method according to any one of claims 2-11, characterized in that, The biological sample is a nucleic acid molecule, and the first reaction and the second reaction are base extension reactions performed on the surface of the biological sample on the solid support.

20. A microscopic imaging device, characterized in that, The system includes a laser and a first optical parameter determination system, which is used to determine the first optical parameters when performing microscopic imaging on a biological sample on the surface of a solid carrier, wherein the first optical parameters are the laser power and / or laser time that meet the brightness requirements. The first optical parameter determination system includes: The first brightness determination unit is used to perform a first reaction on the biological sample based on initial optical parameters, collect the first fluorescence signal on the surface of the solid support after the first reaction, and determine the first brightness of the first fluorescence signal in at least a part of the solid support. The first brightness judgment unit is used to compare the first brightness with the preset standard brightness to determine whether the first brightness meets the standard. If so, then the initial optical parameter is determined to be the first optical parameter; If not, adjust the initial optical parameters until the brightness meets the standard, and then determine the first optical parameters.

21. The apparatus according to claim 20, characterized in that, The first optical parameter determination system further includes: If the first brightness does not meet the standard, the initial optical parameters are adjusted based on a preset function to determine the second optical parameters that meet the preset standard brightness. The first brightness determination unit and the first brightness judgment unit are repeated with the second optical parameters instead of the initial optical parameters until the brightness meets the standard, and then the first optical parameters are determined. The preset function is a function established based on the second brightness and third optical parameters of the second fluorescence signal in at least a part of the solid carrier when the biological sample undergoes a second reaction and the second fluorescence signal is collected on the surface of the solid carrier after the second reaction. The second optical parameters are the second laser power and / or the second laser irradiation time.

22. The apparatus according to claim 21, characterized in that, The initial optical parameters are calculated using the formula: Initial optical parameters = A × b, where A represents the initial laser power or initial laser irradiation time of the laser, or the initial laser parameters determined based on the initial laser power and the initial laser irradiation time, and b represents the ratio of the quality control optical parameters to the average optical parameters of the solid-phase carrier. The quality control optical parameter is the quality control laser power, and the average optical parameter is the average laser power, or The quality control optical parameter is the quality control laser irradiation time, and the average optical parameter is the average irradiation time, or The quality control optical parameters are the initial laser parameters for quality control, and the average optical parameters are the average initial laser parameters.

23. The apparatus according to claim 21, characterized in that, The steps of performing a first reaction on the biological sample, collecting a first fluorescence signal on the surface of the solid support after the first reaction, and determining the first brightness of the first fluorescence signal in at least a portion of the solid support include: The biological sample undergoes a first reaction, and images of at least one field of view (FOV) on the surface of the solid support where the first reaction occurs are acquired. The average brightness is estimated based on the brightness of each bright spot in the image, and the average brightness is output as the first brightness.

24. The apparatus according to claim 23, characterized in that, The image was captured from a field of view (FOV).

25. The apparatus according to claim 23, characterized in that, The first reaction is one of the first N reactions performed on the biological sample.

26. The apparatus according to claim 25, characterized in that, The value of N ranges from 1 to 5.

27. The apparatus according to claim 23, characterized in that, A method for estimating the average brightness based on the brightness of each bright spot in the image includes: Based on images from multiple detection channels within a single field of view (FOV), identify signal peaks and background peaks. Based on the signal peak and the background peak, the gray values ​​of all pixels in the image are fitted respectively to determine the signal peak distribution curve and the background peak distribution curve; The average brightness is determined based on the signal peak distribution curve and the background peak distribution curve.

28. The apparatus according to claim 27, characterized in that, Determining the average brightness based on the signal peak distribution curve and the background peak distribution curve includes: Based on the signal peak distribution curve, determine the median value of the signal peak; Based on the background peak distribution curve, determine the median value of the background peak; Based on the median value of the signal peak and the median value of the background peak, the difference between the two is determined, and the difference is used as the average brightness.

29. The apparatus according to claim 21, characterized in that, The preset standard brightness is a preset standard brightness range.

30. The apparatus according to claim 29, characterized in that, By comparing the first brightness with a preset standard brightness, it is determined whether the first brightness meets the standard, including: Compare the first brightness with the preset standard brightness. If the first brightness is within the preset standard brightness range, then the first brightness is determined to meet the standard. If the first brightness is not within the preset standard brightness range, then the first brightness is determined to be substandard.

31. The apparatus according to any one of claims 20-30, characterized in that, The preset function includes at least one selected from the brightness-laser power function, brightness-laser irradiation time function, and brightness-laser power-laser irradiation time function.

32. The apparatus according to claim 21, characterized in that, The method for establishing the preset function is as follows: Multiple third optical parameters are set, and reactions are carried out based on different third optical parameters. The third fluorescence signal on the surface of the solid support after the third reaction is collected, and the second brightness of the third fluorescence signal in each round is determined. The third optical parameters are preset laser power, preset laser irradiation time, or preset laser power-laser irradiation time array. Based on the third optical parameter and the second brightness, a preset function is obtained by fitting.

33. The apparatus according to claim 21, characterized in that, The step of adjusting the initial optical parameters based on a preset function to determine the second optical parameters includes: Based on the preset function and preset standard brightness, the fourth optical parameter is determined; The initial optical parameters are adjusted to the fourth optical parameter, and the fourth optical parameter is determined as the second optical parameter.

34. The apparatus according to claim 21 or 23, characterized in that, When repeating the first brightness determination unit and the first brightness judgment unit, in each first brightness determination unit, the first brightness is respectively derived from the first brightness of different FOVs in the image generated by the same first reaction, or The first brightness is derived from the first brightness of the same FOV in different first reactions, or The first brightness is derived from the first brightness of different FOVs of different first reactions.

35. The apparatus according to claim 34, characterized in that, The number of times the first brightness determination unit and the first brightness judgment unit are repeated is less than or equal to 3.

36. The apparatus according to any one of claims 20-30, characterized in that, The microscopic imaging device further includes a storage unit connected to the first brightness determination unit, and the storage unit is used to record and store the first optical parameters.

37. The apparatus according to any one of claims 20-30, characterized in that, The CV value of the first brightness in different regions of the solid support is less than or equal to 5%.

38. The apparatus according to any one of claims 21-30, characterized in that, The biological sample is a nucleic acid molecule, and the first reaction and the second reaction are base extension reactions performed on the surface of the biological sample on the solid support.

39. Use of the microscopic imaging apparatus according to any one of claims 20-38 in sequencing.

40. A method for obtaining preset optical parameters of a sequencing chip, characterized in that, The method includes obtaining a first optical parameter using the method for determining a first optical parameter in any one of the microscopic imaging methods of claims 1-19, and using the value of the first optical parameter as a preset optical parameter of the sequencing chip.

41. A sequencing method, characterized in that, The sequencing method includes performing microscopic optical imaging on the fluorescence signal generated by the biological sample on the sequencing chip using the microscopic imaging method according to any one of claims 1-19, or performing microscopic optical imaging on the fluorescence signal generated by the biological sample on the sequencing chip using the microscopic imaging device according to any one of claims 20-38, and obtaining the specific sequence of the biological sample on the sequencing chip by analyzing the imaging results.

42. An electronic device for determining optical parameters during sequencing, characterized in that, Including memory and processor; The processor reads executable program code stored in the memory to run a program corresponding to the executable program code, so as to implement the microscopic imaging method according to any one of claims 1-19.

43. A computer-readable storage medium storing a computer program, characterized in that, When the program is executed by the processor, it implements the microscopic imaging method according to any one of claims 1-19.

Images