Automatic focusing system and method, gene sequencing system and medium

Abstract

The embodiment of the invention provides an automatic focusing system, and the system comprises a light source which is used for transmitting a focusing light beam; the diaphragm is provided with a light through hole used for limiting the focusing light beam, so that the focusing light beam is in a specific shape; the focusing light beam penetrating through the diaphragm irradiates a position, deviating from the center, on the horizontal symmetry axis of the dichroscope, and at least part of the focusing light beam penetrating through the diaphragm is reflected by the dichroscope; the objective lens can move up and down, converges the focusing beam reflected by the dichroscope to the chip, and transmits at least part of the focusing beam reflected by the chip through the dichroscope; the acquisition device is used for generating an image formed by the focusing light beam transmitted by the dichroscope; and the control device adjusts the position of the objective lens according to the position of the light spot centroid of the image so as to realize automatic focusing. When the automatic focusing system and method are applied to a sequencing system, the image definition and the focusing accuracy can be solved, and the image acquisition efficiency in the sequencing process is improved.

Description

Technical Field

[0001] This application relates to the field of optical imaging technology, and in particular to autofocus systems and methods, gene sequencing systems and media. Background Technology

[0002] In high-precision instruments such as gene sequencing and biochip analysis based on fluorescence imaging, the optical detection system needs to operate stably for extended periods to acquire high-quality fluorescence images. The quality of the image, especially its sharpness and contrast, directly determines the accuracy and reliability of subsequent data analysis, such as base identification. Therefore, maintaining the imaging system at the optimal focal plane and achieving fast, accurate, and stable autofocus is one of the key technologies for these devices.

[0003] Traditional autofocus methods, such as search methods based on image sharpness evaluation functions (e.g., gradient, variance, spectral analysis), require driving the objective lens to scan a certain range and calculating the sharpness evaluation value for each frame to find the position with the highest evaluation value as the focus. While this method can achieve accurate focusing under ideal conditions, the process is often time-consuming and computationally resource-intensive, making it difficult to meet the demands of high-throughput, rapid imaging applications.

[0004] Another common autofocus technology is active focusing, which combines a separate focusing light source (such as a semiconductor laser LD) with the imaging optical path. Its basic principle is: the focusing laser illuminates the sample surface through a specific optical path and is reflected; an image sensor (such as an industrial camera) collects the reflected light spot; by analyzing the changes in the characteristics of this light spot in the image (such as the position and shape of the centroid), the offset of the objective lens relative to the sample's focal plane can be calculated, thereby driving an actuator (such as a voice coil motor) to move the objective lens until the optimal focal plane is restored.

[0005] However, applying such active focusing systems to temperature-sensitive equipment (such as gene sequencing systems) presents new challenges. During operation, factors such as heat generation from internal electronic components and ambient temperature fluctuations can cause minute thermal deformations or angular deflections in optical components, particularly dichroic mirrors, which are key elements for beam splitting and deflection. Dichroic mirrors are typically fixed using adhesive bonding, and their bonding areas are highly sensitive to temperature changes. Even micrometer- or submicrometer-level deflections can alter the incident and reflection paths of the laser beam on its surface, leading to unexpected drifts in the final spot position on the image sensor. This temperature-induced systematic drift interferes with the autofocus algorithm based on a fixed spot position model. If the system fails to distinguish between these "false" position signals caused by thermal effects and genuine defocus signals, it will directly lead to misjudgments, decreased focusing accuracy, or even focusing failure, ultimately affecting the clarity of the acquired fluorescence images and the quality of the sequencing data.

[0006] To address the temperature drift problem, some existing solutions attempt to mitigate it by enhancing system temperature control, using materials with low thermal expansion coefficients, or employing more complex mechanical stabilization structures. However, these methods often increase system complexity, cost, and power consumption, and their compensation effectiveness may still be limited under high-precision requirements.

[0007] Therefore, there is an urgent need for an autofocus system that can improve the robustness of the autofocus system to changes in ambient temperature and suppress the drift of the focusing reference signal (center of light spot) caused by thermal deformation of optical elements, so as to ensure that precision optical detection applications such as gene sequencing can still obtain high-quality imaging results in a long-term and stable manner under varying temperature environments. Summary of the Invention

[0008] This application provides an autofocus system and method that can be applied to sequencing systems to improve image clarity and focusing accuracy, and increase the efficiency of image acquisition during sequencing.

[0009] In a first aspect, this application provides an autofocus system, comprising: Light source, used to emit a focusing beam; An aperture stop, wherein the aperture stop is provided with a light-passing hole for limiting the focusing beam, so that the focusing beam is in a specific shape; A dichroic mirror, wherein the focusing beam passing through the aperture illuminates a position on the horizontal axis of the dichroic mirror that is off-center, and the dichroic mirror reflects at least part of the focusing beam passing through the aperture. An objective lens, which can move up and down, converges the focusing beam reflected by the dichroic mirror onto the chip, and transmits at least a portion of the focusing beam reflected by the chip through the dichroic mirror. An acquisition device that generates an image formed by the focusing beam transmitted through the dichroic mirror; A control device that adjusts the position of the objective lens to achieve automatic focusing based on the position of the centroid of the light spot in the image.

[0010] Furthermore, the light-transmitting hole is positioned off-center from the horizontal axis of the aperture.

[0011] Furthermore, the horizontal axis of the dichroic mirror is the horizontal axis of symmetry of the dichroic mirror.

[0012] Furthermore, the light-transmitting aperture is positioned off-center from the horizontal axis of symmetry of the aperture, so that the focusing beam passing through the aperture illuminates the position off-center from the horizontal axis of symmetry of the dichroic mirror.

[0013] Furthermore, it also includes a shaping component disposed between the aperture and the dichroic mirror to collimate the focusing beam passing through the aperture.

[0014] Furthermore, it also includes a dimming element disposed between the aperture stop and the shaping element, for adjusting the intensity of the focusing beam passing through the aperture stop.

[0015] Secondly, this application provides an autofocus method, which applies the autofocus system described above, including the following: Obtain a linear relationship model between the position of the spot centroid and the position of the objective lens, and the ideal centroid position; Based on the current centroid position of the light spot, calculate the deviation between the current centroid position and the ideal centroid position; The distance to be moved by the objective lens is calculated based on the deviation and the linear relationship model. Adjust the objective lens according to the calculated distance to be moved.

[0016] Furthermore, the linear relationship model between the position of the light spot centroid and the position of the objective lens, and the ideal centroid position, include: At approximately the focal plane position, the objective lens is moved step by step along the optical axis. After each movement, the sample is illuminated with light to obtain a fluorescence image, and the chip is illuminated with the focusing beam to obtain the image formed by the focusing beam. The linear relationship model is established based on the position of the centroid of the light spot in the image formed by the focusing beam after a single objective lens movement and the corresponding position of the objective lens. After a single movement, the fluorescence image is analyzed for sharpness, and the centroid position of the spot in the image formed by the focused beam corresponding to the fluorescence image with the highest sharpness is stored as the ideal centroid position.

[0017] Furthermore, the step of calculating the deviation between the current centroid position and the ideal centroid position of the current light spot based on the current centroid position includes: The objective lens is placed in any position, and the image formed by the focusing beam is obtained by illuminating the chip. The current centroid position of the spot in the image formed by the focusing beam is calculated. The current centroid position of the spot is substituted into the linear relationship model to obtain the deviation from the ideal centroid position.

[0018] Further, adjusting the objective lens according to the calculated distance to be moved includes: When the objective lens position is adjusted according to the calculated distance to be moved until the current spot centroid position is consistent with the ideal centroid position, the distance between the objective lens and the object being photographed is the optimal focal plane position.

[0019] Thirdly, this application provides a gene sequencing system, including: a sequencing chip, a chip platform, a reagent storage container, a liquid path system, an optical detection system, a computer system, and a waste liquid storage container; wherein, The sequencing chip is configured to provide reaction regions for amplification and sequencing reactions. The chip platform is configured to fix and support the chip; The reagent storage container is configured to store one or more mixed sample libraries and one or more reagents; The liquid circuit system is configured to controllably deliver one or more mixed sample libraries and one or more reagents from the reagent storage container to the sequencing chip for amplification and sequencing reactions in the sequencing chip, and to controllably deliver the waste liquid after the reaction to the waste liquid storage container. The optical detection system is configured to acquire sequencing images corresponding to the sequencing reactions within the sequencing chip. The computer system is configured to acquire sequencing images from the optical detection system and identify the nucleic acid sequence of the sequencing object based on the sequencing images; The waste liquid storage container is configured to store the waste liquid generated after the reaction. The optical detection system includes the autofocus system described above.

[0020] Fourthly, this application provides a readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the autofocus method as described above.

[0021] As can be seen from the above technical solutions, the autofocus system, method, gene sequencing system, and medium provided in this application significantly improve the focusing system's resistance to environmental interference and long-term stability: by setting the aperture of the aperture off-center from its horizontal axis of symmetry, the position of the focusing beam incident on the dichroic mirror is also located on the horizontal axis of symmetry of the dichroic mirror and off-center. This design utilizes the characteristics of optical devices; when the dichroic mirror undergoes a slight deflection around its mounting axis (usually approximately horizontal) due to temperature changes, the impact of the beam's positional change in that specific incident area on the centroid of the final spot formed on the acquisition device is minimized. This effectively suppresses, in physical principle, the unexpected drift of the focusing reference signal (spot centroid) caused by thermal drift of optical components due to temperature fluctuations, making the entire focusing system highly robust to changes in the external environment, especially temperature changes. This ensures that the device can maintain a high-precision focusing reference even during long-term operation or when the ambient temperature is unstable, improving the system's reliability and stability over its service life. This application achieves fast, accurate autofocus without complex searches: Based on the stable hardware system described above, the proposed autofocus method establishes a linear relationship model between the objective lens position and the centroid position of the focusing beam spot through pre-calibration. During focusing, only one image of the spot in the current state needs to be acquired, its centroid position calculated, and compared with the stored ideal centroid position. Based on this linear model, the objective lens movement distance and direction required to eliminate deviation can be directly and accurately calculated. This method significantly shortens focusing time, achieving "one-step" fast and accurate focusing, and significantly improves the overall efficiency of the image acquisition process. It is particularly suitable for high-throughput applications requiring rapid and continuous imaging, such as gene sequencing. It also ensures and improves image quality and the accuracy of subsequent analysis: Due to the high stability and precision of the autofocus system, it ensures that the imaging objective lens continuously and accurately focuses on the sample (such as a sequencing chip) surface. This directly results in higher clarity, contrast, and signal-to-noise ratio in the acquired fluorescence images. In fields such as gene sequencing that rely on high-precision optical detection, high-quality images are a fundamental prerequisite for accurate signal identification and data analysis. Therefore, the implementation of this application fundamentally ensures focusing quality, providing a more reliable data foundation for subsequent analytical steps such as fluorescence signal extraction and base sequence identification, thereby indirectly improving the accuracy and reliability of the overall detection or sequencing results. It also enhances the system's practicality and overall performance: this application does not rely on a complex active temperature control system to forcibly maintain the temperature of optical elements to suppress thermal drift, but instead maintains focusing stability under conditions that allow for certain temperature fluctuations through ingenious optomechanical design. This simplifies the system structure, reduces energy consumption and cost, and improves the system's adaptability to different working environments.Furthermore, the aforementioned autofocus method has a clear logic and is easy to achieve fully automated control through software programs, making the operation of devices integrating the system (such as gene sequencing systems) simpler and more intelligent, which is conducive to achieving unattended or large-scale automated operation.

[0022] In summary, this application, through an innovative heat-drift-resistant optical path design at the hardware level combined with a fast focusing algorithm based on a pre-calibrated linear model at the software level, provides a stable, fast, and high-precision autofocus solution that remains effective even under varying temperature conditions. It effectively solves the problem of focusing inaccuracies and image quality degradation caused by thermal deflection of core optical components in precision optical imaging equipment, especially in gene sequencing systems operating for extended periods. This results in significant benefits in improving image acquisition efficiency, ensuring data quality, and enhancing the environmental adaptability of the equipment.

[0023] To make the features and advantages of this application more apparent and understandable, some embodiments are described below in detail with reference to the accompanying drawings. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly described below. These drawings are incorporated in and constitute a part of this specification. They illustrate embodiments conforming to this application and, together with the specification, serve to explain the technical solutions of this application. It should be understood that the following drawings only show some embodiments of this application and should not be considered as limiting the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0025] Figure 1 This is a block diagram of the sequencing system structure provided in the embodiments of this application; Figure 2 This is a block diagram of the sequencing chip structure provided in the embodiments of this application; Figure 3 This is a block diagram of the optical detection system provided in the embodiments of this application; Figure 4 A schematic diagram of the light transmission path before the autofocus system was improved; Figure 5 Schematic diagram of the improved front aperture vent layout for an autofocus system; Figure 6 Schematic diagram for improving the aperture position of the front dichroic mirror in an autofocus system; Figure 7 To improve the previously acquired spot images for the autofocus system; Figure 8This is a schematic diagram of the improved light transmission path of the autofocus system provided in the embodiments of this application; Figure 9 This is a schematic diagram of the improved aperture layout of the autofocus system provided in the embodiments of this application; Figure 10 A schematic diagram of the improved aperture position of the dichroic mirror in the autofocus system provided in this application embodiment; Figure 11 A side view of the improved light transmission path of the autofocus system provided in this application embodiment; Figure 12 The improved light spot image acquired by the autofocus system provided in this application embodiment; Figure 13 This is a flowchart of an autofocus method provided in an embodiment of this application. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. The components of the embodiments of this application described and shown herein can generally be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely represents selected embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0027] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0028] In this document, the term "and / or" merely describes a relationship, indicating that three relationships can exist. For example, A and / or B can represent three cases: A alone, A and B simultaneously, and B alone. Furthermore, the term "at least one" in this document means any combination of at least two of any one or more elements. For example, including at least one of A, B, and C can mean including any one or more elements selected from the set consisting of A, B, and C.

[0029] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0030] For gene sequencing, such as high-throughput sequencing, the reaction process is as follows: Four fluorescent dNTPs are added to the sequencing chip through a liquid circuit system. Each dNTP can only be synthesized with one of the four bases ATCG, and the 3' end of each dNTP is blocked by a blocking group (including but not limited to azide). Then, polymerase is added to the sequencing chip through the liquid circuit system. Through the action of polymerase, one of the four dNTPs will be synthesized with a complementary base on the single strand being sequenced. Since the 3' end of the dNTP is blocked by a blocking group, only one dNTP can be extended on the single strand being sequenced at a time. After synthesis, specific chemical reagents are added to the sequencing chip through the liquid circuit system to flush away the excess dNTPs and polymerase. Next, the fluorescent groups of the dNTPs synthesized on the single strands can be excited by the optical detection system, causing the fluorescent groups to emit fluorescent signals. Since the fluorescent groups of the dNTPs on each single strand in a cluster will emit the same fluorescent signal, the fluorescent signal is amplified. Therefore, the optical detection system can collect the fluorescent signal and generate a fluorescent image.

[0031] Furthermore, by processing and analyzing the fluorescence images using a computer system, it can be determined which type of dNTP was synthesized onto the sequenced single strand. Then, based on the complementarity principle, it can be deduced which base on the sequenced single strand was synthesized with the dNTP. This completes one sequencing cycle.

[0032] Next, specific chemical reagents are added to the sequencing chip via a liquid circuit system to remove the blocking and fluorescent groups, thereby exposing the 3'-terminal hydroxyl groups of the dNTPs.

[0033] Next, proceed to the next sequencing cycle and repeat the above process.

[0034] As we can understand, one sequencing cycle can detect one base, and through multiple sequencing cycles, multiple bases in the sequenced single strand can be detected. Specifically, the number of sequencing cycles can be determined based on the set sequencing read length, for example, 150 or 300 sequencing cycles.

[0035] Of course, it should also be noted that the above sequencing reactions are merely illustrative examples. The technical solutions (automatic focusing system and method) proposed in this application can be applied to various gene sequencing reaction processes, as well as various fluorescence imaging-based instruments and reaction processes.

[0036] See Figure 1 As shown, a sequencing system suitable for gene sequencing includes: a sequencing chip 10, a chip platform 20, a reagent storage container 30, a liquid path system 40, an optical detection system 50, a computer system 60, and a waste liquid storage container 70. Among them: Sequencing chip 10 is configured to provide reaction regions for amplification and sequencing reactions; Chip platform 20, configured to fix and support sequencing chip 10; The reagent storage container 30 is configured to store one or more mixed sample libraries and one or more reagents; The liquid circuit system 40 is configured to controllably deliver one or more mixed sample libraries and one or more reagents from the reagent storage container 30 to the sequencing chip 10 for amplification and sequencing reactions in the sequencing chip 10, and to controllably deliver the waste liquid after the reaction from the sequencing chip 10 to the waste liquid storage container 70. The optical detection system 50 is configured to excite and acquire fluorescence signals during the sequencing reaction, and generate a fluorescence image based on the fluorescence signals; Computer system 60 is configured to acquire fluorescence images from optical detection system 50 and identify the base sequences of sample libraries based on the fluorescence images; Waste liquid storage container 70 is configured to store waste liquid generated after the reaction.

[0037] Sequencing chips, serving as carriers for amplification and sequencing reactions, provide reaction regions, known as channels. Typically, a sequencing chip 10 may contain one or more channels 11 (e.g., 2, 4, 6, or 8 channels), which are isolated from each other. See also... Figure 2 As shown, taking four channels as an example, each channel 11 has a small hole 12 at each end for the flow of fluids (e.g., biological samples, reaction reagents) into and out. The upper and lower surfaces of each channel are chemically modified and covalently seeded with two amplification primers. These two amplification primers are complementary to the adapters at both ends of the library to achieve amplification of the library.

[0038] For details, please refer to the optical inspection system. Figure 3 As shown, taking a dual-color channel as an example, the optical detection system 50 includes at least a light source assembly and an imaging assembly. The light source assembly includes at least light sources 5101 and 5102, field stops 5201 and 5202, a dichroic mirror 5301, and a filter element 540.

[0039] In one embodiment, light sources 5101 and 5102 can be light-emitting diodes (LEDs), and the LEDs can be aspherical mirrors to diffuse the LED point light source into parallel light. Of course, other forms of point or area light sources can also be used besides LEDs. In another embodiment, a collimating element can be provided after light sources 5101 and 5102. Figure 3Not shown, it is used to collimate the light emitted by light sources 5101 and 5102 into a parallel beam. The collimating element may include one or more lenses, including but not limited to any one or any combination of single lenses, cemented lenses, spherical lenses, and aspherical lenses.

[0040] Parallel light beams are emitted through field stops 5201 and 5202, which define the field of view of the light emitted from light sources 5101 and 5102, thereby defining the field of view of the excitation light illuminating the sequencing chip 10. The light passing through field stops 5101 and 5102 reaches a dichroic mirror 5301. The dichroic mirror 5301 can transmit light emitted from one of the light sources 5101 and 5102 and reflect light emitted from the other. The light passing through the dichroic mirror 5301 further passes through a filter element 540. The filter element 540 allows light of a certain wavelength emitted from light sources 5101 and 5102 to pass through and be used as excitation light, while blocking light of other wavelengths from passing through. For example, it blocks light of the same wavelength as the fluorescence emitted by the fluorescent group, ensuring that the fluorescence emitted by the fluorescent group does not contain stray light introduced by light sources 5101 and 5102, thus helping to improve optical imaging performance.

[0041] In one embodiment, the dichroic mirror 5301 can be fixed at a certain angle; for example, the dichroic mirror 5301 is fixed by dispensing adhesive. In another implementation, the field stops 5201 and 5202 can be, but are not limited to, rectangular or circular stops; the field stops 5201 and 5202 can be single-aperture or multi-aperture stops; the field stops 5201 and 5202 can be made of opaque material; for example, they can be metal sheets.

[0042] It should be noted that in the optical detection system 50, light sources 5101 and 5102 emit light alternately, not simultaneously. By operating light sources 5101 and 5102 in a time-division manner—that is, by emitting light with different wavelengths—and turning them on alternately during use, with only one light source activated at a time, the optical power of the excitation light illuminating the chip is reduced, which helps protect the fluorescence lifetime of the phosphor.

[0043] Furthermore, in order to obtain the best fluorescence image through the imaging component, the imaging component also needs to be focused. The light source component also includes a light source 5103, a field stop 5203, an attenuator 550, and a dichroic mirror 5302. In one embodiment, since LED light is relatively divergent, while the focusing beam is relatively convergent, in order to make the light spot acquired by the imaging component clearer, the light source 5103 during focusing is generally a semiconductor laser (LD), and the focusing beam is generally a laser. Hereinafter, "laser" or "LD laser" will be used to refer to the focusing beam.

[0044] When focusing the imaging component, the laser emitted by the semiconductor laser passes through the field stop 5203. The field stop 5203 defines the field of view of the laser emitted by the semiconductor laser, thereby defining the field of view of the excitation light illuminating the sequencing chip 10. The field stop 5203 can be a single-aperture or multi-aperture stop. The field stop 5203 can be made of an opaque material, for example, a metal sheet. The laser can only pass through the light-transmitting portion of the field stop corresponding to it, while the laser is blocked from the non-light-transmitting portions of the field stop. The laser passing through the field stop 5203 reaches the attenuator 550. After being attenuated by the attenuator 550, the laser reaches the dichroic mirror 5302, which can transmit the laser and reflect the LED light. The dichroic mirror 5302 can be fixed at a certain angle, for example, by dispensing adhesive. The laser light transmitted through the dichroic mirror 5302 or the LED light reflected by it reaches the convex lens 560, which collimates the laser light or LED light into a parallel beam.

[0045] The imaging assembly includes at least a dichroic mirror 5303, an objective lens 570, a tube lens 580, and an image sensor 590. Laser and LED light, collimated into parallel beams, pass through the dichroic mirror 5303, which reflects the laser and LED light towards the sequencing chip 10. The reflected laser and LED light pass through the objective lens 570 and illuminate the sequencing chip 10, exciting a fluorescent group to emit a fluorescence signal. The objective lens 570 collects the fluorescence signal, which then passes through the objective lens 570 to the dichroic mirror 5303. The dichroic mirror 5303 transmits the fluorescence signal to the tube lens 580, which projects the fluorescence signal onto the image sensor 590. The image sensor 590 acquires the fluorescence signal and generates a fluorescence image. The computer system 60 identifies the base sequence based on the fluorescence image. In one embodiment, a filter element may be disposed between the tube lens 580 and the dichroic mirror 5303. Figure 2 (Not shown) A filter element may be disposed between the tube lens 580 and the image sensor 590. Figure 2 Not shown in the image.

[0046] In one embodiment, the image sensor 590 may be, exemplarily, an industrial camera. In another embodiment, the collimating element may include one or more lenses, including but not limited to any one or any combination of a single lens, a cemented lens, a spherical lens, and an aspherical lens.

[0047] To achieve focusing of the imaging component, a motor 600 is arranged on one side of the objective lens 570. In one implementation, the motor can be, for example, a voice coil motor or other linear motor. Based on the image quality of the fluorescence image identified by the computer system 60, the voice coil motor can be controlled by a driver to move the objective lens 570 up and down, so that the objective lens 570 reaches the optimal focal plane and ultimately obtains the best fluorescence image.

[0048] Figure 4 The solid line represents the optical path of the LD laser 1 before improvement. The light from the LD laser 1 hits the upper end of the dichroic mirror 5. After the dichroic mirror 5 is deflected by high temperature, the light is deflected, as shown by the dashed line. At this time, the centroid of the light spot of the LD laser 1 is shifted, which will lead to a decrease in focusing stability and accuracy.

[0049] For LD focusing systems, a convex illumination lens is used to collimate the single point light source of the LD into parallel light, which is then reflected by a dichroic mirror and converged onto the sample surface by the objective lens. Precise focusing is achieved by moving the objective lens up and down and monitoring the position of the light spot. For example... Figure 4 As shown, before the improvement, the laser emitted by the LD laser 1 passes through the aperture 2 and then illuminates the dichroic mirror 5. Part of the laser is reflected by the dichroic mirror 5, passes through the objective lens 6, and is reflected by the sequencing chip 7. The reflected laser then passes through the objective lens 6 again, partially passing through the dichroic mirror 5 and the tube lens 8 before entering the acquisition device 9. The acquisition device 9 can be an industrial camera. Figure 5 As shown, the light-passing aperture on aperture 2 is located slightly above its vertical axis of symmetry H2. Therefore, the laser light passing through aperture 2 is tilted upwards, as... Figure 6 As shown, the position illuminating the dichroic mirror 5 is also located slightly above the vertical axis of symmetry H5 of the dichroic mirror 5. At this time, the light spot formed after reflection by the sequencing chip 7 is as follows: Figure 7 As shown. Figure 7 The sequencing chip 7 has four light spots because it has a double-layered glass structure with liquid or gas between the layers. The laser light is reflected from each surface of the double-layered glass and enters the acquisition device 9 to form a light spot. During focusing, a light spot with high brightness, clarity, and a small area is generally used. Figure 7 The second light spot from the left in the middle makes it easy to identify its centroid.

[0050] However, in actual experiments, it was found that because gene sequencing systems are high-end precision instruments, the precision requirements of each component are extremely high. During operation, the internal temperature of the gene sequencing system will change. The 5303 dichroic mirror will be slightly deflected after the temperature changes, causing the centroid position of the LD spot to jitter, affecting the focusing accuracy, causing focusing failure and affecting the sequencing quality.

[0051] Research revealed that in the aforementioned problem, the slight deflection of the five points of the dichroic mirror at the adhesive site due to temperature changes caused a shift in the position of the spot centroid in the image. However, its position within the image remained unchanged. Figure 4 The change in the vertical direction is much smaller than the change in the horizontal direction. Based on this, the applicant of this invention proposes the autofocus system and autofocus method of this invention, which can greatly improve focusing accuracy and reduce the impact on focusing caused by the slight deflection of the five points of the dichroic mirror due to temperature changes, which causes the centroid of the light spot to change position in the image.

[0052] Example 1

[0053] refer to Figures 8-10 An autofocus system includes: a light source 1 for emitting a focusing beam; an aperture 2 having a light-passing hole 21 to limit the focusing beam, causing the focusing beam to form a specific shape; a dichroic mirror 5, through which the focusing beam passing through the aperture 2 illuminates a position on the horizontal axis of the dichroic mirror 5 off-center, the dichroic mirror 5 reflecting at least part of the focusing beam passing through the aperture 2; an objective lens 6 movable up and down, converging the focusing beam reflected by the dichroic mirror 5 onto a chip, and transmitting at least part of the focusing beam reflected by the chip through the dichroic mirror 5; a data acquisition device 9 generating an image formed by the focusing beam transmitted by the dichroic mirror 5; and a control device adjusting the position of the objective lens 6 according to the position of the centroid of the light spot in the image to achieve autofocus.

[0054] First, let's explain the meaning of the horizontal axis of dichroic mirror 5, as well as the horizontal axis of symmetry of dichroic mirror 5, the horizontal axis of aperture 2, and the horizontal axis of symmetry of aperture 2, which appear later. (Reference) Figure 9 and Figure 10 The aperture 2 and dichroic mirror 5 in the middle, Figure 9 and Figure 10 These are the front views of their respective horizontal directions. The horizontal axis of the dichroic mirror 5 is the horizontal axis, which has countless axes, such as... Figure 10 p5 in the diagram. The horizontal axis of symmetry of the dichroic mirror 5 refers to the axis within p5 that makes the dichroic mirror horizontally symmetrical around it; in fact, it is its central axis of symmetry in the horizontal direction. Figure 10 L5 in the diagram. It can be understood that the horizontal axis p5 of the dichroic mirror 5 is parallel to its horizontal axis of symmetry L5 and is distributed on both sides of L5. For the same reason, the horizontal axis of the aperture stop 2, that is, the horizontal axis, has countless axes, such as... Figure 9 p2 in the above-mentioned horizontal axis. The horizontal axis of symmetry of aperture 2 refers to the axis in the aforementioned horizontal axis p2 that makes aperture 2 horizontally symmetrical around it, which is actually its central axis of symmetry in the horizontal direction, i.e. Figure 9 L2 in the diagram. It can be understood that the horizontal axis p2 of aperture 2 is parallel to the horizontal axis of symmetry L2 of aperture 2 and is distributed on the upper and lower sides of the horizontal axis of symmetry L2 of aperture 2.

[0055] As previously mentioned, the light source 1 includes at least a point-source LD laser 1 for emitting laser light. The focusing beam is the laser, providing a reference light source for focusing detection. The beam emitted by the point-source LD laser 1 has divergent characteristics, and its spot size is small, close to an ideal point source. This ensures that a small focused spot with a size close to the diffraction limit can be formed on the sample surface after passing through the subsequent optical detection system. This is the basis for achieving high-precision centroid positioning. As for the laser, the dichroic mirror 5 is chosen so that approximately 50% of the laser light is reflected and 50% is transmitted. This allows the laser light to be reflected through the objective lens and reach the chip, and also allows the laser light reflected back from the chip to be transmitted again through the dichroic mirror 5 to the acquisition device 9 for imaging.

[0056] In this application, by setting the position of the laser spot illuminating the dichroic mirror 5 to be on the horizontal axis of the dichroic mirror but off-center, even if the dichroic mirror 5 rotates slightly, the influence of temperature-induced deflection on the centroid of the spot in the image can be reduced by utilizing the characteristic that the slight deflection at the adhesive dot of the dichroic mirror 5 due to temperature changes results in a much smaller change in the centroid of the spot in the horizontal direction compared to the vertical direction. This improves the stability and accuracy of focusing. In this system, the dichroic mirror 5 serves as both an optical path hub (achieving illumination / imaging coupling) and a thermal drift conduction node. Through its collaborative design with the asymmetric aperture, traditional defects are transformed into controllable parameters, ultimately ensuring the system maintains focusing accuracy and providing key technical support for gene sequencing.

[0057] It should be noted that the reason why the position of the laser spot illuminating the dichroic mirror 5 needs to be offset from the center of the horizontal axis is that if the position of the laser spot illuminating the dichroic mirror 5 is at the center of the horizontal axis of the dichroic mirror, then after the laser is reflected by the dichroic mirror 5, the objective lens, the chip, and other components, the reflection on the upper and lower glass surfaces of the chip will form a pattern on the acquisition device as shown in the image. Figure 7 or Figure 12 The four light spots will overlap, making it impossible to extract the smallest and clearest light spot needed for focusing.

[0058] Optionally, the laser wavelength of LD laser 1 is specially designed to avoid the autofluorescence band that the sample itself may generate, thereby minimizing the interference of background noise on the detection. At the same time, the power of LD laser 1 is adjustable, enabling it to adapt to samples with different reflectivity, and it works in conjunction with attenuator 3 to ensure that the light intensity incident on camera 9 is within the optimal linear response range.

[0059] In this embodiment, in the optical autofocus system, the point source characteristics of the point LD (Laser Diode) laser include spatial characteristics and directional control. The spatial characteristics include an emission spot diameter ≤ 5 μm (typically 1-3 μm), approaching the diffraction-limited point source, ensuring a sub-micron spot is formed after focusing by the objective lens. Directional control: asymmetric fast-axis / slow-axis divergence angles (typically 120° × 40°) provide optimized input for the subsequent collimating lens.

[0060] In this embodiment, the coordination mechanism with system components includes aperture coordination: matching the point source size with the asymmetric aperture to improve energy utilization. The point-source LD laser is the physical basis for the system's high-precision focusing. The sub-micron point source ensures the geometric accuracy of the objective lens's focused spot; narrow-spectrum monochromatic light eliminates chromatic aberration interference and matches the response of optical components; low coherence suppresses the influence of speckle noise on centroid detection; and power controllability balances detection sensitivity and sample safety. This provides a high signal-to-noise ratio, low-noise optical signal source for the centroid-based focusing algorithm, enabling the system to maintain focusing accuracy even under temperature-changing environments. Optionally, to position the laser beam off-center from the horizontal axis of the dichroic mirror 5, the position of the aperture 21 of the aperture 2 can be adjusted so that the aperture 21 is positioned off-center from the horizontal axis of the aperture 2. In this case, when installing the components of the focusing system, as long as the centers of each component, such as the aperture 2 and the dichroic mirror 5, are aligned, the light-transmitting aperture 21 will be positioned off-center from the horizontal axis of the aperture 2. Therefore, the light spot formed on the dichroic mirror 5 will naturally be on the horizontal axis of the dichroic mirror and off-center. Of course, in other embodiments, to make the laser irradiate the dichroic mirror 5 off-center from the horizontal axis, it can be achieved without the above method. For example, the light-transmitting aperture 21 can be positioned at the center of the aperture 2, but the center of the aperture 2 and the center of the dichroic mirror can be offset relative to each other in the horizontal direction.

[0061] Figure 9 yes Figure 8 A front view of the circular aperture 2 shows a small circular hole, approximately 1 mm in diameter, located to the left of the center, serving as the output aperture for the LD laser 1. (See image below.) Figure 9As shown, the light-passing aperture is set at a position off-center from the horizontal axis of symmetry of the aperture 2, so that the laser light passing through the aperture 2 illuminates the position off-center from the horizontal axis of symmetry of the dichroic mirror 5. This aperture is used to limit the imaging beam, so that the beam is shaped in a specific way and hits the corresponding exit position.

[0062] In one embodiment, the horizontal axis of the dichroic mirror is the horizontal axis of symmetry of the dichroic mirror 5, that is... Figure 10 L5 in the diagram. By further defining the position of the laser irradiation on the dichroic mirror 5 as a position off-center from the horizontal axis of symmetry, the characteristic that the dichroic mirror 5 has the smallest deflection of its central axis position when it is slightly deflected due to temperature changes at the dispensing point can be further utilized to improve focusing accuracy.

[0063] In one embodiment, the horizontal axis of the aperture stop 2 is the horizontal axis of symmetry of the aperture stop, that is... Figure 9 L2 in this case. When installing the components of the focusing system, as long as the centers of each component, such as aperture 2 and dichroic mirror 5, are aligned, the light-passing hole 21 is set at a position off-center from the horizontal axis of symmetry of aperture 2. Therefore, the light spot formed on dichroic mirror 5 is naturally on the horizontal axis of symmetry of dichroic mirror and off-center.

[0064] In one embodiment, the aperture 2 is a circular substrate made of hard anodized aluminum alloy, and the light-passing hole is treated with black oxidation to reduce stray light; a transversely asymmetrical light-passing hole is provided in the center, and the offset d between the center of the light-passing hole and the geometric center of the aperture satisfies: 0.2R≤d≤0.4R, where R is the radius of the aperture, and optical thermal drift is compensated by the transverse offset.

[0065] In this embodiment, in the autofocus system, the aperture stop 2 is the core mechatronic element for achieving high stability. It performs spatial modulation of the beam and suppresses thermal drift, forcing the beam to deviate from the optical axis and project onto the edge of the dichroic mirror 5; the output spot position is precisely controllable. The aperture stop 2 is the physical hub for thermal drift management in this system. Its geometrically asymmetrical design reduces the sensitivity of the dichroic mirror 5 to deflection; the ultra-black micro-aperture structure eliminates stray light interference with the centroid algorithm; and the rigid thermally stable material ensures minimal deformation in high-temperature environments. This provides a spatially stable and background-clean optical input for the LD spot centroid-based focusing algorithm, improving focusing accuracy without adding active temperature control.

[0066] like Figure 10 As shown, the laser light passing through the aperture 2 illuminates the aperture of the dichroic mirror 5 at a position that is also located on the horizontal axis of symmetry of the dichroic mirror 5 and off-center. The dichroic mirror 5 will reflect at least part of the laser light passing through the aperture 2. This improved optical path can reduce the problem of the centroid shift of the LD laser 1 caused by the deflection of the dichroic mirror 5.

[0067] Optionally, the system also includes a dimming element, which is disposed between the aperture 2 and the illumination convex lens 4, and is used to adjust the intensity of the laser beam transmitted through the aperture 2.

[0068] In this embodiment, as Figure 8 As shown, the dimming element is attenuator 3, which is used to reduce the intensity of the laser beam, protect the camera, and avoid overexposure.

[0069] In one embodiment, the attenuator 3 is used for precise control of laser energy. The energy attenuation mechanism includes using an optical density (OD) coating (e.g., OD1.0-OD2.0) to exponentially reduce the laser intensity. In this embodiment, the attenuator 3 can attenuate the LD laser output to the linear response range of the industrial camera, adapting to intensity requirements and preventing sensor saturation. This ensures focusing accuracy and stabilizes the spot energy distribution. It also extends hardware lifespan, allowing the camera sensor to operate within a safe range. Furthermore, it provides cross-scenario compatibility and allows for the replacement of OD value filters (e.g., OD1.0 / 1.8 / 2.3) to adapt to LD lasers of different power.

[0070] As an example, attenuator 3 must be placed after aperture 2 (sequence: aperture → attenuator → convex lens). If placed in front, stray light will not be blocked by the aperture, and attenuator 3 will absorb heat, causing thermal distortion. The distance between attenuator 3 and dichroic mirror 5 is greater than 10mm to avoid secondary incident reflection of the reflected light, which could lead to abnormal attenuation. Attenuator 3 is the core hub of energy management in this system. Through precise optical attenuation, it balances the triple requirements of laser intensity, camera safety, and signal quality, providing stable and controllable optical input for the focusing algorithm based on the centroid of the LD spot.

[0071] Optionally, the system also includes a shaping component, which is disposed between the aperture 2 and the dichroic mirror 5, for collimating the laser transmitted through the aperture 2.

[0072] In this embodiment, as Figure 8 As shown, the shaping component is the illumination convex lens 4, used for beam collimation and energy optimization, collimating the diverging LD laser source into a parallel beam and correcting the diverging light. In this system, the LD laser 1 emits a beam with a high divergence angle (typically a 120° × 40° elliptical cone), which would cause beam dispersion if directly projected. The illumination convex lens 4 converts the diverging beam into collimated parallel light through a positive optical power design. This improves beam uniformity and eliminates elliptical distortion. An aspherical lens is used to compensate for the difference in divergence angles between the fast and slow axes of the LD, improving the roundness of the output beam. A wavelength-selective anti-reflection coating is applied to eliminate central hot spots and optimize energy distribution.

[0073] As an example, the illumination convex lens 4 prevents focusing failure in collimated scenarios: the diverging beam forms a blur spot (diameter > 20 μm) on the rear focal plane of the objective lens, increasing the error in the centroid detection of the spot. The position sequence of the illumination convex lens 4 includes: aperture 2, attenuator 3, and illumination convex lens 4 arranged in sequence, working in conjunction with aperture 2. Aperture 2 first limits the beam aperture, preventing edge aberrations of the illumination convex lens 4 from affecting the collimation quality. Placing the illumination convex lens 4 at the rear maximizes the utilization of the light-transmitting aperture and improves energy utilization. In this embodiment, the illumination convex lens 4 is the core of the beam shaping of the autofocus system, ensuring the focusing capability of the objective lens 6 through divergence angle compression, and its thermal stability design supports reliable operation in temperature-changing environments. It provides highly stable, low-noise optical input for the autofocus algorithm.

[0074] The microscope assembly includes a microscope objective lens 6 and a tube lens 8, used to image and magnify the specific shape of an object in order to study the object's structure and specific internal features.

[0075] In one embodiment, such as Figure 8 As shown, the objective lens 6 can move up and down to focus the laser reflected by the dichroic mirror 5 onto the chip and transmit at least a portion of the laser reflected by the chip through the dichroic mirror 5.

[0076] Optionally, in the autofocus system, the microscope objective 6 is the core optical hub for imaging and focusing, used for optical path conversion and signal generation, including focusing the LD optical path focal point source into a focused spot. Specifically, it focuses the collimated parallel light output from the illumination convex lens 4 into a sub-micron spot. This minimizes the spot size reflected from the surface of the sequencing chip 7, ensuring accurate centroid positioning. It collects the imaging optical path, using a high numerical aperture to collect the spot signal reflected from the sequencing chip 7 and transmits it to the tube lens 8 to form a microscopic image. This ensures spatial resolution, meeting the base cluster resolution requirements of gene sequencing. Overcoming the physical limitations of focusing sensitivity, the microscope objective 6 plays a dual role in the autofocus system, including converting the LD point source into a sub-micron high-contrast spot and efficiently capturing the spot signal reflected from the sequencing chip 7, ensuring reliable base identification.

[0077] In one embodiment, Figure 11 yes Figure 8 Side view, such as Figure 11 As shown, the tube lens 8 and the objective lens 6 form an infinity correction optical path.

[0078] Optionally, in the optical autofocus system, the tube lens 8 is the core relay element of the imaging optical path, used for optical relay and image quality optimization. Specifically, it performs optical path conversion for infinity-based optical path relay, including converting the infinity-based parallel beam output from the objective lens 6 into a converging beam to form an image on the target surface of the industrial camera 9. In this embodiment, the low-distortion design of the tube lens 8 ensures stable spot geometry. Through planar field design and chromatic aberration correction, it ensures uniformity of resolution across the entire field of view. It suppresses the effects of temperature variations, improving image plane stability. It provides a zero-drift, low-distortion imaging reference surface for the autofocus algorithm, supporting the autofocus system to maintain focusing accuracy even in high-temperature environments.

[0079] This autofocus system also includes a sequencing chip 7, into which the sample to be tested can be filled.

[0080] This autofocus system also includes an acquisition device for generating an image formed by the laser transmitted through the dichroic mirror 5.

[0081] In one embodiment, such as Figure 8 As shown, the data acquisition device is an industrial camera 9.

[0082] Optionally, in the optical autofocus system, the industrial camera 9 serves as the system's visual perception center and algorithm execution carrier. This includes performing optical signal conversion and algorithms, capturing the focusing spot, detecting the high-precision centroid, capturing the LD laser spot on the surface of the sequencing chip 7 via a global shutter CMOS sensor, and achieving a positioning accuracy of ±0.1 pixels (corresponding to a 0.1μm displacement of the objective lens 6) using a sub-pixel centroid algorithm (grayscale weighted method). High-speed response capability: frame rate ≥100fps (resolution 1280×1024), single-frame exposure time ≤1ms, real-time tracking of spot displacement (delay <10ms).

[0083] In this embodiment, Figure 12 The light spot image captured by industrial camera 9 shows that, because the light aperture on the circular aperture 2 is located off-axis, the light spot formed after reflection by sequencing chip 7 is as follows: Figure 12 As shown.

[0084] In this embodiment, the industrial camera 9 plays a core role in the system, including: capturing the centroid of the LD spot with a spot detector at an accuracy of ±0.1 pixels, thus establishing the input basis for the focusing algorithm; and implementing microsecond-level centroid calculation using an algorithm embedded in the computing accelerator FPGA, breaking through the speed bottleneck of traditional sharpness scoring methods. Ultimately, the system maintains fast and high-precision focusing capabilities even in high-temperature and variable environments, improving efficiency and accuracy compared to traditional solutions, and providing a reliable visual perception foundation for gene sequencing.

[0085] The control device adjusts the position of the objective lens 6 according to the position of the centroid of the light spot in the image to achieve automatic focusing.

[0086] The autofocus system of this invention follows a defined optical path sequence, including: the laser emitted by the light source component first undergoes beam spatial shaping via the aperture 2 in the imaging assembly; subsequently, the beam intensity is precisely controlled by the attenuator 3; next, the illumination convex lens 4 collimates the diverging laser into parallel light; this parallel beam is reflected and redirected by the dichroic mirror 5, and then converged onto the surface of the sequencing chip 10 carrying the sample by the microscope objective lens 6, which can move along the optical axis; the laser signal reflected from the surface of the sequencing chip 10 passes through the objective lens 6 again, and through the dichroic mirror 5, is imaged onto the target surface of the industrial camera 9 via the tube lens 8. The control device (not shown in the figure) analyzes the image acquired by the camera 9 in real time, calculates the centroid position of the light spot, and generates control commands accordingly to drive the objective lens 6 to move, ultimately completing closed-loop autofocus.

[0087] Example 2

[0088] The optical autofocus method disclosed herein utilizes the optical autofocus system described above, such as... Figure 13 As shown, it includes: In step 101, a linear relationship model between the position of the spot centroid and the position of the objective lens 6 and the ideal centroid position are obtained.

[0089] Optionally, obtaining the linear relationship model between the centroid position of the light spot and the objective lens position, and the ideal centroid position, includes: moving the objective lens stepwise along the optical axis at approximately the focal plane position; obtaining a fluorescence image by illuminating the sample with light after each movement; obtaining a laser-generated image by illuminating the sequencing chip 10 with laser light; establishing a linear relationship model based on the correspondence between the centroid position of the light spot in the laser-generated image after each objective lens movement and the objective lens position; performing a sharpness analysis on the fluorescence image after each movement, and storing the centroid position of the light spot in the laser-generated image corresponding to the fluorescence image with the highest sharpness as the ideal centroid position.

[0090] In this embodiment, a point laser emitter 1 is used to illuminate the sequencing chip 10 being imaged, and the laser spot is observed in the image. The focal plane distance d and the centroid position of the spot are fixed. Since the distance d between the objective lens 6 and the sample 7 being imaged is fixed, the position of the laser spot (i.e., the centroid of the spot) in the image should also be fixed. By changing the distance between the objective lens 6 and the sample, the centroid position of the spot is recorded at each position, and a linear relationship model y=ax is established, where y is the position of the centroid of the spot, x is the distance between the objective lens and the sample, and a is a coefficient.

[0091] In this embodiment, in the optical detection system, when the laser emitted by the point-shaped LD laser 1 is collimated, reflected, and focused onto the sample surface, the centroid is fixed when the distance is constant. If the distance d between the sequencing chip 10 and the focal plane of the objective lens 6 remains constant, the centroid position of the spot formed by the reflected laser on the target surface of the industrial camera 9 will remain strictly unchanged. Physical essence: The distance d between the objective lens and the sample determines the focusing state of the beam on the camera image plane. When d is at the optimal focal plane, the energy distribution of the reflected spot is most concentrated, and the centroid coordinates are stable at the theoretical position of the geometric optical path. This characteristic is used to construct a linear formula to describe the relationship between the position d and the centroid position of the spot. The objective lens displacement Δd is strictly linearly proportional to the centroid coordinate offset Δy: when the objective lens moves away from the sample (d increases), the spot moves in the positive direction on the camera target surface; when the objective lens moves closer to the sample (d decreases), the spot moves in the negative direction.

[0092] As an example, at the optimal focal plane position, the sequencing chip 10 is fixed in place, and the LD laser 1 is turned on to illuminate the surface. The coordinates of the centroid of the light spot are calculated at this moment. The objective lens 6 is moved in 2µm increments, acquiring 5 images from both above and below. The coordinates y of the centroid of the light spot and the corresponding distance x from the objective lens to the sample in each image are observed and recorded, thus constructing a fitted linear equation: y = ax, where y represents the position of the centroid of the light spot, x represents the actual position d, and a is a constant related to the system characteristics. According to the linear formula, the optimal focal plane position d can be quickly and accurately located in actual operation.

[0093] This embodiment transforms the physical property that "a constant distance d between the objective lens and the sample results in a fixed centroid of the light spot" into a strongly linear mapping relationship applicable to engineering applications by designing the optical path geometric stability (asymmetric aperture) and optimizing optical materials (rigid, low-expansion substrate). This property becomes the core physical basis of the autofocus algorithm, enabling the system to maintain focusing accuracy even in harsh environments and improving reliability compared to traditional methods.

[0094] In step 102, the deviation between the current centroid position and the ideal centroid position of the current light spot is calculated based on the current centroid position of the light spot.

[0095] Optionally, the objective lens 6 is placed in any position, a spot is generated using the point-shaped LD laser 1, and the centroid position of the current spot is calculated. The current centroid position is then substituted into the linear model to calculate the deviation from the ideal centroid position.

[0096] As an example, a new sequencing chip is placed on the sequencing system, and its position may differ from the previous one. In this case, objective lens 6 is first positioned approximately, and a light spot is generated by illuminating it with LD laser 1. The centroid position of the newly generated light spot is analyzed, and if it deviates from the preset ideal centroid position, the actual distance deviation is calculated using the previously established linear formula.

[0097] In this embodiment, real-time deviation calculation and compensation includes initial positioning and spot generation. Initial coarse positioning of objective lens 6 involves moving it to a safe initial position (typically set within ±50μm of the historically optimal focal plane to avoid collision risks) after placing the new sequencing chip. The point-like LD laser 1 is activated, and the beam is reflected sequentially by the asymmetric aperture 2, attenuator 3, collimating lens, and dichroic mirror 5 before being focused onto the surface of the sequencing chip 10 by objective lens 6. Spot acquisition and centroid calculation include capturing the reflected spot using an industrial camera 9 in a local ROI mode (200×200 pixels).

[0098] Optionally, the deviation calculation includes setting the current centroid coordinate y c With respect to the preset ideal centroid coordinates y i Comparison: Δy = y i -y c (Unit: pixels)

[0099] In step 103, the distance to be moved for objective lens 6 is calculated based on the obtained deviation and linear relationship model; Optionally, calculating the objective lens to be moved includes: based on the pre-calibrated linear relationship model y=ax (a: system sensitivity coefficient); converting the centroid deviation Δy (the difference between the current centroid and the ideal centroid coordinates) into the objective lens to be moved, and calculating the theoretical distance the objective lens needs to move: Δx=Δy / a.

[0100] In step 104, the objective lens 6 is adjusted according to the calculated distance to be moved.

[0101] Optionally, based on the objective lens to be moved distance Δx calculated by the model, adjust the position of objective lens 6 until the position of the light spot centroid is consistent with the ideal centroid position. At this time, the distance between objective lens 6 and the object being photographed is the optimal focal plane position d.

[0102] In this embodiment, the control device adjusts the position of the objective lens 6 according to the position of the centroid of the image spot to achieve autofocus. The mechanism for adjusting the position of the objective lens 6 is existing technology, such as a voice coil motor. Based on the specific movement required calculated above, the control device can send a signal to the actuator, such as the aforementioned voice coil motor, to perform the adjustment of the objective lens position.

[0103] In another embodiment, this application also provides a gene sequencing system, including: a sequencing chip 10, a chip platform 20, a reagent storage container 30, a liquid system 40, an optical detection system 50, a computer system 60, and a waste liquid storage container 70; wherein, The sequencing chip 10 is configured to provide reaction regions for amplification and sequencing reactions. The chip platform 20 is configured to fix and support the chip; The reagent storage container 30 is configured to store one or more mixed sample libraries and one or more reagents; The liquid circuit system 40 is configured to controllably deliver one or more mixed sample libraries and one or more reagents from the reagent storage container to the sequencing chip for amplification and sequencing reactions in the sequencing chip, and to controllably deliver the waste liquid after the reaction to the waste liquid storage container. The optical detection system 50 is configured to acquire sequencing images corresponding to sequencing reactions within the sequencing chip. The computer system 60 is configured to acquire sequencing images from the optical detection system and identify the nucleic acid sequence of the sequencing object based on the sequencing images; The waste liquid storage container 70 is configured to store the waste liquid generated after the reaction; The optical detection system 50 includes the autofocus system described above.

[0104] In another embodiment, this application provides a readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the autofocus method as described above.

[0105] This disclosure discloses an autofocus system and method that, when applied to sequencing systems, can improve image clarity and focusing accuracy, and enhance image acquisition efficiency during sequencing. By introducing a laterally asymmetric aperture 2, the incident conditions of the illumination beam on the dichroic mirror 5 are altered, adjusting the beam incident point to a mirror region insensitive to thermal deflection. This significantly suppresses, from a physical perspective, the centroid drift of the light spot caused by thermal deflection of the dichroic mirror 5. This hardware improvement, combined with an autofocus algorithm based on a linear model of the light spot centroid, greatly enhances the robustness of the entire autofocus system to external temperature changes. Even in environments with fluctuating temperatures, the system can maintain sub-micron-level focusing accuracy and rapid convergence speed without complex active temperature control, significantly improving the operational stability, efficiency, and reliability of precision optical equipment such as gene sequencing systems.

[0106] The embodiments described above are some, but not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0107] In the description of the embodiments of this application, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship commonly used when the product of this application is in use. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the electric vehicle or component referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the present invention. In addition, the terms "first," "second," and "third," etc., are only used to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0108] In the description of the embodiments of this application, it should also be noted that, unless otherwise explicitly specified and limited, the terms "set," "install," "connect," and "link" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0109] Finally, it should be noted that the above-described embodiments are merely specific implementations of this application, used to illustrate the technical solutions of this application, and not to limit them. The protection scope of this application is not limited thereto. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any person skilled in the art can still modify or easily conceive of changes to the technical solutions described in the foregoing embodiments, or make equivalent substitutions for some of the technical features, within the technical scope disclosed in this application. Such modifications, changes, or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application, and should all be covered within the protection scope of this application. Therefore, the protection scope of this application should be determined by the protection scope of the claims.

Claims

1. An autofocus system, characterized in that, include: A light source used to emit a focusing beam; An aperture is provided on which a light-transmitting hole is provided to limit the focusing beam, so that the focusing beam is in a specific shape; A dichroic mirror, wherein the focusing beam passing through the aperture illuminates a position on the horizontal axis of the dichroic mirror that is off-center, and the dichroic mirror reflects at least part of the focusing beam passing through the aperture. An objective lens, which can move up and down, converges the focusing beam reflected by the dichroic mirror onto the chip, and transmits at least a portion of the focusing beam reflected by the chip through the dichroic mirror. An acquisition device that generates an image formed by the focusing beam transmitted through the dichroic mirror; A control device that adjusts the position of the objective lens to achieve automatic focusing based on the position of the centroid of the light spot in the image.

2. The autofocus system according to claim 1, characterized in that, The light-transmitting aperture is positioned off-center from the horizontal axis of the aperture.

3. The autofocus system according to claim 1, characterized in that, The horizontal axis of the dichroic mirror is the horizontal axis of symmetry of the dichroic mirror.

4. The autofocus system according to claim 3, characterized in that, The light-transmitting aperture is positioned off-center from the horizontal axis of symmetry of the aperture, so that the focusing beam passing through the aperture illuminates the dichroic mirror at a position off-center from the horizontal axis of symmetry.

5. The autofocus system according to claim 1, characterized in that, It also includes a shaping component disposed between the aperture and the dichroic mirror to collimate the focusing beam passing through the aperture.

6. The autofocus system according to claim 5, characterized in that, It also includes a dimming element, which is disposed between the aperture stop and the shaping element, for adjusting the intensity of the focusing beam passing through the aperture stop.

7. An autofocus method, employing an autofocus system as described in any one of claims 1-6, characterized in that, Including the following: Obtain a linear relationship model between the position of the spot centroid and the position of the objective lens, and the ideal centroid position; Based on the current centroid position of the light spot, calculate the deviation between the current centroid position and the ideal centroid position; The distance to be moved by the objective lens is calculated based on the deviation and the linear relationship model. Adjust the objective lens according to the calculated distance to be moved.

8. The autofocus method according to claim 7, characterized in that, The linear relationship model between the position of the light spot centroid and the position of the objective lens, and the ideal centroid position, include: At approximately the focal plane position, the objective lens is moved step by step along the optical axis. After a single movement, the sample is irradiated with excitation light to obtain a fluorescence image, and the chip is irradiated with the focusing beam to obtain the image formed by the focusing beam. The linear relationship model is established based on the position of the centroid of the light spot in the image formed by the focusing beam after a single objective lens movement and the corresponding position of the objective lens. After a single movement, the fluorescence image is analyzed for sharpness, and the centroid position of the spot in the image formed by the focused beam corresponding to the fluorescence image with the highest sharpness is stored as the ideal centroid position.

9. The autofocus method according to claim 7, characterized in that, The step of calculating the deviation between the current centroid position and the ideal centroid position of the current light spot, based on the current centroid position, includes: The objective lens is placed in any position, and the image formed by the focusing beam is obtained by illuminating the chip. The current centroid position of the spot in the image formed by the focusing beam is calculated. The current centroid position of the spot is substituted into the linear relationship model to obtain the deviation from the ideal centroid position.

10. The autofocus method according to claim 7, characterized in that, The excitation light source is an LED light source.

11. A gene sequencing system, characterized in that, include: Sequencing chips, chip platforms, reagent storage containers, liquid path systems, optical detection systems, computer systems, and waste liquid storage containers; among which, The sequencing chip is configured to provide reaction regions for amplification and sequencing reactions. The chip platform is configured to fix and support the chip; The reagent storage container is configured to store one or more mixed sample libraries and one or more reagents; The liquid circuit system is configured to controllably deliver one or more mixed sample libraries and one or more reagents from the reagent storage container to the sequencing chip for amplification and sequencing reactions in the sequencing chip, and to controllably deliver the waste liquid after the reaction to the waste liquid storage container. The optical detection system is configured to acquire sequencing images corresponding to the sequencing reactions within the sequencing chip. The computer system is configured to acquire sequencing images from the optical detection system and identify the nucleic acid sequence of the sequencing object based on the sequencing images; The waste liquid storage container is configured to store the waste liquid generated after the reaction. The optical detection system includes the autofocus system as described in any one of claims 1-6.

12. A readable storage medium having a computer program stored thereon, characterized in that, When the computer program is executed by the processor, it implements the autofocus method as described in any one of claims 7 to 10.

Images