Single cell shooting method and device, storage medium and computer equipment

Abstract

According to the single cell shooting method and device, the storage medium and the computer equipment, firstly, the actual liquid level height in the target hole is accurately determined, the influence of liquid level height deviation caused by factors such as liquid volatilization and sample adding errors on imaging is avoided, it is ensured that the focal length of an imaging system can be accurately aligned with the liquid level, and the imaging efficiency is improved; and a foundation is laid for subsequent clear shooting of liquid level background images. And secondly, a liquid level background image is shot before the single cell is printed, and pre-scanning monitoring is performed on the basis of the image, so that the falling condition of the single cell liquid drops can be quickly identified, and compared with a traditional scanning mode depending on a fixed time point, the timeliness and accuracy of liquid drop monitoring are greatly improved, and the misjudgment probability is reduced. Moreover, when the drop is monitored, the dynamic position change of the drop in the liquid is covered through continuous zooming, so that the definition and the capture success rate of the single cell image are effectively improved, and the single cell screening efficiency is further improved.

Description

Technical Field

[0001] This application relates to the field of single-cell screening technology, and in particular to a single-cell imaging method, apparatus, storage medium, and computer equipment. Background Technology

[0002] In single-cell screening technology, accurate identification and imaging of single cells are crucial for subsequent cell analysis, screening, and culture. However, traditional single-cell imaging methods suffer from several technical bottlenecks: Firstly, the liquid level in a transparent plate is easily affected by factors such as liquid evaporation and sample loading errors. If the focal length of the imaging system relies solely on the theoretical design height of the plate, the actual liquid level deviates from the theoretical height, leading to blurred background images and subsequent misjudgments in cell monitoring due to focal inaccuracies. Secondly, when a single-cell droplet falls into a transparent liquid, its position changes dynamically as it falls. If the imaging system uses a fixed focal length, it is often difficult to accurately capture clear images of the single cells within the droplet, resulting in a low cell capture success rate. Furthermore, existing technologies, which use a bottom-mounted camera combined with printed single-cell fall times to scan the upper and lower regions of the liquid surface multiple times, typically require at least 15 images to increase the probability of cell detection. However, this process still requires the user to manually set the starting position and interval area for imaging, which demands a high level of user experience and results in a low probability of successfully capturing cells. Summary of the Invention

[0003] The purpose of this application is to at least solve one of the above-mentioned technical defects, in particular the technical defects in the prior art when imaging single cells, where the liquid level in the transparent well plate cannot be accurately determined, and the focus position and timing during imaging cannot be precisely controlled, resulting in a low cell capture success rate and low single cell screening efficiency.

[0004] This application provides a single-cell imaging method, the method comprising:

[0005] Determine the actual liquid level height in the target hole containing the transparent liquid, wherein the target hole is any selected hole in the transparent orifice plate;

[0006] The imaging system's focal length is aligned with the actual liquid level height, and before single-cell printing, the imaging system is controlled to capture the liquid surface background image of the target hole at the aligned focal length.

[0007] During single-cell printing, the imaging system is controlled to perform pre-scan monitoring of the target pore, using the liquid surface background image as a reference.

[0008] When a single-cell droplet is detected falling into the transparent liquid, the lens of the imaging system is controlled to continuously zoom within a preset zoom range. After each zoom, the imaging system is controlled to synchronously acquire the target image of the target hole until a preset number of photos is taken, thus completing cell capture.

[0009] Optionally, controlling the imaging system to perform pre-scan monitoring of the target hole based on the liquid surface background image includes:

[0010] The lens of the imaging system is controlled to zoom downwards from the actual liquid level height within a preset zoom duration by a preset distance, and to simultaneously capture real-time images of the liquid surface of the target hole during the zoom process.

[0011] The real-time image of the liquid surface is compared with the background image of the liquid surface to achieve pre-scan monitoring.

[0012] Optionally, comparing the real-time image of the liquid surface with the background image of the liquid surface to achieve pre-scan monitoring includes:

[0013] The first pixel matrix corresponding to the real-time image of the liquid surface is compared with the second pixel matrix corresponding to the background image of the liquid surface to determine the difference pixel matrix between the first pixel matrix and the second pixel matrix;

[0014] Valid difference regions in the difference pixel matrix are selected, and the area of ​​the valid difference regions is compared with a preset area threshold to achieve pre-scan monitoring.

[0015] Optionally, before detecting a single-cell droplet falling into the transparent liquid, the method further includes:

[0016] If the comparison result between the real-time image of the liquid surface and the background image of the liquid surface meets the preset difference condition, then it is determined that a single-cell droplet has been detected falling into the transparent liquid.

[0017] If the comparison result between the real-time liquid surface image and the liquid surface background image does not meet the preset difference condition, it is determined that a single-cell droplet has not fallen into the transparent liquid.

[0018] Optionally, the method further includes:

[0019] If it is detected that the single-cell droplet has not fallen into the transparent liquid, the system returns to controlling the lens of the imaging system to zoom below the liquid surface by a preset distance within a preset zoom duration, starting from the actual liquid level height, until it is determined that the single-cell droplet has fallen into the transparent liquid.

[0020] Optionally, controlling the lens of the imaging system to continuously zoom within a preset zoom range includes:

[0021] The lens of the imaging system is controlled to continuously zoom within a preset zoom range from below the actual liquid level to the actual liquid level, according to a preset zoom interval.

[0022] Optionally, the preset zoom range is consistent with the scanning range when the imaging system performs pre-scan monitoring of the target aperture.

[0023] This application also provides a single-cell imaging device, comprising:

[0024] The liquid level determination module is used to determine the actual liquid level in a target hole containing transparent liquid, wherein the target hole is any selected hole in the transparent orifice plate.

[0025] The imaging preparation module is used to align the focal length of the imaging system with the actual liquid level height, and before single-cell printing, control the imaging system to capture the liquid background image of the target hole at the aligned focal length.

[0026] A pre-scanning module is used to control the imaging system to pre-scan and monitor the target pores based on the liquid surface background image during single-cell printing.

[0027] The cell imaging module is used to control the lens of the imaging system to continuously zoom within a preset zoom range when a single cell droplet is detected falling into the transparent liquid, and to control the imaging system to synchronously acquire the target image of the target hole after each zoom, until a preset number of photos is taken to complete cell capture.

[0028] This application also provides a computer-readable storage medium storing computer-readable instructions that, when executed by one or more processors, cause the one or more processors to perform the steps of the single-cell imaging method as described in any of the above embodiments.

[0029] This application also provides a computer device, including: one or more processors, and memory;

[0030] The memory stores computer-readable instructions, which, when executed by the one or more processors, perform the steps of the single-cell imaging method as described in any of the above embodiments.

[0031] As can be seen from the above technical solutions, the embodiments of this application have the following advantages:

[0032] The single-cell imaging method, apparatus, storage medium, and computer equipment provided in this application firstly, by accurately determining the actual liquid level height within the target well, avoids the impact of liquid level deviations caused by factors such as liquid evaporation and sample addition errors on imaging, ensuring that the focal length of the imaging system can be accurately aligned with the liquid surface, laying the foundation for subsequent clear imaging of the liquid surface background image. Secondly, by capturing the liquid surface background image before single-cell printing and using it as a benchmark for pre-scanning monitoring, the entry of single-cell droplets can be quickly identified. Compared to traditional methods that rely on scanning at fixed time points, this greatly improves the timeliness and accuracy of droplet monitoring and reduces the probability of misjudgment. Furthermore, once a droplet is detected, the lens of the imaging system is controlled to continuously zoom within a preset zoom range and simultaneously acquire images. This eliminates the need for users to manually set the shooting position and interval, reducing reliance on user experience. At the same time, by continuously zooming to cover the dynamic positional changes of the droplet in the liquid, the clarity and capture success rate of the single-cell image are effectively improved, thereby increasing the efficiency of single-cell screening. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 A flowchart illustrating a single-cell imaging method provided in an embodiment of this application;

[0035] Figure 2 This is a schematic diagram illustrating the process of the control imaging system provided in this application performing pre-scan monitoring of the target hole;

[0036] Figure 3 A schematic diagram illustrating the process of determining whether a single-cell droplet falls into a transparent liquid, as provided in an embodiment of this application.

[0037] Figure 4 This is a schematic diagram of the structure of a single-cell imaging device provided in an embodiment of this application;

[0038] Figure 5 This is a schematic diagram of the internal structure of a computer device provided in an embodiment of this application. Detailed Implementation

[0039] 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 some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0040] In one embodiment, such as Figure 1 As shown, Figure 1 This is a flowchart illustrating a single-cell imaging method provided in an embodiment of this application; this application provides a single-cell imaging method, which may include:

[0041] S110: Determine the actual liquid level in the target hole containing the transparent liquid. The target hole is any selected hole in the transparent orifice plate.

[0042] In this step, before imaging single cells, this application can first determine the actual liquid level in the target well containing the transparent liquid. This target well is a specific well in the transparent plate selected for single-cell screening. It is understood that when culturing single cells, the transparent plate is typically used as a container, with a certain amount of transparent liquid (such as culture medium) pre-added to each well to provide a cell growth environment. Because the transparent liquid may evaporate naturally over time, or due to factors such as pipette accuracy errors or differences in operating techniques during sample addition, the actual liquid level in the target well may deviate from the theoretical design height of the well plate (such as the standard liquid level height indicated in the well plate's instruction manual). If the theoretical height is directly used as the reference for subsequent imaging, focal length inaccuracies are highly likely to occur. Therefore, this application provides an accurate basis for the focal length calibration of the subsequent imaging system by precisely determining the actual liquid level.

[0043] Specifically, the actual liquid level height inside the target hole can be determined in various ways, such as through an optical ranging component or by capturing multiple sets of liquid level images of the target hole. The specific method can be chosen according to the actual situation, and no limitation is imposed here. For example, this application can use a laser ranging sensor to perform non-contact measurement of the liquid level inside the target hole: the laser ranging sensor emits a laser pulse towards the liquid surface, and by recording the time difference between the laser emission and its reflection back to the sensor from the liquid surface, combined with the propagation speed of the laser in the air, the vertical distance between the sensor and the liquid surface is calculated. Then, the fixed installation height of the sensor from the bottom of the target hole is subtracted to obtain the actual liquid level height.

[0044] If image analysis is used, the imaging system can be controlled to capture multiple images of the liquid surface at different focal lengths from directly above the target hole. By analyzing the sharpness characteristics (such as edge gradient values) of the reflective areas of the liquid surface in each image, the object distance corresponding to the focal length that makes the reflective areas of the liquid surface the sharpest can be determined, and then converted into the actual liquid surface height. Alternatively, the imaging system can be controlled to capture images of the liquid surface from the bottom of the target hole upwards. By analyzing the position of the boundary line between the liquid surface and the hole wall in the image, combined with the known dimensional parameters of the orifice plate (such as the depth and inner diameter of the hole), the actual liquid surface height can be calculated. For example, if the depth of the target hole is H, and the pixel distance from the liquid surface boundary line to the bottom of the hole in the captured liquid surface image is P, and the total pixel length from the bottom to the top of the hole is T, then the actual liquid surface height can be calculated using the formula (P / T) × H.

[0045] By accurately obtaining the actual liquid level, the problem of discrepancies between the theoretical and actual liquid levels caused by factors such as liquid evaporation leading to a drop in liquid level and liquid volume deviation during sample addition can be avoided, providing an accurate basis for the focal length alignment of the subsequent imaging system.

[0046] S120: Align the focal length of the imaging system with the actual liquid level and, before single-cell printing, control the imaging system to capture the liquid background image of the target hole at the aligned focal length.

[0047] In this step, after determining the actual liquid level in the target hole through S110, this application can accurately align the focal length of the imaging system with the actual liquid level to eliminate the influence of liquid level deviation on imaging clarity.

[0048] The focal length of the imaging system can be adjusted via an electric focusing mechanism connected to the lens group. This mechanism drives the lens group to move along the optical axis based on the actual liquid level height until the focal point of the imaging system falls on the plane containing the actual liquid surface. Once the focal length is aligned, the imaging system can capture a background image of the liquid surface containing only transparent liquid within the target hole before the single-cell printing operation begins. Because the focal length is accurate, the background image captured at this time clearly shows the fine textures of the liquid surface area and the interior of the liquid (such as surface tension ripples and tiny air bubbles remaining on the hole walls), thus serving as a reference for subsequent pre-scan monitoring.

[0049] For example, if the actual liquid level height measured by this application is 3.2 mm, the electric focusing mechanism can drive the lens group to move to the corresponding position, so that the focus of the imaging system is focused on the liquid level at a height of 3.2 mm. Then, the camera of the imaging system takes a liquid background image with a resolution of 2048×2048 pixels with a preset exposure time (e.g., 10 ms) and sensitivity (e.g., ISO 100). This image will be used as a reference for subsequent pre-scan monitoring.

[0050] S130: During single-cell printing, the imaging system is controlled to perform pre-scan monitoring of the target pore, using the liquid surface background image as a reference.

[0051] In this step, after the imaging system is controlled by S120 to capture the background image of the liquid surface of the target hole at the aligned focal length, during single-cell printing, this application can use the background image of the liquid surface as a reference and control the imaging system to perform pre-scan monitoring of the target hole to determine whether the single-cell droplet has fallen.

[0052] Specifically, the core logic of the pre-scan monitoring in this application is to capture the surface disturbance or visual feature changes caused by a single-cell droplet falling into the liquid by comparing real-time images with background images. After the single-cell printing device starts the printing program, the print head will spray a droplet containing a single cell into the target hole according to preset parameters (such as droplet volume, jet pressure, etc.). At this time, the imaging system will enter a continuous pre-scan state with the liquid surface background image as a reference. During the pre-scan, the lens of the imaging system will move a preset distance below the liquid surface within a preset zoom time, starting from the actual liquid surface height, and simultaneously capture multiple real-time images of the liquid surface. After comparing the real-time liquid surface images with the liquid surface background images, it is determined whether a droplet has been detected. This dynamic monitoring method based on the background image does not require the user to manually set the scanning parameters, reducing the reliance on user experience. At the same time, it can quickly identify the timing of droplet falling by real-time comparison, which greatly improves the accuracy and timeliness of droplet monitoring compared to the traditional fixed-time-point scanning method.

[0053] S140: When a single cell droplet is detected falling into a transparent liquid, the lens of the imaging system is controlled to continuously zoom within a preset zoom range. After each zoom, the imaging system is controlled to synchronously acquire the target image of the target hole until the preset number of photos is reached, thus completing cell capture.

[0054] In this step, the imaging system controlled by S130 performs a pre-scan monitoring of the target well. Once a single-cell droplet is detected falling into the transparent liquid within the target well, the cell capture and imaging process can be initiated immediately. At this time, the lens of the imaging system will continuously zoom within a preset zoom range, simultaneously acquiring the target image of the target well after each zoom, until the preset number of images is completed.

[0055] The preset zoom range refers to a focal length range that is pre-set based on the physical properties of the transparent liquid (such as refractive index and viscosity), the volume of a single-cell droplet, and its falling velocity. This range covers the dynamic position of the droplet within the liquid after it falls in. For example, if the transparent liquid is a cell culture medium (refractive index approximately 1.33), the volume of a single-cell droplet is 10 pL, and the falling velocity is 2 m / s, and experiments have verified that the droplet will complete its deceleration, suspension, or initial sedimentation process within the range of 0.5 mm to 2.0 mm below the liquid surface, then the preset zoom range can be set to the focal length range corresponding to 0.5 mm to 2.0 mm below the actual liquid surface height.

[0056] Continuous zooming is performed by an electric focusing mechanism according to preset zoom steps and intervals, such as adjusting the focal length every 50ms. Each adjustment step corresponds to a 0.1mm interval in the liquid, ensuring coverage of the droplet's dynamic trajectory within the liquid. After each zoom, the imaging system's camera immediately acquires the target image. Parameters such as exposure time and sensitivity can be adjusted according to actual conditions. For example, to capture the dynamic details of the droplet, the exposure time can be shortened to 5ms to avoid motion blur. The preset number of shots is determined based on the total length of the zoom range and the zoom step. For instance, in the 0.5mm to 2.0mm range, the total length is 1.5mm, and the step is 0.3mm; only 5 shots are needed to completely record the droplet's positional changes in the liquid. This continuous zoom shooting method eliminates the need for manual prediction of the droplet's final position, automatically covering the droplet's dynamic movement range, effectively improving the success rate of single-cell image capture, ensuring clear single-cell morphology images, and providing reliable image data support for subsequent cell analysis (such as morphological observation and activity detection).

[0057] In the above embodiments, firstly, by accurately determining the actual liquid level height within the target well, the impact of liquid level deviations caused by factors such as liquid evaporation and sample addition errors on imaging is avoided, ensuring that the focal length of the imaging system can be accurately aligned with the liquid surface, laying the foundation for subsequent clear capture of the liquid surface background image. Secondly, capturing the liquid surface background image before single-cell printing and using it as a benchmark for pre-scanning monitoring can quickly identify the entry of single-cell droplets. Compared to the traditional method relying on fixed-time-point scanning, this greatly improves the timeliness and accuracy of droplet monitoring and reduces the probability of misjudgment. Furthermore, once a droplet is detected, the lens of the imaging system is controlled to continuously zoom within a preset zoom range and simultaneously acquire images. This eliminates the need for users to manually set the shooting position and interval, reducing reliance on user experience. At the same time, by continuously zooming to cover the dynamic positional changes of the droplet in the liquid, the clarity and capture success rate of the single-cell image are effectively improved, thereby increasing the efficiency of single-cell screening.

[0058] In one embodiment, such as Figure 2 As shown, Figure 2 This is a schematic diagram illustrating the process of the controlled imaging system performing pre-scan monitoring of the target hole according to an embodiment of this application; S130, using the liquid surface background image as a reference, controls the imaging system to perform pre-scan monitoring of the target hole, which may include:

[0059] S131: The lens of the control imaging system starts from the actual liquid level height, zooms to a preset distance below the liquid level within a preset zoom duration, and simultaneously captures a real-time image of the liquid level of the target hole during the zoom process.

[0060] S132: Compare the real-time image of the liquid surface with the background image of the liquid surface to achieve pre-scan monitoring.

[0061] In this embodiment, when the imaging system performs pre-scan monitoring of the target hole, the lens of the imaging system can be controlled to move a preset distance (e.g., 1.0 mm) below the liquid surface within a preset zoom duration (e.g., 200 ms). During this process, the imaging system will continuously capture multiple real-time images of the liquid surface at fixed time intervals (e.g., 20 ms / frame).

[0062] The preset zoom duration refers to the total time required for the imaging system to complete the zoom operation from the actual liquid surface height to a preset distance below the liquid surface. Its setting needs to comprehensively consider factors such as the viscosity of the transparent liquid and the falling speed of the single-cell droplet to ensure that a complete pre-scan is completed within the short time it takes for the droplet to fall into the liquid. For example, if the time from the printhead ejection of a single-cell droplet to its fall into the liquid surface is approximately 300 ms, the preset zoom duration can be set to 200 ms, ensuring that the pre-scan frequency covers the possible time window of the droplet's fall. The preset distance is determined based on the initial settling depth range of the droplet after it falls. If experiments show that the droplet settles to 0.8 mm below the liquid surface within 0.5 s after falling, the preset distance can be set to 1.0 mm to ensure that the pre-scan range covers the initial position of the droplet after its fall.

[0063] After capturing a real-time image of the liquid surface, this application can compare it with the background image of the liquid surface acquired in S120 to identify whether any droplets have fallen in. The comparison process can be implemented using an image difference algorithm, and to further improve the accuracy of the comparison, the texture features of the images can also be analyzed. For example, the local binary pattern (LBP) texture features of the liquid surface area in the background image and the real-time image are extracted, and the texture similarity between the two is calculated. If the similarity is lower than a preset threshold (e.g., 0.7), it is determined that a droplet has fallen in. This comparison method, which combines gray-scale difference and texture features, can effectively distinguish between the real changes caused by the falling droplets and the interference caused by small fluctuations in the liquid surface (such as ripples caused by environmental vibrations), reducing the probability of misjudgment.

[0064] Through the above operations, this application realizes dynamic pre-scan monitoring of the target well. Compared with the traditional fixed-time-point scanning method, it can not only capture the moment when the droplet falls in more timely, but also expand the monitoring range through continuous zoom, improve the accuracy of droplet identification, and provide a precise trigger signal for subsequent single-cell capture and imaging.

[0065] In one embodiment, comparing the real-time image of the liquid surface with the background image of the liquid surface in step S132 to achieve pre-scan monitoring may include:

[0066] S1321: Compare the first pixel matrix corresponding to the real-time liquid surface image with the second pixel matrix corresponding to the liquid surface background image to determine the difference pixel matrix between the first pixel matrix and the second pixel matrix.

[0067] S1322: Filter out the effective difference regions in the difference pixel matrix, and compare the area of ​​the effective difference regions with a preset area threshold to achieve pre-scan monitoring.

[0068] In this embodiment, after converting the real-time liquid surface image and the liquid surface background image into grayscale images, the corresponding first pixel matrix and second pixel matrix can be extracted respectively, where the element value of each pixel matrix is ​​the grayscale value (range 0-255) of the pixel at the corresponding position. This application can generate a difference pixel matrix by calculating the absolute difference between the first pixel matrix and the second pixel matrix pixel by pixel. Specifically, if the grayscale difference of a pixel at a certain position is greater than a preset grayscale threshold (e.g., 15), the difference pixel value at that position is recorded as 1; otherwise, it is recorded as 0. For example, the grayscale value of a pixel in the liquid surface background image is 120, and the grayscale value of the corresponding position in the real-time liquid surface image is 140. The difference between the two is 20, which is greater than the grayscale threshold of 15. Therefore, the position at that position is recorded as 1 in the difference pixel matrix.

[0069] After generating the difference pixel matrix, this application can further perform morphological processing to remove noise interference. Specifically, a 3×3 structuring element can be used to perform an opening operation (erosion followed by dilation) on the difference pixel matrix to eliminate isolated noise points (such as grayscale abrupt changes in a single pixel) caused by image sensor noise and minor vibrations of the liquid surface. Subsequently, connected regions in the difference pixel matrix are identified through a connected region analysis algorithm (such as eight-neighbor connectivity analysis), with each connected region corresponding to a potential droplet landing area.

[0070] When screening for valid difference regions, this application can calculate the area of ​​each connected region (in pixels) and compare it with a preset area threshold. The preset area threshold can be set based on the actual size of the single-cell droplet: if the minimum pixel area of ​​a single-cell droplet in the image after falling into the liquid is 50 pixels (derived from previous experimental statistics), then the preset area threshold can be set to 40 pixels. When the area of ​​a connected region is greater than 40 pixels, it is determined to be a valid difference region, that is, it is considered that a single-cell droplet has been detected; if the area of ​​all connected regions is less than 40 pixels, it is determined to be invalid interference (such as tiny tension ripples on the liquid surface). For example, in a pre-scan, after morphological processing, the difference pixel matrix yields a connected region with an area of ​​65 pixels, which is greater than the preset threshold of 40 pixels. Therefore, it is determined that a droplet has been detected, triggering the subsequent cell capture and imaging process.

[0071] By using this method of filtering based on pixel matrix difference and connected region area, it is possible to accurately distinguish between significant image changes caused by droplet intrusion and minor noise caused by environmental interference, greatly improving the accuracy of pre-scan monitoring and avoiding invalid or missed shots due to misjudgment.

[0072] In one embodiment, such as Figure 3 As shown, Figure 3 This is a schematic diagram of the process for determining whether a single-cell droplet has fallen into a transparent liquid, provided in an embodiment of this application; in S140, before detecting that a single-cell droplet has fallen into the transparent liquid, the method may further include:

[0073] S133: If the comparison result between the real-time liquid surface image and the liquid surface background image meets the preset difference condition, then it is determined that a single-cell droplet has been detected falling into the transparent liquid.

[0074] S134: If the comparison result between the real-time liquid surface image and the liquid surface background image does not meet the preset difference condition, it is determined that a single-cell droplet has not fallen into the transparent liquid.

[0075] In this embodiment, the preset difference conditions can be set as a single-dimensional judgment index according to the actual application scenario, or multiple-dimensional judgment indexes can be set, such as the comprehensive difference pixel ratio, the morphological features of the effective difference region and the grayscale change gradient, etc.

[0076] Specifically, the percentage of difference pixels refers to the proportion of pixels with a value of 1 in the difference pixel matrix to the total number of pixels in the liquid surface area. If this proportion exceeds a preset percentage threshold (e.g., 5%), and the ratio of the major axis to the minor axis of the effective difference region is within a preset range (e.g., 1.0-1.5, consistent with the approximately circular or elliptical region formed when a droplet falls in), and the grayscale gradient within the effective difference region is greater than a preset gradient threshold (e.g., 20 grayscale values / pixel, reflecting the significant grayscale changes caused by liquid disturbance when a droplet falls in), then the preset difference condition is satisfied. Conversely, if the percentage of difference pixels is less than 5%, or the effective difference region has an irregular strip shape (possibly liquid surface vibration ripples), or the grayscale gradient is less than 20 grayscale values / pixel, then the preset difference condition is not satisfied.

[0077] For example, after comparing a real-time image of a liquid surface with a background image, if the percentage of differing pixels is 7%, the ratio of the major axis to the minor axis of the effective difference region is 1.2, and the grayscale gradient within the region is 25 grayscale values ​​per pixel, all three indicators meet the preset conditions, thus confirming that a droplet has been detected. If, however, the percentage of differing pixels in another real-time image is only 3%, and the effective difference region is elongated and thin, with a major axis to minor axis ratio of 3.0, then it is determined that no droplet has been detected. This multi-dimensional, preset difference condition judgment further refines the identification criteria for droplet entry, reduces errors caused by judging a single indicator, and ensures the accuracy of the pre-scan monitoring results.

[0078] In one embodiment, the method may further include:

[0079] S135: When it is detected that a single-cell droplet has not fallen into the transparent liquid, return to the execution control imaging system and zoom the lens below the liquid surface by a preset distance within a preset zoom duration, starting from the actual liquid level height, until it is determined that a single-cell droplet has fallen into the transparent liquid.

[0080] In this embodiment, when a single-cell droplet is detected not falling into the transparent liquid, the system triggers a cyclic pre-scan mechanism. Specifically, after completing a zoom scan from the actual liquid level to a preset distance below the liquid level and determining that no droplet has fallen in, the imaging system's lens automatically resets to the actual liquid level. After waiting for a preset interval (e.g., 50ms), a new round of pre-scanning is initiated, repeating steps S131 to S134. This interval setting needs to balance scanning efficiency and system load. If the interval is too short, the imaging system may overheat due to continuous operation; if the interval is too long, the time window for droplet falling may be missed. Experiments have verified that a 50ms interval can ensure stable system operation and cover most of the single-cell droplet ejection cycle.

[0081] During the pre-scan cycle, the system can also record the number of scans and timestamps in real time. For example, if no droplet is detected after five consecutive scans, the system will automatically adjust the preset zoom parameters: if the initial preset zoom duration is 200ms and the preset spacing is 1.0mm, the zoom duration can be extended to 250ms and the preset spacing expanded to 1.2mm to accommodate situations where the droplet jet velocity may fluctuate, leading to longer fall times or increased settling depths. Simultaneously, the system can send low-priority alerts to the user terminal, informing them of the current droplet monitoring status. This allows users to promptly check for issues such as printhead blockage or abnormal droplet generation, without interrupting the pre-scan cycle, ensuring continuous monitoring of droplet entry.

[0082] Furthermore, to avoid wasting system resources due to infinite loops, this application also sets a maximum loop count threshold (e.g., 20 times). When no droplet is detected after 20 pre-scan loops, the system can pause the pre-scan and trigger a high-level alarm, prompting the user to check the operating status of the single-cell printing device, including key parameters such as the printhead jet pressure and the temperature control of the droplet generation module. After the user has troubleshooted and resolved the issue, the pre-scan process can be resumed manually or automatically by the system, thus ensuring continuous monitoring while maintaining system stability and maintainability.

[0083] This cyclic pre-scanning mechanism, combined with adaptive parameter adjustment and anomaly alarm functions, effectively improves the robustness of single-cell droplet monitoring, ensuring that even when there are fluctuations in droplet ejection, the timing of droplet landing can still be accurately captured.

[0084] In one embodiment, controlling the lens of the imaging system to continuously zoom within a preset zoom range in S140 may include:

[0085] The lens of the imaging system is controlled to continuously zoom within a preset zoom range from below the actual liquid level to the actual liquid level, according to a preset zoom interval.

[0086] In this embodiment, when controlling the lens of the imaging system to continuously zoom within a preset zoom range, the preset zoom interval setting needs to balance the image acquisition density and the system response speed. Assuming the preset zoom range is 1mm, this application can set the preset zoom interval to 0.2mm. That is, the lens of the imaging system starts from the starting position below the actual liquid surface height, moves 0.2mm, pauses, and simultaneously acquires an image, until it covers the entire 1mm zoom range, ultimately forming a sequence of single-cell images at five different focal lengths. This equidistant continuous zooming method ensures full coverage capture of droplets at different depths in the liquid, avoiding the loss of clear imaging positions of droplets due to excessively large focal length intervals.

[0087] Furthermore, the preset zoom distance of this application can be dynamically adjusted according to the size of the single-cell droplet and the liquid environment: if the droplet volume is small (e.g., diameter less than 50μm), the preset zoom distance can be reduced to 0.1mm to improve the resolution of image acquisition; if the liquid viscosity is high, resulting in a slow droplet settling speed, the preset zoom distance can be appropriately increased to 0.3mm to improve imaging efficiency while ensuring the capture effect. For example, for a single-cell droplet with a diameter of 30μm, setting the preset zoom distance to 0.1mm, while maintaining a zoom range of 1mm, allows for the acquisition of 10 images at different focal lengths, further improving the clarity and detail capture capability of the single-cell image.

[0088] Furthermore, during continuous zooming, the imaging system's control module sends pulse signals to the stepper motor in real time, driving the lens to complete the zooming action at a constant speed. The zoom speed can be set to 2.0 mm / s to ensure the stability of the lens movement and avoid image blurring due to speed fluctuations. For example, when the preset zoom range is 1 mm and the preset zoom interval is 0.2 mm, the total time for the lens to move from the starting point to the ending point is 0.5 s. The dwell time for each 0.2 mm movement can be set to 50 ms to complete the exposure and acquisition of a single frame image, ultimately completing the continuous acquisition of 5 frames within 0.5 s. This control method, combining constant zooming with fixed dwell time, ensures consistent image acquisition conditions at each focal length, providing reliable basic data for subsequent image sharpness evaluation. In addition, the imaging system also caches the image after each zoom in real time to avoid image loss due to data transmission delays, ensuring the integrity of the image sequence during continuous zooming.

[0089] In one embodiment, the preset zoom range is consistent with the scanning range when the imaging system performs pre-scan monitoring of the target aperture.

[0090] In this embodiment, the preset zoom range is consistent with the scanning range of the pre-scan monitoring in order to achieve a "seamless connection" between the pre-scan and the subsequent capture and shooting.

[0091] Specifically, the scanning range in the pre-scanning stage is determined based on the initial settling depth of the droplet (e.g., a settling depth of 0.8 mm below the liquid surface corresponds to a scanning range of 1.0 mm). The preset zoom range in the capture and imaging stage directly uses this range, meaning that after the imaging system triggers the capture, the zoom range of the lens completely overlaps with the monitoring range during the pre-scan. The core advantage of this design is that the pre-scanning stage has already covered the possible location range of the droplet through continuous zoom. Subsequent capture and imaging only requires finer focus adjustments within the same range to quickly locate a clear imaging plane of the droplet, avoiding the problems of "range omission" or "repeated scanning" caused by inconsistent zoom ranges.

[0092] For example, if the pre-scanning range is 1.0 mm below the liquid surface, and the preset zoom range for capture is also set to 1.0 mm, then once the pre-scan detects a droplet falling into this range, the lens in the capture stage does not need to adjust the zoom range separately. It can directly perform continuous zooming to acquire images within the already covered 1.0 mm range, ensuring full coverage of the droplet's location and shortening the response time from detection to capture. Simultaneously, this range consistency simplifies the system's parameter setting process, eliminating the need to separately adjust different zoom ranges for pre-scanning and capture, reducing operational complexity and parameter matching errors, and further improving the consistency and stability of the single-cell imaging process.

[0093] The single-cell imaging device provided in the embodiments of this application is described below. The single-cell imaging device described below can be referred to in correspondence with the single-cell imaging method described above.

[0094] In one embodiment, such as Figure 4 As shown, Figure 4 This is a schematic diagram of a single-cell imaging device provided in an embodiment of this application; this application also provides a single-cell imaging device, which may include a liquid level determination module 210, an imaging preparation module 220, a pre-scanning module 230, and a cell imaging module 240, specifically including the following:

[0095] The liquid level determination module 210 is used to determine the actual liquid level in a target hole containing transparent liquid, wherein the target hole is any selected hole in the transparent orifice plate.

[0096] The imaging preparation module 220 is used to align the focal length of the imaging system with the actual liquid level height, and before single-cell printing, control the imaging system to capture the liquid background image of the target hole at the aligned focal length.

[0097] The pre-scan module 230 is used to control the imaging system to perform pre-scan monitoring of the target pore during single-cell printing, based on the liquid surface background image.

[0098] The cell imaging module 240 is used to control the lens of the imaging system to continuously zoom within a preset zoom range when a single cell droplet is detected falling into the transparent liquid, and to control the imaging system to synchronously acquire the target image of the target hole after each zoom, until a preset number of photos is taken to complete cell capture.

[0099] In the above embodiments, firstly, by accurately determining the actual liquid level height within the target well, the impact of liquid level deviations caused by factors such as liquid evaporation and sample addition errors on imaging is avoided, ensuring that the focal length of the imaging system can be accurately aligned with the liquid surface, laying the foundation for subsequent clear capture of the liquid surface background image. Secondly, capturing the liquid surface background image before single-cell printing and using it as a benchmark for pre-scanning monitoring can quickly identify the entry of single-cell droplets. Compared to the traditional method relying on fixed-time-point scanning, this greatly improves the timeliness and accuracy of droplet monitoring and reduces the probability of misjudgment. Furthermore, once a droplet is detected, the lens of the imaging system is controlled to continuously zoom within a preset zoom range and simultaneously acquire images. This eliminates the need for users to manually set the shooting position and interval, reducing reliance on user experience. At the same time, by continuously zooming to cover the dynamic positional changes of the droplet in the liquid, the clarity and capture success rate of the single-cell image are effectively improved, thereby increasing the efficiency of single-cell screening.

[0100] In one embodiment, this application also provides a computer-readable storage medium storing computer-readable instructions that, when executed by one or more processors, cause the one or more processors to perform the steps of the single-cell imaging method as described in any of the above embodiments.

[0101] In one embodiment, this application also provides a computer device, including: one or more processors, and memory.

[0102] The memory stores computer-readable instructions, which, when executed by the one or more processors, perform the steps of the single-cell imaging method as described in any of the above embodiments.

[0103] Indicatively, such as Figure 5 As shown, Figure 5 This is a schematic diagram of the internal structure of a computer device 300 provided in an embodiment of this application. The computer device 300 can be provided as a server. (Refer to...) Figure 5 The computer device 300 includes a processing component 302, which further includes one or more processors, and memory resources represented by memory 301 for storing instructions, such as application programs, that can be executed by the processing component 302. The application programs stored in memory 301 may include one or more modules, each corresponding to a set of instructions. Furthermore, the processing component 302 is configured to execute instructions to perform the single-cell imaging method of any of the above embodiments.

[0104] The computer device 300 may also include a power supply component 303 configured to perform power management of the computer device 300, a wired or wireless network interface 304 configured to connect the computer device 300 to a network, and an input / output (I / O) interface 305. The computer device 300 may operate on an operating system stored in memory 301, such as Windows Server™, Mac OS X™, Unix™, Linux™, Free BSD™, or similar.

[0105] Those skilled in the art will understand that Figure 5 The structure shown is merely a block diagram of a portion of the structure related to the present application and does not constitute a limitation on the computer device to which the present application is applied. Specific computer devices may include more or fewer components than those shown in the figure, or combine certain components, or have different component arrangements.

[0106] Finally, it should be noted that in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0107] The various embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. The various embodiments can be combined as needed, and the same or similar parts can be referred to each other.

[0108] The above description of the disclosed embodiments enables those skilled in the art to make or use this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A single-cell imaging method, characterized in that, The method includes: Determine the actual liquid level height in the target hole containing the transparent liquid, wherein the target hole is any selected hole in the transparent orifice plate; The imaging system's focal length is aligned with the actual liquid level height, and before single-cell printing, the imaging system is controlled to capture the liquid surface background image of the target hole at the aligned focal length. During single-cell printing, the imaging system is controlled to perform pre-scan monitoring of the target pore, using the liquid surface background image as a reference. When a single-cell droplet is detected falling into the transparent liquid, the lens of the imaging system is controlled to continuously zoom within a preset zoom range. After each zoom, the imaging system is controlled to synchronously acquire the target image of the target hole until a preset number of photos is taken, thus completing cell capture.

2. The single-cell imaging method according to claim 1, characterized in that, The step of controlling the imaging system to perform pre-scan monitoring of the target hole based on the liquid surface background image includes: The lens of the imaging system is controlled to zoom downwards from the actual liquid level height within a preset zoom duration by a preset distance, and to simultaneously capture real-time images of the liquid surface of the target hole during the zoom process. The real-time image of the liquid surface is compared with the background image of the liquid surface to achieve pre-scan monitoring.

3. The single-cell imaging method according to claim 2, characterized in that, The step of comparing the real-time image of the liquid surface with the background image of the liquid surface to achieve pre-scan monitoring includes: The first pixel matrix corresponding to the real-time image of the liquid surface is compared with the second pixel matrix corresponding to the background image of the liquid surface to determine the difference pixel matrix between the first pixel matrix and the second pixel matrix; Valid difference regions in the difference pixel matrix are selected, and the area of ​​the valid difference regions is compared with a preset area threshold to achieve pre-scan monitoring.

4. The single-cell imaging method according to claim 2 or 3, characterized in that, Before detecting a single-cell droplet falling into the transparent liquid, the method further includes: If the comparison result between the real-time image of the liquid surface and the background image of the liquid surface meets the preset difference condition, then it is determined that a single-cell droplet has been detected falling into the transparent liquid. If the comparison result between the real-time liquid surface image and the liquid surface background image does not meet the preset difference condition, it is determined that a single-cell droplet has not fallen into the transparent liquid.

5. The single-cell imaging method according to claim 4, characterized in that, The method further includes: If it is detected that the single-cell droplet has not fallen into the transparent liquid, the system returns to controlling the lens of the imaging system to zoom below the liquid surface by a preset distance within a preset zoom duration, starting from the actual liquid level height, until it is determined that the single-cell droplet has fallen into the transparent liquid.

6. The single-cell imaging method according to claim 1, characterized in that, The control of the lens of the imaging system to continuously zoom within a preset zoom range includes: The lens of the imaging system is controlled to continuously zoom within a preset zoom range from below the actual liquid level to the actual liquid level, according to a preset zoom interval.

7. The single-cell imaging method according to any one of claims 1-6, characterized in that, The preset zoom range is consistent with the scanning range when the imaging system performs pre-scan monitoring of the target aperture.

8. A single-cell imaging device, characterized in that, include: The liquid level determination module is used to determine the actual liquid level in a target hole containing transparent liquid, wherein the target hole is any selected hole in the transparent orifice plate. The imaging preparation module is used to align the focal length of the imaging system with the actual liquid level height, and before single-cell printing, control the imaging system to capture the liquid background image of the target hole at the aligned focal length. A pre-scanning module is used to control the imaging system to pre-scan and monitor the target pores based on the liquid surface background image during single-cell printing. The cell imaging module is used to control the lens of the imaging system to continuously zoom within a preset zoom range when a single cell droplet is detected falling into the transparent liquid, and to control the imaging system to synchronously acquire the target image of the target hole after each zoom, until a preset number of photos is taken to complete cell capture.

9. A computer-readable storage medium, characterized in that, The computer-readable storage medium stores computer-readable instructions that, when executed by one or more processors, cause the one or more processors to perform the steps of the single-cell imaging method as described in any one of claims 1 to 7.

10. A computer device, characterized in that, include: One or more processors, and memory; The memory stores computer-readable instructions, which, when executed by the one or more processors, perform the steps of the single-cell imaging method as described in any one of claims 1 to 7.

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