Target selection and culture system and method for tiny microorganisms

Abstract

The invention discloses a system and a method for target selection and culture of tiny microorganisms. The system comprises a machine vision module, a pattern design module, a contact microneedle module and a three-dimensional control module, wherein the machine vision module is used for identifying and positioning a tiny microorganism colony; the pattern design module is used for designing a microorganism distribution pattern; the contact microneedle module is used for dipping and transferring the tiny microorganism colonies; and the three-dimensional control module is used for moving the contact microneedle module. According to the invention, through positioning and identification of the machine vision module, randomly distributed tiny microbial colonies can be grabbed; by means of flexible dipping of the contact microneedle module, complex, changeable and undulating thalli can be grabbed, the thalli are different in texture, viscosity and size, the inoculation strength and height control are flexibly controlled, the number of cells dipped each time is small, the resolution ratio is high, the disinfection and heat dissipation efficiency is high, and the patterned granularity is higher.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemistry, and particularly relates to a system and method for target selection and cultivation of minute microorganisms. Background Art

[0002] In the fields of biochemistry, analytical chemistry, microbiology, etc., the construction of many complex compositions or microenvironments is involved. For example, the combination of drugs or reagents, the combination of microorganisms, and the mixing of organic-inorganic composites. It is extremely difficult to micro-dip some surface substances.

[0003] Some existing methods, such as inkjet printing technology and microcontact printing technology (μCP). The microcontact printing technology can generate sub-micron-sized protein patterns in high throughput, and can construct protein microarrays with sizes of 10 microns to 100 microns that are usually required in single-cell related experiments. Moreover, the process and the required equipment are relatively simple. However, it is difficult to construct a multiple protein array. In addition, scanning probe lithography (SPL) such as dip-pen nanolithography (DPN), polymer pen lithography (PPL), and microchannel cantilever spotting (μCS) can construct high-resolution protein arrays. Some existing microcontacts are mainly micro-needles, or even nanowires, etc. However, this kind of contact is often direct contact. For the contact in the Z-axis, either extremely complex microscopic sensing and other problems are involved. The flexibility is also poor. These technologies all need to rely on a microscope or even an atomic force microscope to achieve printing. The atomic force microscope is expensive, large in volume, complex in operation, and has high requirements for the use environment; and it is time-consuming and laborious when constructing a large-scale protein array, thus limiting the application scenarios of these technologies. On the other hand, it is often difficult to have both large-scale and small-scale precise patterning. For example, in the patterning of microorganisms, the existing needles are often relatively thick. In the dipping of microorganisms, only colonies with a relatively large diameter can be dipped. Smaller colonies are either difficult to touch and are easily lost, or are easily contaminated with miscellaneous bacteria. If the diameter of the needle is made small, the general recognition accuracy is also poor. In addition, the existing inoculation needles are all relatively thick, and the cooling during the continuous inoculation sterilization process is slow, significantly reducing the speed of microorganism inoculation. Therefore, it is very difficult to quickly grab existing minute colonies both in the XY-axis and the Z-axis. Generally speaking, there are technical problems of insufficient flexibility and adaptability in the in-situ dipping and transfer of trace surface substances. Summary of the Invention

[0004] The purpose of the present application is to overcome the above technical deficiencies, and propose a system and method for target selection and cultivation of minute microorganisms, so as to solve the technical problems of insufficient flexibility and adaptability in the prior art.

[0005] To achieve the above technical purpose, the present application adopts the following technical solutions: In a first aspect, the present application provides a selection and culture system for tiny microbial targets, including a machine vision module, a pattern design module, a contact microneedle module, and a three-dimensional control module: The machine vision module is used to identify and locate tiny microbial colonies; The pattern design module is used to design microbial distribution patterns; The contact microneedle module is used to dip and transfer the tiny microbial colonies; The three-dimensional control module is used to move the contact microneedle module.

[0006] In some embodiments of the present application, the contact microneedle module includes a microneedle, a needle cap, and a support tube. The needle cap and the support tube are respectively sleeved outside the microneedle. The support tube is located below the needle cap, and the outer diameter of the needle cap is greater than the inner diameter of the support tube.

[0007] In some embodiments of the present application, the contact microneedle module includes a microneedle and a support tube. The support tube is sleeved outside the microneedle. The upper end of the microneedle has a bent portion, and the upper end of the support tube has an open end. The cross-sectional profile of the open end is O-shaped, and the longitudinal-sectional profile is V-shaped. The tip of the microneedle is circular, annular, horizontally sheet-shaped, or water-drop-shaped, and the bent portion of the microneedle abuts against the open end of the support tube. The inner diameter of the support tube is greater than the outer diameter of the microneedle. The microneedle can move upward relative to the support tube, and the outer diameter of the microneedle is below 0.2 cm, preferably 0.1 cm.

[0008] In some embodiments of the present application, the contact microneedle module further includes a coil. The material of the coil includes metal or plastic. The coil is wound around the periphery of the microneedle or the needle cap, and the length of the coil is changed to control the magnitude of the sum of the gravity of the coil and the microneedle.

[0009] In some embodiments of the present application, the contact microneedle module further includes an inductive coil and magnetic particles. The inductive coil is wound around the periphery of the microneedle and is located inside the support tube. The magnetic particles are filled between the support tube and the microneedle, and magnetism is formed by controlling the current to control the rise and fall of the microneedle.

[0010] In some embodiments of the present application, the machine vision module includes a large-field camera and a small-field camera, and the field of view of the large-field camera covers the field of view of the small-field camera.

[0011] In some embodiments of the present application, multiple contact microneedle modules are distributed in an array to improve the speed of picking bacteria.

[0012] In some embodiments of the present application, the three-dimensional control module includes a three-dimensional controller, the three-dimensional controller is independently connected to each of the contact microneedle modules, and the contact microneedle module is signal-connected to the small field of view camera.

[0013] In a second aspect, the present application further provides a method for selecting and culturing a target of minute microorganisms, using the system for selecting and culturing a target of minute microorganisms according to any one of the embodiments in the first aspect, including the following steps: The machine vision module identifies and locates the minute microorganism colonies; The pattern design module designs the microorganism distribution pattern; The three-dimensional control module drives the contact microneedle module to flexibly dip the microorganism colonies and transfer them to the target area.

[0014] In some embodiments of the present application, the machine vision module identifies and locates the minute microorganism colonies, including: The large field of view camera quickly locates the microorganism colonies and guides the three-dimensional control module to move above the microorganism colonies; The small field of view camera accurately identifies the microorganism colonies according to the similarity and minimum distance principles and guides the contact microneedle module to fine-tune and correct.

[0015] In some embodiments of the present application, the three-dimensional control module drives the contact microneedle module to flexibly dip the microorganism colonies and transfer them to the target area, including: Receiving the digital coordinates converted by the machine vision module; Planning the movement trajectory of the contact microneedle module; Outputting a movement signal to the three-dimensional control module.

[0016] In some embodiments of the present application, the three-dimensional control module drives the contact microneedle module to flexibly dip the microorganism colonies and transfer them to the target area, including: In the initial state, the open end of the support tube bears the microneedles so that the microneedles are in a suspended state; In the sampling state, the robotic arm drives the microneedles to move to a preset position, the tips of the microneedles are lifted by the microorganism colonies, and the microneedles are displaced upward relative to the support tube; The robotic arm moves upward, the microneedles are displaced downward relative to the support tube, and the relative position between the microneedles and the support tube returns to the initial state.

[0017] In some embodiments of the present application, the three-dimensional control module drives the contact microneedle module to flexibly dip the microorganism colonies and transfer them to the target area, including: Controlling the individual protrusions of each contact microneedle module.

[0018] In some embodiments of the present application, the three-dimensional control module drives the contact microneedle module to flexibly dip the microbial colony and transfer it to the target area, including: Controlling the inductance of the inductance coil in the contact microneedle module; Controlling the number of microporous wafers in the contact microneedle module.

[0019] Compared with the prior art, the beneficial technical effects brought by the technical solution provided by the present application include: Through the positioning and recognition of the machine vision module, the present application can capture randomly distributed microbial colonies; through the flexible dipping of the contact microneedle module, it can capture complex, variable, undulating bacterial bodies with different textures, viscosities, and sizes, and can achieve flexible control in terms of inoculation force and height control. The number of cells dipped each time is small, the resolution is high, the efficiency is high, and the granularity of the patterning is higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the present application, the following will briefly introduce the drawings required for the embodiments: Figure 1 It is a schematic structural diagram of a system for selecting and culturing tiny microbial targets provided by an embodiment of the present application; Figure 2 It is a schematic structural diagram of a contact needle module provided by an embodiment of the present application; Figure 3 It is another schematic structural diagram of a contact needle module provided by an embodiment of the present application; Figure 4 It is a schematic flowchart of a method for selecting and culturing tiny microbial targets provided by an embodiment of the present application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0021] In order to make the objectives, technical solutions and advantages of the present application more clear and understandable, the following further details the present application in conjunction with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0022] Those skilled in the art can understand that in this specification, the term "including" is an open-ended expression, meaning that the stated features exist but do not exclude other features. The orientation terms such as "upper", "lower", "left", and "right" are exemplary directions based on the figures. Features defined with "first" and "second" implicitly include one or more of such features. The singular form can also be used for the plural form. The meaning of "a plurality" is two or more. The terms "install", "connect", and "couple" can be fixed connections, detachable connections, or integral connections; they can be directly connected or indirectly connected through an intermediate medium, and can be the communication inside two components. In addition, "connection" can include wireless connection.

[0023] The purpose of this application is to overcome the above technical deficiencies and propose a system 1 and a method for selecting and culturing target micro - microorganisms, so as to solve the technical problems of insufficient flexibility and adaptability in the prior art.

[0024] To achieve the above technical objectives, this application adopts the following technical solutions: In a first aspect, this application also provides a system 1 for selecting and culturing target micro - microorganisms, as Figure 1 shown, Figure 1 which is a schematic structural diagram of a system 1 for selecting and culturing target micro - microorganisms provided by an embodiment of this application.

[0025] A system 1 for selecting and culturing target micro - microorganisms includes: A machine vision module 11, used to identify and locate micro - microorganism colonies; A pattern design module 12, used to design microorganism distribution patterns; A contact micro - needle module 14, used to dip and transfer the micro - microorganism colonies; A three - dimensional control module 13, used to move the contact micro - needle module 14.

[0026] As Figure 2 shown, Figure 2 which is a schematic structural diagram of a contact needle module provided by an embodiment of this application.

[0027] In some embodiments of this application, the contact micro - needle module 14 includes a micro - needle 141, a needle head cap 142, and a support tube 143. The needle head cap 142 and the support tube 143 are respectively sleeved outside the micro - needle 141. The support tube 143 is located below the needle head cap 142, and the outer diameter of the needle head cap 142 is greater than the inner diameter of the support tube 143.

[0028] In this embodiment, the contact microneedle 141 is inserted into the support tube 143, the needle cap 142 is larger than the tube mouth of the support tube 143, the lower section of the contact microneedle 141 is smaller than the outlet of the support tube 143, the support tube 143 carries the needle cap 142, the lower end of the microneedle 141 passes through the support tube 143, and the upper end of the microneedle 141 is fixedly connected to the needle cap 142, and the needle cap 142 cannot pass through the support tube 143, causing the microneedle 141 to be suspended in the support tube 143, and can move up and down relative to the support tube 143, flexibly control the Z-axis distance, and its own gravity is the force of contact with the surface of the target object.

[0029] The device cleverly uses the weight of the contact needle as the contact force, overcomes the Z-axis rigid contact problem of traditional printing technology or contact technology, greatly simplifies the control problem of contact distance, protects the structure of the contact surface and the transfer surface, and at the same time, improves the resolution of the plane. The device is simple and lightweight, suitable for the transfer, inoculation and other light contact transfer needs of various solid liquids and high-viscosity substances, and provides low-cost, portable automation methods in the fields of high-throughput material transfer, droplet reactors, microbial culture and other analysis and detection. More importantly, the traditional sampling needle has low precision requirements and the diameter of the needle is relatively large. Another technical problem is that high-temperature disinfection and sterilization are slow, and it takes a long time for the temperature to drop from hundreds of degrees to 40 degrees. In this embodiment, the microneedle pattern can increase the accuracy of inoculation through the modification of the needle tip. At the same time, because the diameter of the microneedle is small, it cools very quickly. The tip of the microneedle is designed to be round, drop-shaped, horizontal sheet or ring-shaped, which is also for rapid heat dissipation and can increase the reliability of grasping. In high-throughput rapid bacteria picking, the speed advantage is obvious.

[0030] The diameter of the contact microneedle 141 is 0.05mm~1mm, and the needle length is 1cm~5cm. Steel with good high temperature resistance can be selected; the front end of the support tube 143 contains a microhole with a diameter of 0.05mm~1mm. The contact microneedle 141 is slightly smaller than the microhole, and the microneedle 141 can easily penetrate up and down. There is a needle cap 142 at the upper end of the microneedle 141, and its diameter is larger than the supporting microhole. By inserting the microneedle 141 with the needle cap 142 into a tube slightly larger than the diameter of the microneedle 141, the microneedle 141 is lifted by the needle cap 142 and the outer support tube 143. The microneedle 141 can move up and down in the support tube 143 without obvious obstruction. The needle assembly is fixed on a telescopic rod or a three-axis mobile platform, and the microneedle 141 moves up and down at a designated location through position movement. The contact with the surface material is achieved by its own weight without the need for additional Z-axis up and down precise control. Since the diameter of the microneedle 141 is relatively small, the weight of the microneedle 141 itself can achieve adhesion sampling at a specific depth on the surface of the target object.

[0031] Through the movable cantilever needle assembly, the tip of the needle can dip ultra-trace substances. This contact process can directly penetrate a certain surface and come into contact with the substances or liquids on the surface, meeting the problem of ultra-high-precision substance sampling in specific scenarios. Through the movable cantilever needle method, the force of the needle tip penetrating can be effectively controlled, which is especially suitable for high-density sampling on some soft surfaces. For some complex uneven interfaces, the contact needle module can maintain the same contact area and force, and will not cause differences in the contact area or force of the collected samples due to the unevenness of the surface. These advantages will significantly improve the defects of existing fixed-needle sampling and achieve the flexibility problems that cannot be achieved by traditional methods, such as flexible arms or nano-arms.

[0032] Different media and properties can also be fixed at the top of the contact needle to achieve control of different media and areas. A soft sponge can be put on the needle tip to achieve the transfer of a thin liquid layer. The microneedle 141 can be specifically modified, such as being modified with PDMS to increase its surface hydrophilicity or hydrophobicity, which can increase its ability to adhere to specific substances. The needle tip can also be ground flat to form a specific size area. This will significantly improve the problem that the dipping area of the traditional fixed needle tip is uneven due to the uneven contact force. More importantly, we provide a movable cantilever needle assembly with low cost and easy installation. The Z-axis positioning mechanism is extremely significantly simplified. In high-frequency sampling, the collected samples can be contact-printed simultaneously instead of being executed individually each time, which will greatly improve the micro-dipping speed and efficiency of different substances on complex interfaces.

[0033] As Figure 3 shown. In some embodiments of the present application, the contact microneedle module includes a microneedle 141 and a support tube 143. The support tube 143 is sleeved outside the microneedle 141. The upper end of the microneedle 141 has a bent portion. The upper end of the support tube 143 has an open end. The cross-sectional contour of the open end is O-shaped, and the longitudinal-sectional contour is V-shaped. The tip of the microneedle 141 is sheet-shaped or water-drop-shaped. The bent portion of the microneedle 141 abuts against the open end of the support tube 143, and the microneedle 141 can move upward relative to the support tube 143.

[0034] The upper end of the support tube 143 has an open end, and the open end is set as an O or V-shaped notch. The tip of the microneedle 141 is circular, annular, horizontally sheet-shaped or water-drop-shaped. The connecting end of the microneedle 141 is bent and connected to the open end of the support tube 143. When the microneedle 141 moves downward, the bent portion will hang on the open end to prevent the microneedle 141 from slipping out of the support tube 143. However, the support tube 143 does not restrict the upward movement of the microneedle 141.

[0035] In some embodiments of the present application, the contact microneedle module 14 further includes a coil 144. The material of the coil 144 includes metal or plastic. The coil 144 is wound around the periphery of the microneedle 141, and the length of the coil 144 is changed to control the magnitude of the sum of the gravity of the coil and the microneedle 141.

[0036] In an alternative embodiment of the present application, the contact microneedle module 14 further includes magnetic particles 145. The coil 144 is an inductor coil 144. The inductor coil 144 is wound around the periphery of the microneedle 141 and is located inside the support tube 143. The magnetic particles 145 are filled between the support tube 143 and the microneedle 141.

[0037] In some embodiments of the present application, the contact microneedle module 14 further includes a microporous wafer 146. The microporous wafer 146 is sleeved outside the microneedle 141, and the microporous wafer is located above the needle cap 142.

[0038] The core of the device is to provide a gentle contact with the surface target through the microgravity of the microneedle 141, so as to achieve ultra-micro sampling and transfer of trace surface samples. The weight of the needle cap 142 can be controlled by stacking the microporous wafers 146, and the weight of the microneedle 141 itself can energize the inductor coil 144. The surface of the microneedle 141 has magnetism to adsorb the magnetic particles 145 carried by the support tube 143. At this time, the force exerted by the microneedle 141 on the target is the sum of the weights of the microneedle 141, the needle cap 142, and the magnetic particles 145. By adjusting the magnetic magnitude of the inductor coil 144, the weight of the adsorbed magnetic particles 145 can be changed, thereby changing the magnitude of the force exerted by the microneedle 141.

[0039] The support tube 143 and the microneedle 141 are high-temperature resistant metal components, and can be moved to a specified position through a three-axis controller during the dipping of organic samples such as microorganisms, and can be subjected to aqueous solution cleaning, high-temperature sterilization treatment, etc.

[0040] In some embodiments of the present application, multiple contact microneedle modules 14 are arranged in an array to improve the speed of picking bacteria.

[0041] The three-dimensional control module 13 includes a three-axis controller, and each contact microneedle module 14 is independently connected to the three-axis controller.

[0042] Continuous inoculation and control of microneedles 141. Fix the above-mentioned needle assembly on a retractable controller and connect it to three control systems. Through the movement of the XYZ axes, the coordinate movement of the microneedles 141 is achieved. The movement on the Z axis only needs to consider the contact within the effective thread range, and without special settings, it can meet the contact problems of extremely complex interfaces. Further, multiple (4 - 30) of these microneedle 141 assemblies can be combined side by side to achieve batch and high-throughput combination of the needle tips, significantly improving the efficiency of sample dipping and transfer. By moving the coordinates, contact at different positions can be achieved without additional attention to changes in the reverse direction of the Z axis. This will significantly reduce the design and manufacturing difficulty of surface sampling and significantly improve the accuracy and efficiency of interface sampling.

[0043] The robotic arm can be single or can form a single-row or multi-row array. Through the three-dimensional controller, the contact needle is pushed out and restored up and down to make a certain microneedle 141 protrude for individual microneedle 141 to perform contact sampling. Or, by using the method of the robotic arm protruding up and down, sequential protrusion and sampling of the microneedles 141 are achieved.

[0044] The robotic arm assembly is detachably fixed on the three-axis controller, and the other end is connected to the support tube 143. During the movement of the robotic arm, by controlling the forward and backward or up and down movement of the end of a single robotic arm, the microneedles 141 on a single robotic arm protrude, and then through moving the three-axis system to reach the designated location to complete the gentle contact of the microneedles 141. In addition, controls are set between the robotic arms to achieve control of the spatial distance during the contact printing of the microneedles 141.

[0045] Multi-mode motion control. Through auxiliary designs such as machine vision, after completing the recognition of objects on the surface, it can achieve extensive dipping and re-inoculation of surface substances (such as microorganisms, liquids or powders, etc.), and can complete multi-mode material taking, transferring, printing, combination, etc. such as solid-liquid and liquid-liquid.

[0046] Integrated system of needle combinations. Further, we can further increase the throughput and efficiency of the sampling needle through the combination of needle assemblies. Options of 8 groups and 12 groups are available to meet the rapid high-throughput combined dipping of 96-well plates.

[0047] The present invention adopts movable microneedles 141, without the need for complex Z-axis control equipment, greatly overcoming the high difficulty and complexity of difficult-to-control contact force in the traditional contact needle process. It is easy to operate and simple to manufacture. It can achieve high-throughput rapid contact and dipping at the millinewton level. These microneedles 141 are thinner than traditional inoculation needles, can provide higher-resolution contact, are easier to clean during the cleaning and disinfection process, and have less contamination. In short, our system has low cost, simple and efficient functions, and is suitable for the high-throughput rapid surface dipping requirements of trace substances.

[0048] In some embodiments of the present application, the machine vision module includes a large field of view camera and a small field of view camera, and the field of view of the large field of view camera covers the field of view of the small field of view camera.

[0049] The large market camera is used for rapid positioning to guide the three-axis movement to the target colony; The small field of view camera captures the target colony, further adjusts the microscopic position and completes precise picking, and is used for precise identification and positioning fine-tuning of the microscopic area.

[0050] Optionally, the tip of the contact needle can be within the small field of view camera. By the principles of similarity and minimum distance, the target bacterium is locked, and the coordinate fine-tuning correction of the absolute position is completed.

[0051] In a second aspect, the present application provides a method for picking and culturing tiny microbial targets, as Figure 4 shown, Figure 4 is a schematic flowchart of a method for picking and culturing tiny microbial targets provided by an embodiment of the present application.

[0052] A method for picking and culturing tiny microbial targets includes the following steps: S1. The machine vision module 11 identifies and locates the tiny microbial colony; S2. The pattern design module 12 designs the microbial distribution pattern; S3. The three-dimensional control module 13 drives the contact micro-needle module 14 to flexibly dip the microbial colony and transfer it to the target area.

[0053] Through positioning and identification, the present application can capture randomly distributed microbial colonies; through flexible dipping, it can capture complex, variable, undulating bacterial bodies with different textures, viscosities, and sizes, and achieve flexible control in terms of inoculation force and height control. The number of cells dipped each time is small, the resolution is high, the efficiency is high, and the granularity of the patterning is higher.

[0054] Identifying microbial colonies: Image recognition technology can be used, combined with machine learning algorithms, to automatically identify and classify microbial colonies.

[0055] Designing the microbial distribution pattern: According to experimental requirements, a specific microbial distribution pattern is designed for subsequent cultivation and application.

[0056] Transferring to the target area: Using a precision robotic arm and dipping device, precise positioning and flexible dipping of the microbial colony are achieved. The dipped microbial colony is transferred to the target area according to the designed pattern, such as a culture dish, a chip, etc.

[0057] By identifying microbial colonies and designing microbial distribution patterns, the distribution of microorganisms can be customized according to different culture requirements, improving the flexibility of the selection and culture method. Locating and flexibly dipping the microbial colonies can, while ensuring cell viability, precisely control the inoculation force and height, resulting in a small number of cells being dipped each time and a high resolution. This method is applicable to complex and variable, undulating microbial cells, as well as microorganisms with different textures, viscosities, and sizes, greatly improving the adaptability of the selection and culture method. Through the patterned inoculation method, the inoculation efficiency is improved, and at the same time, the granularity of the microbial distribution is higher, which is beneficial for subsequent experimental operations and analysis.

[0058] In some embodiments of the present application, the machine vision module 11 identifies and locates minute microbial colonies, including: The large field-of-view camera quickly locates the microbial colonies and guides the three-dimensional control module 13 to move above the microbial colonies; The small field-of-view camera accurately identifies the microbial colonies through the principles of similarity and minimum distance and guides the contact microneedle module 14 for fine-tuning and correction.

[0059] In some embodiments of the present application, the three-dimensional control module 13 drives the contact microneedle module to flexibly dip the microbial colonies and transfer them to the target area, including: Collecting the image of the microbial colonies; Identifying the type of the microorganism; Generating the microbial distribution pattern; Performing digital coordinate conversion through the image and machine vision; Planning the movement trajectory of the contact microneedle module 14; Outputting a movement signal to the three-dimensional control module 13.

[0060] Collecting the image of the microbial colonies: Using a high-resolution camera or microscope to capture the image of the microbial colonies. The image acquisition can be static or dynamic to obtain detailed information of the colonies, such as morphology, size, color, etc.

[0061] Identifying the type of the microorganism: Analyzing the collected image using image processing techniques and machine learning algorithms. Through feature extraction, such as edge detection, color analysis, texture analysis, etc., the microbial colonies are classified and identified. A database containing the characteristics of various microorganisms can be established to assist in the identification process.

[0062] Generating the microbial distribution pattern: Designing a specific microbial distribution pattern according to the identified microorganism type and experimental requirements. The design software can generate the pattern according to preset rules or user-defined parameters to ensure that the pattern meets the experimental requirements.

[0063] By automating the acquisition and analysis of images, manual operations are reduced, and the accuracy and repeatability of the experiment are improved. The high-precision image recognition technology can accurately identify the types of microorganisms, providing a reliable basis for subsequent experiments. Designing specific distribution patterns according to the microorganism types and experimental purposes can better control the experimental conditions and results.

[0064] Through machine vision and three-dimensional coordinate programming control, the contact needle is moved to the specified position and then moved up and down to achieve a controllable force contact and adhesion between the contact needle and the target surface, complete the gentle dipping of the micro target, and then transfer it to other interface positions to achieve the controllable gentle contact transfer and control of the target.

[0065] Obtain the three-dimensional coordinates of the microbial colony: Use a machine vision system to identify the position of the microbial colony through image processing technology and calculate its coordinates in three-dimensional space. This usually involves image acquisition from multiple angles and complex algorithm processing to reconstruct the three-dimensional position of the colony.

[0066] Horizontally move the contact micro-needle module 14 according to the three-dimensional coordinates: After receiving the three-dimensional coordinate information, the control system commands the robotic arm or the moving platform to accurately horizontally move the contact micro-needle module 14 directly above the microbial colony. The accuracy of the horizontal movement needs to be very high to ensure that the contact needle can accurately align with the target colony.

[0067] Vertically move the contact micro-needle module 14 to gently dip the target: After the contact needle reaches the specified position, the control system will vertically move the contact micro-needle module 14 to gently touch the surface of the colony. By finely controlling the contact force, gentle dipping is achieved, that is, the microorganism is adhered to the contact needle without damaging the colony structure.

[0068] The combination of obtaining three-dimensional coordinates and machine vision greatly improves the positioning accuracy of microbial colonies. By programming to control the vertical movement of the contact needle, the dipping force can be precisely controlled to avoid damaging the microorganisms. The flexible design of the contact needle can adapt to microbial colonies of different shapes and hardnesses, ensuring gentle treatment of the microorganisms during the dipping process. This method is applicable to microbial colonies of different sizes, shapes, and viscosities, with wide applicability.

[0069] Perform digital coordinate conversion through images and machine vision: Use a high-resolution camera or microscope to capture the two-dimensional image of the microbial colony. Utilize machine vision technology, including image processing and pattern recognition algorithms, to analyze the image and identify the specific position of the colony. Through techniques such as feature point matching, stereo vision, or structured light, convert the position of the colony in the two-dimensional image into coordinates in three-dimensional space. This conversion involves complex mathematical models and algorithms to ensure the accuracy of the coordinate conversion.

[0070] Plan the movement trajectory of the contact microneedle module 14 based on digital coordinates: Once the three-dimensional coordinates of the microbial colony are obtained, the control system will plan the movement trajectory of the contact microneedle module 14 according to these coordinates. The trajectory planning takes into account the starting position, target position of the contact needle, and possible obstacles to ensure the optimization of the movement path. During the planning process, the movement speed, acceleration of the contact needle, and any possible vibrations are also considered to ensure the smoothness and accuracy of the operation.

[0071] The position of the microneedle 141 is digitally coordinate-converted through camera photography and machine vision, and then transmitted to the host computer for specifying and screening the spatial movement positions of the needle arrangement on the three-axis controller. Through software control, the individual protrusion of each single needle is controlled to contact the specified position surface to complete the dipping of the needle tip. Then it is transferred to the set interface for controllable contact to achieve simultaneous printing of multiple targets, which doubles the contact efficiency compared with the traditional single-needle fixed contact.

[0072] Machine vision and digital processing technology can provide high-precision coordinate conversion to ensure that the microbial colony can be accurately positioned. Automated trajectory planning reduces human error and improves the accuracy and repeatability of experimental operations. By pre-planning the movement trajectory of the contact needle, the operation efficiency can be significantly improved and the experimental preparation time can be reduced.

[0073] In some embodiments of the present application, the three-dimensional control module drives the contact microneedle module 14 to flexibly dip the microbial colony and transfer it to the target area, including: In the initial state, the open end of the support tube 143 bears the microneedle 141 so that the microneedle 141 is in a suspended state; In the sampling state, the robotic arm drives the microneedle 141 to move to a preset position, the tip of the microneedle 141 is lifted by the microbial colony, and the microneedle 141 is displaced upward relative to the support tube 143; The robotic arm moves upward, the microneedle 141 is displaced downward relative to the support tube 143, and the relative position between the microneedle 141 and the support tube 143 returns to the initial state.

[0074] When the needle is fixed, under the action of gravity, the notch of the support tube 143 always fixes the needle in a suspended state at a fixed position, maintaining the relative stability of the needle tip position. When sampling, the needle tip is lifted by contact with the colony, the needle moves upward in the support tube 143, and when the robotic arm is lifted, the needle falls in the support tube 143. The V-shaped tube of the support tube 143 will restrict the movement of the needle in the tube and finally return to the lowest original position, so as to ensure that during each sampling process, the sampling microneedle 141 is at the same position, ensuring the dipping accuracy of the microneedle 141.

[0075] In addition, more importantly, after sampling and inoculation are completed, sterilization is a crucial process. By changing the needle tip to a dome-shaped sheet structure, the accuracy of inoculating bacteria can be significantly increased. At the same time, the time for flame sterilization can be significantly reduced, and it can quickly cool down in the flame for the next inoculation of bacteria.

[0076] In some embodiments of the present application, vertically moving the contact microneedle module 14 to flexibly dip the target includes: Controlling the individual protrusion of the contact microneedle module 14; Adjusting the single-needle contact force of the contact microneedle module 14.

[0077] Controlling the individual protrusion of the contact microneedle module 14 according to the digital coordinates: Using the instructions of the control system, according to the previously obtained three-dimensional digital coordinates, precisely control the vertical movement of the contact microneedle module 14. Each needle in the contact microneedle module 14 may protrude individually or as a unit one by one to approach or contact the microbial colony. This way of controlling the individual protrusion can ensure that each needle can accurately reach the predetermined position, and the force can be adjusted individually.

[0078] Adjusting the single-needle contact force of the contact microneedle module 14: When the contact microneedle module 14 approaches the microbial colony, the control system will adjust the contact force of each needle according to the characteristics of the colony and the required dipping force. This involves delicate force control techniques, such as using piezoelectric actuators or other precision force control devices to achieve high-precision control of the force. Adjusting the contact force can prevent damage to the microbial colony while ensuring sufficient adhesion force to complete the dipping process.

[0079] Through the individual protrusion and separate adjustment of the contact force, a very precise and gentle dipping of the microbial colony can be achieved, avoiding damage to cells. The flexible dipping method helps to maintain the activity of microbial cells because the force can be precisely controlled, avoiding excessive force from damaging the cell structure. Since the single-needle contact force can be adjusted, this method can adapt to microbial colonies with different hardness, viscosity, and size.

[0080] Compared with the prior art, the beneficial technical effects brought by the technical solution provided by the present application include: Through positioning and recognition, the present application can capture randomly distributed microbial colonies; through flexible dipping, it can capture complex, variable, undulating bacteria with different textures, viscosities, and sizes, and can achieve flexible control in terms of inoculation force and height control. The number of cells dipped each time is small, with high resolution, high efficiency, and higher patterning granularity.

[0081] Those skilled in the art of the present technology can understand that the various operations, methods, steps, measures, and solutions in the processes discussed in the present application can be alternated, changed, rearranged, decomposed, combined, or deleted.

[0082] The specific embodiments of the present application described above do not constitute a limitation on the protection scope of the present application. Any other corresponding changes and deformations made according to the technical concept of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. A selection and culture system for tiny microbial targets, characterized in that Comprising: A machine vision module for identifying and positioning minute microbial colonies; A pattern design module for designing microbial distribution patterns; A contact microneedle module for dipping and transferring the minute microbial colonies; A three-dimensional control module for moving the contact microneedle module.

2. The a target selection and culture system for minute microorganisms according to claim 1, wherein The contact microneedle module includes a microneedle, a needle cap, and a support tube. The needle cap and the support tube are respectively sleeved outside the microneedle. The support tube is located below the needle cap, and the outer diameter of the needle cap is greater than the inner diameter of the support tube.

3. The a target selection and culture system for minute microorganisms according to claim 1, wherein, The contact microneedle module includes a microneedle and a support tube. The support tube is sleeved outside the microneedle. The upper end of the microneedle has a bent portion. The upper end of the support tube has an opening. The cross-sectional profile of the opening is O-shaped, and the longitudinal-sectional profile is V-shaped. The tip of the microneedle is circular, annular, horizontally flaky, or water-drop-shaped. The bent portion of the microneedle abuts against the opening of the support tube, and the microneedle can move upward relative to the support tube.

4. A culturing system for selecting a target of minute microorganisms according to claim 1 or 2, characterized in that The contact microneedle module further includes a coil. The material of the coil includes metal or plastic. The coil is wound around the periphery of the microneedle or the needle cap, and the length of the coil is changed to control the magnitude of the sum of the gravity of the coil and the microneedle.

5. The pick-and-culture system for tiny microorganism targets according to claim 1, characterized in that, The machine vision module includes a large-field camera and a small-field camera. The field of view of the large-field camera covers the field of view of the small-field camera.

6. The pick and culture system for minute microorganism targets according to claim 5, wherein, A plurality of the contact microneedle modules are arrayed. The three-dimensional control module includes a three-dimensional controller. The three-dimensional controller is independently connected to each contact microneedle module, and the contact microneedle module is signal-connected to the small-field camera.

7. A method for selectively culturing minute microbial targets, characterized in that, Adopting the culturing system for selecting minute microbial targets as described in any one of claims 1 to 6, comprising the following steps: The machine vision module identifies and positions minute microbial colonies; The pattern design module designs microbial distribution patterns; The three-dimensional control module drives the contact microneedle module to flexibly dip the microbial colonies and transfer them to the target area.

8. A method for selectively culturing a target of minute microorganisms according to claim 7, characterized in that, The machine vision module identifies and positions minute microbial colonies, including: The large-field camera quickly locates the microbial colonies and guides the three-dimensional control module to move above the microbial colonies; The small-field camera accurately identifies the minute microbial colonies through the similarity and minimum distance principles and guides the contact microneedle module to fine-tune and correct.

9. A method for selecting and culturing a target of minute microorganisms according to claim 7, characterized in that, The three-dimensional control module drives the contact microneedle module to flexibly dip the microbial colonies and transfer them to the target area, including: Receiving the digital coordinates converted by the machine vision module; Planning the movement trajectory of the contact microneedle module; Outputting a movement signal to the three-dimensional control module.

10. A method for selecting and culturing a target of minute microorganisms according to claim 7, characterized in that, The three-dimensional control module drives the contact microneedle module to flexibly dip the microbial colonies and transfer them to the target area, including: In the initial state, the opening of the support tube bears the microneedle so that the microneedle remains in a suspended state; In the sampling state, the robotic arm drives the microneedle to move to a preset position. The tip of the microneedle is lifted by the microbial colonies, and the microneedle moves upward relative to the support tube; The robotic arm moves upward, the microneedle moves downward relative to the support tube, and the relative position between the microneedle and the support tube returns to the initial state.

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