A method for synergistic double-needle injection into the yolk sac of zebrafish juveniles based on posture analysis.

Abstract

This invention relates to a dual-needle synergistic injection method for the yolk sac of zebrafish juveniles based on posture analysis, belonging to the field of zebrafish injection. The method comprises the following steps: Step 1) Arranging zebrafish regularly in a culture dish covered with agarose; Step 2) Ensuring the left and right needles are symmetrical; Step 3) Moving the stage to bring the zebrafish into the microscopic field of view, automatically determining the tail direction based on target detection results; Step 4) Moving the zebrafish yolk sac into the microscopic field of view and to the injection position; Step 5) Processing the target detection results and automatically determining the yolk sac direction; Step 6) If the yolk sac faces right, the injection is performed from right to left using the right needle; if the yolk sac faces left, the injection is performed from left to right using the left needle; Step 7) Moving the stage to the next zebrafish. This invention combines target detection and posture analysis, going beyond simply adjusting the zebrafish to a uniform posture to achieve more efficient and flexible automated batch injection.

Description

Technical Field

[0001] This invention belongs to the field of zebrafish injection, specifically relating to a method for dual-needle synergistic injection of yolk sac in zebrafish juveniles based on posture analysis. Background Technology

[0002] Zebrafish share a high degree of genetic similarity with humans, making them important model organisms frequently used in biomedical research. Common sites for microinjection in zebrafish include the yolk sac, main vein, caudal vein, and heart. Among these, yolk sac injection is the simplest and fastest, making it an ideal injection site for achieving highly efficient, automated batch injections.

[0003] However, in traditional automated batch injection, all zebrafish typically need to maintain the same posture and be injected along the same path. Therefore, manual arrangement of the zebrafish is required before injection, involving not only placing them in the grooves but also repeatedly flipping them. This flipping operation is often time-consuming and labor-intensive, and can easily damage the zebrafish. Operators need extensive experience, and arranging a single fish can take longer than the injection itself, significantly hindering the overall efficiency of batch injection. Therefore, designing an automated injection method with greater posture tolerance is essential. Summary of the Invention

[0004] This invention addresses the technical problems existing in the prior art by providing a dual-needle synergistic injection method for the yolk sac of zebrafish juveniles based on posture analysis. This invention combines target detection and posture analysis, and is not limited to adjusting zebrafish to a uniform posture, but achieves more efficient and flexible batch automated injection.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution: a method for synergistic double-needle injection of yolk sac in zebrafish juveniles based on attitude analysis, which is carried out according to the following steps:

[0006] Step 1) Arrange the zebrafish regularly in a petri dish covered with agarose, and let them lie on their sides randomly in four positions: tail up, tail down, yolk sac facing left, and yolk sac facing right.

[0007] Step 2) Initialize the two needles on the left and right sides according to the height of the yolk sac to make them symmetrical;

[0008] Step 3) Move the stage according to the set path so that the zebrafish appears in the microscopic field of view, perform target detection on the microscopic image, and automatically determine the direction of the zebrafish's tail based on the target detection results;

[0009] Step 4) The stage is automatically adjusted according to the direction of the tail to move the zebrafish yolk sac into the microscopic field of view and to the injection site;

[0010] Step 5) Process the target detection results and automatically determine the direction of the yolk sac;

[0011] Step 6) If the yolk sac is facing right, the injection is completed from right to left using the right needle; if the yolk sac is facing left, the injection is completed from left to right using the left needle.

[0012] Step 7) Move the platform to the next zebrafish.

[0013] Preferably, in step 1), the zebrafish are arranged in regular grooves and lie on their sides in random postures. A coordinate system is established with the fish's body along the Y-direction. Based on the direction of the tail and yolk sac, four postures are identified: tail facing positive y-direction, yolk sac facing positive x-direction; tail facing positive y-direction, yolk sac facing negative x-direction; tail facing negative y-direction, yolk sac facing positive x-direction; and tail facing negative y-direction, yolk sac facing negative x-direction. This arrangement allows for multiple postures, reducing the need for manual arrangement and thus significantly reducing the time required for manual placement.

[0014] Preferably, in step 2), the needle tip contacts the outer epidermis of the yolk sac for height positioning; the yolk sac of any zebrafish is positioned in the center of the microscopic field of view, and the needle tip descends vertically from the center of the microscopic field of view. After contacting the epidermis and forming a visible deformation, the right needle is withdrawn a certain distance along the upper right, and the left needle is withdrawn a certain distance along the upper left.

[0015] Preferably, the specific method for determining the direction of the zebrafish's tail in step 3) is as follows:

[0016] Based on the arrangement of the grooves, each movement is a fixed distance so that the upper part of the agarose groove appears in the microscopic field of view. At this time, the zebrafish appears in the microscopic field of view. The zebrafish's tail is facing upward or downward in the field of view. Two situations will appear in the microscopic field of view: only the tail or the yolk sac and part of the tail. Dual-target detection can determine these two situations, thereby determining the direction of the zebrafish's tail.

[0017] Preferably, in step 4), when the tail is facing down, the yolk sac is already in the microscopic field of view; when the tail is facing up, the yolk sac will not appear in the microscopic field of view. At this time, it is necessary to continue moving the stage along the direction of the tail so that the lower half of the groove appears in the microscopic field of view, thereby making the yolk sac appear in the microscopic field of view. In order to be compatible with the four postures, the center of the yolk sac is selected as the injection point, and the yolk sac is moved to the position to be injected, that is, the stage is moved so that the point in the target frame of the yolk sac is located in the center of the microscopic field of view.

[0018] Preferably, the method for determining the direction of the yolk sac in step 5) is as follows:

[0019] Of the four poses of zebrafish, two have the yolk sac facing the positive x-direction and two have the yolk sac facing the negative x-direction, which correspond to two situations in the microscopic field of view: the tail is to the left of the yolk sac and the tail is to the right of the yolk sac. Therefore, by determining the relative position of the two target boxes, the orientation of the yolk sac can be determined.

[0020] Preferably, in step 7), the stage is moved according to the zebrafish arrangement, which can quickly bring the next fish into the microscopic field of view.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0022] 1. This invention reduces the requirements for the zebrafish's posture, unlike the fixed posture required for conventional injections. It accommodates many different postures, significantly reducing the time spent on manual zebrafish placement. In previous automated injection experiments, experienced operators spent approximately 15 seconds placing a single zebrafish. This invention only requires placing the zebrafish in the groove, requiring less operator experience, and reducing placement time to less than 6 seconds. This greatly reduces the overall automated injection process time and improves injection efficiency.

[0023] 2. This invention employs a dual-needle synergistic injection method, where the two injection needles do not interfere with each other. Combined with stage movement, target detection, and posture determination, it achieves automated injection of zebrafish in batches, capable of injecting zebrafish in four different postures, offering high flexibility. Every step of this invention is automated, resulting in a smooth and highly efficient injection process. Attached Figure Description

[0024] Figure 1 This is a flowchart of the present invention.

[0025] Figure 2 This describes the arrangement of zebrafish in this invention.

[0026] Figure 3 This describes the establishment of the coordinate system in this invention.

[0027] Figure 4 This is the platform movement planning in this invention.

[0028] Figure 5 This is the result of the stage movement in this invention.

[0029] Figure 6 This is the result of attitude determination in this invention.

[0030] Figure 7 This is the needle tip movement path of the present invention.

[0031] Figure 8 This is the injection result in this invention. Detailed Implementation

[0032] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0033] This invention discloses a method for synergistic double-needle injection of yolk sac into zebrafish larvae based on posture analysis. The method uses wild-caught AB-type zebrafish, with embryos obtained through natural spawning. The zebrafish embryos are cultured in water at 28°C in E3 medium. Experiments are conducted when the larvae reach 3 days post-flop (dpf). The procedure is as follows: Figure 1 As shown, it includes the following steps:

[0034] Step 1) Placing the zebrafish:

[0035] Zebrafish were arranged in regular grooves, lying randomly on their sides in four positions: tail up, tail down, yolk sac facing left, and yolk sac facing right. Figure 2 As shown. Establish a coordinate system, as follows. Figure 3 As shown, the fish body is along the y-direction, and the yolk sac is along the x-direction. Based on the direction of the tail and the yolk sac, there are four postures. Posture 1 is when the tail faces the positive y-direction and the yolk sac faces the positive x-direction; Posture 2 is when the tail faces the positive y-direction and the yolk sac faces the negative x-direction; Posture 3 is when the tail faces the negative y-direction and the yolk sac faces the positive x-direction; and Posture 4 is when the tail faces the negative y-direction and the yolk sac faces the negative x-direction.

[0036] Step 2) Planning the movement of the stage: Initialize the two needles on the left and right sides according to the height of the yolk sac to make them symmetrical;

[0037] Step 3) Move the stage along the set path to bring the zebrafish into the microscopic field of view, perform target detection on the microscopic image, and automatically determine the direction of the zebrafish's tail based on the target detection results; for example... Figure 4 As shown:

[0038] In this embodiment, according to the arrangement rules of the agarose grooves, the stage moves 5300 nanometers each time, moving from the upper half of one groove to the upper half of the next. Since the zebrafish tail may face either the positive or negative y-direction, each time it moves to a new groove, two situations will appear in the microscopic field of view: when the tail faces the negative y-direction, the yolk sac and a small portion of the tail will be visible, such as... Figure 5 (a); When the tail points in the positive y direction, only the tail appears, such as Figure 5 (c) The first case corresponds to zebrafish poses 3 and 4, and the second case corresponds to zebrafish poses 1 and 2. Target detection is performed using Yolov7. If the yolk sac and tail are detected, it is the first case; if only the tail is detected, it is the second case.

[0039] Step 4) The stage is automatically adjusted according to the tail direction to move the zebrafish yolk sac into the microscopic field of view and to the injection site.

[0040] In the second scenario, if the stage continues to move 1500 nanometers, bringing the lower half of the groove into view, the first scenario will occur, as shown below. Figure 5 (d) Move the yolk sac to the center of the field of view, such as... Figure 5 (b) and 5(e), and record the positions of the detected yolk sac and tail target boxes.

[0041] Step 5) Posture assessment and needle selection, such as Figure 6 As shown:

[0042] Since the yolk sac may be facing either the positive or negative x-direction, two scenarios will appear in the field of view: when the yolk sac is facing the positive x-direction, the tail will be on the left side of the yolk sac; when the yolk sac is facing the negative x-direction, the tail will be on the right side of the yolk sac. The first scenario corresponds to zebrafish poses 1 and 3, and the second scenario corresponds to zebrafish poses 2 and 4. Calculate the center coordinates of the two target boxes and determine their relative positions based on their size. If the tail is on the left side of the yolk sac, select right needle injection. Figure 6 (a) and 6(c); if the tail is on the right side of the yolk sac, choose left-hand injection, such as Figure 6 (b) and 6(d).

[0043] Step 6) Injection point selection and needle tip movement path, such as Figure 7 As shown:

[0044] To accommodate four different postures, the center of the yolk sac was chosen as the injection point. In all four postures, the injection point is the midpoint of the yolk sac target box, indicated by the asterisk in the diagram, allowing for quick and easy detection. Experiments confirmed that choosing the center of the yolk sac as the injection point makes microneedle insertion easier and minimizes the impact on the heart. The stage had already been moved to center the yolk sac in the field of view, ensuring the injection point was located at the center. The two injection needles used in the experiment were symmetrical and worked collaboratively. Before injection, the needle tip position needed to be initialized based on the height of the yolk sac. The yolk sac of any zebrafish was positioned in the center of the microscopic field of view, while the needle tip descended vertically from the center. After the needle tip contacted the epidermis at P0 and formed a visible deformation, the right needle was withdrawn to point P1 along the upper right, and the left needle was withdrawn to point P1 along the upper left, serving as the initial positions for both needles. The needle tip contacted the epidermis at the height positioning point P0, moved 600 micrometers along the x-direction, and 340 micrometers along the y-direction to reach the initial point p1, completing the height positioning. Then, it moves 600 micrometers along both the x and y directions, from point P1 through the insertion point P2 to the injection point P3. After injection, the microneedle will be withdrawn along the reverse path.

[0045] Step 7) Continue moving the stage according to the zebrafish arrangement to quickly bring the next fish into the microscopic field of view.

[0046] Injection results as follows Figure 8 As shown, in this embodiment, the microneedle used has an inner diameter of 8 micrometers and an outer diameter of 10 micrometers. Physiological saline is injected into the yolk sac of zebrafish. The injection pulse time is 0.2s and the injection pressure is 500hPa. Figure 8 The left side shows the result before injection. Figure 8 The right side shows the injection site, where a clump of substance, which is saline solution, is visible after the injection.

[0047] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for synergistic double-needle injection of yolk sac into zebrafish juveniles based on attitude analysis, characterized in that: Follow these steps: Step 1) Arrange the zebrafish regularly in a petri dish covered with agarose, and let them lie on their sides randomly in four positions: tail up, tail down, yolk sac facing left, and yolk sac facing right. In step 1), the zebrafish are arranged in regular grooves and lie on their sides in random postures. A coordinate system is established with the fish body along the Y direction. Based on the direction of the tail and the yolk sac, there are four postures: tail facing the positive y direction and yolk sac facing the positive x direction; tail facing the positive y direction and yolk sac facing the negative x direction; tail facing the negative y direction and yolk sac facing the positive x direction; and tail facing the negative y direction and yolk sac facing the negative x direction. Step 2) Initialize the two needles on the left and right sides according to the height of the yolk sac to make them symmetrical; In step 2), the needle tip contacts the outer epidermis of the yolk sac to perform height positioning; the yolk sac of any zebrafish is positioned in the center of the microscopic field of view, and the needle tip descends vertically from the center of the microscopic field of view. After contacting the epidermis and forming a visible deformation, the right needle is withdrawn a certain distance along the upper right, and the left needle is withdrawn a certain distance along the upper left. Step 3) Move the stage along the set path to bring the zebrafish into the microscopic field of view, perform target detection on the microscopic image, and automatically determine the direction of the zebrafish's tail based on the target detection results; The specific method for determining the direction of the zebrafish's tail in step 3) is as follows: Based on the arrangement of the grooves, each movement is a fixed distance so that the upper part of the agarose groove appears in the microscopic field of view. At this time, the zebrafish appears in the microscopic field of view. The zebrafish's tail is facing upward or downward in the field of view. Two situations will appear in the microscopic field of view: only the tail or the yolk sac and part of the tail. Dual-target detection is performed to judge these two situations, thereby determining the direction of the zebrafish's tail. Step 4) The stage is automatically adjusted according to the direction of the tail to move the zebrafish yolk sac into the microscopic field of view and to the injection site; In step 4), when the tail is facing down, the yolk sac is already in the microscopic field of view; When the tail is facing upwards, the yolk sac will not appear in the microscopic field of view. At this time, it is necessary to continue moving the stage along the direction of the tail so that the lower half of the groove appears in the microscopic field of view, thereby making the yolk sac appear in the microscopic field of view. Select the center of the yolk sac as the injection point and move the yolk sac to the position to be injected, that is, move the stage so that the point in the target frame of the yolk sac is located in the center of the microscopic field of view. Step 5) Process the target detection results and automatically determine the direction of the yolk sac; Step 6) If the yolk sac is facing right, the injection is completed from right to left using the right needle; if the yolk sac is facing left, the injection is completed from left to right using the left needle. Step 7) Move the platform to the next zebrafish.

2. The method for dual-needle synergistic injection of zebrafish yolk sac based on attitude analysis according to claim 1, characterized in that: The method for determining the direction of the yolk sac in step 5) is as follows: Of the four poses of zebrafish, two have the yolk sac facing the positive x-direction and two have the yolk sac facing the negative x-direction, corresponding to two situations in the microscopic field of view: the tail is on the left side of the yolk sac and the tail is on the right side of the yolk sac. By judging the relative position of the two target boxes, the orientation of the yolk sac can be determined.

3. The method for dual-needle synergistic injection of zebrafish yolk sac based on attitude analysis according to claim 1, characterized in that: Step 7) Continue moving the platform according to the zebrafish arrangement.

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