阅读说明：本技术 一种机器人化的斑马鱼主静脉显微注射方法 (Robotized zebra fish main vein microinjection method ) 是由 孙明竹 李璐 赵新 姚亚彤 王一雯 龚慧颖 于 2021-01-25 设计创作，主要内容包括：本发明涉及一种机器人化的斑马鱼主静脉显微注射方法,包括以下步骤：S1,建立斑马鱼放置空间坐标系X-Y-Z；S2,通过光流法进行斑马鱼主静脉检测,以斑马鱼前主静脉与后主静脉的交点为目标注射点Q-t,确定目标注射点的空间坐标；S3,通过椭圆拟合的方式获取斑马鱼卵黄囊的长度L,并计算斑马鱼的脊柱厚度D；S4,设定注射针以目标注射点斜上方45°刺入,计算刺入点Q-p的空间坐标；S5,设定注射针的运动轨迹,完成斑马鱼主静脉注射。本发明所述机器人化的斑马鱼主静脉显微注射方法可以有效克服了人工显微注射方法中,存在的操作难度大以及易对斑马鱼造成伤害等问题。(The invention relates to a motorized zebra fish main vein microinjection method, which comprises the following steps: s1, establishing a zebra fish placing space coordinate system X-Y-Z; s2, detecting the main vein of the zebra fish by an optical flow method, and taking the intersection point of the front main vein and the rear main vein of the zebra fish as a target injection point Q t Determining the space coordinate of the target injection point; s3, obtaining the length L of the zebra fish yolk sac in an ellipse fitting mode, and calculating the thickness D of the spinal column of the zebra fish; s4, setting the injection needle to penetrate at 45 degrees obliquely above the target injection point, and calculating the penetration point Q p The spatial coordinates of (a); and S5, setting the motion track of the injection needle, and finishing the main vein injection of the zebra fish. The invention relates to a robot zebra fish ownerThe intravenous microinjection method can effectively solve the problems of great operation difficulty, easy damage to zebra fish and the like in the manual microinjection method.)

1. A motorized zebra fish main vein microinjection method is characterized by comprising the following steps:

s1, placing the zebra fish in a culture dish covered with agarose in a lateral posture, establishing a zebra fish placing space coordinate system X-Y-Z, and taking the fish body as the X direction and the fish tail as the X positive direction;

s2, detecting the main vein of the zebra fish by an optical flow method, and taking the intersection point of the front main vein and the rear main vein of the zebra fish as a target injection point Q_tSetting a target injection point Q_tIs consistent with the plane position of the origin of the space coordinate system, and the target injection point Q_tBy determining the vertical position within a spatial coordinate systemDetermining the height h of the center of the fish spine, wherein the space coordinate of the target injection point is Q_t＝[0 0 h]^T；

S3, obtaining the length L of the zebra fish yolk sac by means of ellipse fitting, and determining the spine thickness D of the zebra fish according to the proportional relation between the length L of the zebra fish yolk sac and the spine thickness D obtained by a calibration experiment;

s4, setting the injection needle to penetrate at 45 degrees obliquely above the target injection point, and setting the contact position of the injection needle and the zebra fish as a penetration point Q_pSpatial coordinate Q of the point of penetration_p＝[d 0 h+d]^TWherein D is half of the thickness D of the spine of the zebra fish;

s5, setting the movement track of the injection needle to make the needle point reach the puncture point Q from the initial position_pThen from the point of penetration Q_pMove to the target injection point Q_tAnd finishing the main vein injection of the zebra fish.

2. The method according to claim 1, wherein in step S2, the height h of the center of the spine of the zebra fish is the same as the height of the edge of the spine of the zebra fish in the microscopic field of view of the zebra fish lying on the side, the edge of the spine of the zebra fish and the injection needle are automatically focused by an energy gradient function, so that the edge of the spine of the zebra fish and the injection needle are at the same height, and the height of the edge of the spine of the zebra fish is obtained by obtaining the height information of the injection needle; wherein, the energy gradient function calculation formula is as follows:

i (x, y) is the intensity of the pixel at (x, y), and I (x +1, y) and I (x, y +1) are the intensities of the lower right corner neighbors, respectively.

3. The method for robotic main vein microinjection of zebrafish of claim 1, wherein in step S3, the proportional relationship between the length L of the yolk sac and the thickness D of the spine of zebrafish is calculated by a calibration experiment, and the calculation formula is:

wherein: n is the number of the calibrated zebra fish, and R is an average value calculated after summing the ratio of the yolk sac length L to the spinal column thickness D of the n zebra fish.

4. The method for robotic main vein microinjection of zebrafish of claim 3, wherein the specific method of the calibration experiment is as follows: keeping the zebra fish in a supine position, and obtaining the yolk sac length L of the zebra fish in an ellipse fitting mode, wherein the yolk sac length L is the length of the long axis of the ellipse obtained by fitting; two spinal column edges of the zebra fish are obtained through a straight line fitting mode, and the spinal column thickness D of the zebra fish is obtained through calibrating the distance between the two spinal column edges.

5. The method for robotic zebrafish mainvein microinjection according to claim 1, wherein in step S5, the movement locus of the injection needle is set as follows:

(1) adjusting the needle tip of the injection needle to an initial position; the location and the target injection point Q_tX, Z are the same, the distance w from the target injection point in the Y direction, and the coordinate is marked as P₀＝[0 -w h]^T；

(2) Simultaneously moves a distance d along the positive directions of the X axis and the Z axis, and the needle point position of the injection needle is P₁＝[d -w h+d]^T；

(3) Move w along the positive direction of the Y axis to reach the puncturing point Q_pThe needle tip position is Q_p＝[d 0 h+d]^T；

(4) Moving the distance d to the X-axis and Z-axis negative directions at high speed, puncturing the general main vein, and reaching the target injection point Q_t＝[0 0 h]^T；

(5) Inject and withdraw the needle.

Technical Field

The invention relates to the field of microinjection, in particular to a motorized zebra fish main vein microinjection method.

Background

The zebra fish has a complex circulatory system similar to mammals due to high similarity of genes and human genes, is small in size and rapid in development, and is an important model organism for researching vascular biology, transgenic technology and pathogenesis of certain diseases.

At present, the mode of introducing exogenous substances into the body of the zebra fish is mainly to expose the zebra fish to a solution added with the exogenous substances, directly inject the exogenous substances into the zebra fish body by a microinjection technology, and the like. The microinjection is rapid and efficient, and can directly influence zebra fish, so the microinjection is widely applied to the directions of tumor xenograft, drug research and the like. Among the microinjection of zebrafish, the vascular injection is most effective. The injection can directly participate in blood circulation, and further quickly influence the development process of the zebra fish. Wherein, the main intravenous injection can simultaneously meet the requirements of minimum damage to the fish and the highest efficiency. However, the injection part is close to the head of the zebra fish, and high-precision injection is needed, so that the manual operation difficulty is high, the time consumption is long, the injection success rate is low, and the application of the technology is limited to a great extent. Therefore, a need exists for developing a robotic zebra fish main vein microinjection method to overcome the difficulties in manual microinjection and the damage to zebra fish.

Disclosure of Invention

Based on the defects existing in the prior zebra fish main vein microinjection adopting a manual operation mode, the invention provides a robotized zebra fish main vein microinjection method, which combines motion analysis and calibration of zebra fish spine thickness to perform online three-dimensional positioning on the key position of an injection needle in the puncture process, automatically performs three-dimensional path planning on the needle point of the injection needle before injection, and realizes the robotized zebra fish main vein injection.

The technical scheme adopted by the invention for solving the technical problems is as follows: a motorized zebra fish main vein microinjection method comprises the following steps:

s1, placing the zebra fish in a culture dish covered with agarose in a lateral posture, establishing a zebra fish placing space coordinate system X-Y-Z, and taking the fish body as the X direction and the fish tail as the X positive direction;

s2, detecting the main vein of the zebra fish by an optical flow method, and taking the intersection point of the front main vein and the rear main vein of the zebra fish as a target injection point Q_tSetting a target injection point Q_tIs consistent with the plane position of the origin of the space coordinate system, and the target injection point Q_tThe vertical position in a space coordinate system is determined by measuring the height h of the center of the spinal column of the zebra fish, and the space coordinate of the target injection point is Q_t＝[0 0 h]^T；

S3, obtaining the length L of the zebra fish yolk sac by means of ellipse fitting, and determining the spine thickness D of the zebra fish according to the proportional relation between the length L of the zebra fish yolk sac and the spine thickness D obtained by a calibration experiment;

s4, setting the injection needle to penetrate at 45 degrees obliquely above the target injection point, and setting the contact position of the injection needle and the zebra fish as a penetration point Q_pSpatial coordinate Q of the point of penetration_p＝[d 0 h+d]^TWherein D is half of the thickness D of the spine of the zebra fish;

s5, setting the movement track of the injection needle to make the needle point reach the puncture point Q from the initial position_pThen from the point of penetration Q_pMove to the target injection point Q_tAnd finishing the main vein injection of the zebra fish.

Further, in step S2, the height h of the center of the zebra fish spine is the same as the height of the zebra fish spine edge in the lateral microscopic field of view of the zebra fish, the zebra fish spine edge and the injection needle are automatically focused through an energy gradient function, so that the zebra fish spine edge and the injection needle are located at the same height, and the height information of the injection needle is obtained to obtain the height of the zebra fish spine edge; wherein, the energy gradient function calculation formula is as follows:

i (x, y) is the intensity of the pixel at (x, y), and I (x, +1, y) and I (x, y +1) are the intensities of the lower right corner neighbors, respectively.

Further, in step S3, the proportional relationship between the zebra fish yolk sac length L and the spine thickness D is calculated by a calibration experiment, and the calculation formula is as follows:

wherein: n is the number of the calibrated zebra fish, and R is an average value calculated after summing the ratio of the yolk sac length L to the spinal column thickness D of the n zebra fish.

Further, the specific method of the calibration experiment is as follows: keeping the zebra fish in a supine position, and obtaining the yolk sac length L of the zebra fish in an ellipse fitting mode, wherein the yolk sac length L is the length of the long axis of the ellipse obtained by fitting; two spinal column edges of the zebra fish are obtained through a straight line fitting mode, and the spinal column thickness D of the zebra fish is obtained through calibrating the distance between the two spinal column edges.

Further, in step S5, the movement locus of the injection needle is set as follows:

(1) adjusting the needle tip of the injection needle to an initial position; the location and the target injection point Q_tX, Z coordinates are the same, the distance w from the target injection point in the Y direction, and the coordinates are marked as P₀＝[0 -w h]^T；

(2) Simultaneously moves a distance d along the positive directions of the X axis and the Z axis, and the needle point position of the injection needle is P₁＝[d -w h+d]^T；

(3) Move w along the positive direction of the Y axis to reach the puncturing point Q_pThe needle tip position is Q_p＝[d 0 h+d]^T；

(4) Moving the distance d to the X-axis and Z-axis negative directions at high speed, puncturing the general main vein, and reaching the target injection point Q_t＝[0 0 h]^T；

(5) Inject and withdraw the needle.

Compared with the prior art, the invention has the following advantages and effects:

1. the robot zebra fish main vein microinjection method obtains the proportional relation between the zebra fish yolk sac length L and the spine thickness D through a calibration experiment before injection, and the spine thickness D of the zebra fish can be obtained by measuring the zebra fish yolk sac length L to be injected in the injection process. On one hand, the operation method solves the problem that the thickness of the lateral spine of the zebra fish cannot be directly obtained by a visual detection method in the injection process; on the other hand: when having avoided the injection, need keep this consuming time of dorsal position measurement backbone thickness D with the zebra fish, loaded down with trivial details operation process has effectively shortened the injection time, has improved injection efficiency.

2. The robotized zebra fish main vein microinjection method estimates the three-dimensional positions of key points such as a target injection point, a puncture point and the like in a multi-view image through motion analysis and calibration of spine thickness, quantifies the puncture position, and reduces the dependence on operators.

3. The zebra fish main intravenous injection method controls the micro-operation arm to carry out zebra fish main intravenous injection, and the process is robotized, so that the damage of manual operation to zebra fish is reduced, and the efficiency and repeatability of the zebra fish main intravenous injection are improved.

4. The invention realizes the online visual detection and path planning of the injection needle in the zebra fish blood vessel injection process, reduces the injection time and further improves the injection efficiency.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a flow chart of the steps of the robotic zebra fish main vein microinjection method of the present invention.

FIG. 2 shows a zebra fish placement space coordinate system X-Y-Z and an image coordinate system Xc-Yc-Zc.

Fig. 3 is a schematic view of a main vein plane position detection flow based on an optical flow method.

FIG. 4 is a calibration schematic diagram of zebra fish yolk sac length L and spine thickness D; FIG. 4(a) is a proportional relation between a calibration yolk sac length L and a vertebral thickness D of a zebra fish supine view diagram shot in advance in a calibration experiment; fig. 4(b) is a diagram in which the yolk sac length L is obtained by ellipse fitting in the zebra fish lateral view.

FIG. 5 is a schematic diagram of the principle explanation and detection of the height position of the main vein of zebra fish focused on the spinal column edge of zebra fish; fig. 5(a) is a microscopic image of zebrafish in lateral decubitus; FIG. 5(b) is a cross-sectional view of spinal anatomy of zebrafish; FIG. 5(c) energy gradient function during focusing; FIG. 5(d) is the sharpest ROI at the edge of the spine, which is also a focused image of the injection needle at the same height as the edge of the spine; fig. 5(e) is an image in which the ROI region at the edge of the spine is blurred.

Fig. 6 shows the position of the injection site and the puncture site of the zebrafish primary intravenous injection target in the front view (a) and the side view (b).

Fig. 7 is a movement trace of the puncturing process of the injection needle in the present invention.

FIG. 8 is a graph showing the results of injections of the present invention, wherein (a) is zebrafish immediately after completion of the injections, and (b) is zebrafish 12 hours after the injections.

Detailed Description

The present invention will be described in further detail with reference to examples, which are illustrative of the present invention and are not to be construed as being limited thereto.

Example 1: calibration experiment: calibration of proportional relation R between zebra fish yolk sac length L and spine thickness D

As shown in FIG. 6, to obtain a target injection point Q_t(star mark in the figure) and the puncture point Q_pThe relative distance D (marked by a dot in the figure) is required to obtain the thickness D of the spine of the zebra fish. However, during the injection process, the zebra fish lies on the side, and the thickness D of the vertebral column cannot be directly obtained by a visual detection method. The zebra fish of the same species and the same development degree have the same body structure, and the proportional relation of the sizes of all parts of the body is basically consistent although the sizes of all parts of the body are slightly different. Therefore, the proportional relation R of the yolk sac length L and the spine thickness D is calibrated through a plurality of pre-shot zebra fish supine view diagrams. (as shown in fig. 4 (a)).

In the calibration process, the yolk sac length L of the zebra fish is obtained in an ellipse fitting mode, and the yolk sac length L is the length of the long axis of the ellipse obtained through fitting; two spinal column edges of the zebra fish are obtained through a straight line fitting mode, and the spinal column thickness D of the zebra fish is obtained through calibrating the distance between the two spinal column edges.

R is calculated according to the proportional relation of the yolk sac length L and the spinal thickness D of n (n ═ 10) wild zebra fishes, and the expression is as follows:

and R is an average value calculated by summing the ratio of the yolk sac length L to the spinal column thickness D of the n zebra fish, and the value in the experiment is 0.29.

Example 2: as shown in fig. 1, a method for robotized zebra fish main vein microinjection specifically comprises the following steps:

s1, placing the zebra fish in a culture dish covered with agarose in a lateral posture, establishing a zebra fish placing space coordinate system X-Y-Z, and taking the fish body as the X direction and the fish tail as the X positive direction;

s2, detecting the main vein of the zebra fish by an optical flow method, and taking the intersection point of the front main vein and the rear main vein of the zebra fish as a target injection point Q_tSetting a target injection point Q_tIs consistent with the plane position of the origin of the space coordinate system, and the target injection point Q_tBy determining the vertical position in a spatial coordinate systemDetermining the height h of the center of the spinal column of the zebra fish, wherein the space coordinate of the target injection point is Q_t＝[0 0 h]^T；

S3, obtaining the length L of the zebra fish yolk sac by means of ellipse fitting according to the calculation formula in the embodiment 1:determining the thickness D of the spine of the zebra fish;

s4, setting the injection needle to penetrate at 45 degrees obliquely above the target injection point, and setting the contact position of the injection needle and the zebra fish as a penetration point Q_pSpatial coordinate Q of the point of penetration_p＝[d 0 h+d]^TWherein D is half of the thickness D of the spine of the zebra fish;

s5, setting the movement track of the injection needle to make the needle point reach the puncture point Q from the initial position_pThen from the point of penetration Q_pMove to the target injection point Q_tAnd finishing the main vein injection of the zebra fish.

In examples 1 and 2 of the present invention, zebrafish of wild type AB was used.

In example 2, zebrafish were kept in circulating water at 28 ℃ for 14 hours during the day and 10 hours during the night. Embryos were obtained by natural oviposition and were raised to the desired embryonic stage in E4 medium at 28 ℃. The experiments were performed using 48 hour post-fertilization embryos for injection.

Specifically, as shown in fig. 2, in step S1, the zebra fish is placed in the space coordinate system X-Y-Z as shown in fig. 2 on the left, with the spine of the zebra fish being forward, the yolk sac being backward, and the body of the zebra fish lying on its side in the culture dish, in the X direction. The distribution of the main veins of zebra fish is shown in the right panel 2 by the coordinate system Xc-Yc, and the anterior main vein and the posterior main vein are merged into a total main vein.

As shown in fig. 3, in step S2, the main vein detection by the optical flow method is performed under a 10-fold microscope. Fig. 3(a) -3 (i) are processes of main vein detection. Specifically, fig. 3(a) is a microscopic image of the zebra fish under a 10-fold microscope, fig. 3(b) is a next image in the image sequence, and fig. 3(c) is an optical flow detection result; FIG. 3(d) is the optical flow fusion result of the image sequence; FIG. 3 (e)) The method is a result of binarizing the image by adopting an OTSU threshold method; FIG. 3(f) is an image smoothed with a morphological opening operation; FIG. 3(g) shows the vein skeleton extraction result; FIG. 3(h) is a straight line fitting result of the vein skeleton based on Hough transform; FIG. 3(i) is the intersection of two fitted straight lines, i.e., the target injection point Q_tPosition in the X-Y plane. In this embodiment, a target injection point Q is set_tIs coincident with the plane position of the origin of the space coordinate system.

Further, in step S2, as shown in fig. 4(a), the target injection point is set as an intersection of two venous blood vessels, and the position of the injection point can be determined by blood flow when the zebra fish is in a supine view, that is, the star position in the figure is the target injection point, and the target injection point is consistent with the central position of the spinal column of the zebra fish through observation, verification and analysis. However, in the actual injection, the zebrafish should be kept in a lateral decubitus position (as shown in fig. 5 (a)). In the microscopic field of view, as the zebra fish body has a certain thickness, the tissues are overlapped, and the central position of the spinal column of the zebra fish (namely the target injection point Q)_tHeight) cannot be obtained by visual inspection methods. On the other hand, as can be seen from the spinal anatomical cross-sectional view of zebrafish (fig. 5(b)), when the microscope is focused on the edge of the spinal column, that is, at the same time, the microscope is focused on the center of the spinal column (the position of the arrow in fig. 5(b)), the height of the target injection point can be indirectly obtained. The specific operation method comprises the following steps:

as shown in fig. 5(c-d), the zebra fish spinal column edge and the injection needle are automatically focused through an energy gradient function, so that the zebra fish spinal column edge and the injection needle are located at the same height, and the height of the zebra fish spinal column edge is obtained by obtaining the height information of the injection needle; the energy gradient function can evaluate the definition of the image in real time by calculating the horizontal gradient and the vertical gradient change of the image, and the calculation formula is as follows:

i (x, y) is the intensity of the pixel at (x, y), and I (x +1, y) and I (x, y +1) are the intensities of the lower right corner neighbors, respectively.

Further, as shown in fig. 4(b), in step S3, the yolk sac length L is obtained by ellipse fitting, in this example 2, the yolk sac length L of one zebra fish to be injected is detected to be 579 μm, and the spine thickness D of the zebra fish is calculated to be 168 μm.

As shown in FIG. 6, in step S4, the star shape is the target injection point Q_tThe dots being piercing points Q_p. The point of penetration is the position where penetration begins, and the angle between the injection needle and the horizontal plane is 45 degrees. The needle is inserted downwards along the 45-degree direction in the insertion process, and the insertion point Q_pThe distance d in the positive direction of X and Z of the target injection point. According to the geometric relationship, the relative distance D between the target injection point and the puncture point can be determined to be half of the spinal thickness D of the zebra fish, and in step S2, when the spinal thickness D of the zebra fish is 168 μm, D is 84 μm.

As shown in fig. 7, in step S5, the movement locus of the injection needle is designed according to the following principle:

setting the initial position P of the needle tip of the injection needle for the injection needle to accurately penetrate into the zebra fish₀And the target injection point Q_tThe plane position relation of (A) is as follows: the positions of the X axis and the Z axis are the same, and the distance between the Y axis and the Y axis is w; in order to reduce the damage of the injection needle to the zebra fish caused by the friction of the injection needle on the fish body and facilitate the subsequent needle insertion, the needle is moved from the initial position P₀Moving a distance d to reach P in an inclined upward direction₁＝[d -w h+d]^T(ii) a Then moves the distance w to the positive direction of the Y axis to reach the puncturing point Q_p＝[d 0 h+d]^T(ii) a Finally, quickly puncturing at 45 degrees obliquely downwards on an X-Z plane to reach a target injection point Q_t。

In conclusion, in the process of zebra fish main vein microinjection, the zebra fish juvenile fish is about 3 mm long and about 580 micrometers thick, the thickness of the zebra fish juvenile fish exceeds the depth of field range of an inverted microscope, the height of a target injection point cannot be obtained through a visual detection method, the position of a puncture point of an injection needle cannot be determined, and great difficulty is caused to injection. The invention provides a motorized zebra fish main vein microinjection method, which adjusts the position of an injection needle according to the relative position relationship between the edge of a zebra fish spinal column and a target injection point through the three-dimensional movement of the injection needle, ensures that the needle tip of the injection needle penetrates into the zebra fish body from a penetration point and finally reaches the set target injection point.

Example 3: and (3) verification and analysis:

in the embodiment 2 of the invention, 1-2nL of distilled water containing fluorescent particles with the diameter of 1 μm is injected into the main vein of the zebra fish, 20 zebra fish are injected in total, 17 zebra fish are injected successfully, 16 successfully injected zebra fish still survive after 12 hours, and the experimental result proves the high efficiency and repeatability of the method.

The results of the injection are shown in FIG. 8, where FIG. 8(a) is a zebrafish just after the injection, where it is evident that the fluorescent particles have entered the blood circulation; fig. 8(b) is a graph of zebrafish after 12 hours of injection, which survived within 12 hours.

In addition, it should be noted that the specific embodiments described in the present specification may differ in the shape of the components, the names of the components, and the like. All equivalent or simple changes of the structure, the characteristics and the principle of the invention which are described in the patent conception of the invention are included in the protection scope of the patent of the invention. Various modifications, additions and substitutions for the specific embodiments described may be made by those skilled in the art without departing from the scope of the invention as defined in the accompanying claims.