Zebra fish long-time micro-vision device and use method

Abstract

The invention discloses a zebra fish long-time micro-vision device and a use method, and relates to the technical field of zebra fish observation, and the device comprises a zebra fish water circulation device and a zebra fish fixing chip; the zebra fish water circulation device comprises a piezoelectric diaphragm pump, a piezoelectric diaphragm pump control panel, a test tube and a water circulation device shell, the zebra fish fixing chip comprises a fixing chip shell, a threaded pressing ring, a liquid inlet needle head, an observation cavity, a liquid channel and a chip liquid outlet. By constructing a water circulation loop of the piezoelectric diaphragm pump, the test tube, the liquid inlet needle head, the observation cavity, the liquid channel, the chip liquid outlet and the threaded pressing ring and fixing the juvenile zebrafish through the zebrafish fixing chip, a stable and appropriate water circulation environment and a fixed observation position can be provided for the juvenile zebrafish; therefore, long-time continuous observation of the juvenile zebrafish is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of zebrafish observation, and in particular to a zebrafish long-time micro-vision device and a use method thereof. BACKGROUND

[0002] In zebrafish experimental research, especially when microscopically observing 3-5-day-old zebrafish larvae, stable water environment and long-time observation conditions are the key to the success of the experiment. However, the existing experimental devices and methods have many problems.

[0003] The traditional manual water replenishment method needs to be checked regularly and water is added manually, which is not only time-consuming and laborious, but also prone to cause water replenishment to be not timely due to human operation errors, thereby affecting the survival environment of zebrafish and the accuracy of experimental results. In addition, most of the existing water replenishment devices cannot achieve precise flow control, which leads to poor stability of the water environment, which may affect the physiological state of zebrafish larvae and the reliability of experimental data for zebrafish larvae experiments that need long-time microscopic observation. During the microscopic observation process, zebrafish larvae need to be fixed at a stable position for continuous observation. However, the traditional fixing method may not provide sufficient stability.

[0004] The existing zebrafish observation technology does not solve or comprehensively consider the above problems, and it is difficult to continuously observe zebrafish larvae for a long time. SUMMARY

[0005] The present application provides a zebrafish long-time micro-vision device and a use method, which solve the problem that the existing zebrafish observation technology is difficult to continuously observe zebrafish larvae for a long time.

[0006] In one aspect, the present application provides a zebrafish long-time micro-vision device, comprising: a zebrafish water circulation device and a zebrafish fixing chip.

[0007] The zebrafish water circulation device comprises a piezoelectric diaphragm pump, a piezoelectric diaphragm pump control board, a test tube and a water circulation device shell, wherein the piezoelectric diaphragm pump, the piezoelectric diaphragm pump control board and the test tube are fixedly arranged in the interior of the water circulation device shell.

[0008] The side surface of the water circulation device shell is provided with a power supply hole and a communication interface, wherein the power supply hole and the communication interface are connected with the piezoelectric diaphragm pump control board, the piezoelectric diaphragm pump control board is connected with the piezoelectric diaphragm pump, and the power supply hole and the communication interface are respectively connected with a power source and an external computer control device.

[0009] The zebrafish fixing chip comprises a fixing chip shell, a threaded compression ring, a liquid inlet needle, an observation cavity, a liquid channel and a chip liquid outlet.

[0010] The observation cavity, the liquid channel and the chip liquid outlet are sequentially communicated and are all arranged in the interior of the fixed chip shell, the liquid inlet needle and the threaded compression ring are arranged on the upper side of the fixed chip shell, the outlet of the liquid inlet needle is located above the observation cavity, and the chip liquid outlet is communicated with the threaded compression ring.

[0011] The piezoelectric diaphragm pump, the test tube and the liquid inlet needle are sequentially communicated, and the threaded compression ring is communicated with the piezoelectric diaphragm pump.

[0012] In a possible implementation, the top of the water circulation device shell is provided with a transparent cover plate.

[0013] In a possible implementation, the transparent cover plate is made of ABS plastic material.

[0014] In a possible implementation, the piezoelectric diaphragm pump is provided with an output end, an input end and a control interface.

[0015] The test tube is provided with a liquid outlet and a liquid inlet.

[0016] The output end of the piezoelectric diaphragm pump is communicated with the liquid inlet of the test tube through a pipeline, and the liquid outlet of the test tube is communicated with the liquid inlet needle through a pipeline.

[0017] The input end of the piezoelectric diaphragm pump is communicated with the threaded compression ring through a pipeline.

[0018] The control interface is connected with the control panel of the piezoelectric diaphragm pump.

[0019] In a possible implementation, the upper side of the fixed chip shell is provided with a slide, and a needle head fixing slide table is slidably arranged on the slide, and the liquid inlet needle is fixed on the needle head fixing slide table.

[0020] In a possible implementation, the fixed chip shell and the needle head fixing slide table are made of photosensitive resin material.

[0021] In a possible implementation, the bottom of the fixed chip shell is attached with a PCR film for sealing the bottom.

[0022] In a possible implementation, the water circulation device shell is made of any one of photosensitive resin, aluminum alloy or ABS plastic material.

[0023] In a possible implementation, collagen gel is placed in the observation cavity for fixing zebrafish larvae.

[0024] The collagen gel is prepared by mixing collagen, pbs buffer and 0.75% sodium bicarbonate solution according to a volume ratio of 15:2:1.

[0025] In another aspect, the application provides a method for using the zebrafish long-time micro-vision device, which comprises the following steps: Step one, the output end of the piezoelectric diaphragm pump is connected to the liquid inlet of the test tube through a pipeline, the liquid outlet of the test tube is connected to the liquid inlet needle through a pipeline, and the input end of the piezoelectric diaphragm pump is connected to the threaded compression ring through a pipeline, forming a water circulation loop.

[0026] Step two, the PCR film is pasted on the bottom of the fixed chip shell.

[0027] Step three, collagen, pbs buffer and 0.75% sodium bicarbonate solution are prepared into collagen gel according to the volume ratio of 15:2:1, and 1mL is placed in the observation cavity.

[0028] Step four, the 3-5 day old zebrafish larvae are placed above the collagen gel according to the method of side body.

[0029] Step five, the power supply hole and the communication interface are connected to the power supply and the external computer control device respectively, and the frequency and flow of the piezoelectric diaphragm pump are set.

[0030] Step six, the zebrafish larvae in the observation cavity are observed and photographed for a long time under the microscope and the data are collected.

[0031] The zebrafish long-time micro-vision device and the method for using the same have the following advantages: By constructing the water circulation loop of piezoelectric diaphragm pump, test tube, liquid inlet needle, observation cavity, liquid channel, chip liquid outlet and threaded compression ring, and fixing the zebrafish larvae by the zebrafish fixed chip, a stable and suitable water circulation environment and a fixed observation position can be provided for the zebrafish larvae, so that long-time continuous observation of the zebrafish larvae can be realized.

[0032] The piezoelectric diaphragm pump dynamically replenishes water for the survival environment of zebrafish. The system can accurately adjust the replenishment speed and water flow parameters, continuously provide suitable water supply according to the physiological needs of zebrafish, effectively maintain the stability of key environmental indicators such as water temperature and water quality, create a comfortable living environment for zebrafish, and ensure that zebrafish maintain good physiological state during long-time experiment.

[0033] The zebrafish fixed chip includes a fixed chip shell, a threaded compression ring, a liquid inlet needle, an observation cavity, a liquid channel, and a chip liquid outlet. This integrated and lightweight compact design can perfectly adapt to various types of microscopes on the market, and is compatible with different magnification observation lenses. Whether it is low-magnification panoramic observation or high-magnification detail analysis, it can easily cope with it, greatly expanding its application range in various experimental scenarios.

[0034] At the same time, the integrated zebrafish fixation chip can conveniently replace the water in the experimental system with a solution containing a specific drug, realizing precise drug intervention on the zebrafish. This function expansion makes the chip not only meet the basic observation needs, but also become an efficient tool for studying the influence of drugs on the physiological functions of zebrafish.

[0035] The bottom of the fixation chip shell is pasted with a PCR film, which has the convenient characteristics of one-time use. It can not only avoid the risk of cross contamination and ensure the accuracy of experimental results, but also greatly simplify the cleaning process after the experiment. Without complex cleaning steps, the equipment can be arranged, significantly improving the efficiency of experimental operation and reducing the workload of researchers.

[0036] The observation cavity is placed with collagen gel for fixing zebrafish larvae; the collagen gel is prepared by mixing collagen, pbs buffer and 0.75% sodium bicarbonate solution according to the volume ratio of 15:2:1. This improvement effectively avoids the hydrolysis problem commonly existing in the use of methyl cellulose gel in the past. It not only can form a stable and reliable fixation effect on zebrafish, prevent observation deviation caused by biological activity during the experiment, but also can provide a bionic living microenvironment for zebrafish, significantly improve its survival time in the experiment, and ensure the authenticity and continuity of the observation data. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without creating any creative labor.

[0038] Figure 1 A structural schematic diagram of a zebrafish long-time micro-vision device and a use method provided by an embodiment of the present application; Figure 2 A structural schematic diagram of a zebrafish water circulation device provided by an embodiment of the present application; Figure 3 A structural schematic diagram of a piezoelectric diaphragm pump provided by an embodiment of the present application; Figure 4 A structural schematic diagram of a zebrafish fixation chip provided by an embodiment of the present application; Figure 5 A bottom structural schematic diagram of a zebrafish fixation chip provided by an embodiment of the present application.

[0039] Explanation of reference signs: 1 - zebra fish water circulation device, 2 - zebra fish fixed chip, 3 - piezoelectric diaphragm pump, 4 - transparent cover plate, 5 - piezoelectric diaphragm pump control board, 6 - test tube, 7 - liquid outlet, 8 - liquid inlet, 9 - water circulation device shell, 10 - cover plate fixing hole, 11 - control board fixing hole, 12 - power supply hole, 13 - communication interface, 14 - piezoelectric diaphragm pump fixing hole, 15 - input end, 16 - output end, 17 - control interface, 18 - threaded compression ring, 19 - fixed chip shell, 20 - PCR film, 21 - slide, 22 - liquid inlet needle, 23 - needle fixed slide, 24 - observation cavity, 25 - liquid channel, 26 - chip liquid outlet. DETAILED DESCRIPTION

[0040] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0041] As shown in Figure 1 , Figure 2 , Figure 4 , Figure 5 The zebra fish long-time micro-vision device provided by the embodiments of the present application comprises a zebra fish water circulation device 1 and a zebra fish fixed chip 2.

[0042] The zebra fish water circulation device 1 comprises a piezoelectric diaphragm pump 3, a piezoelectric diaphragm pump control board 5, a test tube 6 and a water circulation device shell 9, wherein the piezoelectric diaphragm pump 3, the piezoelectric diaphragm pump control board 5 and the test tube 6 are fixedly arranged inside the water circulation device shell 9.

[0043] The side surface of the water circulation device shell 9 is provided with a power supply hole 12 and a communication interface 13, wherein the power supply hole 12 and the communication interface 13 are connected with the piezoelectric diaphragm pump control board 5, the piezoelectric diaphragm pump control board 5 is connected with the piezoelectric diaphragm pump 3, and the power supply hole 12 and the communication interface 13 are respectively connected with a power supply and an external computer control device.

[0044] The zebra fish fixed chip 2 comprises a fixed chip shell 19, a threaded compression ring 18, a liquid inlet needle 22, an observation cavity 24, a liquid channel 25 and a chip liquid outlet 26.

[0045] The observation cavity 24, the liquid channel 25, and the chip liquid outlet 26 are sequentially communicated and are all arranged inside the fixed chip shell 19. The liquid inlet needle 22 and the threaded compression ring 18 are arranged on the upper side of the fixed chip shell 19. The outlet of the liquid inlet needle 22 is located above the observation cavity 24. The chip liquid outlet 26 is in communication with the threaded compression ring 18.

[0046] The piezoelectric diaphragm pump 3, the test tube 6, and the liquid inlet needle 22 are sequentially communicated. The threaded compression ring 18 is in communication with the piezoelectric diaphragm pump 3.

[0047] Specifically, in the embodiment, the piezoelectric diaphragm pump 3 is fixedly installed on the piezoelectric diaphragm pump fixing hole 14 arranged inside the water circulation device shell 9. The piezoelectric diaphragm pump control panel 5 is fixedly installed on the control panel fixing hole 11 arranged inside the water circulation device shell 9.

[0048] In the embodiment, the chip liquid outlet 26 is in communication with the threaded compression ring 18 through the threaded pipeline of the fixed chip shell 19.

[0049] Specifically, in the embodiment, the path of the water circulation loop is as follows: the liquid sequentially passes through the piezoelectric diaphragm pump 3, the test tube 6, the liquid inlet needle 22, the observation cavity 24, the liquid channel 25, the chip liquid outlet 26, the threaded compression ring 18, and then is circulated back to the piezoelectric diaphragm pump 3 through the pipeline from the threaded compression ring 18.

[0050] In the embodiment, the zebrafish fixed chip 2 is 49 mm long, 40 mm wide, and only 3 mm high. Such a compact structure makes it have strong equipment compatibility.

[0051] Illustratively, the top of the water circulation device shell 9 is provided with a transparent cover plate 4.

[0052] Specifically, in the embodiment, the transparent cover plate 4 is fixedly installed on the cover plate fixing hole 10 arranged on the top of the water circulation device shell 9.

[0053] Illustratively, the transparent cover plate 4 is made of ABS plastic material.

[0054] As shown in the figure, illustratively, the piezoelectric diaphragm pump 3 is provided with an output end 16, an input end 15, and a control interface 17. Figure 3

[0055] The test tube is provided with a liquid outlet 7 and a liquid inlet 8.

[0056] The output end 16 of the piezoelectric diaphragm pump 3 is in communication with the liquid inlet 8 of the test tube 6 through a pipeline. The liquid outlet 7 of the test tube 6 is in communication with the liquid inlet needle 22 through a pipeline.

[0057] ​The input end 15 of the piezoelectric diaphragm pump 3 is communicated with the threaded compression ring 18 through a pipeline.

[0058] The control interface 17 is connected with the piezoelectric diaphragm pump control panel 5.

[0059] Exemplarily, the upper side of the fixed chip shell 19 is provided with a sliding channel 21, and a needle fixing sliding table 23 is slidingly arranged on the sliding channel 21, and the liquid inlet needle 22 is fixed on the needle fixing sliding table 23.

[0060] Specifically, the needle fixing sliding table 23 can drive the liquid inlet needle 22 to slide on the sliding channel 21, so as to adjust the outlet of the liquid inlet needle 22 to above the observation cavity 24, and the outlet of the liquid inlet needle 22 can also be moved away from above the observation cavity 24 according to needs.

[0061] Exemplarily, the fixed chip shell 19 and the needle fixing sliding table 19 adopt a photosensitive resin material.

[0062] Exemplarily, the bottom of the fixed chip shell 19 is attached with a PCR film 20 for sealing the bottom.

[0063] Specifically, by attaching the PCR film 20 to the bottom of the fixed chip shell 19, water leakage at the bottom of the fixed chip shell 19 is avoided.

[0064] Exemplarily, the water circulation device shell 9 adopts any one of a photosensitive resin, an aluminum alloy or an ABS plastic material.

[0065] Specifically, the water circulation device shell 9 in the embodiment adopts a photosensitive resin material.

[0066] Exemplarily, collagen gel is placed in the observation cavity 24 for fixing zebrafish larvae.

[0067] The collagen gel is prepared by mixing collagen, pbs buffer and 0.75% sodium bicarbonate solution according to a volume ratio of 15:2:1.

[0068] The application also provides a use method of the zebrafish long-time micro-vision device, which adopts the above-mentioned zebrafish long-time micro-vision device and includes the following steps. Step one, the output end 16 of the piezoelectric diaphragm pump 3 is communicated with the liquid inlet 8 of the test tube 6 through a pipeline, the liquid outlet 7 of the test tube 6 is communicated with the liquid inlet needle 22 through a pipeline, and the input end 15 of the piezoelectric diaphragm pump 3 is communicated with the threaded compression ring 18 through a pipeline, so as to form a water circulation loop.

[0069] Step two, the PCR film 20 is attached to the bottom of the fixed chip shell 19.

[0070] Step three, collagen, pbs buffer and 0.75% sodium bicarbonate solution are prepared into collagen gel according to the volume ratio of 15:2:1, and 1 mL is placed in the observation chamber 24.

[0071] Step four, 3-5 day old zebrafish larvae are placed above the collagen gel by laying on their side.

[0072] Step five, the power supply hole 12 and the communication interface 13 are connected to the power supply and the external computer control device respectively, and the frequency and flow of the piezoelectric diaphragm pump 3 are set.

[0073] Step six, under the microscope, the zebrafish larvae in the observation chamber 24 are observed for a long time, photographed and data are collected.

[0074] Specifically, in this embodiment, the frequency of the piezoelectric diaphragm pump 3 is set to 50 Hz, and the corresponding flow is 2 ml / min, so that the liquid realizes circulating flow to supply water to the zebrafish.

[0075] The embodiment of the present application can provide a stable and suitable water circulation environment and a fixed observation position for zebrafish larvae by constructing a water circulation loop of the piezoelectric diaphragm pump 3, the test tube 6, the liquid inlet needle 22, the observation chamber 24, the liquid channel 25, the chip liquid outlet 26 and the threaded compression ring 18, and fixing the zebrafish larvae by the zebrafish fixing chip 2, so as to realize long-time continuous observation of the zebrafish larvae.

[0076] The piezoelectric diaphragm pump 3 dynamically supplies water to the living environment of the zebrafish. The system can accurately adjust the water supply speed and water flow parameters, continuously provide suitable water supply according to the physiological needs of the zebrafish, effectively maintain the stability of key environmental indicators such as water temperature and water quality, create a comfortable living environment for the zebrafish, and ensure that the zebrafish maintains a good physiological state during the long-time experiment.

[0077] The zebrafish fixing chip 2 includes a fixing chip shell 19, a threaded compression ring 18, a liquid inlet needle 22, an observation chamber 24, a liquid channel 25 and a chip liquid outlet 26. This integrated and lightweight compact design can perfectly adapt to various types of microscopes on the market, and is compatible with different magnification observation lenses. Whether it is low-magnification panoramic observation or high-magnification detail analysis, it can easily cope with it, which greatly expands its application range in various experimental scenarios.

[0078] At the same time, the integrated zebrafish fixing chip 2 can conveniently replace the water in the experimental system with a solution containing a specific drug, realizing precise drug intervention on the zebrafish. This function expansion enables the chip to not only meet the basic observation needs, but also become an efficient tool for studying the influence of drugs on the physiological functions of zebrafish.

[0079] The bottom of the fixed chip shell 19 is pasted with a PCR film 20, which has the convenient characteristics of one-time use, can avoid the risk of cross contamination, ensure the accuracy of experimental results, and greatly simplify the cleaning process after the experiment. Without complex cleaning steps, the device can be arranged, which significantly improves the experimental operation efficiency and reduces the work burden of scientific researchers.

[0080] The collagen gel in the observation cavity 24 is used to fix the zebrafish larvae; the collagen gel is prepared by mixing collagen, pbs buffer and 0.75% sodium bicarbonate solution according to the volume ratio of 15:2:1. This improvement effectively avoids the hydrolysis problem of methyl cellulose gel in the past, not only can form a stable and reliable fixing effect on zebrafish, prevent observation deviation caused by biological activity in the experimental process, but also can provide a biomimetic survival microenvironment for zebrafish, significantly improve its survival time in the experiment, and ensure the authenticity and continuity of the observation data.

[0081] Although the preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to the embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to be interpreted as including all the preferred embodiments and all the changes and modifications falling within the scope of the present application.

[0082] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and variations.

Claims

1. A zebrafish long-term micro-vision device, characterized in that, The application relates to a zebra fish water circulation device and a zebra fish fixing chip. The zebra fish water circulation device comprises a piezoelectric diaphragm pump, a piezoelectric diaphragm pump control board and a test tube, and the piezoelectric diaphragm pump, the piezoelectric diaphragm pump control board and the test tube are fixedly arranged in the interior of a water circulation device shell. A power supply hole and a communication interface are arranged on the side of the water circulation device shell, and the power supply hole and the communication interface are connected with the piezoelectric diaphragm pump control board; the piezoelectric diaphragm pump control board is connected with the piezoelectric diaphragm pump; the power supply hole and the communication interface are respectively connected with a power supply and an external computer control device. The zebra fish fixing chip comprises a fixing chip shell, a threaded compression ring, a liquid inlet needle, an observation cavity, a liquid channel and a chip liquid outlet. The observation cavity, the liquid channel and the chip liquid outlet are sequentially communicated and arranged in the interior of the fixing chip shell; the liquid inlet needle and the threaded compression ring are arranged on the upper side of the fixing chip shell; the outlet of the liquid inlet needle is located above the observation cavity; and the chip liquid outlet is communicated with the threaded compression ring. The piezoelectric diaphragm pump, the test tube and the liquid inlet needle are sequentially communicated; and the threaded compression ring is communicated with the piezoelectric diaphragm pump. A transparent cover plate is arranged on the top of the water circulation device shell.

2. The zebrafish long-term micro-viewing device according to claim 1, wherein, The transparent cover plate is made of ABS plastic material.

3. The long-term micro-viewing device for zebrafish according to claim 2, characterized in that, The piezoelectric diaphragm pump is provided with an output end, an input end and a control interface.

4. The long-term micro-viewing device for zebrafish according to claim 1, characterized in that, The test tube is provided with a liquid outlet and a liquid inlet. The output end of the piezoelectric diaphragm pump is communicated with the liquid inlet of the test tube through a pipeline; the liquid outlet of the test tube is communicated with the liquid inlet needle through a pipeline; and the input end of the piezoelectric diaphragm pump is communicated with the threaded compression ring through a pipeline. The control interface is connected with the piezoelectric diaphragm pump control board. A slide is arranged on the upper side of the fixing chip shell, and a needle fixing slide table is slidably arranged on the slide; and the liquid inlet needle is fixed on the needle fixing slide table. The fixing chip shell and the needle fixing slide table are made of photosensitive resin material.

5. The zebrafish long-term microvision device according to claim 1, wherein, A PCR film is attached to the bottom of the fixing chip shell for sealing the bottom.

6. The zebrafish long-term microvision device according to claim 5, wherein, The water circulation device shell is made of any one of photosensitive resin, aluminum alloy or ABS plastic material.

7. The zebrafish long-term microvision device according to claim 1, wherein, Collagen gel is placed in the observation cavity for fixing zebra fish larvae.

8. The zebrafish long-term microvision device according to claim 1, wherein, The collagen gel is prepared by mixing collagen, pbs buffer and 0.75% sodium bicarbonate solution in a volume ratio of 15:2:

1.

9. The zebrafish long-term microvision device according to claim 1, wherein, The application further discloses a preparation method of the zebra fish water circulation device and the zebra fish fixing chip. Step one: the output end of the piezoelectric diaphragm pump is communicated with the liquid inlet of the test tube through a pipeline; the liquid outlet of the test tube is communicated with the liquid inlet needle through a pipeline; and the input end of the piezoelectric diaphragm pump is communicated with the threaded compression ring through a pipeline to form a water circulation loop; 10. A method of using a zebrafish long-term micro-vision device, using a zebrafish long-term micro-vision device according to any one of claims 1 to 9, characterized in that, Step two: a PCR film is attached to the bottom of the fixing chip shell; Step three: collagen, pbs buffer and 0.75% sodium bicarbonate solution are mixed in a volume ratio of 15:2:1 to prepare collagen gel, and 1 mL of the collagen gel is placed in the observation cavity; Step four: 3-5 day old zebra fish larvae are placed above the collagen gel in a side-lying manner. ​ ​ Step five, the power supply hole, communication interface is connected to the power supply and external computer control device, set the frequency and flow of piezoelectric diaphragm pump; Step six, under the microscope to observe the zebrafish larvae in the long time observation chamber and collect data.

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