Zebrafish long-term micro-vision device and method of use

Abstract

The application discloses a zebra fish long-time micro-vision device and a use method, relates to the technical field of zebra fish observation, and 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 board, a test tube and a water circulation device shell. 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 application can provide a stable and suitable water circulation environment and a fixed observation position for zebra fish larvae by constructing a water circulation loop of the piezoelectric diaphragm pump, the test tube, the liquid inlet needle, the observation cavity, the liquid channel, the chip liquid outlet and the threaded compression ring and fixing the zebra fish larvae through the zebra fish fixing chip, so that long-time continuous observation of the zebra fish larvae can be realized.

Description

Technical Field

[0001] This application relates to the field of zebrafish observation technology, and in particular to a long-term microscopic device for zebrafish and its usage method. Background Technology

[0002] In zebrafish experimental research, especially when conducting microscopic observations of 3-5 day old zebrafish juveniles, a stable aquatic environment and long-term observation conditions are crucial for experimental success. However, existing experimental apparatus and methods have many problems.

[0003] Traditional artificial water replenishment methods require regular checks and manual water additions, which are not only time-consuming and labor-intensive but also prone to human error leading to delayed water replenishment, affecting the zebrafish's living environment and the accuracy of experimental results. Furthermore, most existing water replenishment devices cannot achieve precise flow control, resulting in poor water environment stability. This may affect the physiological state of zebrafish juveniles and the reliability of experimental data, especially for experiments requiring long-term microscopic observation. During microscopic observation, zebrafish juveniles need to be fixed in a stable position for continuous observation. However, traditional fixation methods may not provide sufficient stability.

[0004] Existing zebrafish observation techniques have not addressed or comprehensively considered the above problems, making it difficult to conduct long-term continuous observation of zebrafish juveniles. Summary of the Invention

[0005] This application provides a long-term microscopic device for zebrafish and a method for using it, in order to solve the problem that existing zebrafish observation techniques are difficult to use for long-term continuous observation of zebrafish juveniles.

[0006] On the one hand, this application provides a zebrafish long-term microscopic device, including: a zebrafish water circulation device and a zebrafish fixation chip.

[0007] The zebrafish water circulation device includes: a piezoelectric diaphragm pump, a piezoelectric diaphragm pump control board, a test tube, and a water circulation device housing. The piezoelectric diaphragm pump, the piezoelectric diaphragm pump control board, and the test tube are all fixedly installed inside the water circulation device housing.

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

[0009] The zebrafish-fixed chip includes: a fixed chip shell, a threaded pressure ring, a liquid inlet needle, an observation chamber, a liquid channel, and a chip liquid outlet.

[0010] The observation chamber, the liquid channel, and the chip outlet are sequentially connected and all located inside the fixed chip housing. The liquid inlet needle and the threaded pressure ring are located on the upper side of the fixed chip housing. The outlet of the liquid inlet needle is located above the observation chamber, and the chip outlet is connected to the threaded pressure ring.

[0011] The piezoelectric diaphragm pump, the test tube, and the inlet needle are connected in sequence, and the threaded pressure ring is connected to the piezoelectric diaphragm pump.

[0012] In one possible implementation, a transparent cover is provided on the top of the housing of the water circulation device.

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

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

[0015] The test tube is equipped with an outlet and an inlet.

[0016] The output end of the piezoelectric diaphragm pump is connected to the inlet of the test tube via a pipeline, and the outlet of the test tube is connected to the inlet needle via a pipeline.

[0017] The input end of the piezoelectric diaphragm pump is connected to the threaded pressure ring via a pipeline.

[0018] The control interface is connected to the piezoelectric diaphragm pump control board.

[0019] In one possible implementation, a slide is provided on the upper side of the fixed chip housing, and a needle fixing slide is slidably disposed on the slide, with the liquid injection needle fixed on the needle fixing slide.

[0020] In one possible implementation, the fixed chip housing and the needle fixing slide are made of photosensitive resin material.

[0021] In one possible implementation, a PCR membrane is attached to the bottom of the fixed chip housing to seal the bottom.

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

[0023] In one possible implementation, collagen gel is placed in the observation cavity to immobilize juvenile zebrafish.

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

[0025] On the other hand, this application provides a method of using a zebrafish long-term microscopic device, which includes the following steps: Step 1: Connect the output end of the piezoelectric diaphragm pump to the inlet of the test tube via a pipeline, connect the outlet of the test tube to the inlet needle via a pipeline, and connect the input end of the piezoelectric diaphragm pump to the threaded pressure ring via a pipeline to form a water circulation loop.

[0026] Step 2: Attach the PCR membrane to the bottom of the chip housing.

[0027] Step 3: Prepare a collagen gel by mixing collagen, PBS buffer and 0.75% sodium bicarbonate solution in a volume ratio of 15:2:1, and place 1 mL of the gel into the observation chamber.

[0028] Step 4: Place the 3-5 day old zebrafish fry on top of the collagen gel, positioning them sideways.

[0029] Step 5: Connect the power supply port and communication interface to the power supply and external computer control equipment respectively, and set the frequency and flow rate of the piezoelectric diaphragm pump.

[0030] Step six: Observe and photograph the zebrafish juveniles in the observation chamber under a microscope for an extended period of time and collect data.

[0031] The zebrafish long-term microscopic device and its usage method disclosed in this application have the following advantages: By constructing a water circulation loop consisting of a piezoelectric diaphragm pump, test tube, inlet needle, observation chamber, liquid channel, chip outlet, and threaded pressure ring, and fixing zebrafish larvae with a zebrafish fixing chip, a stable and suitable water circulation environment and fixed observation position can be provided for zebrafish larvae, thereby enabling long-term continuous observation of zebrafish larvae.

[0032] The system dynamically replenishes water to the zebrafish's habitat using a piezoelectric diaphragm pump. It precisely adjusts the replenishment rate and flow rate, continuously providing suitable water according to the zebrafish's physiological needs. This effectively maintains the stability of key environmental indicators such as water temperature and quality, creating a comfortable living environment for the zebrafish and ensuring they maintain good physiological condition during long-term experiments.

[0033] The proposed zebrafish fixation chip includes: a chip housing, a threaded retaining ring, a liquid inlet needle, an observation chamber, a liquid channel, and a chip outlet. This integrated, lightweight, and compact design is perfectly compatible with various types of microscopes on the market and also supports observation lenses of different magnifications. It can easily handle both low-magnification panoramic observation and high-magnification detailed analysis, greatly expanding its application range in various experimental scenarios.

[0034] Meanwhile, the integrated zebrafish immobilization chip allows for the convenient replacement of the water in the experimental system with a solution containing specific drugs, enabling precise drug intervention in zebrafish. This functional expansion allows the chip not only to meet basic observation needs but also to become a highly efficient tool for studying the effects of drugs on zebrafish physiological functions.

[0035] The proposed fixed chip casing has a PCR membrane attached to its bottom, offering the convenience of single-use. This avoids the risk of cross-contamination, ensures the accuracy of experimental results, and greatly simplifies the post-experiment cleaning process. Equipment preparation can be completed without complicated cleaning steps, significantly improving experimental efficiency and reducing the workload of researchers.

[0036] The proposed observation chamber contains collagen gel to immobilize juvenile zebrafish. The collagen gel is prepared by mixing collagen, PBS buffer, and 0.75% sodium bicarbonate solution in a volume ratio of 15:2:1. This improvement effectively avoids the hydrolysis problem commonly found when using methylcellulose gel. It not only provides a stable and reliable fixation effect for zebrafish, preventing observational biases caused by biological activity during the experiment, but also provides a biomimetic microenvironment for zebrafish, significantly increasing their survival time in the experiment and ensuring the authenticity and continuity of the observation data. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 This is a schematic diagram of the structure of a zebrafish long-term microscopic device and its usage method provided in an embodiment of this application; Figure 2 This is a schematic diagram of the zebrafish water circulation device provided in the embodiments of this application; Figure 3 This is a schematic diagram of the structure of the piezoelectric diaphragm pump provided in the embodiments of this application; Figure 4 This is a schematic diagram of the structure of the zebrafish fixing chip provided in the embodiments of this application; Figure 5 This is a schematic diagram of the bottom structure of the zebrafish fixing chip provided in an embodiment of this application.

[0039] Explanation of reference numerals in the attached figures: 1-Zebrafish water circulation device, 2-Zebrafish fixed chip, 3-Piezoelectric diaphragm pump, 4-Transparent cover, 5-Piezoelectric diaphragm pump control board, 6-Test tube, 7-Outlet, 8-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 pressure ring, 19-Fixed chip shell, 20-PCR membrane, 21-Slide, 22-Inlet needle, 23-Needle fixing slide, 24-Observation chamber, 25-Liquid channel, 26-Chip outlet. Detailed Implementation

[0040] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0041] like Figure 1 , Figure 2 , Figure 4 , Figure 5 As shown in the figure, this application provides a zebrafish long-term microscopic device, including: a zebrafish water circulation device 1 and a zebrafish fixing chip 2.

[0042] The zebrafish water circulation device 1 includes: a piezoelectric diaphragm pump 3, a piezoelectric diaphragm pump control board 5, a test tube 6, and a water circulation device housing 9. The piezoelectric diaphragm pump 3, the piezoelectric diaphragm pump control board 5, and the test tube 6 are all fixedly installed inside the water circulation device housing 9.

[0043] The water circulation device housing 9 has a power supply hole 12 and a communication interface 13 on its side. The power supply hole 12 and the communication interface 13 are both connected to the piezoelectric diaphragm pump control board 5. The piezoelectric diaphragm pump control board 5 is connected to the piezoelectric diaphragm pump 3. The power supply hole 12 and the communication interface 13 are respectively connected to a power supply and an external computer control device.

[0044] The zebrafish-fixed chip 2 includes: a fixed chip housing 19, a threaded pressure ring 18, a liquid inlet needle 22, an observation chamber 24, a liquid channel 25, and a chip liquid outlet 26.

[0045] The observation chamber 24, the liquid channel 25, and the chip outlet 26 are sequentially connected and all located inside the fixed chip housing 19. The liquid inlet needle 22 and the threaded pressure ring 18 are located on the upper side of the fixed chip housing 19. The outlet of the liquid inlet needle 22 is located above the observation chamber 24, and the chip outlet 26 is connected to the threaded pressure ring 18.

[0046] The piezoelectric diaphragm pump 3, the test tube 6, and the liquid inlet needle 22 are connected in sequence, and the threaded pressure ring 18 is connected to the piezoelectric diaphragm pump 3.

[0047] Specifically, in this embodiment, the piezoelectric diaphragm pump 3 is fixedly installed on the piezoelectric diaphragm pump fixing hole 14 provided inside the housing 9 of the water circulation device, and the piezoelectric diaphragm pump control board 5 is fixedly installed on the control board fixing hole 11 provided inside the housing 9 of the water circulation device.

[0048] In this embodiment, the chip outlet 26 is connected to the threaded pressure ring 18 through a threaded pipe that fixes the chip housing 19.

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

[0050] In this embodiment, the zebrafish fixing chip 2 is 49mm long, 40mm wide, and only 3mm high. This compact structure gives it excellent device compatibility.

[0051] For example, a transparent cover plate 4 is provided on the top of the housing 9 of the water circulation device.

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

[0053] For example, the transparent cover 4 is made of ABS plastic material.

[0054] like Figure 3 As shown, exemplarily, the piezoelectric diaphragm pump 3 is provided with an output terminal 16, an input terminal 15, and a control interface 17.

[0055] The test tube is equipped with an outlet 7 and an inlet 8.

[0056] The output end 16 of the piezoelectric diaphragm pump 3 is connected to the inlet 8 of the test tube 6 via a pipeline, and the outlet 7 of the test tube 6 is connected to the inlet needle 22 via a pipeline.

[0057] The input end 15 of the piezoelectric diaphragm pump 3 is connected to the threaded pressure ring 18 via a pipeline.

[0058] The control interface 17 is connected to the piezoelectric diaphragm pump control board 5.

[0059] For example, a slide 21 is provided on the upper side of the fixed chip housing 19, and a needle fixing slide 23 is slidably disposed on the slide 21, and the liquid injection needle 22 is fixed on the needle fixing slide 23.

[0060] Specifically, by setting up the slide 21 and the needle fixing slide 23, the needle fixing slide 23 can drive the liquid inlet needle 22 to slide on the slide 21, adjust the outlet of the liquid inlet needle 22 to the top of the observation chamber 24, or move the outlet of the liquid inlet needle 22 away from the top of the observation chamber 24 as needed.

[0061] For example, the fixed chip housing 19 and the needle fixing slide 19 are made of photosensitive resin material.

[0062] For example, a PCR membrane 20 is attached to the bottom of the fixed chip housing 19 to seal the bottom.

[0063] Specifically, by attaching the PCR membrane 20 to the bottom of the chip housing 19, water leakage at the bottom of the chip housing 19 is prevented.

[0064] For example, the housing 9 of the water circulation device is made of any one of photosensitive resin, aluminum alloy or ABS plastic material.

[0065] Specifically, in this embodiment, the outer shell 9 of the water circulation device is made of photosensitive resin material.

[0066] For example, collagen gel is placed in the observation cavity 24 to fix the zebrafish fry.

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

[0068] This application embodiment also provides a method for using a zebrafish long-term microscopic device, which includes the following steps: Step 1: Connect the output end 16 of the piezoelectric diaphragm pump 3 to the inlet 8 of the test tube 6 through a pipeline, connect the outlet 7 of the test tube 6 to the inlet needle 22 through a pipeline, and connect the input end 15 of the piezoelectric diaphragm pump 3 to the threaded pressure ring 18 through a pipeline to form a water circulation loop.

[0069] Step 2: Attach the PCR membrane 20 to the bottom of the chip housing 19.

[0070] Step 3: Prepare a collagen gel by mixing collagen, PBS buffer and 0.75% sodium bicarbonate solution in a volume ratio of 15:2:1, and place 1 mL of the gel in observation chamber 24.

[0071] Step 4: Place the 3-5 day old zebrafish fry on top of the collagen gel, positioning them sideways.

[0072] Step 5: Connect the power supply port 12 and the communication interface 13 to the power supply and the external computer control equipment, respectively, and set the frequency and flow rate of the piezoelectric diaphragm pump 3.

[0073] Step 6: Observe and photograph the zebrafish juveniles in observation chamber 24 under a microscope for an extended period of time and collect data.

[0074] Specifically, in this embodiment, the frequency of the piezoelectric diaphragm pump 3 is set to 50Hz, corresponding to a flow rate of 2ml / min, so that the liquid can circulate and replenish the zebrafish with water.

[0075] This embodiment of the application constructs a water circulation loop consisting of a piezoelectric diaphragm pump 3, a test tube 6, an inlet needle 22, an observation chamber 24, a liquid channel 25, a chip outlet 26, and a threaded pressure ring 18. By fixing the zebrafish larvae with the zebrafish fixing chip 2, a stable and suitable water circulation environment and a fixed observation position can be provided for the zebrafish larvae, thereby enabling long-term continuous observation of the zebrafish larvae.

[0076] The zebrafish's living environment is dynamically replenished using a piezoelectric diaphragm pump 3. This system can precisely adjust the replenishment rate and flow rate parameters, continuously providing suitable water supply according to the zebrafish's physiological needs. This effectively maintains the stability of key environmental indicators such as water temperature and water quality, creating a comfortable living environment for the zebrafish and ensuring that they maintain a good physiological state during long-term experiments.

[0077] The proposed zebrafish fixation chip 2 includes: a chip fixation shell 19, a threaded pressure ring 18, a liquid inlet needle 22, an observation chamber 24, a liquid channel 25, and a chip liquid outlet 26. This integrated, lightweight, and compact design can perfectly adapt to various types of microscopes on the market, while also being compatible with observation lenses of different magnifications. It can easily handle both low-magnification panoramic observation and high-magnification detailed analysis, greatly expanding its application range in various experimental scenarios.

[0078] Meanwhile, the integrated zebrafish immobilization chip 2 allows for convenient replacement of the water in the experimental system with a solution containing specific drugs, enabling precise drug intervention in zebrafish. This functional expansion allows the chip not only to meet basic observation needs but also to become a highly efficient tool for studying the effects of drugs on zebrafish physiological functions.

[0079] The proposed fixed chip casing 19 has a PCR membrane 20 attached to its bottom, offering the convenience of single-use. This avoids the risk of cross-contamination, ensures the accuracy of experimental results, and greatly simplifies the post-experiment cleaning process. Equipment preparation can be completed without complex cleaning steps, significantly improving experimental efficiency and reducing the workload of researchers.

[0080] The proposed observation chamber 24 contains collagen gel for fixing zebrafish juveniles. The collagen gel is prepared by mixing collagen, PBS buffer, and 0.75% sodium bicarbonate solution in a volume ratio of 15:2:1. This improvement effectively avoids the hydrolysis problem commonly found when using methylcellulose gel. It not only provides a stable and reliable fixation effect for zebrafish, preventing observational biases caused by biological activity during the experiment, but also provides a biomimetic microenvironment for zebrafish, significantly improving their survival time in the experiment and ensuring the authenticity and continuity of the observation data.

[0081] Although preferred embodiments of this application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this application.

[0082] Obviously, those skilled in the art can make various modifications and variations to this application without departing from the spirit and scope of this application. Therefore, if such modifications and variations fall within the scope of the claims of this application and their equivalents, this application also intends to include such modifications and variations.

Claims

1. A long-term microscopic device for zebrafish, characterized in that, include: Zebrafish water circulation device and zebrafish fixation chip; The zebrafish water circulation device includes: a piezoelectric diaphragm pump, a piezoelectric diaphragm pump control board, a test tube, and a water circulation device housing. The piezoelectric diaphragm pump, the piezoelectric diaphragm pump control board, and the test tube are all fixedly installed inside the water circulation device housing. The side of the water circulation device housing is provided with a power supply hole and a communication interface. The power supply hole and the communication interface are both connected to the piezoelectric diaphragm pump control board. The piezoelectric diaphragm pump control board is connected to the piezoelectric diaphragm pump. The power supply hole and the communication interface are respectively connected to a power supply and an external computer control device. The zebrafish fixation chip includes: a fixation chip shell, a threaded pressure ring, a liquid inlet needle, an observation chamber, a liquid channel, and a chip liquid outlet; The observation chamber, the liquid channel, and the chip outlet are sequentially connected and all located inside the fixed chip housing. The liquid inlet needle and the threaded pressure ring are located on the upper side of the fixed chip housing. The outlet of the liquid inlet needle is located above the observation chamber, and the chip outlet is connected to the threaded pressure ring. The piezoelectric diaphragm pump, the test tube, and the inlet needle are connected in sequence, and the threaded pressure ring is connected to the piezoelectric diaphragm pump; The piezoelectric diaphragm pump is equipped with an output end, an input end, and a control interface; The test tube is equipped with an outlet and an inlet; The output end of the piezoelectric diaphragm pump is connected to the inlet of the test tube via a pipeline, the outlet of the test tube is connected to the inlet needle via a pipeline, and the input end of the piezoelectric diaphragm pump is connected to the threaded pressure ring via a pipeline, forming a water circulation loop; The control interface is connected to the piezoelectric diaphragm pump control board; A slide rail is provided on the upper side of the fixed chip shell, and a needle fixing slide is slidably disposed on the slide rail, and the liquid injection needle is fixed on the needle fixing slide. A PCR membrane is attached to the bottom of the fixed chip casing to seal the bottom; Collagen gel was placed in the observation chamber to fix the zebrafish fry. The collagen gel is prepared by mixing collagen, PBS buffer, and 0.75% sodium bicarbonate solution in a volume ratio of 15:2:

1.

2. The zebrafish long-term microscopic device according to claim 1, characterized in that, The top of the water circulation device housing is provided with a transparent cover.

3. The zebrafish long-term microscopic device according to claim 2, characterized in that, The transparent cover is made of ABS plastic.

4. The zebrafish long-term microscopic device according to claim 1, characterized in that, The chip housing and the needle fixing slide are made of photosensitive resin material.

5. The zebrafish long-term microscopic device according to claim 1, characterized in that, The outer shell of the water circulation device is made of any one of photosensitive resin, aluminum alloy or ABS plastic material.

6. A method of using a zebrafish long-term microscopic device, comprising the zebrafish long-term microscopic device as described in any one of claims 1 to 5, characterized in that, Includes the following steps: Step 1: Connect the output end of the piezoelectric diaphragm pump to the inlet of the test tube through a pipeline, connect the outlet of the test tube to the inlet needle through a pipeline, and connect the input end of the piezoelectric diaphragm pump to the threaded pressure ring through a pipeline to form a water circulation loop. Step 2: Attach the PCR membrane to the bottom of the chip housing; Step 3: Prepare a collagen gel by mixing collagen, PBS buffer and 0.75% sodium bicarbonate solution in a volume ratio of 15:2:1, and place 1 mL of the gel into the observation chamber. Step 4: Place the 3-5 day old zebrafish fry on top of the collagen gel, positioning them sideways. Step 5: Connect the power supply port and communication interface to the power supply and external computer control equipment respectively, and set the frequency and flow rate of the piezoelectric diaphragm pump; Step six: Observe and photograph the zebrafish juveniles in the observation chamber under a microscope for an extended period of time and collect data.

Images