High-compatibility imaging and temperature control system for ovarian simulated organ and zebra fish early embryo

Abstract

The utility model discloses a high-compatibility imaging and temperature control system for an ovarian simulated organ and a zebra fish early embryo, which comprises a mold and a culture dish, agarose is placed in the culture dish, the mold is used for rubbing the ovarian simulated organ and an embryo imaging dish on the agarose, and the system further comprises an electric heating film and a temperature controller, the electric heating film is arranged below the culture dish, the input end of the electric heating film is electrically connected with the temperature controller, and an inductive probe of the temperature controller is arranged in the agarose for temperature monitoring; the mold comprises an ovarian organ simulating mold and a zebra fish early embryo mold. The mold is more fit in design and good in stability, 63 ovarian simulated organs or 30 early embryos can be contained at the same time, the mold is used in cooperation with a temperature controller and an electric heating film, imaging is stable, development temperature control is accurate, the operation difficulty is low, and repeatability is high.

Description

Technical Field

[0001] The utility model belongs to the technical field of ovarian organoids and embryo microscopic imaging, and in particular relates to a highly compatible imaging and temperature control system for ovarian organoids and zebrafish early embryos. Background Art

[0002] Ovarian organoids are a combination of oocytes and several types of somatic cells, and have some key biological characteristics in the process of ovarian development and oogenesis, such as hormone secretion and oocyte meiosis. Ovarian organoids are not a single cell line, but a three-dimensional structure of multicellular tissues. They are dynamic, and their structure and function may change under different physiological and pathological conditions. Imaging technology can provide real-time quantitative observation of the tissue, helping biologists to solve more biological problems. Because the state of cells is affected by temperature, keeping it consistent with the body temperature will provide more reliable observation results.

[0003] As a popular model species in developmental biology, zebrafish has many advantages, such as short reproductive cycle, fast development, small size, and easy breeding. More importantly, the eggs are fertilized in vitro, the fertilized eggs develop in vitro, and the early stage relies on yolk for nutrition to avoid interference from feeding. In addition, the early embryos are almost transparent, which greatly facilitates microscopic imaging and observation, and can be used to conduct in-depth research on a variety of in vivo systems, such as the cardiovascular system and reproductive system. Developmental biologists use 28.5˚C as the gold standard for development and divide the specific timeline of the early development stage of zebrafish embryos. Maintaining the ideal development temperature of the embryo is crucial to research.

[0004] Since the imaging instrument needs to be kept in a constant temperature environment of 20-22˚C, an additional temperature control system is required for imaging biological samples to maintain the standard development speed and living conditions. At present, the temperature control incubation systems of mainstream confocal microscope imaging systems are mostly expensive, and each microscope imaging platform such as upright and inverted has its own characteristics. A highly applicable, ready-to-use temperature control system can greatly save costs. In addition, since biological samples such as ovarian organoids and zebrafish embryos have three-dimensional shapes, it is difficult to place and fix the position and angle of the samples. Since the imaging working distance of the high-magnification microscope lens is as short as 0.2mm, which is smaller than the sample size, the placement and angle of the sample will affect the quality, stability and repeatability of the imaging data. Therefore, there is an urgent need for a highly compatible imaging and temperature control system with an adaptable mold that can adapt to a variety of upright and inverted large-scale microscopic platforms, and provide an accurate, efficient and stable sample placement and fixing device. Utility Model Content

[0005] In order to make up for the shortcomings of the existing technology, the utility model provides a highly compatible imaging and temperature control system for ovarian organoids and early zebrafish embryos. The system has high compatibility and can be applied to mainstream confocal imaging platforms (including upright and inverted imaging) such as Leica, Olympus and Zeiss. It can be freely disassembled, has strong portability, low operation difficulty, stable imaging, and precise developmental temperature control.

[0006] The highly compatible imaging and temperature control system for ovarian organoids and early zebrafish embryos comprises a mold and a culture dish, agarose is placed in the culture dish, and the mold is used to print the ovarian organoids and embryo imaging dish on the agarose. The system also comprises an electric heating film and a temperature controller, wherein the electric heating film is arranged below the culture dish, and its input end is electrically connected to the temperature controller, and the induction probe of the temperature controller is placed in the agarose for temperature monitoring; the mold comprises an ovarian organoid mold and a zebrafish early embryo mold.

[0007] Furthermore, the ovarian organoid mold comprises a second base and a plurality of independent second model slots arranged in an array on the second base. The second model slots are mainly composed of a truncated cone and can accommodate the ovarian organoid and culture medium.

[0008] Furthermore, the early zebrafish embryo mold includes a first base, and a plurality of model groups are arranged in an array on the first base, each model group includes two first model slots arranged in a mirror image, and the first model slot includes a head slot, a trunk slot and a tail slot that are connected in an integral manner, which are respectively used to accommodate the embryo's head and round yolk, the central trunk and the tail, and the two mirror image tail slots are connected.

[0009] Furthermore, the depth of the head groove is greater than that of the trunk groove and the tail groove, and the widths of the head groove, the trunk groove and the tail groove decrease successively. The head groove is a cubic structure, the trunk groove is a trapezoidal structure, the tail groove is a rectangular structure, and the head groove is provided with a slope extending outward on the side away from the trunk groove, and the tail groove is provided with a wedge-shaped slope with an arc extending outward on the side away from the trunk groove.

[0010] Furthermore, air guide grooves are arranged between each row of model groups on the first base.

[0011] Furthermore, the air guide groove is an equilateral triangular groove.

[0012] Furthermore, when the imaging platform adopts an upright objective lens, the diameter of the culture dish is 30 to 55 mm, and the culture dish is fixed by a metal clamp mounted on a microscope;

[0013] When the imaging platform adopts an inverted objective lens, the culture dish adopts a confocal glass bottom dish with a glass bottom thickness of 0.17 mm, a glass bottom diameter of 30 mm, and an outer diameter of less than 55 mm.

[0014] Furthermore, the electric heating film is a circular ring structure, which is closely attached to the bottom of the culture dish, and the zebrafish embryos in the culture dish are aligned with the central circle of the electric heating film.

[0015] Furthermore, the outer diameter of the electric heating film is 60-80 mm, the inner diameter is 20-40 mm, and the power is 10-15 watts.

[0016] Furthermore, the maximum output power of the thermostat is 60 watts, the temperature setting accuracy is 0.1°C, the minimum upper and lower fluctuation range is 0.2°C, and it can be calibrated according to the measured temperature.

[0017] Compared with the prior art, the utility model has the following advantages: the mold in the present application is designed to be used for placing a model slot for zebrafish embryos from 1 to 7 days old, including an integrally connected head slot, trunk slot and tail slot, which are used to accommodate the head and round yolk, central trunk and tail of the embryo respectively, which is more in line with the body shape of the early embryo, has good stability, and can accommodate 30 early embryos at the same time; and a mold for placing ovarian organoids is designed, which can perform multi-point multi-operation imaging observation while briefly cultivating ovarian organoids, and can accommodate 63 ovarian organoids at the same time. The zebrafish early embryo mold and ovarian organoid mold are used in conjunction with the temperature control system of the present application, with excellent imaging stability, precise development temperature control, low operating difficulty, and strong repeatability. In addition, with the ring-shaped electric heating film, the center circle is aligned with the embryo or pseudo-ovarian sample in the culture dish, which can ensure accurate and stable temperature control of the culture dish without affecting the observation of the sample and blocking the light path. The electric heating film has a low production cost, a temperature error of less than 0.2˚C, and can be installed and used at any time, which greatly facilitates the imaging of pseudo-ovarian organs and early embryos. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a structural schematic diagram of the utility model system;

[0019] Figure 2 Schematic diagram of the structure of the zebrafish early embryo mold of the utility model, (a) is the main view, (b) and (c) are side views;

[0020] Figure 3 This is a schematic diagram of the model slot structure of the zebrafish early embryo mold of the utility model;

[0021] Figure 4 This is a schematic diagram of the structure of the ovarian organoid mold of the utility model, (a) is the main view, (b) and (c) are side views.

[0022] In the figure: 1-mold, 10-zebrafish early embryo mold, 11-first base, 12-model group, 121-first model slot, 1211-head slot, 1212-trunk slot, 1213-tail slot, 1214-slope, 1215-wedge slope, 13-air guide slot, 20-ovarian organoid mold, 21-second base, 22-second model slot, 2-culture dish, 3-agarose, 4-electric heating film, 5-temperature controller, 51-sensing probe, 6-metal clamp. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the present invention is further described below in conjunction with the accompanying drawings.

[0024] like Figure 1-Figure 4 As shown, a highly compatible imaging and temperature control system for ovarian organoids and zebrafish early embryos includes a mold 1 and a culture dish 2, agarose 3 is placed in the culture dish 2, and the mold 1 is used to print ovarian organoids and embryo imaging dishes on the agarose 3. The system also includes an electric heating film 4 and a temperature controller 5. The electric heating film 4 is arranged below the culture dish 2, and its input end is electrically connected to the temperature controller 5. The induction probe 51 of the temperature controller 5 is placed in the agarose 3 for temperature monitoring; the mold 1 includes an ovarian organoid mold 20 and a zebrafish early embryo mold 10. Specifically, the ovarian organoid mold 20 includes a second base 21 and a plurality of independent second model slots 22 arranged in an array on the second base 21. The second model slot 22 is mainly composed of a truncated cone and can accommodate ovarian organoids and culture medium.

[0025] The zebrafish early embryo mold 10 includes a first base 11, and a plurality of model groups 12 are arranged in an array on the first base 11. Each model group 12 includes two first model slots 121 arranged in a mirror image, and the first model slot 121 includes a head slot 1211, a trunk slot 1212 and a tail slot 1213 that are connected to each other, which are respectively used to accommodate the head and round yolk, the central trunk and the tail of the embryo, and the two mirror image tail slots 1213 are connected. Among them, the depth of the head groove 1211 is greater than the trunk groove 1212 and the tail groove 1213, and the widths of the head groove 1211, the trunk groove 1212 and the tail groove 1213 decrease in sequence, the head groove 1211 is a cubic structure, the trunk groove 1212 is a trapezoidal structure, the tail groove 1213 is a rectangular parallelepiped structure, and the head groove 1211 extends outwardly from the side of the trunk groove 1212 to set a slope 1214, and the tail groove 1213 extends outwardly from the side of the trunk groove 1212 to set a curved wedge slope 1215. In addition, an air guide groove 13 is set between each row of model groups 12 on the base 11, and the air guide groove 13 is an equilateral triangular groove, which is convenient for taking out the zebrafish early embryo mold 10 after the agarose 3 is finalized, and can also stabilize the water surface, similar to a breakwater, to prevent the longitudinal displacement of the horizontal embryo caused by the up and down disturbance.

[0026] The system of the present application can be used for a confocal imaging platform (including upright and inverted imaging). When the imaging platform adopts an upright objective lens, the diameter of the culture dish 2 is 30 to 55 mm, and the culture dish 2 is fixed using a microscope-mounted metal clamp 6; when the imaging platform adopts an inverted objective lens, the culture dish 2 adopts a confocal glass-bottomed small dish with a glass bottom thickness of 0.17 mm, a glass bottom diameter of 30 mm, and an outer diameter of less than 55 mm.

[0027] As shown in the figure, the electric heating film 4 is a circular ring structure, close to the bottom of the culture dish 2, the zebrafish embryo in the culture dish 2 is aligned with the center circle of the electric heating film 4, and the center circle is aligned with the light path, so that the electric heating film 4 does not block the embryo sample and the observation light path. The outer diameter of the electric heating film 4 is 60-80 mm, the inner diameter is 20-40 mm, and the power is 10-15 watts. The maximum output power of the thermostat 5 is 60 watts, the temperature setting accuracy is 0.1°C, the minimum upper and lower fluctuation range is 0.2°C, and it can be calibrated according to the measured temperature. Example 1

[0028] Preparation of zebrafish early embryo mold 10:

[0029] like Figure 3 The figure shows a top view and a side view of the first model groove 121. In this embodiment, the cube of the head groove 1211 (accommodating the head and the round yolk) is 1.05 mm long, 0.7 mm wide and 0.3 mm high; the trapezoidal body of the trunk groove 1212 (accommodating the central trunk including the yolk extension tube) is 0.75 mm long, 0.7 mm wide at the top, close to the width of the cube, 0.5 mm wide at the bottom and 0.1 mm high; the rectangular area of ​​the tail groove 1213 (accommodating the tail) is made into a curved wedge-shaped slope 1215, which is 1.4 mm long, 0.5 mm wide, close to the width at the bottom of the trapezoidal body, and the height increases from 0.1 mm according to the slope and finally decreases to zero; the head groove 1211 is extended outwardly from one side of the trunk groove 1212 to set a slope 1214, the slope is extended outward at 45 degrees, and the height is 0.3 mm, it is convenient to slide from the shallow to the deep when placing the embryo. Each right angle of the base 11 is rounded to facilitate the production and removal of the zebrafish early embryo mold 10. The rectangular surface of the first base 11 is 24.25 mm long and 13.68 mm wide. The conical surface with an inclination of 60 degrees is extended outward to form a three-dimensional shape with a height of 2 mm. The tail of the first model groove 121 on the first base 11 is connected as a pair. In this embodiment, there are 3 pairs of horizontal rows and 5 pairs of vertical rows on the first base 11, a total of 15 pairs, and up to 30 embryos can be placed at the same time for stable imaging. An air guide groove 13 is set between each row of model groups 12 on the first base 11, with a side length of 0.5 mm, which is convenient for taking out the zebrafish early embryo mold 10 after finalization, and can also stabilize the water flow to prevent up and down fluctuations from affecting the horizontal embryo.

[0030] Figure 2 Shown as including Figure 3 The top view and side view of the first base 11 of the model group 12, the zebrafish early embryo mold 10 is finally made of red wax through 3D printing.

[0031] The steps of printing a zebrafish embryo imaging dish with the zebrafish early embryo mold 10 on the agarose 3 include: dissolving agarose 3 in water at 1.5%, and heating it to 100 ˚C until it is completely dissolved to form a liquid agarose solution, then pouring it into the culture dish 2, waiting for the high-temperature agarose solution to cool to about 60 ˚C, and placing the zebrafish early embryo mold 10 upside down on the agarose solution so that the lower surface is completely immersed and can just float on the solution, and try to make the mold 1 at the center of the culture dish 2, and let the culture dish 2 gradually cool at room temperature, or place it on an ice box to quickly cool and solidify, and then gently lift the zebrafish early embryo mold 10 from one side with tweezers. Due to the unique design of the air guide groove 13, the solidified agarose gel is easier to separate from the zebrafish early embryo mold 10, and the molded embryo fixation imaging dish can carry up to 30 zebrafish early embryos of 1 to 7 days.

[0032] Before the liquid agarose solidifies, the induction probe 51 of the temperature controller 5 or a metal rod of the same thickness can be inserted beside the mold 1 to form a reserved hole for temperature measurement. Example 2

[0033] Preparation of ovarian organoid mold 20:

[0034] like Figure 4 The figure shows the structure of the second model slot 22. In this embodiment, the second base 21 is 25 mm long, 20 mm wide and 1.5 mm high. The independent second model slot 22 is a truncated cone with a diameter of 2 mm on the side close to the second base 21 and a diameter of 0.7 mm and a height of 1.5 mm on the side away from the second base 21. There is no spacing between the independent second model slots 22. The ovarian organoid mold 20 is finally made of red wax material by 3D printing.

[0035] The steps of printing the zebrafish ovary pseudo-mold 20 on the agarose 3 include: dissolving 1.5% agarose 3 in water, heating it to 100 ˚C until it is completely dissolved to form a liquid agarose solution, then pouring it into the culture dish 2, waiting for the high-temperature agarose solution to cool to about 60 ˚C, and Figure 4The ovarian organoid mold 20 is inverted on the agarose solution, so that the lower surface is completely immersed and just floats on the solution. The mold 1 is placed at the center of the culture dish 2 as much as possible, and the culture dish 2 is allowed to gradually cool at room temperature, or placed on an ice box to quickly cool and solidify. Then, the ovarian organoid mold 20 is gently lifted from one side with tweezers. The ovarian imaging dish after molding can carry up to 63 ovarian organoids. The culture medium of the ovarian organoid is a non-CO2 dependent culture medium, and the ovarian pseudo-ovary can live in it for at least 2 days.

[0036] Before the liquid agarose solidifies, the induction probe 51 of the temperature controller 5 or a metal rod of the same thickness can be inserted beside the ovarian organoid mold 20 to form a reserved hole for temperature measurement. Example 3

[0037] Preparation steps of imaging temperature control system:

[0038] Prepare 1% low melting point agarose 3, dispense 1 ml into 1.5 ml centrifuge tubes and place in a 4 degree refrigerator as stock. When using, take out one tube and place it in a 70 degree water bath to melt. After melting, add 0.5% tricaine anesthetic solution at a ratio of 24:1, mix well and transfer to a 40 degree water bath for use.

[0039] The embryos after membrane stripping are placed in 0.015% tricaine anesthetic solution for anesthesia. At the same time, 500 μl of low-melting-point agarose is dripped into the culture tank of the fixed imaging dish prepared in Example 1 (i.e., the culture tank for embryo imaging formed by rubbing the first model tank 121), ensuring that all embryo placement culture tanks are covered. At this time, the required number of embryos are sucked with a dropper, and the embryos are waited to sink and gather at the slender opening of the dropper. They are dripped into each first model tank 121 at one time and dispersed as much as possible. The embryo angle is quickly adjusted with a line drawing brush so that the head yolk falls completely into the deep head tank 1211, and the trunk is allowed to lie flat in the trunk tank 1212 and the tail tank 1213 as much as possible. After all is completed, wait for a few minutes until the low-melting-point agarose solution is completely solidified, and then add about 5 ml of anesthetic solution (0.015% tricaine).

[0040] Continue reading Figure 1When the embryo fixation culture dish 2 is placed on the stage (not shown in the figure), the edge can be fixed with a common metal clamp 6, or other imaging platform supporting clamps can be used for simple fixation. A customized annular electric heating film 4 is inserted between the culture dish 2 and the stage. The electric heating film 4 is close to the culture dish 2, ensuring that the observed sample is aligned with the center circle of the electric heating film 4, and the center circle is aligned with the light path, so that the electric heating film 4 does not block the sample and the light path. The electric heating film 4 is connected to the output end of the temperature controller 5 (in this embodiment, the temperature controller 5 selects a microcomputer digital temperature controller), the temperature controller 5 is turned on, and the temperature is adjusted to 28.5 ˚C, and the upper and lower temperature fluctuations are between 0.2 ˚C; the induction probe 51 connected to the temperature controller 5 is inserted into the reserved hole for temperature measurement in Example 1; while waiting for the temperature to stabilize, the microscope imaging platform is turned on at the same time to perform a series of subsequent imaging operations.

[0041] After the imaging is completed, the temperature controller 5 is turned off, the metal clamp 11 is unfastened, and the sensing probe 51 and the electric heating film 4 under the culture dish 2 are taken out for next use. The culture dish 2 can be kept to continue observing the embryonic development or directly discarded.

[0042] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the utility model, rather than to limit it. Although the utility model has been described in detail with reference to the aforementioned embodiments, ordinary technicians in this field should understand that they can still modify the technical solutions recorded in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solution deviate from the scope of the technical solution of the embodiments of the utility model.

Claims

1. A highly compatible imaging and temperature control system for ovarian organoids and zebrafish early embryos, comprising a mold (1) and a culture dish (2), wherein agarose (3) is placed in the culture dish (2), and the mold (1) is used to print ovarian organoids and embryo imaging dishes on the agarose (3), characterized in that: The system also includes an electric heating film (4) and a temperature controller (5). The electric heating film (4) is arranged below the culture dish (2), and its input end is electrically connected to the temperature controller (5). The sensing probe (51) of the temperature controller (5) is placed in the agarose (3) for temperature monitoring. The mold (1) includes an ovarian organoid mold (20) and a zebrafish early embryo mold (10).

2. A highly compatible imaging and temperature control system for ovarian organoids and zebrafish early embryos according to claim 1, characterized in that: The ovarian organoid mold (20) comprises a second base (21) and a plurality of independent second model slots (22) arranged in an array on the second base (21); the second model slots (22) are mainly truncated cones and can accommodate the ovarian organoid and culture medium.

3. A highly compatible imaging and temperature control system for ovarian organoids and zebrafish early embryos according to claim 1, characterized in that: The zebrafish early embryo mold comprises a first base (11), and a plurality of model groups (12) arranged in an array on the first base (11), each model group (12) comprising two first model slots (121) arranged in a mirror image, and the first model slots (121) comprise a head slot (1211), a trunk slot (1212) and a tail slot (1213) which are connected in an integral manner, and are respectively used to accommodate the head and round yolk, the central trunk and the tail of the embryo, and the two tail slots (1213) arranged in a mirror image are connected.

4. A highly compatible imaging and temperature control system for ovarian organoids and zebrafish early embryos according to claim 3, characterized in that: The depth of the head groove (1211) is greater than that of the trunk groove (1212) and the tail groove (1213), and the widths of the head groove (1211), the trunk groove (1212) and the tail groove (1213) decrease in sequence; the head groove (1211) is a cubic structure, the trunk groove (1212) is a trapezoidal structure, and the tail groove (1213) is a rectangular parallelepiped structure; a slope (1214) is provided on a side of the head groove (1211) away from the trunk groove (1212), and a wedge-shaped slope (1215) with an arc is provided on a side of the tail groove (1213) away from the trunk groove (1212).

5. A highly compatible imaging and temperature control system for ovarian organoids and zebrafish early embryos according to claim 4, characterized in that: Air guide grooves (13) are arranged between each row of model groups (12) on the first base (11).

6. A highly compatible imaging and temperature control system for ovarian organoids and zebrafish early embryos according to claim 5, characterized in that: The air guide groove (13) is an equilateral triangular groove.

7. A highly compatible imaging and temperature control system for ovarian organoids and zebrafish early embryos according to any one of claims 1 to 6, characterized in that: When the imaging platform uses an upright objective lens, the culture dish (2) has a diameter of 30 to 55 mm, and a metal clamp (6) mounted on a microscope is used to fix the culture dish (2); When the imaging platform uses an inverted objective lens, the culture dish (2) uses a confocal glass bottom dish with a glass bottom thickness of 0.17 mm, a glass bottom diameter of 30 mm, and an outer diameter of less than 55 mm.

8. A highly compatible imaging and temperature control system for ovarian organoids and zebrafish early embryos according to claim 7, characterized in that: The electric heating film (4) is a circular ring structure and is closely attached to the bottom of the culture dish (2); the zebrafish embryos in the culture dish (2) are aligned with the center circle of the electric heating film (4).

9. A highly compatible imaging and temperature control system for ovarian organoids and zebrafish early embryos according to claim 8, characterized in that: The electric heating film (4) has an outer diameter of 60 to 80 mm, an inner diameter of 20 to 40 mm, and a power of 10 to 15 watts.

10. A highly compatible imaging and temperature control system for ovarian organoids and zebrafish early embryos according to claim 9, characterized in that: The maximum output power of the thermostat (5) is 60 watts, the temperature setting accuracy is 0.1°C, the minimum upper and lower fluctuation range is 0.2°C, and it can be calibrated according to the actual measured temperature.

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