KR102617905B1 - Electrophoresis device for tissue clearing and tissue dlearing method using the same - Google Patents

Abstract

The electrophoresis device (1) for biological tissue transparency according to the present invention is configured as follows.
It includes a chamber 10 that is open upward, and a cap 50 that covers the chamber 10, wherein the chamber 10 protrudes upward from the bottom and is formed to place biological tissue (L). It includes a seating portion 11, and a cathode electrode 13 and an anode electrode 15 that are respectively disposed at lower portions of both sides of the seating portion 11 and connected to a power source.
In addition, the method of making biological tissue transparent using the electrophoresis apparatus 1 for making biological tissue transparent includes the following process.
Opening the cap 50 and placing biological tissue (L) on the seating portion 11; filling the chamber 10 with a buffer solution (Q) as an electrolyte; closing the cap 50 and It includes applying electricity to the cathode electrode 13 and the anode electrode 15.

Description

Electrophoresis device for tissue clearing and tissue clearing method using the same {Electrophoresis device for tissue clearing and tissue clearing method using the same}

The present invention relates to an electrophoresis device for making biological tissue transparent and a method for making biological tissue transparent using the same. More specifically, the present invention relates to an electrophoresis device for making biological tissue transparent, and more specifically, to improve the density of the electric field acting on biological tissue so that lipids can be easily and quickly discharged from the biological tissue. The present invention relates to an electrophoresis device for transparent biological tissue, which is configured so that the transparency process can be easily confirmed with the naked eye and the buffer solution can be maintained at a specified constant temperature, and a method for transparent biological tissue using the same.

Below, we will look at the electrophoresis device for biological tissue transparency according to the background technology along with the attached drawings.

Figure 1 is a photograph showing an electrophoresis device for making biological tissue transparent using background technology, and Figure 2 is a flowchart showing the principle of an electrophoresis device for making biological tissue transparent using background technology. In addition, Figure 3 is an example diagram showing the process of biological tissue becoming transparent through an electrophoresis device for biological tissue transparency according to background technology, and Figure 4 is an exemplary diagram showing the process of biological tissue becoming transparent through an electrophoresis device for biological tissue transparency according to background technology. This is an example diagram showing a state where lipids remain in biological tissue even though the transparency process has been completed.

In order to closely observe the internal tissues by taking three-dimensional images of biological tissues such as the brain, liver, lungs, kidneys, stomach, large intestine, heart, muscles, blood vessels, etc. of living organisms using an electrophoresis device, the lipids (lipoids) inside the biological tissues are captured. There is a need to release it and make it transparent.

For this purpose, biological tissue is accommodated inside the chamber of the electrophoresis device shown in FIG. 1 and filled with a buffer solution, which is an electrolyte. Then, electricity is applied to the anode and cathode electrodes placed on both sides of the tissue to cause electrophoresis to separate lipids from biological tissues. As the negative charge generated from the cathode electrode moves to the anode electrode, it penetrates into the biological tissue and separates the lipids. By surrounding it, micelles are formed. And since the micelles break away from the biological tissue and move toward the anode electrode, lipids can be discharged.

In addition, an inlet and an outlet are formed in the chamber to allow the buffer solution to circulate, and cooling is possible through an externally installed cooling device.

However, this electrophoresis device had the following problems.

First, as shown in FIG. 3, when the biological tissue (L) is immersed in the buffer solution (Q), current flows through the electrodes (S, T) on both sides through the buffer solution (Q), which is an electrolyte. Accordingly, a magnetic field is formed through each electrode, and negative charges move along the equipotential line (D) forming the magnetic field, causing the lipid (O) inside the biological tissue (L) to escape. However, if the equipotential line (D) is not dense, the lipid (O) inside the biological tissue (L) cannot be completely discharged. In other words, when the equipotential line (D) densely passes through the biological tissue (L), the lipid (O) can be easily and quickly removed.

Therefore, in order to prevent the phenomenon where the equipotential line (D) is not dense, the intensity of the current may be increased, but in this case, the temperature of the buffer solution (Q) is increased, causing deterioration of the biological tissue, so it is not used. . Therefore, even after going through the transparency process, lipids remain inside the biological tissue as shown in FIG. 4, making it difficult to produce 3D images. Therefore, there was the inconvenience of having to go through the electrophoresis process for a long time (5 to 9 days) while rotating the biological tissue.

Second, since the transparency process cannot be confirmed in real time, a camera is installed inside the chamber and observed from the outside through a monitor. However, since additional cameras and monitors must be provided, small and lightweight design is difficult and manufacturing costs are high. .

Third, since a structure for circulating the buffer solution and a cooling device are installed on the outside to cool the buffer solution, there is a problem in that it is difficult to design a compact and lightweight product and the manufacturing cost is high.

Korean Patent Publication No. 10-2018-0118514 (Publication date: October 31, 2018)

https://lifecanvastech.com/products/tissue-clearing-labeling/

The problems to be solved through the electrophoresis device for biological tissue transparency according to the present invention and the biological tissue transparency method using the same are as follows.

First, as shown in Figure 3, when the biological tissue is immersed in the buffer solution, current flows through the buffer solution, which is an electrolyte, to both electrodes. Accordingly, an electric field is formed through each electrode, and negative charges move along the equipotential lines forming the electric field, causing the lipids inside the biological tissue to escape. However, if the equipotential lines are not dense, we would like to solve the problem that lipids inside biological tissues cannot be completely discharged.

Second, since the transparency process cannot be confirmed in real time, a camera is installed inside the chamber and observed from the outside through a monitor. However, since additional cameras and monitors must be provided, small and lightweight design is difficult and manufacturing costs are high, solving the problem. I want to do it.

Third, since a structure for circulating the buffer solution and a cooling device are provided on the outside to cool the buffer solution, it is intended to solve the problem of difficulty in designing a small and lightweight device and high manufacturing costs.

In order to solve the above problems, the electrophoresis device for making biological tissue transparent according to the present invention may include the following configuration.

It includes a chamber open upward and a cap covering the chamber,

The chamber is,

It may include a seating portion that protrudes upward from the bottom surface and is formed to place biological tissue on it, and a cathode electrode and an anode electrode that are disposed on lower sides of both sides of the seating portion and are connected to a power source.

Additionally, the cap may include holes drilled upward and downward, and a fan mounted in the hole.

Additionally, the chamber may be made of a transparent material.

Additionally, when the chamber is made of a transparent material, it may include a light emitting means mounted on the chamber to irradiate light into the interior.

Additionally, the light emitting means may be mounted on the inside of the cap and configured to emit light downward.

In addition, it includes a block that is seated on the upper part of the seating portion and holds the biological tissue, the block is porous and has a space inside for accommodating the biological tissue, and is connected from the outer surface of the block to the space to accommodate the biological tissue. It may include possible inflow means.

In order to solve the above problem, a method for making biological tissue transparent using the electrophoresis device for making biological tissue transparent according to the present invention will be examined as follows.

It includes a chamber open upward and a cap covering the chamber,

The chamber is,

Electrophoresis for making biological tissue transparent, comprising a seating portion that protrudes upward from the bottom surface and is formed to place biological tissue, and a cathode electrode and an anode electrode disposed on both lower sides of the seating portion and connected to a power source. A method of making biological tissue transparent using a device,

It may include opening the cap and placing a biological tissue on the seating portion, filling the chamber with a buffer solution that is an electrolyte, and closing the cap and applying electricity to the cathode electrode and the anode electrode.

In addition, the cap includes a hole drilled in the upper and lower directions, and a fan mounted in the hole, and the step of applying electricity may include cooling the buffer solution by driving the fan. .

In addition, the chamber is made of a transparent material, includes a light emitting means that irradiates light into the chamber, and includes the step of applying electricity to the light emitting means to observe the inside of the chamber after the step of applying electricity. can do.

In addition, it includes a block that is seated on the upper part of the seating portion and holds the biological tissue, the block is porous and has a space inside for accommodating the biological tissue, and is connected from the outer surface of the block to the space to accommodate the biological tissue. It may include an inflow means that can open the cap and place the biological tissue on the seating portion, and the step of receiving the biological tissue in the block through the inflow means and then placing it on the seating portion. .

The electrophoresis device for biological tissue transparency according to the present invention and the method for biological tissue transparency using the same have the following effects.

First, because the density of equipotential lines passing through biological tissue can be increased compared to background technology, biological tissue can be made transparent more easily and quickly than compared to background technology. According to the experiment of the present invention, the mouse brain was able to become completely transparent in 8 hours at 80V and 10mA. This is an encouraging phenomenon compared to the fact that it took 5 to 9 days for background technology to make a mouse's brain transparent under the same electrical conditions.

Second, the transparency process of biological tissue can be easily observed with the naked eye by using a chamber made of transparent material and a light emitting means. As in the background technology, there is no need to have a camera or monitor to penetrate the inside of the chamber, so a small, lightweight, and compact design is possible.

Third, in order to increase the density of the equipotential lines , the present invention can naturally improve the density of the equipotential lines by using the protruding shape of the seating portion without increasing the intensity of the current. Therefore, the temperature of the buffer solution due to the intensity of the current is maintained lower than that of the background technology. For example, the room temperature is maintained at 25 ℃ to 30 ℃, and when the temperature rises further, the temperature is lowered to 20 ℃ to 26 ℃ through the fan, so the biological temperature is maintained. It can prevent the tissue from deteriorating at high temperatures. At this time, a small, lightweight and compact design is possible because a complex structure for circulating a buffer solution is not used as in the background technology.

Figure 1 is a photograph depicting an electrophoresis device for making biological tissue transparent using background technology.
Figure 2 is a flowchart showing the principle of an electrophoresis device for transparent biological tissue according to background technology.
Figure 3 is an exemplary diagram showing the process of making biological tissue transparent through an electrophoresis device for making biological tissue transparent according to background technology.
Figure 4 is an example diagram showing a state in which lipids remain in the biological tissue even though the biological tissue has gone through a process of becoming transparent through an electrophoresis device for biological tissue transparency according to background technology.
Figure 5 is a perspective view showing an electrophoresis device for making biological tissue transparent according to the present invention.
Figure 6 is a perspective view showing the cap separated from the chamber in the electrophoresis device for transparent biological tissue according to the present invention.
FIG. 7 is a cross-sectional view taken along line F-F' in FIG. 6.
Figure 8 is a perspective view showing an example in which a light emitting means is mounted on the inside of the cap of the electrophoresis device for making biological tissue transparent according to the present invention.
FIG. 9 is a cross-sectional view showing a biological tissue placed on the seating portion and filled with a buffer solution in the state of FIG. 7 and then undergoing a transparency process.
Figure 10 is a cross-sectional view showing the process of housing biological tissue in a porous block to place it in the electrophoresis device for transparent biological tissue according to the present invention.
Figure 11 is an exemplary diagram showing the process of making biological tissue transparent by the electrophoresis device for making biological tissue transparent according to the present invention, showing a state in which no lipids remain in the biological tissue.

Hereinafter, various embodiments of this document are described with reference to the attached drawings. However, this is not intended to limit the technology described in this document to specific embodiments, and should be understood to include various modifications, equivalents, and/or alternatives to the embodiments of this document. In connection with the description of the drawings, similar reference numbers may be used for similar components.

Additionally, expressions such as “first,” “second,” etc. used in this document may modify various components regardless of order and/or importance, and may be used to distinguish one component from another component. It is only used and does not limit the corresponding components. For example, 'first part' and 'second part' may refer to different parts, regardless of order or importance. For example, a first component may be renamed a second component without departing from the scope of rights described in this document, and similarly, the second component may also be renamed to the first component.

Additionally, the terms used in this document are merely used to describe specific embodiments and may not be intended to limit the scope of other embodiments. Singular expressions may include plural expressions, unless the context clearly indicates otherwise. Terms used herein, including technical or scientific terms, may have the same meaning as commonly understood by a person of ordinary skill in the technical field described in this document. Among the terms used in this document, terms defined in general dictionaries may be interpreted to have the same or similar meaning as the meaning they have in the context of related technology, and unless clearly defined in this document, have an ideal or excessively formal meaning. It is not interpreted as In some cases, even terms defined in this document cannot be interpreted to exclude embodiments of this document.

Hereinafter, a detailed embodiment of the electrophoresis device 1 for biological tissue transparency according to the present invention will be described along with the accompanying drawings.

Figure 5 is a perspective view showing the electrophoresis device for making biological tissue transparent according to the present invention, and Figure 6 is a perspective view showing the cap separated from the chamber in the electrophoresis device for making biological tissue transparent according to the present invention. In addition, Figure 7 is a cross-sectional view taken along line F-F' in Figure 6, and Figure 8 is a perspective view showing an example in which a light emitting means is mounted on the inside of the cap of the electrophoresis device for making biological tissue transparent according to the present invention. In addition, Figure 9 is a cross-sectional view showing the biological tissue placed on the mounting portion and filled with a buffer solution in the state of Figure 7 and then undergoing a transparency process, and Figure 10 shows the biological tissue being placed on the electrophoresis device for transparent biological tissue according to the present invention. This is a cross-sectional view showing the process of accommodating biological tissue into a porous block. In addition, Figure 11 is an example showing the process of making biological tissue transparent by the electrophoresis device for making biological tissue transparent according to the present invention, showing a state in which no lipids remain in the biological tissue.

A chamber 10 is formed that opens upward and is filled with a buffer solution (Q), which is an electrolyte, and a cap 50 covers the chamber 10.

The chamber 10 is formed with a seating portion 11 that protrudes upward from the bottom and has a flat upper surface so that biological tissue can be placed thereon, and a cathode electrode is disposed on both lower sides of the seating portion 11 and connected to a power source. (13) and the anode electrode (15) are comprised. The cathode electrode 13 and the anode electrode 15 have a wire shape and are preferably made of platinum for smooth electrical conduction. Then, it extends outward through the chamber 10 and is exposed, and an electric wire is connected to the extended portion to enable supply of direct current power. Of course, the portion where the cathode electrode 13 and the anode electrode 15 pass through the chamber 10 is finished to be watertight.

The cap 50 has a hole 51 drilled to open upward and downward, and a fan 55 is mounted in the hole 51. Accordingly, the buffer solution Q is configured to be cooled by blowing external air into the chamber 10 through the fan 55. In addition, a temperature sensor (not shown) is mounted on the cap 50 or chamber 10, and a control unit (not shown) is connected to the fan 55, so that the temperature of the buffer solution (Q) can be measured from the temperature sensor. It is configured to apply electricity to the fan 55 only when the received temperature is above a specified temperature.

The chamber 10 is made of a transparent material such as synthetic resin such as acrylic or glass so that the interior can be observed with the naked eye.

In addition, as shown in FIGS. 7 and 9, a light emitting means 20 is mounted on the chamber 10 and irradiates light into the chamber 10. The light emitting means 20 is capable of receiving electricity and emitting light, and may be configured as an LED, for example. In addition, an accommodating part 12 is formed in the lower part of the chamber 10, and the light emitting means 20 is configured to be accommodated in the accommodating part 12. Accordingly, light is irradiated from the lower part of the chamber 10, and at this time, the seating portion 11 is also formed of the transparent material so that the light can also shine on the biological tissue (L). Alternatively, as shown in FIG. 8, the light emitting means 20 may be mounted inside the cap 50. At this time, it is mounted on the inner side of the cap 50 so as not to block the fan 55 and is configured to illuminate downward.

In addition, as shown in FIG. 10, a block 30 is formed that is seated on the upper part of the seating portion 11 and holds the biological tissue (L). The block 30 is made of a porous material such as a sponge or scrubber and is made of a flexible material with a space 31 inside to accommodate the biological tissue (L), and is connected from the outer surface to the space 31 to allow the biological tissue (L) to flow in. An inflow means 33 that can be used is formed. The inflow means 33 may be formed as a slit, for example. Therefore, the slit can be opened to accommodate the biological tissue (L). Instead of the slit, it may be formed as a hole.

The method for making biological tissue transparent using the electrophoresis device for making biological tissue transparent according to the present invention is as follows.

The drawings refer to FIGS. 5 to 11 and illustrate a process using the electrophoresis device 1 for making biological tissue transparent.

The step of opening the cap 50 and placing the biological tissue (L) on the seating portion 11 is performed. At this time, the biological tissue (L) is placed in the block 30 in a housed state. That is, the slit, which is the inlet means 33, is opened and the biological tissue L is accommodated in the space 31 inside the block 30. Accordingly, the biological tissue (L) is floating on the upper part of the seating portion (11). If the biological tissue (L) contacts the seating portion (11) without the block (30), lipid (O) remains in the area adjacent to the contact portion.

Next, the step of filling the chamber 10 with a buffer solution (Q), which is an electrolyte, is performed. At this time, the buffer solution used is the composition for making biological tissue transparent presented in Korean Patent Registration No. 10-2296381. That is, a compound containing 4 to 50 w/v% of the compound represented by the chemical symbol below or its hydrate and 20 to 60 w/v% of urea is used.

[Chemical formula]

Fill the buffer solution (Q) so that the block 30 can be sufficiently submerged. At this time, since the block 30 is porous, the biological tissue (L) is in the same state as if it were immersed in the buffer solution (Q).

Next, the cap 50 is closed and direct current electricity is applied to the cathode electrode 13 and the anode electrode 15. Then, a current flows from the anode electrode 15 to the cathode electrode 13 due to the buffer solution (Q), which is an electrolyte, and as shown in FIG. 9, the cathode electrode 13 and the anode electrode 15 form a concentric equipotential line ( M) is generated, which spreads outward like a wave, forming an electric field inside the chamber 10. However, at this time, the equipotential line (M) cannot spread out in its original concentric shape because the seating portion (11) becomes an obstacle, and the density of the equipotential lines (M) increases as the spacing between the equipotential lines (M) near the seating portion (11) narrows. Accordingly, the equipotential lines M close to the vertical line are densely formed on the upper part of the seating portion 11. When the equipotential lines (M) pass through the biological tissue (L) in a dense state, the negative charge passes through the block 30 along the plurality of equipotential lines (M), penetrates the biological tissue (L), and then surrounds the lipids. They are wrapped to form micelles and move out of the biological tissue (L) to the anode electrode (15), making the biological tissue (L) transparent. Therefore, lipid (O) can be removed more easily and quickly compared to the background technology.

In addition, the step of applying electricity to the light emitting means 20 to illuminate the inside of the chamber 10 with light for observation may be further included.

Next, when the temperature of the buffer solution (Q) is above the specified temperature, the fan 55 is driven by the temperature sensor and the control unit. When the temperature falls below the specified temperature, a cooling step is performed in which the fan 55 is turned off.

According to the present invention described above, the density of the equipotential line (M) passing through the biological tissue (L) can be increased compared to the background technology, so that the biological tissue (L) can be made transparent more easily and quickly than the background technology. . According to the experiment of the present invention, the mouse brain was able to become completely transparent in 8 hours at 80V and 10mA. This is an encouraging phenomenon compared to the fact that it took 5 to 9 days for background technology to make a mouse's brain transparent under the same electrical conditions.

In addition, the transparent process of the biological tissue (L) can be easily observed with the naked eye by the chamber 10 and the light emitting means 20 made of transparent material. As in the background technology, there is no need to have a camera or monitor to penetrate the inside of the chamber, so a small, lightweight, and compact design is possible.

In addition, the present invention can naturally improve the density of the equipotential lines (M) by using the protruding shape of the seating portion 11 without increasing the intensity of the current in order to increase the density of the equipotential lines (M) . Therefore, the temperature of the buffer solution (Q) due to the intensity of the current is maintained lower than that of the background technology, for example, maintained at 25°C to 30°C, and when the temperature rises further, it is increased to 20°C to 26°C through the fan 55. By lowering the temperature, it is possible to prevent the biological tissue (L) from being deteriorated due to high temperature. At this time, a small, lightweight and compact design is possible because a complex structure for circulating the buffer solution (Q) is not used as in the background technology.

1: Electrophoresis device for biological tissue transparency 10: Chamber
11: Seating part 12: Storage part
13: cathode electrode 15: anode electrode
20: light emitting means 30: block
31: Space 33: Inflow means
50: cap 51: hole
55: Pan Q: Buffer solution
M: Equipotential line L: Biological tissue
O: lipid

Claims (10)

a chamber open upward,
Includes a cap covering the chamber,
The chamber is,
A seating portion that protrudes upward from the bottom surface and is formed to place biological tissue on it,
It includes a cathode electrode and an anode electrode disposed on both lower sides of the seating portion and connected to a power source,
The cap has holes drilled in the upper and lower directions,
Including a fan mounted in the hole,
The chamber is made of a transparent material,
It includes a light emitting means mounted on the chamber and irradiating light into the chamber,
It includes a light emitting means mounted on the inside of the cap and emitting light downward,
It includes a block that is seated on the upper part of the seating portion and holds biological tissue,
The block is porous and has a space inside for accommodating biological tissue,
An electrophoresis device for making biological tissue transparent, comprising an inlet means connected from the outer surface of the block to the space to accommodate biological tissue.
delete delete delete delete delete a chamber open upward,
Includes a cap covering the chamber,
The chamber is,
A seating portion that protrudes upward from the bottom surface and is formed to place biological tissue on it,
A method of making biological tissue transparent using an electrophoresis device for making biological tissue transparent, comprising a cathode electrode and an anode electrode disposed on both lower sides of the seating portion and connected to a power source,
Opening the cap and placing biological tissue on the seating portion;
Filling the chamber with a buffer solution, which is an electrolyte;
Closing the cap and applying electricity to the cathode electrode and the anode electrode ,
The cap has holes drilled in the upper and lower directions,
Including a fan mounted in the hole,
In the step of applying electricity, driving the fan to cool the buffer solution,
The chamber is made of a transparent material,
It includes a light emitting means that irradiates light into the chamber,
After the step of applying electricity, it includes the step of applying electricity to the light emitting means to observe the inside of the chamber,
It includes a block that is seated on the upper part of the seating portion and holds biological tissue,
The block is porous and has a space inside for accommodating biological tissue,
It includes an inlet means connected from the outer surface of the block to the space to accommodate biological tissue,
An electrophoresis device for making biological tissue transparent, comprising the step of opening the cap and placing the biological tissue on the seating portion, receiving the biological tissue in the block through the inlet means, and then placing the biological tissue on the seating portion. A method of making biological tissue transparent using . delete delete delete

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