Staining apparatus and staining method

Abstract

To provide a staining device capable of speeding up the staining of a biological tissue.SOLUTION: The staining apparatus 1 includes a cathode chamber 2, an anode chamber 4, a stain solution 21 containing an ionic dye 23 and contained in one of the cathode chamber 2 and the anode chamber 4, a buffer solution 22 contained in the other of the cathode chamber 2 and the anode chamber 4, a first electrode 11 immersed in the stain solution 21, a second electrode 12 immersed in the buffer solution 22, and a communicating tube 3 connecting the cathode chamber 2 and the anode chamber 4. When the living tissue 20 is arranged so as to block the cross section of the communicating tube 3 and electricity is made to flow between the first electrode 11 and the second electrode 12, the dye 23 is electrophoresed so as to pass through the living tissue 20.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a dyeing apparatus and a dyeing method. [Background technology]

[0002] Micro-Computed Tomography (Micro-CT) is used for non-destructive imaging of three-dimensional microstructures in both industrial products and biological tissues. When imaging soft tissues such as biological tissues and blood vessels, the difference in X-ray attenuation coefficients between tissues is small, so the contrast of the micro-CT images of soft tissues is significantly lower than that of industrial products.

[0003] One way to improve contrast is to use stains containing molecules or ions with high X-ray attenuation coefficients. These stains, such as Lugol's solution or phosphotungstic acid solution, have the advantage of being inexpensive and easy to use.

[0004] However, to obtain micro-CT images with sufficient contrast, the specimen must be immersed in the staining solution for a long period of time, which can take anywhere from half a day to several days. Considering future applications in large-scale data analysis of biological tissues and clinical applications, it is desirable to speed up the staining time.

[0005] In technical fields other than micro-CT, a method has been proposed for unblocking staining, which is performed as a preliminary step to electron microscopy. This involves immersing a tissue sample in a staining solution in a tube, such as a tube cell electrophoresis assembly, and staining it by electrophoresis (see, for example, Patent Document 1). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 6,409,774 Summary of the Invention [Problem to be solved by the invention]

[0007] However, the technique described in Patent Document 1 is designed for antibody staining, and although it can stain samples smaller than a tube for use with an electron microscope, it is difficult to stain biological tissues of a size suitable for micro-CT. Furthermore, because the dye precursor and the dye molecule or the second dye precursor, which move in opposite directions due to electrophoresis, bind to each other within the sample and develop color, there are significant limitations on the options for antibodies, etc.

[0008] The present invention has been made in view of the above-mentioned problems, and has an object to provide a staining apparatus and a staining method that can increase the speed of staining biological tissue. [Means for solving the problem]

[0009] A staining device according to one aspect of the present invention includes a cathode chamber, an anode chamber, a staining solution containing an ionic dye and contained in one of the cathode chamber and the anode chamber, a buffer solution contained in the other of the cathode chamber and the anode chamber, a first electrode immersed in the staining solution, a second electrode immersed in the buffer solution, and a connecting tube connecting the cathode chamber and the anode chamber. When biological tissue is placed so as to block the cross section of the connecting tube and electricity is applied between the first electrode and the second electrode, the dye is electrophoresed so as to pass through the biological tissue.

[0010] A staining method according to another aspect of the present invention includes placing biological tissue so as to block a cross section of a connecting pipe connecting a cathode chamber and an anode chamber; pouring a staining solution containing an ionic dye into one of the cathode chamber and the anode chamber; pouring a buffer solution into the other of the cathode chamber and the anode chamber; and passing electricity between a first electrode immersed in the staining solution and a second electrode immersed in the buffer solution to cause the dye to permeate the biological tissue by electrophoresis.

[0011] According to these aspects, the ionic dye penetrates into the biological tissue due to the electric field generated between the first electrode and the second electrode, which allows for faster staining of the biological tissue compared to conventional techniques in which the dye penetrates into the biological tissue solely by the diffusion of the dye without using an electric field.

[0012] In the above embodiment, the staining solution may be Lugol's solution, and the first electrode may be a cathode.

[0013] According to this aspect, it is possible to speed up staining of biological tissue using an easily available and inexpensive staining solution.

[0014] In the above aspect, the apparatus may further include a cooling device that cools at least one of the staining solution and the buffer solution.

[0015] According to this aspect, even if Joule heat is generated by passing electricity through the staining solution or buffer solution, the temperature rise can be suppressed so that the staining solution or buffer solution does not become excessively hot, and biological tissue can be prevented from being heated and deformed by the staining solution or buffer solution.

[0016] In the above embodiment, at least one of the first electrode and the second electrode may be a carbon electrode or a platinum electrode.

[0017] According to this embodiment, even when electricity is applied to the staining solution or buffer solution, the first electrode or the second electrode is unlikely to dissolve.

[0018] In the above aspect, the communicating pipe may be configured to be detachable from the cathode chamber and the anode chamber.

[0019] According to this aspect, when inserting biological tissue into the communicating tube or cleaning the communicating tube, the communicating tube can be removed from the cathode chamber or the anode chamber for the work. Old communicating tubes can also be replaced with new ones.

[0020] In the above aspect, another communicating pipe having a cross-sectional shape different from that of the communicating pipe may be further provided, and the other communicating pipe may be configured to be attachable to and detachable from the cathode cell and the anode cell in place of the communicating pipe.

[0021] According to this aspect, it is possible to change the size or shape of the biological tissue by replacing the communicating tube.

[0022] In the above aspect, a gel-like filler may be filled in the gap between the inner wall of the communicating tube and the biological tissue.

[0023] According to this aspect, even if a gap occurs between the inner wall of the communicating tube and the biological tissue, the outer shape of the biological tissue can be complemented with the filler, and the biological tissue can be positioned so as to block the cross section of the communicating tube. [Effects of the Invention]

[0024] According to the present invention, it is possible to provide a staining device and a staining method that can speed up staining of biological tissue. [Brief explanation of the drawings]

[0025] [Figure 1] FIG. 1 is a perspective view showing an example of a staining apparatus according to an embodiment of the present invention. [Figure 2] FIG. 2 is a perspective view showing a modification of the staining apparatus shown in FIG. [Figure 3] FIG. 3 is a flow chart showing an example of a staining method according to one embodiment of the present invention. [Figure 4] FIG. 4 is a diagram showing a CT image obtained in Example 1. [Figure 5] FIG. 5 is a diagram showing a CT image obtained in Example 2. [Figure 6] FIG. 6 is a diagram showing a CT image obtained in Example 3. [Figure 7] FIG. 7 is a diagram showing a CT image obtained in Example 4. [Figure 8] FIG. 8 is a diagram showing a CT image obtained in Comparative Example 1. [Figure 9] FIG. 9 is a diagram showing a CT image obtained in Comparative Example 2. [Figure 10] FIG. 10 is a diagram showing the CT values ​​of the CT images shown in FIGS. DETAILED DESCRIPTION OF THE INVENTION

[0026] A preferred embodiment of the present invention will be described with reference to the accompanying drawings. In each drawing, components with the same reference numerals have the same or similar configurations. Fig. 1 is a perspective view showing an example of a staining apparatus 1 according to one embodiment of the present invention.

[0027] As shown in FIG. 1, the dyeing apparatus 1 includes a cathode chamber 2, an anode chamber 4, a dyeing solution 21 contained in either the cathode chamber 2 or the anode chamber 4, a buffer solution 22 contained in the other of the cathode chamber 2 or the anode chamber 4, a first electrode 11 immersed in the dyeing solution 21, a second electrode 12 immersed in the buffer solution 22, and a connecting pipe 3 connecting the cathode chamber 2 and the anode chamber 4.

[0028] An example of the buffer solution 22 is a Tris-HCl buffer solution. The type of buffer solution 22 is not particularly limited, and any known buffer solution can be appropriately selected. The staining solution 21 contains an ionic dye 23. The dye 23 may be a dye containing an anionic atom or atomic group, or a dye containing a cationic atom or atomic group.

[0029] In the illustrated example, the staining solution 21 is Lugol's solution, and the ionic dye 23 is triiodide ion I3 - The staining solution 21 is not limited to Lugol's solution, but may be a polyacid such as a phosphotungstic acid solution. If the staining solution 21 is Lugol's solution and the first electrode 11 is a cathode, the staining of the biological tissue 20 can be accelerated using the staining solution 21, which is easy to obtain and inexpensive.

[0030] Dye 23 is a polyatomic anion, triiodide ion I3 - In the case of dyes such as those mentioned above, the first electrode 11 immersed in the dye solution 21 is connected to the negative pole of the external power supply 10 and serves as a cathode. In the case of dyes such as those mentioned above, the first electrode 11 immersed in the dye solution 21 is connected to the positive pole of the external power supply 10 and serves as an anode.

[0031] In this specification, in a configuration in which the first electrode 11 serves as a cathode, the electrode tank containing the first electrode 11 is referred to as a cathode tank. In a configuration in which the first electrode 11 serves as an anode, the electrode tank containing the first electrode 11 is referred to as an anode tank.

[0032] The first and second electrodes 11, 12 are made of, for example, carbon or platinum electrodes. Such electrodes make it difficult for the first and second electrodes 11, 12 to dissolve even when electricity is passed through the staining solution 21 or the buffer solution 22. In the illustrated example, both the first and second electrodes 11, 12 are made of carbon electrodes.

[0033] In the illustrated example, the staining apparatus 1 further includes a cooling device 5 that cools at least one of the staining liquid 21 and the buffer solution 22. When electricity is passed through the staining liquid 21 or the buffer solution 22, Joule heat is generated, causing the liquid temperature to rise. With the cooling device 5, even if Joule heat is generated when electricity is passed through the staining liquid 21 or the buffer solution 22, the temperature rise can be suppressed so that the staining liquid 21 or the buffer solution 22 does not become excessively hot. This can prevent biological tissue from being heated and deformed by the staining liquid 21 or the buffer solution 22.

[0034] The communicating tube 3 is made of an insulating material such as a resin material. FIG. 2 is a perspective view showing a modified example of the staining apparatus 1 shown in FIG. 1. In this modified example, the communicating tube 3A is configured to be detachable from the cathode chamber 2 and the anode chamber 4. According to this modified example, when inserting the biological tissue 20 into the communicating tube 3 or cleaning the communicating tube 3, the communicating tube 3 can be removed from the cathode chamber 2 or the anode chamber 4. An old communicating tube 3 can also be replaced with a new communicating tube 3.

[0035] Furthermore, a modified example may further include another communicating pipe 3B having a cross-sectional shape different from that of the communicating pipe 3A, and the other communicating pipe 3B may be configured to be attachable to and detachable from the cathode chamber 2 and the anode chamber 4 instead of the communicating pipe 3A. In the illustrated example, the communicating pipe 3A has a circular cross-section, and the other communicating pipe 3B has a square cross-section.

[0036] The cathode chamber 2 and the anode chamber 4 are formed with openings 13A and 13B corresponding to the respective communicating tubes 3A and 3B. Unused openings 13A and 13B are closed by lids 14A and 14B. The size and shape of the biological tissue 20 can be changed by replacing the communicating tube 3. The cross sections of the communicating tubes 3A, 3B, etc. may be circular with various diameters or may have other shapes. In this case, multiple openings 13A, 13B, etc. may be formed in a line.

[0037] If a gap occurs between the inner wall of the communicating tube 3A and the biological tissue 20, the gap may be filled with a gel-like filler 24. The filler 24 complements the outer shape of the biological tissue 20, and the biological tissue 20 can be arranged so as to close the cross section of the communicating tube 3A. An example of the filler 24 is polyacrylamide gel.

[0038] 3 is a flow chart showing an example of a staining method according to one embodiment of the present invention. In the present invention, first, biological tissue 20 is placed so as to block the cross section of the communicating tube 3 connecting the cathode chamber 2 and the anode chamber 4 (Step S1). Next, a staining solution containing an ionic dye is poured into either the cathode chamber 2 or the anode chamber 4 (Step S2). A buffer solution 22 is poured into the other of the cathode chamber 2 or the anode chamber 4 (Step S3). Electricity is applied between a first electrode 11 immersed in the staining solution 21 and a second electrode 12 immersed in the buffer solution 22, causing the dye 23 to permeate the biological tissue 20 by electrophoresis (Step S4). [Example]

[0039] The present invention will be described below based on examples and comparative examples, but the present invention is not limited to the following examples.

[0040] [Example 1] 1. Sample Preparation A porcine liver block (refrigerated) was cut into pieces approximately 50 mm wide and fixed in 10% neutral buffered formalin. After fixation, the pieces were washed with tap water for 1 hour, and then cylindrically cut using an 8 mm diameter biopsy trephine. These pieces were then cut into pieces approximately 14 mm long to prepare biological tissue samples. The samples were then immersed in 70% ethanol and stored refrigerated.

[0041] 2. Preparation of Staining Solutions and Buffer Solutions Lugol's solution was used as the staining solution. First, potassium iodide was dissolved in ultrapure water to prepare a potassium iodide solution, and then half the amount of iodine was dissolved in the potassium iodide to prepare 1.0% Lugol's solution and 3.0% Lugol's solution, respectively. The prepared Lugol's solution was placed in a light-shielding bottle and stored in a dark place. 100 mM Tris-HCl buffer was also prepared.

[0042] 3. Preparation of electrophoresis tank An electrophoresis chamber equipped with the aforementioned cathode chamber, anode chamber, and connecting tube was produced using a 3D printer. The inner dimensions of the cathode chamber and anode chamber are 30 mm wide, 40 mm deep, and 25 mm high. The connecting tube is cylindrical, with an inner diameter of 8 mm and a length of 20 mm. A cathode and an anode were placed in the cathode chamber and the anode chamber, respectively, and connected to an external power supply.

[0043] 4. Staining by Electrophoresis The prepared sample was inserted into the connecting tube, and 3.0% Lugol's solution was added to the cathode chamber, and 100 mM Tris-HCl buffer solution was added to the anode chamber. Electrophoresis was performed at a maximum voltage of 100 V for 20 minutes to stain the sample.

[0044] 5. Micro-CT Imaging To prevent saturation of the CT value on the sample surface, the specimen was washed with tap water for five minutes after staining and then imaged using a CosomoScan GX (Rigaku Corporation) (tube voltage 50 kV, tube current 160 μA, FOV 36 mm, pixel size 36 μm). The projection data obtained from the image were reconstructed using analytical reconstruction methods to obtain the CT image shown in Figure 4. The CT values ​​plotted on the straight line in Figure 4 are shown in Figure 10.

[0045] [Example 2] Staining was performed in the same manner as in Example 1, except that 3.0% Lugol's solution was added to the cathode chamber instead of 1.0%, and the CT image shown in Fig. 5 was obtained by micro-CT photography in the same manner as in Example 1. The CT values ​​plotted on the straight line in Fig. 5 are shown in Fig. 10.

[0046] [Example 3] Staining was performed in the same manner as in Example 1, except that 3.0% Lugol's solution instead of 1.0% was added to the cathode chamber, and the cathode chamber, the anode chamber, and the connecting tube were cooled in an ice-water bath. The CT image shown in FIG. 6 was obtained by micro-CT photography in the same manner as in Example 1.

[0047] [Example 4] Staining was performed in the same manner as in Example 1, except that 5.0% Lugol's solution instead of 1.0% was added to the cathode chamber, and the cathode chamber, the anode chamber, and the connecting tube were cooled in an ice-water bath. The CT image shown in FIG. 7 was obtained by micro-CT photography in the same manner as in Example 1.

[0048] [Comparative Example 1] The sample was immersed in 1.0% Lugol's solution for 24 hours, and the CT image shown in Fig. 8 was obtained by micro-CT photography in the same manner as in Example 1. The CT values ​​plotted on the straight line in Fig. 8 are shown in Fig. 10.

[0049] Comparative Example 2 The sample was immersed in 1.0% Lugol's solution for 20 minutes, and the CT image shown in Fig. 9 was obtained by micro-CT photography in the same manner as in Example 1. The CT values ​​plotted on the straight line in Fig. 9 are shown in Fig. 10.

[0050] As shown in Figure 10, in Example 1, the CT value increases even after 20 minutes of staining, approaching the CT value of Comparative Example 1, which was stained for 24 hours using a conventional method. Therefore, a micro-CT image with sufficient contrast can be obtained. In Example 4, which uses a higher concentration of Lugol's solution than in Example 1, the CT value approaches that of Comparative Example 1 even more. Increasing the concentration of Lugol's solution tends to increase the staining efficiency. Example 2 and Comparative Example 2 use the same concentration of Lugol's solution and have the same immersion time. Despite this, Example 2 has a larger CT value than Comparative Example 2.

[0051] In Examples 2 and 3, the Lugol's solution was used at the same concentration and for the same immersion time. As shown in Figures 5 and 6, the specimen in Example 3 showed less deformation and almost no damage compared to Example 2. Cooling the staining apparatus can prevent specimen deterioration.

[0052] According to the staining apparatus 1 of the present invention configured as described above and staining methods S1 to S4 using the apparatus, the ionic dye 23 is permeated into the biological tissue 20 by the electric field generated between the first electrode 11 and the second electrode 12. This allows for faster staining of biological tissue compared to conventional techniques in which the dye 23 is permeated into the biological tissue 20 solely by the diffusion action of the dye 23 without using an electric field. The present invention is particularly suitable for micro-CT imaging because it can stain biological tissue that is larger than that of a tube cell.

[0053] The above-described embodiments are intended to facilitate understanding of the present invention and are not intended to limit the present invention. The elements of the embodiments, as well as their arrangement, materials, conditions, shapes, sizes, etc., are not limited to those illustrated and can be modified as appropriate. Furthermore, configurations shown in different embodiments can be partially substituted or combined with each other. [Explanation of symbols]

[0054] 1...staining device, 2...cathode chamber, 3, 3A...connecting pipe, 3B...other connecting pipe, 4...anode chamber, 5...cooling device, 10...external power supply, 11...first electrode, 12...second electrode, 13A, 13B...opening, 14A, 14B...lid, 20...biological tissue, 21...staining solution, 22...buffer solution, 23...dye, 24...filler

Claims

1. a cathode chamber; an anode cell; a dyeing solution containing an ionic dye and placed in one of the cathode chamber and the anode chamber; a buffer solution placed in the other of the cathode chamber and the anode chamber; a first electrode immersed in the dye solution; a second electrode immersed in the buffer solution; a communicating pipe connecting the cathode chamber and the anode chamber, When a biological tissue is placed so as to block the cross section of the communicating tube and an electric current is applied between the first electrode and the second electrode, the dye is electrophoresed so as to penetrate the biological tissue. Dyeing equipment.

2. the staining solution is Lugol's solution, and the first electrode is a cathode; The dyeing apparatus according to claim 1 .

3. The apparatus further includes a cooling device for cooling at least one of the staining solution and the buffer solution. The dyeing apparatus according to claim 1 .

4. At least one of the first electrode and the second electrode is a carbon electrode or a platinum electrode. The dyeing apparatus according to claim 1 .

5. The communicating pipe is configured to be detachably attached to the cathode chamber and the anode chamber. The dyeing apparatus according to claim 1 .

6. Further provided is another communicating pipe having a cross-sectional shape different from that of the communicating pipe, The other communicating pipe is configured to be detachably attached to the cathode cell and the anode cell in place of the communicating pipe. The dyeing apparatus according to claim 5.

7. A gel-like filler is filled in the gap between the inner wall of the communicating tube and the biological tissue. The dyeing apparatus according to claim 1 .

8. Placing the biological tissue so as to block the cross section of the communicating tube connecting the cathode tank and the anode tank; placing a dyeing solution containing an ionic dye in either the cathode chamber or the anode chamber; placing a buffer solution in either the cathode chamber or the anode chamber; and and passing an electric current between a first electrode immersed in the staining solution and a second electrode immersed in the buffer solution to cause the dye to permeate the biological tissue by electrophoresis. Dyeing method.

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