High-speed fluorescent labeling technique

Abstract

The present invention relates to a high-speed human tissue cell fluorescence-labeling technique for fluorescence imaging of human tissue and, more particularly, to a method capable of obtaining fluorescence imaging information by rapidly staining cell nuclei and cell substrates of excised tissue (ex vivo) through a rapid fluorescence labeling technique within a few minutes. The method according to the present invention can provide a high-speed fluorescence labeling technique capable of overcoming the limitations of a conventional H&E staining method, greatly shortening the time required for staining to within a few minutes, and lowering the risk of tissue loss.

Description

High-speed fluorescent labeling technique

[0001] This application claims priority to Korean Patent Application No. 10-2024-0202101 filed on December 31, 2024, and the entire specification is a reference to this application. The present invention relates to a high-speed fluorescent labeling technique for human tissue cells for fluorescent imaging of human tissue, and in particular to a method for obtaining fluorescent image information by rapidly staining the cell nucleus and cytoplasm of excised tissue (ex vivo) within minutes using a rapid fluorescent labeling technique.

[0002]

[0003] In tissue cell staining, the traditional H&E (hematoxylin & eosin) staining method is a widely used tissue staining method in pathological analysis. Hematoxylin stains the cell nuclei of excised tissue purple, and eosin stains the cytoplasm pink, providing useful information for observing the respective tissue structures. Depending on the sectioning method used, this H&E staining method is classified into two types: Frozen H&E and Formalin Fixed Paraffin-Embedded H&E (FFPE H&E). These two methods differ in the time required and quality according to their respective advantages and disadvantages.

[0004] First, Frozen H&E is a method that involves rapidly freezing and fixing tissue for staining after sectioning. Specifically, the excised tissue is rapidly frozen at -80 to -20°C using an OCT compound, and then very thin section slides with a thickness of 4 to 10 μm are prepared using a cryosection instrument. Next, the section slides are immersed in a formalin solution for several minutes to fix the tissue, and after washing, the cell nuclei are stained with hematoxylin. After washing again and dehydrating by immersion in ethanol, the cytoplasm is stained with eosin and dehydrated once more by immersion in ethanol. After washing, the tissue section is placed on a slide glass, covered with a cover glass, and observed under a microscope; this process takes at least 30 minutes.

[0005] Next, FFPE H&E is a method that involves creating paraffin blocks after tissue fixation and staining after sectioning. Specifically, the tissue to be observed is fixed with formalin, embedded in paraffin, and sectioned. Then, a microtome is used to prepare very thin section slides with a thickness of 4 to 10 μm. The section slides are immersed in a formalin solution for several minutes to fix the tissue, and after washing, the cell nuclei are stained with hematoxylin. After washing, the slides are immersed in ethanol for dehydration, the cytoplasm is stained with eosin, and then immersed in ethanol for dehydration again. After washing, the sectioned tissue is placed on a slide glass, covered with a cover glass, and observed under a microscope; however, the fixation and paraffin embedding processes themselves can take anywhere from several hours to several days.

[0006] To summarize the above, it takes anywhere from 30 minutes to several days to stain excised tissue with H&E, and the process of slicing the tissue into very thin sections using sectioning equipment requires specialized skills. In other words, since the section preparation process is influenced by the researcher's proficiency, there is a very high risk of tissue loss if performed by an inexperienced person. These issues highlight the need for a rapid and high-quality staining method.

[0007] The inventors have developed a high-speed fluorescent labeling technique that improves upon the problems of traditional H&E staining methods, reduces time consumption and the risk of tissue loss, eliminates a series of labor-intensive processes requiring specialized skills to enable rapid staining even by unskilled workers, and demonstrates superior effects compared to existing staining methods.

[0008]

[0009] The present invention aims to provide a high-speed fluorescent labeling technique that improves upon the problems of the traditional H&E staining method described above, significantly reduces the time required for staining, and lowers the risk of tissue loss.

[0010]

[0011] To achieve the above objective, the present invention provides a high-speed fluorescence labeling technique comprising: (S1) a step of preparing a mixture by mixing a tissue fixative, a cell nucleus label, and a cell matrix label; (S2) a step of immersing or applying a tissue to be observed in the mixture; and (S3) a step of washing the tissue to be observed in 1XPBS.

[0012] In the present invention, the tissue fixative may be acetone.

[0013] In addition, in the present invention, the cell nucleus label may be SYTO9, Acridine Orange, or DAPI, wherein the concentration of SYTO9 may be 0.05 to 0.1 mM, the concentration of Acridine Orange may be 0.005 to 0.1 w / v%, or the concentration of DAPI may be 2 to 5 mg / ml.

[0014] In addition, in the present invention, the cell substrate label may be Rhodamine B, and the concentration of Rhodamine B may be 0.1 to 0.3 mg / ml.

[0015] In addition, steps (S1) to (S3) in the present invention may be performed for 30 seconds to 5 minutes.

[0016]

[0017] The method according to the present invention can overcome the limitations of conventional H&E staining methods, significantly reduce the time required for staining to within a few minutes, and provide a high-speed fluorescent labeling technique that can lower the risk of tissue loss.

[0018]

[0019] Figures 1a to 1c show a comparison of the respective fluorescence signals when formalin, methanol, ethanol, or acetone were used as tissue fixatives.

[0020] Figure 2 shows the time required for tissue staining commonly used in stages.

[0021] Figure 3 shows the time required for the high-speed fluorescent labeling technique according to the present invention in steps.

[0022] Figures 4a and 4b show a comparison of fluorescence signals according to the concentration of the fluorescent label.

[0023] Figures 5a to 5c show a comparison of fluorescence signals according to staining time for each fluorescent label.

[0024] Figures 6a to 6c show a comparison of fluorescence signals according to staining time when a mixture of cell nuclear labeling solution and cell matrix labeling solution is used.

[0025]

[0026] The terms used in this invention are used merely to describe specific embodiments and are not intended to limit the invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this application, terms such as "to constitute," "to provide," "to include," or "to have" are intended to specify the existence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0027] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which this invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this invention.

[0028]

[0029] In one embodiment according to the present invention, the present invention provides a high-speed fluorescence labeling technique comprising: (S1) a step of preparing a mixture by mixing a tissue fixative, a cell nucleus labeling solution, and a cell matrix labeling solution; (S2) a step of immersing or applying a tissue to be observed in the mixture; and (S3) a step of washing the tissue to be observed in 1XPBS.

[0030] The present invention enables high-speed fluorescence staining by omitting or shortening parts of the processes required in conventional tissue cell staining. Specifically, it eliminates essential processes such as paraffin embedding and ethanol dehydration found in conventionally used staining methods, and also allows for the omission of the section preparation process. To explain the omission of the section preparation process in more detail, in the case of Frozen H&E, tissues must be frozen to prepare sections, and in the case of FFPE H&E, sections must be prepared after tissue fixation and paraffin block formation. Therefore, the section preparation process is mandatory in conventional technology. However, since this process requires specialized skills, the quality of the tissue sections is determined by proficiency, and there is a significant risk of tissue loss during the preparation process. In contrast, the present invention does not require section preparation, and the excised tissue itself can be stained directly.

[0031] In another embodiment according to the present invention, the tissue fixative may be acetone.

[0032] In the case of the present invention, staining efficiency can be improved by using acetone to resolve tissue fixation and permeabilization. To explain in more detail, conventional staining methods require a fixation process before staining the tissue, and typically, formalin, PFA, methanol, ethanol, etc., are used for tissue fixation. When the target to be fluorescently labeled is an intracellular component such as a cell nucleus, a permeabilization process and a solvent are required to allow the fluorescent label to penetrate into the cell; in this case, additional time is required and the total staining time is extended. However, when acetone is used as in the present invention, not only fixation but also permeabilization is possible simultaneously, so a separate permeabilization step is not required.

[0033] In another embodiment according to the present invention, the cell nucleus label may be SYTO9, Acridine Orange, or DAPI, wherein the concentration of SYTO9 may be 0.05 to 0.1 mM, the concentration of Acridine Orange may be 0.005 to 0.1 w / v%, or the concentration of DAPI may be 2 to 5 mg / ml.

[0034] In another embodiment according to the present invention, the cell substrate label may be Rhodamine B, and the concentration of Rhodamine B may be 0.1 to 0.3 mg / ml.

[0035] In conventional tissue staining, histological analysis was performed by observing the cell nucleus and cytoplasm using hematoxylin, which labels the cell nucleus, and eosin, which labels the cytoplasm. However, in the present invention, various fluorescence labels for the cell nucleus and cytoplasm that can be used in a fluorescence microscope can be used to obtain image information of the cell nucleus and cytoplasm that is superior to or equivalent to that of conventional staining.

[0036] In another embodiment according to the present invention, steps (S1) to (S3) may be performed for 30 seconds to 5 minutes.

[0037] In conventional staining methods, the process involved sequentially staining with formalin, hematoxylin, and eosin, but in the present invention, the staining time is shortened by applying a method of immersing or applying a mixture of acetone, a cell nucleus labeling solution, and a cell matrix labeling solution to the tissue. Accordingly, the total staining time, which took at least 30 minutes to several days in conventional technology, can be significantly reduced to 1 to 2 minutes in the present invention.

[0038]

[0039] The present invention will be described in more detail below through specific examples. These examples are intended solely to illustrate the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples. Additionally, the unit of additives not specifically stated in the specification may be w / v%.

[0040]

[0041] Example 1. Selection of Tissue Fixative

[0042] When tissue fixation was performed using various solvents such as formalin, paraformaldehyde (PFA), methanol, ethanol, and acetone, which are used as solutions for fixing tissues, it was confirmed that when formalin, methanol, and ethanol were used as tissue fixatives, the fluorescence signal of the cell nucleus was very weak compared to acetone (Figs. 1a to 1c). This means that in the case of formalin, methanol, and ethanol, the cell nucleus label did not penetrate into the cell, so the cell nucleus was not stained. Accordingly, in order to fluorescently label intracellular components such as the cell nucleus after tissue fixation, an additional permeabilization step and solvent are required to allow the fluorescence label to penetrate into the cell. However, since acetone possesses both fixation and permeabilization functions and has the advantage of not requiring a separate permeabilization step, acetone was applied as the tissue fixative for tissue fixation in this invention.

[0043]

[0044] Example 2. Comparison of time required for general dyeing and high-speed fluorescent labeling techniques

[0045] The time required for each step of the tissue staining commonly used is shown in Figure 2.

[0046] On the other hand, the high-speed fluorescent labeling technique according to the present invention consists of a total of two steps: a tissue staining step and a washing step. The reagents required for tissue staining are acetone, a cell nucleus labeling solution, and a cell matrix labeling solution, and the specific fluorescent labeling method is shown in Fig. 3.

[0047] First, the excised tissue can be immersed or coated in acetone for 30 seconds to 5 minutes, then immersed or coated in a cell nucleus labeling solution for 30 seconds to 5 minutes, then immersed or coated in a cell matrix labeling solution for 30 seconds to 5 minutes, and then washed with 1XPBS to complete the fluorescence labeling step (Fig. 3).

[0048] Furthermore, the fluorescent labeling technique according to the present invention can also be performed by first mixing acetone, a cell nucleus labeling solution, and a cell matrix labeling solution, then immersing or applying the excised tissue in the mixture for 30 seconds to 5 minutes, and then washing it with 1XPBS (Fig. 3).

[0049]

[0050] Example 3. Confirmation of effects according to fluorescent label concentration

[0051] Figures 4a and 4b show the results of cell observation according to the concentration of the fluorescent label. When SYTO9 is used as the nuclear label, the cell morphology can be clearly identified at a concentration of 0.05 to 0.1 mM; when Acridine Orange is used as the nuclear label, the cell morphology can be clearly identified at a concentration range of 0.005 to 0.1 w / v%; when DAPI is used as the nuclear label, the best cell morphology can be observed at a concentration range of 2 to 5 mg / ml; and when Rhodamine B is used as the cytoplasm label, the cell morphology can be clearly identified at a concentration range of 0.1 to 0.3 mg / ml. When the concentration is higher than the above range, the fluorescent signal becomes saturated, making it difficult to identify the cell shape, and when the concentration is lower than the above range, the fluorescent signal is weak, making it difficult to observe the cell morphology clearly (Figures 4a and 4b).

[0052]

[0053] Example 4. Confirmation of effects according to dyeing time

[0054] The results of observing tissue cells according to staining time for each fluorescent label are shown in Figures 5a to 5c. In the case of staining tissue cells using the high-speed fluorescent staining technique according to the present invention (Figure 5a), the clear morphology of the cell can be observed with only a staining time of about 0.5 minutes (30 seconds), whereas when using the conventional tissue staining method, it can be seen that at least 10 minutes are required to identify the cell morphology (Figure 5b). Furthermore, the fluorescence intensity of the high-speed fluorescent staining technique according to the present invention and the conventional tissue staining method are compared (Figure 5c). It can be confirmed through the fluorescence intensity that the high-speed fluorescent staining technique according to the present invention can not only significantly reduce the staining time compared to the conventional tissue staining method but also allow for more clear observation of the cell morphology resulting from the staining.

[0055]

[0056] Example 5. Comparison of effects of using a mixture of nuclear label and cytoplasm label

[0057] Figures 6a to 6c show the results of observing tissue cells according to staining time when using a mixture of nuclear labeling solution (DAPI, SYTO9, Acridine Orange) and cytoplasmic labeling solution (Rhodamine B). According to Figures 6a to 6c, when using the high-speed fluorescent staining technique according to the present invention (Figure 6a), cell morphology can be clearly observed with only a very short staining time (30 seconds to 5 minutes) compared to the conventional tissue staining method (Figure 6b), and this can also be clearly confirmed by the difference in fluorescence intensity between the high-speed fluorescent staining technique according to the present invention and the conventional tissue staining method (Figure 6c).

[0058]

[0059] Optimal embodiments have been disclosed in the drawings and specification as described above. Specific terms have been used herein, but they are used only for the purpose of describing the invention and are not intended to limit the meaning or the scope of the invention as described in the claims. Therefore, those skilled in the art will understand that various modifications and equivalent alternative embodiments are possible therefrom. Accordingly, the true technical scope of protection of the invention should be determined by the technical spirit of the appended claims.

Claims

1. (S1) A step of preparing a mixture by mixing a tissue fixative, a cell nuclear labeling solution, and a cell matrix labeling solution; (S2) A step of immersing or applying the tissue to be observed in the above mixture; (S3) A step of washing the tissue to be observed in 1XPBS; a high-speed fluorescence labeling technique comprising.

2. A high-speed fluorescent labeling method according to claim 1, characterized in that the tissue fixative is acetone.

3. A high-speed fluorescent labeling method according to claim 1, characterized in that the cell nuclear label is SYTO9, Acridine Orange, or DAPI.

4. A high-speed fluorescent labeling method according to claim 3, characterized in that the concentration of SYTO9 is 0.05 to 0.1 mM.

5. A high-speed fluorescent labeling method according to claim 3, characterized in that the concentration of Acridine Orange is 0.005 to 0.1 w / v%.

6. A high-speed fluorescent labeling method according to claim 3, characterized in that the concentration of DAPI is 2 to 5 mg / ml.

7. A high-speed fluorescent labeling method according to claim 1, characterized in that the cell matrix label is Rhodamine B.

8. A high-speed fluorescent labeling method according to claim 7, characterized in that the concentration of Rhodamine B is 0.1 to 0.3 mg / ml.

9. A high-speed fluorescent labeling technique characterized in that steps (S1) to (S3) of claim 1 are performed for 30 seconds to 5 minutes.

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