JP7660821B2 - Compound, mitochondrial staining agent, and method for fluorescent staining of mitochondria - Google Patents

Description

The present invention relates to compounds, mitochondrial stains and methods for fluorescently staining mitochondria.

Mitochondria are intracellular organelles that eukaryotic cells use to synthesize ATP, a form of bioenergy, using oxygen. Therefore, mitochondrial dysfunction is directly linked to cell death and causes various diseases (JP 2019-112338 A). In addition, since mitochondrial dysfunction is also related to mitochondrial morphology, observation of mitochondrial morphology is an important indicator when investigating the state and function of cells. Therefore, methods using various fluorescent low molecular weight compounds and fluorescent proteins have been developed to observe mitochondrial morphology (JP 2020-098199 A; JP 2017-48268 A).

The use of fluorescent low molecular weight compounds in mitochondria has the advantage of being simple and capable of staining all cells, whereas the use of fluorescent proteins is cumbersome, as it requires procedures such as genetic recombination and the introduction of expression vectors into cells. On the other hand, when labeling mitochondria with fluorescent low molecular weight compounds, there is a problem in that the localization of the fluorescent low molecular weight compounds in the mitochondrial membrane is unstable because the fluorescent low molecular weight compounds bind to the mitochondrial lipid membrane. The main cause of this problem is that when used in combination with other staining methods such as fluorescent antibody techniques, the mitochondrial membrane is destroyed by treatment with detergents to increase the cell membrane permeability of cell fixatives or antibodies, causing the fluorescent low molecular weight compounds to fall off from the mitochondrial membrane.

In addition, the emission wavelength of the fluorescent small molecule compounds can be problematic; for example, in multiple staining, overlap with emission wavelengths from other target factors or target intracellular organelles can make it impossible to specifically detect mitochondria.

The objective of the present invention is to provide a novel mitochondrial staining agent and a method for fluorescent staining of mitochondria.

The embodiments of the present invention are as follows.
[1] A compound represented by the following formula (I), or a salt thereof:

（式中、
Ｒ^１が、水素、Ｃ１～Ｃ６アルキル、－ＮＨ２、－ＯＲ^３、－ＣＯＯＨ、－ＣＯＮＨ_２、－ＣＯＯＮＨ_２、－Ｎ＝Ｃ＝Ｏ、もしくは－Ｎ＝Ｃ＝Ｓであって、Ｒ^２が、－Ｎ＝Ｃ＝Ｏ、もしくは－Ｎ＝Ｃ＝Ｓであるか、または
Ｒ^２が、水素、Ｃ１～Ｃ６アルキル、－ＮＨ２、－ＯＲ^３、－ＣＯＯＨ、－ＣＯＮＨ_２、－ＣＯＯＮＨ_２、－Ｎ＝Ｃ＝Ｏ、もしくは－Ｎ＝Ｃ＝Ｓであって、Ｒ¹が、－Ｎ＝Ｃ＝Ｏ、もしくは－Ｎ＝Ｃ＝Ｓであり、
Ｒ^３が、水素またはＣ１～Ｃ６アルキルである。）
［２］Ｒ^２が－Ｎ＝Ｃ＝Ｏである、［１］項に記載の化合物、またはその塩。
［３］Ｒ¹が－Ｎ＝Ｃ＝Ｏである、［２］項に記載の化合物、またはその塩。
［４］Ｒ¹が－ＣＯＮＨ_２である、［２］項に記載の化合物、またはその塩。
［５］［１］～［４］項のいずれか一項に記載の化合物またはその塩を含む、ミトコンドリア染色剤。
［６］［１］～［４］項のいずれか一項に記載の化合物またはその塩を含む、細胞マーキング剤。
［７］［１］～［４］項のいずれか一項に記載の化合物またはその塩を含む、細胞状態評価剤。
［８］細胞のオートファジーを検出するための薬剤であって、
［１］～［４］項のいずれか一項に記載の化合物またはその塩を含む薬剤。
［９］生細胞を死細胞から識別するための薬剤であって、
［１］～［４］項のいずれか一項に記載の化合物またはその塩を含む薬剤。

(Wherein,
R ¹ is hydrogen, C1 to C6 alkyl, -NH2, -OR ³ , -COOH, -CONH ₂ , -COONH ₂ , -N=C=O or -N=C=S and R ² is -N=C=O or -N=C=S, or R ² is hydrogen, C1 to C6 alkyl, -NH2, -OR ³ , -COOH, -CONH ₂ , -COONH ₂ , -N=C=O or -N=C=S and R ¹ is -N=C=O or -N=C=S;
^R3 is hydrogen or C1-C6 alkyl.
[2] The compound according to item [1], wherein R ² is —N═C═O, or a salt thereof.
[3] The compound according to item [2], wherein R ¹ is -N=C=O, or a salt thereof.
[4] The compound according to [2], wherein R ¹ is -CONH ₂ , or a salt thereof.
[5] A mitochondrial staining agent comprising the compound or a salt thereof according to any one of [1] to [4].
[6] A cell marking agent comprising the compound or a salt thereof according to any one of [1] to [4].
[7] An agent for evaluating a cell state, comprising the compound or a salt thereof according to any one of [1] to [4].
[8] A drug for detecting cellular autophagy, comprising:
A pharmaceutical agent comprising the compound or a salt thereof according to any one of [1] to [4].
[9] A drug for distinguishing live cells from dead cells, comprising:
A pharmaceutical agent comprising the compound or a salt thereof according to any one of [1] to [4].

[10] A method for staining mitochondria, comprising a step of fluorescently staining mitochondria using the compound or a salt thereof according to any one of [1] to [4].
[11] A method for detecting cells that have undergone autophagy, comprising:
A step of fluorescently staining mitochondria of a cell to be examined with the compound according to any one of items [1] to [4];
observing the fluorescently stained mitochondria;
A detection method comprising:
[12] A step of fluorescently staining mitochondria using the compound or a salt thereof according to any one of [1] to [4];
identifying cells having the fluorescently stained mitochondria;
A method for identifying a cell, comprising:
[13] A method for identifying a first cell from a cell group including a first cell having mitochondria fluorescently stained with the compound or a salt thereof according to any one of [1] to [4], and a second cell having mitochondria not fluorescently stained with any of the compounds according to [1] to [4], comprising:
A method for identifying cells, the method comprising the step of identifying cells in the cell group that have fluorescently stained mitochondria.
[14] A method for identifying a first cell having mitochondria that are fluorescently stained with the compound or a salt thereof according to any one of [1] to [4] above, and a second cell having mitochondria that are not fluorescently stained with any of the compounds according to [1] to [4] above, by fluorescent staining the first cell and the second cell in a cell group comprising the first cell and the second cell;
evaluating the transfer of material or information between the first cell and the second cell;
A method comprising:
[15] A method for evaluating a test compound as a cosmetic, comprising the steps of:
A step of fluorescently staining either keratinocytes or melanocytes in vitro with the compound or a salt thereof according to any one of items [1] to [4];
culturing a cell population comprising the melanocytes and the keratinocytes in the presence of the compound to be tested;
A step of distinguishing the keratinocytes from the melanocytes by the fluorescent staining; and a step of examining whether or not melanosomes have been transferred from the melanocytes to the keratinocytes.
evaluating the test compound that alters the melanosome migration as a cosmetic product;
A method comprising:
[16] A method for identifying tumor cells in a non-human animal, comprising:
A step of fluorescently staining the tumor cells in vitro with the compound or salt thereof according to any one of items [1] to [4];
implanting the fluorescently stained tumor cells into the animal;
identifying the fluorescently stained tumor cells in the animal body;
A method comprising:
[17] A first step of imaging a cell having one or more mitochondria fluorescently stained with the compound or a salt thereof according to any one of [1] to [4] to obtain a cell image;
A method for analyzing a cell image comprising: a second step of obtaining information on the one or more mitochondria from the cell image; and a third step of evaluating the health state of the cell based on the obtained information on the one or more mitochondria.
[18] In the first step, the fluorescent image of the cell is a microscopic image,
In the second step, the information on the one or more mitochondria is a statistical quantity obtained by statistically processing at least one of the following values of the one or more mitochondria: area, length, width, aspect ratio, circularity, elongation, shape factor, roughness, shortest distance to adjacent mitochondria, number of branching points, distance from the nucleus, distance from the cell membrane, and distance from the cell body;
In a third step, the health of the cells is evaluated based on the statistics.
The analysis method according to item [17].
[19] In the first step, the fluorescent image of the cell is a microscopic image,
In the second step, the information on the one or more mitochondria is an occupancy rate of a region in which the two or more mitochondria are aggregated at a predetermined density or more in the cell relative to an area of the cell,
In a third step, the health of the cells is evaluated based on the occupancy rate.
The analysis method according to item [17].
[20] The analysis method described in [18], further comprising a fourth step of acquiring a bright-field image of the cell and recognizing the cell in the bright-field image.
[21] The method further comprises a fourth step of acquiring a bright field image of the cell,
The analysis method according to item [19], wherein the area of the cell is calculated from the bright-field image.
[22] The analysis method described in [18] or [20], wherein in the third step, the health state of the cell is evaluated by comparing information on one or more fluorescently stained mitochondria in the second cell with statistics obtained by statistical processing.
[23] The analysis method described in [19] or [21], wherein in the third step, the health state of the cell is evaluated by comparing the occupancy rate of an area in which two or more fluorescently stained mitochondria in the second cell are aggregated at a predetermined density or higher in the second cell with the area of the second cell.
[24] The analysis method according to [22] or [23], wherein the second cell is a normal cell.

==Cross references to related literature==
This application claims priority based on Japanese Patent Application No. 2021-070021 filed on April 16, 2021, the basic application of which is incorporated herein by reference.

FIG. 1 is a schematic diagram of a microscope apparatus used in one embodiment of the present invention. FIG. 2 is a diagram showing a flow chart of the computer-controlled operation of the microscope apparatus used in one embodiment of the present invention. FIG. 3 is a flowchart illustrating a method for analyzing images of cells stained for mitochondria in accordance with one embodiment of the present invention. FIG. 4 shows images observed by a fluorescence microscope when mitochondria were labeled with compound 3 (compound (II)) (FIG. 4A) or compound 4 (compound (III)) (FIG. 4B) in Example 3. Fig. 5 shows images observed by a fluorescence microscope when mitochondria were labeled with compound 4, cells were fixed, and then triple stained with COX4-Alexa488 and DAPI in Example 4. Fig. 5A shows an image of the fluorescence due to compound 4, Fig. 5B shows an image of the fluorescence due to Alexa488, and Fig. 5C shows an image in which the fluorescence due to compound 4, the fluorescence due to Alexa488, and the fluorescence due to DAPI are superimposed. Fig. 5D is a bright field image. 6A is a fluorescent microscope image showing the results of Example 5. Fig. 6A is a fluorescent microscope image showing the results of staining with compound 4, Fig. 6B is a fluorescent microscope image showing the results of staining with compound 4 and then treatment with a mitochondrial uncoupler, and Fig. 6C is a fluorescent microscope image showing the results of treatment with a mitochondrial uncoupler first and then staining with compound 4. 7 is a fluorescent microscope image showing the results of Example 6. The figure at the top of the image is a schematic diagram showing the staining process. Fig. 7A is a bright field image of the area where cells are present. Fig. 7B is an observation image of the fluorescence emitted by compound 4 (described as compound 4 in the figure). Fig. 7C is an image in which the bright field image and the fluorescent image are superimposed. 8 shows images observed by a fluorescence microscope, showing the results of Example 7. Fig. 8A is an image of the fluorescence emitted by compound 4. Fig. 8B is an image of the fluorescence staining using LC3B antibody. Fig. 8C is an image obtained by superimposing the fluorescence by compound 4, the fluorescence staining using LC3B antibody, and the fluorescence by DAPI. Fig. 8D is a bright field image. 9A and 9B are images observed by a fluorescence microscope showing the results of Example 8. Fig. 9A is an image of the fluorescence emitted by compound 1. Fig. 9B is an image of staining using LC3B antibody. Fig. 9C is an image in which the fluorescence by compound 1, the fluorescent staining using LC3B antibody, and the fluorescence by DAPI are superimposed. Fig. 9D is a bright field image. 10A and 10B are images observed by a fluorescence microscope showing the results of Example 9. Fig. 10A is a bright field image. Fig. 10B is an image of the fluorescence emitted by compound 3. FIG. 11 shows an image (A) of HeLa cells stained with compound (III) in Example 9, and an image (B) in which a region with continuously high brightness values is detected. FIG. 12 shows an image (A) of normal mesenchymal stem cells stained with compound (III) in Example 9, an image (B) of an area where mitochondria have a density equal to or higher than a predetermined level, and an image (C) of an area where the entire cell is present. FIG. 13 shows an image (A) of aged mesenchymal stem cells stained with compound (III) in Example 9, an image (B) of an area where mitochondria have a density equal to or higher than a predetermined level, and an image (C) of an area where the entire cell is present.

The objectives, features, advantages, and ideas of the present invention will be clear to those skilled in the art from the description in this specification, and those skilled in the art can easily reproduce the present invention from the description in this specification. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention, and are shown for illustrative or explanatory purposes, and do not limit the present invention thereto. It will be clear to those skilled in the art that various changes and modifications can be made based on the description in this specification within the intent and scope of the present invention disclosed in this specification.

Compound
A compound according to one embodiment disclosed herein is a compound represented by the following formula (I) or a salt thereof (in this specification, a compound represented by the following formula (I) or a salt thereof is referred to as compound (I)).

（式中、
Ｒ^１が、水素、Ｃ１～Ｃ６アルキル、－ＮＨ２、－ＯＲ^３、－ＣＯＯＨ、－ＣＯＮＨ_２、－ＣＯＯＮＨ_２、－Ｎ＝Ｃ＝Ｏ、もしくは－Ｎ＝Ｃ＝Ｓであって、Ｒ^２が、－Ｎ＝Ｃ＝Ｏ、もしくは－Ｎ＝Ｃ＝Ｓであるか、または
Ｒ^２が、水素、Ｃ１～Ｃ６アルキル、－ＮＨ２、－ＯＲ^３、－ＣＯＯＨ、－ＣＯＮＨ_２、－ＣＯＯＮＨ_２、－Ｎ＝Ｃ＝Ｏ、もしくは－Ｎ＝Ｃ＝Ｓであって、Ｒ¹が、－Ｎ＝Ｃ＝Ｏ、もしくは－Ｎ＝Ｃ＝Ｓであり、
Ｒ^３が、水素またはＣ１～Ｃ６アルキルである。）
Ｒ^２が－Ｎ＝Ｃ＝Ｏであることが好ましく、下記式（ＩＩ）または（ＩＩＩ）で表されるように、Ｒ¹が－Ｎ＝Ｃ＝Ｏまたは－ＣＯＮＨ_２であることがより好ましい。（本明細書において、以下の化合物を化合物（ＩＩ）および化合物（ＩＩＩ）と称する。）

(Wherein,
R ¹ is hydrogen, C1 to C6 alkyl, -NH2, -OR ³ , -COOH, -CONH ₂ , -COONH ₂ , -N=C=O or -N=C=S and R ² is -N=C=O or -N=C=S, or R ² is hydrogen, C1 to C6 alkyl, -NH2, -OR ³ , -COOH, -CONH ₂ , -COONH ₂ , -N=C=O or -N=C=S and R ¹ is -N=C=O or -N=C=S;
^R3 is hydrogen or C1-C6 alkyl.
It is preferable that ^R2 is -N=C=O, and it is more preferable that ^R1 is -N=C=O or _-CONH2 as represented by the following formula (II) or (III). (In this specification, the following compounds are referred to as compound (II) and compound (III).)

When compound (I) is irradiated with excitation light of 520 nm to 590 nm, it emits fluorescence of 590 nm to 660 nm.

Compound (I) can be easily produced by referring to the methods described in the examples and making appropriate modifications using common general technical knowledge.

==Mitochondrial staining agent and staining method using the same==
Since compound (I) binds to mitochondria, it can be used as a dye for fluorescently staining mitochondria. The solvent for the dye is not particularly limited as long as it can dissolve the compound, and examples thereof include acetone, DMSO, ethanol, acetonitrile, and aqueous solutions thereof.

In addition to compound (I), the mitochondrial stain may contain a fluorescent compound for fluorescently staining mitochondria and cellular components other than mitochondria, such as cell membranes and intracellular organelles. This allows multiple elements to be stained simultaneously. In addition, the interaction between compound (I) and the fluorescent compound can enhance the luminescence of compound (I) and improve the specificity of compound (I) to mitochondria. Examples of fluorescent compounds that enhance the luminescence of compound (I) include (Mito Tracker), and examples of fluorescent compounds that improve the specificity of compound (I) include (DAPI: 4,6-Diamidino-2-phenylindole, dihydrochloride).

Staining of mitochondria using the mitochondrial staining agent disclosed herein can be performed by a routine method for those skilled in the art. For example, when staining mitochondria of cultured cells, compound (I) may be added to the medium during culture. The concentration of compound (I) is not particularly limited, but is preferably 0.001 μM to 10 μM, and more preferably 0.01 μM to 0.1 μM. The staining time is also not particularly limited, but is preferably (1 minute) to (1 week), and more preferably (5 minutes) to (1 hour). The staining temperature is also not particularly limited, but is preferably (10°C) to (45°C), and more preferably (30°C) to (40°C). The lower the staining temperature, the longer the staining time is, and specific examples of conditions include (37)°C and (15) minutes. Thereafter, the medium is removed and the cells may be washed, or the next step may be proceeded to without washing. When washing, cell culture medium, phosphate buffer, etc. can be used as the washing solution. The washing solution may contain a surfactant.

Alternatively, to stain mitochondria in cultured cells, the cultured cells may be harvested, centrifuged, the supernatant removed, suspended in a buffer or medium, and compound (I) added to the cell suspension. The staining conditions are the same as above. After staining, the cells may be seeded on a plate or slide glass, or may proceed to the next step while still suspended.

After the mitochondria are fluorescently stained, the cells are irradiated with light of an excitation wavelength and the fluorescence emitted from the stain is detected, thereby allowing the detection of mitochondria within the cells. The excitation light can be irradiated using the same irradiation means as in general fluorescence detection, for example, a specific wavelength can be selected as necessary from a laser light source provided in a fluorescence microscope. The fluorescence can be detected from the lens barrel of the fluorescence microscope, or an image captured by a camera or the like installed in the fluorescence microscope can be displayed on a display means such as a monitor. A filter that selectively passes specific wavelengths can be used as necessary.

An example of how to analyze images of cells with stained mitochondria is described below.

==Use of mitochondrial stains==
Agents for distinguishing live from dead cells
Compound (I) stains mitochondria depending on the membrane potential of mitochondria, and therefore stains live cells but not dead cells. Therefore, by staining a cell population containing live and dead cells with compound (I), it is possible to stain only live cells, thereby making it possible to distinguish dead cells from live cells.

<Cell marking agent>
Compound (I) can be used as a very convenient cell marking agent.

For example, by marking the mitochondria of tumor cells taken from a human with compound (I), transplanting them into a mouse, and tracing the fluorescence emission, it is possible to determine the behavior of tumor cells within the mouse (e.g., their propensity to metastasize, etc.).

Alternatively, in a cell group including a first cell having mitochondria fluorescently stained by compound (I) and a second cell having mitochondria that are not fluorescently stained by compound (I), the first cell can be identified by detecting the fluorescent staining from compound (I) and identifying the cell having the fluorescently labeled mitochondria.

Such a method for distinguishing between a first cell and a second cell can be used to evaluate the substance transfer or information transfer between the first cell and the second cell. For example, a method including the steps of fluorescently staining either keratinocytes or melanocytes with compound (I) in vitro, mixing the melanocytes and keratinocytes and culturing a cell group containing both in the presence of a test compound and compound (I), distinguishing between keratinocytes and melanocytes by fluorescent staining, and examining whether melanosomes have moved from melanocytes to keratinocytes makes it possible to evaluate a test compound that changes the movement of melanosomes as a cosmetic. In particular, a compound that inhibits the movement of melanosomes from melanocytes to keratinocytes can be evaluated as a cosmetic with a whitening effect because it can inhibit the movement of melanin to the body surface.

<Cell state evaluation agent>
Compound (I) can also be used as an agent for evaluating cell states. Examples of cell states include cell differentiation stages, apoptosis, hypoxic conditions, cell proliferation inhibition, proliferation promotion, etc.

For example, in fibroblasts, mitochondria have an elongated shape, but when reprogrammed into iPS cells, the mitochondria become fragmented, and when redifferentiated into differentiated cells, they regain their elongated shape (Biochem Biophys Res Commun. 2018 vol.500(1) pp.59-64.). Therefore, by fluorescently staining mitochondria with compound (I) and identifying their shape, it is possible to identify the stage at which the cells are in the reprogramming or redifferentiation process.

In addition, when melanocytes are at an advanced stage of differentiation and melanin synthesis is active, mitochondria accumulate around the nucleus, but when they are at a low stage of differentiation and melanin synthesis is not active, mitochondria are scattered within the cells. Therefore, the differentiation stage of melanocytes can be evaluated by fluorescently staining mitochondria with compound (I) and observing the intracellular localization of mitochondria.

In addition, when pulmonary artery endothelial cells are exposed to hypoxia, retrograde migration of mitochondria is induced via microtubules and the microtubule motor protein dynein, causing mitochondria to form clusters around the nucleus (Sci. Signal., 2012vol.5(231) p.ra47). At that time, accumulation of ROS is observed in the nucleus, but when mitochondrial clustering around the nucleus is inhibited, accumulation of ROS in the nucleus is not observed. Therefore, by fluorescently staining mitochondria with compound (I) and observing the intracellular localization of mitochondria, it is possible to determine the hypoxic state of cells and accumulation of ROS in the nucleus.

Furthermore, when cells undergo autophagy, it is known that excess mitochondria are selectively broken down, a phenomenon known as mitophagy. When mitochondria are fluorescently stained with compound (I) and the state of fluorescent staining within the cells is examined, in cells that have undergone mitophagy, the fluorescence is finely scattered and a fluorescent signal specific to mitophagy is observed. Therefore, by fluorescently staining mitochondria with compound (I) and observing the distribution of the fluorescence, it is possible to identify whether a cell is undergoing autophagy.

<Multiple staining with fluorescence>
Since compound (I) binds tightly to mitochondria, even if the compound is fixed with a fixative or the membrane is permeabilized with a surfactant after fluorescent staining with compound (I), the fluorescence intensity of compound (I) is not as weak as that of conventional fluorescent dyes. Moreover, the peak width of the emitted fluorescent wavelength is narrow (i.e., 590 nm to 660 nm), so there is little overlap with the fluorescent wavelengths of other fluorescent dyes. For these reasons, compound (I) can be suitably used for multiple staining with other fluorescent dyes.

For example, after fluorescently staining mitochondria in cells with compound (I), the cells can be fixed and multi-stained by histochemical techniques using antibodies. In this case, the fluorescent dye used at the same time can be any dye that does not overlap in wavelength with the fluorescence of compound (I), such as FITC, ATTO 488, Alexa Fluor 488, and Cy5.

Alternatively, it can be used simultaneously with a fluorescent dye that binds to other organelles such as the nucleus or endosomes. In this case, an example of the fluorescent dye is DAPI.

<Evaluation methods for cosmetics and pharmaceuticals>
In addition to the above-mentioned autophagy, the morphology and distribution of mitochondria reflect the health of cells. For example, the morphology and distribution of mitochondria, such as fragmentation or accumulation near the nucleus, reflect the health of cells. Therefore, the toxicity of cosmetics and medicines can be evaluated by treating cultured cells with cosmetics or medicines in advance and fluorescently staining the mitochondria of the cells with compound (I).

<Disease markers>
The shape of mitochondria changes depending on the state of cells and the pathology. For example, it is known that mitochondria become round when malnutrition occurs. Alternatively, it is known that abnormalities in mitochondrial fission and fusion are the cause of cardiovascular disease, neurological diseases such as Hattington's disease and ALS, and diabetes (Japanese Pharmacological Society, Journal of Japanese Pharmacology 149: 269-273 (2017) "Ecopharma of Approved Drugs Focusing on Mitochondrial Fission"). Therefore, compound (I) can also be used as a diagnostic marker for diseases, etc. For example, compound (I) can be used as a diagnostic marker by a method including a step of collecting biological tissue from a living body, a step of staining the mitochondria of the collected biological tissue with compound (I), and a step of evaluating the disease.

== Method for analyzing cell images ==
The method for analyzing a cell image according to the present embodiment includes a first step of capturing an image of a cell having mitochondria fluorescently stained with a compound disclosed herein or a salt thereof to obtain a cell image, a second step of obtaining mitochondrial information from the cell image, and a third step of evaluating the health of the cell based on the obtained mitochondrial information. This analysis method makes it possible to investigate the state of the cell based on mitochondrial morphology.

<First step>
First, a cell having one or more mitochondria fluorescently stained with a compound disclosed herein or a salt thereof is imaged to obtain a cell image.

The cell image is preferably a microscope image. The microscope image is preferably captured by a camera equipped with an image sensor attached to a microscope, such as a stereomicroscope or an electron microscope, and is obtained as an image file. The cell image may include an image of one cell or multiple cells.

<Second step>
In this step, information on one or more mitochondria present in a single cell is obtained from the cell image obtained in the first step.

Mitochondrial information refers to numerical values indicating the characteristics of a mitochondrion obtained from an image of the mitochondrion, such as a statistical quantity obtained by statistically processing at least one of the following numerical values: area, length, width, aspect ratio, circularity, elongation, shape factor, roughness, shortest distance to adjacent mitochondria, number of branching points, distance from the nucleus, distance from the cell membrane, and distance from the cell body, or the occupancy rate within the cell of the area where fluorescently stained mitochondria are aggregated at a specified density or more.

The length of a mitochondrion refers to the longest line segment connecting two points on the perimeter of a mitochondrion. The width of a mitochondrion refers to the shortest line segment connecting two points on the perimeter of a mitochondrion. The circularity of a mitochondrion refers to the result of dividing the square of the circumference of a circle with the same area as the mitochondrial region by the square of the circumference of the area. The elongation of a mitochondrion refers to the ratio of the length of a mitochondrion to its width. The shape factor of a mitochondrion refers to the ratio of the square of the perimeter of a circle with the same area as the mitochondrial region to the square of the perimeter of a shape that fills in the unevenness of the mitochondrion region. The shortest distance between a mitochondrion and an adjacent mitochondrion refers to either the shortest line segment connecting a point on the perimeter of the mitochondrion and a point on the perimeter of the adjacent mitochondrion, or the distance between the center of gravity of the mitochondrion and the center of gravity of the adjacent mitochondrion. The number of branching points of a mitochondrion refers to the number of branched parts when the mitochondrion has a branched shape. The distance from the mitochondrial nucleus refers to either the shortest line segment length connecting a point on the periphery of the mitochondrion to a point on the periphery of an adjacent mitochondrion, or the distance between the center of gravity of the mitochondrion and the center of gravity of the nucleus. The distance from the mitochondrial cell membrane refers to the shortest line segment length connecting a point on the periphery of the mitochondrion to a point on the cell membrane. The distance from the mitochondrion to the cell body refers to the distance between the mitochondria present in the neurite and the connection point between the neurite and the cell body, the center of gravity of the cell body, or the center of gravity of the nucleus in a nerve cell.

In the case of a single mitochondrion, the obtained values can be used as is, or can be subjected to statistical processing such as rounding to a specified number of digits to calculate the statistical quantity.

In the case of two or more mitochondria, after obtaining the above information for each mitochondria, the obtained values may be used for statistical processing to calculate statistics. Examples of statistical processing include calculating the mean, median, variance, quartiles, etc.

Alternatively, instead of statistics, the occupancy rate of the area in the cell where fluorescently stained mitochondria are aggregated at a predetermined density or higher may be calculated. Specifically, for example, first, the area of the cell in which the mitochondria are present is calculated. Next, the area of the area in which the mitochondria are aggregated at a predetermined density or higher is calculated. Finally, the ratio of the area in which the mitochondria are aggregated to the area of the cell is calculated, and this ratio may be used as the occupancy rate.

<Third step>
In this step, the health state of the cells is evaluated based on the mitochondrial information obtained in the second step.

The health of a cell refers to whether the cell is in an abnormal state or in an aged state. An abnormal state of a cell refers to, for example, the occurrence of necrosis, apoptosis, a faster or slower proliferation rate than normal, a change in cell morphology, the occurrence of transdifferentiation such as mesenchymal-epithelial transdifferentiation, an imbalance between mitochondrial division and fusion, or the accumulation of abnormal proteins. In addition, when a cell ages, for example, the collagen production function decreases, the proliferation rate slows, telomeres shorten, cell-specific or cell-intrinsic functions are lost, the cytoplasm increases, the cell becomes multinucleated, or mitochondria become fragmented. This health of a cell also includes the state of an individual, for example, the state of cells in a patient with bipolar disorder.

When statistics are used as mitochondrial information, the health of the cell may be evaluated by comparing the statistics obtained by statistically processing information on one or more fluorescently stained mitochondria in a second cell different from the cell whose cell image is analyzed with the statistics obtained in the second step. When occupancy is used as mitochondrial information, the health of the cell may be evaluated by comparing the occupancy of a region in which two or more fluorescently stained mitochondria in a second cell different from the cell whose cell image is analyzed are aggregated at a predetermined density or higher in the second cell with respect to the area of the second cell.

Here, the second cell is preferably a cell of the same type as the cell whose cell image is to be analyzed, and is preferably a normal cell. Here, "normal" means not being in an abnormal state as described above. For example, this includes cells whose morphology, function, and proliferation are normal.

<Fourth step>
In this step, a bright-field image of the cell from which mitochondrial information has been obtained is acquired. The acquired bright-field image can be used, for example, to calculate the area of the cell.

<Specific image analysis procedure>
With reference to FIG. 3, an example of a specific image analysis procedure for analyzing an image of a cell containing stained mitochondria will be described.

First, a fluorescent image of one or more cells containing one or more stained mitochondria is acquired using a fluorescence microscope equipped with an image sensor (S21). Then, shot noise is removed from the acquired captured image (S21). Specifically, the shot noise can be removed from the captured image by averaging the brightness values in a region of a certain size within the image or by removing high-frequency components.

Next, the entire cell is stained with a reagent such as HCS CellMask (registered trademark), and the fluorescent image obtained using a fluorescent microscope with an image sensor is subjected to binarization processing to detect the cell area (hereinafter referred to as the "cell area") within the image (S22).

Alternatively, nuclei are detected by binarizing the fluorescent image of a nuclear staining reagent such as DAPI, and then the cellular regions are detected from the nuclei using the Watershed algorithm on the fluorescent image of the cytoplasmic staining. A mitochondrial fluorescent image may be used instead of the cytoplasmic staining reagent.

Alternatively, cell regions may be detected using phase contrast images. For example, a closed-open filter may be applied to the phase contrast image to obtain the image difference and edge information, and a GVF (Gradient Vector Flow) may be created based on the obtained edge information to obtain the cell contours and detect the cell regions. Furthermore, in addition to processing the GVF, the cell contours may be finally obtained from the fluorescence image to detect the cell regions.

From the captured image of each cell from which shot noise has been removed, regions with continuously high brightness values are detected based on the brightness value information using the Detect Peaks algorithm implemented in Nikon Corporation's NIS Elements software, and the brightness is further enhanced to a high brightness value (S23). As a result, the outline of the thread-like structure is emphasized as the internal structure of each cell.

The brightness values thus obtained are binarized using a predetermined threshold value to obtain a binary image of mitochondria having a thread-like structure, which can be recognized as mitochondria (S24).
Using this binary image, a numerical value indicating the characteristics of each mitochondrion is obtained (S25).

Next, areas in the cells where mitochondria are aggregated at a predetermined density or higher are detected (S26). In this case, a binary image in which mitochondria can be identified may be used, or an image of the cells from which shot noise has been removed may be used. In the latter case, for example, the sum of the brightness values for each divided area may be calculated, and areas with relatively high brightness values may be identified to detect areas where mitochondria are aggregated.

The area of the cell region detected in S22 and the area of the region where mitochondria are present detected in S26 are measured, and the occupancy rate of the region where mitochondria are aggregated at a predetermined density or higher within the cell is calculated relative to the cell area (S27).

Statistical processing is performed on the numerical values indicating the mitochondrial characteristics obtained in S25, and statistics are calculated (S28).

Evaluate the cell health based on the occupancy rate obtained in S27 and/or the statistics obtained in S28.

The evaluation method is not particularly limited, but for example, the health of the cells may be evaluated by obtaining mitochondrial information of the second cell and comparing it with the mitochondrial information of the second cell using the learning device described below.

First, abnormal or aging cells or normal cells are used as the second cells, and one or more pieces of information obtained from these cells are mapped into an n-dimensional vector space and trained using techniques such as deep learning, support vector machines, and decision trees. When information about the target mitochondria is input into this learning machine, it is possible to determine whether abnormality or aging is occurring.

==Microscope Equipment==
An example of a microscope apparatus used in the analysis method of the present disclosure will be described below with reference to FIGS. 1 and 2. FIG.

1, the microscope device 1 includes a device body 10, a computer 170, a monitor 160 serving as a display unit, and an input device 180. The device body 10 includes a microscope unit 150, a transmitted illumination unit 40, an imaging device (hereinafter referred to as a digital camera) 300, an excitation light source 70, and an optical fiber 7. Examples of the digital camera include a CCD camera and a CMOS camera.

The microscope section 150 comprises a stage 23, an objective lens section 27, a fluorescence filter section 34, an imaging lens section 38, a deflection mirror 452, a field lens 411, and a collector lens 41, and the transmitted illumination section 40 comprises a transmitted illumination light source 47, a field lens 44, and a deflection mirror 45.

A chamber 100 containing a transparent culture vessel 20 is placed on the stage 23. In the culture vessel 20, cultured cells whose mitochondria are labeled with a fluorescent substance are grown in a medium.
In order to observe the cultured cells from outside the chamber 100, a part a of the bottom surface of the chamber 100 and a part b of the top surface of the chamber 100 are each transparent. Here, a case where the top surface of the culture vessel 20 is open will be described, but if necessary, the top surface of the culture vessel 20 may be covered with a lid made of the same material as the culture vessel 20.

The objective lens unit 27 is fitted with multiple types of objective lenses arranged in the X direction in Fig. 1, and the type of objective lens arranged in the optical path of the device body 10 is switched by moving the objective lens unit 27 in the X direction. This switching is performed under the control of the computer 170.

The fluorescent filter unit 34 is fitted with multiple types of filter blocks arranged along the X direction in Fig. 1, and the type of filter block arranged in the optical path of the device body 10 is switched by moving the fluorescent filter unit 34 along the X direction. This switching is also performed under the control of the computer 170.

The computer 170 switches the combination of the type of objective lens placed in the optical path and the type of filter block placed in the optical path according to the observation method to be set in the device body 10. In this embodiment, this switching switches the observation method by the device body 10 between phase contrast observation and two types of fluorescence observation.

Phase contrast observation and fluorescence observation differ in the type of filter block placed in the light path and the type of objective lens that can be placed in the light path, whereas the two types of fluorescence observation differ only in the type of filter block placed in the light path, and the types of objective lenses that can be placed in the light path are the same. Phase contrast observation and fluorescence observation also differ in the illumination methods.

During phase contrast observation, the computer 170 uses the transmitted illumination light source 47 to use the optical path of the transmitted illumination unit 40, and during fluorescence observation, the computer 170 uses the excitation light source 70 to use the optical path of the epi-illumination unit (i.e., the optical path passing through the excitation light source 70, optical fiber 7, collector lens 41, field lens 411, deflection mirror 452, fluorescence filter unit 34, and objective lens unit 27 in that order). Note that the excitation light source 70 is turned off when the transmitted illumination light source 47 is used, and the transmitted illumination light source 47 is turned off when the excitation light source 70 is used.

During phase-contrast observation, which is a bright-field observation, the light emitted from the transmitted illumination light source 47 illuminates the observation point c in the culture vessel 20 through the field lens 44, the deflection mirror 45, and the transparent part b of the chamber 100. The light transmitted through the observation point c reaches the light receiving surface of the digital camera 300 via the bottom surface of the culture vessel 20, the transparent part a of the chamber 100, the objective lens part 27, the fluorescent filter part 34, and the imaging lens 38, forming a phase-contrast image of the observation point c. When the digital camera 300 is operated in this state, a phase-contrast image is captured and phase-contrast image data is generated. This image data is transmitted to the computer 170 and stored there.

Note that bright-field images obtained by bright-field observation include images obtained by bright-field observation other than fluorescence observation, such as images obtained by dark-field observation, phase contrast observation, and differential interference observation.

During fluorescence observation, the light emitted from the excitation light source 70 illuminates the observation point c in the culture vessel 20 through the optical fiber 7, the collector lens 41, the field lens 411, the deflection mirror 452, the fluorescence filter unit 34, the objective lens unit 27, the transparent part a of the chamber 100, and the bottom surface of the culture vessel 20. This excites the fluorescent substance present at the observation point c, and the fluorescent substance emits fluorescence. This fluorescence reaches the light receiving surface of the digital camera 130 through the bottom surface of the culture vessel 20, the transparent part a of the chamber 100, the objective lens unit 27, the fluorescence filter unit 34, and the imaging lens 38, forming a fluorescent image of the observation point c. When the digital camera 300 is operated in this state, a fluorescent image is captured and image data is generated. This image data is also transmitted to the computer 170 and stored there.

In addition, the computer 170 controls the position of the observation point c in the culture vessel 20 in the XYZ coordinates by controlling the XY coordinates of the stage 23 and the Z coordinate of the objective lens section 27.

A humidifier may be connected to the chamber 100 via a silicone tube, and the humidity and CO2 concentration inside the chamber 100 may be controlled to a predetermined value. The air around the chamber 100 may be circulated appropriately by a heat exchanger, thereby controlling the internal temperature of the chamber 100 to a predetermined value. The humidity, CO2 concentration, and temperature inside the chamber 100 may be measured by a sensor. The measurement results may be sent to the computer 170 and stored there. Meanwhile, the digital camera 300 may be housed in a housing separate from the other parts of the device main body 10, and may be kept at about the same temperature as the outside air temperature of the device main body 10 regardless of the internal temperature of the chamber 100.

Next, the operation of computer 170 regarding time lapse photography will be described.

An observation program is installed in the computer 170, and the computer 170 operates according to the program. All information input from the user to the computer 170 is performed via the input device 180.

2 is a flowchart of the operation of the computer 170. As shown in Fig. 2, the computer 170 first sets the observation method of the device main body 10 to low-magnification phase-contrast observation, acquires image data of a bird's-eye view image in this state, and displays it on the monitor 160 (S11). A bird's-eye view image is an image of a relatively wide area of the culture vessel 20.

When acquiring image data of the bird's-eye view image, the computer 170 repeatedly acquires image data of the phase contrast image while moving the observation point c in the culture vessel 20 in the XY direction, and synthesizes the acquired multiple image data into image data of a single image. Each piece of image data is the image data of a so-called tile image, and the synthesized image data is the image data of the bird's-eye view image.

The user determines the conditions for time lapse photography (e.g., interval, number of rounds, observation points, observation method, etc.) while observing the bird's-eye view image displayed on the monitor 160.

When the conditions are input by the user (S12: YES), the computer 170 creates a recipe with the conditions written in (S13). The recipe is stored, for example, on the hard disk of the computer 170. Hereinafter, the interval, number of rounds, observation point, and observation method written in the recipe are referred to as the "designated interval," "designated number of rounds," "designated point," and "designated observation method," respectively.

Thereafter, when an instruction to start time lapse photography is input by the user (S14), the computer 170 starts time lapse photography (S15).
In time lapse photography, the computer 170 matches the observation point c of the incubation container 20 with the designated point, sets the observation method of the device body 10 to the designated observation method, and acquires image data in this state. If there are three designated observation methods, the observation method of the device body 10 is switched between the three designated observation methods while three types of image data are acquired continuously. This completes the first round of photography.

Then, the computer 170 waits for the specified interval from the start time of the first round of shooting, and starts the second round of shooting. The shooting method for the second round is the same as the shooting method for the first round.

Furthermore, the following shooting is repeated until the number of rounds performed reaches the specified number of rounds (S16: YES).

During the time lapse photography period (S16: NO), the computer 170 sequentially accumulates the image data captured from the device main body 10 in an implementation progress file. This implementation progress file is stored, for example, on the hard disk of the computer 170.

Furthermore, when a confirmation instruction is input by the user during the time lapse photography period or after the time lapse photography is completed, the computer 170 refers to the contents of the implementation progress file at that time and, based on that, displays the implementation progress confirmation screen described below on the monitor 160.

For example, an image of each cell in the bird's-eye view image acquired by this microscope device 1 may be input to an image processing unit of the computer 170, where the above-mentioned image analysis process is executed and the analysis results are output.

Example 1 Synthesis of Compound (II) Compound (II) was synthesized according to the procedure described below.

ATTO565-free COOH (1 mg) was dissolved in acetonitrile (0.5 mL) to obtain a solution, to which 2 equivalents of DPPA and 1 equivalent of triethylamine were added and reacted at room temperature for 30 minutes. 1 mL of water and 2 mL of hexane were added to the reaction solution, mixed, and allowed to stand, and the hexane layer was collected. The mixture was then concentrated by degassing, and finally replaced with 0.1 mL of acetonitrile, and the target product was purified by passing it through a silica gel column equilibrated with acetonitrile. All of the eluate was collected, dried by degassing under reduced pressure, and dissolved in DMSO before use.
1H-NMR (CDCl ₃ , 400 MHz) δ: 1.23 (t, 3H, J = 14 Hz), 2.25 (broad s, 4H), 3.00 (m, 2H),7.06 (m, 1H), 7.22 (s, 1H).

The product was confirmed by UPLC-MS/MS (Waters) analysis. MS: 505.22, MS/MS: 489.19, 477.19, 461.16 were detected, confirming that the desired product had been obtained.

Example 2 Synthesis of Compound (III) Compound (III) was synthesized according to the procedure described below.

ATTO565-HNS ester (1 mg) was dissolved in acetonitrile (0.5 mL) to obtain a solution, to which 1 equivalent of DPPA and 1 equivalent of triethylamine were added and reacted at room temperature for 30 minutes. 1 mL of water and 2 mL of hexane were added to the reaction solution, mixed, and allowed to stand, and the hexane layer was collected. The mixture was then concentrated by degassing, and finally replaced with 0.1 mL of acetonitrile. The target product was purified by passing through a silica gel column equilibrated with acetonitrile. All of the eluate was collected, dried by degassing under reduced pressure, and dissolved in DMSO before use.

The product was confirmed by UPLC-MS/MS (Waters) analysis. MS: 507.25, MS/MS: 479.24, 449.19, 435.18 were detected, confirming that the desired product had been obtained.

Example 3 Fluorescent staining of mitochondria in living cells Fluorescent staining of intracellular mitochondria was carried out using compound (II) and compound (III) synthesized in Example 1 and Example 2. The procedure is as follows.

(1) 3 x ¹⁰⁴ mouse malignant melanoma cells B16F10 (obtained from Riken Cell Bank) were seeded on a glass bottom dish (manufactured by Matsunami Co., Ltd.) and cultured for 24 hours. The medium used was Dulbecco's modified Eagle's medium (DMEM) high glucose (D6046 (Merck)) supplemented with 1x penicillin-streptomycin (26252-94 (Nacalai Tesques)) and 10% fetal bovine serum (Nichirei). Culture was performed at 37°C and 5% _CO2 .
(2) Compound (II) and compound (III) were added to the medium to a final concentration of 0.01 μM.
(3) After 15 minutes, the medium was removed by aspiration and replaced with fresh medium.
(4) Using a fluorescence microscope (BZ-9000, manufactured by Keyence Corporation), compound (II) and compound (III) were excited at 560 nm, the emitted fluorescence was observed (wavelength: 620 nm), and the cells were imaged. The results are shown in FIG. 4. FIG. 4A is an image of the whole cell with compound (II). FIG. 4B is an image of the whole cell with compound (III). As shown in FIG. 4, compound (II) and compound (III) caused fluorescent staining of mitochondria having a linear structure in the cells.
Thus, compound (II) and compound (III) can stain intracellular mitochondria.

Example 4: Triple staining with DAPI and anti-COX4 antibody In this example, intracellular mitochondria were fluorescently stained with compound (III) synthesized in Example 2, and then fixed, and the cells were stained with DAPI and an antibody against COX4, a mitochondrial marker. The procedure is as follows.

(1) 3 x ¹⁰⁴ HEK293A cells (obtained from Thermofisher) were seeded on a glass bottom dish (Matsunami) and cultured for 24 hours. The medium used was Dulbecco's modified Eagle's medium (DMEM) high glucose (D6046 (Merck)) supplemented with 1x penicillin-streptomycin (26252-94 (Nacalai Tesques)) and 10% fetal bovine serum (Nichirei). Culture was performed at 37°C and 5% _CO2 .
(2) Compound (III) was added to the medium to a final concentration of 0.01 μM.
(3) After 5 minutes, the medium was removed by aspiration, and 1 mL of 4% paraformaldehyde/phosphate buffer (09154-14 (Nacalai Tesques)) was added, followed by fixation at room temperature for 15 minutes.
(4) The 4% paraformaldehyde/phosphate buffer solution was removed by suction, and 1 mL of phosphate buffer solution (containing 0.1% Tween 20 (Kanto Chemical), hereinafter referred to as PBST) was added.
(5) 1 μL of DAPI (Cellstain (registered trademark) DAPI solution (Dojindo Laboratories)) was added and left for 15 minutes to fluorescently stain the nuclei.
(6) The cells were washed three times with PBST.
(7) After washing, 1 mL of a reaction solution prepared by diluting anti-COX4 antibody (PM063MS (MBL)) at 1/1000 in 10% BlockingOne (Nacalai Tesque)/phosphate buffer was added, and the plate was left at room temperature for 1 hour.
(8) The cells were washed three times with PBST.
(9) After washing, 1 mL of a reaction solution prepared by diluting anti-mouse IgG H&L (Alexa Fluor488) (ab150113 (Abcam)) at 1/1000 in ( ) mL of 10% BlockingOne/phosphate buffer was added, and the plate was left at room temperature for 1 hour.
(10) The cells were washed four times with PBST.
(11) The cells were observed under a fluorescence microscope. During the observation (wavelength: 620 nm), compound (III) was excited at 560 nm, the antibody bound to COX4 was excited at 470 nm, and DAPI was excited at 360 nm.

The results are shown in Figure 5. Figure 5A is a fluorescence microscope image of the whole cell with compound (III). Figure 5B shows the accumulation site of the anti-COX4 antibody. Figure 5C is a superposition of the fluorescence images of Figures 5A, 5B and the nucleus (DAPI). Figure 5D is a bright field image of the whole cell. As shown in Figures 5A and B, fluorescence was emitted from compound (III) and the antibody bound to COX4, and as shown in Figure 5C, the fluorescence from compound (III) and the antibody bound to COX4 were overlapped.

In this way, compound (III) can be fixed after staining and used for multiplex staining with DAPI or antibodies.

Example 5 Dependence of fluorescent staining on mitochondrial membrane potential Mitochondrial membranes have stronger membrane potentials than membranes of other intracellular organelles, and most of the mitochondria-accumulating compounds are localized in mitochondria in a manner dependent on the mitochondrial membrane potential.
In this example, we show that the staining of mitochondria by compound (III) depends on the membrane potential, and that once stained, the staining remains even if the membrane potential is lost. The procedure is shown below, but the details of the staining of mitochondria by compound (III) are the same as in Example 4.

(1) 3 × 10 ⁴ HEK293A cells (obtained from Thermofisher) were seeded on a glass bottom dish and cultured for 24 hours.
(2) Compound (III) was added to a final concentration of 0.01 μM.
(3) The cells were observed under a fluorescence microscope, where compound (III) was excited at 560 nm.
(4) Thereafter, a mitochondrial membrane potential inhibitor (uncoupling agent: carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP)) (C2902 (Merck)) was added to the medium to a final concentration of 10 μM, and the cells were cultured for 1 hour.
(5) The specimen was observed again using a fluorescent microscope.
(6) Next, a test was carried out in which FCCP was pretreated and then compound (III) was added.
(7) Observation was performed using a fluorescent microscope.

The results are shown in Figure 6. Figure 6A is an image of the fluorescence emitted by treatment with compound (III) only. Figure 6B is an image of the fluorescence emitted by treatment with compound (III) followed by FCCP treatment. Figure 6C is an image of the fluorescence emitted by treatment with compound (III) followed by FCCP treatment.

As shown in Figures 6A and 6B, once compound (III) stained mitochondria, it remained accumulated in mitochondria even after the mitochondrial membrane potential was lost. Also, as shown in Figure 6C, the accumulation of compound (III) in mitochondria required the mitochondrial membrane potential.

Thus, mitochondrial membrane potential is required for the staining of mitochondria with compound (III). Therefore, dead cells that lack mitochondrial membrane potential are not stained by compound (III), and compound (III) can stain only live cells, so compound (III) can be used to distinguish between live and dead cells.

Example 6 Selection of Multiple Types of Cells by Fluorescent Staining of Mitochondria In this example, the transfer of substances between two types of cells was evaluated by fluorescent staining of mitochondria using compound (III).

Compound (III) was used to stain mitochondria of human keratinocyte PSVK1 cells, and to identify which cells were keratinocytes when evaluating melanosome transfer from mouse melanoma B16F10 cells to PSVK1 cells. The procedure is shown below, but the details of staining mitochondria with compound (III) are the same as in Example 4.
(1) 1 x ¹⁰ PSVK1 cells (obtained from the National Institutes of Biomedical Innovation, Health and Nutrition) were seeded on a glass bottom dish and cultured for 24 hours. The medium used was Keratinocyte Growth Medium 2 Kit (C-20111 (TAKARA Bio)).
(2) Compound (III) was added to a final concentration of 0.01 μM, and the mixture was incubated for 5 minutes.
(3) The cells were washed three times with medium.
(4) 1 x 10 ⁴ B16F10 cells were seeded and further cultured for 24 hours.
(5) Fluorescence observation and bright field observation of the cells were performed using a fluorescence microscope. Fluorescence observation was performed with excitation at 560 nm. The results are shown in FIG.

Figure 7A is a bright-field image showing the coexistence of B16F10 cells and PSVK1 cells. The black vesicles are melanosomes. Figure 7B is a fluorescent image detecting cells stained with compound (III), and the three cells in the center are identified as PSVK1 cells. Figure 7C is an image obtained by superimposing Figures 7A and 7B.

Although melanosomes were only produced in B16F10 cells, melanosomes were present in all cells in the field of view in Figure 7A. On the other hand, as shown in Figures 7B and 7C, the cell population also contained PSVK1 cells stained with compound (III), indicating that the melanosomes present in PSVK1 cells were melanosomes transferred from B16F10 cells.

Thus, fluorescent staining of mitochondria using compound (III) enables the selection of multiple types of cells, which can be used to evaluate interactions between cells.

Example 7 Detection of Mitophagy In this example, mitophagy was detected by staining mitochondria with compound (III).

First, in a steady state in which autophagy was not occurring, triple staining was performed using compound (III), an antibody against LC3B, an autophagy marker, and DAPI for nuclear staining. The procedure is as follows.

(1) HEK293A cells were seeded on a glass bottom dish and cultured for 24 hours. The medium used was Dulbecco's modified Eagle's medium (DMEM) high glucose (D6046 (Merck)) supplemented with 1x penicillin-streptomycin (26252-94 (Nacalai Tesques)) and 10% fetal bovine serum (Nichirei). Culture was performed at 37°C and 5% _CO2 .
(2) Compound (III) was added to a final concentration of 0.01 μM.
(3) After 15 minutes, the medium was removed by aspiration, and 1 mL of 4% paraformaldehyde/phosphate buffer (09154-14 (Nacalai Tesques)) was added, followed by fixation at room temperature for 15 minutes.
(4) The 4% paraformaldehyde/phosphate buffer solution was removed by aspiration, and 1 mL of PBST was added.
(5) 1 μL of DAPI was added and left for 15 minutes to fluorescently stain the nuclei.
(6) The cells were washed three times with PBST.
(7) After washing, 1 mL of a reaction solution prepared by diluting anti-LC3B antibody (GTX127375 (GENETEX)) at 1/1000 in 10% BlockingOne (Nacalai Tesque)/phosphate buffer was added, and the plate was left at room temperature for 1 hour.
(8) The cells were washed three times with PBST.
(9) After washing, 1 mL of a reaction solution prepared by diluting anti-rabbit IgG H&L (Alexa Fluor 488) (ab150077 (Abcam)) at 1/1000 in 10% Blocking One/phosphate buffer was added, and the plate was left at room temperature for 1 hour.
(10) The cells were washed four times with PBST.
(11) The cells were observed under a fluorescence microscope. In the measurement, compound (III) was excited at 560 nm, the antibody bound to LC3B was excited at 470 nm, and DAPI was excited at 360 nm.

The results are shown in Figure 8. Figure 8A is a fluorescent image of fluorescent staining with compound (III). Figure 8B is a fluorescent image of fluorescent staining with LC3B antibody. Figure 8C is an image in which Figures 8A and 8B are superimposed with nuclear staining with DAPI. Figure 8D is a bright field image.

As shown in Figures 8A, 8B, and 8C, autophagy did not occur in live cells, so the fluorescent signal of LC3B did not overlap with the fluorescent signal of mitochondria induced by compound (III).

Next, triple staining was performed in the same manner after inducing autophagy by culturing in a nutrient-free medium. Specifically, 5 minutes after adding compound (III) during the above procedure, the medium was aspirated and replaced with a medium that did not contain glutamine, glucose, pyruvate, or fetal bovine serum, and the cells were further cultured for 24 hours. The medium was then aspirated and fixed with 4% paraformaldehyde/phosphate buffer, and the cells were treated in the same manner from (4) onwards.

The results are shown in Figure 9. Figure 9A is a fluorescent image of staining with compound (III). Figure 9B is a fluorescent image of staining with LC3B antibody. Figure 9C is an image obtained by superimposing Figures 9A and 9B with nuclear staining with DAPI. Figure 9D is a bright field image.

As shown in Figures 9A, 9B, and 9C, when autophagy was induced, the mitochondrial fluorescent signal from compound (III) was diffused throughout the cells, unlike in live cells. Furthermore, the mitochondrial fluorescent signal from compound (III) was overlapped with the fluorescent signal from LC3B.

In this way, by staining mitochondria with compound (III), it is possible to determine whether cells are undergoing autophagy from the pattern of the fluorescent signal.

Example 8: Method for evaluating the differentiation stage of melanocytes As the differentiation stage of melanocytes progresses, the number of melanosomes increases and mitochondria become localized near the nucleus. In this example, the differentiation stage of melanocyte B16F10 cells was evaluated by the localization of mitochondria using compound (II). The procedure is as follows.

(1) B16F10 cells were seeded on a glass bottom dish and cultured for 24 hours. The medium used was Dulbecco's modified Eagle's medium (DMEM) high glucose (D6046 (Merck)) supplemented with 1x penicillin-streptomycin (26252-94 (Nacalai Tesques)) and 10% fetal bovine serum (Nichirei). Culture was performed at 37°C and 5% _CO2 .
(2) Compound (II) was added to a final concentration of 0.01 μM.
(3) After 15 minutes, the medium was removed by suction and replaced with fresh medium, and then the cells were observed under a microscope.

A bright-field image is shown in Figure 10A. The black particles are melanosomes. Figure 10B is a fluorescent image of mitochondria stained with compound (II). In B16F10 cells, which are at an advanced stage of differentiation and have many melanosomes, mitochondria are localized near the nucleus. On the other hand, in B16F10 cells, which are at a lower stage of differentiation and have fewer melanosomes, mitochondria are present even at the ends of the cells.

In this way, compound (II) can be used to evaluate the differentiation stage of melanocytes.

Example 9: Image analysis (1) Detection of mitochondria HeLa cells were stained with compound (III) in the same manner as in Example 3. The stained cells were photographed using a confocal microscope (FIG. 11A). Based on the brightness value information, a region with a continuous distribution of high brightness values was detected using the Detect Peaks algorithm implemented in NIS Elements software manufactured by Nikon Corporation (FIG. 11B). The image was binarized to obtain the image shown in FIG. 11C.

This method allows the clear detection of mitochondrial shape automatically from images of cells with stained mitochondria.

(2) Detection of areas with mitochondria at a certain density or higher Aged mesenchymal stem cells and normal mesenchymal stem cells were stained with compound (III) in the same manner as in Example 3 (FIGS. 12A and 13A). Areas with mitochondria at a certain density or higher were detected by detecting areas with relatively high brightness value information compared to the areas around the mitochondria in the cells (FIGS. 12B and 13B). Meanwhile, areas where the entire cell was present were detected by binarizing the mitochondrial fluorescence image (FIGS. 12C and 13C).

Thus, in aged cells, the ratio of the area of the region with mitochondria above a certain density to the total area of the cell decreases.

The present invention provides a novel mitochondrial staining agent and a method for fluorescent staining of mitochondria.

Claims (15)

A compound represented by the following formula (I), or a salt thereof:
(Wherein,
R ¹ is hydrogen, C1 to C6 alkyl, -NH2, -OR ³ , -COOH, -CONH ₂ , -COONH ₂ , -N=C=O or -N=C=S and R ² is -N=C=O or -N=C=S, or R ² is hydrogen, C1 to C6 alkyl, -NH2, -OR ³ , -COOH, -CONH ₂ , -COONH ₂ , -N=C=O or -N=C=S and R ¹ is -N=C=O or -N=C=S;
^R3 is hydrogen or C1-C6 alkyl. The compound of claim 1, or a salt thereof, wherein ^R2 is -N=C=O. The compound according to claim 2, or a salt thereof, wherein R ¹ is -N=C=O. 3. The compound according to claim 2, wherein R ¹ is -CONH ₂ , or a salt thereof. A mitochondrial staining agent comprising the compound or salt thereof according to any one of claims 1 to 4. A cell marking agent comprising the compound or salt thereof according to any one of claims 1 to 4. A cell state evaluation agent comprising the compound or salt thereof according to any one of claims 1 to 4. An agent for detecting autophagy in a cell, comprising:
A pharmaceutical agent comprising the compound according to any one of claims 1 to 4 or a salt thereof. A method for detecting cells that have undergone autophagy, comprising:
A step of fluorescently staining mitochondria of a cell to be examined with a compound according to any one of claims 1 to 4;
observing the fluorescently stained mitochondria;
A detection method comprising: A step of fluorescently staining mitochondria using the compound or a salt thereof according to any one of claims 1 to 4;
identifying cells having the fluorescently stained mitochondria;
A method for identifying a cell, comprising: A method for identifying a first cell from a cell group including a first cell having mitochondria fluorescently stained with the compound according to any one of claims 1 to 4 or a salt thereof, and a second cell having mitochondria not fluorescently stained with any of the compounds according to claims 1 to 4, comprising:
A method for identifying cells, the method comprising the step of identifying cells in the cell group that have fluorescently stained mitochondria. A method for determining whether a substance has been transferred between a first cell and a second cell, comprising:
A step of distinguishing the first cells and the second cells by the fluorescent staining in a cell group including the first cells having mitochondria fluorescently stained with the compound according to any one of claims 1 to 4 or a salt thereof and the second cells having mitochondria not fluorescently stained with any of the compounds according to claims 1 to 4;
determining whether a substance has been transferred between the first cell and the second cell;
A method comprising: 1. A method for evaluating a test compound as a cosmetic product, comprising:
A step of fluorescently staining either keratinocytes or melanocytes in vitro with the compound according to any one of claims 1 to 4 or a salt thereof;
culturing a cell population comprising the melanocytes and the keratinocytes in the presence of the compound to be tested;
A step of distinguishing the keratinocytes from the melanocytes by the fluorescent staining; and a step of examining whether or not melanosomes have been transferred from the melanocytes to the keratinocytes.
evaluating the test compound that alters the melanosome migration as a cosmetic product;
A method comprising: 1. A method for identifying tumor cells in a non-human animal, comprising:
A step of fluorescently staining the tumor cells in vitro with the compound according to any one of claims 1 to 4 or a salt thereof;
implanting the fluorescently stained tumor cells into the animal;
identifying the fluorescently stained tumor cells in the animal body;
A method comprising: A first step of imaging a cell having one or more mitochondria fluorescently stained with the compound or salt thereof according to any one of claims 1 to 4 to obtain a cell image;
A second step of obtaining information on the one or more mitochondria from the cell image ;
A method for analyzing a cell image comprising :