WO2022220163A1 - 化合物、ミトコンドリア染色剤およびミトコンドリアの蛍光染色方法 - Google Patents

化合物、ミトコンドリア染色剤およびミトコンドリアの蛍光染色方法 Download PDF

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WO2022220163A1
WO2022220163A1 PCT/JP2022/016836 JP2022016836W WO2022220163A1 WO 2022220163 A1 WO2022220163 A1 WO 2022220163A1 JP 2022016836 W JP2022016836 W JP 2022016836W WO 2022220163 A1 WO2022220163 A1 WO 2022220163A1
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mitochondria
compound
cell
cells
image
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French (fr)
Japanese (ja)
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美保 古江
純子 坂神
憲太郎 大橋
茂雄 高島
美和 前田
洋 竹森
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Nikon Corp
Tokai National Higher Education and Research System NUC
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Tokai National Higher Education and Research System NUC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
    • C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
    • C07D311/82—Xanthenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/12—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains three hetero rings
    • C07D491/14—Ortho-condensed systems
    • C07D491/153—Ortho-condensed systems the condensed system containing two rings with oxygen as ring hetero atom and one ring with nitrogen as ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B57/00—Other synthetic dyes of known constitution

Definitions

  • the present invention relates to a compound, a mitochondrial stain, and a fluorescent staining method for mitochondria.
  • Mitochondria are intracellular organelles used by eukaryotic cells to synthesize ATP, a bioenergy, using oxygen. Therefore, mitochondrial dysfunction is directly linked to cell death and the like, causing various diseases (Japanese Patent Application Laid-Open No. 2019-112338). In addition, since mitochondrial dysfunction is also associated with mitochondrial morphology, observation of mitochondrial morphology is an important indicator when examining cell states and functions. Therefore, methods using various fluorescent low-molecular-weight compounds and fluorescent proteins have been developed for morphological observation of mitochondria (Japanese Patent Application Laid-Open No. 2020-098199; Japanese Patent Application Laid-Open No. 2017-48268).
  • fluorescent low-molecular-weight compounds in mitochondria is simple and has the advantage of being able to stain all cells, whereas the use of fluorescent proteins requires manipulations such as genetic recombination and introduction of expression vectors into cells. It is complicated because On the other hand, when mitochondria are labeled with a fluorescent low-molecular-weight compound, the fluorescent low-molecular-weight compound binds to the mitochondrial lipid membrane, resulting in unstable localization of the fluorescent low-molecular-weight compound on the mitochondrial membrane.
  • the emission wavelength of fluorescence from fluorescent small-molecular-weight compounds can be a problem. .
  • An object of the present invention is to provide a novel mitochondrial stain and a fluorescent staining method for mitochondria.
  • Embodiments according to the present invention are as follows. [1] A compound represented by the following formula (I) or a salt thereof.
  • R 1 is hydrogen, C1-C6 alkyl, —NH2, —OR 3 , —COOH, —CONH 2 , —COONH 2 , —N ⁇ C ⁇ O, or —N ⁇ C ⁇ S
  • R 3 is hydrogen or C1-C6 alkyl.
  • a method for staining mitochondria comprising the step of fluorescently staining mitochondria using the compound or salt thereof according to any one of [1] to [4].
  • a method for detecting cells that have undergone autophagy comprising: A step of fluorescently staining the mitochondria of the cells to be examined with the compound according to any one of [1] to [4]; a step of observing the fluorescently stained mitochondria; A method of detection, including [12] A step of fluorescently staining mitochondria using the compound or salt thereof according to any one of [1] to [4]; identifying cells having the fluorescently stained mitochondria; A method of identifying a cell, comprising: [13] A first cell having mitochondria fluorescently stained with the compound or salt thereof according to any one of [1] to [4] and any compound of [1] to [4]
  • a method of distinguishing a first cell from a cell population comprising a second cell having mitochondria that are not fluorescently stained comprising: A method for identifying cells, comprising the step of identifying cells having the fluorescently stained
  • a second step of obtaining information on the one or more mitochondria from the cell image When, a second step of obtaining information on the one or more mitochondria from the cell image; and a third step of evaluating the state of health of the cells based on the obtained information on the one or more mitochondria.
  • a method for analyzing cell images including [18]
  • the fluorescence image of the cell is a microscope image
  • the information of the one or more mitochondria is the area, length, width, aspect ratio, circularity, Elongation, Shape Factor, Roughness, and relationship with adjacent mitochondria of the one or more mitochondria.
  • the health of the cells is evaluated based on the statistics.
  • the analysis method according to the item. [19] In the first step, the fluorescence image of the cell is a microscope image, In the second step, the information of the one or more mitochondria is the occupancy rate of the area where the two or more mitochondria are aggregated at a predetermined density or higher in the cell, with respect to the area of the cell, In a third step, the health of the cells is evaluated based on the occupancy. [17] The analysis method according to the item.
  • the area in which two or more mitochondria fluorescently stained in the second cell are aggregated at a predetermined density or higher in the second cell, relative to the area of the second cell The analysis method according to item [19] or [21], wherein the health condition of the cells is evaluated by comparing with the occupancy rate. [24] The analysis method according to [22] or [23], wherein the second cells are normal cells.
  • 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 computer controlled operation of a microscope apparatus for use in an embodiment of the present invention.
  • FIG. 3 shows a flow chart of a method for analyzing images of cells stained for mitochondria in one embodiment of the present invention.
  • FIG. 4 shows images observed with 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 with a fluorescence microscope when mitochondria were labeled with compound 4, fixed, and triple-stained with COX4-Alexa488 and DAPI in Example 4.
  • FIG. 5A is an image of compound 4 fluorescence
  • FIG. 5B is Alexa488 fluorescence
  • FIG. 5C is a superimposed image of compound 4 fluorescence, Alexa488 fluorescence, and DAPI fluorescence.
  • FIG. 5D is a bright field image.
  • 10 is an image observed with a fluorescence microscope showing the results of Example 5.
  • FIG. 6A is stained with compound 4
  • FIG. 6B is stained with compound 4 and then treated with mitochondrial uncoupler
  • FIG. 6C is stained with mitochondrial uncoupler first and then stained with compound 4.
  • FIG. 10 is an image observed with a fluorescence microscope showing the results of Example 6.
  • FIG. The figure at the top of the image is a schematic diagram showing the dyeing process.
  • FIG. 7A is a brightfield image of where cells are present.
  • FIG. 7B is an observation image of fluorescence emitted by Compound 4 (denoted as Chemical Compound 4 in the figure).
  • FIG. 7C is an image in which the bright field image and the fluorescence image are superimposed.
  • 10 is an image observed with a fluorescence microscope showing the results of Example 7.
  • FIG. 8A is an observation image of fluorescence emitted by Compound 4.
  • FIG. FIG. 8B is an observation image of fluorescence staining using the LC3B antibody.
  • FIG. 8C is a superimposed image of fluorescence from compound 4, fluorescence staining with the LC3B antibody, and fluorescence from DAPI.
  • FIG. 8D is a bright field image.
  • FIG. 10 is an image observed with a fluorescence microscope showing the results of Example 8.
  • FIG. 9A is an observation image of fluorescence emitted by compound 1.
  • FIG. 9B is an observation image of staining using the LC3B antibody.
  • FIG. 9C is a superimposed image of fluorescence from Compound 1, fluorescence staining with the LC3B antibody, and fluorescence from DAPI.
  • FIG. 9D is a bright field image.
  • 10 is an image observed with a fluorescence microscope showing the results of Example 9.
  • FIG. 10A is a brightfield image.
  • 10B is an observation image of 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 in which luminance values are continuously distributed at high levels is detected. It is a diagram showing.
  • FIG. 12 shows an image of normal mesenchymal stem cells stained with compound (III) in Example 9 (A), and an image (B) of a region in which mitochondria have a predetermined density or higher. , and an image (C) in which a region in which whole cells are present is detected.
  • FIG. 13 shows an image (A) obtained by imaging aged mesenchymal stem cells stained with compound (III) in Example 9, and an image (B) obtained by detecting a region in which mitochondria have a predetermined density or more. , and an image (C) in which a region in which whole cells are present is detected.
  • a compound according to one embodiment disclosed in the present specification 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).
  • the salt is called compound (I)).
  • R 1 is hydrogen, C1-C6 alkyl, —NH2, —OR 3 , —COOH, —CONH 2 , —COONH 2 , —N ⁇ C ⁇ O, or —N ⁇ C ⁇ S
  • R 3 is hydrogen or C1-C6 alkyl.
  • formula (II) or (III) represented by formula (II) or (III) below .
  • compound (I) 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 method described in the Examples and appropriately modifying it using common general technical knowledge.
  • a solvent for the dye is not particularly limited as long as it can dissolve the above compound, and examples thereof include acetone, DMSO, ethanol, acetonitrile, and aqueous solutions thereof.
  • the mitochondrial stain may contain, for example, a fluorescent compound for fluorescently staining mitochondria and cellular constituents different from mitochondria, such as cell membranes and intracellular organelles. This allows multiple elements to be dyed simultaneously. Further, the interaction between the compound (I) and the fluorescent compound can sensitize the luminescence of the compound (I) and improve the specificity of the compound (I) to mitochondria. Examples of fluorescent compounds that sensitize the emission 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), etc.
  • Staining of mitochondria using the mitochondrial staining agent disclosed herein can be performed by a routine method for those skilled in the art.
  • compound (I) when staining mitochondria in cultured cells, compound (I) may be added to the medium during culture.
  • concentration of compound (I) is not particularly limited, it is preferably 0.001 ⁇ M to 10 ⁇ M, more preferably 0.01 ⁇ M to 0.1 ⁇ M.
  • the dyeing time is also not particularly limited, but is preferably from (1 minute) to (1 week), more preferably from (5 minutes) to (1 hour).
  • the dyeing temperature is not particularly limited, it is preferably (10°C) to (45°C), more preferably (30°C) to (40°C).
  • the dyeing time is preferably longer, and specific examples include conditions such as (37)° C. and (15) minutes.
  • the medium may be removed and the cells may be washed, or may proceed to the next step without washing.
  • a cell culture medium, a phosphate buffer, or the like can be used as a washing solution.
  • the cleaning liquid may contain a surfactant.
  • the cultured cells may be collected, 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 a slide glass, or may proceed to the next step while being suspended.
  • the cells are irradiated with light of the excitation wavelength, and the fluorescence emitted from the staining agent is detected to detect the mitochondria in the cells.
  • irradiation means similar to those used for general fluorescence detection may be used.
  • a predetermined wavelength may be selected from a laser light source provided in a fluorescence microscope, if necessary. Fluorescence may 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 may be displayed on display means such as a monitor.
  • a filter that selectively passes a predetermined wavelength may be used as needed.
  • Compound (I) can be used as a very convenient cell marking agent.
  • the mitochondria of tumor cells collected from humans are marked with compound (I), transplanted into mice, and fluorescence emission is traced to determine the behavior of tumor cells in the body of mice (e.g., ease of metastasis, etc.). ) can be known.
  • a cell group comprising a first cell having mitochondria fluorescently stained with compound (I) and a second cell having mitochondria not fluorescently stained with compound (I)
  • fluorescence from compound (I) The first cell can be identified by detecting staining and identifying cells with fluorescently labeled mitochondria.
  • substance transfer or information transfer between the first cell and the second cell can be evaluated.
  • a step of fluorescently staining either keratinocytes or melanocytes with compound (I) mixing these melanocytes and keratinocytes, and a cell group containing both, a compound to be tested and compound (I) culturing in the presence of; distinguishing between keratinocytes and melanocytes by fluorescent staining; and examining whether melanosomes have migrated from melanocytes to keratinocytes.
  • the compound to be tested as a cosmetic product.
  • compounds that inhibit the movement of melanosomes from melanocytes to keratinocytes can inhibit the movement of melanin to the body surface, so they can be evaluated as cosmetics with a whitening effect.
  • Compound (I) can also be used as a cell state-evaluating agent.
  • Examples of cell states include cell differentiation stage, apoptosis, hypoxia, cell growth inhibition, growth promotion, and the like.
  • mitochondria have an elongated shape, but when reprogrammed into iPS cells, the mitochondria become fragmented, and when redifferentiated into differentiated cells, the mitochondria assume an elongated shape again. become (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 of reprogramming or regeneration of cells.
  • the differentiation stage of melanocytes can be evaluated by fluorescently staining mitochondria with compound (I) and observing the intracellular localization of mitochondria.
  • mitophagy a phenomenon known as mitophagy in which surplus mitochondria are selectively degraded when cells undergo autophagy. Therefore, when mitochondria are fluorescently stained with compound (I) and the state of intracellular fluorescent staining is examined, fluorescence is finely scattered in mitophagy-induced cells, and fluorescence signals specific to mitophagy are observed. Therefore, by fluorescently staining mitochondria with compound (I) and observing the fluorescence distribution state, it is possible to identify whether or not the cells are undergoing autophagy.
  • compound (I) strongly binds to mitochondria, compound (I) is as strong as a conventional fluorescent dye, even if it is fixed with a fixative after fluorescent staining with compound (I) or the membrane is permeabilized with a surfactant. ) does not weaken the fluorescence intensity. Moreover, since the peak width of the emitted fluorescence wavelength is narrow (ie, 590 nm to 660 nm), there is little overlap with the fluorescence wavelengths of other fluorescent dyes. From these facts, compound (I) can be suitably used for multiple staining with other fluorescent dyes.
  • the cells after fluorescently staining the mitochondria of cells with compound (I), the cells can be fixed and multi-stained by histochemical techniques using antibodies.
  • the fluorescent dye to be used at the same time may have a wavelength that does not overlap with the fluorescence of compound (I), and examples thereof include FITC, ATTO 488, Alexa Fluor 488, and Cy5.
  • DAPI DAPI
  • mitochondrial morphology and distribution reflect cell health.
  • mitochondrial morphology and distribution such as fragmentation and clustering near the nucleus, reflect cellular health. Therefore, the toxicity of cosmetics or pharmaceuticals can be evaluated by treating cultured cells with cosmetics or pharmaceuticals in advance and fluorescently staining the mitochondria of the cells with compound (I).
  • ⁇ Disease marker> The shape of mitochondria changes according to cell conditions and pathologies. For example, mitochondria are known to curl up when undernourished. Alternatively, cardiovascular diseases, neurological diseases such as Hutchinton's disease, ALS, and diabetes are known to be triggered by abnormal mitochondrial fission/fusion (Japanese Pharmacological Society, Japanese Pharmacological Journal 149: 269-273 ( 2017) “Ecopharma, an Approved Drug Focusing on Mitochondrial Fission”). Therefore, compound (I) can also be used as a diagnostic marker for diseases and the like. For example, a method comprising the steps of collecting biological tissue from a living body, staining the mitochondria of the collected biological tissue with compound (I), and evaluating a disease, using compound (I) as a diagnostic marker. can be used.
  • the cell image analysis method includes a first step of acquiring a cell image by imaging a cell having mitochondria fluorescently stained with the compound disclosed herein or a salt thereof, and from the cell image, A second step of obtaining mitochondrial information and a third step of assessing cell health based on the obtained mitochondrial information. This analysis method allows investigation of the state of cells based on mitochondrial morphology.
  • a cell image is obtained by imaging a cell having one or more mitochondria fluorescently stained with a compound disclosed herein or a salt thereof.
  • This cell image is preferably a microscope image.
  • a microscope image is preferably captured by a camera with an image sensor attached to a microscope including a stereomicroscope and an electron microscope, and obtained as an image file.
  • a single cell may be captured in the cell image, or a plurality of cells may be captured.
  • ⁇ Second step> information on one or more mitochondria present in one cell is obtained from the cell image obtained in the first step.
  • Mitochondrial information refers to numerical values obtained from the mitochondrial image that indicate the characteristics of the mitochondrion, such as area, length, width, aspect ratio, circularity, elongation, shape factor, roughness, shortest distance to adjacent mitochondria.
  • Statistical value obtained by statistically processing at least one of the distance, the number of branching points, the distance from the nucleus, the distance from the cell membrane, and the distance from the cell body, or the number of fluorescently stained mitochondria in the cell It is the occupancy rate in the cell of the area aggregated at a predetermined density or more in the cell.
  • the length of mitochondria is the longest line segment connecting two points on the circumference of mitochondria.
  • the width of mitochondria is the shortest of the lengths of line segments connecting two points on the circumference of mitochondria.
  • the circularity of mitochondria refers to the result of dividing the square of the circumference of a circle having the same area as the mitochondrial region by the square of the circumference of the area.
  • Mitochondrial elongation refers to the ratio of mitochondrial length to width.
  • the shape factor of mitochondria refers to the ratio of the square of the perimeter of a circle having the same area as the mitochondrial region to the square of the perimeter of a shape that fills the unevenness of the mitochondrial region.
  • the shortest distance between a mitochondrion and an adjacent mitochondrion is the shortest length of a line segment connecting a point on the periphery of the mitochondrion and a point on the periphery of the adjacent mitochondrion, or between the center of gravity of the mitochondrion and the distance between the adjacent mitochondria It refers to either of the distances from the center of gravity.
  • the number of branching points of mitochondria refers to the number of branching portions when mitochondria have a branched shape.
  • the distance from the mitochondrial nucleus is the shortest of the lengths of the line segments connecting the points on the circumference of the mitochondria and the points on the circumference of the adjacent mitochondria, or the distance between the centroid of the mitochondria and the centroid of the nucleus.
  • the distance between the mitochondria and the cell membrane is the shortest of the lengths of the line segments connecting the points on the periphery of the mitochondria and the points on the cell membrane.
  • the distance of mitochondria from 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 centroid of the cell body, or the centroid of the nucleus in nerve cells.
  • the obtained value may be used as is, or statistical processing such as rounding may be performed so that the value is rounded to a predetermined number of digits to calculate the statistic.
  • statistical processing may be performed using the obtained numerical values to calculate the statistic.
  • Examples of statistical processing include obtaining average values, median values, variance values, quartiles, and the like.
  • the occupancy rate of the area of the cell where fluorescently-stained mitochondria are aggregated at a predetermined density or higher in the cell may be obtained. Specifically, for example, first, the area of the cell in which the mitochondria are present is obtained. Next, the area of the region where mitochondria are aggregated at a predetermined density or higher is determined. Finally, the ratio of the area of the mitochondria-aggregated region to the area of the cells is obtained, and this ratio may be used as the occupancy rate.
  • ⁇ Third step> the health condition of the cells is evaluated from the mitochondrial information obtained in the second step.
  • the state of health of cells means that cells are in an abnormal state or in an aged state.
  • Cells are in an abnormal state, e.g. undergoing necrosis, undergoing apoptosis, exhibiting faster or slower than normal proliferation rates, altered cell morphology, differentiation such as mesenchymal-epithelial transdifferentiation It refers to the occurrence of transformations, disruption of the balance between mitochondrial fission and fusion, and accumulation of abnormal proteins.
  • cells age for example, their ability to produce collagen decreases, their proliferation rate slows down, their telomeres shorten, they lose cell-specific or intrinsic functions, and their cytoplasm increases. , multinucleation, or fragmentation of mitochondria.
  • This cellular health state also includes the state of the individual, for example the state of cells in patients with bipolar disorder.
  • two or more mitochondria fluorescently stained in a second cell different from the cell for which the cell image is analyzed are aggregated at a predetermined density or more in the second cell. The health of the cells may be evaluated by comparing the occupancy ratio of the area in which the cells are located to the area of the second cells and the occupancy ratio obtained in the second step.
  • the second cells are preferably cells of the same type as the cells whose cell images are to be analyzed, and are preferably normal cells.
  • normal means not being in an abnormal state as described above. Examples include normal cell morphology, function, and proliferation.
  • ⁇ Fourth step> a bright-field image of the cell from which information on mitochondria was obtained is obtained. Acquired bright-field images can be used, for example, to determine cell area.
  • a fluorescence image of one or more cells containing one or more stained mitochondria is acquired using a fluorescence microscope with an image sensor (S21). Then, shot noise is removed from the captured image (S21). Specifically, shot noise can be removed from the captured image by averaging luminance values or removing high-frequency components in a region of a certain size in the captured image.
  • cell area the cell region in the image (hereinafter referred to as "cell area") is detected (S22).
  • the nucleus is detected by binarizing the fluorescent image of a nuclear staining reagent such as DAPI, and the cell area is detected using the Watershed algorithm for the fluorescent image of cytoplasmic staining using that as a starting point.
  • Mitochondrial fluorescence imaging may be used instead of cytoplasmic staining reagents.
  • a cell area may be detected using a phase contrast image.
  • a close/open filter is applied to the phase contrast image to obtain the difference between the images, acquire edge information, and create a GVF (Gradient Vector Flow) based on the acquired edge information to extract the outline of the cell. It is also good to acquire and detect the cell area.
  • the cell area can be detected by finally obtaining cell contours from the fluorescence image.
  • the Detect Peaks algorithm implemented in NIS Elements software manufactured by Nikon Corporation is used to detect a continuous high brightness value distribution. Detect the area where it is, and further increase the luminance to a high luminance value (S23). As a result, the outline of the filamentous structure is emphasized as the internal structure of each cell.
  • areas where mitochondria are aggregated to a predetermined density or higher within the cell are detected (S26).
  • a binary image from which mitochondria can be identified may be used, but a captured image of cells from which shot noise has been removed may also be used. In the latter case, for example, by calculating the sum of luminance values for each divided region and identifying regions with relatively high luminance values, regions where mitochondria are aggregated may be detected.
  • the area of the cell region detected in S22 and the area of the region where mitochondria are detected in S26 are measured, and the area where mitochondria are aggregated at a predetermined density or more in the cell is compared to the area of the cell.
  • the occupancy rate is calculated (S27).
  • the evaluation method is not particularly limited, for example, the mitochondrial information of the second cell is obtained, the learning device described below is used, and the health state of the cell is evaluated by comparing it with the mitochondrial information of the second cell. You may
  • abnormal or senescent cells or normal cells are used, and one or two or more pieces of information obtained from these cells are mapped to an n-dimensional vector space, and deep learning, support vector machine , decision tree, etc. By inputting information on the target mitochondria into this learning machine, it is possible to determine whether abnormalities or aging have occurred.
  • FIG. 1 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 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 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 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.
  • the microscope apparatus 1 includes an apparatus main body 10, a computer 170, a monitor 160 that is a display section, and an input device 180.
  • the device body 10 includes a microscope section 150 , a transmitted illumination section 40 , an imaging device (hereinafter referred to as a digital camera) 300 , an excitation light source 70 and an optical fiber 7 .
  • digital cameras include CCD cameras and CMOS cameras.
  • the microscope section 150 includes 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. , a light source 47 for transmitted illumination, a field lens 44 and a deflection mirror 45 .
  • a chamber 100 containing a transparent culture container 20 is mounted on the stage 23 .
  • cultured cells whose mitochondria are labeled with a fluorescent substance grow in a medium.
  • a portion a of the bottom surface of the chamber 100 and a portion b of the top surface of the chamber 100 are transparent.
  • the top surface of the culture container 20 is open will be described, but the top surface of the culture container 20 may be covered with a lid of the same quality as the culture container 20 if necessary.
  • a plurality of types of objective lenses are attached to the objective lens section 27 along the X direction in FIG.
  • the type of objective lens is switched. This switching is performed under the control of computer 170 .
  • a plurality of types of filter blocks are attached to the fluorescence filter section 34 along the X direction in FIG.
  • the type of filter block is switched. This switching is also performed under the control of the computer 170 .
  • the computer 170 switches the combination of the type of objective lens arranged in the optical path and the type of filter block arranged in the optical path according to the observation method to be set in the apparatus main body 10 .
  • the observation method by the apparatus main body 10 is switched between phase contrast observation and two types of fluorescence observation by this switching.
  • Phase contrast observation and fluorescence observation differ in the type of filter block placed in the optical path and the type of objective lens that can be placed in the optical path. are different, and the types of objective lenses that can be placed in the optical path are the same.
  • the illumination method differs between phase contrast observation and fluorescence observation.
  • the computer 170 uses the transmitted illumination light source 47 to use the optical path of the transmitted illumination unit 40 during phase contrast observation, and uses the optical path of the epi-illumination unit (that is, the excitation light source 70, the optical fiber 7, the collector An optical path passing through the lens 41, the field lens 411, the deflection mirror 452, the fluorescence filter section 34, and the objective lens section 27 in that order) is used, so the excitation light source 70 is used.
  • 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.
  • phase contrast observation which is bright field observation
  • the light emitted from the transmission illumination light source 47 illuminates the observation point c in the culture vessel 20 via the field lens 44, the deflection mirror 45, and the transparent portion b of the chamber 100. do.
  • 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 container 20, the transparent portion a of the chamber 100, the objective lens portion 27, the fluorescence filter portion 34, and the imaging lens 38, and is observed.
  • a phase contrast image of point c When the digital camera 300 operates in this state, a phase contrast image is captured and phase contrast image data is generated. This image data is transmitted to computer 170 and stored there.
  • the bright-field image obtained by bright-field observation includes 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.
  • the light emitted from the excitation light source 70 passes through the optical fiber 7, the collector lens 41, the field lens 411, the deflection mirror 452, the fluorescence filter section 34, the objective lens section 27, the transparent section a of the chamber 100, the culture vessel.
  • Observation point c in culture vessel 20 is illuminated through the bottom surface of 20 .
  • This fluorescence reaches the light-receiving surface of the digital camera 130 via the bottom surface of the culture container 20, the transparent portion a of the chamber 100, the objective lens portion 27, the fluorescence filter portion 34, and the imaging lens 38, and a fluorescence image at the observation point c is obtained.
  • a fluorescence image is captured and image data is generated. This image data is also transmitted to computer 170 and stored there.
  • the computer 170 controls the XYZ coordinate position of the observation point c in the culture container 20 by controlling the XY coordinates of the stage 23 and the Z coordinate of the objective lens unit 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 predetermined values.
  • the air around the chamber 100 may be appropriately circulated by a heat exchanger, thereby controlling the internal temperature of the chamber 100 to a predetermined value.
  • Humidity, CO2 concentration, and temperature inside such chamber 100 may be measured by sensors. The measurement results may be transmitted to computer 170 and stored there.
  • the digital camera 300 may be housed in a housing separate from the other parts of the device main body 10 and maintained at the same temperature as the outside air temperature of the device main body 10 regardless of the internal temperature of the chamber 100 .
  • a program for observation is installed in the computer 170, and the computer 170 operates according to the program. Any information input from the user to the computer 170 is performed through the input device 180 .
  • FIG. 2 is an operation flowchart of the computer 170.
  • the computer 170 first sets the observation method of the apparatus main body 10 to low-magnification phase-contrast observation, acquires the image data of the bird's-eye view image in this state, and displays it on the monitor 160.
  • S11 A bird's-eye view image refers to an image of a relatively wide area of the culture container 20 .
  • the computer 170 In acquiring the image data of the bird's-view image, the computer 170 repeatedly acquires the image data of the phase-contrast image while moving the observation point c in the culture container 20 in the XY directions. composited with the image data of the image of Each piece of image data is image data of a so-called tile image, and image data after combining is image data of a bird's-eye view image.
  • the user While observing the bird view image displayed on the monitor 160, the user determines the conditions for time-lapse photography (for example, interval, number of rounds, observation point, observation method, etc.).
  • the computer 170 When the user inputs the conditions (S12: YES), the computer 170 creates a recipe in which the conditions are written (S13).
  • the storage destination of the recipe is, for example, the hard disk of the computer 170 .
  • the interval, the number of rounds, the observation point, and the observation method written in the recipe will be referred to as "designated interval,” “designated number of rounds,” “designated point,” and “designated observation method,” respectively.
  • the computer 170 receives an instruction to start time-lapse photography from the user (S14), the computer 170 starts time-lapse photography (S15).
  • the computer 170 matches the observation point c of the culture container 20 with the designated point, sets the observation method of the apparatus main body 10 to the designated observation method, and acquires image data in this state.
  • three designated observation methods three types of image data are continuously acquired while switching the observation method of the device main body 10 among the three designated observation methods. This completes the shooting of the first round.
  • the computer 170 waits for a specified interval from the first round shooting start time, and starts shooting the second round.
  • the imaging method for the second round is the same as the imaging method for the first round.
  • the computer 170 sequentially accumulates the image data captured from the apparatus main body 10 in the execution progress file.
  • the storage destination of this implementation progress file is, for example, the hard disk of the computer 170 .
  • the computer 170 refers to the contents of the implementation progress file at that point in time, and based on that, displays the implementation progress confirmation screen described below. is displayed on the monitor 160 .
  • the captured image of each cell in the bird's-eye view image acquired by the microscope apparatus 1 is input to the image processing unit of the computer 170, where the above-described image analysis processing is performed, and the analysis result is output. good.
  • Example 3 Fluorescent Staining of Mitochondria in Live Cells Using compound (II) and compound (III) synthesized in Examples 1 and 2, intracellular mitochondria were fluorescently stained. The procedure is shown below.
  • FIG. 4A is an image of whole cells by compound (II).
  • FIG. 4B is a whole-cell image with compound (III).
  • compound (II) and compound (III) fluorescently stained mitochondria having linear structures in cells.
  • compound (II) and compound (III) can stain intracellular mitochondria.
  • Example 4 Triple staining with DAPI and anti-COX4 antibody
  • intracellular mitochondria were fluorescently stained with compound (III) synthesized in Example 2, fixed, and further stained with DAPI and mitochondrial markers. Cells were stained with an antibody against COX4. The procedure is shown below.
  • HEK293A cells obtained from Thermofisher were seeded in a glass bottom dish (Matsunami) and cultured for 24 hours.
  • the medium used was Dulbecco's Modified Eagle's Medium (DMEM) with high glucose (D6046 (Merck)) added with 1 ⁇ penicillin/streptomyne (26252-94 (Nacalai Techs)) and 10% fetal bovine serum (Nichirei). . Cultivation was performed at 37° C., 5% CO 2 .
  • Compound (III) was added to the medium to a final concentration of 0.01 ⁇ M.
  • FIG. 5A is a fluorescence microscope image of whole cells by compound (III).
  • FIG. 5B is the accumulation site of the anti-COX4 antibody.
  • FIG. 5C is a superimposition of FIGS. 5A, 5B and the fluorescence image of the nucleus (DAPI).
  • FIG. 5D is a bright-field image of the whole cell. Fluorescence was emitted from compound (III) and antibody bound to COX4, as shown in FIGS. 5A and B, and fluorescence from compound (III) and antibody bound to COX4, as shown in FIG. 5C. fluorescence was superimposed.
  • compound (III) can be fixed after staining and subjected to multiple staining with DAPI and antibodies.
  • Example 5 Dependence of fluorescent staining on mitochondrial membrane potential
  • the mitochondrial membrane has a stronger membrane potential than the membranes of other intracellular organs, and most of the compounds with mitochondrial accumulation are dependent on the mitochondrial membrane potential. Localized in mitochondria.
  • This example shows that staining of mitochondria by compound (III) is dependent on membrane potential and that once stained, staining remains even with loss of membrane potential. The procedure is shown below, but the details of staining mitochondria with compound (III) are the same as in Example 4.
  • FIG. 6A is an image of fluorescence emitted by treatment with compound (III) alone.
  • FIG. 6B is an image after FCCP treatment after treatment with compound (III).
  • FIG. 6C is an image of compound (III) treatment after FCCP treatment.
  • Example 6 Selection of multiple types of cells by fluorescent staining of mitochondria
  • transfer of substances between two types of cells was evaluated by fluorescent staining of mitochondria using compound (III).
  • Compound (III) was used to stain the mitochondria of human keratinocyte PSVK1 cells to distinguish which were keratinocytes when assessing melanosome transport 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 ⁇ 10 4 PSVK1 cells obtained from National Institute of Biomedical Innovation, Health and Nutrition
  • Keratinocyte Growth Medium 2 Kit C-20111 (TAKARA Bio)
  • Compound (III) was added to a final concentration of 0.01 ⁇ M and incubated for 5 minutes.
  • FIG. 7A is a bright field image showing the coexistence of B16F10 cells and PSVK1 cells.
  • the black grains that are vesicles are melanosomes.
  • FIG. 7B is a fluorescence image detecting cells stained with compound (III), and the three cells in the central portion are identified as PSVK1 cells.
  • FIG. 7C is a superimposed image of FIGS. 7A and 7B.
  • fluorescence staining of mitochondria using compound (III) enables selection of multiple types of cells, which can be used to evaluate interactions between cells.
  • Example 7 Detection of mitophagy
  • mitophagy was detected by staining mitochondria with compound (III).
  • HEK293A cells were seeded in a glass bottom dish and cultured for 24 hours.
  • the medium used was Dulbecco's Modified Eagle's Medium (DMEM) with high glucose (D6046 (Merck)) added with 1 ⁇ penicillin/streptomyne (26252-94 (Nacalai Techs)) and 10% fetal bovine serum (Nichirei). .
  • Cultivation was performed at 37° C., 5% CO 2 .
  • Compound (III) was added to a final concentration of 0.01 ⁇ M.
  • FIG. 8A is a fluorescence image of fluorescence staining with compound (III).
  • FIG. 8B is a fluorescence image of fluorescent staining with the LC3B antibody.
  • FIG. 8C is an image obtained by superimposing the nuclear staining with DAPI on FIGS. 8A and 8B.
  • FIG. 8D is a bright field image.
  • FIG. 9A is a fluorescent image of staining with compound (III).
  • FIG. 9B is a fluorescent image of staining with the LC3B antibody.
  • FIG. 9C is an image obtained by superimposing the nuclear staining with DAPI on FIGS. 9A and 9B.
  • FIG. 9D is a bright field image.
  • Example 8 Method for evaluating the differentiation stage of melanocytes As the differentiation stage of melanocytes progresses, melanosomes increase and mitochondria are localized near the nucleus.
  • compound (II) was used to assess the differentiation stage of melanocyte B16F10 cells by mitochondrial localization. The procedure is shown below.
  • B16F10 cells were seeded in a glass bottom dish and cultured for 24 hours.
  • the medium used was Dulbecco's Modified Eagle's Medium (DMEM) with high glucose (D6046 (Merck)) added with 1 ⁇ penicillin/streptomyne (26252-94 (Nacalai Techs)) and 10% fetal bovine serum (Nichirei). .
  • Cultivation was performed at 37° C., 5% CO 2 .
  • Compound (II) was added to a final concentration of 0.01 ⁇ M.
  • the medium was removed by aspiration and replaced with new medium, followed by microscopic observation.
  • FIG. 10A A bright-field image is shown in FIG. 10A. Black grains are melanosomes.
  • FIG. 10B is a fluorescence image of mitochondria stained with compound (II). Mitochondria were localized in the vicinity of the nucleus in B16F10 cells, which advanced in the differentiation stage and contained many melanosomes. On the other hand, B16F10 cells with low differentiation stage and few melanosomes had mitochondria at the cell ends.
  • compound (II) can be used to evaluate the differentiation stage of melanocytes.
  • Example 9 Image analysis (1) Detection of mitochondria HeLa cells were stained in the same manner as in Example 3 using compound (III). Using the Detect Peaks algorithm implemented in NIS Elements software manufactured by Nikon Corporation, based on the brightness value information for the image (Fig. 11A) of the stained cells captured using a confocal microscope. , a region where luminance values are continuously distributed at high levels was detected (FIG. 11B). The image was binarized to obtain the image shown in FIG. 11C.
  • the shape of mitochondria can be automatically and clearly detected from images of cells stained with mitochondria.
  • the ratio of the area of mitochondria with a predetermined density or higher to the total area of the cell has decreased.
  • the present invention can provide a novel mitochondrial staining agent and a fluorescent staining method for mitochondria.

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006056885A (ja) * 1999-11-03 2006-03-02 Applera Corp 水溶性ローダミン色素およびその結合体
JP2010180409A (ja) * 1997-09-23 2010-08-19 Molecular Probes Inc スルホン化キサンテン誘導体
JP2011506673A (ja) * 2007-12-14 2011-03-03 バイオティウム， インコーポレイテッド 蛍光性化合物
WO2013035767A1 (ja) * 2011-09-07 2013-03-14 国立大学法人 東京大学 酸性環境検出蛍光プローブ
JP2018502581A (ja) * 2015-01-06 2018-02-01 ルテリオン カンパニー リミテッドＬｕｔｅｒｉｏｎ Ｃｏ．， Ｌｔｄ． ルテリオンおよびその分離・培養方法
CN111004246A (zh) * 2019-12-13 2020-04-14 山西大学 监测线粒体自噬的罗丹明类pH荧光探针及制备和应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010180409A (ja) * 1997-09-23 2010-08-19 Molecular Probes Inc スルホン化キサンテン誘導体
JP2006056885A (ja) * 1999-11-03 2006-03-02 Applera Corp 水溶性ローダミン色素およびその結合体
JP2011506673A (ja) * 2007-12-14 2011-03-03 バイオティウム， インコーポレイテッド 蛍光性化合物
WO2013035767A1 (ja) * 2011-09-07 2013-03-14 国立大学法人 東京大学 酸性環境検出蛍光プローブ
JP2018502581A (ja) * 2015-01-06 2018-02-01 ルテリオン カンパニー リミテッドＬｕｔｅｒｉｏｎ Ｃｏ．， Ｌｔｄ． ルテリオンおよびその分離・培養方法
CN111004246A (zh) * 2019-12-13 2020-04-14 山西大学 监测线粒体自噬的罗丹明类pH荧光探针及制备和应用

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