WO2015068329A1 - 細胞分析システム、細胞分析プログラム及び細胞分析方法 - Google Patents
細胞分析システム、細胞分析プログラム及び細胞分析方法 Download PDFInfo
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- G06T7/0002—Inspection of images, e.g. flaw detection
- G06T7/0012—Biomedical image inspection
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- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
- G01N33/5029—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on cell motility
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
- G01N33/5035—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on sub-cellular localization
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
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- G06T7/20—Analysis of motion
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
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- G06T2207/30004—Biomedical image processing
- G06T2207/30024—Cell structures in vitro; Tissue sections in vitro
Definitions
- the present technology relates to a cell analysis system, a cell analysis program, and a cell analysis method suitable for analyzing movement of ions or molecules through a cell membrane.
- ion transport between cell membranes The movement of molecules and ions through the cell membrane (hereinafter referred to as ion transport between cell membranes) is related to the formation and fluctuation of membrane potential important for cell function. Ion transport between cell membranes is performed by specific molecules (proteins) such as ion pumps and ion channels provided in the cell membrane.
- An ion pump is a molecule that consumes energy and moves ions, and an ion channel opens and closes due to membrane potential or the binding of specific molecules, and moves ions according to the concentration difference inside and outside the cell.
- Intercellular membrane ion transport is very important for cell function, and if it can be understood, various applications are possible.
- ion channels are important as targets for drug discovery.
- ion transport between cell membranes is generally evaluated by a method using a patch clamp (for example, see Patent Document 1) or a method for measuring an extracellular electric field (for example, see Patent Document 2).
- a method of measuring intracellular ion concentration using fluorescent staining is also used.
- the spatial resolution depends on the electrode size and the number of electrodes.
- the electrode size is large and the number of electrodes is small, the spatial resolution is low.
- the number of electrodes is large, a complicated device configuration is required. Necessary.
- an object of the present technology is to provide a cell analysis system, a cell analysis program, and a cell analysis method suitable for analyzing the movement of ions or molecules through a cell membrane.
- a cell analysis system includes a motion information extraction unit and a motion characteristic calculation unit.
- the motion information extraction unit extracts motion information resulting from the movement of ions or molecules through the cell membrane from a cell image obtained by imaging a cell over time.
- the motion characteristic calculation unit calculates a motion characteristic of the motion information.
- a cell analysis program operates an information processing apparatus as a motion information extraction unit and a motion characteristic calculation unit.
- the motion information extraction unit extracts motion information resulting from the movement of ions or molecules through the cell membrane from a cell image obtained by imaging a cell over time.
- the motion characteristic calculation unit calculates a motion characteristic of the motion information.
- a cell analysis method is such that the motion information extraction unit performs a motion caused by movement of ions or molecules through a cell membrane from a cell image obtained by imaging a cell over time. Extract information.
- a motion characteristic calculator calculates a motion characteristic of the motion information.
- the present technology it is possible to provide a cell analysis system, a cell analysis program, and a cell analysis method suitable for analyzing movement of ions or molecules through a cell membrane.
- the effects described here are not necessarily limited, and may be any of the effects described in the present disclosure.
- a cell analysis system includes a motion information extraction unit and a motion characteristic calculation unit.
- the motion information extraction unit extracts motion information resulting from the movement of ions or molecules through the cell membrane from a cell image obtained by imaging a cell over time.
- the motion characteristic calculation unit calculates a motion characteristic of the motion information.
- the movement information extracted from the cell image includes information resulting from the movement of ions or molecules. Yes. Therefore, it is possible to evaluate the movement of ions or molecules through the cell membrane (hereinafter, ion transport between cell membranes) based on the motion characteristics that are the characteristics of motion information. According to the above configuration, it is not necessary to stain cells, and it is possible to prevent the stain from affecting the cells. Furthermore, since the cell analysis system can evaluate ion transport between cell membranes by image processing on a cell image, it can analyze a wide area with high resolution.
- the cell analysis system is You may further comprise the image generation part which superimposes the said motion characteristic and the said cell image, and produces
- the cell analysis system can display the motion characteristics in a form that is easy to visually grasp for the user, and the user can refer to the motion characteristics display image to determine the ions or molecules through the cell membrane. It is possible to evaluate movement.
- the cell analysis system is An evaluation unit that evaluates the movement of ions or molecules through the cell membrane based on the motion characteristics may be further provided.
- the evaluation unit can evaluate the movement of ions or molecules through the cell membrane based on the movement characteristics. is there.
- the motion characteristic calculation unit calculates a motion amount as the motion characteristic,
- the evaluation unit may evaluate the movement amount of ions or molecules based on the movement amount.
- the amount of movement increases or decreases depending on the amount of ions or molecules flowing into the cell or the amount flowing out of the cell. For this reason, it is possible to evaluate the amount of movement of ions or molecules based on the amount of movement.
- the motion characteristic calculation unit calculates a motion amount as the motion characteristic,
- the evaluation unit may evaluate the activity of an ion channel or an ion channel receptor based on the amount of movement.
- the activity of an ion channel or ion channel receptor that allows ions or molecules to pass through is evaluated based on the amount of movement. It becomes possible.
- the movement characteristic calculation unit calculates a movement change before and after the administration of the activator or inhibitor of the ion channel or ion channel receptor as the movement characteristic,
- the evaluation unit may evaluate the presence or absence of an ion channel or an ion channel type receptor based on the movement change.
- the ion channel or ion channel receptor activator or inhibitor When an ion channel or ion channel receptor activator or inhibitor is administered to a cell, the ion channel or ion channel receptor is suppressed or activated. Thereby, since the inflow amount of ions or molecules into the cell or the outflow amount to the outside of the cell varies, the movement of the cell membrane changes. Therefore, it is possible to evaluate the presence / absence or abundance of an ion channel or an ion channel type receptor based on a change in motion.
- the movement characteristic calculation unit calculates a movement change before and after administration of the inhibitor of the ion channel receptor as the movement characteristic,
- the evaluation unit may evaluate the type or strength of a synapse formed between cells based on the movement change.
- the synapse (intercellular connection) formed between cells stimulates connected cells using a specific chemical substance (neurotransmitter) and transmits signals. Therefore, if migration of a neurotransmitter through a cell membrane is suppressed by administration of an inhibitor to a receptor for a specific neurotransmitter, it can be evaluated that a synapse using the neurotransmitter is formed. . Since the movement of the neurotransmitter through the cell membrane is reflected in the movement of the cell membrane, the type or strength of the synapse can be evaluated from the change in movement before and after the administration of the inhibitor.
- the motion characteristic calculation unit calculates a motion amount as the motion characteristic,
- the evaluation unit may evaluate the type of substance acting on the ion channel or the ion channel type receptor or the strength of the effect based on the amount of movement.
- the movement of ions through the ion channel or the ion channel type receptor can be evaluated based on the amount of movement. Therefore, based on the amount of movement when a specific substance is administered to a cell, it is possible to evaluate the type of substance acting on the ion channel or ion channel receptor or the strength of the effect.
- the motion characteristic calculation unit calculates a motion amount as the motion characteristic
- the evaluation unit may evaluate the movement, expansion, contraction, and vibration of the cell membrane due to the movement of ions through the cell membrane or the accompanying movement of water based on the amount of movement.
- the cell membrane When the ion or water flows into the cell, the cell membrane expands, and when the ion or water flows out of the cell, the cell membrane contracts. Moreover, the cell membrane is vibrated by repeating the inflow and outflow of ions or water in a short time. Since the movement of these cell membranes is reflected in the amount of movement, it is possible to evaluate the movement, expansion, contraction, and vibration of the cell membrane due to the movement of ions or water based on the amount of movement.
- the motion characteristic calculator calculates a motion direction as a motion characteristic
- the movement characteristic calculation unit may evaluate the direction of ion or molecule flow through the cell membrane based on the movement direction.
- the motion information in the cell image has information on the direction of motion, evaluate whether ions or molecules are flowing into the cell or out of the cell from the amount of motion in the specific motion direction. Is possible.
- the motion characteristic calculation unit calculates the duration or spatial distribution of the motion as the motion characteristic
- the evaluation unit may evaluate the movement time or spatial distribution of ions or molecules through the cell membrane based on the duration or spatial distribution of the movement.
- the cell analysis system is In the cell image, further comprising a range designating unit for designating an extraction range using the luminance difference of the cell image,
- the motion information extraction unit may extract the motion information from the extraction range.
- the cell included in the cell image is a nerve cell
- the cell body of the nerve cell and the neurite are greatly different in luminance. Therefore, it is possible to detect the cell body using the luminance difference and set it as the extraction range. For this reason, according to the said structure, it becomes possible to calculate a motion characteristic only for a cell body as an extraction range, and to utilize for an evaluation.
- a cell analysis program causes an information processing apparatus to operate as a motion information extraction unit and a motion characteristic calculation unit.
- the motion information extraction unit extracts motion information resulting from the movement of ions or molecules through the cell membrane from a cell image obtained by imaging a cell over time.
- the motion characteristic calculation unit calculates a motion characteristic of the motion information.
- the motion information extraction unit extracts motion information resulting from movement of ions or molecules through the cell membrane from a cell image obtained by imaging the cell over time.
- a motion characteristic calculator calculates a motion characteristic of the motion information.
- FIG. 1 is a schematic diagram showing a cell analysis system 100 according to the present embodiment.
- the cell analysis system includes an image acquisition unit 101, a range specification unit 102, a motion information extraction unit 103, a motion characteristic calculation unit 104, an evaluation unit 105, and an image generation unit 106.
- Each of these configurations is a functional configuration realized by the information processing apparatus.
- the image acquisition unit 101 acquires a “cell image”.
- the cell image is an image obtained by imaging a cell or a group of cells to be analyzed over time, and can be a moving image or a plurality of still images taken continuously.
- the imaging speed can be set to 1 to 30 fps (frame / s), for example.
- the cell image is a variety of optical imaging such as bright field imaging, dark field imaging, phase difference imaging, fluorescence imaging, confocal imaging, multiphoton excitation fluorescence imaging, absorption light imaging, and irregular light imaging. It can be an image taken using the method.
- FIG. 2 is an example of a cell image, which is an image including a plurality of nerve cells.
- the image acquisition unit 101 may acquire a cell image from an imaging device (microscope imaging device) (not shown), or may acquire an image stored in a storage or an image supplied from a network as a cell image. Is possible.
- the image acquisition unit 101 supplies the acquired cell image to the range specification unit 102.
- the range designation unit 102 designates an “extraction range” in the cell image.
- the extraction range is a range in which a motion information extraction unit 103 described later extracts motion information.
- FIG. 3 is an example of an extraction range (within a square frame in the figure) designated by the range designation unit 102 in the cell image.
- the range designation unit 102 can designate the extraction range according to the analysis target (cell unit, cell group, etc.) designated by the user.
- the range designating unit 102 may designate the entire cell image as the extraction range when only the analysis target cell or cell group is included in the cell image. Moreover, the range designation
- the range specification unit 102 may specify an extraction range upon receiving an instruction input from the user, or may detect cells by image processing and specify the extraction range.
- the range designation unit 102 can detect cells using the luminance difference of the cell image.
- the cell included in the cell image is a nerve cell
- the cell body of the nerve cell and the neurite are greatly different in luminance, so that the cell body can be detected using the luminance difference and set as the extraction range.
- the range designation unit 102 supplies the extraction range designated together with the cell image to the motion information extraction unit 103.
- the motion information extraction unit 103 extracts “motion information” from the extraction range specified by the range specification unit 102.
- the motion information is information on the motion in the cell image captured over time, and specifically, it can be a motion vector with the lapse of time of the feature point in the cell image.
- the motion information extraction unit 103 can extract motion information by image processing such as block matching for cell images.
- FIG. 4 is a schematic diagram of ion transport between cell membranes.
- ions K + , Na + and the like
- molecules H 2 O and the like
- the channel C is an ion channel, a molecular channel, or an ion channel type receptor.
- the channel C may be an ion pump or a molecular pump.
- ions or molecules transported by ion channels or the like are referred to as ions or the like.
- the present inventors have found that the cell membrane moves to an observable level due to the ion transport between the cell membranes. This is due to cell expansion caused by inflow of ions or the like into cells, contraction of cells due to outflow of ions or the like out of cells, or vibration due to inflow or outflow of ions or the like. These movements are minute and high-speed movements observed at intervals of several seconds or less, unlike movements due to movement of the cells themselves observed at intervals of several hours.
- the movement information includes the influence of ion transport between the cell membranes, and it is possible to evaluate the ion transport between the cell membranes by analyzing the motion information.
- the motion information extraction unit 103 supplies the extracted motion information to the motion characteristic calculation unit 104.
- the motion characteristic calculation unit 104 calculates a “motion characteristic” from the motion information.
- the motion characteristics are characteristics of motion information, and are calculated from motion information such as motion speed, motion amount, motion change, motion direction, motion duration, motion spatial distribution, motion suppression ratio, or motion region amount.
- the motion region amount is a ratio of a region where the amount of motion is equal to or greater than a certain amount in a region where cells exist in the extraction range.
- the motion characteristic may include an average value of these in the extraction range and a median value of them in a certain time.
- the motion characteristic calculation unit 104 can calculate a difference in motion before and after administration as a motion characteristic. Specifically, the motion characteristic calculation unit 104 can calculate a change in a motion speed, a motion amount, a motion direction, a spatial distribution of motion, or a motion region amount before and after administration as a motion property.
- FIG. 5 is a graph showing the change over time in the amount of motion, which is an example of motion characteristics.
- the vertical axis represents the amount of movement, and the horizontal axis represents time (time for capturing a cell image).
- the figure is an example of the amount of movement calculated for a cell administered with a specific ion channel inhibitor, and shows that the amount of movement has decreased from the administration time of the ion channel inhibitor (the left end of the horizontal axis).
- the motion characteristic calculation unit 104 calculates such a motion characteristic.
- the motion characteristic calculation unit 104 may calculate a plurality of types of motion characteristics from the motion information.
- the motion information extraction unit 103 supplies the calculated motion characteristics to the evaluation unit 105.
- the motion information extraction unit 103 may supply the calculated motion characteristics to the image generation unit 106.
- Evaluation unit 105 evaluates ion transport between cell membranes based on motion characteristics. For example, in the example of FIG. 5, it can be seen from the decrease in the amount of movement immediately after the ion channel inhibitor administration that the function of the ion channel is inhibited by the ion channel inhibitor, and from the increase in the amount of movement over time, It can be seen that the function of the ion channel is gradually recovering. In addition, the evaluation unit 105 can evaluate ion transport between cell membranes based on various motion characteristics.
- the evaluation unit 105 can evaluate the movement amount of ions and the like based on the movement amount. Since the amount of movement increases or decreases depending on the amount of ions or the like flowing into the cell or the amount of outflow outside the cell, the amount of movement of ions or the like can be evaluated based on the amount of movement.
- the evaluation unit 105 can evaluate the activity of the ion channel or the like based on the amount of movement. Since the amount of movement increases or decreases depending on the amount of ions flowing into or out of the cell, the activity of the ion channel is high, and if the amount of ions flowing in or out is large, the amount of movement is reflected in the amount of movement. The For this reason, the evaluation unit 105 can evaluate the activity of the ion channel or the like based on the amount of movement.
- the evaluation unit 105 can evaluate the presence / absence or abundance of an ion channel or the like based on a change in motion before and after administration of an activator or inhibitor such as an ion channel.
- an activator or inhibitor such as an ion channel
- the ion channel or the like is suppressed or activated.
- the inflow amount of ions or the like into the cell or the outflow amount to the outside of the cell changes, so that the movement of the cell membrane changes. Therefore, it is possible to evaluate the presence / absence or abundance of an ion channel or an ion channel-type receptor based on the movement change.
- the evaluation unit 105 can evaluate the type or strength of the synapse formed between cells based on the movement change before and after the administration of the inhibitor of the ion channel receptor.
- Synapses cell-cell junctions formed between cells stimulate the connected cells using specific chemical substances (neurotransmitters) and transmit signals. Therefore, if migration of a neurotransmitter through a cell membrane is suppressed by administration of an inhibitor to a receptor for a specific neurotransmitter, it can be evaluated that a synapse using the neurotransmitter is formed. . Since the movement of the neurotransmitter through the cell membrane is reflected in the movement of the cell membrane, it is possible to evaluate the type or strength of the synapse from the movement change before and after the administration of the inhibitor.
- the evaluation unit 105 can evaluate the type of substance acting on the ion channel or the like or the strength of the effect based on the amount of movement. Since the movement of ions, etc. through ion channels, etc. can be evaluated based on the amount of movement, the type or effect of substances that act on ion channels, etc., based on the amount of movement when a specific substance is administered to cells. It is possible to evaluate the strength of
- the evaluation unit 105 can evaluate the movement, expansion, contraction, and vibration of the cell membrane due to the movement of ions through the cell membrane or the accompanying movement of water based on the amount of movement.
- the cell membrane expands, and when ions or water flows out of the cell, the cell membrane contracts. Further, when the inflow / outflow of ions or water is repeated in a short time, the cell membrane vibrates. Since the movement of these cell membranes is reflected in the amount of movement, the movement, expansion, contraction, and vibration of the cell membrane due to the movement of ions or water can be evaluated based on the amount of movement.
- the evaluation unit 105 can evaluate the direction of ion or molecule flow through the cell membrane based on the movement direction. Since the amount of movement in a specific movement direction is caused by the flow of ions or the like moving in that direction, it is possible to evaluate whether ions or the like are flowing into or out of the cells. For example, to evaluate whether ions etc. flowed into the cell or flowed out of the cell depending on whether the movement direction at a specific point of the cell body detected as the extraction range is toward the center point of the cell body Can do.
- the evaluation unit 105 can evaluate the movement time or spatial distribution of ions or molecules through the cell membrane based on the duration or spatial distribution of movement. As described above, movement in the cell image is caused by ion transport between the cell membranes, so that the duration of movement can be regarded as the movement time of ions or molecules. From the spatial distribution of movement, ion movement, ion channel, etc. It is possible to estimate the spatial distribution.
- the evaluation unit 105 can evaluate the ion transport between the cell membranes based on the motion characteristics.
- the evaluation unit 105 supplies the evaluation result to the image generation unit 106.
- the image generation unit 106 generates an image for displaying the evaluation result.
- the image generation unit 106 may generate an image that displays a motion characteristic (for example, the graph of FIG. 5) together with the evaluation result, generates an image that displays only the motion characteristic, and evaluates ion transport between cell membranes. You may leave it to the user.
- a motion characteristic for example, the graph of FIG. 5
- the image generation unit 106 may generate a motion characteristic display image by superimposing the cell image and the movement characteristic. Specifically, the image generation unit 106 adds a color according to the magnitude of the motion characteristic, or adds a shade and superimposes it on a corresponding position of the cell image to generate a motion characteristic display image. Is possible.
- the image generation unit 106 overlaps the cell image with the rate of movement suppression by the ion channel inhibitor as a color map, the user can evaluate the presence / absence and expression level of the ion channel of each cell. Become. Further, the image generation unit 106 overlaps the cell image as a color map with the rate of movement suppression by the ion channel receptor inhibitor, so that the user can input the type and strength of the synapse formed by individual cells. It becomes possible to evaluate the spatial distribution.
- the cell analysis system 100 is configured as described above. As described above, the cell analysis system 100 can evaluate the ion transport between the cell membranes from the motion characteristics of the cell image. Therefore, the user does not need to stain the cells by using the cell analysis system 100, and can prevent the stain from affecting the cells. Furthermore, since the cell analysis system 100 can evaluate ion transport between cell membranes by image processing on a cell image, it can analyze a wide area with high resolution.
- the activity of the ion channel or the like is relatively quantified without staining, or the physiologically active substance or drug acting on the ion channel or the like is screened without staining and the effect thereof. Can be quantified relative to each other. Furthermore, by quantifying the presence or absence and expression level of ion channels or the like expressed in individual cells, profiling of cell types becomes possible without staining.
- nonstaining synaptic connection profiling is possible by relative quantification of the type and amount of synaptic ion channel receptors that are input to individual neurons. It becomes.
- a voltage-gated ion channel is a molecule that opens and closes depending on the membrane potential and is responsible for ion migration according to the ion concentration gradient between the membranes. In many nerve cells, the membrane potential is not static and vibrates, and voltage-gated channels repeatedly open and close.
- An ion channel inhibitor inhibits the opening and closing of an ion channel and inhibits ion transfer between membranes.
- iPS cell-derived neuron iCell Neuron manufactured by Cellular Dynamics International
- a moving image of 260 frames was imaged with an objective lens 20 times at 7.5 fps (frame / second) to obtain a cell image.
- Various ion channel inhibitors were added to the cell culture medium.
- the ion channel inhibitor was any one of Na + channel inhibitor TTX (tetrodotoxin), K + channel inhibitor TEA (tetraethylammonium) and Ca 2+ channel inhibitor Nifedipine.
- FIG. 6 is calculated from iCellonNeuron added with 100 nm of TTX
- FIG. 7 is calculated from iCell Neuron added with 1 mM TEA
- FIG. 8 is calculated from iCell Neuron added with 10 ⁇ M Nifedipine.
- the amount of movement shown in each figure is an average value of the amount of movement calculated from a plurality (six) nerve details.
- the horizontal axis 0 min is the addition time of the ion channel inhibitor
- the vertical axis is the relative value of the amount of movement with 1 (0 min) before administration of the ion channel inhibitor.
- An ion channel type receptor is a molecule that opens and closes depending on the binding of a ligand (substance that binds to a receptor) and is responsible for ion transfer according to the ion concentration between membranes.
- Many of the neurotransmitters act as ligands for ion channel receptors, and are released from the presynapse at the tip of the axon of the neuron, and stimulate or inhibit the neuron connected at the synapse via the receptor at the postsynapse. Each neuron releases only one type of neurotransmitter, and only one neurotransmitter is involved in a synapse.
- An ion channel receptor inhibitor suppresses ion movement between synaptic membranes by inhibiting ligand binding to the ion channel receptor and channel opening / closing.
- iPS cell-derived neuronal cells iCell Neuron (Cellular Dynamics International) and rat cerebral cortex primary culture cells (Lonza) are set in a microscope with a culture chamber, respectively, and objective lens 20 times, 7.5 fps (iCell Neuron) or A moving image of 260 frames was taken at 5 fps (rat cerebral cortex primary cultured cells) to obtain cell images.
- Various ion channel receptor inhibitors were added to the culture medium of each cell.
- An ion channel receptor inhibitor is GABA (gamma-aminobutyrate) receptor inhibitor Bicuculline or Glu (glutamate) receptor (non-NMDA type) inhibitor CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) ).
- FIG. 9 is calculated from iCell Neuron to which 5 ⁇ M of Bicucullin is added
- FIG. 10 is calculated from iCell Neuron to which 5 ⁇ M of CNQX is added
- FIG. 11 is calculated from rat cerebral cortex primary culture cells added with 5 ⁇ M Bicucullin
- FIG. 12 is calculated from rat cerebral cortex primary culture cells added with 5 ⁇ M CNQX.
- the amount of movement shown in each figure is an average value of the amount of movement calculated from a plurality of nerve details.
- the horizontal axis 0 min is the addition time of the ion channel inhibitor
- the vertical axis is the relative value of the amount of movement with the ion channel receptor inhibitor before administration (0 min) being 1.
- ICell Neuron is a mixture of cells that release various neurotransmitters, most of which are GABAergic neurons or Gluergic neurons.
- GABA receptor inhibitor which is an inhibitory neurotransmitter
- Glu glutamic acid
- FIG. 11 shows that there is almost no change in the amount of movement due to the addition of the GABA receptor inhibitor, and no synapses via GABA are formed.
- FIG. 12 shows that the amount of movement is reduced by the addition of a Glu receptor inhibitor, and a synapse via Glu is formed. In this way, by using the cell analysis system, it is possible to evaluate whether or not the analysis target cell has a specific neurotransmitter receptor.
- FIG. 13 is a graph in which the amount of movement calculated from primary cultured cells of rat cerebral cortex to which 5 ⁇ M of CNQX (Glu receptor inhibitor) is added is plotted for each cell.
- the primary cultured cells of rat cerebral cortex include cells that are affected by the Glu receptor inhibitor and decreased in movement amount, and cells that are not affected by the inhibitor and do not decrease movement amount. You can see that That is, it can be said that by using a cell analysis system, it is possible to evaluate each cell whether or not it is a cell in which a synapse having a receptor for a specific neurotransmitter is input.
- Example 3 Evaluation of water movement by osmotic pressure change accompanying ion movement between membranes
- aquaporin is a passive channel molecule that opens and closes due to osmotic pressure difference and is responsible for water movement.
- aquaporins open and the cell volume increases or decreases. It has not been clarified so far that the volume of cells increases or decreases due to local and transient osmotic pressure difference due to opening and closing of ion channels and ion channel receptors.
- Rat primary cerebral cortex cultured cells (manufactured by Lonza) were set in a microscope with a culture chamber, and a moving image of 260 frames was imaged at an objective lens of 20 ⁇ and 5 fps to obtain cell images. A final concentration of 2.5% galactose was added to the cell culture.
- FIG. 14 shows the median value of the motion amount in the extraction range. The graph on the left is the value when galactose is not added, and the graph on the right is the value when galactose is added.
- rat cerebral cortex primary culture cells (manufactured by Lonza) are set in a microscope equipped with a culture chamber, and a 260-frame video is obtained with an objective lens 20 times, 7.5 fps (iCell Neuron) or 5 fps (rat cerebral cortex primary culture cells). Images were taken to obtain cell images. 5 ⁇ M of aquaporin inhibitor HgCl 2 was added to the cell culture medium.
- FIG. 15 is a graph showing the calculation result of the motion amount.
- the horizontal axis 0 min is the time of addition of the ion channel inhibitor
- the vertical axis is the relative value of the amount of movement with 1 before administration of HgCl 2 (0 min).
- HgCl 2 which is an aquaporin inhibitor
- HgCl 2 which is an aquaporin inhibitor
- the amount of movement was temporarily suppressed. It has been confirmed that addition of 5 ⁇ M HgCl 2 does not suppress cell metabolism. Therefore, it was confirmed that when water transfer between cell membranes is inhibited by an aquaporin inhibitor, cell movement is suppressed. That is, it can be seen that the movement of water between cell membranes can be evaluated by using a cell analysis system.
- Example 4 Correlation between amount of movement and extracellular electric field
- Rat cerebral cortical neurons manufactured by Lonza
- Rat cerebral cortical neurons were cultured in a multi-electrode array (Alpha Med Scientific), and the extracellular electric field (field potential) was measured at a sampling frequency of 20 kHz.
- FIG. 16 shows the measured extracellular electric field. After extracting 200 Hz data from the measured value and making it an absolute value, an average of 0.2 seconds was taken to obtain 5 Hz data (Field potential thin line in FIG. 16).
- a moving image of cells around the electrode was taken at 5 fps and 260 frames to obtain cell images.
- the cell image was analyzed using the cell analysis system. Specifically, motion information was extracted by block matching using individual nerve cells included in the cell image as an extraction range, and a motion speed average per cell was calculated as motion characteristics from the motion information (Motion thin line in FIG. 16).
- FIG. 17 is a graph showing the correlation between the extracellular electric field shown in FIG. 16 and the moving average of the amount of motion, and plots the values at the time of the measurement. As shown in the figure, it can be seen that there is a certain correlation between the extracellular electric field and the amount of movement.
- Example 5 Evaluation of ion inflow into cells
- Rat cerebral cortical neurons (manufactured by Lonza) were set in a microscope with a chamber, and a moving image of 260 frames was imaged at an objective lens of 20 times and 5 fps (frame / second) to obtain cell images.
- the culture medium of rat cerebral cortical neurons is a normal culture medium, a HEPES buffer solution containing Na + (containing NaCl, KCl, MgCl 2 CaCl 2 and glucose) or a HEPES buffer solution containing no Na + (instead of NaCl). N-methyl-D-glucamine included).
- HEPES buffer without Na +, due N- methyl -D- glucamine, are adjusted in HEPES buffer and osmotic pressure comparable containing Na +.
- FIG. 18 shows the median value of the motion amount in the extraction range. The amount of movement is standardized for each culture solution.
- the amount of movement was the same as in the case of a normal culture solution (Medium in the figure).
- the culture solution was a HEPES buffer solution not containing Na + (HEPES-Na in the figure)
- the amount of movement was greatly reduced. Thereby, it can be said that the movement of the cells is increased by the inflow of Na + into the cells.
- a movement information extraction unit that extracts movement information resulting from the movement of ions or molecules through the cell membrane from a cell image obtained by imaging cells over time;
- a cell analysis system comprising: a motion characteristic calculator that calculates a motion characteristic of the motion information.
- a cell analysis system further comprising: an image generation unit that generates a motion characteristic display image by superimposing the motion characteristic and the cell image.
- a cell analysis system further comprising an evaluation unit that evaluates the movement of ions or molecules through the cell membrane based on the movement characteristics.
- the motion characteristic calculation unit calculates a motion amount as the motion characteristic
- the evaluation unit is a cell analysis system that evaluates the amount of movement of ions or molecules based on the amount of movement.
- the motion characteristic calculation unit calculates a motion amount as the motion characteristic, The cell evaluation system, wherein the evaluation unit evaluates the activity of an ion channel or an ion channel receptor based on the amount of movement.
- the movement characteristic calculation unit calculates a movement change before and after the administration of the activator or inhibitor of the ion channel or ion channel receptor as the movement characteristic,
- the cell evaluation system wherein the evaluation unit evaluates the presence / absence or abundance of an ion channel or an ion channel receptor based on the movement change.
- the movement characteristic calculation unit calculates a movement change before and after administration of the inhibitor of the ion channel receptor as the movement characteristic
- the evaluation unit evaluates the type or strength of a synapse formed between cells based on the movement change.
- the motion characteristic calculation unit calculates a motion amount as the motion characteristic
- the cell evaluation system wherein the evaluation unit evaluates the type of substance acting on the ion channel or the ion channel receptor based on the amount of movement or the strength of the effect.
- the motion characteristic calculation unit calculates a motion amount as the motion characteristic
- the cell evaluation system wherein the evaluation unit evaluates movement, expansion, contraction, and vibration of the cell membrane due to the movement of ions through the cell membrane or the accompanying movement of water based on the amount of movement.
- the movement characteristic calculation unit calculates a movement direction as the movement characteristic
- the cell analysis system wherein the motion characteristic calculation unit evaluates a flow direction of ions or molecules through the cell membrane based on the motion direction.
- the motion characteristic calculation unit calculates a duration or spatial distribution of motion as the motion characteristic
- the evaluation unit evaluates the movement time or spatial distribution of ions or molecules through the cell membrane based on the duration or spatial distribution of the movement.
- a movement information extraction unit that extracts movement information resulting from the movement of ions or molecules through the cell membrane from a cell image obtained by imaging cells over time;
- a cell analysis program for operating an information processing apparatus as a motion characteristic calculation unit that calculates a motion characteristic of the motion information.
- the movement information extraction unit extracts movement information resulting from the movement of ions or molecules through the cell membrane from the cell image obtained by imaging the cells over time, A cell analysis method in which a motion characteristic calculation unit calculates a motion characteristic of the motion information.
- DESCRIPTION OF SYMBOLS 100 ... Cell analysis system 101 ... Image acquisition part 102 ... Range designation
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Abstract
Description
上記動き情報抽出部は、細胞を経時的に撮像した細胞画像から、細胞膜を介したイオン又は分子の移動に起因する動き情報を抽出する。
上記動き特性算出部は、上記動き情報の動き特性を算出する。
上記動き情報抽出部は、細胞を経時的に撮像した細胞画像から、細胞膜を介したイオン又は分子の移動に起因する動き情報を抽出する。
上記動き特性算出部は、上記動き情報の動き特性を算出する。
動き特性算出部が、上記動き情報の動き特性を算出する。
なお、ここに記載された効果は必ずしも限定されるものではなく、本開示中に記載されたいずれかの効果であってもよい。
上記動き情報抽出部は、細胞を経時的に撮像した細胞画像から、細胞膜を介したイオン又は分子の移動に起因する動き情報を抽出する。
上記動き特性算出部は、上記動き情報の動き特性を算出する。
上記動き特性と上記細胞画像を重畳させ、動き特性表示画像を生成する画像生成部
をさらに具備してもよい。
上記動き特性に基づいて、細胞膜を介したイオン又は分子の移動を評価する評価部
をさらに具備してもよい。
上記評価部は、上記動き量に基づいてイオン又は分子の移動量を評価してもよい。
上記評価部は、上記動き量に基づいてイオンチャネル又はイオンチャネル型受容体の活性を評価してもよい。
上記評価部は、上記動き変化に基づいてイオンチャネル又はイオンチャネル型受容体の有無又は存在量を評価してもよい。
上記評価部は、上記動き変化に基づいて細胞間で形成されているシナプスの種類又は強さを評価してもよい。
上記評価部は、上記動き量に基づいてイオンチャネル又はイオンチャネル型受容体に作用する物質の種類又は効果の強さを評価してもよい。
上記評価部は、上記動き量に基づいて細胞膜を介したイオンの移動又はそれに伴なう水の移動による細胞膜の動き、膨張、収縮及び振動を評価してもよい。
上記動き特性算出部は、上記動き方向に基づいて細胞膜を介したイオン又は分子の流れの方向を評価してもよい。
上記評価部は、上記動きの継続時間又は空間的分布に基づいて細胞膜を介したイオン又は分子の移動時間又は空間的分布を評価してもよい。
上記細胞画像において、上記細胞画像の輝度差分を利用して抽出範囲を指定する範囲指定部をさらに具備し、
上記動き情報抽出部は、上記抽出範囲から上記動き情報を抽出してもよい。
上記動き情報抽出部は、細胞を経時的に撮像した細胞画像から、細胞膜を介したイオン又は分子の移動に起因する動き情報を抽出する。
上記動き特性算出部は、上記動き情報の動き特性を算出する。
動き特性算出部が、上記動き情報の動き特性を算出する。
図1は、本実施形態に係る細胞分析システム100を示す模式図である。同図に示すように細胞分析システムは、画像取得部101、範囲指定部102、動き情報抽出部103、動き特性算出部104、評価部105及び画像生成部106を有する。これらの各構成は、情報処理装置によって実現されている機能的構成である。
電位依存性イオンチャネルは膜電位に依存して開閉し、膜間のイオン濃度勾配に応じたイオン移動を担う分子である。神経細胞の多くは、膜電位が静止しておらず振動しており、電位依存性チャネルも開閉を繰返している。イオンチャネル阻害剤は、イオンチャネルの開閉を阻害し、膜間のイオン移動を阻害する。
イオンチャネル型受容体はリガンド(受容体に結合する物質)の結合に依存して開閉し、膜間のイオン濃度に応じたイオン移動を担う分子である。神経伝達物質の多くはイオンチャネル型受容体のリガンドとなり、神経細胞の軸索先端の前シナプスから放出され、後シナプスの受容体を介してシナプスで接続された神経細胞を刺激、又は抑制する。個々の神経細胞は一種類のみの神経伝達物質を放出し、一つのシナプスで関与する神経伝達物質は一つのみである。
細胞膜間では、水チャネルであるアクアポリンを介してのみ、水の移動が行われる。アクアポリンは浸透圧差により開閉し、水移動を担う受動的なチャネル分子である。細胞全体で内外の浸透圧差が生じた場合、アクアポリンが開き、細胞の体積が増減する。イオンチャネルやイオンチャネル型受容体の開閉等による局所的かつ一過性の浸透圧差により、細胞の体積が増減し、あるいは細胞膜が動くことは、これまで明らかになっていない。
多電極アレイ(アルファメッドサイエンティフィック社)にラット大脳皮質神経細胞(Lonza社製)を培養し、20kHzのサンプリング周波数で細胞外電場(field potential)を測定した。図16に測定した細胞外電場を示す。測定値から200Hzのデータを抽出し、絶対値にした後、0.2秒間の平均をとり、5Hzのデータ(図16中Field potential細線)とした。
ラット大脳皮質神経細胞(Lonza社製)をチャンバー付き顕微鏡にセットし、対物レンズ20倍、5fps(フレーム/秒)で260フレームの動画を撮像し、細胞画像とした。ラット大脳皮質神経細胞の培養液は、通常の培養液、Na+を含むHEPES緩衝液(NaCl、KCl、MgCl2CaCl2及びグルコースを含む)又はNa+を含まないHEPES緩衝液(NaClの代わりにN-メチル-D-グルカミンを含む)とした。Na+を含まないHEPES緩衝液は、N-メチル-D-グルカミンにより、Na+を含むHEPES緩衝液と同程度の浸透圧に調整されている。
細胞を経時的に撮像した細胞画像から、細胞膜を介したイオン又は分子の移動に起因する動き情報を抽出する動き情報抽出部と、
上記動き情報の動き特性を算出する動き特性算出部と
を具備する細胞分析システム。
上記(1)に記載の細胞分析システムであって、
上記動き特性と上記細胞画像を重畳させ、動き特性表示画像を生成する画像生成部
をさらに具備する細胞分析システム。
上記(1)又は(2)に記載の細胞分析システムであって、
上記動き特性に基づいて、細胞膜を介したイオン又は分子の移動を評価する評価部
をさらに具備する細胞分析システム。
上記(1)から(3)のうちいずれか1つに記載の細胞分析システムであって、
上記動き特性算出部は、上記動き特性として動き量を算出し、
上記評価部は、上記動き量に基づいてイオン又は分子の移動量を評価する
細胞分析システム。
上記(1)から(4)のうちいずれか1つに記載の細胞分析システムであって、
上記動き特性算出部は、上記動き特性として動き量を算出し、
上記評価部は、上記動き量に基づいてイオンチャネル又はイオンチャネル型受容体の活性を評価する
細胞分析システム。
上記(1)から(5)のうちいずれか1つに記載の細胞分析システムであって、
上記動き特性算出部は、上記動き特性としてイオンチャネル又はイオンチャネル型受容体の活性化剤又は阻害剤の投与前後における動き変化を算出し、
上記評価部は、上記動き変化に基づいてイオンチャネル又はイオンチャネル型受容体の有無又は存在量を評価する
細胞分析システム。
上記(1)から(6)のうちいずれか1つに記載の細胞分析システムであって、
上記動き特性算出部は、上記動き特性としてイオンチャネル型受容体の阻害剤の投与前後における動き変化を算出し、
上記評価部は、上記動き変化に基づいて細胞間で形成されているシナプスの種類又は強さを評価する
細胞分析システム。
上記(1)から(7)のうちいずれか1つに記載の細胞分析システムであって、
上記動き特性算出部は、上記動き特性として動き量を算出し、
上記評価部は、上記動き量に基づいてイオンチャネル又はイオンチャネル型受容体に作用する物質の種類又は効果の強さを評価する
細胞分析システム。
上記(1)から(8)のうちいずれか1つに記載の細胞分析システムであって、
上記動き特性算出部は、上記動き特性として動き量を算出し、
上記評価部は、上記動き量に基づいて細胞膜を介したイオンの移動又はそれに伴なう水の移動による細胞膜の動き、膨張、収縮及び振動を評価する
細胞分析システム。
上記(1)から(9)のうちいずれか1つに記載の細胞分析システムであって、
上記動き特性算出部は、上記動き特性として動き方向を算出し、
上記動き特性算出部は、上記動き方向に基づいて細胞膜を介したイオン又は分子の流れの方向を評価する
細胞分析システム。
上記(1)から(10)のうちいずれか1つに記載の細胞分析システムであって、
上記動き特性算出部は、上記動き特性として動きの継続時間又は空間的分布を算出し、
上記評価部は、上記動きの継続時間又は空間的分布に基づいて細胞膜を介したイオン又は分子の移動時間又は空間的分布を評価する
細胞分析システム。
上記(1)から(11)のうちいずれか1つに記載の細胞分析システムであって、
上記細胞画像において、上記細胞画像の輝度差分を利用して抽出範囲を指定する範囲指定部をさらに具備し、
上記動き情報抽出部は、上記抽出範囲から上記動き情報を抽出する
細胞分析システム。
細胞を経時的に撮像した細胞画像から、細胞膜を介したイオン又は分子の移動に起因する動き情報を抽出する動き情報抽出部と、
上記動き情報の動き特性を算出する動き特性算出部と
として情報処理装置を動作させる細胞分析プログラム。
動き情報抽出部が、細胞を経時的に撮像した細胞画像から、細胞膜を介したイオン又は分子の移動に起因する動き情報を抽出し、
動き特性算出部が、上記動き情報の動き特性を算出する
細胞分析方法。
101…画像取得部
102…範囲指定部
103…動き情報抽出部
104…動き特性算出部
105…評価部
106…画像生成部
Claims (14)
- 細胞を経時的に撮像した細胞画像から、細胞膜を介したイオン又は分子の移動に起因する動き情報を抽出する動き情報抽出部と、
前記動き情報の動き特性を算出する動き特性算出部と
を具備する細胞分析システム。 - 請求項1に記載の細胞分析システムであって、
前記動き特性と前記細胞画像を重畳させ、動き特性表示画像を生成する画像生成部
をさらに具備する細胞分析システム。 - 請求項1に記載の細胞分析システムであって、
前記動き特性に基づいて、細胞膜を介したイオン又は分子の移動を評価する評価部
をさらに具備する細胞分析システム。 - 請求項3に記載の細胞分析システムであって、
前記動き特性算出部は、前記動き特性として動き量を算出し、
前記評価部は、前記動き量に基づいてイオン又は分子の移動量を評価する
細胞分析システム。 - 請求項3に記載の細胞分析システムであって、
前記動き特性算出部は、前記動き特性として動き量を算出し、
前記評価部は、前記動き量に基づいてイオンチャネル又はイオンチャネル型受容体の活性を評価する
細胞分析システム。 - 請求項3に記載の細胞分析システムであって、
前記動き特性算出部は、前記動き特性としてイオンチャネル又はイオンチャネル型受容体の活性化剤又は阻害剤の投与前後における動き変化を算出し、
前記評価部は、前記動き変化に基づいてイオンチャネル又はイオンチャネル型受容体の有無又は存在量を評価する
細胞分析システム。 - 請求項3に記載の細胞分析システムであって、
前記動き特性算出部は、前記動き特性としてイオンチャネル型受容体の阻害剤の投与前後における動き変化を算出し、
前記評価部は、前記動き変化に基づいて細胞間で形成されているシナプスの種類又は強さを評価する
細胞分析システム。 - 請求項3に記載の細胞分析システムであって、
前記動き特性算出部は、前記動き特性として動き量を算出し、
前記評価部は、前記動き量に基づいてイオンチャネル又はイオンチャネル型受容体に作用する物質の種類又は効果の強さを評価する
細胞分析システム。 - 請求項3に記載の細胞分析システムであって、
前記動き特性算出部は、前記動き特性として動き量を算出し、
前記評価部は、前記動き量に基づいて細胞膜を介したイオンの移動又はそれに伴なう水の移動による細胞膜の動き、膨張、収縮及び振動を評価する
細胞分析システム。 - 請求項3に記載の細胞分析システムであって、
前記動き特性算出部は、前記動き特性として動き方向を算出し、
前記動き特性算出部は、前記動き方向に基づいて細胞膜を介したイオン又は分子の流れの方向を評価する
細胞分析システム。 - 請求項3に記載の細胞分析システムであって、
前記動き特性算出部は、前記動き特性として動きの継続時間又は空間的分布を算出し、
前記評価部は、前記動きの継続時間又は空間的分布に基づいて細胞膜を介したイオン又は分子の移動時間又は空間的分布を評価する
細胞分析システム。 - 請求項1に記載の細胞分析システムであって、
前記細胞画像において、前記細胞画像の輝度差分を利用して抽出範囲を指定する範囲指定部をさらに具備し、
前記動き情報抽出部は、前記抽出範囲から前記動き情報を抽出する
細胞分析システム。 - 細胞を経時的に撮像した細胞画像から、細胞膜を介したイオン又は分子の移動に起因する動き情報を抽出する動き情報抽出部と、
前記動き情報の動き特性を算出する動き特性算出部と
として情報処理装置を動作させる細胞分析プログラム。 - 動き情報抽出部が、細胞を経時的に撮像した細胞画像から、細胞膜を介したイオン又は分子の移動に起因する動き情報を抽出し、
動き特性算出部が、前記動き情報の動き特性を算出する
細胞分析方法。
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