JP6609967B2 - Determination device, determination system, determination program, and cell manufacturing method - Google Patents

Description

The present invention, determination device, determination system, determination program, and those related to the cell production how.

In general, for example, a technique for evaluating a cell culture state has become a basic technique in a wide range of fields including advanced medical fields such as regenerative medicine and drug screening. In the process of evaluating the cell culture state, it is required to accurately determine the cell state. As an example, a method for determining whether a cell is viable or not by staining a cell to be determined with a dye that permeates the cell membrane of a dead cell preferentially over the cell membrane of a living cell such as trypan blue is disclosed (Patent Document). 1).
In addition to the field of cell culture state evaluation, a technique for evaluating the state of an observation object from images obtained by imaging various observation objects is an important technique in various fields.

Special table 2014-505465 gazette

  In recent years, it has been desired to determine the state of an object having a complicated structure with high accuracy from an image obtained by imaging the observation object.

  Therefore, the present invention has been made in view of the above problems, and an object of the present invention is to provide a determination device that can improve the accuracy of state determination of an observation object using an image obtained by capturing the observation object. .

In order to solve the above problem, an aspect of the present invention provides an image acquisition unit that acquires a phase difference image of an observation object including cells, which is observed by phase difference observation , and a cell region of the cell in the phase difference image. Based on the filter section for removing the halo region from the cell region, the entire circumference of the outline of the cell region from which the halo region is removed, and the length of the cell membrane obtained based on the phase difference image And a determination unit that determines the state of the cell .

In order to solve the above problem, one embodiment of the present invention includes the above-described determination device, a phase difference imaging unit that generates the phase difference image, and an output unit that outputs a determination result of the determination device. It is the determination system characterized by providing.

In order to solve the above problem, according to one embodiment of the present invention, an image acquisition procedure for acquiring a phase difference image of an observation object including a cell and a cell region of the cell in the phase difference image are extracted from a computer. The cell based on the procedure for deleting the halo region from the cell region, the entire circumference of the outline of the cell region from which the halo region has been removed, and the length of the cell membrane obtained based on the phase contrast image And a determination program for executing the determination procedure for determining the state of.

In order to solve the above problem, one embodiment of the present invention is a cell manufacturing method using the above-described determination apparatus, in which the image acquisition unit acquires a phase difference image of an observation object including cells. A procedure, the filter unit extracts a cell region of the cell in the phase difference image, and deletes the halo region from the cell region; and the determination unit includes the cell region from which the halo region is removed. and the entire circumference of the contour, the phase difference image based on the length of the cell membrane obtained on the basis of the procedure for determining the state of the cells, cells producing method that have a.

  ADVANTAGE OF THE INVENTION According to this invention, the precision of the state determination of the object imaged from the imaged image can be improved.

It is a schematic diagram which shows the outline | summary of a structure of the determination system of this embodiment. It is a block diagram which shows the function structure of the determination apparatus of this embodiment. It is a flowchart which shows an example of the flow of the process by the control part of this embodiment. It is a schematic diagram which shows an example of the image which the determination system of this embodiment handles. It is a schematic diagram which shows the example of determination of the living cell by the determination part of this embodiment. It is a schematic diagram which shows the example of determination of the dead cell by the determination part of this embodiment. It is a schematic diagram which shows an example of the change curve of the phase difference in the modification of this embodiment.

[Embodiment]
Embodiments of the present invention will be described below with reference to the drawings. First, the configuration of the determination system 1 according to the embodiment of the present invention will be described with reference to FIG.
FIG. 1 is a schematic diagram showing an outline of the configuration of the determination system 1 of the present embodiment. The determination system 1 includes a determination device 10, a display unit 20, and a phase contrast microscope device 30.

[Configuration of phase contrast microscope apparatus]
The phase-contrast microscope apparatus 30 irradiates the subject S to be observed with the illumination light L, and enlarges the subject S obtained by converting the phase difference of the transmitted light Lp from the subject S into a light / dark difference. Get a statue. A specific configuration of the phase contrast microscope apparatus 30 will be described.

  The phase contrast microscope apparatus 30 forms an image of the light source 31 that emits the illumination light L, the illumination optical system 32 that irradiates the subject S with the illumination light L from the light source 31, and the transmitted light Lp from the subject S. An image optical system 33 and a solid-state imaging device 34 that receives the transmitted light Lp imaged by the imaging optical system 33 and converts it into an electrical signal to generate an image of the subject S are provided.

A stage 36 is disposed between the illumination optical system 32 and the imaging optical system 33.
The stage 36 has a placement surface 36a on which the subject S is placed. Further, the stage 36 is moved and operated in two directions (X-axis direction and Y-axis direction shown in FIG. 1) perpendicular to each other in the plane. Thereby, it is possible to arbitrarily change the observation position of the subject S. Further, the stage 36 may be configured to be moved in the height direction (Z-axis direction shown in FIG. 1).

  In the following, the optical axis (the central axis of the light beam) of the illumination light L emitted from the light source 31 is defined as the Z-axis direction, and two directions orthogonal to each other in a plane orthogonal to the Z-axis are defined as the X-axis direction and the Y-axis. The axial direction will be described. The illumination light L emitted from the light source 31 is schematically shown by a broken line in FIG.

  The light source 31 irradiates, as illumination light L, visible light such as white light or light in a wavelength region in the vicinity thereof. As the light source 31, natural light or light from an external light source such as a white fluorescent lamp or a white light bulb can be used as the illumination light L using a reflecting mirror or the like. Further, as the light source 31, light from an internal light source such as a halogen lamp or a tungsten lamp can be used as the illumination light L.

  The light source 31 may be a light emitting diode (LED) or the like. In this case, the light source 31 can be configured by a combination of LEDs that emit light of each wavelength of red, blue, and green, for example. Further, since the wavelength of illumination light emitted from the light source 31 can be variably controlled by controlling the turning on and off of the LEDs having different wavelengths, when such an LED is used as the light source 31, the wavelength of a wavelength filter or the like is used. It is possible to omit the conversion member.

  In the illumination optical system 32, a first condenser lens 37, a first spatial light modulator 38, and a second condenser lens 39 are arranged in this order from the light source 31 side. Among these, the first condenser lens 37 and the second condenser lens 39 condense the illumination light L emitted from the light source 31 onto the subject S on the stage 36.

  The first spatial light modulator 38 is arranged at a position that is conjugate with the pupil position of the imaging optical system 33. The first spatial light modulation element 38 variably adjusts the light intensity distribution of the illumination light L irradiated to the subject S (aperture), and the aperture (area through which the illumination light L passes) 38a. It is possible to freely change the shape, size, etc.

  In the imaging optical system 33, an objective lens 40 and a second spatial light modulator 41 are arranged in this order from the stage 36 side.

  The objective lens 40 images the transmitted light Lp from the subject S on the light receiving surface of the solid-state imaging device 34.

  The second spatial light modulator 41 is disposed at or near the pupil position of the imaging optical system 33. Further, the first spatial light modulation element 38 and the second spatial light modulation element 41 are arranged at conjugate positions to each other.

The second spatial light modulator 41 variably adjusts the spatial distribution of the phase added to the transmitted light Lp from the subject S, and adjusts the phase added to the transmitted light Lp to 0 ° or ± 90 °. To do.
Specifically, the second spatial light modulation element 41 uses a quarter wavelength (± 90 °) of direct light (zero-order light) that has passed through the subject S out of the transmitted light Lp from the subject S. And a diffracted light transmission region 41b for transmitting the diffracted light diffracted by the subject S with the same phase (0 °) around the phase modulation region 41a. doing.

  The second spatial light modulation element 41 can freely change the shape and size of the phase modulation region 41a with respect to the diffracted light transmission region 41b. In addition, as the second spatial light modulation element 41, for example, a liquid crystal panel (liquid crystal element) or the like can be used.

  Furthermore, it is preferable that the second spatial light modulator 41 has a function of variably adjusting the spatial distribution of the transmittance for transmitting the transmitted light Lp from the subject S together with the spatial distribution of the phase described above. In general, of the transmitted light Lp that passes through the second spatial light modulator 41, the direct light that passes through the phase modulation region 41a has higher light intensity than the diffracted light that passes through the diffracted light transmission region 41b. Make adjustments to reduce the light intensity using a filter or the like.

  As such an ND filter, for example, an optical element capable of variably adjusting the spatial distribution of transmittance as disclosed in JP 2010-507119 A can be used. Further, as the second spatial light modulation element 41, an element to which such an optical element or the like is added can be used.

  The solid-state imaging device 34 includes a plurality of light receiving elements having different light receiving wavelengths, such as a CCD image sensor and a CMOS image sensor, and receives the transmitted light Lp imaged by the imaging optical system 33 described above to receive an electrical signal ( Image signal) and output to the determination apparatus 10.

  In the phase contrast microscope apparatus 30 having the above-described structure, the illumination light L emitted from the light source 31 passes through the first condenser lens 37 and is converted into parallel illumination light L, and then the parallel light The illumination light L is incident on the first spatial light modulator 38. The illumination light L that has passed through the opening 38a of the first spatial light modulator 38 is converted into parallel illumination light L by passing through the second condenser lens 39, and then the parallel illumination light L. Is irradiated onto the subject S placed on the placement surface 36 a of the stage 36.

  Then, the transmitted light Lp from the subject S passes through the objective lens 40 and then enters the second spatial light modulation element 41. At this time, after the direct light transmitted through the phase modulation region 41a out of the transmitted light Lp from the subject S is attenuated by the ND filter in a state where the phase is shifted by a quarter wavelength, the solid-state imaging device An image is formed on the light receiving surface 34. On the other hand, the diffracted light transmitted through the diffracted light transmitting region 41b is imaged on the light receiving surface of the solid-state imaging device 34 with the same phase (0 °). In the phase contrast microscope, it is possible to observe the change in the phase as the brightness of light by the interference between the straight light and the diffracted light.

[About cells to be judged]
Here, a specific example of the subject S of the phase-contrast microscope apparatus 30, that is, the determination target cell of the determination system 1 will be described. In this example, the subject S is a stained cell. This staining process will be described by taking an example in which the cells are animal cells. In the staining process, for example, a dye such as trypan blue is dropped on the cells. Since the cell membrane of dead cells is damaged, the dye is likely to penetrate inside the cells compared to live cells. For this reason, trypan blue selectively permeates dead cells among live cells and dead cells. That is, when cells are stained with trypan blue, live cells are not stained and dead cells are stained blue. By counting the number of cells stained in blue, the number of dead cells can be calculated.
In addition, although dyeing | staining by trypan blue was demonstrated as an example of a dyeing | staining process here, it is not restricted to this. As long as it is a dye that can selectively dye live cells and dead cells, it may be dyed with a dye other than trypan blue.

  Here, if the cell to be stained is a cell having no pigment, it can be easily determined whether the cell is a living cell or a dead cell by determining the color of the cell. However, if the cell to be stained is a cell having a pigment, that is, a pigment cell (or colored cell), it is determined whether it is the original color of the cell or the color by the staining process. May be difficult to do. In particular, the color of pigment cells such as retinal pigment epithelial cells (hereinafter also referred to as RPE cells) changes from light to dark as the cells mature. Therefore, when trypan blue staining is performed on RPE cells, it may be difficult to determine whether the color of the cells is the original color of the RPE cells or the color obtained by the staining process. Hereinafter, the configuration of the determination apparatus 10 that can determine whether a pigment cell such as an RPE cell is alive or dead will be described.

[Configuration of judgment device]
Next, the configuration of the determination apparatus 10 will be described with reference to FIG.
FIG. 2 is a block diagram illustrating a functional configuration of the determination apparatus 10 according to the present embodiment. The determination device 10 includes a control unit 11 and a storage unit 12.
The storage unit 12 stores in advance a determination program used for determining whether a cell is alive or not, and a control program for the phase-contrast microscope apparatus 30.
The control unit 11 includes a CPU (Central Processing Unit), and drives each unit of the phase-contrast microscope apparatus 30 according to a control program stored in the storage unit 12. Since the specific content of the control of the phase contrast microscope apparatus 30 by the control unit 11 is known, the description thereof is omitted.

The control unit 11 includes an image acquisition unit 111, a filter unit 112, and a determination unit 113 as functional units.
The image acquisition unit 111 acquires a phase difference image captured by the phase contrast microscope apparatus 30.
The filter unit 112 performs a filtering process on the phase difference image acquired by the image acquisition unit 111. This filter processing includes integration processing and differentiation processing of pixel values with respect to the phase difference image. By this integration processing, the color unevenness of the phase difference image is smoothed. Further, the edge portion where the pixel value of the phase difference image changes suddenly by the differentiation process is emphasized. In addition, the filter unit 112 extracts an image indicating the outer shape of the cells included in the phase difference image. In the following description, an image showing the outer shape of a cell extracted by the filter unit 112 is also referred to as a cell region ROI.
The determination unit 113 determines whether the cell is a living cell or a dead cell based on the cell image included in the phase difference image acquired by the image acquisition unit 111. Details of the determination process performed by the determination unit 113 will be described with reference to FIGS.

[Judgment process flow]
FIG. 3 is a flowchart illustrating an example of a process flow by the control unit 11 of the present embodiment.
The image acquisition unit 111 acquires a phase difference image captured by the phase contrast microscope apparatus 30 (step S10). An example of the phase difference image acquired in step S10 is shown in FIG.

FIG. 4 is a schematic diagram illustrating an example of an image handled by the determination system 1 of the present embodiment. The image acquisition unit 111 acquires the phase difference image illustrated in FIG. This phase difference image includes an image of the cell C1, an image of the cell C2, and an image of the cell C3. Among these cells, the cell C1 and the cell C2 are both living cells. The cell C3 is a dead cell. The image acquisition unit 111 outputs the acquired phase difference image to the filter unit 112.
In FIG. 4 (A), for example, in the example of the cell C1, a ring-shaped portion (partially interrupted) expressed in a slightly darker color on the drawing is a portion estimated to be a cell membrane existence region.

  Returning to FIG. 3, the filter unit 112 performs a filtering process on the phase difference image acquired in step S10 (step S20). In this example, the filter unit 112 applies an integration filter and a differentiation filter to the phase difference image acquired in step S10 to make it easy to extract a cell outline. Note that the filtering process in step S20 is not essential. The filter unit 112 may not perform the filtering process in step S20 depending on the state of the phase difference image.

  Next, the filter unit 112 extracts the cell region ROI (step S30). An example of the cell region ROI extracted by the filter unit 112 is shown in FIG. The filter unit 112 extracts the outline of the cell included in the phase difference image shown in FIG. In this example, the filter unit 112 extracts the outline of the cell C1 as the cell region ROI1. Further, the filter unit 112 extracts the outline of the cell C2 as the cell area ROI2 and the outline of the cell C3 as the cell area ROI3. A known method such as edge detection is applied to the contour extraction processing performed by the filter unit 112. The filter unit 112 outputs the image after extracting the cell region ROI to the determination unit 113.

  Returning to FIG. 3, the determination unit 113 determines the viability state of the cell for each cell region ROI extracted in step S <b> 30. Specifically, the determination unit 113 determines a determination line JL in the radial direction of the cell region ROI (step S40). Here, an example of the determination line JL defined by the determination unit 113 when the determination target cell is a living cell will be described with reference to FIG.

[When the judgment target is a living cell]
FIG. 5 is a schematic diagram illustrating an example of determination of a living cell by the determination unit 113 of the present embodiment. An example in the case where the determination unit 113 determines the image PCell1 of a certain living cell is shown in FIG. The image PCell1 is a phase difference image in which live cells are imaged. The image PCell1 includes images of the cell nucleus Cn, the cell membrane Cm, and the cytoplasm Cy. In FIG. 5A, the phase difference Δp is shown by blackening the portion where the phase difference Δp of the phase difference image is large and whitening the portion where the phase difference Δp is small.

  The determination unit 113 determines a determination line JL in the radial direction of the image PCell1. In the example shown in FIG. 5A, the radial direction of the image PCell1 is the X-axis direction of FIG. That is, in this example, the radial direction of the image PCell1 is the radial direction of the image PCell1.

  Returning to FIG. 3, the determination unit 113 obtains the number of changes of the phase difference Δp on the determination line JL (step S50). Specifically, the determination unit 113 calculates the number of changes of the phase difference Δp on the determination line JL by calculating the number of intersections between the phase difference threshold ThL1 and the change curve W1 of the phase difference Δp. Ask.

  More specifically, the determination unit 113 obtains a change curve of the phase difference Δp in the X-axis direction along the determination line JL shown in FIG. In the example shown in FIG. 5A, the phase difference Δp between the position x2 to position x3, the position x5 to position x7, and the position x9 to position x10 on the determination line JL is relatively large. Further, in the example shown in FIG. 5A, the phase difference Δp between the position x1, the position x4 to the position x5, the position x7 to the position x8, and the position x11 on the determination line JL is relatively small. A change curve W1 of the phase difference Δp along the determination line JL is shown in FIG.

  Here, when the cell stained with trypan blue is a living cell, a change in the phase difference Δp indicating the cell membrane Cm appears in the phase difference image of the living cell. That is, the phase difference Δp of the cell membrane Cm portion of the living cell is relatively large. Further, the phase difference Δp between the cytoplasmic Cy portion and the extracellular portion of the living cell is relatively small. Here, a position x1 on the determination line JL shown in FIG. 5A indicates an extracellular portion. A portion from position x2 to position x3 on the determination line JL indicates a cell membrane Cm. The portion from position x4 to position x5 on this determination line JL indicates cytoplasm Cy.

A change curve such that the change curve W1 of the phase difference Δp exceeds the threshold ThL1 between the extracellular position x1 on the determination line JL shown in FIG. 5A and the position x2 indicating the cell membrane Cm. W1 and threshold value ThL1 intersect. An intersection between the change curve W1 and the threshold value ThL1 between the position x1 and the position x2 indicates the position of the outer peripheral portion of the cell membrane Cm.
Further, on the determination line JL, between the position x3 indicating the cell membrane Cm and the position x4 indicating the cytoplasm Cy, the change curve W1 and the threshold ThL1 are set such that the change curve W1 of the phase difference Δp is equal to or less than the threshold ThL1. And intersect. An intersection between the change curve W1 and the threshold value ThL1 between the position x3 and the position x4 indicates the position of the inner peripheral portion of the cell membrane Cm.
That is, the two intersections of the change curve W1 and the threshold value ThL1 indicate the position of the outer peripheral portion and the position of the inner peripheral portion of the cell membrane Cm.

  Here, of the positions on the determination line JL, the negative direction side of the X axis from the center line CL, that is, the range from the position x1 to the position x6 has been described. That is, the range of less than half of the radial length has been described. Of the positions on the determination line JL, the same applies to the positive direction side of the X axis from the center line CL, that is, the range from the position x6 to the position x11. That is, the intersection between the change curve W1 and the threshold value ThL1 between the position x8 and the position x9 indicates the position of the inner periphery of the cell membrane Cm. Further, the intersection between the change curve W1 and the threshold value ThL1 between the position x10 and the position x11 indicates the position of the outer peripheral portion of the cell membrane Cm.

  Here, among the positions on the determination line JL, a change in the phase difference Δp indicating the cell membrane Cm at any position on the negative direction side of the X axis from the center line CL or on the positive direction side of the X axis from the center line CL. Is present, the determination unit 113 determines that the determination target cell is a living cell. That is, if there is a change in the phase difference Δp indicating the cell membrane Cm within a range of half or less of the length in the radial direction of the cell, the determination unit 113 determines that the determination target cell is a living cell.

[When the judgment target is dead cells]
Next, the case where the cell to be determined by the determination unit 113 is a dead cell in step S40 and step S50 described above will be described with reference to FIG.
FIG. 6 is a schematic diagram illustrating an example of determining a dead cell by the determination unit 113 according to the present embodiment. An example in the case where the determination unit 113 determines the image PCell2 of a certain dead cell is shown in FIG. The image PCell2 is a phase difference image in which dead cells are imaged. In this image PCell2, the cell nucleus Cn, the cell membrane Cm, and the cytoplasm Cy are difficult to distinguish. In FIG. 6 (A), as in FIG. 5 (A) described above, the portion where the phase difference Δp of the phase difference image is black, the portion where the phase difference Δp is small is white, and the phase difference Δp is set. Show.

  The determination unit 113 determines a determination line JL in the radial direction of the image PCell2. In the example shown in FIG. 6A, the radial direction of the image PCell2 is the X-axis direction of FIG. That is, in this example, the radial direction of the image PCell2 is the radial direction of the image PCell2.

  In step S50 described above, the determination unit 113 obtains the number of changes in the phase difference Δp on the determination line JL. Specifically, the determination unit 113 calculates the number of changes in the phase difference Δp on the determination line JL by calculating the number of intersections between the phase difference threshold ThL1 and the change curve W2 of the phase difference Δp. Ask.

  More specifically, the determination unit 113 obtains a change curve of the phase difference Δp in the X-axis direction along the determination line JL shown in FIG. In the example shown in FIG. 6A, the phase difference Δp between the position x2 and the position x10 on the determination line JL is relatively large. In the example shown in FIG. 6A, the phase difference Δp between the position x1 on the determination line JL and the position x11 is relatively small. FIG. 6B shows a change curve W2 of the phase difference Δp along the determination line JL.

  Here, when a cell stained with trypan blue is a dead cell, a change in the phase difference Δp indicating the cell membrane Cm hardly appears in the phase difference image of the dead cell. That is, in dead cells, the difference between the phase difference Δp in the cell membrane Cm portion and the phase difference Δp in the cytoplasm Cy portion is small. This is different from the fact that in a living cell, the phase difference Δp of the cell membrane Cm portion is relatively large, and the phase difference Δp of the cytoplasmic Cy portion and the extracellular portion is relatively small.

More specifically, the change curve W2 of the phase difference Δp exceeds the threshold ThL1 between the extracellular position x1 on the determination line JL shown in FIG. 6A and the position x2 indicating the cell membrane Cm. Thus, the change curve W2 and the threshold value ThL1 intersect. An intersection between the change curve W2 and the threshold value ThL1 between the position x1 and the position x2 indicates the position of the outer peripheral portion of the cell membrane Cm.
Further, the change curve W2 of the phase difference Δp does not become equal to or less than the threshold value ThL1 between the position x2 and the position x6 on the determination line JL. This is different from the case where the change curve W2 of the phase difference Δp is equal to or less than the threshold value ThL1 in the living cell.

  Here, of the positions on the determination line JL, the negative direction side of the X axis from the center line CL, that is, the range from the position x1 to the position x6 has been described. That is, the range of half or less of the length in the radial direction has been described. Of the positions on the determination line JL, the same applies to the positive direction side of the X axis from the center line CL, that is, the range from the position x6 to the position x11.

Here, among the positions on the determination line JL, a change in the phase difference Δp indicating the cell membrane Cm at any position on the negative direction side of the X axis from the center line CL or on the positive direction side of the X axis from the center line CL. If no exists, the determination unit 113 determines that the cell to be determined is a dead cell. That is, if there is no change in the phase difference Δp indicating the cell membrane Cm within a range of half or less of the length in the radial direction of the cell, the determination unit 113 determines that the determination target cell is a dead cell.
In the present embodiment, the information on the cell region ROI that is a specific region extracted by the filter unit 112 and the image information on the cell membrane that is a specific region different from the above are used to calculate the length or area of the cell membrane and the cell area. It is possible to pay attention and to determine the state of an object, for example, whether a cell is alive or dead.
First, the cell C1 in FIG. 4A will be described as an example.
The same processing as described above is performed on the cell C1, and the cell region ROI1 is extracted as shown in FIG. As shown in FIG. 4A, the cell region ROI1 is an outer region that is assumed to be a cell membrane illustrated by a ring-shaped portion (partially interrupted) expressed in a slightly darker color on the drawing. There is a halo region (noise component) that is white-bleeded. This halo region is a region where cells are not present. Therefore, the outline of the cell C1 can be extracted by deleting the halo region. This contour can be assumed to be the outer wall portion of the cell membrane. The halo region can be specified by image recognition based on the luminance information of the phase difference image. Alternatively, it is possible to store dimension information of the halo region in advance for each cell type, for example, length information indicating that there is a halo from the outer periphery of the halo region toward the inside of the cell, and removing the halo region based on this information It is. As described above, the outline of the cell C1 excluding the halo region can be defined as a region in which the equivalent exists in the outer peripheral portion of the cell membrane, and the entire circumference of the cell membrane of the cell C1 can be measured.
Next, the same processing as described above is performed, and as shown in FIG. 4C, for example, when the cell C1 is targeted, the cell membrane images CT11 to CT14 are recognized and extracted. Attention is paid to the circumferential lengths of the cell membrane images CT11 to CT14. As described above, the entire length of the cell membrane can be measured. By calculating the ratio (ratio) of the sum of the lengths of the cell membrane images CT11 to CT14 with respect to the total length of the cell membrane, it can be used as a reference for cell viability determination. This criterion also varies depending on the cell type. Therefore, it is also possible to store a life / death determination reference value for each cell type in advance and determine whether or not this reference value is satisfied. In the above example, the life / death judgment is made based on the ratio of the actual cell membrane length to the entire length of the cell membrane. However, the present invention is not limited to this. For example, it is possible to determine whether the life or death is based on the absolute value of the actual cell membrane length. In this case, it is also possible to determine in advance a life / death criterion for each cell type, culture condition, etc., and make a determination according to this.
Furthermore, it is also possible to determine using the area of the cell and the area of the cell membrane.
In this example, first, the cell region ROI is extracted by the filter unit 112 as shown in FIG. 4B by the same processing as described above (step S30). Next, the area of the cell region ROI is calculated. For example, when the cell C1 is targeted, the area of the cell region ROI1 is calculated. Next, cell membrane images CT11 to CT14 are recognized and extracted in the same manner as described above. Paying attention to the area of the cell membrane images CT11 to CT14, the area is calculated. By calculating the sum of the areas of the cell membrane images CT11 to CT14 and the ratio (ratio) of the area of the cell region ROI1, it can be used as a reference for cell viability determination. This criterion also varies depending on the cell type. Therefore, it is also possible to store a life / death determination reference value for each cell type in advance and determine whether or not this reference value is satisfied. In addition, for example, life / death can be determined by the absolute value of the actual cell membrane area. In this case, it is also possible to determine in advance a life / death criterion for each cell type, culture condition, etc., and make a determination according to this.
The length and area of the cell membrane and cell region as described above can be calculated based on the number of pixels using, for example, pixels of the image sensor.
The invention of the present application extracts at least one cell from a plurality of cultured cells, and can perform the image analysis as described above. Can determine the cell state without adverse effects.
Observables applicable to the present invention are not limited to the cells described in the present embodiment, and for example, various cells such as stem cells, nerve cells, cardiomyocytes, skin cells, hepatocytes, blood cells, immune cells, etc. In addition, the present invention can be applied to living organisms such as microorganisms, tissues, and the like, and can be applied to all observation objects capable of phase difference observation.
Moreover, it can be used not only for cell viability determination but also for cell state determination such as differentiation state, differentiation time, drug efficacy in screening, toxicity and the like. Also. It is applicable not only to cells but also to the growth status and function detection of microorganisms. By adopting the condition determination method of the present invention in such screening, drug toxicity test, drug efficacy test or regenerative medicine processing process, more efficient screening, efficient drug production, or efficient regenerative cells Can be made.
It is also possible to construct an observation device, a screening device, and a cell culture device that can determine the state of an object to be observed by driving the observation device with a control device programmed with the method of the present invention.

[Determination of live and dead cells]
As described above, the change curve of the phase difference Δp on the determination line JL is different between the living cell and the dead cell. If the change curve of the phase difference Δp is a change curve indicating a live cell (for example, the change curve W1 shown in FIG. 5B), the determination unit 113 determines that the determination target cell is a live cell. . If the change curve of the phase difference Δp is a change curve indicating a dead cell (for example, the change curve W2 shown in FIG. 6B), the determination unit 113 determines that the determination target cell is a dead cell. judge.

Here, the determination unit 113 determines whether the cell to be determined is based on the number of intersections between the change curve of the phase difference Δp and the threshold value ThL1 in a range that is less than or equal to half the radial length of the cell to be determined. It is determined whether the cell is a live cell or a dead cell (step S60).
As a specific example, a case where the determination target cell is a living cell will be described. As shown in FIG. 5B, in the range from the position x1 to the position x6, the number of intersections between the change curve W1 and the threshold value ThL1 is two. In this case, the determination unit 113 calculates that the number of intersections between the change curve W1 and the threshold value ThL1 is two.
As another specific example, a case where the determination target cell is a dead cell will be described. As shown in FIG. 6B, in the range from the position x1 to the position x6, the number of intersections between the change curve W2 and the threshold value ThL1 is one. In this case, the determination unit 113 calculates that the number of intersections between the change curve W2 and the threshold value ThL1 is one.

  In this example, if the number of intersections between the change curve of the phase difference Δp and the threshold value ThL1 is two or more, the determination unit 113 determines that the determination target cell is a living cell (step S60; YES). Determination is made and the process proceeds to step S70. If the number of intersections between the change curve of the phase difference Δp and the threshold value ThL1 is 1 or less, the determination unit 113 determines that the determination target cell is a dead cell (step S60; NO). Then, the process proceeds to step S80.

  Next, the determination unit 113 determines whether or not all the cell regions ROI to be determined have been determined to be alive (step S90). If the determination unit 113 determines that all the cell regions ROI to be determined are not determined to be alive (step S90; NO), the process returns to step S40 to continue the process for the next cell region ROI. To do. If the determination unit 113 determines that all the cell regions ROI to be determined have been determined to be alive (step S90; YES), the series of processing ends.

  As described above, the determination system 1 of this embodiment includes the determination unit 113. The determination unit 113 determines whether the cell is a live cell or a dead cell based on the phase difference image acquired by the image acquisition unit 111. As described above, according to the phase difference image, the physical structure of the subject S is likely to appear in the image as compared with an image not using the phase difference. For example, in an animal cell, the physical structure of the cell membrane tends to appear in a phase difference image. Therefore, by performing the determination based on the phase difference image, even when the determination target cell is a pigment cell, the influence of the color of the cell on the determination can be reduced. That is, according to the determination unit 113 of the present embodiment, it is possible to reduce the influence of the color of the cell on the determination by determining based on the phase difference image.

  In addition, the determination unit 113 according to the present embodiment determines whether the cell is a live cell or a dead cell based on a change in the radial phase difference Δp of the image showing the cell. As described above, when the dead cells are selectively stained, the dye penetrates into the dead cells. Thereby, in the dead cell, the physical structure inside the cell is less likely to appear in the phase difference image. In other words, in a living cell, the phase difference Δp is likely to change according to the physical structure inside the cell. On the other hand, in a dead cell, the phase difference Δp hardly changes depending on the physical structure inside the cell. That is, the change state of the phase difference Δp of the phase difference image is different between the living cell and the dead cell. The determination unit 113 determines whether the cell to be determined is a living cell or a dead cell by utilizing the difference in the change state of the phase difference Δp of the phase difference image. Therefore, according to the determination unit 113 of the present embodiment, it is possible to reduce the influence of cell color on determination. That is, according to the determination system 1 of the present embodiment, it is possible to improve the accuracy of cell life / death determination.

  In addition, the determination unit 113 of the present embodiment determines whether the cell is a live cell or a dead cell based on a change in the phase difference Δp in a range that is half or less of the length in the radial direction of the cell. That is, the determination unit 113 of the present embodiment determines whether a cell is a live cell or a dead cell based on a change in the phase difference Δp in a part of the range, not the entire range in the radial direction of the cell. judge. Thereby, the determination part 113 can determine whether a cell is a living cell or a dead cell, if the image which shows a cell membrane is contained in the phase difference image at least.

  Further, the determination system 1 of the present embodiment includes a filter unit 112. The filter unit 112 extracts the outer shape of the cell. This filter unit 112 can facilitate the determination in the determination unit 113.

[Modification]
In addition, although the case where the determination part 113 determined based on threshold value ThL1 was demonstrated as an example so far, it is not restricted to this. The determination unit 113 may determine whether the cell is alive or dead based on the threshold value ThL2. According to the threshold value ThL2, as shown in FIG. 5B, in addition to the change in the phase difference Δp caused by the cell membrane Cm, the change in the phase difference Δp caused by the cell nucleus Cn can also be detected.

In the above description, the cell phase difference image has been described as having a large phase difference Δp in the cell membrane portion, but the present invention is not limited to this. As illustrated in FIG. 7, the phase difference may be reversed from the phase difference image illustrated in FIGS. 5A and 6A.
FIG. 7 is a schematic diagram illustrating an example of a change curve of the phase difference Δp in a modification of the present embodiment. Here, the change curve W3 shown in FIG. 7 (A) is a curve obtained by inverting the change curve W1 of the living cells shown in FIG. 5 (A). Moreover, the change curve W4 shown in FIG. 7B is a curve obtained by inverting the change curve W2 of the living cell shown in FIG.

  Further, the filter unit 112 described above emphasizes the cell membrane portion as shown in FIG. 4C by performing integration processing and differentiation processing of the pixel value on the phase difference image acquired by the image acquisition unit 111. An image may be generated. In this case, the filter unit 112 is configured to output an image highlighting the cell membrane portion shown in FIG. 4C to the determination unit 113 together with the image showing the cell region ROI shown in FIG. Good. In the example shown in FIG. 4C, the filter unit 112 outputs an image in which the cell membrane images CT11 to CT14 are emphasized for the cell C1 to the determination unit 113. The filter unit 112 outputs an image in which the cell membrane images CT21 to CT23 are emphasized for the cell C2 to the determination unit 113.

  Further, by recording a program for executing each process of the determination system 1 in the present embodiment on a computer-readable recording medium, causing the computer system to read and execute the program recorded on the recording medium, You may perform the various process mentioned above.

  Here, the “computer system” may include an OS and hardware such as peripheral devices. Further, the “computer system” includes a homepage providing environment (or display environment) if a WWW system is used. The “computer-readable recording medium” means a flexible disk, a magneto-optical disk, a ROM, a writable nonvolatile memory such as a flash memory, a portable medium such as a CD-ROM, a hard disk built in a computer system, etc. This is a storage device.

  Further, the “computer-readable recording medium” means a volatile memory (for example, DRAM (Dynamic DRAM) in a computer system that becomes a server or a client when a program is transmitted through a network such as the Internet or a communication line such as a telephone line. Random Access Memory)), etc., which hold programs for a certain period of time. The program may be transmitted from a computer system storing the program in a storage device or the like to another computer system via a transmission medium or by a transmission wave in the transmission medium. Here, the “transmission medium” for transmitting the program refers to a medium having a function of transmitting information, such as a network (communication network) such as the Internet or a communication line (communication line) such as a telephone line. The program may be for realizing a part of the functions described above. Furthermore, what can implement | achieve the function mentioned above in combination with the program already recorded on the computer system, what is called a difference file (difference program) may be sufficient.

  As mentioned above, although embodiment of this invention was explained in full detail with reference to drawings, the concrete structure is not restricted to this embodiment, The design etc. of the range which does not deviate from the summary of this invention are included.

  DESCRIPTION OF SYMBOLS 1 ... Determination system, 10 ... Determination apparatus, 111 ... Image acquisition part, 112 ... Filter part, 113 ... Determination part, 20 ... Display part, 30 ... Phase contrast microscope apparatus

Claims (11)

An image acquisition unit for acquiring a phase difference image of an observation object including cells, which is observed by phase difference observation;
A filter unit for extracting a cell region of the cell in the phase difference image and deleting a halo region from the cell region;
A determination unit that determines the state of the cell based on the entire circumference of the outline of the cell region from which the halo region has been removed, and the length of the cell membrane obtained based on the phase difference image ,
A determination apparatus comprising:   The length of the cell membrane is the sum of the lengths of the portions where the cell membrane is broken into at least two in the phase difference image,
  The determination unit uses a ratio of the sum of the lengths of the cell membrane to the entire circumference of the outline of the cell region as a reference for determining the state of the cell.
  The determination apparatus according to claim 1.   The determination unit determines the state of the cell according to whether the criteria different for each cell type is satisfied,
  The determination apparatus according to claim 2.   The determination unit includes determining a life / death state of the cell,
  The determination apparatus according to any one of claims 1 to 3.   The filter unit extracts the cell region of the cell from the phase difference image by edge detection;
  The determination apparatus according to any one of claims 1 to 4.   The filter unit performs integration processing and differentiation processing of pixel values on the phase difference image.
The determination apparatus according to any one of claims 1 to 5.   The filter unit deletes the halo region based on luminance information of the phase difference image.
  The determination apparatus according to any one of claims 1 to 6.   The cells are pigment cells;
  The determination apparatus according to any one of claims 1 to 7. The determination device according to any one of claims 1 to 8 ,
A phase difference imaging unit for generating the phase difference image;
An output unit for outputting a determination result of the determination device;
A determination system comprising: On the computer,
An image acquisition procedure for acquiring a phase difference image of an observation object including cells;
Extracting a cell region of the cell in the phase contrast image, and removing a halo region from the cell region;
A determination procedure for determining the state of the cell based on the entire circumference of the outline of the cell region from which the halo region is removed, and the length of the cell membrane obtained based on the phase difference image ,
Judgment program for executing A method for producing a cell using the determination apparatus according to any one of claims 1 to 8,
The image acquisition unit acquires a phase difference image of an observation object including cells;
The filter unit extracts a cell region of the cell in the phase difference image, and deletes a halo region from the cell region;
The determination unit determines the state of the cell based on the entire circumference of the outline of the cell region from which the halo region is removed, and the length of the cell membrane obtained based on the phase difference image,
Cells method of manufacturing that have a.