JP6284832B2 - Cell evaluation apparatus, method and program - Google Patents

Description

  The present invention relates to a cell evaluation apparatus, method, and program for detecting scattered light generated in a cell group and evaluating the cell group based on the intensity of the scattered light.

  Conventionally, a method has been proposed in which pluripotent stem cells such as ES cells, iPS cells, and STAP cells and differentiation-induced cells are cultured, imaged with a microscope, and the state of the cells is evaluated by capturing the characteristics of the images. ing.

  In addition, the method for evaluating the state of the cell is not limited to the image observation as described above, but the scattered light generated in the cell by the irradiation of the light to the cell is detected, and the state of the cell is determined based on the intensity of the scattered light. Various methods have also been proposed for evaluating the above.

  For example, Patent Document 1 proposes that scatter of sperm in an image is evaluated by capturing scattered light of sperm with an image sensor and evaluating a time correlation function of the captured image.

  Patent Document 2 discloses that blood scattered light is detected and information is obtained from the distribution of the angle of the scattered light. Patent Document 3 discloses scattered light from biologically active substances derived from living organisms. It is disclosed to detect and acquire temporal fluctuations of the scattered light. Patent Document 4 discloses that a change in the state of a cell is obtained from a temporal change in Raman scattered light.

JP 2008-527359 A Japanese Patent Laid-Open No. 08-054388 JP 2010-185849 A JP 2011-257691 A

  Here, the above-described stem cells and differentiation-induced cells reach colony as the culture progresses, and proliferate over a large area. The size of the cell colony is on the order of several millimeters to several centimeters, and the cell colony may grow three-dimensionally by stacking cells.

  When cell colonies grow three-dimensionally in this way, multiple scattering, refraction, and diffraction by cells within the cell colony are affected even if a phase contrast image or bright field image of the cell colony is captured and observed. From these images, it is difficult to observe the state of individual cells in the cell colony.

  Therefore, in order to evaluate the state of the cell colony, it is conceivable to detect and analyze the scattered light generated in the cell colony. When detecting the scattered light of the cell colony, for example, the cell colony is cultured. If scattered light is detected while scanning the entire area with light, scanning will be performed with light up to an area where there are no cell colonies, the detection time will be longer, and the amount of data that detects the scattered light will be enormous. The analysis also takes time.

  Patent Documents 1 to 4 disclose that the scattered light of individual cells is detected, but nothing is shown about a specific method for detecting the scattered light of cell colonies. Absent.

  In view of the above problems, an object of the present invention is to provide a cell evaluation apparatus, method, and program capable of more efficiently detecting and analyzing scattered light generated in a cell colony.

  The cell evaluation apparatus of the present invention includes an illumination light irradiating unit that irradiates a cell group with illumination light for capturing an image of the cell group, and transmitted light that has passed through the cell group by irradiating the cell group with illumination light. Detecting a cell group image, detecting an image of the cell group, a cell group region specifying unit for specifying a cell group region in the cell group image detected by the image detecting unit, and detecting scattered light in the cell group Detected by a detection light irradiating unit that irradiates the cell group with detection light to detect, a scattered light detection unit that detects scattered light generated in the cell group by irradiating the cell group with detection light, and a scattered light detection unit And a cell evaluation unit that evaluates a cell group based on the spatial spread or temporal fluctuation of the intensity of the scattered light.

  The cell evaluation apparatus of the present invention includes an irradiation range determination unit that determines the irradiation range of the detection light based on the region of the cell group specified by the cell group region specification unit, and the detection light irradiation unit includes the irradiation range. Detection light can be irradiated to the irradiation range determined by the determination unit.

  Further, a wavelength separation optical system that separates the transmitted light and the scattered light, guides the transmitted light to the image detection unit, and guides the scattered light to the scattered light detection unit can be provided.

  In addition, an illumination optical system that reflects one of the illumination light and the detection light to irradiate the cell group and transmits the other to the cell group can be provided.

  The illumination light irradiation unit can irradiate the cell group with illumination light for phase difference measurement, and the image detection unit can detect a phase difference image of the cell group.

  Further, the image detection unit can detect a bright field image of the cell group.

  The illumination light irradiation unit can irradiate the cell group with illumination light for differential interference measurement, and the image detection unit can detect the differential interference image of the cell group.

  The detection light irradiation unit can irradiate the non-cell group region where the cell group does not exist, and the scattered light detection unit can detect the scattered light generated in the non-cell group region where the cell group does not exist as a noise component. The cell evaluation unit can evaluate the cell group by removing noise components from the spatial spread or temporal fluctuation of the intensity of scattered light generated in the cell group.

  In addition, laser light can be used as detection light, and the cell evaluation unit can evaluate a cell group based on temporal fluctuation of the intensity of scattered light.

  Moreover, parallel light can be used as the detection light, and the cell evaluation unit can acquire the angular distribution of the scattered light and can evaluate the cell group based on the angular distribution.

  Further, convergent light can be used as detection light, and the cell evaluation unit can acquire the spatial distribution of scattered light and can evaluate a cell group based on the spatial distribution.

  Moreover, based on the image of the cell group detected by the image detection part, the conditions for detecting scattered light can be determined.

  The cell evaluation method of the present invention irradiates a cell group with illumination light for capturing an image of the cell group, detects transmitted light transmitted through the cell group by irradiation of the illumination light, and detects an image of the cell group Is detected, the region of the cell group is identified in the image of the cell group, the detection light for detecting the scattered light in the cell group is irradiated to the cell group, and the detection light irradiation generates in the cell group The scattered light is detected, and the cell group is evaluated on the basis of the spatial spread or temporal fluctuation of the intensity of the detected scattered light.

  The cell evaluation program of the present invention includes a computer, an image acquisition unit that acquires an image of a cell group in which transmitted light transmitted through the cell group is detected by irradiation of illumination light to the cell group, and a cell group in the image of the cell group Acquired by a scattered light information acquisition unit, a scattered light information acquisition unit that acquires information on the intensity of scattered light generated in the cell group by irradiation of detection light to the cell group, and a scattered light information acquisition unit It is made to function as a cell evaluation part which evaluates a cell group based on the spatial spread or temporal fluctuation of the intensity of scattered light.

  According to the cell evaluation apparatus, method, and program of the present invention, the illumination light for illuminating the cell group with the illumination light for capturing an image of the cell group separately from the irradiation of the detection light for detecting the scattered light, and the illumination light By detecting the transmitted light transmitted through the cell group by irradiation of the cell group and detecting the cell group image, the region of the cell group in the image of the cell group is specified. Based on this, it is possible to determine the irradiation range of the detection light, and irradiate the determined irradiation range with the detection light to detect the scattered light of the cell group, that is, limit the irradiation of the detection light to the region of the cell group. Therefore, irradiation of detection light and detection of scattered light can be performed more efficiently.

  In addition, when evaluating a cell group based on the spatial spread or temporal fluctuation of the intensity of scattered light generated in the cell group, the intensity of the scattered light generated in the region of the cell group specified as described above. If the analysis is limited to the above, the intensity of the scattered light can be efficiently analyzed, and the influence of the noise of the scattered light other than the region of the cell group can be suppressed.

The figure which shows schematic structure of the microscope system using one Embodiment of the cell evaluation apparatus of this invention. Diagram showing the configuration of the slit plate Diagram showing the configuration of the phase difference lens The figure which shows an example of the time fluctuation of the intensity of the scattered light which generate | occur | produced in the cell colony The flowchart for demonstrating the effect | action of the microscope system using one Embodiment of the cell evaluation apparatus of this invention. The figure which shows schematic structure of the modification of the microscope system shown in FIG. The figure which shows an example of the histogram regarding the cell size calculated based on an example of the angular distribution of scattered light when a cell colony is irradiated with parallel light, and the angular distribution The figure which shows an example of the spatial distribution of the scattered light when a cell colony is irradiated with convergent light Diagram for explaining scattered light when parallel light is irradiated to undifferentiated cell colonies and differentiated cell colonies The figure which shows an example of angle distribution of the light intensity of the scattered light at the time of irradiating a parallel light to the undifferentiated cell colony and the differentiated cell colony Diagram for explaining scattered light when undifferentiated cell colonies and differentiated cell colonies are irradiated with convergent light The figure which shows an example of the spatial distribution of the light intensity of the scattered light at the time of irradiating convergent light to the undifferentiated cell colony and the differentiated cell colony The figure which shows schematic structure of the other modification of the microscope system shown in FIG. The figure for demonstrating the method of calculating the noise component resulting from dust etc. based on the information of a cell colony area

  Hereinafter, a microscope system 1 using an embodiment of a cell evaluation apparatus and method and a program of the present invention will be described in detail with reference to the drawings. FIG. 1 is a diagram showing a schematic configuration of a microscope system 1 of the present embodiment.

  As shown in FIG. 1, the microscope system 1 of the present embodiment includes an illumination light irradiation unit 10, an image detection unit 20, a detection light irradiation unit 30, a scattered light detection unit 40, an illumination optical system 50, and a wavelength. A separation optical system 60, a cell evaluation device 70, and a display 80 are provided.

  The illumination light irradiation unit 10 emits illumination light for so-called phase difference measurement to the culture dish 15 on which cell colonies (cell groups) are cultured. In the present embodiment, the illumination for phase difference measurement is performed. The ring-shaped illumination light L1 is emitted as light. Specifically, the illumination light irradiation unit 10 according to the present embodiment includes a white light source 11 that emits white light and a ring-shaped slit, and the white light emitted from the white light source 11 is incident on the ring light. The slit lens 12 that emits the light L1 and the ring-shaped illumination light L1 emitted from the slit plate 12 are incident, and the objective lens that irradiates the incident ring-shaped illumination light L1 to the cell colonies in the culture dish 15 13.

  FIG. 2 is a diagram showing a specific configuration of the slit plate 12. As shown in FIG. 2, the slit plate 12 is provided with a ring-shaped slit 12 a that transmits white light to a light shielding plate 12 b that blocks white light emitted from the white light source 11. When the light passes through the slit 12a, ring-shaped illumination light L1 is formed.

  In the present embodiment, the ring-shaped illumination light L1 is formed using the slit plate 12 as described above. However, the method for forming the ring-shaped illumination light L1 is not limited to this. The ring-shaped illumination light L1 may be formed using a light modulation element or the like.

  In the present embodiment, the ring-shaped illumination light L1 is used as the phase difference measurement illumination light. However, illumination light having a structure other than the ring shape may be used, and the shape is conjugate to a phase plate described later. Other shapes such as a triangular shape or a rectangular shape may be used.

  The image detection unit 20 includes a phase difference lens 21, an imaging lens 24, and an image sensor 25.

  The phase difference lens 21 includes an objective lens 22 and a phase plate 23. FIG. 3 is a diagram showing a specific configuration of the phase plate 23. As shown in FIG. 3, the phase plate 23 is obtained by forming a phase ring 23a on a transparent plate 23b that is transparent with respect to the wavelength of the ring-shaped illumination light L1. Note that the size of the slit 12a described above is in a conjugate relationship with the phase ring 23a.

  The phase ring 23a is a ring in which a phase film that shifts the phase of incident light by a quarter wavelength and a neutral density filter that attenuates incident light are formed. The direct light incident on the phase difference lens 21 is collected by the objective lens 22 and passes through the phase ring 23a so that the phase is shifted by a quarter wavelength and the brightness is weakened. On the other hand, most of the diffracted light diffracted by the cell colonies in the culture dish 15 passes through the transparent plate 23b of the phase plate 23, and its phase and brightness do not change.

  Note that the phase difference lens 21 of the present embodiment also receives scattered light generated in the cell colony by irradiation of detection light, which will be described later, onto the cell colony, and most of the scattered light is transparent to the phase plate 23. It passes through the plate 23b.

  The imaging lens 24 receives direct light and diffracted light that have passed through the phase difference lens 21 and the wavelength separation optical system 60 and forms an image of these lights. The imaging element 25 is connected by the imaging lens 24. The imaged phase difference image is picked up. As the image sensor 25, a charge-coupled device (CCD) image sensor, a complementary metal-oxide semiconductor (CMOS) image sensor, or the like is used.

  The detection light irradiation unit 30 irradiates the cell colonies in the culture dish 15 with detection light for detecting scattered light generated in the cell colonies. The detection light irradiation unit 30 of the present embodiment includes a laser light source, and the cell colony is irradiated with the laser light L2 emitted from the laser light source.

  The scattered light detection unit 40 detects scattered light generated in the cell colonies in the culture dish 15. The scattered light detection unit 40 of the present embodiment has an imaging lens 41 through which the scattered light that has been transmitted through the phase difference lens 21 and reflected by the wavelength separation optical system 60 is incident, and the scattering imaged by the imaging lens 41. And a photodetector 42 for detecting the light L3.

  The photodetector 42 of the present embodiment detects the scattered light L3 and outputs the intensity signal thereof, and an image sensor such as a CCD or CMOS may be used in the same manner as the imaging device 25 described above. A one-dimensional sensor such as a line sensor may be used, or a single photodiode may be used.

  The illumination optical system 50 transmits the ring-shaped illumination light L1 emitted from the illumination light irradiation unit 10 and reflects the detection light L2 emitted from the detection light irradiation unit 30 in a direction perpendicular to the incident direction. It has characteristics. Specifically, a half mirror, a prism, or the like can be used as the illumination optical system 50. Further, other known optical systems may be used.

  The wavelength separation optical system 60 transmits the transmitted light transmitted through the cell colony by irradiation of the ring-shaped illumination light L1 to the cell colony, and the scattered light generated from the cell colony by irradiation of the detection light L2 to the cell colony. It has an optical characteristic of reflecting in a direction perpendicular to the incident direction. Specifically, a half mirror, a prism, or the like can be used as the wavelength separation optical system 60. Further, other known optical systems may be used.

  The culture dish 15 on which the cell colonies are cultured is placed on the stage 16. The stage 16 is configured to be movable in the X direction and the Y direction orthogonal to each other within the installation surface of the culture dish 15. The stage 16 moves based on a control signal output from the irradiation range control unit 77 of the cell evaluation apparatus 70 described later, and thereby, the detection light is irradiated to the cell colony region in the culture dish 15.

  The cell evaluation apparatus 70 is obtained by installing an embodiment of the cell evaluation program of the present invention on a computer.

  The cell evaluation apparatus 70 includes a central processing unit, a semiconductor memory, a hard disk, and the like, and one embodiment of the cell evaluation program of the present invention is installed on the hard disk. Then, the cell evaluation program is executed by the control unit 76 having a central processing unit, whereby an image acquisition unit 71, a cell colony region specifying unit 72 (corresponding to a cell group region specifying unit) as shown in FIG. The irradiation range determination unit 73, the scattered light information acquisition unit 74, the cell evaluation unit 75, and the irradiation range control unit 77 operate.

  The image acquisition unit 71 acquires the phase difference image output from the image sensor 25 of the image detection unit 20 and stores it.

  The cell colony region specifying unit 72 acquires the phase difference image read from the image acquisition unit 71 and specifies a cell colony region in the phase difference image. As a method for specifying a cell colony region, for example, after converting a phase difference image captured by the image detection unit 20 into a binary image, a cell colony region is automatically extracted and specified by template matching or the like. You can do it. Moreover, about automatic extraction of the area | region of a cell colony, you may make it use not only the method mentioned above but another well-known method.

  Not only automatic extraction but also a phase difference image is displayed on the display 80 by the control unit 76, and a user designates a region of a cell colony in the phase difference image using a predetermined input device (not shown), and the designation The cell colony region specifying unit 72 may acquire the information such as the coordinates, and thereby specify the cell colony region.

  The irradiation range determination unit 73 determines the irradiation range of the laser light L2 as the detection light emitted from the detection light irradiation unit 30 based on the cell colony region specified by the cell colony region specification unit 72. . As the irradiation range of the laser beam L2, the entire cell colony region may be operated with the laser beam L2, the central portion of the cell colony region may be set as the irradiation range, or the peripheral portion of the cell colony region is set as the irradiation range. It is good. Which range in the cell colony region is irradiated with the laser beam L2 may be set in advance, or may be set and input by a user using a predetermined input device (not shown).

  The scattered light information acquisition unit 74 acquires the intensity signal output from the photodetector 42 of the scattered light detection unit 40 and stores it.

  The cell evaluation unit 75 acquires the intensity signal of the scattered light read from the scattered light information acquisition unit 74, and evaluates the state of the cell colony by analyzing the temporal fluctuation of the intensity signal. .

  The upper diagram of FIG. 4 shows an example of temporal fluctuation of the intensity signal of the scattered light L3 when each cell in the cell colony is actively moving. The lower diagram of FIG. It shows an example of temporal fluctuation of the intensity signal of the scattered light L3 when each cell is not moving very actively. The horizontal axis of the graph shown in FIG. 4 is time t, and the vertical axis indicates the magnitude of the intensity signal I of the scattered light L3.

  As the intensity signal I of the scattered light L3, for example, when the photodetector 42 is an image sensor or a line sensor, statistical values such as an average value, a maximum value, and a minimum value of signals detected by a plurality of photodiodes. May be used as the intensity signal I. If the photodetector 42 is composed of one photodiode, the detected signal may be used as the intensity signal I as it is.

  In the upper graph of FIG. 4, since the cells in the cell colony are actively moving, the temporal fluctuation of the intensity signal I is large, and in the lower graph of FIG. 4, the cells in the cell colony move very actively. Therefore, the temporal fluctuation of the intensity signal I is smaller than the graph in the upper diagram.

  As described above, the cell evaluation unit 75 analyzes the temporal fluctuation of the intensity signal I of the scattered light L3 as shown in FIG. 4 and evaluates the state of the cell colony. Specifically, for example, an analysis value of temporal fluctuation is obtained by calculating a time correlation function or differential value of the intensity signal I, a complexity of the graph of the intensity signal I, and the like, and based on the magnitude of the analysis value. To evaluate the activity of cell colonies step by step. Further, based on the analysis value, the proliferation rate, migration rate, or motility of the microorgan in the cell may be evaluated.

  Also, undifferentiated cell colonies are densely packed with highly active cells, and differentiated cell colonies are dispersed with low activity cells. The cell may be in an undifferentiated state or a differentiated state, or the degree of differentiation of a cell colony may be evaluated. The analysis value calculation method is not limited to the time correlation function, the differential value, and the complexity, and other known calculation methods can be used.

  Returning to FIG. 1, the control unit 76 controls the entire cell evaluation device 70. In particular, the control unit 76 of the present embodiment includes an irradiation range control unit 77. The irradiation range control unit 77 controls and outputs a control signal to a moving mechanism (not shown) of the stage 16 based on the irradiation range of the laser light L2 determined by the irradiation range determination unit 73. By the movement control of the stage 16 by the irradiation range control unit 77, the laser beam L2 is scanned over the entire cell colony region as described above, the laser beam L2 is irradiated to the central part of the cell colony region, or the cell colony region The laser light L2 is irradiated to the peripheral part of the.

  Further, the control unit 76 causes the display 80 to display the phase difference image of the cell colony imaged by the image detection unit 20 and the evaluation result in the cell evaluation unit 75.

  Next, the operation of the microscope system 1 shown in FIG. 1 will be described.

  First, the culture dish 15 in which cell colonies are cultured is placed on the stage 16 (S10). Examples of cells to be cultured include stem cells such as ES cells and iPS cells, and cells obtained by inducing differentiation of stem cells.

  Next, the ring-shaped illumination light L1 is irradiated with respect to the cell colony in the culture dish 15, and the phase contrast image of the cell colony by this is imaged (S12). Specifically, the ring-shaped illumination light L <b> 1 is emitted from the illumination light irradiation unit 10, passes through the illumination optical system 50, and is irradiated to the cell colonies in the culture dish 15.

  The transmitted light that has passed through the cell colony by irradiation with the ring-shaped illumination light L1 passes through the phase difference lens 21 and the wavelength separation optical system 60, is imaged on the image sensor 25 by the imaging lens 24, and is imaged by the image sensor 25. Are obtained.

  The phase difference image captured by the image sensor 25 is acquired and stored by the image acquisition unit 71 of the cell evaluation device 70. The phase difference image read from the image acquisition unit 71 is output to the cell colony region specifying unit 72, and the cell colony region specifying unit 72 specifies the region of the cell colony region in the input phase difference image. (S14).

  The cell colony region information specified by the cell colony region specifying unit 72 is output to the irradiation range determining unit 73, and the irradiation range determining unit 73 uses the laser as detection light based on the input cell colony region information. The light irradiation range is determined (S16).

  The irradiation range of the laser light determined by the irradiation range determination unit 73 is output to the irradiation range control unit 77, and the irradiation range control unit 77 determines the moving mechanism of the stage 16 based on the input irradiation range of the laser light. A control signal is output, and the stage 16 is moved so that the laser beam is irradiated in the laser beam irradiation range described above (S18).

  The cell colony is irradiated with the laser light L2 as detection light, and the scattered light L3 generated in the cell colony is detected by the irradiation of the laser light L2 (S20). Specifically, the laser light L <b> 2 is emitted from the detection light irradiation unit 30, reflected by the illumination optical system 50, and irradiated to the cell colonies in the culture dish 15.

  The scattered light L3 generated in the cell colony by the irradiation of the laser light L2 passes through the phase difference lens 21, is reflected by the wavelength separation optical system 60, is imaged on the photodetector 42 by the imaging lens 41, and is detected by light. The scattered light L3 is detected by the device 42.

  The intensity signal of the scattered light L3 detected by the photodetector 42 is acquired by the scattered light information acquisition unit 74 and stored. Then, the intensity signal of the scattered light L3 read from the scattered light information acquisition unit 74 is output to the cell evaluation unit 75, and the cell evaluation unit 75 analyzes the temporal fluctuation of the intensity signal of the scattered light L3 to analyze the cell. The state of the colony is evaluated (S22).

  The evaluation result of the cell colony in the cell evaluation unit 75 is output to the control unit 76, and the control unit 76 displays the input evaluation result on the display 80 (S24).

  According to the microscope system 1 of the above-described embodiment, separately from the irradiation of the detection light for detecting the scattered light, the cell colony is irradiated with the illumination light for capturing the image of the cell colony. Detect the transmitted light that has passed through the cell colony to detect the image of the cell colony, identify the area of the cell colony in the detected image of the cell colony, and irradiate the detection light based on the area of the identified cell colony Since the range is determined and the detection light is irradiated to the determined irradiation range to detect the scattered light of the cell colony, the irradiation of the detection light can be limited to the area of the cell colony and more efficient Therefore, it is possible to irradiate detection light and detect scattered light.

  Next, a modification of the microscope system of the above embodiment will be described with reference to FIG. In this modification, in the microscope system of the above-described embodiment, a region where no cell colony exists in the culture dish 15 (hereinafter referred to as a non-cell group region) is also irradiated with laser light as detection light. Scattered light generated in the group region is detected as a noise component, and the noise component is removed to evaluate a cell colony. In addition, this noise component originates in the component of the culture solution which culture | cultivates a cell colony, the garbage which floats in the culture solution, or a dead cell.

  More specifically, after the scattered light generated in the cell colony by the irradiation of the laser beam to the cell colony is detected and the intensity signal is acquired by the cell evaluation unit 75, based on the control signal from the control unit 76 The moving mechanism (not shown) of the stage 16 is controlled to move the stage 16. At this time, the control unit 76 moves the stage 16 based on the cell colony region information specified by the cell colony region specifying unit 72 so that the non-cell group region in which no cell colony exists is irradiated with laser light. .

  Then, laser light is emitted from the detection light irradiation unit 30, and the laser light reflected by the illumination optical system 50 is irradiated to the non-cell group region. Scattered light generated in the non-cell group region by the irradiation of the laser beam to the non-cell group region is transmitted through the phase difference lens 21, reflected by the wavelength separation optical system 60, and applied to the photodetector 42 by the imaging lens 41. An image is formed and scattered light is detected by the photodetector 42.

  The intensity signal of the scattered light detected at this time is acquired by the scattered light information acquisition unit 74 and stored. The intensity signal of the scattered light generated in the non-cell region group is read from the scattered light information acquisition unit 74 and output to the cell evaluation unit 75. The cell evaluation unit 75 converts the intensity signal of the scattered light into a noise component. Get as. The cell evaluation unit 75 removes the intensity signal of the scattered light of the non-cell group region from the intensity signal of the scattered light of the cell colony, analyzes the temporal fluctuation of the intensity signal after the removal, and evaluates the state of the cell colony To do. As a method for removing the intensity signal of scattered light from the non-cell group region, for example, the intensity signal may be logarithmically converted and subtracted.

  In addition, as a non-cell group region, by specifically extracting the region of floating substances such as dead cells and dust from the phase contrast image, specifying the position, and irradiating the specified floating region with laser light You may make it detect a noise component. The floating material region may be extracted based on, for example, the size, shape, or luminance value of a predetermined pattern extracted from the phase difference image. Further, the cell colony region and the suspended solid region may be distinguished and specified based on the movement of the pattern of each cell including the phase difference image, the growth rate, or the like.

  In the above description, the noise component in the culture solution in the culture dish 15 is removed. However, the present invention is not limited to this. For example, a noise component based on a scratch on the culture dish 15 itself is acquired in advance. The cell colony may be evaluated by removing the noise component of the dish 15 itself from the intensity signal of the scattered light of the cell colony.

  As described above, the accuracy of cell colony evaluation can be improved by removing noise components.

  In the microscope system 1 of the above embodiment, the laser light is irradiated as the detection light. However, the present invention is not limited to the laser light, and the parallel light or the convergent light is irradiated as the detection light. Scattered light generated in the cell colony by irradiation of the cell colony may be detected by the photodetector 42. When the laser light is irradiated as the detection light, the cell colony is evaluated based on the temporal fluctuation of the intensity of the scattered light as described above, but when the parallel light or the convergent light is irradiated. Can evaluate cell colonies based on the spatial spread of scattered light.

  For example, when irradiating a cell colony with parallel light as detection light, an image sensor is used as the photodetector 42, and the state of the cell colony is analyzed by analyzing the angular distribution of the detection image of the scattered light detected by the image sensor. Can be evaluated.

  Specifically, as shown in the upper diagram of FIG. 7, the cell evaluation unit 75 acquires the detection image detected by the photodetector 42 as the angular distribution (θx−θy axis) of the scattered light, and this angular distribution. Based on the above, the size of each cell (particle) in the cell colony is estimated from the scattering theory, and a histogram relating to the cell size as shown in the lower diagram of FIG. 7 is calculated. Here, an undifferentiated cell colony is a state in which undifferentiated cells smaller than differentiated cells are densely packed. On the contrary, a differentiated cell colony is a state in which differentiated cells larger than undifferentiated cells are distributed. Therefore, for example, when the frequency of relatively small cells is higher than the frequency of relatively large cells, the cell colony is evaluated as being undifferentiated, and conversely, the frequency of relatively large cells is When the frequency is relatively higher than the frequency of relatively small cells, the cell colony may be evaluated as being in a differentiated state.

  For example, when convergent light is irradiated on the cell colony as detection light, the state of the cell colony can be evaluated by analyzing the spatial distribution of the detection image of the scattered light detected by the photodetector 42.

  Specifically, as shown in FIG. 8, the cell evaluation unit 75 acquires the detection image detected by the photodetector 42 as a spatial distribution (XY axis) of scattered light, and based on this spatial distribution. Then, the size of each cell (particle) in the cell colony is estimated from the scattering theory, and a histogram relating to the cell size is calculated in the same manner as in the case of the parallel light irradiation described above. The cell colony evaluation based on the histogram is the same as described above.

  Since the calculation of the histogram based on the scattering theory described above is a known calculation method, detailed description thereof is omitted.

  In the above description, a histogram related to the size of individual cells included in a cell colony is calculated. However, the present invention is not limited to such a method, and the spread of the spatial distribution of the light intensity of the detection image is not limited. May be evaluated to determine whether the cell colony is in an undifferentiated state or a differentiated state.

  Specifically, as shown in FIG. 9, an undifferentiated cell colony is a state in which a large number of cells are densely stacked, whereas a differentiated cell colony has a cytoplasm of each cell containing a medium. It deforms like a wrinkle and becomes a state where it is formed as a single layer.

  Therefore, when parallel light is irradiated to an undifferentiated cell colony, scattered light is emitted from each individual cell, and the scattered light generated in each individual cell is repeatedly reflected within the cell colony. Compared with the case where the colony is irradiated with parallel light, scattered light having a spread angle of various sizes is generated. On the other hand, when collimated light is irradiated to a differentiated cell colony, only scattered light is generated from individual cells in a single layer, so the spread angle is larger than when collimated light is irradiated to an undifferentiated cell colony. Small scattered light.

  That is, when the undifferentiated cell colony is irradiated with parallel light, the angular distribution of the light intensity of the scattered light has a peak at the light intensity of the scattered light having spread angles θx and θy = 0 ° as shown in FIG. The distribution is such that the light intensity gradually decreases as the spread angles θx and θy increase. In contrast, as shown in FIG. 10, the angular distribution of the light intensity of the scattered light when the differentiated cell colony is irradiated with parallel light has a peak light intensity of the scattered light with spread angles θx and θy = 0 °. However, most of the light intensity is distributed in a range where the spread angles θx and θy are small.

  Therefore, the angle distribution of the light intensity of the scattered light when the cell colony is irradiated with parallel light is acquired, and the angle distribution is the angle distribution of the undifferentiated cell colony and the angle of the cell colony of the differentiated cell shown in FIG. By analyzing which of the distributions is close, it is possible to evaluate whether the cell colony is in an undifferentiated state or a differentiated state. As an evaluation method, a correlation function of the angle distribution may be obtained, or a half value width of the angle distribution as shown in FIG. 10 is obtained, and when the half value width is equal to or greater than a threshold value, the undifferentiated state is obtained. It may be evaluated that there is a differentiation state when it is less than a threshold value.

  In addition, as shown in FIG. 11, when the undifferentiated cell colony is irradiated with convergent light, the scattered light generated in each cell repeats reflection within the cell colony as described above. Compared with the case of irradiating with convergent light, the scattered light spreads uniformly over a wide range. On the other hand, when the converged light is irradiated to the differentiated cell colony, the scattered light generated in the cell colony as described above is only in a narrow range compared to the case where the converged light is irradiated to the undifferentiated cell colony. It becomes scattered light that does not spread.

  That is, the spatial distribution of the light intensity of the scattered light when the undifferentiated cell colony is irradiated with convergent light, as shown in FIG. 12, is the light of the scattered light with X = Y = 0 (near the center of the detection image). With the intensity as a peak, the light intensity gradually decreases with the spatial spread. On the other hand, the spatial distribution of the light intensity of the scattered light when the differentiated cell colony is irradiated with the convergent light, as shown in FIG. 12, is X = Y = 0 (near the center of the detected image). Although the light intensity has a peak, most of the light intensity is distributed in a narrow range.

  Therefore, the spatial distribution of the light intensity of the scattered light when the convergent light is irradiated to the cell colony is obtained, and the spatial distribution is the spatial distribution of the undifferentiated cell colony and the space of the differentiated cell colony shown in FIG. By analyzing which of the distributions is close, it is possible to evaluate whether the cell colony is in an undifferentiated state or a differentiated state. As an evaluation method, a correlation function of the spatial distribution may be obtained, or a half-value width of the spatial distribution as shown in FIG. 12 is acquired, and in the undifferentiated state when the half-value width is equal to or greater than a threshold value. It may be evaluated that there is a differentiation state when it is less than a threshold value.

  Next, another modification of the microscope system 1 of the above embodiment will be described. FIG. 13 is a diagram illustrating a schematic configuration of a microscope system 2 according to another modification. In the microscope system 1 of the above embodiment, the scattered light of the cell colonies that are planarly cultured in the culture dish 15 is detected. However, the microscope system 2 shown in FIG. The scattered light of the cell colony is detected. In addition, the illumination light irradiation unit 10, the image detection unit 20, the scattered light detection unit 40, the illumination optical system 50, and the wavelength separation optical system 60 in the microscope system 2 illustrated in FIG. 13 are the same as the microscope system of the above embodiment. The detection light irradiation unit 30 irradiates convergent light as detection light.

  As shown in FIG. 13, the microscope system 2 includes a culture tank 90 in which cell colonies are suspended and cultured, a transparent tube 91 from which cell colonies flow together with the culture solution, and a state of cell colonies flowing in the transparent tube 91. Based on the evaluation results, the cell colony is sorted into the high-quality cell tank side flow path 93 or the waste tank side flow path 95 and passed, and the high-quality cell tank side flow path 93 sorted by the sorting section 92 is selected. A high-quality cell tank 94 in which high-quality cell colonies that have been flown into the reservoir are stored, and a waste tank 96 in which cell colonies that have been sorted by the sorting unit 92 and have been flowed into the waste tank-side flow path 95 are stored.

  The transparent tube 91 is formed of a material that transmits the illumination light L1 emitted from the illumination light irradiation unit 10 and the detection light L2 emitted from the detection light irradiation unit 30.

  Then, as shown in FIG. 13, the ring-shaped illumination light L <b> 1 and the convergent light L <b> 2 as the detection light are simultaneously irradiated to the cell colony that has flowed from the culture tank 90 to the transparent tube 91. As in the above embodiment, a phase difference image of the cell colony is captured by the image detection unit 20 by irradiation of the ring-shaped illumination light L1 on the cell colony, and is generated in the cell colony by irradiation of the convergent light L2 on the cell colony. The scattered light thus detected is detected by the photodetector 42 of the scattered light detector 40.

  The phase difference image captured by the image detection unit 20 and the intensity of the scattered light detected by the scattered light detection unit 40 are output to the cell evaluation apparatus 100 shown in FIG.

  As shown in FIG. 13, the cell evaluation apparatus 100 includes an image acquisition unit 101, a cell colony region specifying unit 102, a scattered light information acquisition unit 103, a cell evaluation unit 104, and a control unit 105. The control unit 105 includes a selection control unit 106. The image acquisition unit 101 and the cell colony region specifying unit 102 are the same as the image acquisition unit 71 and the cell colony region specifying unit 72 of the above-described embodiment, and the scattered light information acquisition unit 103 of this modification is an intensity signal of scattered light. The spatial distribution (XY axis) is acquired and stored.

  The cell evaluation unit 104 evaluates a cell colony based on the spatial distribution of the intensity signal of the scattered light read from the scattered light information acquisition unit 103. At this time, the cell evaluation unit 104 specifies the cell colony. The spatial distribution of the intensity signal of the scattered light is analyzed using the information of the obtained cell colony region.

  Specifically, for example, as shown in the right diagram of FIG. 14, only cell colonies appear in the phase contrast image, but as shown in the left diagram of FIG. In addition to the scattered light intensity distribution, there may be included the scattered light intensity distribution of floating substances such as dust and dead cells other than cell colonies. In such a case, if the analysis is performed over the entire detected image, the intensity distribution of the scattered light of the suspended matter may be a noise component and accurate evaluation may not be performed.

  Therefore, in this modification, the cell colony region specified in the phase contrast image is associated with the scattered light detection image, thereby detecting the detection image of the cell colony region and the region other than the cell colony region from the detection image of the scattered light. The size of each cell (particle) in each cell colony is estimated from the scattering theory as described above using only the detection image of the cell colony region, and a histogram relating to the cell size for each cell colony. Is calculated. Then, in the same manner as described above, whether each cell colony is in a differentiated state or an undifferentiated state is evaluated from the histogram relating to the cell size.

  In the above description, only the detection image of the cell colony region is analyzed and the histogram is calculated. At this time, the noise component is calculated based on the detection image of the region other than the cell colony region, and the noise component is calculated. Alternatively, the histogram may be calculated by analyzing the result of subtracting from the detection image of the cell colony region.

  Specifically, for example, a floating image area such as dead cells or dust is extracted based on the size, brightness value, or shape of a predetermined pattern included in the phase difference image, and a detection image corresponding to the floating image area. Alternatively, the spatial distribution or angular distribution of the region may be acquired as a noise component, and the result of subtracting the noise component from the spatial distribution or angular distribution of the detection image of the cell colony region may be analyzed to calculate a histogram.

  The control unit 105 controls the entire cell evaluation apparatus 100. The control unit 105 causes the display 80 to display the phase difference image detected by the image detection unit 20 and the evaluation result in the cell evaluation unit 101. Further, the selection control unit 106 in the control unit 105 outputs a control signal to the above-described selection unit 92 based on the evaluation result in the cell evaluation unit 104, and the selection unit 92 selects the cell colony to be evaluated as a good quality cell tank side flow path. 93 or the waste tank side flow path 95 is controlled. The selection unit 92 has, for example, a valve mechanism, and moves the valve in the direction of the arrow A shown in FIG. However, the configuration for selecting cell colonies is not limited to this configuration, and other known configurations can be used.

  More specifically, when the cell evaluation unit 104 evaluates that the cell colony is in an undifferentiated state, the selection control unit 106 selects the cell colony so that it flows into the good quality cell tank side channel 93. When the cell evaluation unit 104 evaluates that the cell colony is in a differentiated state, the selection unit 92 is controlled so that the cell colony flows into the waste tank side flow path 95.

  By controlling the selection unit 92 based on the evaluation result of the cell colony as described above, it is possible to appropriately select the undifferentiated cell colony and the differentiated cell colony, and only the undifferentiated cell colony Can be taken out. In the above description, the cell evaluation unit 104 evaluates whether the cell colony is in an undifferentiated state or a differentiated state. However, the present invention is not limited to this. For example, when culturing differentiation-induced cell colonies. Evaluates the degree of differentiation of cell colonies, stores only good quality cell colonies in which differentiation has progressed appropriately in the quality cell tank 94, and discards cell colonies in which differentiation has not progressed properly or abnormal cell colonies 96 may be stored.

  Moreover, in the said modification, although convergent light was irradiated as detection light, you may make it irradiate a parallel colony to a cell colony.

  Moreover, in the said embodiment and modification, it irradiated the ring-shaped illumination light to the cell colony, and picked up the phase-contrast image of the cell colony, but it is not restricted to this, Uniform illumination light with respect to a cell colony The bright field image of the cell colony may be imaged by illuminating the cell colony, or the differential interference image of the cell colony may be imaged by irradiating the cell colony with illumination light for differential interference measurement. Also good.

  Moreover, in the said embodiment and modification, you may make it determine the conditions at the time of detecting scattered light based on the phase difference image of a cell colony, a bright field image, and a differential interference image. For example, the irradiation range of the detection light when detecting scattered light, the wavelength of the detection light, and the like may be determined based on the uniformity of brightness, density, color, or the like such as a phase contrast image of a cell colony. More specifically, in order to generate more scattered light when the detection light irradiation range is narrowed down to a high uniformity range within the cell colony or when the overall uniformity is high such as a phase contrast image The wavelength of the detection light may be shortened.

DESCRIPTION OF SYMBOLS 1, 2 Microscope system 10 Illumination light irradiation part 11 White light source 12 Slit plate 13 Objective lens 15 Culture dish 16 Stage 20 Image detection part 21 Phase difference lens 22 Objective lens 23 Phase plate 24 Imaging lens 25 Imaging element 30 Detection light irradiation part 40 Scattered Light Detection Unit 41 Imaging Lens 42 Photodetector 50 Illumination Optical System 60 Wavelength Separation Optical System 70 Cell Evaluation Device 71 Image Acquisition Unit 72 Cell Colony Region Identification Unit 73 Irradiation Range Determination Unit 74 Scattered Light Information Acquisition Unit 75 Cell Evaluation Unit 76 Control unit 77 Irradiation range control unit 80 Display 90 Culture tank 91 Micro channel 92 Sorting unit 93 Culture tank side channel 94 Waste tank side channel 95 Waste tank 100 Cell evaluation device 101 Image acquisition unit 102 Cell colony region specifying unit 103 scattered light information acquisition unit 104 cell evaluation unit 105 control unit 106 sorting system Part

Claims (13)

An illumination light irradiation unit that emits illumination light for capturing an image of the cell group on the cell group;
An image detector that detects transmitted light transmitted through the cell group by irradiation of the illumination light to the cell group, and detects an image of the cell group;
A cell group region specifying unit for specifying a region of the cell group in the image of the cell group detected by the image detection unit;
A detection light irradiation unit for irradiating the cell group with detection light for detecting scattered light in the cell group;
A scattered light detection unit for detecting scattered light generated in the cell group by irradiation of the detection light to the cell group;
A cell evaluation unit that evaluates the cell group based on a spatial spread or temporal fluctuation of the intensity of the scattered light detected by the scattered light detection unit ;
An irradiation range determining unit that determines an irradiation range of the detection light based on the region of the cell group specified by the cell group region specifying unit;
The cell evaluation apparatus in which the detection light irradiation unit irradiates the detection light to the irradiation range determined by the irradiation range determination unit . A wavelength separation optical system is provided that separates the transmitted light and the scattered light, guides the transmitted light to the image detection unit, and guides the scattered light to the scattered light detection unit. claim 1 Symbol placement cell evaluation device. Reflects one of the illumination light and the detection light is irradiated to the cell group, cells evaluation apparatus according to claim 1 or 2 Claims comprising a passes through the other illumination optical system for irradiating the cell group . The illumination light irradiation unit irradiates the cell group with illumination light for phase difference measurement,
The image detection unit, the cell evaluation apparatus of claims 1 detects a phase difference image 3 set forth in any one said group of cells. The image detection unit, the cell evaluation apparatus of a cell group detects the bright-field image is claims 1 to 3 any one of claims. The illumination light irradiation unit irradiates the cell group with illumination light for differential interference measurement,
The image detection unit, the cell evaluation device cell groups differential interference image according any of the preceding claims 1 to detects a. The detection light irradiation unit irradiates the detection light to a non-cell group region where the cell group does not exist,
The scattered light detection unit detects scattered light generated in a non-cell group region where the cell group does not exist as a noise component,
The cell evaluation unit, the spatial 6 claim 1 is spread or by removing the noise component from the temporal fluctuations evaluates the cell group of the intensity of scattered light generated in the cell group The cell evaluation apparatus according to 1. The detection light is laser light;
The cell evaluation apparatus according to any one of claims 1 to 7 , wherein the cell evaluation unit evaluates the cell group based on temporal fluctuation of the intensity of the scattered light. The detection light is parallel light;
The cell evaluation apparatus according to any one of claims 1 to 7 , wherein the cell evaluation unit acquires an angular distribution of the scattered light and evaluates the cell group based on the angular distribution. The detection light is convergent light;
The cell evaluation unit, wherein the spatial distribution of the scattered light acquired cell evaluation device intended to evaluate a claimed any one of claims 1 to 7 the cell group based on the spatial distribution. The cell evaluation apparatus according to any one of claims 1 to 10 , wherein a condition for detecting the scattered light is determined based on an image of the cell group detected by the image detection unit. Illuminating the cell group with illumination light for capturing an image of the cell group,
Detecting transmitted light transmitted through the cell group by irradiation of the illumination light to detect an image of the cell group;
Identifying the region of the cell group in the detected image of the cell group;
Determining an irradiation range of detection light for detecting scattered light in the cell group based on the identified region of the cell group;
Irradiating the determined irradiation range with the detection light,
Detecting scattered light generated in the cell group by irradiation of the detection light;
Spatial extent or cells evaluated how to evaluate the cell group based on temporal fluctuations of the intensity of the scattered light the detected. An image acquisition unit for acquiring an image of the cell group in which transmitted light that has passed through the cell group by irradiation of illumination light to the cell group is detected;
A cell group region specifying unit for specifying a region of the cell group in the image of the cell group;
An irradiation range determining unit that determines an irradiation range of detection light for detecting scattered light in the cell group based on the region of the cell group specified by the cell group region specifying unit;
A scattered light information acquisition unit for acquiring information on the intensity of scattered light generated in the cell group by irradiation of detection light to the cell group in the irradiation range determined by the irradiation range determination unit ;
A cell evaluation program that functions as a cell evaluation unit that evaluates the cell group based on a spatial spread or temporal fluctuation of the intensity of the scattered light acquired by the scattered light information acquisition unit.