JPWO2017195794A1 - Cell observation device and program - Google Patents

Abstract

The present invention provides a cell observation device capable of quantitatively evaluating the state of cultured cells. A cell observation device includes a first imaging process (S11) for acquiring a first photograph of the culture medium according to the first imaging condition, and a second imaging process for acquiring the second photograph of the culture medium according to the second imaging condition. A photographing process (S12), a first identification process (S14, S15) for identifying a cell area presumed to be an area in which the cultured cells are present in the first picture, a position of cell nucleus of the cultured cells in the second picture and Of the second identification process (S17 to S20) for identifying the estimated cell location and the cell location identified in the second identification process, the number of cell locations located in the cell area identified in the first identification process Are counted as the number of cells of cultured cells (S21). [Selected figure] Figure 2

Description

  The present invention relates to a cell observation device for observing the state of cells in a culture medium.

  Conventionally, cell culture has been performed for the treatment of diseases or for the production and evaluation of medicines. Culturing of cells is carried out in an incubator placed in a sterile room, with temperature and humidity kept constant. In addition, a worker performing cell culture needs to periodically observe the state of cells in culture for the purpose of, for example, determining the passage timing.

  Here, if it is necessary to remove the petri dish from the incubator every time the state of the cultured cells is observed, the cultured cells may be damaged. Therefore, for example, in Patent Document 1, a photographing means provided in the incubator for photographing the cultured cells, and a display means provided on the outer surface of the incubator for displaying the image of the cultured cells photographed by the photographing means And an incubator comprising the This allows observation without removing cultured cells from the incubator.

JP 2005-65567 A

  The worker who observes the cultured cells determines the passage timing, for example, by determining the number of cultured cells in the petri dish, the area occupied by the cultured cells in the petri dish, and the like. However, in the incubator described in Patent Document 1, since it is necessary to determine the state of the cultured cells from the image captured by the imaging means, there is a problem that the determination requires skill.

  The present invention has been made in view of the above-described circumstances, and an object thereof is to provide a cell observation device capable of quantitatively evaluating the state of cultured cells.

  (1) A cell observation device according to an aspect of the present invention includes a photographing unit that takes a picture, and a control unit. The control unit causes the imaging unit to capture a culture medium containing cultured cells according to a first imaging condition, thereby performing a first imaging process for acquiring a first photograph, and a second imaging process in which the culture medium is different from the first imaging condition A second photographing process for acquiring a second photograph by causing the photographing unit to photograph according to the condition, and a first specifying process for specifying a cell area which is presumed to be an area in which the cultured cell is present in the first picture And a second specifying process for specifying a cell position estimated to be a position of a cell nucleus of the cultured cell in the second photograph, and the first specifying process among the cell positions specified in the second specifying process. The number of the cell positions located in the cell area identified by the treatment is counted as the number of cells of the cultured cells.

  As described above, by counting the number of cultured cells, the state of the cultured cells can be quantitatively evaluated. As a result, even a worker who is not skilled in the determination of the state of cultured cells can appropriately determine, for example, the timing of passage.

  (2) Preferably, in the first identification process, the control unit replaces a pixel value less than a first threshold value with a first value in the first photo data indicating the first photo, and the first threshold value. The generation process for generating binary image data in which the above pixel values are replaced with the second value, and the area of the pixel of the first value in the binary image represented by the binary image data is specified as the cell area To execute the area identification process.

  According to the above configuration, it is possible to distinguish the area in which the cultured cell is present from the area of the culture medium by the difference in pixel value (typically, the brightness value).

  (3) Preferably, in the area specifying process, the control unit specifies an area in which pixels of the first value continue a threshold number or more as the cell area.

  The generation process may assign the first value to the position of the noise included in the first photo. However, since noise is small compared to cells, the influence of noise can be reduced by making a region where pixels of the first value continue more than a threshold number as a cell region.

  (4) Preferably, in the second identification process, the control unit performs an extraction process of extracting a specific frequency component from second photo data indicating the second photo, and the second process in which the specific frequency component is extracted. When a pixel value of a photograph is scanned, a position specifying process is performed to specify, as the cell position, a pixel position at a boundary where a change in pixel value shifts from one of decrease and increase to the other.

  The component higher in frequency than the specific frequency component corresponds to, for example, an outline of a cell, noise due to thermal noise of a camera, or an image such as small dust. On the other hand, components lower in frequency than the specific frequency component correspond to, for example, images of the cytoplasm and the area of the culture medium. Therefore, by removing these from the second photographic data, an image of cell nuclei can be extracted, so that the number of cells can be counted accurately.

  (5) Preferably, in the extraction process, the control unit applies a conversion process for FFT-converting the second picture data, and a filter for causing the specific frequency component to pass through the second picture data after the conversion process. Filter processing and inverse conversion processing for performing FFT inverse conversion on the second photo data after the filter processing are performed.

  As in the above configuration, by using FFT (Fast Fourier Transform) transform, band pass filter, and FFT inverse transform, an image of cell nucleus can be extracted simply and accurately.

  (6) Preferably, the control unit executes a calculation process for calculating the area of the cell area identified in the first identification process.

  As described above, by calculating the area of the cell region, the state of the cultured cell can be further quantitatively evaluated. The area calculated by the calculation process may be used to evaluate the proportion of cultured cells in a petri dish, or may be used to evaluate the size of cultured cells by dividing the area by the number of cells. It is also good.

  (7) Preferably, the cell observation device further includes an output unit. The control unit executes an output process to output the number of cells counted in the counting process to the output unit.

  Although the specific example of the output process is not particularly limited, for example, the number of cells may be displayed on the display unit, or the number of cells may be printed on a printer. Further, the output target is not limited to only the number of cells, and the area of the cell area calculated by the calculation processing may be further output.

  (8) As one example, the cell observation device includes an irradiation unit that irradiates the culture medium with light. The first photographing condition indicates that the first photograph is photographed with the light of the first wavelength output from the culture medium among the light irradiated by the irradiation unit. The second photographing condition indicates that the second photograph is photographed with the light of the second wavelength longer than the first wavelength outputted from the culture medium.

  (9) For example, the first wavelength is 400 nm or more and 700 nm or less. The second wavelength is 700 nm or more and is longer than the first wavelength.

  (10) As another example, the imaging unit includes an adjustment unit that adjusts the position of the focus. The first photographing condition indicates that the first photograph is photographed in a state in which the outline of the cultured cell is focused. The second photographing condition is photographing the third photograph in a state in which the second photographing condition is focused on a position closer to the photographing unit than the first photographing condition, and is focused on a position distant from the photographing unit than the first photographing condition. It shows taking the fourth picture in the combined state. Then, in the second photographing process, the control unit acquires a difference between pixel values of the third and fourth photographs as the second photograph.

  (11) A cell observation device according to another aspect of the present invention includes an imaging unit that takes a picture, and a control unit. The control unit causes the imaging unit to capture a culture medium containing cultured cells according to a first imaging condition, thereby performing a first imaging process for acquiring a first photograph, and a second imaging process in which the culture medium is different from the first imaging condition A second photographing process for acquiring a second photograph by causing the photographing unit to photograph according to the condition, and a first specifying process for specifying a cell area which is presumed to be an area in which the cultured cell is present in the first picture And a calculation process for calculating the area of the cell area identified in the first identification process.

  (12) The program according to the present invention is executable by a computer. The program acquires a first picture of a culture medium containing cultured cells according to a first photographing condition and a photographic data showing a second picture of the culture medium according to a second photographing condition different from the first photographing condition In the first picture, a first identification process for identifying the cell area presumed to be the area in which the cultured cell is present, and in the second picture, the position of the cell nucleus of the cultured cell is presumed The number of cells in the cultured cell or the number of cells in the cultured cell is determined based on at least one of a second identification process for identifying a cell position, the cell area identified in the first identification process, and the cell position identified in the second identification process. The computer is caused to execute calculation processing for calculating the area of the cell area.

  According to the present invention, by counting the number of cultured cells, the state of the cultured cells can be quantitatively evaluated.

FIG. 1 is a functional block diagram of a cell observation device 10 according to the present embodiment. FIG. 2 is a flowchart of the cell observation process according to the present embodiment. FIG. 3 is a view showing an example of the first and second photographs taken in steps S11 and S12. FIG. 4 is an example of the binary image generated in step S14. FIG. 5 is an example of the binary image from which the noise 34 has been removed in step S15. FIG. 6 is an example of a second image in which the cell nucleus 31 and the noise 34 are extracted in steps S17 to S19. FIG. 7 is a diagram schematically showing changes in luminance values of pixels arranged in a line in the second photograph after FFT conversion. FIG. 8 is an example of the second image in which the plot 50 is set in step S20. FIG. 9 is an example of an image in which the plot 50 is superimposed on the binary image shown in FIG.

  Hereinafter, preferred embodiments of the present invention will be described. It is needless to say that the present embodiment is only one embodiment of the present invention, and the embodiment can be modified without departing from the scope of the present invention.

  As shown in FIG. 1, the cell observation device 10 according to the present embodiment includes the irradiation unit 11, the first polarizing plate 12A, and the second polarizing plate 12B (these are collectively referred to as "polarizing plate 12". And the imaging unit 13, the control unit 14, and the display unit 15 are mainly included. In addition, an optical system such as a collimator lens or an imaging lens (not shown) is appropriately disposed on an optical path from the irradiation unit 11 to the photographing unit 13 via the polarizing plate 12. In FIG. 1, broken arrows indicate the traveling direction of light, and solid arrows indicate the traveling direction of the signal.

  The irradiation unit 11 outputs white light including at least the first wavelength and the light of the second wavelength longer than the first wavelength. Although the specific example of the irradiation part 11 is not specifically limited, For example, LED (Light Emitting Diode) etc. can be used. The first wavelength is selected, for example, from 400 nm to 700 nm. Preferably, the first wavelength is selected from 500 nm to 550 nm. The second wavelength is, for example, 700 nm or more and a wavelength longer than the first wavelength is selected. Preferably, the second wavelength is selected from 700 nm to 900 nm.

  The first polarizing plate 12A transmits only the light of the first wavelength in the white light emitted from the irradiation unit 11, and blocks the light of the other wavelengths. The second polarizing plate 12B transmits only the light of the second wavelength in the white light emitted from the irradiation unit 11, and blocks the light of the other wavelengths. Although the specific structure of the polarizing plate 12 is not particularly limited, for example, a general polarizing film in which iodine compound molecules are adsorbed and oriented to polyvinyl alcohol as a main component can be used.

  The photographing unit 13 photographs a picture with light emitted from the irradiation unit 11 and having passed through the polarizing plate 12. More specifically, the photographing unit 13 receives light that has passed through the polarizing plate 12 and generates photographic data by photoelectric conversion. Then, the photographing unit 13 outputs the generated photograph data to the control unit 14. Although the specific example of the imaging unit 13 is not particularly limited, for example, the imaging unit 13 is a camera including an imaging device such as a CCD (Charge Coupled Device) and a lens unit (an example of an adjustment unit) that adjusts the position of the focus. Moreover, the imaging | photography part 13 which concerns on this embodiment shall be able to image | photograph a color photograph.

  The control unit 14 controls the operations of the irradiation unit 11, the polarizing plate 12, the imaging unit 13, and the display unit 15, and executes image processing on the photographic data acquired from the imaging unit 13. The operation of the control unit 14 will be described later with reference to FIG. The control unit 14 includes, for example, a storage unit that stores a program, and a CPU (Central Processing Unit) that reads and executes the program from the storage unit.

  The display unit 15 displays information in accordance with the control of the control unit 14. Although the specific configuration of the display unit 15 is not particularly limited, for example, a liquid crystal display (abbreviation of Liquid Crystal Display), an organic EL display (abbreviation of Organic Electro-Luminescence Display) or the like can be adopted. The display unit 15 is an example of an output unit.

[Cell observation processing]
The cell observation process will be described with reference to FIGS. The following processing may be realized by a program executed by the CPU, may be realized by a hardware circuit mounted in the control unit 14, or may be realized by combining these.

  First, on the light path from the irradiation unit 11 to the imaging unit 13 of the cell observation device 10, a petri dish 20 containing a culture medium 21 for culturing cells is disposed. Thus, part of the light emitted from the irradiation unit 11 passes through the culture medium 21 and reaches the imaging unit 13. The petri dish 20 may be set in the cell observation device 10 by a worker. Alternatively, the irradiation unit 11 and the imaging unit 13 of the cell observation device 10 incorporated in the incubator may be disposed opposite to each other across the mounting portion of the petri dish 20.

  The control unit 14 causes the irradiating unit 11 to emit white light, and causes the photographing unit 13 to photograph the first photo (S11). The first photograph is a photograph taken by the photographing unit 13 with the light of the first wavelength that has passed through the first polarizing plate 12A among the light transmitted through the culture medium 21. That is, in step S11, the first polarizing plate 12A is disposed on the optical path from the irradiation unit 11 to the imaging unit 13. Then, the control unit 14 acquires, from the photographing unit 13, first photograph data indicating the first photograph. Step S11 is an example of a first imaging process and an acquisition process. The wavelength of light to be incident on the imaging unit 13 in step S11 is an example of the first imaging condition.

  Next, the control unit 14 causes the irradiating unit 11 to emit white light, and causes the photographing unit 13 to take a second photograph (S12). The second photograph is a photograph taken by the photographing unit 13 with the light of the second wavelength that has passed through the second polarizing plate 12 </ b> B among the light transmitted through the culture medium 21. That is, in step S12, the second polarizing plate 12B is disposed on the optical path from the irradiation unit 11 to the imaging unit 13. Then, the control unit 14 acquires, from the photographing unit 13, second photograph data indicating the second photograph. Step S12 is an example of a second imaging process and an acquisition process. Further, the wavelength of light to be incident on the imaging unit 13 in step S12 is an example of a second imaging condition.

  FIG. 3 shows an example of the first and second photographs taken in steps S11 and S12. The first photograph and the second photograph have the same shooting conditions such as the field of view, the angle of view, and the magnification, and have different wavelengths of light reaching the shooting unit 13 at the time of shooting. The first and second photographs show the cells 30 cultured in the medium 21. The cell 30 is composed of a cell nucleus 31, a cytoplasm 32 surrounding the cell nucleus 31, and a cell wall 33 forming a contour of the cell 30. In addition, the noise 34 is included in the first and second photographs. Although the cause of the noise 34 is not particularly limited, for example, noise due to thermal noise of the imaging unit 13, noise due to small dust attached to a lens of the imaging unit 13 or the like may be considered.

  Next, the control unit 14 converts the first color photo data and the second color photo data (hereinafter, these may be collectively referred to as "photo data") into grayscale photo data. (S13). The photograph data generated in step S13 is not limited to grayscale as long as each pixel represents the magnitude of the luminance value. For example, it may be a luminance image in which photographic data is converted from the RGB color space to the YUV color space and only the luminance signal is extracted. In addition, in steps S11 and S12, when the photographing unit 13 generates gray scale photographic data, step S13 can be omitted.

  Next, the control unit 14 generates binary image data from the gray scale first photo data (S14). Specifically, the control unit 14 replaces the pixel value less than the first threshold value in the gray scale first photo data with the first value (for example, "0") 41, and the pixel value greater than or equal to the first threshold value. Is replaced by a second value (e.g., "1") 42 to generate binary image data. The process of step S14 is an example of a generation process.

  FIG. 4 is an example of a binary image represented by binary image data generated in step S14. In the binary image shown in FIG. 4, the pixel value corresponding to the position of the cell 30 and the noise 34 in the first picture is the first value (white area in FIG. 4) 41, and the pixel value corresponding to the position of the culture medium 21 Is the second value (black area in FIG. 4) 42. That is, in the binary image shown in FIG. 4, the culture medium 21 and the cells 30 are distinguished, and the cell nucleus 31, the cytoplasm 32, and the cell wall 33 are not distinguished.

  In the present embodiment, it is assumed that the pixel of the cell 30 and the noise 34 is smaller than the pixel of the culture medium 21. However, when the relationship of the pixel values is reversed, the control unit 14 replaces the pixel value of the first threshold or more with the first value and replaces the pixel value of the first threshold or less with the second value in step S14. Just do it.

  Next, the control unit 14 removes the noise 34 from the binary image data generated in step S14 (S15). Specifically, the control unit 14 changes the pixel value of the pixel from the first value 41 to the second value 42 in response to the fact that the consecutive first value pixels are less than the threshold number. In other words, the control unit 14 replaces the block of pixels of the first value 41 sufficiently smaller than the general size of the cell 30 with the pixels of the second value 42.

  FIG. 5 is an example of a binary image in which the second value 42 is set to the pixel corresponding to the noise 34. The binary image shown in FIG. 5 is different from the binary image shown in FIG. 4 in that the second value 42 is set to the pixel at the position of the noise 34. Then, the control unit 14 describes an area of the pixel in which the first value 41 is set in the binary image shown in FIG. 5 as an area where the cell 30 is presumed to exist (hereinafter, referred to as a “cell area”. Identify as.). The process of step S15 is an example of the area specifying process. Steps S14 and S15 are an example of the first identification process.

  Next, the control unit 14 calculates the area of the pixel of the first value 41 in the binary image shown in FIG. 5 (S16). Specifically, the control unit 14 counts the number of pixels of the first value 41 included in the binary image, and calculates the area of the pixel of the first value 41 by multiplying the number of pixels by the area of one pixel. Do. The area calculated in step S16 corresponds to the area of the cell area included in the first photograph. The process of step S16 is an example of a calculation process.

  Next, the control unit 14 performs FFT (Fast Fourier Transform) on the gray scale second photo data (S17). That is, the control unit 14 converts the second picture data of the space area into the second picture data of the frequency area. The process of step S17 is an example of the conversion process. In addition, the specific example of the process applied to 2nd photography data by step S17 is not limited to FFT, For example, a discrete cosine transform (Discrete Cosine Transform) etc. may be sufficient.

  Next, the control unit 14 applies a band pass filter to the FFT-transformed second photo data (S18). The band pass filter passes a specific frequency component of the frequency components of the second photographic data after conversion processing, and passes a frequency component higher than the specific frequency component (hereinafter referred to as "high frequency component") and a specific frequency component. It is a filter that blocks (attenuates) lower frequency components (hereinafter referred to as "low frequency components"). The process of step S18 is an example of the filtering process.

  The specific frequency component is, for example, a frequency in a predetermined range corresponding to the complexity of the image representing the cell nucleus 31. Also, the high frequency component corresponds, for example, to the complexity of the image representing the contour of the cell 30 (ie, the cell wall 33) and the noise 34 and the like. Furthermore, the low frequency component corresponds to, for example, the complexity of the image that represents the brightness unevenness and the like due to the bias of the light irradiated from the culture medium 21, the cytoplasm 32, and the irradiation unit 11.

  Next, the control unit 14 performs FFT inverse transform on the second photo data to which the band pass filter is applied (S19). That is, the control unit 14 converts the second picture data in the frequency domain into the second picture data in the spatial domain. However, when the second photograph data is discrete cosine transformed in step S17, the second photograph data is discrete cosine inverse transformed in step S19. The process of step S19 is an example of the inverse conversion process. The processes of steps S17 to S19 are an example of an extraction process of extracting a specific frequency component from the second picture data.

  FIG. 6 is an example of the second photograph indicated by the second photograph data after the extraction processing. The second picture shown in FIG. 6 corresponds to an image showing a part of the culture medium 21, the cytoplasm 32, the cell wall 33 and the noise 34 removed from the second picture shown in FIG. However, the second picture shown in FIG. 6 may still include part of the noise 34. Further, in the second photograph shown in FIG. 6, the pixels at the cell nucleus 31 and the noise 34 have pixel values smaller than those at the other positions.

  Next, the control unit 14 sets a plot 50 at the pixel position of the extremum pixel in the second photograph after the inverse FFT transform (S20). The extremum pixel refers to, for example, a pixel at the boundary where the change in luminance changes from one of decrease and increase to the other when the pixel of the second photograph is scanned in the left-right direction (or in the up-down direction). For example, as shown in FIG. 7, in the extremum pixel when the pixel at the position of cell nucleus 31 and noise 34 is smaller in pixel value than the pixels at other positions, the pixel at the boundary where the change in luminance value turns from decrease to increase ( That is, it refers to a minimal pixel) in which the pixel value is minimal. On the other hand, the extremum pixels where the pixel values at the cell nucleus 31 and the noise 34 position are larger than the pixels at the other positions are boundary pixels where the change in luminance value turns from increase to decrease (ie, the pixel value is maximum Point), which means that

  FIG. 7 is a diagram schematically showing changes in luminance values of pixels arranged in a line in the second photograph after FFT conversion. In step S20, the control unit 14 sets only pixels (solid line portions) whose luminance value is less than the second threshold among the pixels illustrated in FIG. 7 as scanning targets. Then, the control unit 14 sets the plot 50 at the pixel position of the minimal pixel A1 at which the luminance value of the continuous scanning target pixel row A is minimum. In addition, when the maximum pixel B1 exists in the continuous scan target pixel row B, the control unit 14 determines the pixel position of the minimum pixel B2 whose brightness value is minimum on one side in the scanning direction from the maximum pixel B1, and the maximum pixel B1. Further, the plot 50 is set at the pixel position of the minimal pixel B3 at which the luminance value is minimum on the other side in the scanning direction.

  FIG. 8 is an example of the second photograph in which the plot 50 is set. In the second picture shown in FIG. 8, a plot 50 is set at the position of the cell nucleus 31 and the noise 34 included in the second picture shown in FIG. Further, in FIG. 8, the cell nucleus 31 and the noise 34 are not shown. The process of step S20 is an example of the position specifying process. The process of steps S17 to S20 is an example of a second specifying process for specifying a position estimated to be the position of cell nucleus 31 (hereinafter referred to as "cell position"). That is, the position of the plot 50 corresponds to the cell position.

  Next, the control unit 14 superimposes the plot 50 shown in FIG. 8 on the first photo shown in FIG. 4 as shown in FIG. Then, the control unit 14 counts the number of plots 50 overlapping the area of the first value 41 in the image shown in FIG. 9 as the number of cells 30 (hereinafter referred to as “the number of cells”) (S21). ). That is, in the image shown in FIG. 9, the control unit 14 indicates that the plot 50 overlapping the area of the first value 41 indicates the position of the cell nucleus 31, and the plot 50 overlapping on the area of the second value 42 indicates the position of the noise 34. It is determined that Step S21 is an example of the counting process.

  Next, the control unit 14 causes the display unit 15 to display the area of the cell area calculated in step S16 and the number of cells counted in step S21 (S22). The control unit 14 may further cause the display unit 15 to display the average value of the area of the cells 30 obtained by dividing the area of the cell area by the number of cells. The process of step S22 is an example of the output process.

[Operation and effect of this embodiment]
As in the above embodiment, the area of the cell area is calculated and the number of cells of the cell 30 is counted by performing image processing on the first photograph and the second photograph taken using two light beams different in wavelength. Can. As a result, since the state of the cell 30 can be quantitatively evaluated without being a skilled worker, for example, it is possible to appropriately determine the passage timing and the doubling time of the culture medium. In addition, although said example demonstrated the example which performs both calculation of an area, and the count of a cell number, only any one may be performed. When the count of the number of cells is omitted, the processes of steps S17 to S21 are omitted.

  The determination of the pass-through timing and the doubling time may be performed by the control unit 14 instead of the worker. Generally, passage is performed when the number of cells reaches a predetermined number. Also, the doubling time is longer as the area of the cell is larger, and the doubling time tends to be shorter as the area of the cell is smaller. Therefore, by giving the control unit 14 in advance the number of cells when performing passaging, the relationship between the area of cells and the doubling time, etc. to the control unit 14, the control unit 14 can draw up a culture schedule. it can. Then, the prepared schedule may be displayed on the display unit 15 in step S22.

  Further, according to the above embodiment, the culture medium 21 and the cells 30 are distinguished by the difference in pixel value in the first photograph (S14), and the cells 30 and the noise 34 are determined according to the size of the pixel block of the first value 41. And (S15). Thereby, the cell area in the first photograph can be appropriately identified.

  Further, according to the above-described embodiment, the band pass filter extracts the specific frequency component (that is, the image indicating the cell nucleus 31) from the first photograph (S17 to S19), and the noise 34 is generated by overlapping with the binary image. It is removed (S21). Thereby, the cell position in the 2nd photograph can be specified appropriately.

  In addition, the cell observation process which concerns on said embodiment may be performed with respect to the some location on the Petri dish 20, respectively. More specifically, the control unit 14 may execute the processing of steps S11 to S21 on different imaging ranges on the petri dish 20. Then, in step S22, the control unit 14 displays the total value (or the average value) of the areas calculated in step S16 and the total value (or the average value) of the number of cells counted in step S21. It may be displayed on the part 15.

  Moreover, the specific structure of the cell observation apparatus 10 shown by FIG. 1 is not limited to the above-mentioned example. For example, the irradiation unit 11 may selectively output the monochromatic light of the first wavelength and the monochromatic light of the second wavelength according to the control of the control unit 14. In this case, the polarizer 12 can be omitted. Further, the light reaching the imaging unit 13 from the irradiation unit 11 via the culture medium 21 is not limited to transmitting through the culture medium 21, and may be reflected by the culture medium 21. Further, the specific example of the output unit is not limited to the display unit 15, and may be a printer or the like that records information on a sheet.

  Moreover, the imaging conditions in steps S11 and S12 are not limited to the difference in the wavelength of light to be incident on the imaging unit 13, but may be different in the position of the focal point. The position of the focus is changed by operating the lens unit. The lens unit may be operated manually by the operator of the cell observation device 10 or may be operated by an actuator (not shown) controlled by the control unit 14. As still another example, in steps S11 and S12, imaging conditions may be adopted in which both the wavelength of light and the position of the focal point are different. The processes after step S13 are the same as those in the above-described example.

  More specifically, in step S11, the control unit 14 may cause the imaging unit 13 to capture the first photograph in a state in which the outline of the cell 30 is focused. The position of the focus at this time (hereinafter referred to as “first focus position”) is an example of the first imaging condition. The first picture in this case is a picture in which the outline of the cell 30 is clearly seen.

  On the other hand, in step S12, the control unit 14 causes the photographing unit 13 to execute the third process while focusing on a position closer to the photographing unit 13 than the first focus position (hereinafter referred to as "second focus position"). The fourth photographing is performed by the photographing unit 13 in a state in which a photograph is taken and focused on a position (hereinafter, referred to as “third focal position”) distant from the photographing unit 13 from the first focal position. May be The position of the focus at this time is an example of the second imaging condition. That is, the first focal position is located between the second focal position and the third focal position. The distance between the first focal position and the second focal position, and the distance between the first focal position and the third focal position are, for example, 10 μm to 30 μm.

  When the third and fourth photographs are compared, the contrast of the position of the cell nucleus 31 is relatively high, and the contrast of the position different from the cell nucleus 31 is relatively low. Therefore, in step S12, the control unit 14 may acquire the difference between the pixel values of the third and fourth photographs as the second photograph. More specifically, the control unit 14 sets the difference between the pixel values of the same position (for example, the left end) of the third and fourth photographs as the pixel value of the corresponding position (for example, the left end) in the second photograph. Good. The second picture in this case is a picture in which the cell nucleus 31 is clearly seen.

  In addition, the cell observation device 10 according to the above embodiment is not limited to one in which all the components shown in FIG. 1 are integrated. In the cell observation device 10, for example, an imaging device including the irradiation unit 11, the polarizing plate 12, and the imaging unit 13 and an image processing device including the control unit 14 and the display unit 15 may be connected through a communication network. The image processing apparatus in this case may be, for example, a general PC (Personal Computer).

  Furthermore, the execution order of each step of the cell observation process is not limited to the example of FIG. For example, the process of step S12 may be performed between steps S16 and S17. However, steps S11, S13, S14, S15, and S16 are performed in this order, and steps S12, S13, S17, S18, S19, S20, and S21 are performed in this order.

DESCRIPTION OF SYMBOLS 10 ... Cell observation apparatus 11 ... Irradiation part 12A, 12B ... Polarizing plate 13 ... Photography part 14 ... Control part 15 ... Display part

Claims (12)

A shooting unit that takes a picture,
And a control unit,
The control unit
A first imaging process for acquiring a first picture by causing the imaging unit to capture a culture medium containing cultured cells according to a first imaging condition;
Second imaging processing for acquiring a second photograph by causing the imaging unit to capture the culture medium according to a second imaging condition different from the first imaging condition;
A first identification process for identifying a cell area presumed to be an area in which the cultured cell is present in the first picture;
A second identification process for identifying the position of the cell nucleus of the cultured cell and the estimated cell position in the second photograph;
And counting the number of cell positions located in the cell area specified in the first specific process among the cell positions specified in the second specific process as the cell number of the cultured cells , To perform cell observation equipment. The control unit is configured to:
In the first photograph data indicating the first photograph, pixel values less than the first threshold are replaced with the first value, and pixel values equal to or more than the first threshold are replaced with the second value to generate binary image data Generation process,
The cell observation device according to claim 1, wherein an area identification process of identifying an area of a pixel of the first value as the cell area in the binary image represented by the binary image data is executed.   The cell observation device according to claim 2, wherein the control unit specifies, as the cell region, a region in which pixels of the first value continue a threshold number or more in the region specifying process. The control unit is configured to:
An extraction process for extracting a specific frequency component from second picture data representing the second picture;
A position specifying process of specifying, as the cell position, a pixel position at a boundary where a change in pixel value changes from one of decrease and increase to the other when scanning the pixel value of the second photo from which the specific frequency component is extracted; The cell observation device according to any one of claims 1 to 3, wherein In the extraction process, the control unit
Conversion processing for FFT converting the second picture data;
A filter process for applying a filter that passes the specific frequency component to the second photo data after the conversion process;
The cell observation device according to claim 4, wherein an inverse conversion process is performed to perform an FFT inverse conversion on the second photographic data after the filtering process.   The cell observation device according to any one of claims 1 to 5, wherein the control unit executes a calculation process for calculating an area of the cell area identified in the first identification process. It further comprises an output unit,
The cell observation device according to any one of claims 1 to 6, wherein the control unit executes an output process to output the number of cells counted in the counting process to the output unit. The cell observation apparatus includes an irradiation unit that irradiates the culture medium with light,
The first photographing condition indicates that the first photograph is photographed with the light of the first wavelength output from the culture medium among the light irradiated by the irradiation unit,
The cell observation device according to any one of claims 1 to 7, wherein the second photographing condition indicates that the second photograph is photographed with the light of the second wavelength longer than the first wavelength outputted from the culture medium. The first wavelength is 400 nm or more and 700 nm or less,
The cell observation device according to claim 8, wherein the second wavelength is 700 nm or more and is longer than the first wavelength. The imaging unit includes an adjustment unit that adjusts the position of the focal point,
The first photographing condition indicates that the first picture is photographed while focusing on the contour of the cultured cell,
The second photographing condition is photographing the third photograph in a state in which the second photographing condition is focused on a position closer to the photographing unit than the first photographing condition, and is focused on a position distant from the photographing unit than the first photographing condition. Indicates that the fourth photo is taken in the combined state,
The cell observation device according to any one of claims 1 to 9, wherein the control unit acquires a difference between pixel values of the third photograph and the fourth photograph in the second photographing process as the second photograph. A shooting unit that takes a picture,
And a control unit,
The control unit
A first imaging process for acquiring a first picture by causing the imaging unit to capture a culture medium containing cultured cells according to a first imaging condition;
Second imaging processing for acquiring a second photograph by causing the imaging unit to capture the culture medium according to a second imaging condition different from the first imaging condition;
A first identification process for identifying a cell area presumed to be an area in which the cultured cell is present in the first picture;
A cell observation device that executes a calculation process of calculating an area of the cell area identified in the first identification process; A computer executable program,
An acquisition process for acquiring photographic data indicating a first photograph of the culture medium containing cultured cells according to a first imaging condition, and a second photograph of the culture medium according to a second imaging condition different from the first imaging condition;
A first identification process for identifying a cell area presumed to be an area in which the cultured cell is present in the first picture;
A second identification process for identifying the position of the cell nucleus of the cultured cell and the estimated cell position in the second photograph;
Calculating the number of cells of the cultured cell or the area of the cell area based on at least one of the cell area specified in the first specific process and the cell position specified in the second specific process; , A program that causes the above computer to execute.

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