WO2017221616A1 - 培養細胞の撮像方法及び装置 - Google Patents

培養細胞の撮像方法及び装置 Download PDF

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WO2017221616A1
WO2017221616A1 PCT/JP2017/019233 JP2017019233W WO2017221616A1 WO 2017221616 A1 WO2017221616 A1 WO 2017221616A1 JP 2017019233 W JP2017019233 W JP 2017019233W WO 2017221616 A1 WO2017221616 A1 WO 2017221616A1
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observation image
imaging
brightness
image
focal position
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PCT/JP2017/019233
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English (en)
French (fr)
Japanese (ja)
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正広 村井
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東洋製罐グループホールディングス株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements

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  • the present invention relates to an imaging method and apparatus for cultured cells, and more particularly to an imaging method and apparatus capable of optimizing imaging conditions for cultured cells.
  • time-lapse imaging in which cells in culture are imaged every predetermined time may be performed.
  • Image processing is performed on the captured observation image in order to extract various information about the cultured cells.
  • the observation image needs to have an image brightness and focus suitable for image processing.
  • Patent Document 1 describes an imaging condition for adjusting the brightness of an observation image and a technique for imaging by adjusting a focal position.
  • luminance control S64 in FIG. 13
  • autofocus control S65 in FIG. 13
  • LED control LED control or the like is performed so that the average luminance of the observation image falls within a predetermined range set in advance (FIG. 14).
  • the observed image may not be suitable for image processing even if the average luminance of the observed image is within a predetermined range.
  • the turbidity and color tone of the culture solution also change because the cell concentration and the pH of the culture solution change.
  • the observed image may not be suitable for image processing even if the average luminance of the observed image is within a predetermined range.
  • the focal position of the observation image changes.
  • imaging is performed with the cultured cells settled in the culture solution, but it is not necessarily located on the same plane in the culture solution, and it may be difficult to focus on all the cultured cells at the same time. Can occur.
  • the present invention has been made in view of the above circumstances, and an object thereof is to provide a culture cell imaging method and apparatus capable of optimizing the culture cell imaging conditions.
  • the first cultured cell imaging method of the present invention is an imaging method for imaging an observation image of a cultured cell in a culture vessel, and sets imaging conditions for adjusting the brightness of the observation image
  • Imaging the cultured cells calculating the average luminance and average saturation of the observation image in the HLS color space for each of the captured observation images, and calculating the average luminance and the average saturation for each observation image.
  • a step of setting an imaging condition for adjusting brightness when an observation image having the maximum value is taken as an optimum value.
  • the second cultured cell imaging method of the present invention is an imaging method for imaging an observation image of a cultured cell in a culture container, and includes a focal position setting step for setting a focal position of the observation image, and an observation image.
  • An image acquisition step of acquiring, and the focus position setting step includes: a step of imaging the cultured cells by changing the focus position within a predetermined change range with a predetermined step size; and observing each of the captured observation images.
  • the method includes a step of measuring the number of particles in the image and a step of setting the focal position when an observation image having the maximum number of particles is captured as an optimum value.
  • the third cultured cell imaging method of the present invention is an imaging method for capturing an observation image of a cultured cell in a culture vessel, and a brightness setting step for setting an imaging condition for adjusting the brightness of the observation image And a focus position setting step for setting the focus position of the observation image, and an image acquisition step for acquiring the observation image, wherein the brightness setting step changes the imaging condition for adjusting the brightness of the observation image with a predetermined change.
  • the step of imaging the cultured cells with a predetermined step size within the range, the step of calculating the average luminance and the average saturation of the observation image in the HLS color space for each of the captured observation images, and the observation image A step of calculating an index based on the average luminance and the average saturation, and a step of setting an imaging condition for adjusting brightness when an observation image having the maximum value of the index is captured as an optimum value.
  • the focal position setting step A step of imaging the cultured cells by changing the focal position by a predetermined step within a predetermined change range, a step of measuring the number of particles in the observation image for each of the captured observation images, and the maximum number of particles And a step of setting, as an optimum value, a focal position when an observation image that is a value is captured.
  • the first cultured cell imaging apparatus of the present invention is an imaging apparatus that captures an observation image of a cultured cell in a culture container, and illuminates the cultured cell and images the cultured cell.
  • An imaging unit that generates an observation image; and a brightness control unit that sets an imaging condition for adjusting the brightness of the observation image.
  • the brightness control unit sets an imaging condition for adjusting the brightness of the observation image to a predetermined value.
  • the imaging unit is caused to capture the cultured cells with a predetermined step size within a change range, and the average luminance and average saturation of the observation image in the HLS color space are calculated for each observation image captured by the imaging unit.
  • an index is calculated based on the average luminance and the average saturation, and an imaging condition for adjusting brightness when the observation image having the maximum index is captured is set as an optimal value. It is characterized by that.
  • the second cultured cell imaging device of the present invention is an imaging device that captures an observation image of a cultured cell in a culture container, and illuminates the cultured cell and images the cultured cell.
  • An imaging unit that generates an observation image; and a focal position control unit that sets a focal position of the observation image.
  • the focal position control unit changes the focal position within a predetermined change range with a predetermined step size and performs the imaging.
  • Optimum focus position when the number of particles in the observed image is measured for each observation image captured by the imaging unit and the observation image with the maximum number of particles is captured. It is characterized by being set as a value.
  • the third cultured cell imaging device of the present invention is an imaging device that captures an observation image of a cultured cell in a culture container, and illuminates the cultured cell and images the cultured cell.
  • the imaging condition for adjusting the brightness of the observation image is changed within a predetermined change range by a predetermined step size, and the cultured cell is imaged by the imaging unit, and an HLS color is obtained for each observation image captured by the imaging unit.
  • the focal position control unit changes the focal position by a predetermined step size within a predetermined change range, causes the imaging unit to image the cultured cells, and for each observation image captured by the imaging unit, an observation image The number of particles therein is measured, and the focal position when an observation image having the maximum number of particles is captured is set as an optimum value.
  • the average luminance and average color of the observed image in the HLS color space are obtained for each observed image captured by changing the imaging condition for adjusting the brightness of the observed image.
  • An imaging condition for adjusting the brightness when an observation image having a maximum index based on the degree is captured is set as an optimal value, and the observation image is acquired with the optimal value.
  • the focal position when the observation image with the maximum number of particles in the observation image captured by changing the focal position is captured is the optimum value. And an observation image is acquired with the optimum value.
  • FIG. 1 shows a schematic diagram of an imaging device for cultured cells according to the first embodiment of the present invention.
  • FIG. 2 shows a flowchart of a method for imaging cultured cells according to the first embodiment of the present invention.
  • FIG. 3 is a flowchart for setting the imaging condition for adjusting the brightness of the observation image.
  • FIG. 4A to FIG. 4E show observation images picked up by changing the image pickup condition for adjusting the brightness of the observation image.
  • FIG. 5 is a graph showing the relationship between the imaging condition for adjusting the brightness of the observation image and the index.
  • FIG. 6 shows a flowchart for setting the focal position of the observation image.
  • FIGS. 7A to 7E show observation images picked up by changing the focal position.
  • FIG. 8 is a graph showing the relationship between the focal position and the number of particles.
  • FIGS. 9A to 9C show observation images showing the relationship between the focal position and the number of particles.
  • FIG. 10 shows a flowchart of a method for imaging cultured cells according to the second embodiment of the present invention.
  • FIGS. 11A to 11E show observation images obtained by alternately repeating brightness setting and focus position setting.
  • FIG. 12A shows a graph showing the relationship between the imaging condition for adjusting the brightness and the index in the first brightness setting step.
  • FIG. 12B shows a graph showing the relationship between the imaging condition for adjusting the brightness in the second brightness setting step and the index.
  • FIG. 12C is a graph showing the relationship between the imaging condition for adjusting the brightness in the third brightness setting step and the index.
  • FIG. 13A shows a graph showing the relationship between the focal position and the number of particles in the first focal position setting step.
  • FIG. 13B shows a graph showing the relationship between the focal position and the number of particles in the second focal
  • FIG. 1 is a schematic diagram of an imaging apparatus according to the present embodiment.
  • the imaging device performs time-lapse imaging of the cultured cells 11 in the culture solution 10 accommodated in the culture vessel 1 every predetermined time (for example, 60 minutes).
  • the imaging device of the present embodiment is an imaging device that captures an observation image of a cultured cell 11 in a culture vessel 1, and an LED lamp 2 as an illumination unit that illuminates the cultured cell 11;
  • a CCD camera 3 is provided as an imaging unit that images the cultured cells 11 and generates an observation image.
  • the CCD camera 3 is disposed on the opposite side of the LED lamp 2 with the culture vessel 1 in between, and takes a transmission image.
  • the imaging apparatus includes a dimmer 4 that adjusts the brightness of the LED lamp 2, an actuator 5 that moves the CCD camera 3, and a control unit 6 that is a computer that controls the dimmer 4 and the actuator 5. Is further provided.
  • the control unit 6 controls the CCD camera 3 to automatically execute time-lapse imaging.
  • the control unit 6 also functions as a brightness control unit that sets an imaging condition for adjusting the brightness of the observation image.
  • the current value applied to the LED lamp 2 is adjusted, and the brightness of the observation image is adjusted. Therefore, in the present embodiment, the current value of the LED lamp 2 is an imaging condition for adjusting the brightness of the observation image.
  • the brightness of the observation image may be adjusted not only by the current value of the LED lamp 2 but also by the aperture of the diaphragm and the shutter speed of the CCD camera, or may be adjusted in combination.
  • the control unit 6 also functions as a focal position control unit that sets the focal position of the observation image.
  • the position of the CCD camera 3 is adjusted by the control unit 6 controlling the actuator 5.
  • the distance between the CCD camera 3 and the culture vessel 1 is adjusted by moving the CCD camera 3 in the direction of the arrow in FIG. Therefore, in the present embodiment, the focal position is adjusted according to the position of the CCD camera 3.
  • the focal position may be adjusted not only by moving the CCD camera 3, but also by an optical system such as a lens of the CCD camera 3, or may be adjusted by moving the culture vessel 1, or a combination thereof. May be adjusted.
  • Imaging method Next, with reference to FIG. 2, the imaging method by the imaging device of this embodiment is demonstrated.
  • the imaging condition for adjusting the brightness of the observation image the current value of the LED lamp 2 is It is set (S1).
  • the CCD camera 3 is moved to set the focal position (S2).
  • the observation image imaged on the imaging conditions which optimized the electric current value of LED lamp 2, and the focus position of CCD camera 3 is acquired (S3).
  • FIGS. 4A to 4E show five observation images when the current set value is “10” to “50” among the ten observation images.
  • Each observation image shown in FIG. 4 is a black-and-white image, but in the actual image, the culture solution 10 is yellow.
  • the current setting value of the LED lamp 2 is too low, it becomes difficult to distinguish the particles of the cultured cells 11 in the observation image as shown in FIG.
  • the current setting value of the LED lamp 2 is too high, as shown in FIG. 4E, the entire observation image becomes white and small particles disappear, making it difficult to distinguish the particles.
  • the current setting value of the LED lamp 2 is further increased, the entire observation image becomes whiter and particle discrimination becomes more difficult.
  • control unit 6 uses the average luminance (L) and average saturation of the observation image in the HLS color space. (S) is calculated (S12).
  • the HLS color space (also referred to as HSL color space) refers to a color space represented by three components of hue, luminance (lightness / luminance), and saturation.
  • Hue represents the hue with an angle of 360 degrees.
  • Brightness has a different meaning from “luminance” described in Patent Document 1, and represents the brightness of a color with white as a maximum value and black as a minimum value, and when the brightness is half of the maximum value and the minimum value. It becomes a pure color.
  • Saturation represents the vividness of a color, with a pure color as the maximum value and gray as the minimum value.
  • the average luminance (L) and average saturation (S) of the observation image are values obtained by averaging the data for each pixel of the RGB color system representing the received light intensity of the RGB light receiving elements of the CCD camera 3 over the entire observation image. Is converted into the HLS color system. Table 1 below shows the average luminance (L) and average saturation (S) of the entire observation image for each current setting value of the LED lamp 2. In Table 1, the average luminance (L) and the average saturation (S) are represented by 256 gradations from 0 to 255.
  • the control unit 6 calculates an index based on the average luminance (L) and the average saturation (S) (S13).
  • the index is the difference (255 ⁇ L) between the maximum luminance (255), which is the maximum value of the luminance gradation, and the average luminance (L) of the observation image, and the average luminance as in the following equation (1) It includes the product of (L) and the sum (L + S) of average saturation (S).
  • Index (I) (255 ⁇ L) ⁇ (L + S) (1)
  • the above formula (1) has been found empirically by the present inventor through various experiments and studies.
  • Table 1 shows the value of the index (I) for each current setting value of the LED lamp 2. As shown in Table 1, when the current setting value of the LED lamp 2 is “20”, the index (I) is the maximum value “25652”.
  • the index (I) for each current set value of the LED lamp 2 is shown in the graph of FIG. FIG. 5 shows that the index (I) is maximum when the current setting value of the LED lamp 2 is “20”.
  • the current setting value of the LED lamp 2 when the observation image having the maximum index (I) is taken is set as the optimum value (S14).
  • the current set value “20” of the LED lamp 2 is set as the optimum value of the imaging condition for adjusting the brightness.
  • the cultured cell 11 is imaged while changing the focal position within a predetermined change range with a predetermined step size, and a plurality of observation images are generated (S21).
  • the actuator 5 moves the CCD camera 3 to change the Z coordinate (arbitrary unit) representing the focal position in increments of “250” from “0” to “9750”.
  • the cultured cells 11 were imaged at the coordinates, and a total of 40 observation images were generated.
  • FIG. 7A to FIG. 7E show 5 when the Z coordinate of the focal position is “0”, “3000”, “4250”, “6000”, and “9750” out of 40 observation images. A representative observation image is shown.
  • the number of particles in the observation image is measured (S22). Individual particles in the observation image are measured by image processing.
  • the graph of FIG. 8 shows the number of particles for each set value of the Z coordinate.
  • FIG. 8 shows that the number of particles is maximum when the Z coordinate is “4250”.
  • Table 2 shows the number of particles measured from the observed image when the Z coordinate of the focal position is “3000” to “6000”. As shown in Table 2, when the Z coordinate is “4250”, the number of particles is the maximum value “6110”.
  • FIG. 9A and FIG. 9C show an observation image when the image is out of focus
  • FIG. 9B shows an observation image when the image is in focus
  • FIGS. 9 (a) and 9 (c) when the particles are not in focus, the contour of each particle is unclear, so that the contour of each particle is integrated with the adjacent particle. Yes. For this reason, it is difficult to separate and extract individual particles during image processing, and the number of particles to be measured is reduced.
  • FIGS. 7A and 7C when the focus is not at all, it is difficult to determine the particles themselves in the observation image, and thus the number of particles to be measured is large. descend.
  • the focal position when an observation image with the maximum number of particles is captured is set as an optimum value (S23).
  • the Z coordinate “4250” is set as the optimum value of the focal position of the CCD camera 3.
  • Image acquisition process Next, an image captured under the imaging condition of the optimal current setting value of the LED lamp 2 and the optimal focal position of the CCD camera 3 is acquired (S3 in FIG. 2). In acquiring this image, an observation image may be taken again under the optimized imaging conditions, or in the process of setting the imaging conditions, the observation image captured under the same conditions as the optimized imaging conditions may be captured. Data may be selected and used.
  • an index based on the average luminance (L) and average saturation (S) of the observation image in the HLS color space was set. Furthermore, in the present embodiment, an imaging condition is set that maximizes the number of particles in the observed image captured by changing the focal position. Thereby, even if the color tone of the culture solution 10 is changed or the transparency is lowered while the cell culture is continued, the cultured cells 11 are not located on the same plane in the culture solution 10. Even in this case, the imaging conditions of the cultured cells 11 can be optimized.
  • an LED lamp is used as the imaging condition for adjusting the brightness of the observation image when setting the imaging condition when imaging the observation image of the cultured cell 11.
  • the brightness setting step (S1, S3 and S5) for setting the current value of 2 and the focus position setting step (S2 and S4) for setting the focus position by moving the CCD camera 3 are alternately repeated.
  • the observation image imaged on the imaging condition optimized with the electric current value of the LED lamp 2 set last and the focus position of the CCD camera 3 is acquired (S6).
  • FIGS. 11A to 11E show observation images picked up under the image pickup conditions optimized in the steps S1 to S5 in FIG.
  • FIG. 11A shows an observation image captured with the current setting value of the LED lamp 2 optimized in the first brightness setting step (S1 in FIG. 10). At this stage, the particles cannot be identified because they are not yet in focus.
  • the graph of Fig.12 (a) shows the parameter
  • the current setting value of the LED lamp 2 is changed from “2” to “40” in increments of “2”, and the cultured cells 11 are imaged at each current setting value. Twenty observation images were generated.
  • the index (I) is maximized.
  • the observation image shown in FIG. 11A is an image taken under an imaging condition where the current set value is “14”.
  • FIG. 11B shows an observation image captured at the focal position optimized in the first focal position setting step (S2 in FIG. 10).
  • the particles in the observed image can be distinguished because they are in focus.
  • FIG. 13A shows the number of particles for each focal position of the CCD camera 3 in the first focal position setting step (S2).
  • the Z coordinate representing the focus position is changed from “0” to “9750” in increments of “250”, and the cultured cells 11 are imaged at each Z coordinate, for a total of 40 sheets. The observed image was generated.
  • the number of particles is maximum when the Z coordinate is “3500”.
  • the observation image shown in FIG. 11B is an image taken under an imaging condition in which the Z coordinate is “3500” while the current setting value is “14”.
  • FIG. 11 (c) shows an observation image taken with the current setting value of the LED lamp 2 optimized in the second brightness setting step (S3 in FIG. 10). Even if the brightness of the observation image is not optimal as a result of the first focus adjustment, the brightness of the observation image is optimized again by the second brightness adjustment.
  • the graph of FIG.12 (b) shows the parameter
  • the second brightness setting process (S3) is also performed by changing the current setting value of the LED lamp 2 from “2” to “40” in increments of “2”.
  • the cultured cells 11 were imaged at the current setting value, and a total of 20 observation images were generated.
  • the observation image shown in FIG. 11C is an image taken under an imaging condition in which the current set value is “14” while the Z coordinate is “3500”.
  • FIG. 11 (d) shows an observation image captured at the focal position optimized by the second focus adjustment in the second focus position setting step (S4 in FIG. 10).
  • 13B shows the number of particles for each focal position of the CCD camera 3 in the second focal position setting step (S4).
  • the step size of the Z coordinate representing the focus position is set to “100” units narrower than the first time, and the change range of the Z coordinate is “3500, which is the first optimum value.
  • the cultured cells 11 were imaged at each Z coordinate from “2800” to “4200”, which is a narrower range than the first time, and a total of 15 observation images were generated.
  • the number of particles is maximum when the Z coordinate is “3600”.
  • the observation image shown in FIG. 11D is an image taken under an imaging condition in which the Z coordinate is “3600” while the current setting value is “14”.
  • the step size in the second focus position setting is made narrower than that in the first time, and the change range in the second focus position setting is made narrower than that in the first time.
  • the setting accuracy of the imaging condition for the focal position of the CCD camera 3 can be improved.
  • FIG. 11 (e) shows an observation image captured with the current setting value of the LED lamp 2 optimized in the third brightness setting step (S5 in FIG. 10).
  • the graph of FIG.12 (c) shows the parameter
  • the step size of the current setting value of the LED lamp 2 is set to “1” units narrower than the first time, and the change range of the current setting value is the optimum value for the second time. From “9” to “18” including “14”, the cultured cells 11 were imaged at each current setting value, and a total of 10 observation images were generated.
  • the third brightness setting step (S5) when the current setting value of the LED lamp 2 is “15”, the index (I) is maximized.
  • the observation image shown in FIG. 11E is an image taken under an imaging condition where the current set value is “15” while the Z coordinate is “3600”.
  • the current setting value in the third brightness setting step (S5) is set while the step size of the current setting value of the LED lamp 2 in the third brightness setting step (S5) is made narrower than that in the second time.
  • the value change range narrower than that of the second time it is possible to improve the setting accuracy of the imaging condition of the current setting value of the LED lamp 2 while avoiding an increase in the number of captured images.
  • this invention can perform a various change and deformation
  • a CCD camera as an imaging unit is arranged on the opposite side of the LED lamp with the culture vessel interposed therebetween and an observation image in which transmitted light is imaged is generated has been described.
  • the observation image is not limited to the transmitted light image, and may be a reflected light image, for example.
  • the imaging condition for adjusting the brightness of the observation image is
  • the present invention is not limited to this.
  • the brightness may be adjusted by the aperture of the aperture, or the brightness may be adjusted by the shutter speed of the CCD camera.
  • the index may be calculated using the HSV color system.
  • the HSV color system is represented by three components of hue, saturation, and value.
  • Hue represents the hue with an angle of 360 degrees.
  • Saturation represents the vividness of a color, with a pure color as the maximum value and gray as the minimum value.
  • the brightness represents the brightness of the color.
  • the method for adjusting the focal position is not limited to this, and the CCD camera is not limited to this.
  • the focal position may be adjusted by moving the optical system, or the focal position may be adjusted by moving the culture vessel.
  • the illumination unit is not limited to this in the present invention.
  • an example in which a CCD camera is used as the imaging unit has been described.
  • the imaging unit is not limited to this.
  • the present invention is suitable for application to automatic time-lapse imaging of cultured cells.

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JP2015181374A (ja) * 2014-03-24 2015-10-22 東レ株式会社 糸状性細菌検出装置および糸状性細菌の検出方法
JP2015194544A (ja) * 2014-03-31 2015-11-05 富士フイルム株式会社 細胞撮像制御装置および方法並びにプログラム

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