WO2015133187A1 - Dispositif, procédé et programme de commande d'imagerie de cellules - Google Patents

Dispositif, procédé et programme de commande d'imagerie de cellules Download PDF

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Publication number
WO2015133187A1
WO2015133187A1 PCT/JP2015/051075 JP2015051075W WO2015133187A1 WO 2015133187 A1 WO2015133187 A1 WO 2015133187A1 JP 2015051075 W JP2015051075 W JP 2015051075W WO 2015133187 A1 WO2015133187 A1 WO 2015133187A1
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Prior art keywords
cell
region
magnification
partial
area
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PCT/JP2015/051075
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English (en)
Japanese (ja)
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尭之 辻本
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富士フイルム株式会社
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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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/48—Automatic or computerized control
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1429—Signal processing
    • G01N15/1433—Signal processing using image recognition
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology

Definitions

  • the present invention relates to a cell imaging control apparatus, method, and program for controlling imaging of a cell image.
  • Patent Document 3 proposes a method that enables a more detailed observation by specifying the position of a cell colony from an image captured at a low magnification and capturing the identified cell colony at a high magnification. .
  • JP 2011-229410 A JP 2013-236564 A JP 2012-002949 A JP 2013-109119 A Japanese Patent No. 5145487
  • Patent Document 1 and Patent Document 2 when a highly accurate determination is to be made, it is necessary to image the entire dish or the entire cell region in time series with high magnification, and the data to be processed and stored The amount becomes enormous.
  • the position of a cell colony can be specified by an image captured at a low magnification, and only the identified cell colony can be captured at a high magnification. It is necessary to image all of the cell colonies performed at high magnification, and the amount of data to be processed and stored is enormous.
  • Patent Document 4 describes narrowing down cell colonies for detailed observation at high magnification based on morphological characteristics of cell colonies
  • Patent Document 5 describes based on the size of cell colonies. In addition, it is described to narrow down cell colonies for performing detailed observation at high magnification.
  • Patent Document 4 and Patent Document 5 show the observation region in a relatively short culture period. Cannot narrow down. Moreover, since it cannot narrow down to an appropriate observation area
  • Patent Document 4 it is possible to narrow down in units of cell colonies, but detailed observation cannot be performed by narrowing down to a local region in the cell colonies.
  • an object of the present invention is to provide a cell imaging control apparatus, method, and program capable of appropriately setting a region of interest for performing high-magnification imaging even in a relatively short culture period. To do.
  • the cell imaging control device of the present invention sets a plurality of partial areas in a low-magnification image acquisition unit that acquires images obtained by imaging cells at a first magnification in time series, and each partial area.
  • a growth rate acquisition unit that acquires cell growth rates based on a plurality of images captured in time series, and an attention area that determines a region of interest from a plurality of partial regions based on the growth rates of each partial region
  • An area determination unit and an imaging control unit that controls to capture only a region of interest among a plurality of partial regions at a second magnification higher than the first magnification are provided.
  • the attention area determination unit can determine an abnormal area or a normal area as the attention area.
  • the attention area determination unit can determine a partial area whose growth rate is equal to or higher than a threshold value or lower than a threshold value as a normal area.
  • the attention area determination unit can determine the undifferentiated cell area as a normal area.
  • the attention area determination unit can determine the differentiated cell area as a normal area.
  • the attention area determination unit can determine a partial area whose growth rate is equal to or higher than a threshold value or lower than a threshold value as an abnormal area.
  • the attention area determination unit can determine the differentiated cell area as an abnormal area.
  • the attention area determination unit can determine the undifferentiated cell area as an abnormal area.
  • the growth rate acquisition unit can set an area obtained by dividing a colony area of cells included in the image area as the partial area.
  • the cell imaging control method of the present invention acquires an image obtained by imaging cells at a first magnification in time series, sets a plurality of partial areas in the area of the image, and images each of the partial areas in time series. Cell proliferation rates are obtained based on the plurality of images, and a region of interest is determined from the plurality of partial regions based on the growth rate of each partial region. Control is performed so that an image is captured at a second magnification higher than the first magnification.
  • a computer sets a plurality of partial areas in an image area, a low-magnification image acquisition unit that acquires an image obtained by imaging cells in time series at a first magnification, For a partial region, a growth rate acquisition unit that acquires cell growth rates based on a plurality of images captured in time series, and a region of interest from a plurality of partial regions based on the growth rate of each partial region An attention area determination unit to be determined, and an imaging control unit that controls to image only the attention area of the plurality of partial areas at a second magnification higher than the first magnification are characterized.
  • an image obtained by imaging a cell at a low magnification is acquired, a plurality of partial areas are set in the area of the image, and the image of each partial area is obtained. Since each cell growth rate was acquired and the region of interest for high-magnification imaging was determined from the multiple partial regions based on the growth rate of each partial region, even during a relatively short culture period It is possible to appropriately set the attention area of the high magnification imaging target.
  • the block diagram which shows schematic structure of the cell culture observation system using one Embodiment of the cell imaging control apparatus of this invention.
  • Diagram for explaining how to set a partial area Diagram for explaining other methods for setting partial areas The flowchart for demonstrating the effect
  • FIG. 1 is a block diagram showing a schematic configuration of a cell culture observation system.
  • the cell culture observation system includes a cell culture device 1, an imaging device 2, a cell imaging control device 3, a display 4, and an input device 5.
  • the cell culture device 1 is a device for culturing cells.
  • Examples of cells to be cultured include stem cells such as iPS cells and ES cells, cells such as nerves, skin and liver induced by differentiation from stem cells, and cancer cells.
  • stem cells such as iPS cells and ES cells
  • cells such as nerves, skin and liver induced by differentiation from stem cells, and cancer cells.
  • the cell culture device 1 includes a stage 10, a transport unit 11, and a control unit 12.
  • the stage 10 is provided with a culture container to be imaged by the imaging device 2.
  • the transport unit 11 selects a culture container to be imaged from among a plurality of culture containers accommodated at a predetermined position in the cell culture apparatus 1, and transports the selected culture container to the stage 10.
  • the control unit 12 controls the entire cell culture device 1 and controls environmental conditions such as temperature, humidity, and CO 2 concentration in the cell culture device 1 in addition to the operations of the stage 10 and the transport unit 11 described above.
  • the temperature, the configuration for adjusting the humidity and CO 2 concentration can be a known configuration.
  • the imaging device 2 captures images of cells in the culture vessel installed on the stage 10 in time series.
  • the imaging device 2 includes an optical system 20 for imaging and acquiring an image of a cell, an imaging element 21 that photoelectrically converts an image imaged by the optical system 20 and outputs the image signal, an optical system 20, And a control unit 22 that controls the image sensor 21.
  • CMOS Complementary Metal-Oxide Semiconductor
  • CCD charge-coupled device
  • the control unit 22 controls the entire imaging apparatus 2, and in particular, in this embodiment, controls the magnification of the optical system 20 and the position of the imaging region. Then, the control unit 22 sets the magnification of the optical system 20 to a low magnification of about 1 to 4 times and 10 to 20 times based on a control signal output from the imaging control unit 33 in the cell imaging control device 3 described later. The magnification is switched to a high magnification of about twice, and the position of the imaging region is controlled in accordance with the magnification switching. Specific control of the control unit 22 will be described in detail later. In the present embodiment, as described above, the magnification of the optical system 20 is automatically switched based on the control signal output from the imaging control unit 33. However, the present invention is not limited to this, and the user manually performs the magnification. May be switched.
  • the cell imaging control device 3 controls imaging conditions in the imaging device 2. Specifically, in the imaging device 2, first, the cells are imaged at a low magnification, cell proliferation rates are acquired for a plurality of partial regions in the image captured at the low magnification, and based on the proliferation rates. The attention area is determined from the plurality of partial areas, and the imaging device 2 is controlled so as to capture an image of the attention area at a high magnification.
  • the cell imaging control device 3 is obtained by installing an embodiment of the cell imaging control program of the present invention on a computer.
  • the cell imaging control device 3 includes a central processing unit, a semiconductor memory, a hard disk, and the like, and an embodiment of the cell imaging control program is installed on the hard disk. Then, when this program is executed by the central processing unit, the image acquisition unit 30, the growth rate acquisition unit 31, the attention area determination unit 32, the imaging control unit 33, and the display control unit 34 as shown in FIG. 1 operate. .
  • the image acquisition unit 30 acquires and stores a plurality of images (hereinafter, referred to as low-magnification images) captured in time series at a low magnification (first magnification) by the imaging device 2, and high-magnification by the imaging device 2.
  • An image of a region of interest (hereinafter referred to as a high magnification image) captured at (second magnification) is acquired and stored.
  • the image acquisition unit 30 outputs the acquired low-magnification image to the growth rate acquisition unit 31 and outputs the low-magnification image and the high-magnification image to the display control unit 34.
  • the growth rate acquisition unit 31 sets a plurality of partial regions in the region of the low-magnification image, and the proliferation rate of cells in the partial region based on the low-magnification images captured in time series for each partial region. Get each.
  • the partial region may be set by dividing the imaging region obtained by imaging one stem cell colony into a plurality of rectangular regions.
  • Each partial area may be divided and set so as not to overlap as shown in FIG. 2, or may be set so that a part of adjacent partial areas overlap.
  • the growth rate acquisition unit 31 counts the number of cells in each corresponding partial region in a plurality of low magnification images taken at different times, and the increase in the number of cells and the low magnification image It may be calculated based on the imaging interval.
  • cell edges may be detected and individual cells may be detected using pattern matching or the like, or intracellular nuclei or nucleoli are detected.
  • individual cells may be detected, and other known methods can be used.
  • the partial area is set by dividing the imaging area in which one stem cell colony is imaged into a plurality of rectangular areas.
  • the partial area setting method is not limited to this.
  • FIG. 3 a plurality of stem cell colony regions included in the imaging region may be extracted, and the extracted stem cell colony regions may be set as partial regions.
  • the attention area determination unit 32 determines the attention area from among the plurality of partial areas based on the proliferation speed of each partial area acquired by the proliferation speed acquisition section 31.
  • the proliferation rate is determined in advance.
  • a partial region that is equal to or greater than a set threshold value may be determined as a region of interest as an undifferentiated cell region and a normal region.
  • a partial region whose growth rate is equal to or lower than a preset threshold value may be determined as a region of interest as a differentiated cell region and an abnormal region.
  • the partial region in which the growth rate is equal to or less than a preset threshold is determined as undifferentiated.
  • the region of interest may be determined as being a cell region and a normal region.
  • a partial region having a growth rate equal to or higher than a preset threshold value may be determined as a region of interest as a differentiated cell region and an abnormal region.
  • the proliferation rate is equal to or higher than a preset threshold value.
  • the partial area is a differentiated cell area that has been differentiated into a target cell such as skin, for example, and may be determined as an attention area as a normal area.
  • the partial region is an undifferentiated cell region that has not been induced to differentiate into the target cell, and is considered as an abnormal region. You may make it decide to.
  • the proliferation rate is set to a preset threshold value.
  • the partial region described above may be determined as a region of interest as an undifferentiated cell region and an abnormal region.
  • a partial region whose growth rate is equal to or less than a preset threshold value may be determined as a region of interest as a differentiated cell region and a normal region.
  • the attention area determination unit 32 determines the attention area by comparing the growth rate with a threshold value, and this threshold value is changed according to the cell type, the culture condition, or the culture period. It may be.
  • the threshold may be increased as the cell grows faster, the threshold may be increased as the culture conditions grow faster, or the threshold may be decreased as the culture period is longer.
  • the threshold value corresponding to the cell type, culture conditions, or culture period may be set in advance using a table, for example. Moreover, what is necessary is just to make it a user input using the input device 5 about the kind of cell, culture conditions, or a culture
  • the imaging control unit 33 outputs a control signal to the imaging device 2 to control imaging conditions in the imaging device 2.
  • the imaging control unit 33 of the present embodiment outputs a control signal to the control unit 22 of the imaging apparatus 2 to instruct to capture a low-magnification image for acquiring the growth rate, and then acquires the growth rate.
  • a control signal for instructing to image only the attention area determined by the attention area determination section 32 among the plurality of partial areas set in the section 31 is output to the control section 22 of the imaging apparatus 2.
  • the magnification of the optical system 20 when acquiring a low-magnification image, the magnification of the optical system 20 is controlled to about 1 to 4 times, and when acquiring a high-magnification image, the optical system 20 The magnification is controlled to about 10 to 20 times.
  • the imaging control unit 33 outputs a control signal to the control unit 12 of the cell culture device 1 based on the attention area determined by the attention area determination unit 32 so that the attention area is imaged at a high magnification. Is controlled to move in the XY direction (the direction in the installation surface of the culture vessel).
  • the display control unit 34 receives the low-magnification image and the high-magnification image acquired by the image acquisition unit 30 and displays them on the display 4. In addition, the display control unit 34 may display the position (for example, coordinate value) of the partial area determined as the attention area, the number of the cell colony, and the like on the display 4. Further, the display control unit 34 has information indicating that it is a normal region, information indicating that it is an abnormal region, information indicating that it is an undifferentiated cell region, or the region of interest determined by the region of interest determination unit 32 Information indicating a differentiated cell region or the like may be displayed on the display 4.
  • the input device 5 includes a mouse, a keyboard, etc., and accepts an operation input by the user.
  • the input device 5 can accept a setting input when capturing a magnification when capturing a low-magnification image or a high-magnification image.
  • the input device 5 accepts a setting input for the range of the partial region and the setting input such as the above-described cell type, culture condition, and culture period.
  • the culture to be photographed is selected from the plurality of accommodated culture containers by the transport unit 11, and the selected culture container is placed on the stage 10 (S10).
  • a low magnification imaging control signal is output from the imaging control unit 33 of the cell imaging control device 3 to the control unit 22 of the imaging device 2, and the control unit 22 of the imaging device 2 responds to the input control signal.
  • the magnification of the optical system 20 is set to the low magnification (first magnification) described above, and so-called time-lapse imaging is performed to capture time-series low-magnification images (S12).
  • the time-series low-magnification image captured by the imaging device 2 is acquired by the image acquisition unit 30 of the cell imaging control device 3 and output to the growth rate acquisition unit 31.
  • the growth rate acquisition unit 31 sets a plurality of partial regions within the region of the input low-magnification image, and calculates a cell growth rate for each partial region (S14).
  • the growth rate of each partial area acquired in the growth rate acquisition part 31 is output to the attention area determination part 32, and the attention area determination part 32 is based on the input proliferation speed of each partial area.
  • An attention area is determined from the partial areas (S16).
  • the position information of the attention area is output to the imaging control section 33, and the imaging control section 33 instructs to capture the attention area at the above-described high magnification (second magnification) based on the input position information.
  • the control signal to be output is output to the control unit 22 of the imaging device 2 and the control unit 12 of the cell culture device 1.
  • the control unit 22 of the imaging device 2 sets the magnification of the optical system 20 to the high magnification (second magnification) described above according to the input control signal, and the control unit 12 of the cell culture device 1 Is moved in the XY direction, so-called time-lapse imaging is performed, and a time-series high-magnification image is captured (S18).
  • the high-magnification image acquired by the imaging device 2 is acquired by the image acquisition unit 30 of the cell imaging control device 3 and input to the display control unit 34.
  • the display control unit 34 displays the input high-magnification image on the display 4.
  • the user determines the state of the cell by observing the high-magnification image displayed on the display (S20).
  • the determination of the state of the cell based on the high-magnification image may be automatically performed in the determination unit provided in the cell imaging control device 3.
  • whether the stem cells are differentiated or undifferentiated can be determined from, for example, the density or luminance distribution of cells in the high-magnification image, and other known methods may be used.
  • differentiation / undifferentiation may be determined based on the growth rate used when determining the region of interest.
  • whether or not differentiation-induced cells are differentiated into target cells can be determined from, for example, morphological characteristics of the cells. Also in this case, it may be determined whether or not normal differentiation has occurred based on the growth rate used when determining the region of interest.
  • an image obtained by capturing cells at a low magnification is acquired, a plurality of partial regions are set in the region of the image, and cell proliferation is performed based on the images of the partial regions. Since the speed is acquired and the attention area of the high-magnification imaging target is determined from a plurality of partial areas based on the growth rate of each partial area, high-magnification imaging is performed even during a relatively short culture period.
  • the target region of interest can be set appropriately.
  • a region obtained by dividing a cell colony is set as a partial region
  • a local region in the cell colony can be set as a region of interest. Differentiation / undifferentiation can be determined with high accuracy.

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Abstract

L'invention concerne un dispositif, un procédé et un programme d'acquisition d'image de cellules pouvant limiter les cellules sur lesquelles une imagerie est effectuée à un fort grossissement et pouvant réduire la quantité de données devant être traitées et stockées. Le dispositif de commande d'imagerie de cellules est pourvu : d'une unité d'acquisition d'image (30) pour l'acquisition d'images dans lesquelles les cellules sont photographiées à un premier grossissement ; d'une unité d'acquisition de vitesse de croissance (31) pour établir de multiples régions partielles à l'intérieur de la région de prise d'image et pour acquérir les vitesses de croissance cellulaire respectives sur la base des images des différentes régions partielles ; d'une unité de détermination de région d'intérêt (32) pour déterminer une région d'intérêt parmi les multiples régions partielles sur la base des vitesses de croissance respectives des régions partielles ; d'une unité d'acquisition d'image (33) pour exécuter une commande de sorte que seule la région d'intérêt parmi les multiples régions partielles est photographiée à un second grossissement qui est supérieur au premier grossissement.
PCT/JP2015/051075 2014-03-04 2015-01-16 Dispositif, procédé et programme de commande d'imagerie de cellules WO2015133187A1 (fr)

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JP7462265B2 (ja) 2020-03-31 2024-04-05 中国電力株式会社 自動プランクトン検出方法

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JP5760811B2 (ja) 2011-07-28 2015-08-12 ソニー株式会社 固体撮像素子および撮像システム
CN114578536B (zh) * 2020-11-30 2024-03-26 深圳市瑞图生物技术有限公司 图像采集方法、装置、计算机设备和存储介质

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WO2017056945A1 (fr) * 2015-09-29 2017-04-06 富士フイルム株式会社 Dispositif et procédé d'évaluation de cellules
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