JP2010276585A - Device and method for measuring activity - Google Patents

Device and method for measuring activity Download PDF

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JP2010276585A
JP2010276585A JP2009132391A JP2009132391A JP2010276585A JP 2010276585 A JP2010276585 A JP 2010276585A JP 2009132391 A JP2009132391 A JP 2009132391A JP 2009132391 A JP2009132391 A JP 2009132391A JP 2010276585 A JP2010276585 A JP 2010276585A
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activity
cell
cells
feature amount
phase information
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JP5663147B2 (en
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Yoshimizu Sakamoto
宜瑞 坂本
Hiroshi Fukuda
宏 福田
Yoshiaki Okazaki
善朗 岡崎
Masayuki Kobayashi
雅之 小林
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Olympus Corp
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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
    • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability

Abstract

<P>PROBLEM TO BE SOLVED: To measure activity of cells without performing processing having an influence on the cells such as dyeing. <P>SOLUTION: An activity measuring device 1 includes a phase information measuring part 9 for measuring phase information of a cell A group including a plurality of cells A, a characteristic quantity extraction part 10 for extracting a characteristic quantity carried by each cell A from the phase information measured by the phase information measuring part 9, and an activity calculation part 12 for calculating activity of the whole cell A group based on the characteristic quantity extracted by the characteristic quantity extraction part 10. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

本発明は、活性度測定装置および活性度測定方法に関するものである。   The present invention relates to an activity measuring device and an activity measuring method.

従来、細胞のような測定対象の生存率（バイアビリティ）等の活性度を測定する方法が知られている。この方法は、細胞自体または特定の分子を蛍光物質によって染色しておき、励起光を照射して発生する蛍光の強度を測定するものである（例えば、特許文献１〜３参照。）。   2. Description of the Related Art Conventionally, a method for measuring activity such as survival rate (viability) of a measurement object such as a cell is known. In this method, cells themselves or specific molecules are stained with a fluorescent substance, and the intensity of fluorescence generated by irradiating excitation light is measured (see, for example, Patent Documents 1 to 3).

特開平７−１９８７１４号公報JP 7-198714 A 特開２００６−２３０３３３号公報JP 2006-230333 A 特開２００６−３１４２１４号公報JP 2006-314214 A

しかしながら、従来の生存率測定方法は、生存率を測定する測定対象である細胞を染色する必要があるため、一度測定に用いた細胞は、他の用途、例えば、移植等に利用することができず、廃棄しなければならないという不都合がある。   However, since the conventional survival rate measurement method needs to stain the cells to be measured for measuring the survival rate, the cells once used for the measurement can be used for other purposes such as transplantation. There is a disadvantage that it must be discarded.

本発明は上述した事情に鑑みてなされたものであって、染色等の細胞に影響を与える処理を行うことなく細胞の活性度を測定することができる活性度測定装置および活性度測定方法を提供することを目的としている。   The present invention has been made in view of the above-described circumstances, and provides an activity measuring apparatus and an activity measuring method capable of measuring the activity of a cell without performing a treatment that affects the cell such as staining. The purpose is to do.

上記目的を達成するために、本発明は以下の手段を提供する。
本発明は、複数の細胞を含む細胞群の位相情報を測定する位相情報測定部と、該位相情報測定部により測定された位相情報から各前記細胞の有する特徴量を抽出する特徴量抽出部と、該特徴量抽出部により抽出された特徴量に基づいて前記細胞群全体の活性度を算出する活性度算出部とを備える活性度測定装置を提供する。 In order to achieve the above object, the present invention provides the following means.
The present invention provides a phase information measurement unit that measures phase information of a cell group including a plurality of cells, and a feature amount extraction unit that extracts a feature amount of each cell from the phase information measured by the phase information measurement unit. There is provided an activity measuring device including an activity calculating unit that calculates the activity of the entire cell group based on the feature extracted by the feature extracting unit.

本発明によれば、位相情報測定部によって測定された細胞群の位相情報から、特徴量抽出部によって各細胞の有する特徴量が抽出され、その特徴量に基づいて活性度算出部により細胞群全体の活性度が算出される。
細胞群の位相情報は、細胞群に対して焦点位置をずらした複数の画像に基づいて測定することができる。 According to the present invention, the feature amount of each cell is extracted by the feature amount extraction unit from the phase information of the cell group measured by the phase information measurement unit, and the whole cell group is extracted by the activity calculation unit based on the feature amount. The degree of activity is calculated.
The phase information of the cell group can be measured based on a plurality of images whose focal positions are shifted with respect to the cell group.

位相情報は細胞内部の液体の屈折率によって変化する。そして、生細胞は外部の培地と内部の液体とを所定の濃度を保ちながら交換しているため、培地より高い屈折率が維持されているが、死細胞は細胞壁が破れることによって内部の液体が外部に放出されて培地が浸入するために、培地の屈折率に近い屈折率に変化する。したがって、位相情報に基づいて抽出された各細胞の特徴量、例えば、位相値φを任意の屈折率ｎで除算することによって算出された見かけの最大厚さ寸法等は生細胞か死細胞かによって変化しており、これによって細胞群の活性度を精度よく算出することができる。すなわち、本発明によれば、細胞を蛍光染色する等の物理的な処理を施すことなく、細胞の活性度を測定することができる。   The phase information changes depending on the refractive index of the liquid inside the cell. And since living cells exchange the external medium and the internal liquid while maintaining a predetermined concentration, the refractive index higher than that of the medium is maintained, but dead cells have a liquid inside because the cell wall is broken. Since it is released to the outside and the medium enters, it changes to a refractive index close to the refractive index of the medium. Therefore, the feature quantity of each cell extracted based on the phase information, for example, the apparent maximum thickness dimension calculated by dividing the phase value φ by an arbitrary refractive index n depends on whether it is a living cell or a dead cell. Thus, the activity of the cell group can be accurately calculated. That is, according to the present invention, cell activity can be measured without performing physical treatment such as fluorescent staining of cells.

上記発明においては、前記特徴量抽出部が、特徴量として前記細胞の最大位相値を抽出してもよい。
このようにすることで、特徴量抽出部によって抽出された最大位相値を用いて、細胞の活性度を簡易に判定することができる。 In the above invention, the feature amount extraction unit may extract the maximum phase value of the cell as the feature amount.
In this way, it is possible to easily determine the degree of cell activity using the maximum phase value extracted by the feature amount extraction unit.

また、上記発明においては、前記活性度算出部が、前記特徴量抽出部により抽出された最大位相値の出現頻度の分布を示すヒストグラムを生成し、全体頻度に対する所定の閾値以上の最大位相値を有する細胞の出現頻度の割合を活性度として算出してもよい。
細胞の有する特徴量は、細胞の大きさや構造等の細胞の個性によってばらつきがあるので、細胞単独では測定誤差が大きいが、細胞群全体においてその分布を示すヒストグラムを生成することで、生細胞と死細胞との割合を求めることができ、活性度を精度よく測定することができる。 In the above invention, the activity calculation unit generates a histogram indicating the distribution of appearance frequencies of the maximum phase values extracted by the feature amount extraction unit, and sets a maximum phase value equal to or greater than a predetermined threshold with respect to the overall frequency. You may calculate the ratio of the appearance frequency of the cell which has as activity.
The characteristic amount of a cell varies depending on the individuality of the cell, such as the size and structure of the cell, so the measurement error is large for the cell alone, but by generating a histogram showing the distribution of the entire cell group, The ratio of dead cells can be determined, and the activity can be measured with high accuracy.

また、上記発明においては、前記活性度算出部が、前記特徴量抽出部により抽出された最大位相値の出現頻度の分布を示すヒストグラムを生成し、細胞活性度毎に予め計算された少なくとも２つ以上の前記最大位相値の正規分布に基づいて、該正規分布にそれぞれ係数をかけて重畳させたものとヒストグラムとの差分が最小となる各正規分布の前記係数の割合を活性度として算出することとしてもよい。   In the above invention, the activity calculation unit generates a histogram indicating the distribution of the appearance frequency of the maximum phase value extracted by the feature amount extraction unit, and at least two calculated in advance for each cell activity Based on the normal distribution of the maximum phase value described above, the ratio of the coefficient of each normal distribution that minimizes the difference between the normal distribution multiplied by the coefficient and the histogram is calculated as the activity. It is good.

また、上記発明においては、前記活性度算出部が、前記特徴量抽出部により抽出された最大位相値の出現頻度の分布を示すヒストグラムを生成し、該ヒストグラムに現れる極小値以上の最大位相値を有する細胞の出現頻度の割合を活性度として算出してもよい。
このようにすることで、生細胞と死細胞との特徴量が離れた分布を示している場合に、間に極小値が現れるので、これを用いて生細胞と死細胞との割合を求めることができ、活性度を精度よく測定することができる。 Further, in the above invention, the activity calculation unit generates a histogram indicating the distribution of the appearance frequency of the maximum phase value extracted by the feature amount extraction unit, and sets the maximum phase value equal to or greater than the minimum value appearing in the histogram. You may calculate the ratio of the appearance frequency of the cell which has as activity.
By doing this, when the feature quantity of live cells and dead cells shows a distant distribution, a minimum value appears between them, and this can be used to determine the ratio of live cells to dead cells. And the activity can be accurately measured.

また、上記発明においては、前記極小値が複数存在する場合に、各極小値となる最大位相値における出現頻度とその両側に隣接する最大位相値における出現頻度との差分の小さい方の値が、前記ヒストグラム内において最大となる最大位相値における出現頻度を、前記活性度の算出のための極小値としてもよい。   Further, in the above invention, when there are a plurality of the minimum values, the smaller value of the difference between the appearance frequency in the maximum phase value that becomes each minimum value and the appearance frequency in the maximum phase value adjacent to both sides thereof, The appearance frequency at the maximum phase value that is maximum in the histogram may be a minimum value for calculating the activity.

また、本発明は、複数の細胞を含む細胞群の位相情報を測定する位相情報測定ステップと、該位相情報測定ステップにより測定された位相情報から各前記細胞の有する特徴量を抽出する特徴量抽出ステップと、該特徴量抽出ステップにより抽出された特徴量に基づいて前記細胞群全体の活性度を算出する活性度算出ステップとを含む活性度測定方法を提供する。   The present invention also provides a phase information measurement step for measuring phase information of a cell group including a plurality of cells, and a feature amount extraction for extracting a feature amount of each cell from the phase information measured by the phase information measurement step. There is provided an activity measuring method including a step and an activity calculating step for calculating the activity of the entire cell group based on the feature extracted in the feature extracting step.

本発明によれば、染色等の細胞に影響を与える処理を行うことなく細胞の活性度を測定することができるという効果を奏する。   According to the present invention, the cell activity can be measured without performing a treatment that affects the cells such as staining.

本発明の一実施形態に係る活性度測定装置を示す模式図である。It is a schematic diagram which shows the activity measuring apparatus which concerns on one Embodiment of this invention. 図１の活性度測定装置を用いた本実施形態に係る活性度測定方法を示すフローチャートである。It is a flowchart which shows the activity measuring method which concerns on this embodiment using the activity measuring apparatus of FIG. 図１の活性度測定装置の位相情報測定部により生成された位相分布画像の一例を示す図である。It is a figure which shows an example of the phase distribution image produced | generated by the phase information measurement part of the activity measuring device of FIG. 図１の活性度測定装置の特徴量抽出部により最大位相値が抽出された位相分布画像の一例を示す図である。It is a figure which shows an example of the phase distribution image from which the maximum phase value was extracted by the feature-value extraction part of the activity measuring device of FIG. 図１の活性度測定装置のヒストグラム生成部により生成されたヒストグラムであって、所定の閾値を基準として活性度を算出する場合を示す図である。It is a histogram produced | generated by the histogram production | generation part of the activity measuring device of FIG. 1, Comprising: It is a figure which shows the case where activity is calculated on the basis of a predetermined threshold value. 図５の変形例であって、発生頻度が最小値となる最大位相差を基準として活性度を算出する場合を示す図である。FIG. 6 is a modified example of FIG. 5 and shows a case where the activity is calculated based on the maximum phase difference at which the occurrence frequency is a minimum value. 図５の変形例であって、正規分布の合成によって活性度を算出する場合を示す図である。FIG. 6 is a modified example of FIG. 5 and shows a case where the activity is calculated by combining normal distributions. （ａ）図５の方法、（ｂ）図７の方法でそれぞれ活性度を算出した結果を示す図である。(A) It is a figure which shows the result of having calculated activity by the method of FIG. 5, (b) respectively by the method of FIG.

本発明の一実施形態に係る活性度測定装置１および活性度測定方法について、図面を参照して以下に説明する。
本実施形態に係る活性度測定装置は、図１に示されるように、細胞Ａ群を培地Ｂとともに収容したシャーレ２を搭載するステージ３と、該ステージ３の鉛直下方に配置され、所定の波長の光、例えば、近赤外光を発生する光源４と、該光源４から発せられた近赤外光を鉛直方向に配される光軸Ｃに沿って細胞群Ａに照射する照明光学系５と、細胞群Ａを上方に透過した透過光を集光する集光光学系６と、該集光光学系６によって集光された透過光を撮影するＣＣＤのような撮像素子７と、該撮像素子７により取得された画像に基づいて細胞Ａの活性度を判定する細胞活性度判定部８とを備えている。 An activity measuring device 1 and an activity measuring method according to an embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the activity measuring device according to the present embodiment is arranged on a stage 3 on which a petri dish 2 containing a group of cells A together with a culture medium B is mounted, and vertically below the stage 3, and has a predetermined wavelength. Light, for example, a light source 4 that generates near-infrared light, and an illumination optical system 5 that irradiates the cell group A along the optical axis C arranged in the vertical direction with the near-infrared light emitted from the light source 4. A condensing optical system 6 that condenses the transmitted light that has passed through the cell group A, an imaging element 7 such as a CCD that photographs the transmitted light collected by the condensing optical system 6, and the imaging A cell activity determination unit 8 that determines the activity of the cell A based on the image acquired by the element 7 is provided.

ステージ３は、集光光学系６の光軸Ｃ方向に移動可能に設けられており、ステージ３を移動させた複数の位置において撮影することにより、１枚のフォーカス画像と２枚のデフォーカス画像とを取得することを可能にしている。なお、ステージ３を固定して集光光学系６を光軸Ｃ方向に移動可能に設けてもよい。   The stage 3 is provided so as to be movable in the direction of the optical axis C of the condensing optical system 6, and one focus image and two defocus images are obtained by photographing at a plurality of positions where the stage 3 is moved. And make it possible to get to. The stage 3 may be fixed and the condensing optical system 6 may be provided so as to be movable in the direction of the optical axis C.

細胞活性度判定部８は、撮像素子７により取得された１枚のフォーカス画像と２枚のデフォーカス画像とを用いた公知の方法によって、位相情報の２次元的な分布を示す位相分布画像を取得する位相情報測定部９と、取得された位相分布画像に基づいて、該位相分布画像内に含まれる複数の細胞Ａの最大位相値を抽出する特徴量抽出部１０と、抽出された最大位相値の出現頻度の分布を示すヒストグラムを生成するヒストグラム生成部１１と、生成されたヒストグラムの中で、予め定められた閾値を超える頻度で出現する最大位相値の割合に基づいて細胞Ａ群の活性度を算出する活性度算出部１２とを備えている。   The cell activity determination unit 8 generates a phase distribution image indicating a two-dimensional distribution of the phase information by a known method using one focus image and two defocus images acquired by the image sensor 7. A phase information measuring unit 9 to be acquired, a feature amount extracting unit 10 for extracting the maximum phase values of a plurality of cells A included in the phase distribution image based on the acquired phase distribution image, and an extracted maximum phase A histogram generation unit 11 that generates a histogram indicating the distribution of the appearance frequency of the values, and the activity of the cell A group based on the ratio of the maximum phase value that appears with a frequency exceeding a predetermined threshold in the generated histogram And an activity calculation unit 12 for calculating the degree.

このように構成された本実施形態に係る活性度測定装置１を用いた活性度測定方法について以下に説明する。
まず、図２に示されるように、複数の細胞Ａを含む細胞Ａ群を収容したシャーレ２をステージ３に載置し、光源４から発生した近赤外光を照明光学系５を介してステージ３上の細胞Ａ群に照射する。細胞Ａ群を透過した透過光を集光光学系６によって集光し、撮像素子７によって撮影する。この場合には、ステージ３を光軸Ｃ方向に移動して１枚のフォーカス画像と２枚のデフォーカス画像とを取得する（ステップＳ１）。 The activity measuring method using the activity measuring device 1 according to the present embodiment configured as described above will be described below.
First, as shown in FIG. 2, a petri dish 2 containing a group of cells A including a plurality of cells A is placed on a stage 3, and near-infrared light generated from a light source 4 is staged through an illumination optical system 5. Irradiate cell A group 3 above. The transmitted light that has passed through the cell group A is condensed by the condensing optical system 6 and photographed by the image sensor 7. In this case, the stage 3 is moved in the direction of the optical axis C to acquire one focus image and two defocus images (step S1).

次に、取得されたフォーカス画像およびデフォーカス画像に基づいて、位相情報測定部９が、位相情報の２次元的な分布を示す位相分布画像を生成する（ステップＳ２）。そして、特徴量抽出部１０が、位相分布画像に基づいて、各細胞Ａの有する最大位相値を抽出する（ステップＳ３）。特徴量抽出部１０による最大位相値の抽出は、画像処理により各細胞Ａの輪郭形状を抽出するとともに、抽出された各細胞Ａの輪郭形状内において位相値が最大となる箇所を探索することにより行われる。   Next, based on the acquired focus image and defocus image, the phase information measurement unit 9 generates a phase distribution image indicating a two-dimensional distribution of the phase information (step S2). Then, the feature amount extraction unit 10 extracts the maximum phase value of each cell A based on the phase distribution image (step S3). The extraction of the maximum phase value by the feature quantity extraction unit 10 is performed by extracting the contour shape of each cell A by image processing and searching for a location where the phase value is maximum in the extracted contour shape of each cell A. Done.

この後に、ヒストグラム生成部１１が、抽出された各細胞Ａの最大位相値の大きさと出現頻度との関係を示すヒストグラムを生成する（ステップＳ４）。そして、生成されたヒストグラムに基づいて活性度算出部１２が細胞Ａ群全体の活性度を算出する（ステップＳ５）。   Thereafter, the histogram generation unit 11 generates a histogram indicating the relationship between the magnitude of the maximum phase value of each extracted cell A and the appearance frequency (step S4). Then, the activity calculation unit 12 calculates the activity of the entire cell A group based on the generated histogram (step S5).

具体的には、図３に示されるように、ステップＳ２において位相分布画像が生成され、図４に示されるように、ステップＳ３において該位相分布画像を処理して各細胞Ａの有する最大位相値が抽出される。図中、符号＋は、抽出された各細胞Ａの最大位相値の位置を示している。
そして、図５に示されるように、ステップＳ４において、抽出された最大位相値を横軸とし、その出現頻度を縦軸としたヒストグラムが生成される。 Specifically, as shown in FIG. 3, a phase distribution image is generated in step S2, and as shown in FIG. 4, the phase distribution image is processed in step S3 to obtain the maximum phase value of each cell A. Is extracted. In the figure, the sign + indicates the position of the extracted maximum phase value of each cell A.
Then, as shown in FIG. 5, in step S4, a histogram is generated with the extracted maximum phase value on the horizontal axis and the appearance frequency on the vertical axis.

生細胞は細胞膜内に細胞内液を保有し、外部の培地Ｂとは異なる屈折率を保持しているので、細胞を通過する近赤外光と細胞以外の場所を通過する近赤外光とでは、屈折率の相違に基づく位相差が発生し、その位相差の分布が位相分布画像として生成される。
死細胞も外部の培地とは異なる屈折率を有しているものの、細胞膜が破壊されることにより、細胞内液が放出され外部の培地が入り込んで培地に近い屈折率になっている。したがって、生細胞とはその位相差が異なっている。 Since living cells have an intracellular fluid in the cell membrane and have a refractive index different from that of the external medium B, near-infrared light that passes through cells and near-infrared light that passes through places other than cells Then, a phase difference based on the difference in refractive index occurs, and the distribution of the phase difference is generated as a phase distribution image.
Although dead cells also have a refractive index different from that of the external medium, when the cell membrane is destroyed, the intracellular fluid is released and the external medium enters and has a refractive index close to that of the medium. Therefore, the phase difference is different from that of living cells.

このため、生細胞と死細胞とが同じ厚さ寸法を有していても、屈折率の相違により位相分布が異なり、最大厚さ寸法に対応する最大位相値の値も相違してくる。各細胞Ａは、その個体差によって最大厚さ寸法が異なるが、その分布は所定の平均値の周囲に正規分布をなして分散している。したがって、多数の細胞Ａについて最大位相値の出現頻度を示すヒストグラムを作成することにより、生細胞と死細胞とが混在した細胞Ａ群の最大位相値の分布を得ることができる。   For this reason, even if the living cell and the dead cell have the same thickness dimension, the phase distribution differs due to the difference in refractive index, and the maximum phase value corresponding to the maximum thickness dimension also differs. Each cell A has a maximum thickness dimension that varies depending on individual differences, but its distribution is distributed in a normal distribution around a predetermined average value. Therefore, by creating a histogram indicating the appearance frequency of the maximum phase value for a large number of cells A, it is possible to obtain the distribution of the maximum phase value of the group of cells A in which live cells and dead cells are mixed.

細胞内液の屈折率は培地Ｂの屈折率よりも高いので、生細胞の屈折率は死細胞の屈折率より高く、最大位相値の大きい側に生細胞が分布している。したがって、所定の閾値より大きな最大位相値の出現頻度の割合が多い程、生細胞が多いと考えることができる。   Since the refractive index of the intracellular fluid is higher than the refractive index of the medium B, the refractive index of the living cells is higher than that of the dead cells, and the living cells are distributed on the side where the maximum phase value is larger. Therefore, it can be considered that the larger the ratio of the appearance frequency of the maximum phase value larger than the predetermined threshold, the more living cells.

そこで、生成されたヒストグラムにおいて、図５に示すように、所定の閾値を超える最大位相値の出現頻度の全データ数に対する割合を求めることにより、細胞Ａの活性度を算出することができる。逆に、所定の閾値以下の最大位相値の出現頻度の全データ数に対する割合を求めることにより、細胞Ａの死亡率を算出することができる。ここで、所定の閾値は、細胞Ａの種類毎に予めデータを採取することにより求めておくことにすればよい。   Accordingly, in the generated histogram, as shown in FIG. 5, the activity of the cell A can be calculated by obtaining the ratio of the appearance frequency of the maximum phase value exceeding a predetermined threshold to the total number of data. Conversely, the mortality rate of the cell A can be calculated by obtaining the ratio of the appearance frequency of the maximum phase value equal to or less than a predetermined threshold to the total number of data. Here, the predetermined threshold value may be obtained by collecting data in advance for each type of cell A.

本実施形態に係る活性度測定装置１および活性度測定方法によれば、細胞Ａ群に近赤外光を照射して生成された位相分布画像を用いて細胞Ａの活性度を算出するので、従来、染色することによって測定していた細胞Ａの活性度を無線色のまま測定することができる。したがって、活性度の測定に使用した細胞Ａを廃棄することなくそのまま用いることができ、貴重な細胞Ａを無駄にせずに済むという利点がある。   According to the activity measuring device 1 and the activity measuring method according to the present embodiment, the activity of the cell A is calculated using the phase distribution image generated by irradiating the cell A group with near infrared light. Conventionally, the activity of the cell A, which has been measured by staining, can be measured in a wireless color. Therefore, there is an advantage that the cell A used for measuring the activity can be used as it is without being discarded, and the valuable cell A can be saved.

なお、本実施形態においては、予めデータを採取して求めておいた閾値を用いて、当該閾値を超える最大位相値を有する細胞Ａの割合によって細胞Ａ群の活性度を評価することとしたが、これに代えて、図６に示されるように、ヒストグラムに現れる極小値の最大位相値を基準として活性度を評価することにしてもよい。生細胞と死細胞との間の屈折率の差が大きい場合には、ヒストグラムにおける生細胞と死細胞との分布の間に極小値が現れるので、それを基準として活性度を評価することができる。   In the present embodiment, the activity of the cell A group is evaluated based on the ratio of the cells A having the maximum phase value exceeding the threshold using a threshold obtained by collecting data in advance. Instead of this, as shown in FIG. 6, the activity may be evaluated based on the maximum phase value of the minimum value appearing in the histogram. When the difference in refractive index between live and dead cells is large, a minimum value appears between the distribution of live and dead cells in the histogram, and the activity can be evaluated based on that. .

ヒストグラムの中に複数の極小値が現れる場合には、注目極小値の前のデータとの差分と後のデータとの差分を計算し、差分が小さい方を記録する。同様の処理をヒストグラム内の全データで実施する。得られた差分データ内で値が最大となる極小値を閾値とする。   When a plurality of local minimum values appear in the histogram, the difference between the data before the local minimum value of interest and the data after it is calculated, and the smaller difference is recorded. Similar processing is performed on all data in the histogram. The minimum value with the maximum value in the obtained difference data is set as a threshold value.

また、生細胞と死細胞のそれぞれの正規分布を予め用意しておき、それらの正規分布にそれぞれ係数をかけたものを合成した分布が、取得されたヒストグラムに近似するように係数を調節し、得られた係数の割合によって活性度を評価することにしてもよい。
例えば、図７に示す例では、（ａ）に示されるヒストグラムに対し、（ｂ）に示す生細胞の正規分布ＸをＰ倍し、死細胞の正規分布ＹをＱ倍することで、（ｃ）に示されるように取得されたヒストグラムの分布に近似する分布が得られた場合に、細胞Ａ群の活性度はＰ／（Ｐ＋Ｑ）によって算出することができ、細胞群の死亡率は、Ｑ／（Ｐ＋Ｑ）によって算出することができる。 In addition, the normal distribution of each of live cells and dead cells is prepared in advance, and the coefficient is adjusted so that the distribution obtained by multiplying those normal distributions multiplied by the coefficients approximates the acquired histogram, You may decide to evaluate activity by the ratio of the obtained coefficient.
For example, in the example shown in FIG. 7, the normal distribution X of live cells shown in (b) is multiplied by P and the normal distribution Y of dead cells is multiplied by Q with respect to the histogram shown in (a). ), The activity of the cell group A can be calculated by P / (P + Q), and the mortality rate of the cell group is expressed as Q / (P + Q).

ここで、本実施形態に係る活性度測定装置１を用いた細胞Ａ群の活性度測定の実施例について説明する。
まず、シャーレ２内に脂肪由来幹細胞Ａを培地Ｂとともに収容したものを６組用意し、３組の脂肪由来幹細胞Ａには紫外線刺激を４時間行い、他の３組の脂肪由来幹細胞Ａは常温で放置した。 Here, the Example of the activity measurement of the cell A group using the activity measuring apparatus 1 which concerns on this embodiment is demonstrated.
First, six sets of adipose-derived stem cells A and medium B are prepared in the petri dish 2. The three sets of fat-derived stem cells A are subjected to UV stimulation for 4 hours, and the other three sets of fat-derived stem cells A are at room temperature. Left alone.

各シャーレ２内の脂肪由来幹細胞Ａに対して、本実施形態に係る活性度測定装置１を用いて、脂肪由来幹細胞Ａの位相分布画像を、視野を変えながら１０枚取得した。次いで、各位相分布画像から各細胞Ａの最大位相値を抽出した。
最大位相値を横軸、出現頻度を縦軸としてヒストグラムを生成し、予め定めた閾値に基づく方法による細胞死亡率と、正規分布Ｘ，Ｙの係数Ｐ，Ｑを調整する方法による細胞死亡率とを求めた結果、図８（ａ），（ｂ）に示す通りの結果が得られた。 For the adipose-derived stem cells A in each petri dish 10, 10 phase distribution images of the adipose-derived stem cells A were obtained while changing the visual field using the activity measuring device 1 according to this embodiment. Next, the maximum phase value of each cell A was extracted from each phase distribution image.
A histogram is generated with the maximum phase value as the horizontal axis and the appearance frequency as the vertical axis, and the cell mortality rate by a method based on a predetermined threshold and the cell mortality rate by a method of adjusting the coefficients P and Q of the normal distributions X and Y As a result, the results shown in FIGS. 8A and 8B were obtained.

各組の脂肪由来細胞Ａに対しては、比較のために従来と同様のNucleoCounterによる細胞死亡率の測定も合わせて行った。
その結果、本実施形態に係る活性度測定装置１を用いたいずれの方法によっても、従来の測定方法と同様の結果を得ることができた。 For each set of adipose-derived cells A, for the purpose of comparison, measurement of cell mortality using the same NucleoCounter was also performed.
As a result, the same result as that of the conventional measurement method could be obtained by any method using the activity measuring apparatus 1 according to the present embodiment.

Ａ 細胞
１ 活性度測定装置
９ 位相情報測定部
１０ 特徴量抽出部
１２ 活性度算出部
Ｓ２ 位相分布画像生成ステップ（位相情報測定ステップ）
Ｓ３ 最大位相値抽出ステップ（特徴量抽出ステップ）
Ｓ５ 活性度算出ステップ A cell 1 activity measurement device 9 phase information measurement unit 10 feature quantity extraction unit 12 activity calculation unit S2 phase distribution image generation step (phase information measurement step)
S3 Maximum phase value extraction step (feature value extraction step)
S5 Activity calculation step

Claims (7)

複数の細胞を含む細胞群の位相情報を測定する位相情報測定部と、
該位相情報測定部により測定された位相情報から各前記細胞の有する特徴量を抽出する特徴量抽出部と、
該特徴量抽出部により抽出された特徴量に基づいて前記細胞群全体の活性度を算出する活性度算出部とを備える活性度測定装置。 A phase information measurement unit for measuring phase information of a cell group including a plurality of cells;
A feature amount extraction unit that extracts a feature amount of each cell from the phase information measured by the phase information measurement unit;
An activity measuring apparatus comprising: an activity calculating unit that calculates the activity of the entire cell group based on the feature extracted by the feature extracting unit. 前記特徴量抽出部が、特徴量として前記細胞の最大位相値を抽出する請求項１に記載の活性度測定装置。   The activity measuring device according to claim 1, wherein the feature amount extraction unit extracts a maximum phase value of the cell as a feature amount. 前記活性度算出部が、前記特徴量抽出部により抽出された最大位相値の出現頻度の分布を示すヒストグラムを生成し、全体頻度に対する所定の閾値以上の最大位相値を有する細胞の出現頻度の割合を活性度として算出する請求項２に記載の活性度測定装置。   The activity calculation unit generates a histogram indicating the distribution of the appearance frequency of the maximum phase value extracted by the feature amount extraction unit, and the ratio of the appearance frequency of cells having a maximum phase value equal to or greater than a predetermined threshold to the overall frequency The activity measuring device according to claim 2, wherein the activity is calculated as activity. 前記活性度算出部が、前記特徴量抽出部により抽出された最大位相値の出現頻度の分布を示すヒストグラムを生成し、細胞活性度毎に予め計算された少なくとも２つ以上の前記最大位相値の正規分布に基づいて、該正規分布にそれぞれ係数をかけて重畳させたものとヒストグラムとの差分が最小となる各正規分布の前記係数の割合を活性度として算出する請求項２に記載の活性度測定装置。   The activity calculation unit generates a histogram indicating a distribution of appearance frequencies of the maximum phase values extracted by the feature amount extraction unit, and includes at least two or more maximum phase values calculated in advance for each cell activity level. The activity according to claim 2, wherein the activity is calculated based on the normal distribution as a degree of activity of the coefficient of each normal distribution that minimizes a difference between a histogram and a result obtained by superimposing the normal distribution on each of the coefficients. measuring device. 前記活性度算出部が、前記特徴量抽出部により抽出された最大位相値の出現頻度の分布を示すヒストグラムを生成し、該ヒストグラムに現れる極小値以上の最大位相値を有する細胞の出現頻度の割合を活性度として算出する請求項２に記載の活性度測定装置。   The activity calculation unit generates a histogram showing the distribution of the appearance frequency of the maximum phase value extracted by the feature amount extraction unit, and the ratio of the appearance frequency of cells having a maximum phase value equal to or greater than the minimum value appearing in the histogram The activity measuring device according to claim 2, wherein the activity is calculated as activity. 前記極小値が複数存在する場合に、各極小値となる最大位相値における出現頻度とその両側に隣接する最大位相値における出現頻度との差分の小さい方の値が、前記ヒストグラム内において最大となる最大位相値における出現頻度を、前記活性度の算出のための極小値とする請求項５に記載の活性度測定装置。   When there are a plurality of the minimum values, the smaller value of the difference between the appearance frequency at the maximum phase value that is each minimum value and the appearance frequency at the maximum phase value adjacent to both sides thereof is the maximum in the histogram. The activity measuring device according to claim 5, wherein the appearance frequency in the maximum phase value is a minimum value for calculating the activity. 複数の細胞を含む細胞群の位相情報を測定する位相情報測定ステップと、
該位相情報測定ステップにより測定された位相情報から各前記細胞の有する特徴量を抽出する特徴量抽出ステップと、
該特徴量抽出ステップにより抽出された特徴量に基づいて前記細胞群全体の活性度を算出する活性度算出ステップとを含む活性度測定方法。 A phase information measurement step for measuring phase information of a cell group including a plurality of cells;
A feature amount extraction step of extracting a feature amount of each of the cells from the phase information measured by the phase information measurement step;
An activity calculating step including calculating an activity of the entire cell group based on the feature extracted in the feature extracting step.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013517460A (en) * 2010-01-12 2013-05-16 バイオ−ラド ラボラトリーズ インコーポレイテッド Cell characterization using multiple focal planes
WO2016080442A1 (en) * 2014-11-21 2016-05-26 住友電気工業株式会社 Quality evaluation method and quality evaluation device
JP2016192923A (en) * 2015-03-31 2016-11-17 株式会社ニコン Determination apparatus, determination system, determination program, method for producing cells, and cell
WO2020261607A1 (en) * 2019-06-26 2020-12-30 株式会社島津製作所 Three-dimensional cell shape evaluation method
WO2023022091A1 (en) * 2021-08-16 2023-02-23 株式会社ニコン Analysis system, observation container, analysis method and program

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6366465A (en) * 1986-09-08 1988-03-25 Res Dev Corp Of Japan Identification and determination of cell
JPH0227977A (en) * 1988-07-18 1990-01-30 Hitachi Ltd Device, method and examination method for culturing animal cell
JPH04332847A (en) * 1991-05-09 1992-11-19 Hitachi Ltd Analyzing apparatus of white blood corpuscle
JP2928714B2 (en) * 1993-12-28 1999-08-03 アサヒビール株式会社 Cell activity determination method and device
JP2003016555A (en) * 2001-06-29 2003-01-17 Matsushita Electric Works Ltd Wireless sensor system
JP2006230333A (en) * 2005-02-28 2006-09-07 Hitachi Medical Corp Flow site meter, method for analyzing cell, cell-analyzing method, method for setting sensitivity of fluorescent light detector and method for setting standard gate in positive rate-judging method
JP2006314214A (en) * 2005-05-10 2006-11-24 Olympus Corp System, method and program for cell observation
JP2007509314A (en) * 2003-09-23 2007-04-12 イアティア イメージング プロプライアタリー リミティド Method and apparatus for determining sample area or confluence
JP2008212017A (en) * 2007-03-01 2008-09-18 Nikon Corp Apparatus for determining cell state, and method for determining cell state
JP2009106272A (en) * 2007-10-10 2009-05-21 Olympus Corp Method for measuring cellular thickness

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6366465A (en) * 1986-09-08 1988-03-25 Res Dev Corp Of Japan Identification and determination of cell
JPH0227977A (en) * 1988-07-18 1990-01-30 Hitachi Ltd Device, method and examination method for culturing animal cell
JPH04332847A (en) * 1991-05-09 1992-11-19 Hitachi Ltd Analyzing apparatus of white blood corpuscle
JP2928714B2 (en) * 1993-12-28 1999-08-03 アサヒビール株式会社 Cell activity determination method and device
JP2003016555A (en) * 2001-06-29 2003-01-17 Matsushita Electric Works Ltd Wireless sensor system
JP2007509314A (en) * 2003-09-23 2007-04-12 イアティア イメージング プロプライアタリー リミティド Method and apparatus for determining sample area or confluence
JP2006230333A (en) * 2005-02-28 2006-09-07 Hitachi Medical Corp Flow site meter, method for analyzing cell, cell-analyzing method, method for setting sensitivity of fluorescent light detector and method for setting standard gate in positive rate-judging method
JP2006314214A (en) * 2005-05-10 2006-11-24 Olympus Corp System, method and program for cell observation
JP2008212017A (en) * 2007-03-01 2008-09-18 Nikon Corp Apparatus for determining cell state, and method for determining cell state
JP2009106272A (en) * 2007-10-10 2009-05-21 Olympus Corp Method for measuring cellular thickness

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013517460A (en) * 2010-01-12 2013-05-16 バイオ−ラド ラボラトリーズ インコーポレイテッド Cell characterization using multiple focal planes
US9001200B2 (en) 2010-01-12 2015-04-07 Bio-Rad Laboratories, Inc. Cell characterization using multiple focus planes
WO2016080442A1 (en) * 2014-11-21 2016-05-26 住友電気工業株式会社 Quality evaluation method and quality evaluation device
JPWO2016080442A1 (en) * 2014-11-21 2017-10-05 住友電気工業株式会社 Quality evaluation method and quality evaluation apparatus
JP2016192923A (en) * 2015-03-31 2016-11-17 株式会社ニコン Determination apparatus, determination system, determination program, method for producing cells, and cell
WO2020261607A1 (en) * 2019-06-26 2020-12-30 株式会社島津製作所 Three-dimensional cell shape evaluation method
JPWO2020261607A1 (en) * 2019-06-26 2021-10-21 株式会社島津製作所 Cell three-dimensional shape evaluation method and cell observation device
JP7147982B2 (en) 2019-06-26 2022-10-05 株式会社島津製作所 Three-dimensional cell shape evaluation method and cell observation device
WO2023022091A1 (en) * 2021-08-16 2023-02-23 株式会社ニコン Analysis system, observation container, analysis method and program

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