JP2020190459A5 - - Google Patents
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- JP2020190459A5 JP2020190459A5 JP2019095499A JP2019095499A JP2020190459A5 JP 2020190459 A5 JP2020190459 A5 JP 2020190459A5 JP 2019095499 A JP2019095499 A JP 2019095499A JP 2019095499 A JP2019095499 A JP 2019095499A JP 2020190459 A5 JP2020190459 A5 JP 2020190459A5
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- cells
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- cytotoxicity
- objective lens
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- 239000000126 substance Substances 0.000 claims 26
- 238000000034 method Methods 0.000 claims 13
- 230000003013 cytotoxicity Effects 0.000 claims 10
- 231100000135 cytotoxicity Toxicity 0.000 claims 10
- 238000005286 illumination Methods 0.000 claims 5
- 238000003384 imaging method Methods 0.000 claims 4
- 230000003287 optical effect Effects 0.000 claims 4
- 230000001678 irradiating effect Effects 0.000 claims 3
- 238000012258 culturing Methods 0.000 claims 1
- 231100000433 cytotoxic Toxicity 0.000 claims 1
- 230000001472 cytotoxic effect Effects 0.000 claims 1
Claims (13)
前記被験物質の存在下及び非存在下で細胞を培養する工程と、
前記被験物質の存在下の前記細胞を撮像し、第１の画像を得る工程と、
前記被験物質の非存在下の前記細胞を撮像し、第２の画像を得る工程と、
前記第１の画像及び前記第２の画像を比較して、前記被験物質が細胞毒性を有するか否かを判定する工程と、を備え、
前記第１の画像を得る工程及び前記第２の画像を得る工程は、前記第１の画像及び前記第２の画像としてそれぞれ定量位相画像を得るため、顕微鏡装置の対物レンズの光軸上に前記細胞を配置する工程と、前記細胞に照明光を照射する工程と、前記光軸に沿って互いに間隔Δｚ離れた複数の位置のそれぞれに前記対物レンズの焦点を配置して前記細胞からの光を検出する工程と、検出した前記光に基づいて、前記複数の位置のそれぞれに対応する光強度分布データを生成する工程と、前記光強度分布データに基づいて前記定量位相画像を生成する工程と、を含み、
前記間隔Δｚは、前記対物レンズの開口数ＮＡ、前記照明光の波長λ、及び、前記対物レンズと前記細胞との間の屈折率ｎの少なくとも１つに基づいて設定される、方法。 A method for assessing the cytotoxicity of a test substance,
The step of culturing cells in the presence and absence of the test substance, and
The step of imaging the cells in the presence of the test substance to obtain a first image, and
The step of imaging the cells in the absence of the test substance to obtain a second image, and
A step of comparing the first image and the second image to determine whether or not the test substance has cytotoxicity is provided.
The step of obtaining the first image and the step of obtaining the second image are performed on the optical axis of the objective lens of the microscope device in order to obtain quantitative phase images as the first image and the second image, respectively. The step of arranging the cells, the step of irradiating the cells with illumination light, and the steps of arranging the focal points of the objective lens at each of a plurality of positions separated by Δz from each other along the optical axis to emit the light from the cells. A step of detecting, a step of generating light intensity distribution data corresponding to each of the plurality of positions based on the detected light, and a step of generating the quantitative phase image based on the light intensity distribution data. Including
The interval Δz is set based on at least one of the numerical aperture NA of the objective lens, the wavelength λ of the illumination light, and the refractive index n between the objective lens and the cell .
前記被験物質が細胞毒性を有するか否か判定する工程は、前記第１の画像と前記第２の画像の経時的な変化を比較する工程を含む、請求項１に記載の方法。 In the step of obtaining the first image and the step of obtaining the second image, the cells were imaged a plurality of times at different times.
The method according to claim 1, wherein the step of determining whether or not the test substance has cytotoxicity includes a step of comparing changes over time between the first image and the second image.
前記第１の画像における前記被験物質の存在下の細胞の短軸方向の長さに対する前記被験物質の存在下の細胞の長軸方向の長さの比が、前記第２の画像における前記被験物質の非存在下の細胞の短軸方向の長さに対する前記被験物質の非存在下の細胞の長軸の長さの比と比較して低下した場合に、前記被験物質が細胞毒性を有すると判定する、
請求項１～６のいずれか一項に記載の方法。 In the step of determining whether or not the test substance has cytotoxicity,
The ratio of the length of the cell in the presence of the test substance in the minor axis direction to the length of the cell in the presence of the test substance in the first image is the ratio of the length in the major axis direction of the test substance in the second image. The test substance is determined to be cytotoxic when it is reduced compared to the ratio of the length of the cell in the absence of the test substance to the length of the cell in the absence of the test substance in the minor axis direction. do,
The method according to any one of claims 1 to 6 .
前記第１の画像において前記被験物質の存在下の細胞が存在しない領域の面積が、前記第２の画像において前記被験物質の非存在下の細胞が存在しない領域の面積と比較して増加した場合に、前記被験物質が細胞毒性を有すると判定する、
請求項１～７のいずれか一項に記載の方法。 In the step of determining whether or not the test substance has cytotoxicity,
When the area of the region where the cells in the presence of the test substance are not present in the first image is increased as compared with the area of the region in which the cells are not present in the absence of the test substance in the second image. In addition, it is determined that the test substance has cytotoxicity.
The method according to any one of claims 1 to 7 .
被験物質の存在下で培養された細胞を異なる時間において複数回撮像し、定量位相イメージングにより複数の定量位相画像を生成する工程と、
前記複数の定量位相画像における経時的な変化に基づいて、前記被験物質が細胞毒性を有するか否かを判定する工程と、を備え、
前記複数の定量位相画像を生成する工程は、前記定量位相画像を得るため、顕微鏡装置の対物レンズの光軸上に前記細胞を配置する工程と、前記細胞に照明光を照射する工程と、前記光軸に沿って互いに間隔Δｚ離れた複数の位置のそれぞれに前記対物レンズの焦点を配置して前記細胞からの光を検出する工程と、検出した前記光に基づいて、前記複数の位置のそれぞれに対応する光強度分布データを生成する工程と、前記光強度分布データに基づいて前記定量位相画像を生成する工程と、を含み、
前記間隔Δｚは、前記対物レンズの開口数ＮＡ、前記照明光の波長λ、及び、前記対物レンズと前記細胞との間の屈折率ｎの少なくとも１つに基づいて設定される、方法。 A method for assessing the cytotoxicity of a test substance,
A step of imaging cells cultured in the presence of a test substance multiple times at different times and generating multiple quantitative phase images by quantitative phase imaging.
A step of determining whether or not the test substance has cytotoxicity based on the change over time in the plurality of quantitative phase images is provided.
The steps for generating the plurality of quantitative phase images include a step of arranging the cells on the optical axis of the objective lens of the microscope device, a step of irradiating the cells with illumination light, and the steps of irradiating the cells with illumination light in order to obtain the quantitative phase images. A step of arranging the focal points of the objective lens at each of a plurality of positions separated from each other by Δz along the optical axis to detect light from the cells, and a step of detecting the light from the cells, and each of the plurality of positions based on the detected light. Including a step of generating the light intensity distribution data corresponding to the above and a step of generating the quantitative phase image based on the light intensity distribution data.
The interval Δz is set based on at least one of the numerical aperture NA of the objective lens, the wavelength λ of the illumination light, and the refractive index n between the objective lens and the cell .
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2019095499A JP7228189B2 (en) | 2019-05-21 | 2019-05-21 | Method and apparatus for evaluating cytotoxicity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2019095499A JP7228189B2 (en) | 2019-05-21 | 2019-05-21 | Method and apparatus for evaluating cytotoxicity |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| JP2020190459A JP2020190459A (en) | 2020-11-26 |
| JP2020190459A5 true JP2020190459A5 (en) | 2022-05-06 |
| JP7228189B2 JP7228189B2 (en) | 2023-02-24 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2023022091A1 (en) * | 2021-08-16 | 2023-02-23 | 株式会社ニコン | Analysis system, observation container, analysis method and program |
| JP2024022034A (en) * | 2022-08-05 | 2024-02-16 | 浜松ホトニクス株式会社 | A method for determining a region of a cell that has undergone programmed cell death, an apparatus including a determination unit, and an information processing program including a determination step |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP5614928B2 (en) | 2008-12-05 | 2014-10-29 | 国立大学法人 東京医科歯科大学 | Myocardial toxicity test apparatus, myocardial toxicity test chip, and myocardial toxicity test method |
| JP6191888B2 (en) | 2010-10-19 | 2017-09-06 | ソニー株式会社 | Image processing apparatus and method, and program |
| JP5939562B2 (en) | 2010-12-22 | 2016-06-22 | 国立大学法人富山大学 | Non-spherical cell life / death activity determination method and determination apparatus |
| JP6253400B2 (en) | 2013-12-26 | 2017-12-27 | オリンパス株式会社 | Image forming method and image forming apparatus |
| US11884905B2 (en) | 2017-02-09 | 2024-01-30 | University Public Corporation Osaka | Fluidic chip for cell culture use, culture vessel, and culture method |
| EP3712596A4 (en) | 2017-11-14 | 2021-11-24 | Nikon Corporation | Quantitative phase image generating method, quantitative phase image generating device, and program |
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