WO2021192910A1 - 画像生成方法、画像生成装置及びプログラム - Google Patents

画像生成方法、画像生成装置及びプログラム Download PDF

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Publication number
WO2021192910A1
WO2021192910A1 PCT/JP2021/008589 JP2021008589W WO2021192910A1 WO 2021192910 A1 WO2021192910 A1 WO 2021192910A1 JP 2021008589 W JP2021008589 W JP 2021008589W WO 2021192910 A1 WO2021192910 A1 WO 2021192910A1
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Prior art keywords
fluorescence
fluorescent substance
image
time
noise
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PCT/JP2021/008589
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English (en)
French (fr)
Japanese (ja)
Inventor
羚 馮
雄大 ▲高▼橋
秀樹 郷田
藤原　浩次
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Konica Minolta Inc
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Konica Minolta Inc
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    • 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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to an image generation method, an image generation device, and a program.
  • fluorescent bright spots are extracted from a fluorescent image obtained by imaging a tissue sample in which a specific biological substance is stained with fluorescent nanoparticles containing a large number of fluorescent dyes (fluorescent substances), and expression of the specific biological substance in the fluorescent image.
  • Patent Documents 1 and 2 disclose a method of eliminating the influence of noise fluorescence by utilizing the difference in fluorescence wavelength and specifying a fluorescent substance.
  • Fluorescent substance-accumulated nanoparticles are based on particles made of organic or inorganic substances, and a plurality of fluorescent substances (for example, the quantum dots, fluorescent dyes, etc.) are contained therein. It is a nano-sized particle having a structure that is and / or is adsorbed on its surface. As the fluorescent substance-accumulated nanoparticles, it is preferable that the matrix and the fluorescent substance have substituents or sites having opposite charges and electrostatically interact with each other. As the fluorescent substance integrated nanoparticles, quantum dot integrated nanoparticles, fluorescent dye integrated nanoparticles and the like are used.
  • an autoclave As the heating device, an autoclave, a microwave, a pressure cooker, a water bath, or the like can be used.
  • the temperature is not particularly limited, but it can be carried out at room temperature.
  • the temperature can be 50 to 130 ° C. and the time can be 5 to 30 minutes.
  • tissue specimen after the immunostaining step is subjected to treatments such as immobilization / dehydration, permeation, and encapsulation so as to be suitable for observation.
  • the fluorescence emitted from the tissue sample 30 after immunostaining includes fluorescence derived from a fluorescent substance that labels the target substance and fluorescence derived from a substance other than the fluorescent substance (noise fluorescence). Further, as an example of noise fluorescence, there is typically fluorescence (so-called autofluorescence) that is naturally emitted by a fluorescent substance (coenzyme, amino acid, protein, intracellular pigment substance, etc.) that is naturally present in a tissue sample. In addition, for example, substances other than the fluorescent substance contained in the tissue sample and fluorescence emitted by an instrument such as a slide glass can be mentioned.
  • step S15 the number of bright spots is measured for the image from which the bright spot region is extracted. That is, the expression level of the target substance can be quantitatively evaluated by measuring one bright spot as one fluorescent nanoparticle.
  • the number of bright spots is simply measured here, for example, a bright field image on the same plane as the fluorescence image and in the same range is imaged, a cell region or a cell nucleus region is extracted, and the bright spot region image is superimposed. Therefore, the number of bright spots for each cell region or each cell nucleus region can be calculated.
  • the attenuation rate (M%) of noise fluorescence at a predetermined time (t) is calculated in advance by the following equation (1).
  • M% F (t) / F0 ... (1)
  • F0 fluorescence intensity before excitation is stopped
  • F (t) fluorescence intensity at exposure time (t).
  • the pixel values of the original fluorescence image and the fading fluorescence image are compared, and a pixel having a fluorescence intensity attenuation rate of M% or less and M0% or more is determined to contain noise fluorescence, and is M0% or less. Pixels are determined to be free of noise fluorescence.
  • the target fluorescence image from which the fluorescence intensity of the fluorescent substance is extracted is acquired by setting the pixel value of the pixel determined to contain noise fluorescence to 0.
  • the pixel values of the original fluorescence image and the noise fluorescence exclusion image are compared, and it is determined that pixels having a fluorescence intensity attenuation rate of more than N% include noise fluorescence, and pixels of N% or less are noise. Judged as not containing fluorescence.
  • the target fluorescence image from which the fluorescence intensity of the fluorescent substance is extracted is acquired by setting the pixel value of the pixel determined to contain noise fluorescence to 0.
  • the pixel values of the original fluorescence image and the noise fluorescence exclusion image are compared, and it is determined that pixels having a fluorescence intensity attenuation rate of more than N% include noise fluorescence, and pixels of N% or less are noise. Judged as a fluorescent substance containing no fluorescence.
  • the target fluorescence image from which the fluorescence intensity of the fluorescent substance is extracted is acquired by setting the pixel value of the pixel determined to contain noise fluorescence to 0.
  • the processing flow of pattern 7 is as follows. (1) The tissue specimen stained with the fluorescent substance is irradiated with excitation light for a time shorter than a predetermined time (t) (t0, t0 ⁇ x), and the original derived from the fluorescence of a part of the fluorescent substance and the fluorescence of noise fluorescence. Acquire a fluorescence image. (2) The fluorescent substance is completely faded by exposure for a predetermined time (t) in the same field of view as the original fluorescence image, and a noise fluorescence image is acquired.
  • the processing flow of pattern 9 is as follows. (1) The tissue specimen stained with the fluorescent substance is irradiated with excitation light for a time shorter than a predetermined time (t) (t0, x ⁇ t0 ⁇ T1), and is derived from the fluorescence of a part of the fluorescent substance and the fluorescence of noise fluorescence. Acquire the original fluorescence image. (2) In the same visual field as the original fluorescence image, the fluorescence of the fluorescent substance is forcibly faded by exposure for a predetermined time (t) to obtain a faded fluorescence image in which a part of the fluorescence of the fluorescent substance is faded. (3) Using the attenuation curve of the fluorescence intensity of the fluorescent substance, the attenuation rate (N% and N0%) of the fluorescent substance is calculated in advance by the above formula (2) at the exposure time (t and t0).
  • the fluorescence image is faded in a predetermined time (t0) based on the fluorescence intensity of the faded fluorescence image.
  • the target fluorescence image from which the fluorescence intensity of the fluorescent substance is extracted is obtained by supplementing the fluorescence intensity of the fluorescence.
  • the processing flow of pattern 10 is as follows. (1) Tissue specimens stained with a fluorescent substance are irradiated with excitation light for a time shorter than a predetermined time (t) (t0, t0 ⁇ y1), and the fluorescence of the fluorescent substance, the fluorescence of long noise fluorescence, and the fluorescence of short noise fluorescence are performed. Obtain the original fluorescence image derived from. (2) An image excluding short noise fluorescence is acquired by exposure for a predetermined time (t) in the same field of view as the original fluorescence image.
  • the pixel values of the original fluorescence image and the short noise fluorescence exclusion image are compared, and it is determined that pixels having a fluorescence intensity attenuation rate of less than M% contain noise fluorescence, and others are noise fluorescence. Is not included.
  • the target fluorescence image from which the fluorescence intensity of the fluorescent substance is extracted is acquired by setting the pixel value of the pixel determined to contain noise fluorescence to 0.
  • the processing flow of pattern 11 is as follows. (1) Tissue specimens stained with a fluorescent substance are irradiated with excitation light for a time shorter than a predetermined time (t) (t0, T2-1 ⁇ t0 ⁇ x) to obtain fluorescence of the fluorescent substance and fluorescence of long noise fluorescence. Obtain the original fluorescence image (short noise fluorescence exclusion) from which it is derived. (2) A faded fluorescence image is obtained by forcibly fading the fluorescence of a fluorescent substance and long noise fluorescence by exposure for a predetermined time (t) in the same field of view as the original fluorescence image.
  • the attenuation rate (N%) of the fluorescent substance and the attenuation rate (M%) of long noise fluorescence are calculated by a predetermined exposure time (t).
  • the attenuation rate of the fluorescent substance is N%> the attenuation rate of long noise fluorescence is M%. Further, the above equations (1) and (2) can be used.
  • the pixel values of the original fluorescence image and the fading fluorescence image are compared, and it is determined that the pixels having a fluorescence intensity attenuation rate of less than M% contain noise fluorescence, and the others contain noise fluorescence. Judge as not.
  • the target fluorescence image from which the fluorescence intensity of the fluorescent substance is extracted is acquired by setting the pixel value of the pixel determined to contain noise fluorescence to 0.
  • the processing flow of pattern 13 is as follows. (1) Tissue specimens stained with a fluorescent substance are irradiated with excitation light for a time shorter than a predetermined time (t) (t0, T2-1 ⁇ t0 ⁇ x) to obtain fluorescence of the fluorescent substance and fluorescence of long noise fluorescence. Obtain the original fluorescence image (short noise fluorescence exclusion) from which it is derived. (2) A faded fluorescence image in which a part of the fluorescence of the fluorescent substance is faded is obtained by forcibly fading the fluorescence of the fluorescent substance by exposing it for a predetermined time (t) in the same field of view as the original fluorescent image. (3) Using the attenuation curve of the fluorescence intensity of the fluorescent substance, the attenuation rate (N%) of the fluorescent substance is calculated by the above formula (2) according to the predetermined exposure time (t).
  • a tissue sample stained with a fluorescent substance is irradiated with excitation light and exposed for a predetermined time, and a part of the fluorescence generated from the tissue sample is faded. It includes a fading fluorescence image acquisition step of acquiring a fluorescence image and a target fluorescence image acquisition step of acquiring a target fluorescence image from which fluorescence of a fluorescent substance is extracted based on the fading fluorescence image. Therefore, it is possible to obtain a target fluorescence image obtained by extracting only the fluorescence of the fluorescent substance based on the faded fluorescence image in which a part of the fluorescence is forcibly faded.
  • the faded fluorescence image acquisition step a faded fluorescence image obtained by fading the fluorescence of the fluorescent substance is acquired.
  • image processing is performed to remove the fluorescence intensity of the faded fluorescence image from the fluorescence intensity of the original fluorescence image, and the target fluorescence image is acquired.
  • the fading fluorescence image acquisition step after the noise fluorescence fades.
  • a fading fluorescence image is acquired, and in the target fluorescence image acquisition step, the fading fluorescence image is determined as the target fluorescence image. Therefore, the target fluorescence image can be obtained from one faded fluorescence image.
  • the pixel value of the original fluorescence image is corrected to acquire the target fluorescence image. Therefore, even when the fluorescence of the fluorescent substance and the first noise fluorescence and the second noise fluorescence having different time until fading can be mixed, the target fluorescence image is obtained from the original fluorescence image and the fading fluorescence image. Can be obtained.
  • the focusing position of the fluorescence image is specified by using the general-purpose microscope image acquisition device 1A.
  • a known hall slide scanner may be used instead of the microscope image acquisition device 1A. According to the hall slide scanner, not only the focus is automatically adjusted in the thickness direction (Z direction) of the tissue sample, but also the stage can be moved in the length and width direction (XY direction) of the tissue sample. A wide range of fluorescent images can be generated. Even with a hall slide scanner, after the in-focus position of the fluorescent image is specified, the focal position can be automatically moved to the in-focus position of the specified fluorescent image.
  • a tissue section is targeted as a biological sample, and the tissue sample is stained with an immunostaining agent containing fluorescent nanoparticles as a fluorescence marker to identify the in-focus position of the fluorescence image.
  • the target of the biological sample may be a cultured cell or a gene (DNA).
  • a fluorescent dye can be used as a fluorescent marker. Both the fluorescent nanoparticles and the fluorescent dye are examples of fluorescent markers, and other known fluorescent markers may be used.
  • an HDD, a non-volatile memory of a semiconductor, or the like is used as a computer-readable medium for the program according to the present invention, but the present invention is not limited to this example.
  • a portable recording medium such as a CD-ROM can be applied.
  • a carrier wave is also applied as a medium for providing the data of the program according to the present invention via a communication line.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Analytical Chemistry (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
PCT/JP2021/008589 2020-03-23 2021-03-05 画像生成方法、画像生成装置及びプログラム Ceased WO2021192910A1 (ja)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024171922A1 (ja) * 2023-02-15 2024-08-22 横河電機株式会社 画像取得装置、画像取得方法および画像取得プログラム

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013534171A (ja) * 2010-08-16 2013-09-02 コグノプティックス， インコーポレイテッド アミロイドタンパク質を検出するためのシステムおよび方法
JP2014519361A (ja) * 2011-04-28 2014-08-14 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア Ｓｉ含有粒子を用いた時間ゲート蛍光イメージング
US20180172588A1 (en) * 2015-06-30 2018-06-21 Imec Vzw Bleaching of Dyes in Luminescent Detection
JP2019088200A (ja) * 2017-11-10 2019-06-13 オリンパス株式会社 細胞集塊の突起形成能評価方法
JP2019526055A (ja) * 2016-08-01 2019-09-12 コグノプティックス， インコーポレイテッド 眼組織におけるタウタンパク質を検出するためのシステムおよび方法
WO2020022038A1 (ja) * 2018-07-24 2020-01-30 ソニー株式会社 情報処理装置、情報処理方法、情報処理システム、およびプログラム

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013534171A (ja) * 2010-08-16 2013-09-02 コグノプティックス， インコーポレイテッド アミロイドタンパク質を検出するためのシステムおよび方法
JP2014519361A (ja) * 2011-04-28 2014-08-14 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア Ｓｉ含有粒子を用いた時間ゲート蛍光イメージング
US20180172588A1 (en) * 2015-06-30 2018-06-21 Imec Vzw Bleaching of Dyes in Luminescent Detection
JP2019526055A (ja) * 2016-08-01 2019-09-12 コグノプティックス， インコーポレイテッド 眼組織におけるタウタンパク質を検出するためのシステムおよび方法
JP2019088200A (ja) * 2017-11-10 2019-06-13 オリンパス株式会社 細胞集塊の突起形成能評価方法
WO2020022038A1 (ja) * 2018-07-24 2020-01-30 ソニー株式会社 情報処理装置、情報処理方法、情報処理システム、およびプログラム

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024171922A1 (ja) * 2023-02-15 2024-08-22 横河電機株式会社 画像取得装置、画像取得方法および画像取得プログラム
JP2024115986A (ja) * 2023-02-15 2024-08-27 横河電機株式会社 画像取得装置、画像取得方法および画像取得プログラム
JP7845220B2 (ja) 2023-02-15 2026-04-14 横河電機株式会社 画像取得装置、画像取得方法および画像取得プログラム

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