JP6767981B2 - 細胞測定方法 - Google Patents
細胞測定方法 Download PDFInfo
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- 238000000691 measurement method Methods 0.000 title claims description 9
- 201000011510 cancer Diseases 0.000 claims description 73
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- 239000002246 antineoplastic agent Substances 0.000 description 17
- 229940041181 antineoplastic drug Drugs 0.000 description 13
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- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
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- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
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- 229920001817 Agar Polymers 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
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- 239000007995 HEPES buffer Substances 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 239000002696 acid base indicator Substances 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
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- 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
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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
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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
- C12M1/3446—Photometry, spectroscopy, laser technology
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- 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
- C12M1/3446—Photometry, spectroscopy, laser technology
- C12M1/3476—Fluorescence spectroscopy
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/06—Quantitative determination
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
- G01N21/88—Investigating the presence of flaws or contamination
- G01N21/8806—Specially adapted optical and illumination features
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- G—PHYSICS
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- 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
- G01N33/4833—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures
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- 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
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
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- 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
- G01N2021/1734—Sequential different kinds of measurements; Combining two or more methods
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- Biodiversity & Conservation Biology (AREA)
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- Apparatus Associated With Microorganisms And Enzymes (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Description
I/I0=exp(−αL)
の関係がある。ここで、αは染色された癌細胞の吸光係数、Lは癌細胞中を光が通過する距離、すなわち癌細胞の厚さである。各画素の癌細胞による吸光度Aは、
A=−log(I/I0)
=(αL)/2.303
で表されるので、吸光度Aは癌細胞厚さLに比例する。吸光度Aは当該画素の細胞量の指標であり、これを試料の全範囲にわたって積算することにより細胞の体積を求めることができる。なお、logは常用対数である。
A=log{(W−B)/(T1−B)}
で求められる。ここで、Wはブランク画像の画素の明度、Bは暗画像の画素の明度、T1は修正された第1画像の画素の明度である。
V=ΣL=CΣA=CΣ[log{(W−B)/(T1−B)}] (式1)
で求められる。ここで、Cは定数である。このように、各画素の明度から吸光度を求め、吸光度を試料範囲全体にわたって積算することによって、細胞の推定体積値を求めることができる。
Vp=CpAp=Cplog{(ΣW−ΣB)/(ΣT1−ΣB)}
で表される。ここで、Cpは定数、Apは吸光度である。この式は試料範囲全体を一つの領域として吸光度を求めているが、細胞量が多ければ、例えば手術材料を出発材料とするような場合には、十分な精度が得られる。この式を用いる場合でも、上記画像処理によって、ゴミ等に起因するノイズの影響はすでに排除されている。
11 試料ステージ
12 照明
13 LEDパッケージ
14 照明電源
15 光拡散板
16 カラーカメラ
17 画像処理装置
20 試料
Claims (7)
- コラーゲンゲルに包埋して培養された標的細胞を色素で染色する工程と、
前記色素による吸光度が異なる第1の光および第2の光に対する透過像である第1画像および第2画像を取得する工程と、
前記第1画像および前記第2画像のそれぞれを複数の分割領域に分割し、前記分割領域毎に前記第1画像と前記第2画像を比較して、比較した分割領域の明度または吸光度の差または比が所定値未満であるときは、当該分割領域を前記標的細胞量の評価の基になるデータから除外することにより、気泡および不透明なゴミに起因するノイズを排除する工程と、
前記ノイズを排除した画像において前記分割領域毎の細胞量の指標を積算することにより、前記標的細胞量を評価する工程と、
を有する細胞測定方法。 - 前記ノイズを排除する工程は、前記分割領域毎に前記第1画像と前記第2画像を比較して、比較した分割領域の明度の差または比が所定値未満であるときは、当該分割領域を前記標的細胞量の評価の基になるデータから除外する工程である、
請求項1に記載の細胞測定方法。 - 前記ノイズを排除する工程は、前記分割領域毎に前記第1画像と前記第2画像を比較して、比較した分割領域の吸光度の差または比が所定値未満であるときは、当該分割領域を前記標的細胞量の評価の基になるデータから除外する工程である、
請求項1に記載の細胞測定方法。 - 前記標的細胞が癌細胞である、
請求項1〜3のいずれか一項に記載の細胞測定方法。 - 前記第1画像および前記第2画像は、前記第1の光および前記第2の光を同時に照射して、1台のカラーカメラで撮像した画像を色分解することにより取得される、
請求項1〜4のいずれか一項に記載の細胞測定方法。 - 前記第1画像および前記第2画像は、前記第1の光と前記第2の光を順次照射して、1台のカメラで都度撮像することにより取得される、
請求項1〜4のいずれか一項に記載の細胞測定方法。 - 前記標的細胞量の評価は、前記分割領域毎に画像明度から吸光度を算出し、得られた吸光度を複数の該分割領域にわたって積算して、標的細胞の推定体積値を算出することにより行われる、
請求項1〜6のいずれか一項に記載の細胞測定方法。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2015167122 | 2015-08-26 | ||
JP2015167122 | 2015-08-26 | ||
PCT/JP2016/073996 WO2017033809A1 (ja) | 2015-08-26 | 2016-08-17 | 細胞測定方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
JPWO2017033809A1 JPWO2017033809A1 (ja) | 2018-06-14 |
JP6767981B2 true JP6767981B2 (ja) | 2020-10-14 |
Family
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JP2017536770A Expired - Fee Related JP6767981B2 (ja) | 2015-08-26 | 2016-08-17 | 細胞測定方法 |
Country Status (6)
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---|---|
US (1) | US10514334B2 (ja) |
JP (1) | JP6767981B2 (ja) |
CN (1) | CN107922963A (ja) |
CA (1) | CA2993629A1 (ja) |
HK (1) | HK1248774A1 (ja) |
WO (1) | WO2017033809A1 (ja) |
Families Citing this family (1)
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TWI733927B (zh) | 2016-11-01 | 2021-07-21 | 日商倉敷紡績股份有限公司 | 細胞測定方法 |
Family Cites Families (19)
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US3327117A (en) * | 1963-08-12 | 1967-06-20 | Ibm | Cancer cell detector using two wavelengths for comparison |
JPS61226638A (ja) | 1985-03-30 | 1986-10-08 | Shimadzu Corp | 顕微鏡分光像形成装置 |
JPH0728757B2 (ja) | 1990-03-15 | 1995-04-05 | 新田ゼラチン株式会社 | 制癌剤感受性試験方法 |
US5356793A (en) | 1990-03-15 | 1994-10-18 | Nitta Gelatin Inc. | Method for testing the sensitivity of anticancer drug |
DE69430054T2 (de) | 1993-12-30 | 2002-10-24 | Nitta Gelatin Inc., Osaka | Verfahren zum einbetten von tierkulturzellen |
JP3222785B2 (ja) * | 1996-10-11 | 2001-10-29 | 新田ゼラチン株式会社 | 癌細胞の定量測定方法 |
JP2000131322A (ja) * | 1998-10-29 | 2000-05-12 | Matsushita Electric Works Ltd | グルコース濃度の定量方法及びその装置 |
JP3363445B1 (ja) | 2001-04-23 | 2003-01-08 | 新田ゼラチン株式会社 | 生検採取細胞の培養方法および動物細胞培養用キット |
SE0104443D0 (sv) * | 2001-12-28 | 2001-12-28 | Hemocue Ab | Analysis method and cuvette therefor |
JP2003222624A (ja) * | 2002-01-29 | 2003-08-08 | Miura Co Ltd | 5,5−ジブロモ−4,4−ジクロロインジゴの存否確認方法 |
MX345106B (es) * | 2003-03-28 | 2017-01-16 | Inguran Llc * | Método y aparato para orientar esperma en una corriente de líquido. |
US8129105B2 (en) * | 2006-04-13 | 2012-03-06 | Ralph Zuckerman | Method and apparatus for the non-invasive measurement of tissue function and metabolism by determination of steady-state fluorescence anisotropy |
JP4866162B2 (ja) | 2006-07-06 | 2012-02-01 | 倉敷紡績株式会社 | 抗がん作用の評価方法 |
US20110033887A1 (en) * | 2007-09-24 | 2011-02-10 | Fang Nicholas X | Three-Dimensional Microfabricated Bioreactors with Embedded Capillary Network |
ITTO20070771A1 (it) * | 2007-10-29 | 2009-04-30 | Silicon Biosystems Spa | Metodo e apparato per la identificazione e manipolazione di particelle |
CN104094099B (zh) * | 2011-04-15 | 2017-05-03 | 罗氏血液诊断股份有限公司 | 测量细胞体积和成份 |
KR101463005B1 (ko) * | 2013-10-15 | 2014-11-18 | (주)한국해양기상기술 | 특정 파장에 대한 형광 특성을 갖는 미생물 검사방법 |
CN105899936B (zh) * | 2013-11-13 | 2019-12-24 | 贝克顿·迪金森公司 | 光学成像系统及使用其的方法 |
JP2017176811A (ja) * | 2016-03-28 | 2017-10-05 | ソニー株式会社 | 撮像装置、撮像方法及び医療用観察機器 |
-
2016
- 2016-08-17 US US15/754,291 patent/US10514334B2/en not_active Expired - Fee Related
- 2016-08-17 CN CN201680047633.5A patent/CN107922963A/zh active Pending
- 2016-08-17 JP JP2017536770A patent/JP6767981B2/ja not_active Expired - Fee Related
- 2016-08-17 CA CA2993629A patent/CA2993629A1/en not_active Abandoned
- 2016-08-17 WO PCT/JP2016/073996 patent/WO2017033809A1/ja active Application Filing
-
2018
- 2018-06-28 HK HK18108327.7A patent/HK1248774A1/zh unknown
Also Published As
Publication number | Publication date |
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US10514334B2 (en) | 2019-12-24 |
HK1248774A1 (zh) | 2018-10-19 |
WO2017033809A1 (ja) | 2017-03-02 |
JPWO2017033809A1 (ja) | 2018-06-14 |
US20180252636A1 (en) | 2018-09-06 |
CN107922963A (zh) | 2018-04-17 |
CA2993629A1 (en) | 2017-03-02 |
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