JP5496906B2 - 画像から自己蛍光を除去する方法及びシステム - Google Patents
画像から自己蛍光を除去する方法及びシステム Download PDFInfo
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- JP5496906B2 JP5496906B2 JP2010535365A JP2010535365A JP5496906B2 JP 5496906 B2 JP5496906 B2 JP 5496906B2 JP 2010535365 A JP2010535365 A JP 2010535365A JP 2010535365 A JP2010535365 A JP 2010535365A JP 5496906 B2 JP5496906 B2 JP 5496906B2
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Classifications
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- G—PHYSICS
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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
- G01N21/62—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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
- G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels
Description
本実施例では、DAPI、Cy3及びCy5で染色した生体材料の画像が取得されるが、限定的なものではない。これら3つの色素の中で、核のマーカーであるDAPIは、非常に高いシグナル対自己蛍光比(低AF)を有することが知られている。第1の段階において、DAPIに対応するフィルターキューブを用いた材料の画像を取得する。さらに、Cy3及びCy5に対応するフィルターキューブを用いた材料の第1組の画像も取得する。追加の色素を添加する前の、追加の色素に対応するフィルターキューブ画像を用いたこれらの追加の画像は、追加の色素を添加した後に撮影される画像(これは、後に追加の色素に対応する自己蛍光を示すことになる)に対する対照画像又は対照画像としての役割を果たす。さらに、DAPI、Cy3及びCy5との相互干渉が最小限のフィルターキューブを用いた追加の光学画像も取得する。この最小限の相互干渉のフィルターキューブは、シアン蛍光タンパク質(CFP)に対応する。このフィルターキューブは、自己蛍光のみの画像を取得する時に使用され、以下にさらに詳細に記載するように、画像の位置決めのためにのみ使用される。
Claims (22)
- 生体材料に付随する固有の自己蛍光を除去するための方法であって、
a)生体材料の第1の対照画像を取得する段階と、
b)1種以上の情報色素に対応する1以上のフィルターを用いた生体材料の第1組の1以上の画像を取得する段階と、
c)前記1種以上の情報色素を生体材料に添加し、次いで前記情報色素に対応する各々のフィルターを用いた生体材料の別々の画像と生体材料の第2の対照画像とを含む第2組の1以上の画像を取得する段階と、
d)前記第1の対照画像と第2の対照画像を位置合わせする段階と、
e)次いで段階c)で取得した画像にみられる固有の自己蛍光を除去する段階と
を含む方法。 - 前記第1の対照画像と第2の対照画像が、シアン蛍光タンパク質に対応するフィルターを用いて取得される、請求項1記載の方法。
- 段階a)が、高いシグナル対自己蛍光比を有する対照色素を生体材料に添加する段階をさらに含み、前記第1の対照画像と第2の対照画像が、前記対照色素に対応するフィルターを用いて取得される、請求項1記載の方法。
- 段階a)で添加される色素がUVスペクトルに対応する色素を含む、請求項3記載の方法。
- 前記UVスペクトルに対応する段階a)で添加される色素がDAPIである、請求項4記載の方法。
- 段階a)で添加される色素がIRスペクトルに対応する色素を含む、請求項3記載の方法。
- 生体材料の第1の対照画像と第2の対照画像が、シアン蛍光タンパク質に対応するフィルターを用いて取得され、
前記第1の対照画像が座標系を有する静止画像であり、前記第2の対照画像が座標系を有する動画像であり、1以上の根本的変換パラメータを推定し、前記静止画像の座標系の上に前記動画像をマッピングすることによって、前記対照画像が少なくとも部分的に位置合わせされて、座標系を有する合成画像を形成する、請求項1記載の方法。 - 段階a)が高いシグナル対自己蛍光比を有する対照色素を生体材料に添加する段階をさらに含み、
前記第1の対照画像及び第2の対照画像が、前記対照色素に対応するフィルターを用いて取得され、
前記第1の対照画像が座標系を有する静止画像であり、前記第2の対照画像が座標系を有する動画像であり、前記対照画像が、1以上の根本的変換パラメータを推定し、前記静止画像の座標系の上に前記動画像をマッピングすることによって、少なくとも部分的に位置合わせされて、座標系を有する合成画像を形成する、請求項1記載の方法。 - 前記対照画像が、変換、回転及び拡大縮小からなる群から選択される1以上の変換パラメータを用いる相似変換を用いて少なくとも部分的に位置合わせされる、請求項1記載の方法。
- 前記対照画像が、次式に従って1以上の根本的変換パラメータを推定し、静止画像の座標系の上に動画像をマッピングすることによって位置合わせされる、請求項1記載の方法。
- 生体材料に伴う固有の自己蛍光を除去するためのシステムであって、
a)高いシグナル対自己蛍光比を有する1種以上の対照色素の存在を示す生体材料の対照画像を解析するように構成された処理装置と、
b)前記対照色素に対応する1以上のフィルター及び1種以上の情報色素に対応する1以上のフィルターと、
c)前記対照色素及び前記1種以上の情報色素に対応するフィルターと組合せて、生体材料の第1組の1以上の画像を取得するように構成され、前記対照色素及び前記1種以上の情報色素に対応するフィルターと組合せて、生体材料の第2組の1以上の画像を取得するようにさらに構成され、生体材料が1種以上の情報色素の存在をさらに示す、デジタルイメージング装置とを含み、
前記第2組の画像が、前記対照色素と前記1種以上の情報色素に対応する各々のフィルターを用いた生体材料の別々の画像を含み、
前記処理装置が前記対照画像を位置合わせした後、1種以上の情報色素の存在を示す画像から前記固有の自己蛍光を除去するようにさらに構成される、システム。 - 前記対照色素のうちの少なくとも1つが前記UVスペクトルに対応する、請求項11記載のシステム。
- 前記UVスペクトルに対応する色素のうちの少なくとも1つがDAPIである、請求項12記載のシステム。
- 前記対照色素のうちの少なくとも1つが前記IRスペクトルに対応する、請求項11記載のシステム。
- 前記デジタルイメージング装置が、シアン蛍光タンパク質に対応する対照フィルターを用いて生体材料の対照画像を取得するように構成され、
前記プロセッサが、1以上の根本的変換パラメータを推定し、画像の座標系をマッピングすることによって前記対照画像の位置合わせをするように構成される、請求項11記載のシステム。 - 前記対照画像が、強度ベースのパラメータ又は特徴ベースのパラメータを用いて位置合わせされる、請求項11記載のシステム。
- 前記対照画像が、変換、回転及び拡大縮小を組み込んだ相似変換を用いて、少なくとも部分的に位置合わせされる、請求項11記載のシステム。
- 前記対照画像が、次式に従って1以上の根本的変換パラメータを推定し、静止画像の座標系の上に動画像をマッピングすることによって位置合わせされる、請求項11記載のシステム。
- 生体材料に伴う固有の自己蛍光を除去するためのシステムであって、
a)高いシグナル対自己蛍光比を有するシアン蛍光タンパク質に対応する対照フィルターを用いて撮影された生体材料の対照画像を解析するように構成された処理装置と、
b)1種以上の情報色素に対応する1以上のフィルターと、
c)前記対照フィルター及び前記1種以上の情報色素に対応するフィルターと組合せて、生体材料の第1組の1以上の画像を取得するように構成され、前記対照フィルターと前記1種以上の情報色素に対応するフィルターと組合せて、生体材料の第2組の1以上の画像を取得するようにさらに構成され、生体材料が1種以上の前記情報色素の存在をさらに示す、デジタルイメージング装置とを含み、
前記第2組の画像が、前記対照フィルター及び前記1種以上の情報色素に対応する各々のフィルターを用いた生体材料の別々の画像を含み、
前記処理装置が、前記対照画像を位置合わせした後、1種以上の情報色素の存在を示す画像から前記固有の自己蛍光を除去するようにさらに構成される、システム。 - 前記対照画像が、強度ベースのパラメータ又は特徴ベースのパラメータを用いて位置合わせされる、請求項19記載のシステム。
- 前記対照画像が、変換、回転及び拡大縮小を組み込んだ相似変換を用いて、少なくとも部分的に位置合わせされる、請求項19記載のシステム。
- 前記対照画像が、次式に従って1以上の根本的変換パラメータを推定し、静止画像の座標系の上に動画像をマッピングすることによって位置合わせされる、請求項19記載のシステム。
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US20090141959A1 (en) | 2009-06-04 |
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