JP5656920B2 - 蛍光ナノスコピー方法 - Google Patents
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Description
Claims (13)
- 対象物のナノスコピー方法において、
・対象物を非蛍光体により染色し、
・生成プロセスで、前記非蛍光体の分子を、励起されると蛍光することのできる分子に修正し、
・励起プロセスで、前記修正された分子を励起し、これにより蛍光させ、
・記録プロセスで、
−前記染色された対象物をフレームごとに検出装置によって光学的にキャプチャし、
−前記フレーム中に蛍光する分子を少なくとも1つの検出装置によって、回折に依存する大きさを有するスポットとして記録し、
−前記記録されたフレームを記憶媒体に記憶し、
・前記生成プロセス、前記励起プロセス、および前記記録プロセスを周期的に繰り返すナノスコピー方法において、
−光を分割するプリズムと前記少なくとも1つの検出装置としての2つのカメラが、対象物の隣接する2つの平面がそれぞれのカメラの焦点内にあり、1つの対物レンズを通して前記カメラ上に投影されるように配置されており、
−前記各カメラが、各蛍光分子ごとに異なるスポットを検出し、
−コンピュータプログラムが、
・前記カメラにより検出されたスポットの強度分布間の差を使用して、前記蛍光分子の第3の座標を計算し、
・前記対象物の3D画像を形成するために使用される、
ことを特徴とするナノスコピー方法。 - 前記キャプチャされたフレームは、当該フレーム中の他の分子の蛍光よりも弱い残留蛍光を有する分子により形成されたスポットを含み、該残留蛍光は、前記励起プロセスによるサイクル内で色喪失プロセスが生じた結果である、請求項1に記載の方法。
- 前記キャプチャされたフレーム中に検出されたスポットの中心座標がコンピュータプログラムによって決定され、前記中心座標は、対象物を染色する分子の位置に関連しており、
前記検出されたスポットの決定された座標が、回折限界よりも良好な解像度で染色された対象物の提示を形成するために使用される、請求項1または2に記載の方法。 - 前記記録プロセスで、残留蛍光を有するフレームと、該残留蛍光に重なる蛍光分子を有するフレームとが、記録可能媒体に記録され、
前記残留蛍光を有するフレームが、当該残留蛍光に重なる蛍光分子の画像を含むフレームから減算され、減算されたフレームが獲得され、
前記減算されたフレームは、スポット中心とスポット強度分布の計算に基づき分子位置を計算するために使用される、請求項1に記載の方法。 - 前記生成プロセスで、前記修正は、物理的影響または化学反応によって実行される、請求項1から4までのいずれか1項に記載の方法。
- 前記非蛍光体の分子の前記修正が、蛍光をブロックする化学群を光分解することによって実行される、請求項5に記載の方法。
- 前記非蛍光体の分子の前記修正が、当該分子に光またはUV光パルスを照射することによって実行される、請求項5に記載の方法。
- 前記励起プロセスで、前記修正された分子を照射し、蛍光させるためにレーザが使用される、請求項1から7までのいずれか1項に記載の方法。
- 前記レーザは、対物レンズを通してまたは対象物スライドを通して前記修正された分子を備える対象物を照射する、請求項8に記載の方法。
- 前記レーザは、前記修正された分子を備える対象物を、全深度で照射するか、または全反射の角度で照射する、請求項8または9に記載の方法。
- 前記カメラはビデオをキャプチャし、キャプチャされたフレームはビデオフレームである、請求項1に記載の方法。
- 複数のダイクロイックミラーと複数のカメラが、種々の波長で分子の蛍光を検出するために使用される、請求項1または11に記載の方法。
- ・対象物の隣接する2つの平面がそれぞれのカメラの焦点内にあり、1つの対物レンズを通してカメラ上に投影されるように配置される、光を分割するプリズムおよび少なくとも1つの検出装置としての2つのカメラと、なお、前記各カメラは、各蛍光分子ごとに異なるスポットを検出し、
・前記試料の蛍光光をピックアップするために、前記2つのカメラの前方に配置された交換可能なカラーフィルタセットと、
・物理的影響または化学反応により、励起されると蛍光することのできる分子に、前記試料の染色された非蛍光染色分子を周期的に変換するための生成コンポーネントと、
・前記試料内で前記分子の蛍光を励起する光源を含む励起コンポーネントと、
・前記検出装置から受信された画像を記録するための記録媒体と、
・前記カメラにより検出されたスポットの強度分布間の差を使用して、前記蛍光分子の第3の座標を計算し、かつ、前記対象物の3D画像を形成するプロセッサと、
を有する蛍光ナノスコピー装置。
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CN102023148B (zh) | 2013-07-24 |
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CN102023148A (zh) | 2011-04-20 |
WO2006123967A2 (fr) | 2006-11-23 |
US20130099136A1 (en) | 2013-04-25 |
EP1892517A2 (en) | 2008-02-27 |
EP1892517A4 (en) | 2013-03-13 |
WO2006123967A3 (fr) | 2007-03-22 |
RU2005115052A (ru) | 2006-11-27 |
CA2648777A1 (en) | 2006-11-23 |
CN101228428B (zh) | 2012-11-07 |
US9028757B2 (en) | 2015-05-12 |
US20140127079A1 (en) | 2014-05-08 |
JP2012198234A (ja) | 2012-10-18 |
US8668872B2 (en) | 2014-03-11 |
US20150316478A1 (en) | 2015-11-05 |
JP2008541128A (ja) | 2008-11-20 |
US8110405B2 (en) | 2012-02-07 |
US8334143B2 (en) | 2012-12-18 |
US7803634B2 (en) | 2010-09-28 |
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