JP2006258821A - 標識ターゲットを検出するシステムおよび方法 - Google Patents
標識ターゲットを検出するシステムおよび方法 Download PDFInfo
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Abstract
【解決手段】基板の表面上のマークされた領域を検出するシステムは、励起放射源と、該励起放射源からの放射を該基板の該表面の選択領域上にフォーカシングさせる焦点システムであって、フォーカスされたスポット径に対する走査フィールド径の比が約2000より大きく且つ開口数が約0.2よりも大きい対物レンズを含むフォーカシングシステムと、該基板の該表面にわたって少なくとも5画像ライン/秒の速度で該フォーカスされた励起放射を走査する放射指向システムと、 該励起放射に応答して該基板の該表面からの発光を検出する検出器であって、該対物レンズが該発光を受けて該発光を該検出器に伝送する、検出器と、 該検出された発光の量を、該発光が発せられた該基板の該表面上の位置の関数として記録するデータ取得システムを有する。
【選択図】図1
Description
I.定義
II.総論
a.はじめに
b.撮像システムの概観
III.撮像システムの1つの実施形態の詳細な記述
a.検出装置
b.データ獲得
IV.撮像システムの第2の実施形態の詳細な説明
以下の用語は、本明細書で使用される場合には以下の一般的な意味を有するように意図される。
A.はじめに
本発明は、基板を走査して高感度および高解像度の画像を高速で得る方法および装置を提供する。本発明は、広い範囲の用途を有し、特に、100mm2に対して2または3μm2などの、大きな領域内から微視的な領域の定量研究を必要とする場合にも使用され得る。例えば、本発明は、組織学(組織化学的に染色された、および免疫学的に蛍光染色された画像の研究)、または蛍光原位置ハイブリダイゼーションの分野において適用され得る。1つの適用では、本発明は、本明細書で述べるように、支持体上に製造されたプローブ配列アレイを撮像するために使用される。
高解像度の走査システムおよび方法は、微視的、巨視的を問わず、電子産業界、例えば半導体および微細製造業界で、微細製造の電子部品、例えばマイクロプロセッサ、マイクロ回路などを走査するために、日常的に使用されている。しかし、このような走査はまた、化学および遺伝子分析を組み合わせた分野で非常に有用である。特に、高解像度の走査方法および装置は、ポリマーアレイの適用において使用され得る。これらのポリマーアレイは、一般に、典型的には平坦な基板の表面に結合される多くの異なるポリマー配列よりなる。
本発明の装置は概して、基板の表面に亘って活性化放射ビームまたはスポットを迅速に掃引する走査装置を用いる。装置はさらに、励起放射を基板の表面上において、基板上に高解像のフィーチャーを提供するに十分小さくフォーカスさせ、同時に広い走査フィールドを提供する焦点光学系を含む。標識が照射されたときにサンプル上の標識によって発された電磁放射を検出することにより、像が得られる。1つの実施形態において、蛍光発光が焦点光学系によって収集され検出されることによって、基板表面上に蛍光の像が形成される。好適な局面において、本発明の装置はさらに、共焦検出系を用いることにより、励起放射の焦点の面の上方または下方の構造から不要な信号を減少または排除し、さらに自動焦点系を用いることにより基板表面上の活性化放射と表面からの発光放射との両方をフォーカスさせる。概して、励起放射および応答発光は、異なる波長を有する。望まれない発光の検出を阻止するために、標識の発光帯内に対する高透過率と励起波長内に対する低透過率とを有するフィルタが用いられ得る。これらは概して、背景ノイズの潜在的源として、焦点のずれた平面からの発光または散乱励起照射を含む。
A.検出装置
図1は、本発明に基づく撮像システム100を示す光学ブロック図である。典型的には、撮像システム100は、蛍光標識されたDNAまたはRNAが結合したオリゴヌクレオチドプローブアレイの像を得るために用いられる。また、ウェハまたはマスク検査あるいはポリペプチドその他のポリマーアレイ、電気泳動ゲル、あるいは生物学的標本の撮像など、他のアプリケーションにおいても使用され得る。図1に示すように、レーザ102からの励起放射ビーム(例えば488nm光)が、ビームスプリッタ104により部分的に反射されかつ部分的に透過される。ビームの反射された部分は、フォトディテクタ131(オプションである)に入射する。フォトディテクタ131は典型的には、レーザ出力モニタとして用いられるフォトダイオードである。ビームのうちビームスプリッタ104を透過した部分はダイクロイックビームスプリッタ106および108によって反射され、レンズ111および112を透過する。レンズ111および112は、レーザ102から発せられたビームを拡大し、基板表面から得られたコリメートされた蛍光を縮小(demagnify)するためのテレスコープを提供する。一実施態様例において、レンズ111の焦点距離は20mmであり、レンズ112の焦点距離は80mmである。その他の焦点距離および焦点距離比を用いてもよいが、システム性能(共焦性、解像度など)に影響を及ぼす場合がある。拡大されたレーザビームはミラー114によって反射されてレンズ116によってフォーカスされる(以下により詳細に説明する)。
具体的には、レーザビームが集束されるサンプル118上の位置から発せられる戻り光は、最大限ピンホールを透過するのに対して、他の位置から発せられる光はピンホールを透過しない。レンズ134、120および132は、好ましくは、50mmの焦点距離を有する。ピンホール136および122は、好ましくは、100ミクロンの直径を有し、ピンホール128は、好ましくは、50ミクロンの直径を有する。他のレンズの焦点距離およびピンホール直径も使用可能であるが、システムの性能(共焦性、調整不良に対する感度など)に影響し得る。
図3に示すように、撮像システム300は、ガルバノメータ306(ミラー114が取り付けられている)およびGeneral Scanning Inc.(Watertown、MA)から入手できるガルバノメータ駆動盤305を有する。ガルバノメータ306は、モデルM2Tである。他の適切なガルバノメータおよび駆動盤は、例えば、Cambridge Technology Inc.(Watertown、MA)から入手できる。駆動盤305への入力は、コンピュータ302内のISAスロットに設けられた任意の波長生成器304(Keithley Metrabyte Model PCIP−AWFG、Taunton、MA)からの電圧波形である。駆動盤305の回路は、常に、ガルバノメータ306を、波形生成器304によって命令される角度位置(所望の角度位置は、波形電圧に線形に関連する)に付勢する。波形生成器304は、プログラムされた後、コンピュータ302によってさらに干渉されずに、無限に波形を生成し得る。使用される波形は、通常、鋸歯状波形であり、例えば、波形周期が33.3ミリ秒である場合、電圧は、25ミリ秒間線形に上昇(ramp up)し、この間、レーザビームがフィールドわたって通過している間にデータが得られ、次の8.3ミリ秒間で、電圧は、初期の値に戻り、レーザビームはたどり直す。正弦波または対称三角波などの他の波形も使用され得る。PCIP−AWFG波形生成器を必要としない波形生成の様々な方法が公知である。
撮像システム400の第2の実施形態の光学ブロック図が、図4に示される。示されるように、撮像システム400は、図1に示されるシステムと共通の構成要素を含む。このスキャナーでは、レンズ116は、無限共役比(conjugate ratio)ではなく有限共役比で使用されるように意図されるため、レンズ112は省略される。このスキャナーは、ピンホール401を1つしか使用せず、戻り光はすべて、このピンホール401を通過してから、様々なダイクロイックビームスプリッタによって分離される。このスキャナーは、2つの追加のダイクロイックビームスプリッタ、スペクトルフィルタおよび光電子倍増管を有するため、2チャネル蛍光走査ではなく、4チャネル蛍光走査に使用することができる。
Claims (34)
- 基板の表面上のマークされた領域を検出するシステムであって、 励起放射源と、 該励起放射源からの放射を該基板の該表面の選択領域上にフォーカシングさせる焦点システムであって、フォーカスされたスポット径に対する走査フィールド径の比が約2000より大きく且つ開口数が約0.2よりも大きい対物レンズを含むフォーカシングシステムと、 該基板の該表面にわたって少なくとも5画像ライン/秒の速度で該フォーカスされた励起放射を走査する放射指向システム(radiation direction system)と、 該励起放射に応答して該基板の該表面からの発光を検出する検出器であって、該対物レンズが該発光を受けて該発光を該検出器に伝送する、検出器と、 該検出された発光の量を、該発光が発せられた該基板の該表面上の位置の関数として記録するデータ取得システム(data acquisition system)と、を備えた、システム。
- 前記フォーカシングシステムのフォーカスされたスポット径に対する走査フィールド径の比は3000より大きい、請求項1に記載のシステム。
- 前記フォーカシングシステムのフォーカスされたスポット径に対する走査フィールド径の比は4000より大きい、請求項1に記載のシステム。
- 前記フォーカシングシステムは、前記励起放射を直径約10μm未満のスポットにフォーカスさせる、請求項1に記載のシステム。
- 前記フォーカシングシステムは、前記励起放射を前記基板の前記表面上において直径約5μm未満のスポットにフォーカスさせる、請求項1に記載のシステム。
- 前記フォーカシングシステムは、前記励起放射を前記基板の前記表面上において直径約3μmのスポットにフォーカスさせる、請求項1に記載のシステム。
- 前記走査フィールド径は約10mmより大きい、請求項1に記載のシステム。
- 前記走査フィールド径は約14mmである、請求項1に記載のシステム。
- 前記開口数は約0.25より大きい、請求項1に記載のシステム。
- 前記フォーカシングシステムはアクロマチックである、請求項1に記載のシステム。
- 前記放射指向システムは、前記基板にわたって少なくとも10画像ライン/秒の速度でスポットを走査させ得る、請求項1に記載のシステム。
- 前記放射指向システムは、前記基板にわたって少なくとも30画像ライン/秒の速度でスポットを走査させ得る、請求項1に記載のシステム。
- 前記放射指向システムは角振動ミラー(angularly oscillating mirror)または回転多面ミラー(rotating polyhedral mirror)を含む、請求項1に記載のシステム。
- 前記基板が搭載される並進ステージをさらに備え、該並進ステージは前記対物レンズの光学軸に垂直な少なくとも1つの寸法方向(dimension)に可動である、請求項1に記載のシステム。
- 前記基板の前記表面を前記フォーカシングシステムの焦点面に配置するオートフォーカスシステムをさらに備えた、請求項1に記載のシステム。
- 前記基板が搭載される並進ステージをさらに備え、該並進ステージは前記フォーカシングシステムの光学軸に平行な少なくとも1つの寸法方向および該フォーカシングシステムの該光学軸に垂直な少なくとも1つの寸法方向に可動である、請求項1に記載のシステム。
- 集光システムをさらに備え、該集光システムは、 前記基板の前記表面から発光された蛍光を集光し、該基板の該表面から反射した励起放射を集光する集光光学系と、 該発光された蛍光を該基板の該表面から反射した励起放射から分離し、該蛍光を共焦ピンホールを通してフォーカスさせる分離光学系(separation optics)と、 該蛍光に応答して、該共焦ピンホールを通ってフォーカスした該蛍光の量を記録するレコーダと、を備えている、請求項1に記載のシステム。
- 前記基板の前記表面は、該表面上にある複数の異なる既知の位置において、複数の互いに異なるポリマーシーケンス(polymer sequences)を含む、請求項1に記載のシステム。
- 前記異なるポリマーシーケンスのそれぞれは、幅または長さ寸法の少なくとも一方が約50μm未満であるフィーチャー内に含まれる、請求項18に記載のシステム。
- 信号を処理および格納して、これにより、サンプルの2次元画像を生成するプロセッサをさらに備えた、請求項1に記載のシステム。
- サンプルが載った少なくとも第1の表面を有する前記基板を固定化する本体をさらに備え、該本体は、 搭載面と、 該搭載面内のキャビティ(cavity)であって、該第1の表面が該搭載面に結合して該キャビティを封止し、該サンプルは該キャビティと流体連通しており(in fluid communication)、該キャビティは光吸収材料を含む底面を有する、キャビティと、 該キャビティと連通した入口および出口であって、該サンプルに接触するために該キャビティ内へと流れる流体は該入口を通って流れ、該キャビティから流出する流体は該出口を通って流れる、入口および出口と、 該キャビティ内の温度を制御する温度制御装置と、を備えている、請求項1に記載のシステム。
- 前記温度制御装置は熱電冷却器を含む、請求項21に記載のシステム。
- 基板の表面上のマークされた領域を検出するシステムであって、 励起放射源と、 該励起放射を該基板の該表面上で直径5μm未満のスポットにフォーカスさせる第1の焦点光学系であって、対物レンズを含む焦点光学系と、 該スポットを該基板の該表面にわたって直線的に走査させる往復放射指向システムであって、該スポットの移動距離は少なくとも10mmである、往復放射指向システムと、 該基板の該表面からの発光を該基板の該表面から反射した該励起放射から分離する光学トレインであって、該対物レンズを含む光学トレインと、 該基板の該表面を該第1の焦点光学系の焦点面に自動的に配置するオートフォーカスシステムと、を備えた、システム。
- 前記光学トレインは、前記基板の前記表面から発光された蛍光を該基板の該表面から反射した前記励起放射から分離するダイクロイックビームスプリッタを含む、請求項23に記載のシステム。
- 前記オートフォーカスシステムは、 前記基板の前記表面から反射した前記励起放射を共焦ピンホールを通してフォトダイオード上にフォーカシングする第2の焦点光学系と、 該基板が搭載される並進ステージであって、該基板を該第1の焦点光学系の焦点面へと動かすために、該第1の焦点光学系の光学軸に平行な方向に可動である並進ステージと、を備えている、請求項23に記載のシステム。
- 前記励起放射に応答して前記基板の前記表面からの発光を検出する検出器と、該検出された発光量を、該発光が発せられた該基板の該表面上の位置の関数として記録するデータ取得システムとをさらに備えた、請求項23に記載のシステム。
- それぞれ異なる既知のロケーションにおいて基板の表面上に固定化された複数の異なるポリマーシーケンスを有するポリマーアレイを走査し、これにより、該アレイ上のどのポリマーシーケンスが標的分子と結合しているかを特定する方法であって、 対物レンズを用いて励起放射源を該基板の該表面上にフォーカスさせる工程と、 該基板の該表面にわたって少なくとも約5画像ライン/秒の速度で該励起放射を走査させる工程と、 該対物レンズを用いて、該励起放射に応答して該基板の該表面からの発光を集光する工程と、 該基板の該表面上の位置の関数として該発光を記録する工程であって、該位置が標的分子と結合した該アレイ上の該ポリマーシーケンスを示す、工程と、を包含する、方法。
- 前記ポリマーアレイは、異なる既知のロケーションにおいて前記基板の前記表面上に固定化された複数の異なるオリゴヌクレオチドシーケンスを含む、請求項27に記載の方法。
- 前記励起放射源を直径約3μmのスポットにフォーカスさせる工程と、前記基板上で少なくとも14mmにわたって直線方向に該スポットを走査させる工程とをさらに包含する、請求項27に記載の方法。
- 前記基板を本体上に固定化する工程と、 第1の波長を有する電磁放射源からの励起放射で該基板上の前記サンプルを励起する工程と、 該励起放射に応答して第2の波長を有する応答放射を検出する工程であって、該応答放射は前記複数の領域の画像を表す、工程と、 該サンプル上の次の複数の領域を励起する工程と、 該応答放射を処理および格納して、これにより、該サンプルの2次元画像を生成する工程と、 該サンプルを該励起放射の焦点面にオートフォーカスする工程と、をさらに包含する、請求項27に記載の方法。
- 前記本体は上にキャビティのある搭載面を備え、前記基板は、前記サンプルが前記反応チャンバと流体連通するように該搭載面上に固定化され、該反応チャンバは、該反応チャンバ内におよび該反応チャンバを通して、流体を流すための入口および出口を備えている、請求項30に記載の方法。
- 前記本体は、前記キャビティ内の温度を制御するための温度制御装置をさらに備えている、請求項31に記載の方法。
- 前記励起放射を前記基板の前記表面にわたって少なくとも約10画像ライン/秒の速度で走査させる、請求項27に記載の方法。
- 前記励起放射を前記基板の前記表面にわたって少なくとも約30画像ライン/秒の速度で走査させる、請求項27に記載の方法。
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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KR20150041667A (ko) * | 2012-08-24 | 2015-04-16 | 가부시끼가이샤 사따께 | 미생물 검사 방법 및 그 장치 |
KR102024974B1 (ko) | 2012-08-24 | 2019-09-24 | 가부시끼가이샤 사따께 | 미생물 검사 방법 및 그 장치 |
Also Published As
Publication number | Publication date |
---|---|
JP2000512744A (ja) | 2000-09-26 |
EP0902885A1 (en) | 1999-03-24 |
US5981956A (en) | 1999-11-09 |
US20020185610A1 (en) | 2002-12-12 |
WO1997043611A1 (en) | 1997-11-20 |
US6597000B2 (en) | 2003-07-22 |
US20010030290A1 (en) | 2001-10-18 |
EP0902885A4 (en) | 2006-09-27 |
US6207960B1 (en) | 2001-03-27 |
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