JP3828799B2

JP3828799B2 - Thin-layer oblique illumination method for optical systems

Info

Publication number: JP3828799B2
Application number: JP2001380967A
Authority: JP
Inventors: 万喜洋徳永
Original assignee: Japan Science and Technology Agency; National Institute of Japan Science and Technology Agency
Current assignee: Japan Science and Technology Agency; National Institute of Japan Science and Technology Agency
Priority date: 2001-12-14
Filing date: 2001-12-14
Publication date: 2006-10-04
Anticipated expiration: 2021-12-14
Also published as: JP2003185930A

Description

【０００１】
【発明の属する技術分野】
本発明は、物質や分子を光を使って高感度検出する顕微鏡と同等の光学系の薄層斜光照明法に関する。
【０００２】
【従来の技術】
従来、物質や分子を光を使って高感度検出する光学顕微鏡の照明技術は種々のものが提案されており、その内の幾つかを以下に説明する。図９は、蛍光顕微鏡の照明として、現在一般的に使われている落射照明方法の概略図である。図に示すように、光源からの光を、照射光は反射し、蛍光は透過させるダイクロイックミラーで反射させて、対物レンズ中央に入射し、試料観察面を照射するものである。
【０００３】
また、ガラス表面近傍のみを局所的に照明する方法として、全反射照明法がある。全反射した際に生じるエバネッセント光（深さ１００２００ｎｍ程度）を使って照明するものである。プリズムを使う方法と対物レンズを使う方法があるが、図１０に対物レンズ型全反射照明法の概略図を示す。対物レンズの開口数をＮＡ、試料溶液の屈折率をｎとしたとき、開口数（ＮＡ）がＮＡ＞ｎの式を満たす対物レンズにより全反射照明が可能となる。この方法は、従来、極く限られた利用のみであったが、蛍光色素１分子を蛍光顕微鏡で観察する方法として有用であることが判明し、最近１分子イメージング用に使用され始めている。
【０００４】
更に、本発明者が提案し、特許第３０９３1４５号として既に特許されている光照射切り替え方法の概略を図１１に示す。１は試料溶液、２はカバーガラス、３はオイル、４は対物レンズ（レンズ群）、５は照射光、６は照射光、７はダイクロイックミラーである。落射照明から全反射照明の状態への変更を、ミラー等の光学部品を図１１（Ａ）→１１（Ｂ）→図１１（Ｃ）へと移動することのみで実現することができ、余分な光学系を必要とせず、簡単な原理で照射の切替を行えることができる。また、この方法では、入射光位置又は光源位置（例えば光ファイバーの出射位置）をずらすことによっても同等の状態変更を実現することができる。
【０００５】
また、他の方法として、通常の光学顕微鏡や落射照明法による蛍光顕微鏡において、セクショニング像や３次元画像を得る方法としては、焦点を連続的に変化させて得た連続画像からデコンボリューションによって計算する方法が用いられている。これは、試料上の１点から出た光の像が、焦点からはずれた時にどうなるかを予め知っておくことにより、計算によって元の３次元像を計算するものであるが、厚みのある試料や、明るい中にある暗い部分を観察する場合には、この方法では限界がある。
【０００６】
また、他の方法として、セクショニング像や３次元画像を用いる方法としては、現在、共焦点顕微鏡法が広く普及している。この方法は、高感度カメラに比べると感度が劣っており、特に蛍光試料の観察においては、レーザー光を１点に集光して照射するために、蛍光色素の退色が早くなったり、生物試料に損傷を与えるなどの難点がある。
【０００７】
【発明が解決しようとする課題】
このように、従来の方法では、厚みのある試料や明るい中にある暗い部分の観察において、高感度、高Ｓ／Ｎ比を得ることができなかった。そこで本発明は、顕微鏡と同等の光学系を用いて光を使って物質や分子を高感度検出することを可能とし、光学顕微鏡における低背景・高感度観察を可能にする光学系の薄層斜光照明法を提供することを目的とする。
【０００８】
【課題を解決するための手段】
上記課題を解決するために、この発明の請求項１に係る光学系の薄層斜光照明法は、対物レンズを用いたレンズ光学系の斜光照明において、試料観察面での照射半径をｒ、試料における照明光の厚さをｄ、試料における照明光の入射角（又は、照明光と対物レンズ光軸とのなす角）をθとすると、ｄ＝２ｒ・ cos θの式により計算される試料における照明光の厚さｄが２０μｍ以下になるように、対物レンズの縁に光を入射し、試料への照明光の入射角度を対物レンズ光軸に垂直に近い角度にし、照明光の対物レンズ入射の開き角の半分δφを小さくして、照射領域を絞ることにより、薄い層状の光で試料を照明する構成とした。
【０００９】
これにより、対物レンズを用いた斜光照明において、照射領域を絞ることにより、薄い層状の光で試料を照明することができ、光学顕微鏡においては、低背景・高感度のセクショニング像や３次元画像を得ることができる。
【００１０】
この発明の請求項２に係る光学系の薄層斜光照明法は、対物レンズを用いたレンズ光学系の斜光照明であって、試料観察面での照射半径をｒ、試料における照明光の厚さをｄ、試料における照明光の入射角（又は、照明光と対物レンズ光軸とのなす角）をθとすると、ｄ＝２ｒ・ cos θの式により計算される試料における照明光の厚さｄが２０μｍ以下になるように、対物レンズの縁に光を入射し、試料への照明光の入射角度を対物レンズ光軸に垂直に近い角度にし、照明光の対物レンズ入射の開き角の半分δφを小さくして、照射領域を絞ることにより、薄い層状の光で試料を照明する薄層斜光照明法において、試料の屈折率よりも閉口数が大きい対物レンズを用い、試料への照明光入射角度をさらに対物レンズ光軸に垂直に近い角度にし、照射領域を絞ることにより、より薄い層状の光で試料を照明する構成とした。
【００１１】
これにより、開口数の大きい対物レンズを用いるほど照明光を薄くでき、試料の屈折率よりも開口数が大きい対物レンズを用いて、試料への照明光入射角度を更に光軸に垂直に近くすることで、より薄い光で照明することができる。
【００１２】
この発明の請求項３に係る光学系の薄層斜光照明法は、対物レンズを用いたレンズ光学系の斜光照明であって、試料観察面での照射半径をｒ、試料における照明光の厚さをｄ、試料における照明光の入射角（又は、照明光と対物レンズ光軸とのなす角）をθとすると、ｄ＝２ｒ・ cos θの式により計算される試料における照明光の厚さｄが２０μｍ以下になるように、対物レンズの縁に光を入射し、試料への照明光の入射角度を対物レンズ光軸に垂直に近い角度にし、照明光の対物レンズ入射の開き角の半分δφを小さくして、照射領域を絞ることにより、薄い層状の光で試料を照明する光学系の薄層斜光照明において、焦点位置を変えて異なる高さの試料面を観察する場合に、照明光の対物レンズへの入射角を変えることにより、試料観察面における照明光の入射角を一定に保つように構成した。
【００１３】
これにより、対物レンズの焦点位置を変えて、試料観察面のカバーガラス表面からの高さを変えても、対物レンズへの照明光の入射角を変えることにより、同じ入射角で試料を照明することができる。
【００１７】
この発明の請求項４に係る光学系の薄層斜光照明法は、対物レンズを用いたレンズ光学系の斜光照明であって、試料観察面での照射半径をｒ、試料における照明光の厚さをｄ、試料における照明光の入射角（又は、照明光と対物レンズ光軸とのなす角）をθとすると、ｄ＝２ｒ・ cos θの式により計算される試料における照明光の厚さｄが２０μｍ以下になるように、対物レンズの縁に光を入射し、試料への照明光の入射角度を対物レンズ光軸に垂直に近い角度にし、照明光の対物レンズ入射の開き角の半分δφを小さくして、照射領域を絞ることにより、薄い層状の光で試料を照明する光学系の薄層斜光照明において、複数の入射光の使用や、回転等による入射位置の移動によって、偏りのない薄層斜光照明を行う構成とした。
【００１８】
この発明の請求項５に係る光学系の薄層斜光照明法は、上記請求項１乃至請求項４の光学系が蛍光顕微鏡や暗視野顕微鏡などの光学顕微鏡、対物レンズを用いたレンズ光学系にも適用可能な構成とした。
【００１９】
これにより、光学顕微鏡はもとより各種の顕微鏡及び光を用いた検出において、低背景の画像及び低バックグラウンドのシグナルを得ることができる。その結果として、高感度・高いＳ／Ｎ比の画像及びシグナルを得ることができる。
【００２０】
この発明の請求項６に係る光学系の薄層斜光照明法は、上記請求項１乃至請求項４の薄層斜光照明法を用いる蛍光顕微鏡、原子間力顕微鏡、トンネル顕微鏡、又はフォトトンネル顕微鏡の光学系において、薄層光照明を用いた顕微鏡観察を、焦点位置を移動させながら連続画像を得て、デコンボリューションによってセクショニング画像及び３次元画像を得る構成した。
【００２１】
これにより、薄層光照明を用いた顕微鏡観察から、焦点位置を移動させながら連続画像を得て、デコンボリューションによってセクショニング画像及び３次元画像を得ることができ、背景光が低く高画質である。また、試料の照明が薄い層状領域のみの局所的であるので、得られる蛍光像を高感度カメラであるイメージングインテンシファイアーＣＣＤによって観察することにより、蛍光色素１分子を可視化できる。
【００２２】
【発明の実施の形態】
本発明は、対物レンズを用いたレンズ光学系の斜光照明において、試料を薄い層状の光で照明することにより、照明光のあたる領域が局所的に制限されて、背景光を低くすることができ、高感度で高いＳ／Ｎ比の画像およびシグナルを得ることができることに依拠するものである。光学系としては蛍光顕微鏡や暗視野顕微鏡などの光学顕微鏡はもとより、対物レンズを用いたレンズ光学系にも広く適用できるものである。次に、本発明の実施形態を図１乃至図８に基づいて以下に詳述する。
【００２３】
図１に照明光の厚さを求める原理を示す。図において、試料観察面での照射半径をｒ、試料における照明光の厚さをｄ、試料における照明光の入射角（又は、照明光と対物レンズ光軸とのなす角）をθとすると、
【数１】
ｄ＝２ｒ・cosθ
の関係式により、試料における照明光の厚さｄを求めることができる。
【００２４】
図２に光学系の対物レンズに関連する部分拡大図を示す。図において、１は試料媒体（溶液等）、２はカバーガラス、３はオイル、４は対物レンズ（レンズ群）、５は照射光である。光源からの照射光はダイクロイックミラー（図示なし）で直角に屈折されて照射光５となり、オイル３、カバーガラス２を経て試料媒体１に斜光照明される。
【００２５】
図２において、試料における照明光の厚さをｄ、試料における照明光の入射角をθ、レンズへの照明光入射位置、即ち、対物レンズ後焦点面における照明光のレンズ光軸からの距離をＸ、照明光の対物レンズへの入射角をφ、照明光の対物レンズ入射の開き角の半分をδφとすると、試料における照明光の厚さｄを与える式ｄ＝２ｒ・cosθにおいて、試料への照明光の入射角θはＸとφによって決まり、試料観察面での照射半径ｒはδφによって決まる値であることがわかる。従って、照明光の厚さｄを薄くすることは、cosθおよびｒを小さくすることにより実現できる。
【００２６】
cosθを小さくするには、θを９０度に近づければ良いが、そのためにＸを大きくする。即ち、対物レンズの縁に光を入射し、試料において対物レンズ光軸と垂直に近い角度で斜光照明をおこなう。更に、対物レンズへの入射角φを調節して、θを９０度近くにする。
【００２７】
照射半径ｒを小さくするためには、δφを小さくすれば良い。その際、ｒを小さくすると観察視野が狭められるので、観察視野の許す範囲において、ｒを小さくする必要がある。
【００２８】
こうして、対物レンズを用いた斜光照明において、試料への照明光入射角度を対物レンズ光軸に垂直に近い角度にし、照射領域を絞ることにより、薄い層状の光で試料を照明することができる。そして、光学顕微鏡においては、低背景・高感度のセクショニング像や３次元画像を得ることができる。
【００２９】
また、開口数の大きい対物レンズを用いるほど照明光を薄くできるが、試料の屈折率よりも開口数が大きい対物レンズを用いて、試料への照明光入射角度を更に光軸に垂直に近くすることで、より薄い光で照明することができる。対物レンズの開口数（ＮＡ）が大きいほど、図２においてＸを大きくすることができ、照明光の入射角θが大きくなるので、その結果として、照明光の厚さｄを薄くすることができる。
【００３０】
次に、図３を参照して、対物レンズの開口数ＮＡで決まるＸの最大値をＸ_NA、試料の屈折率ｎで決まる全反射が起こるＸの境界値をＸ_n とすると、閉口数ＮＡが試料の屈折率ｎよりも大きいレンズでは、図３（Ａ）に示すように、Ｘを大きくすると全反射が起こり試料を照明できなくなる。
【００３１】
しかし、図３（Ｂ）に示すように、対物レンズへの入射角φを調節することにより、全反射を起こすことなく試料を斜光照明することができる。この方法を用いると、照明光の入射角θを９０度により近い値にすることが可能となり、照明光の厚さｄを更に薄くすることができる。
【００３２】
次に、対物レンズの焦点位置を変えて、試料観察面のカバーガラス表面からの高さＺを変える場合について図４を参照して説明する。図４（Ａ）は対物レンズへの照明光の入射角φでは、試料観察面のカバーガラス表面からの高さはＺであることを示す。
【００３３】
図４（Ｂ）は対物レンズへの照明光の入射角が（φ＋△φ）では、試料観察面のカバーガラス表面からの高さは（Ｚ＋△Ｚ）となることを示す。それ故、対物レンズの焦点位置を変えても対物レンズへの照明光の入射角φを変えることにより、同じ入射角θで試料を照明することができる。
【００３４】
次に、カメラ等の受光素子の受光面を傾ける場合、あるいは結像レンズを傾ける場合について図５を参照して説明する。図５において、１は試料媒体（溶液等）、２はカバーガラス、３はオイル、４は対物レンズ（レンズ群）、５は照射光、８は試料観察面、９は結像レンズ、１０はカメラ等の受光素子である。図では、レンズ系を単純化して描いているが、実際には中間にレンズ群が入って構成されている。
【００３５】
図５（Ａ）はカメラ等の受光素子１０の受光面を傾けることにより、試料観察面８を傾け、斜光照明光５と試料観察面８を平行、若しくはほぼ平行にして観察することができることを示している。
【００３６】
また、図５（Ｂ）は結像レンズ９を傾けることにより、試料観察面８を傾け、斜光照明光５と試料観察面８を平行もしくはほぼ平行にして観察することができることを示している。結像レンズ９を傾ける方法では、中間の光学系の変更によっても同等のことを行うことができる。
【００３７】
図５（Ａ）の試料観察面８を傾ける場合に、対物レンズ４に入射する照明光の形を細長くし、試料を照明する薄層光をさらに薄くすることができる。このことを図６を参照して説明する。符号は上述の例と同一である。
【００３８】
図６（Ａ）は図２と同じ構成であるので説明は省略する。図６（Ｂ）は試料領域の照射光を上側からみた拡大図である。照明光の厚さｄを与える式ｄ＝２ｒ・cosθにおける、ｒはカバーガラスに平行な面による照明光の断面形状の、照明光進行方向の半径である。試料観察面を傾けている場合には、観察視野の大きさはｒには依存せず、ｒに垂直な方向の半径ｒ’によって決まる。従って、ｒ’は大きくし、ｒは小さくした細長い形の入射光を用いると、観察視野を狭めることなく、照明光の厚さｄを薄くすることができる。
【００３９】
こうして、薄層光照明を用いた顕微鏡観察から、焦点位置を移動させながら連続画像を得て、デコンボリューションによってセクショニング画像及び３次元画像を得ることができる。そして、本発明によると従来法とは違って、背景光が低く高画質であることに加え、試料の照明が薄い層状領域のみの局所的である。
【００４０】
それ故に、試料観察面近傍の領域のみをデコンボリューションの計算対象とすれば良いという計算上・画質上ともに大きな利点がある。従って、厚みのある試料の観察、明るい中にある暗い部分の観察、１分子観察のような高感度観察の、セクショニング画像及び３次元画像を行うことを可能にする。
【００４１】
更に、図７のように、複数の入射光または回転対称な入射光の使用や、回転等による入射位置の移動によって、偏りのない薄層光照明を行うことができる。
【００４２】
次に、本発明を適用して蛍光顕微鏡に薄層斜光照明法を用いた例を図８に示す。図において、符号は上述の例と同一物のものには同一符号を付しており、１１は光学フィルタ、１２はミラー、１３は集光用レンズ、Ｒは照明レーザー光可変絞りの内径である。レーザー光を照明光として用い、集光用レンズ１３によって対物レンズ４の後焦点面にレーザー光を集光し、試料における照明光を平行光にする。
【００４３】
可変絞り径Ｒを変えて入射開き角δφを変化させ、照射半径ｒを調節する。ミラー１２と集光用レンズ１３を一体としてｔｘ方向に移動させることにより、入射位置Ｘを調節する。次に、ミラー１２へのレーザー光の入射位置をｔφ方向に移動すると、集光用レンズ１３を通過後の光路の傾きが変化するので、ｔφによって対物レンズ４への入射角φを調節する。以上の調節によって、試料における照明光の層の厚さｄ＝２ｒ・cosθを数ミクロンに設定することができる。
【００４４】
実施例を挙げると、油浸１００倍ＮＡ１.４の対物レンズを用い、試料観察面における照射領域の直径２ｒ＝３０μｍの時、試料における入射角θ＝８０°でｄ＝５μｍ、入射角θ＝８４°でｄ＝３μｍとなる。油浸６０倍ＮＡ１.４の対物レンズを用い、試料観察面における照射領域の直径２ｒ＝４５μｍの時、試料における入射角θ＝８６°でｄ＝３μｍとなる。
【００４５】
こうして、薄層斜光照明によって得られる蛍光像を、高感度カメラであるイメージングインテンシファイアーＣＣＤによって観察することにより、蛍光色素１分子を可視化できる。
【００４６】
また、他の適用例として、蛍光顕微鏡において、試料観察面を傾け、照明光を細長くし、高ＮＡ対物レンズを用いて、薄層照明光を薄くする例を説明する。図８の蛍光顕微鏡において、図５の結像レンズを傾ける方法により試料観察面を傾け、図６の細長い照明光を使う方法を用いる。
【００４７】
カバーガラスでの照明光の断面形状は、短径２ｒ＝３０μｍ、長径２ｒ’＝1００μｍにする。このようにすれば、１辺１００μｍの観察視野を得ることができ、観察視野を狭めることなく、照明光の厚みを薄くできる。
【００４８】
式ｄ＝２ｒ・cｏsθから、試料における照明光の層の厚さｄは、油浸６０倍ＮＡ１.４の対物レンズを用いると、２ｒ＝３０μｍ、試料における照明光の入射角θ＝８６°でｄ＝２μｍとなる。但し、厚さｄが光の波長（可視光で０.４〜０.７μｍ）に近くなるため、回折現象により光の層の上下に光の広がりが見られ始める。
【００４９】
更に、ＮＡの大きい油浸６０倍ＮＡ１.４５の対物レンズを用いると、試料における入射角θ＝８７°、即ち、カバーガラスとのなす角が３°となり、照明光とカバーガラスとがほとんど平行になる。このように平行に近づけることにより、結像レンズを傾けることによる画質への影響が無視できるようになる。光の厚さｄに関しては、理論的には２μｍより薄くなるが、回折光による厚みの広がりも強くなる。
【００５０】
本発明の光学系の薄層斜光照明と従来の斜光照明法による照明光の厚みを実測比較すると、図１２に示すように、例えば開口数ＮＡ＝１.４の対物レンズを用いた場合、従来の斜光照明法による照明光の厚みの約半分の厚みが得られた。
【００５１】
本発明の薄層斜光照明法を用いた蛍光顕微鏡観察によって、細胞内においても明瞭な１分子イメージングが実現した。その結果、分子１個の動きや変化を直接観察できるようになった。それと共に、分子１個の蛍光強度を得ることによって、蛍光強度から細胞内における分子数を定量することも実現できた。更に、分子数の定量から細胞内における分子間相互作用の結合分子数と結合の強さを求めることを実現できた。
【００５２】
現在、ナノテクノロジーのバイオへの応用が強い興味を集めているが、１分子レベルの高感度検出を可能にする本発明の薄層斜光照明法は、この分野における重要な要素技術になるものと考えられる。また、顕微鏡技術としても、新しい顕微鏡法として発展することが期待される。
【００５３】
【発明の効果】
以上のように、本発明の光学系の薄層斜光照明法は、対物レンズを用いた斜光照明において、薄い層状の光で試料を照明することにより、光学顕微鏡はもとより各種の顕微鏡及び光を用いた検出において、低背景の画像及び低バックグラウンドのシグナルを得ることができる。その結果として、高感度・高いＳ／Ｎ比の画像及びシグナルを得ることができる。
【００５４】
また、薄層光照明を用いた顕微鏡観察から、焦点位置を移動させながら連続画像を得て、デコンボリューションによってセクショニング画像及び３次元画像を得ることができ、背景光が低く高画質である。また、試料の照明が薄い層状領域のみの局所的であるので、得られる蛍光像を高感度カメラで観察することにより、蛍光色素１分子を可視化できると共に、分子１個の蛍光強度を得ることによって、蛍光強度から細胞内における分子数を定量することも実現できる。更に、分子数の定量から細胞内における分子間相互作用の結合分子数と結合の強さを求めることを実現できる。
【図面の簡単な説明】
【図１】本発明の照明光の厚さを求める原理図。
【図２】光学系の対物レンズに関連する部分拡大図。
【図３】対物レンズへの入射角を調節方法の概略図。
【図４】対物レンズの焦点位置を変える方法の概略図。
【図５】受光素子の受光面あるいは結像レンズを傾ける方法の概略図。
【図６】対物レンズに入射する照明光の形を細長くする方法の概略図。
【図７】複数の入射光または回転対称な入射光を使用する方法の概略図。
【図８】蛍光顕微鏡の薄層斜光照明法の概略図。
【図９】蛍光顕微鏡の落射照明法の概略図。
【図１０】対物レンズ型全反射照明法の概略図。
【図１１】光照射切り替え方法の概略図。
【図１２】本発明方法と従来方法の照明光の厚めの比較表。
【符号の説明】
１試料媒体（溶液等）
２カバーガラス
３オイル
４対物レンズ（レンズ群）
５，６照射光
７ダイクロイックミラー
８試料観察面
９結像レンズ
１０受光素子
１１光学フィルタ
１２ミラー
１３集光用レンズ[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a thin-layer oblique illumination method of an optical system equivalent to a microscope that detects light with high sensitivity using a light.
[0002]
[Prior art]
Conventionally, various illumination techniques of an optical microscope for detecting substances and molecules with high sensitivity using light have been proposed, some of which will be described below. FIG. 9 is a schematic view of an epi-illumination method that is currently generally used as illumination for a fluorescence microscope. As shown in the figure, the light from the light source is reflected by a dichroic mirror that reflects the irradiation light and transmits the fluorescence, enters the center of the objective lens, and irradiates the sample observation surface.
[0003]
Further, there is a total reflection illumination method as a method of locally illuminating only the vicinity of the glass surface. Illumination is performed using evanescent light (depth of about 100200 nm) generated upon total reflection. There are a method using a prism and a method using an objective lens. FIG. 10 shows a schematic diagram of an objective lens type total reflection illumination method. When the numerical aperture of the objective lens is NA and the refractive index of the sample solution is n, total reflection illumination can be performed by the objective lens having a numerical aperture (NA) satisfying the formula of NA> n. Conventionally, this method has been used only for a limited amount, but it has been found useful as a method for observing one molecule of a fluorescent dye with a fluorescence microscope, and has recently begun to be used for single-molecule imaging.
[0004]
Further, FIG. 11 shows an outline of a light irradiation switching method proposed by the present inventor and already patented as Japanese Patent No. 3093145. 1 is a sample solution, 2 is a cover glass, 3 is oil, 4 is an objective lens (lens group), 5 is irradiation light, 6 is irradiation light, and 7 is a dichroic mirror. The change from epi-illumination to the state of total reflection illumination can be realized only by moving optical components such as mirrors from FIG. 11 (A) → 11 (B) → FIG. 11 (C). Irradiation can be switched by a simple principle without requiring an optical system. Further, in this method, the same state change can be realized by shifting the incident light position or the light source position (for example, the emission position of the optical fiber).
[0005]
As another method for obtaining a sectioning image or a three-dimensional image in a normal optical microscope or a fluorescent microscope using an epi-illumination method, calculation is performed by deconvolution from a continuous image obtained by continuously changing the focus. The method is used. This is to calculate the original three-dimensional image by calculation by knowing in advance what happens when the image of light emitted from one point on the sample deviates from the focus. In addition, this method has a limit when observing a dark part in a bright light.
[0006]
As another method, confocal microscopy is now widely used as a method using a sectioning image or a three-dimensional image. This method is inferior in sensitivity to a high-sensitivity camera. Especially in the observation of a fluorescent sample, the laser light is focused on one point and irradiated, so that the fading of the fluorescent dye is accelerated, or the biological sample There are difficulties such as damaging.
[0007]
[Problems to be solved by the invention]
As described above, the conventional method cannot obtain a high sensitivity and a high S / N ratio in the observation of a thick sample or a dark portion in a bright area. Therefore, the present invention makes it possible to detect substances and molecules with high sensitivity using light using an optical system equivalent to a microscope, and to perform low-level background / high-sensitivity observation in an optical microscope. The purpose is to provide an illumination method.
[0008]
[Means for Solving the Problems]
In order to solve the above-mentioned problem, the thin-layer oblique illumination method of the optical system according to claim 1 of the present invention is such that, in the oblique illumination of the lens optical system using the objective lens , the irradiation radius on the sample observation surface is r, and the sample Where d is the thickness of the illumination light at, and θ is the incident angle of the illumination light at the sample (or the angle between the illumination light and the optical axis of the objective lens), d = 2r · cos θ. As the thickness d of the illumination light becomes 20μm or less in a sample, the light incident on the edge of the objective lens, and the incident angle of the illumination light to the specimen at an angle close to perpendicular to the optical axis of the objective lens, the illumination light objective The configuration is such that the sample is illuminated with a thin layer of light by reducing the half opening angle δφ of the lens incidence and narrowing the irradiation area.
[0009]
This makes it possible to illuminate the sample with thin layered light by narrowing the irradiation area in oblique illumination using an objective lens. In an optical microscope, a low-background / high-sensitivity sectioning image or a three-dimensional image can be displayed. Obtainable.
[0010]
An optical system thin-layer oblique illumination method according to claim 2 of the present invention is oblique illumination of a lens optical system using an objective lens , wherein the irradiation radius on the sample observation surface is r, and the thickness of the illumination light on the sample Where d is the incident angle of the illumination light on the sample (or the angle between the illumination light and the optical axis of the objective lens) is θ, and d = 2r · cos θ. The light is incident on the edge of the objective lens so that the thickness d of the illumination light in the sample is 20 μm or less, and the incident angle of the illumination light to the sample is set to an angle close to the optical axis of the objective lens. In the thin-layer oblique illumination method that illuminates the sample with thin layered light by reducing the half-opening angle δφ of the lens incidence and narrowing the irradiation area, an objective lens that has a larger number of apertures than the refractive index of the sample is used. The sample was illuminated with a thinner layer of light by making the incident angle of illumination light on the sample closer to perpendicular to the optical axis of the objective lens and narrowing the irradiation area.
[0011]
This allows thinner as illumination light using an open number of units of a large objective lens, by using an objective lens numerical aperture is larger than the refractive index of the sample, more nearly perpendicular to the optical axis of the illumination light incident angle to the sample Therefore, it can illuminate with thinner light.
[0012]
The thin-layer oblique illumination method of the optical system according to claim 3 of the present invention is oblique illumination of a lens optical system using an objective lens, wherein the irradiation radius on the sample observation surface is r, and the thickness of the illumination light on the sample Where d is the incident angle of the illumination light on the sample (or the angle between the illumination light and the optical axis of the objective lens) is θ, and d = 2r · cos θ. The light is incident on the edge of the objective lens so that the thickness d of the illumination light in the sample is 20 μm or less, and the incident angle of the illumination light to the sample is set to an angle close to the optical axis of the objective lens. In the thin-layer oblique illumination of the optical system that illuminates the sample with thin layered light by reducing the half-opening angle δφ of the lens incidence and narrowing the irradiation area, the sample surface with different heights can be changed by changing the focal position. In the observation, the incident angle of the illumination light on the sample observation surface is kept constant by changing the incident angle of the illumination light to the objective lens.
[0013]
Thus, even if the focal position of the objective lens is changed and the height of the sample observation surface from the cover glass surface is changed, the sample is illuminated at the same incident angle by changing the incident angle of the illumination light to the objective lens. be able to.
[0017]
The thin-layer oblique illumination method for an optical system according to claim 4 of the present invention is an oblique illumination of a lens optical system using an objective lens, which is illuminated on a sample observation surface. The radius of irradiation is r, the thickness of the illumination light on the sample is d, and the incident angle (or illumination) of the illumination light on the sample. If the angle between the bright light and the optical axis of the objective lens is θ, it is calculated by the formula d = 2r · cos θ. The light is incident on the edge of the objective lens so that the thickness d of the illumination light in the sample is 20 μm or less, and the incident angle of the illumination light to the sample is set to an angle close to the optical axis of the objective lens. In the thin-layer oblique illumination of the optical system that illuminates the sample with thin layered light by reducing the half-opening angle δφ of the objective lens and narrowing the irradiation area , use of multiple incident light, rotation, etc. A thin layer oblique illumination with no bias is performed by moving the incident position.
[0018]
The thin-layer oblique illumination method for an optical system according to claim 5 of the present invention is such that the optical system of claims 1 to 4 is an optical microscope such as a fluorescence microscope or a dark field microscope, or a lens optical system using an objective lens. Is also applicable.
[0019]
Thereby, in the detection using not only an optical microscope but also various microscopes and light, a low background image and a low background signal can be obtained. As a result, an image and a signal with high sensitivity and high S / N ratio can be obtained.
[0020]
A thin layer oblique illumination method of an optical system according to claim 6 of the present invention is a fluorescence microscope, an atomic force microscope, a tunnel microscope, or a photo tunnel microscope using the thin layer oblique illumination method according to any one of claims 1 to 4. In the optical system, a microscope observation using thin-layer light illumination was configured to obtain a continuous image while moving the focal position, and obtain a sectioning image and a three-dimensional image by deconvolution.
[0021]
Thereby, it is possible to obtain a continuous image while moving the focal position from a microscopic observation using thin-layer light illumination, and obtain a sectioning image and a three-dimensional image by deconvolution, and the background light is low and the image quality is high. Further, since the illumination of the sample is local only in a thin layered region, one fluorescent dye molecule can be visualized by observing the obtained fluorescent image with an imaging intensifier CCD which is a high sensitivity camera.
[0022]
DETAILED DESCRIPTION OF THE INVENTION
According to the present invention, in the oblique illumination of the lens optical system using the objective lens, by illuminating the sample with a thin layered light, the area to which the illumination light is applied is locally limited, and the background light can be lowered. It relies on being able to obtain images and signals with high sensitivity and high S / N ratio. The optical system can be widely applied not only to an optical microscope such as a fluorescence microscope or a dark field microscope but also to a lens optical system using an objective lens. Next, an embodiment of the present invention will be described in detail below with reference to FIGS.
[0023]
FIG. 1 shows the principle for determining the thickness of illumination light. In the figure, when the irradiation radius on the sample observation surface is r, the thickness of the illumination light on the sample is d, and the incident angle of the illumination light on the sample (or the angle formed between the illumination light and the objective lens optical axis) is θ.
[Expression 1]
d = 2r · cos θ
From the relational expression, the thickness d of the illumination light in the sample can be obtained.
[0024]
FIG. 2 shows a partially enlarged view related to the objective lens of the optical system. In the figure, 1 is a sample medium (solution or the like), 2 is a cover glass, 3 is oil, 4 is an objective lens (lens group), and 5 is irradiation light. Irradiated light from the light source is refracted at right angles by a dichroic mirror (not shown) to become irradiated light 5, and obliquely illuminates the sample medium 1 through the oil 3 and the cover glass 2.
[0025]
2, the distance of the thickness of the illumination light in the sample d, the incident angle of the illumination light in the sample theta, illumination light incident position on the lens, i.e., the lens optical axis of the illumination light in the focal plane after the objective lenses Is X, the incident angle of the illumination light to the objective lens is φ, and the half of the opening angle of the illumination light incident on the objective lens is δφ, the expression d = 2r · cos θ gives the thickness d of the illumination light in the sample. It can be seen that the incident angle θ of the illumination light on the surface is determined by X and φ, and the irradiation radius r on the sample observation surface is determined by δφ. Therefore, reducing the thickness d of the illumination light can be realized by reducing cos θ and r.
[0026]
In order to reduce cos θ, θ should be close to 90 degrees. For this purpose, X is increased. That is, light is incident on the edge of the objective lens, and oblique illumination is performed on the sample at an angle close to perpendicular to the optical axis of the objective lens. Further, the incident angle φ to the objective lens is adjusted so that θ is close to 90 degrees.
[0027]
In order to reduce the irradiation radius r, δφ may be reduced. At this time, if r is reduced, the observation field of view is narrowed. Therefore, it is necessary to reduce r within the range permitted by the observation field of view.
[0028]
Thus, in oblique illumination using the objective lens, the specimen can be illuminated with thin layered light by setting the incident light incident angle on the specimen to an angle close to perpendicular to the optical axis of the objective lens and narrowing the irradiation area. In the optical microscope, a low background / high sensitivity sectioning image or three-dimensional image can be obtained.
[0029]
Although it thin enough illumination light using an open number of units of a large objective lens, by using an objective lens numerical aperture is larger than the refractive index of the sample, more nearly perpendicular to the optical axis of the illumination light incident angle to the sample Therefore, it can illuminate with thinner light. As the numerical aperture (NA) of the objective lens is increased, X in FIG. 2 can be increased, and the incident angle θ of the illumination light is increased. As a result, the thickness d of the illumination light can be reduced. .
[0030]
Next, referring to FIG. 3, if the maximum value of X determined by the numerical aperture NA of the objective lens is X _NA , and the boundary value of X at which total reflection determined by the refractive index n of the sample occurs is X _n , the closed numerical aperture NA In a lens whose refractive index is larger than the refractive index n of the sample, as shown in FIG. 3A, when X is increased, total reflection occurs and the sample cannot be illuminated.
[0031]
However, as shown in FIG. 3B, by adjusting the incident angle φ to the objective lens, the sample can be illuminated obliquely without causing total reflection. When this method is used, the incident angle θ of the illumination light can be made closer to 90 degrees, and the thickness d of the illumination light can be further reduced.
[0032]
Next, the case where the height Z from the cover glass surface of the sample observation surface is changed by changing the focal position of the objective lens will be described with reference to FIG. FIG. 4A shows that the height of the sample observation surface from the cover glass surface is Z at the incident angle φ of the illumination light to the objective lens.
[0033]
FIG. 4B shows that when the incident angle of the illumination light to the objective lens is (φ + Δφ), the height of the sample observation surface from the cover glass surface is (Z + ΔZ). Therefore, even if the focal position of the objective lens is changed, the sample can be illuminated with the same incident angle θ by changing the incident angle φ of the illumination light to the objective lens.
[0034]
Next, a case where the light receiving surface of a light receiving element such as a camera is tilted or a case where the imaging lens is tilted will be described with reference to FIG. In FIG. 5, 1 is a sample medium (solution or the like), 2 is a cover glass, 3 is oil, 4 is an objective lens (lens group), 5 is irradiation light, 8 is a sample observation surface, 9 is an imaging lens, 10 is It is a light receiving element such as a camera. In the figure, the lens system is depicted in a simplified manner, but in actuality, the lens system is configured with a lens group in the middle.
[0035]
FIG. 5A shows that the sample observation surface 8 can be inclined by tilting the light receiving surface of the light receiving element 10 such as a camera, and the oblique illumination light 5 and the sample observation surface 8 can be observed in parallel or substantially parallel. Show.
[0036]
FIG. 5B shows that the sample observation surface 8 can be inclined by tilting the imaging lens 9 so that the oblique illumination light 5 and the sample observation surface 8 can be observed in parallel or substantially in parallel. In the method of tilting the imaging lens 9, the same thing can be done by changing the intermediate optical system.
[0037]
When the sample observation surface 8 in FIG. 5A is tilted, the shape of the illumination light incident on the objective lens 4 can be elongated, and the thin layer light that illuminates the sample can be further reduced. This will be described with reference to FIG. The reference numerals are the same as in the above example.
[0038]
Since FIG. 6A has the same configuration as FIG. 2, description thereof is omitted. FIG. 6B is an enlarged view of the irradiation light of the sample region viewed from above. In the formula d = 2r · cos θ that gives the thickness d of the illumination light, r is the radius of the illumination light in the traveling direction of the cross-sectional shape of the illumination light by the plane parallel to the cover glass. When the sample observation surface is inclined, the size of the observation visual field does not depend on r, but is determined by the radius r ′ in the direction perpendicular to r. Therefore, when elongated incident light having a larger r 'and a smaller r is used, the thickness d of the illumination light can be reduced without narrowing the observation field.
[0039]
In this way, a continuous image can be obtained from a microscope observation using thin layer light illumination while moving the focal position, and a sectioning image and a three-dimensional image can be obtained by deconvolution. According to the present invention, unlike the conventional method, the background light is low and the image quality is high, and the illumination of the sample is local only in a thin layered region.
[0040]
Therefore, there is a great advantage in terms of both calculation and image quality that only the region near the sample observation surface needs to be a deconvolution calculation target. Accordingly, it is possible to perform sectioning images and three-dimensional images of high-sensitivity observation such as observation of a thick sample, observation of a dark portion in a bright state, and observation of one molecule.
[0041]
Further, as shown in FIG. 7, thin layer light illumination with no bias can be performed by using a plurality of incident light or rotationally symmetric incident light, or by moving the incident position by rotation or the like.
[0042]
Next, an example in which the present invention is applied and a thin layer oblique illumination method is used for a fluorescence microscope is shown in FIG. In the figure, the same reference numerals are attached to the same components as in the above example, 11 is an optical filter, 12 is a mirror, 13 is a condensing lens, and R is the inner diameter of the illumination laser light variable aperture. . The laser light is used as illumination light, the laser light is condensed on the rear focal plane of the objective lens 4 by the condensing lens 13, and the illumination light on the sample is converted into parallel light.
[0043]
The incident aperture angle δ φ is changed by changing the variable aperture diameter R to adjust the irradiation radius r. The incident position X is adjusted by moving the mirror 12 and the condenser lens 13 together in the tx direction. Next, when the incident position of the laser beam on the mirror 12 is moved in the tφ direction, the inclination of the optical path after passing through the condensing lens 13 changes, so the incident angle φ to the objective lens 4 is adjusted by tφ. By the above adjustment, the thickness d = 2r · cos θ of the illumination light in the sample can be set to several microns.
[0044]
In an example, when an objective lens having an oil immersion of 100 times NA 1.4 is used and the diameter of the irradiated region on the sample observation surface is 2r = 30 μm, the incident angle θ = 80 ° at the sample, d = 5 μm, and the incident angle θ = At 84 °, d = 3 μm. When an objective lens with an oil immersion of 60 times NA 1.4 is used and the diameter of the irradiated region on the sample observation surface is 2r = 45 μm, the incident angle θ of the sample is 86 ° and d = 3 μm.
[0045]
In this way, one fluorescent dye molecule can be visualized by observing a fluorescent image obtained by thin-layer oblique illumination with an imaging intensifier CCD, which is a high-sensitivity camera.
[0046]
As another application example, in fluorescence microscopy, tilting the sample viewing surface, and elongated illumination light, using a high NA objective lenses, an example of thin thin layer illumination light. In the fluorescence microscope of FIG. 8, the method of tilting the sample observation surface by the method of tilting the imaging lens of FIG. 5 and using the elongated illumination light of FIG.
[0047]
The cross-sectional shape of the illumination light on the cover glass is such that the minor axis 2r = 30 μm and the major axis 2r ′ = 100 μm. In this way, an observation field having a side of 100 μm can be obtained, and the thickness of the illumination light can be reduced without narrowing the observation field.
[0048]
From the equation d = 2r · c o sθ, the thickness d of the layer of the illumination light in a sample, using the objective lens of the oil immersion 60x NA1.4, 2r = 30μm, the incidence angle of the illumination light in the sample theta = 86 At d, d = 2 μm. However, since the thickness d is close to the wavelength of light (0.4 to 0.7 μm for visible light), the spreading of light begins to be observed above and below the light layer due to the diffraction phenomenon.
[0049]
Further, when an objective lens having a large NA of oil immersion 60 times NA 1.45 is used, the incident angle θ of the sample is 87 °, that is, the angle formed with the cover glass is 3 °, and the illumination light and the cover glass are almost parallel. become. By approaching parallel in this way, the influence on the image quality caused by tilting the imaging lens can be ignored. The light thickness d is theoretically less than 2 μm, but the spread of the thickness due to diffracted light is also strong.
[0050]
When the thickness of the illumination light by the thin oblique illumination of the optical system of the present invention and the conventional oblique illumination method is measured and compared, as shown in FIG. 12, for example, when an objective lens having a numerical aperture NA = 1.4 is used, The thickness of the illumination light by the oblique illumination method was about half the thickness.
[0051]
A clear single-molecule imaging was realized even in cells by observation with a fluorescence microscope using the thin-layer oblique illumination method of the present invention. As a result, it became possible to directly observe the movement and change of one molecule. At the same time, by obtaining the fluorescence intensity of one molecule, it was also possible to quantify the number of molecules in the cell from the fluorescence intensity. Furthermore, it was possible to determine the number of binding molecules and the strength of binding in the intercellular interaction from the quantification of the number of molecules.
[0052]
Currently, the application of nanotechnology to biotechnology is attracting strong interest, but the thin-layer oblique illumination method of the present invention that enables highly sensitive detection at the single molecule level will be an important elemental technology in this field. Conceivable. Moreover, it is expected to develop as a new microscope method as a microscope technique.
[0053]
【The invention's effect】
As described above, the thin-layer oblique illumination method of the optical system according to the present invention uses various microscopes and light in addition to an optical microscope by illuminating a sample with thin layered light in oblique illumination using an objective lens. Detection, a low background image and a low background signal can be obtained. As a result, an image and a signal with high sensitivity and high S / N ratio can be obtained.
[0054]
In addition, a continuous image can be obtained from a microscope observation using thin layer light illumination while moving the focal position, and a sectioning image and a three-dimensional image can be obtained by deconvolution, and the background light is low and the image quality is high. In addition, since the illumination of the sample is local only in the thin layered region, by observing the obtained fluorescent image with a high sensitivity camera, one molecule of the fluorescent dye can be visualized and the fluorescence intensity of one molecule can be obtained. It is also possible to quantify the number of molecules in the cell from the fluorescence intensity. Furthermore, it is possible to determine the number of binding molecules and the strength of binding in the intercellular interaction from the quantification of the number of molecules.
[Brief description of the drawings]
FIG. 1 is a principle diagram for determining the thickness of illumination light according to the present invention.
FIG. 2 is a partially enlarged view related to an objective lens of an optical system.
FIG. 3 is a schematic view of a method for adjusting an incident angle to an objective lens.
FIG. 4 is a schematic view of a method for changing the focal position of an objective lens.
FIG. 5 is a schematic view of a method of tilting a light receiving surface or an imaging lens of a light receiving element.
FIG. 6 is a schematic view of a method for elongating the shape of illumination light incident on an objective lens.
FIG. 7 is a schematic diagram of a method of using multiple incident lights or rotationally symmetric incident light.
FIG. 8 is a schematic view of a thin layer oblique illumination method of a fluorescence microscope.
FIG. 9 is a schematic view of an epi-illumination method of a fluorescence microscope.
FIG. 10 is a schematic view of an objective lens type total reflection illumination method.
FIG. 11 is a schematic diagram of a light irradiation switching method.
FIG. 12 is a comparative table of the illumination light thickness of the method of the present invention and the conventional method.
[Explanation of symbols]
1 Sample medium (solution, etc.)
2 Cover glass 3 Oil 4 Objective lens (lens group)
5, 6 Irradiation light 7 Dichroic mirror 8 Sample observation surface 9 Imaging lens 10 Light receiving element 11 Optical filter 12 Mirror 13 Condensing lens

Claims

In oblique illumination of a lens optical system using an objective lens , the irradiation radius on the sample observation surface is r, the thickness of the illumination light on the sample is d, the incident angle of the illumination light on the sample (or the illumination light and the objective lens optical axis) Is calculated by the following formula : d = 2r · cos θ As the thickness d of the illumination light becomes 20μm or less in a sample, the light incident on the edge of the objective lens, and the incident angle of the illumination light to the specimen at an angle close to perpendicular to the optical axis of the objective lens, the illumination light objective A thin-layer oblique illumination method for an optical system, characterized in that the sample is illuminated with a thin layer of light by reducing the half-opening angle δφ of the lens incidence and narrowing the irradiation area.

2. An objective lens having a numerical aperture larger than the refractive index of the sample is used to illuminate the sample with thinner light by making the incident angle of illumination light on the sample closer to perpendicular to the optical axis. Thin-layer oblique illumination method for optical systems.

An oblique illumination of a lens optical system using an objective lens, wherein the irradiation radius on the sample observation surface is r, the thickness of the illumination light on the sample is d, and the incident angle of the illumination light on the sample (or illumination light and objective lens) When the angle between the optical axis and θ is θ, it is calculated by the formula d = 2r · cos θ. The light is incident on the edge of the objective lens so that the thickness d of the illumination light in the sample is 20 μm or less, and the incident angle of the illumination light to the sample is set to an angle close to the optical axis of the objective lens. In the thin-layer oblique illumination of the optical system that illuminates the sample with thin layered light by reducing the half-opening angle δφ of the lens incidence and narrowing the irradiation area, the sample surface with different heights can be changed by changing the focal position. A thin-layer oblique illumination method for an optical system characterized in that the incident angle of illumination light on the sample observation surface is kept constant by changing the incident angle of illumination light to the objective lens when observing.

Oblique illumination of a lens optical system using an objective lens, and illumination on the sample observation surface The radius of irradiation is r, the thickness of the illumination light on the sample is d, and the incident angle of illumination light on the sample (or illumination If the angle between the bright light and the optical axis of the objective lens is θ, it is calculated by the formula d = 2r · cos θ. The light is incident on the edge of the objective lens so that the thickness d of the illumination light in the sample is 20 μm or less, and the incident angle of the illumination light to the sample is set to an angle close to the optical axis of the objective lens. In the thin-layer oblique illumination of the optical system that illuminates the sample with thin layered light by reducing the half-opening angle δφ of the objective lens and narrowing the irradiation area , use of multiple incident light, rotation, etc. A thin-layer oblique illumination method for an optical system, characterized in that thin-layer oblique illumination with no bias is performed by moving an incident position.

5. The thin-layer oblique illumination method for an optical system according to claim 1, wherein the optical system is an optical microscope such as a fluorescence microscope or a dark field microscope, or a lens optical system using an objective lens.

6. The thin layer oblique light of an optical system according to claim 5, wherein, in microscopic observation using thin layer light illumination, a continuous image is obtained while moving a focal position, and a sectioning image and a three-dimensional image are obtained by deconvolution. Lighting method.