JP4112257B2 - Confocal microscope - Google Patents

Description

（ｑ＝１〜０．２）である。
【００３７】
上記手段を講じた結果、それぞれ以下のような作用を奏する。
【００３８】
（１）本発明の共焦点顕微鏡によれば、ディスク上にパターンを持つ領域を複数設けることで、ディスクを光軸に対し略垂直方向に移動させるといった単純な動作のみで、条件に合った最良の状態で共焦点観察を行なえる。さらに、減光手段を光軸上に挿脱自在とすることで、共焦点観察時、非共焦点観察時とも、観察者が眩しく感じることがなくなる。これにより、他の光学部材の挿脱や、複数の光路を必要とせず、複雑な光学系や電気的な制御も不要であるため、大きなスペースが不要になり、顕微鏡全体をコンパクト且つ簡単な機構のみで構成でき、良好な状態で観察を行なえる。
【００３９】
（２）本発明の共焦点顕微鏡によれば、ディスクの挿脱と同時に減光手段が光軸上に挿脱されるので、減光手段の挿脱機構など新たな機構を必要とせず、機構を簡単にすることができる。
【００４０】
（３）本発明の共焦点顕微鏡によれば、減光手段を設けるためにディスクの径を大きくする必要がないため、さらにスペースが少なくて済み、顕微鏡全体をコンパクトに構成でき、良好な状態で観察を行なえる。
【００４１】
（４）本発明の共焦点顕微鏡によれば、明視野像の観察時には、ディスクのパターンが無く一様に光を透過する部分を通して観察をすることになり、共焦点像の観察時と明視野像の観察時とで、光を同じ素材上に通すことができる。
【００４２】
（５）本発明の共焦点顕微鏡によれば、明視野像の観察時に光が透過率
【数３】

（ｑ＝１〜０．２）の領域を通ることになるので、共焦点像の観察時と比べて明るさがほぼ同じになる。
【００４３】
【発明の実施の形態】
以下、本発明の実施の形態を図面を参照して説明する。
【００４４】
（第１の実施の形態）
図１は、本発明の第１の実施の形態に係る共焦点顕微鏡の構成を示す図である。図１において、例えば水銀又はキセノンの光源１から出射した光は、コレクタレンズ２を介して平行光になり、ビームスプリッタ３に入射する。ビームスプリッタ３に入射した光は９０°曲げられ、ディスク５を照射する。ディスク５は、モータ４の軸４ａに嵌装されていて、モータ４の駆動により常時回転している。
【００４５】
図２の（ａ）は、ラインパターンディスク（共焦点用ディスク）であるディスク５の構成を示す図である。このディスク５には、図２の（ｂ）に示すような透過部５ｘと反射部５ｙをもつラインパターンが複数設けられており、透過部５ｘの幅は反射部５ｙの幅の約１／２０程度である。そしてディスク５は、透過部の幅が小さい領域５ａ、透過部の幅が大きい領域５ｂ、ラインパターンがなく例えばＮＤ６（６％の透過率をもつ減光部材）が設置されている領域５ｃの３つの領域を有している。なお、ディスク５は、対物レンズ１０の像位置、該像位置と共役な位置、又はそれらの近傍に配置されている。
【００４６】
なお、本実施の形態においては、図１に示すように、モータ４は、ディスク５の面が光軸１５に垂直な面に対して５°傾くよう、移動部材６に支持されている。モータ４とディスク５は、移動部材６の移動に伴い、図２に示す３つの領域５ａ、５ｂ、５ｃのうちの１つが光軸１５を通るよう、前記のように傾いた状態で３段階に移動する。移動部材６にはＶ溝６ａ、６ｂ、６ｃとレバー８が設けられ、図示しない顕微鏡本体には位置決め機構７が固定されている。移動部材６は、観察者がレバー８を操作することにより移動される。この移動に伴い、Ｖ溝６ａ、６ｂ、６ｃのいずれかが位置決め機構７に係合することで、３つの領域５ａ、５ｂ、５ｃの１つが光軸１５を通るよう、移動部材６が位置決めされる。
【００４７】
ディスク５に照射された光は、結像レンズ９を通り対物レンズ１０を介して試料１１を照射する。試料１１からの反射光は、対物レンズ１０を介し、結像レンズ９によりディスク５で結像し、このディスク５を通過することで形成される共焦点像は、ビームスプリッタ３を介して結像レンズ１２により結像し、接眼レンズ１３を介して観察者１４により観察される。
【００４８】
次に、上記のような構成をなす共焦点顕微鏡の動作を説明する。まず、例えば低い倍率の対物レンズを使用して共焦点観察をする場合、観察者はレバー８を持って移動部材６を移動させ、領域５ａに光軸１５が通るようにディスク５を位置決めする。この場合、Ｖ溝６ａに位置決め機構７が係合する。
【００４９】
また、例えば高い倍率の対物レンズを使用して共焦点観察をする場合、観察者はレバー８を持って移動部材６を移動させ、領域５ｂに光軸１５が通るようにディスク５を位置決めする。この場合、Ｖ溝６ｂに位置決め機構７が係合する。
【００５０】
次に、非共焦点観察をする場合、観察者はレバー８を持って移動部材６を移動させ、領域５ｃに光軸１５が通るようにディスク５を位置決めする。この場合、Ｖ溝６ｃに位置決め機構７が係合する。
【００５１】
これらのいずれかの動作の後、光源１から出射した光がコレクタレンズ２、ビームスプリッタ３を通ってディスク５を照射し、その後結像レンズ９を通って対物レンズ１０を介して試料１１を照射する。このとき、ディスク５は高速で回転しており、試料１１上では光が高速に走査される。試料１１からの反射光は、対物レンズ１０を通って結像レンズ９によりディスク５又はその近傍で結像し、ディスク５の領域を通過したことで形成される像が結像レンズ１２により再結像され、接眼レンズ１３を介して観察者１４により観察される。
【００５２】
以上のように本第１の実施の形態では、ディスク５のラインパターンが配置されたいずれかの領域５ａ、５ｂが、使用する対物レンズの倍率に応じて光軸１５上に位置するように移動部材６が移動するため、最良の状態で共焦点観察を行なえる。また、ラインパターンが配置されている領域により、光源１からの入射光に対して利用できる試料１１からの反射光は５％程度と低くなるため、水銀又はキセノン光源といった高輝度の光源を使用している。しかし、観察者が非共焦点観察をしようとしたとき、ディスク５のラインパターンのない領域５ｃにＮＤ６や２５といった減光部材が設置されているので、その領域５ｃが光軸１５上に位置するように移動部材６を移動すれば、観察者は眩しく感じずに共焦点観察を行なえる。
【００５３】
（第２の実施の形態）
本第２の実施の形態は、第１の実施の形態に対してディスク上のパターンのみが異なるので、ディスクのパターンについて図３の（ａ），（ｂ）を参照して説明し、他の部分については説明を省略する。
【００５４】
図３の（ａ）は、ラインパターンディスク（共焦点用ディスク）であるディスク２５の構成を示す図である。このディスク２５には、図３の（ｂ）に示すような透過部２５ｘと反射部２５ｙをもつラインパターンが複数設けられており、透過部２５ｘの幅は反射部２５ｙの約１／２０程度である。そしてディスク２５は、透過部の幅が小さく透過部に例えばＮＤ２５（２５％の透過率をもつ減光部材）が設置されている領域２５ａ、透過部の幅が大きい領域２５ｂ、ラインパターンがなく例えばＮＤ６が設置されている領域２５ｃの３つの領域を有している。
【００５５】
上記のような構成をなすディスク２５を備えた共焦点顕微鏡の動作は、第１の実施の形態と同様であるので説明を省略する。
【００５６】
例えば、使用する低倍率の対物レンズと高倍率の対物レンズの倍率の差が大きい場合、低倍率の対物レンズは高倍率の対物レンズに比べて瞳径が著しく大きい。このため、高倍率の対物レンズを使用して観察することに合わせて光源の種類や光量を設定すると、低倍率の対物レンズを使用して共焦点観察をしたときに眩しく感じる。
【００５７】
これに対して本第２の実施の形態では、第１の実施の形態と異なり、透過部の幅が小さい領域にも減光部材が設置されているので、低倍率の対物レンズを使用し透過部の幅の小さい領域２５ａを光軸上に位置させて観察したときに、観察者は眩しく感じることがない。
【００５８】
（第３の実施の形態）
本第３の実施の形態は、第１の実施の形態に対してディスク上の形状、パターンと、移動部材の形状のみが異なるので、それらの部分について図４、図５を参照して説明し、他の部分については説明を省略する。
【００５９】
図４の（ａ）は、移動部材３６とラインパターンディスク（共焦点用ディスク）であるディスク３５の構成を示す上面図である。このディスク３５には、図４の（ｂ）に示すような透過部３５ｘと反射部３５ｙをもつラインパターンが複数設けられており、透過部３５ｘの幅は反射部３５ｙの約１／２０程度である。そしてディスク３５は、透過部の幅が小さい領域３５ａと、透過部の幅が大きい領域３５ｂの２つの領域を有している。
【００６０】
図５は、移動部材３６とディスク３５の構成を示す側面図である。移動部材３６にはＶ溝３６ａ、３６ｂ、３６ｃと孔３６ｌ、３６ｍ、３６ｎが設けられている。移動部材３６の移動に伴い、Ｖ溝３６ａ、３６ｂ、３６ｃの１つが位置決め機構７に係合することで、それぞれ孔３６ｌ、３６ｍ、３６ｎが光軸１５を通るよう、移動部材３６が位置決めされる。また、各孔３６ｌ、３６ｍ、３６ｎの入り口には、それぞれ当て付け３６ｓ、３６ｔ、３６ｕが設置されている。そして孔３６ｌには、ＮＤ６からなる減光部材３０１が当て付け３６ｓに保持されて挿入されている。
【００６１】
上記のような構成をなす共焦点顕微鏡の動作は、低い倍率の対物レンズを使用して共焦点観察をする場合と、高い倍率の対物レンズを使用して共焦点観察をする場合は、第１の実施の形態と同様であるが、非共焦点観察をする場合は異なるので、その場合について以下に述べ、他の観察については説明を省略する。
【００６２】
非共焦点観察をする場合、観察者はレバー８を持って移動部材３６を移動させ、孔３６ｌに光軸１５が通るようにディスク３５を位置決めする。この場合、Ｖ溝３６ａに位置決め機構７が係合する。この動作を行なった後、第１の実施の形態と同様に、観察者１４は接眼レンズ１３を介して観察をする。
【００６３】
以上のように本第３の実施の形態では、ディスク３５のラインパターンが配置されたいずれかの領域３５ａ、３５ｂが、使用する対物レンズの倍率や観察法に応じて光軸１５上に位置するように移動部材６が移動するため、最良の状態で共焦点観察を行なえる。また、ディスク３５外の孔３６ｌに光軸１５が通るように位置決めすれば、孔３６ｌに減光部材３０１が設置されているので、第１の実施の形態と同様に、観察者は眩しく感じずに非共焦点観察を行なえる。また、例えば孔３６ｎにＮＤ２５からなる減光部材を挿入してもよく、この場合、第２の実施の形態と同様に、低倍率の対物レンズを使用したときに観察者が眩しく感じることはない。
【００６４】
上記実施の形態においては、減光手段としてＮＤ６、ＮＤ２５を設置したが、これらと異なる透過率の減光部材を各箇所に配置してもよい。また、上記実施の形態においては、ディスク又は移動部材に例えばＮＤ６を設置したが、光軸に対して垂直な方向へ移動し光軸上の任意の位置に挿脱可能なようＮＤ６を設置してもよい。また、上記実施の形態においては、ディスク上に２種類のラインパターンを配置したが、これは１種類でも３種類以上であってもよい。
【００６５】
また、上記実施の形態においては、観察者が接眼レンズを介して像を観察するようにしているが、ＣＣＤカメラなどの撮像手段で撮像、観察してもよい。また、上記実施の形態においては、光源から試料に照射させるためにビームスプリッタを設置したが、偏光ビームスプリッタを設置し、この偏光ビームスプリッタが光を入射、出射する３方向側に偏光板を配置するように構成してもよい。また、上記実施の形態においては、光源に水銀又はキセノンの光源を使用したが、ハロゲン等その他の光源であってもよい。
【００６６】
また、上記実施の形態においては、ラインパターンの設置されているディスクを使用したが、例えばＮｉｐｋｏｗディスクのようなピンホールが螺旋状に配置されているディスクを使用してもよい。また、上記実施の形態においては、ディスク面から反射した不要な光、いわゆるゴーストが観察者の視野内に戻ってこないよう、ディスク５の面を光軸１５に垂直な面に対して５°傾けた。この傾ける角度は５°に限らず、例えば０°であってもよいが、好ましくは０°〜５°の間が効果的である。また、上記実施の形態においては、移動部材の移動を観察者が手動で行なったが、電動で移動させるようにしてもよい。
【００６７】
（第４の実施の形態）
図６は、本発明の第４の実施の形態に係る共焦点顕微鏡の構成を示す図である。この共焦点顕微鏡では、セクショニング像観察を行う。図６において、例えば水銀又はキセノンの光源１０１から出射される光の光路上に、コンデンサーレンズ１０２、偏光板１０３、ＰＢＳ（偏光ビームスプリッター）１０４が配置されている。ＰＢＳ１０４の反射光路上には、ディスク１０５、第１の結像レンズ１０７、１／４波長板１０８、対物レンズ１０９、及び図示しないステージに載置された試料１１０が配置されている。ＰＢＳ１０４の試料１１０に対する透過光路上には、第２の結像レンズ１１１とＣＣＤカメラ１１２が配置されている。ＣＣＤカメラ１１２の画像出力端子にはモニター１１３が接続されている。モニター１１３は、画像を表示する。
【００６８】
図７は、ディスク１０５の構成を示す図である。ディスク１０５は円盤状をなし、光学ガラスの基板上に図７に示すようなパターンが形成されており、同心円により三つの領域Ａ’、領域Ｂ’、及び領域Ｃ’に区分されている。このうち最内周の領域Ａは、光が一様に透過するよう透明になっている。その他の領域Ｂ’及びＣ’には多数のピンホールが螺旋状に設けられている。各領域Ｂ’、Ｃ’間のピンホールの直径は異なり、領域Ｂ’、Ｃ’の順に大きくなっている。各ピンホールは、互いの間隔がピンホールの直径の４〜１０倍程度となるよう配設されている。なお、図７において網で示された部分は、遮蔽部である。
【００６９】
ディスク１０５は、図示しない駆動機構によって、図６の矢印ａ又は矢印ｂの方向（紙面に対して左右方向）に移動可能である。また、ディスク１０５は、回転軸１０６を介して図示しないモータの軸に連結されており、前記モータの駆動により一定の回転速度で回転する。
【００７０】
次に、上記のような構成をなす共焦点顕微鏡の動作を説明する。
【００７１】
光源１０１から出射された光は、コンデンサーレンズ１０２、偏光板１０３を介し、ＰＢＳ１０４で一定の方向の偏光成分のみが反射され、一定の速度で回転するディスク１０５に入射される。ディスク１０５のピンホールを通過した光は、第１の結像レンズ１０７を通り、１／４波長板１０８で円偏光になって、対物レンズ１０９によって結像され、試料１１０に入射される。
【００７２】
試料１１０から反射された光は、対物レンズ１０９を介し、再度、１／４波長板１０８にて入射時の偏光方向に対して直交する偏光方向になり、第１の結像レンズ１０７によりディスク１０５上に試料像が投影される。投影された試料像のうち焦点の合っている部分はピンホールを通過し、さらにＰＢＳ１０４を第２の結像レンズ１１１の方向に透過して、第２の結像レンズ１１１を介してＣＣＤカメラ１１２で撮像される。ＣＣＤカメラ１１２で撮像された共焦点画像は、モニター１１３に表示される。
【００７３】
適切な共焦点効果を得るためにピンホール径Ｒは、光の波長をλ、観察する試料１１０のディスク１０５上への投影倍率をＭ、対物レンズ１０９の開口数をＮＡとすると、
Ｒ＝ｋλＭ／ＮＡ
の関係で設定される。ｋは係数であり、ｋ＝０．５〜１程度が良く使われる。したがって、対物レンズ１０９を交換すると、ディスク１０５上への投影倍率Ｍと開口数ＮＡが変わってしまうため、共焦点観察をするためのピンホール直径Ｒも変える必要がある。
【００７４】
観察に要する倍率を変えたときには、対物レンズ１０９を交換する。このとき、上記のようにディスク１０５上のピンホール直径も変える必要がある。通常、対物レンズ１０９の倍率が大きくなるにつれて、ＮＡは大きくなることが多いが、倍率が大きくなる割合に対して、ＮＡの大きくなる割合は小さい。よって、倍率の大きい対物レンズ１０９を用いる場合、ディスク１０５を図６の矢印ａの方向に移動させ、ピンホール径の大きい領域Ｃ’を用いるようにする。
【００７５】
また、焦点を合わせたり、観察位置を探したりするために、明視野像（非共焦点像）の観察を行いたいときには、ディスク１０５を図６の矢印ｂの方向に移動させ、パターンが無い素通し（透明）の領域Ａ’を用いるようにする。このとき、ガラス基板で形成されているディスク１０５を光路が通ることになるため、光路長に変化が無く、共焦点像の観察時と明視野像の観察時とで焦点が移動しないことになる。
【００７６】
なお、明視野像の観察時は、ディスク１０５のパターンが無い部分を使用するので、必ずしもディスク１０５を回転させる必要は無い。しかし、ディスク１０５は像面にあり、静止しているとディスク５上に挨等がつき、非常に目立つ。このため明視野像の観察時は、ディスク１０５を回転させることが望ましい。
【００７７】
（第５の実施の形態）
図８は、本発明の第５の実施の形態に係る共焦点顕微鏡の構成を示す図である。例えば水銀又はキセノンの光源３００から出射される光の光路上に、コリメータレンズ３０２、励起フィルター３０３、ダイクロイックミラー３０４が配置されている。ダイクロイックミラー３０４の反射光路上には、ディスク３０５、第１の結像レンズ３０７、対物レンズ３０８、及び図示しないステージに載置された試料３０９が配置されている。ダイクロイックミラー３０４の試料３０９に対する透過光路上には、吸収フィルタ３１０、第２の結像レンズ３１１、及びＣＣＤカメラ３１２が配置されている。
【００７８】
ディスク３０５は、図示しない駆動機構によって、図８の矢印ａ又は矢印ｂの方向（紙面に対して左右方向）に、モータ３０６と共に移動可能である。また、ディスク３０５は、回転軸３０５１を介してモータ３０６の軸に連結されており、モータ３０６の駆動により一定の回転速度で回転する。
【００７９】
図９の（ａ）は、ディスク３０５の構成を示す全体図であり、図９の（ｂ）は、ディスク３０５の部分拡大図である。ディスク３０５は円盤状をなし、図９の（ａ）に示すように、同心円により領域Ａ”、Ｂ”に分けられている。領域Ａ”（パターン部）にはスリット状の透過部が等間隔に配置され、さらに直線パターンを複数配置した一定の幅Ｄの領域４０１が、他の部分と直交するように設けられている。図９の（ｂ）は、図９の（ａ）の領域Ａ”の一部を拡大した図である。領域Ａ”のパターンは、図９の（ｂ）に示すように、直線状の光が通過するスリット部分４０５と光を遮蔽する遮蔽部分４０４とが交互に並んで配置されている。遮蔽部分４０４のラインの幅は、スリット部分４０５の幅より広く、これらは例えば１９：１の比になっている。また、領域Ｂ”は一様な透過率で光を透過するＮＤフィルタとなっており、その透過率は、領域Ａ”の透過率がＴの場合は、
【数４】

（ｑ＝１〜０．２）となっている。例えば上記のように領域Ｂ”の透過率Ｔは５％、すなわち０．０５である。ｑ＝０．８を選べば、ＮＤフィルタの透過率は、
【数５】

であるので、約１８％の透過率のＮＤフィルタを領域Ｂ”に形成しておく。なお、ｑ＝０．２の場合、視野中の１／２５（４％）の範囲で焦点が合っており、ｑ＝１の場合、視野中の全て（１００％）の範囲で焦点が合っている状態にある。共焦点顕微鏡で観察されるほぼ全ての試料は、このｑ＝１〜０．２の範囲で観察可能である。なお、図９の（ａ），（ｂ）において白抜きで示された部分は、遮蔽部である。
【００８０】
次に、上記のような構成をなす共焦点顕微鏡の動作を説明する。
【００８１】
光源３００から出射された光は、コリメータレンズ３０２を通って、励起フィルター３０３で波長選択され、ダイクロイックミラー３０４に入射する。励起フィルター３０３は、図１０の（ａ）に示すような透過特性を有し、光源３００からの光から蛍光を励起する波長の光を選択的に透過し、その他の波長の光を遮断する。また、ダイクロイックミラー３０４は、図１０の（ｂ）に示すように、励起フィルター３０３を透過した波長の光を反射し、図１０の（ｃ）に示すように、試料３０９からの蛍光の波長を透過する特性を有している。また、後述する吸収フィルター３１０は、励起波長を遮断し蛍光波長を透過する、図１０の（ｃ）に示すような透過特性を有している。
【００８２】
これらの波長特性は、使用する蛍光色素により異なる。例えば、ＦＩＴＣを観察する場合は、励起最大波長が４９０ｎｍ、蛍光最大波長が５２０ｎｍである。このため、励起フィルター３０３は透過する光の波長が４６０〜４９０ｎｍであるもの、ダイクロイックミラー３０４は反射波長が４６０〜４９０ｎｍであるもの、ダイクロイックミラー３０４と吸収フィルターは透過する波長が５１０ｎｍであるものが多く使われる。
【００８３】
ダイクロイックミラー３０４で反射された励起光は、一定の速度で回転するディスク３０５に入射される。共焦点像を観察する場合は、直線状のパターンが形成されている図９の（ａ）の領域Ａ”が光路上に位置するようディスク５を移動させておく。このディスク３０５の直線状のスリット部分４０５を透過した光は、第１の結像レンズ３０７を通り、対物レンズ３０８によって結像され、試料３０９に入射される。
【００８４】
励起光により、試料３０９は蛍光発光する。試料３０９からの蛍光は、対物レンズ３０８を通り、第１の結像レンズ３０７を介してディスク３０５に入射し、ディスク３０５上で試料３０９の像が結像される。結像された像のうち、焦点の合っている成分は、ディスク３０５上のスリット部分４０５を通過し、蛍光波長の光がダイクロイックミラー３０４を透過する。吸収フィルター３１０でも、励起波長の光は遮断される。蛍光波長の光は吸収フィルター３１０を透過し、第２結像レンズ３１１を介してＣＣＤカメラ３１２に入射し、ＣＣＤカメラ３１２で試料蛍光像が結像される。
【００８５】
焦点を合わせたり、観察位置を探したりするために、明視野像(非共焦点像)の観察を行いたいときには、ディスク３０５を図８の矢印ａの方向に移動させ、パターンが無い一様な透過用の領域Ｂ”を用いるようにする。ここで、直線状のパターンの領域Ａ”から領域Ｂ”に切り替えたときに、領域Ｂ”が透過率がほぼ１００％である素通しのガラス基板であると、観察像が明るくなり過ぎてしまうため、何らかの手段で減光をする必要がある。共焦点像の観察時には、ディスク３０５の透過率がＴのとき、光源３００からの光のＴ倍の光が試料３０９に照射される。試料３０９からの蛍光は、ディスク３０５を通る。このとき焦点が合っていれば、９０％程度の蛍光を透過するが、焦点が合っていないと、非合焦の度合いに応じて透過する蛍光が減ることになる。数百μｍ以上非合焦であれば、蛍光もディスク３０５によりＴ倍になるが、通常顕微鏡で観察する試料の厚さは数μｍ〜数十μｍであるため、Ｔ倍も暗くはならない。
【００８６】
一様な透過用の領域Ｂ”の透過率を、
【数６】

（ｑ＝１〜０．２）にすると、１００％の透過に対して、観察像の明るさはｑ^２Ｔ倍になる。本実施の形態では、明視野像の明るさが０．１８^２＝０．０３倍になり、共焦点像の明るさは合焦部分のみが０．０５倍であり非合焦像はこれより非常に暗くなっているので、明視野像と共焦点像の明るさはほぼ同じになると考えられる。
【００８７】
また、明視野像の観察時でも第４の実施の形態と同様に、ガラス基板で形成されているディスク３０５を光路が通ることになるため、光路長に変化が無く、共焦点像の観察時と比べて焦点は移動しない。
【００８８】
本第５の実施の形態では、ディスク３０５における直線状の透過部分の幅と遮蔽部分の幅との比を１：１９にしているが、この比はこれより小さくても大きくても良い。例えば、非常に暗い蛍光の場合は、非合焦成分が増えるが、前記比を１：５ほどにすると、像が明るくなる。また、非常に明るい蛍光の場合は１：５０ほどにすると、非合焦成分がほとんど無くなり、焦点の合った像のみのセクショニング画像が得られる。しかしこの場合、像は暗くなる。
【００８９】
また第４の実施の形態では、試料の反射像をピンホールが形成されたＮｉｐｋｏｗディスクで、第５の実施の形態では、蛍光試料を直線状のパターンを有するディスクで観察する例を示したが、ディスクのパターンは第４の実施の形態と第５の実施の形態とを入れ替えてもよい。
【００９０】
また、第５の実施の形態においても、ＮＤフィルタの部分を第４の実施の形態と同様に透明にして、焦点ずれがなくなる効果のみにしてもよい。さらに第５の実施の形態においても、第４の実施の形態と同様に、ディスクを同心円上の領域に分けて、異なったラインパターンを領域ごとに形成し、異なった対物レンズヘの対応を図ることも考えられる。この場合、一様に光を透過する領域を外側の同心円領域に配置してもよい。また、Ｚステージを追加して、試料と対物レンズとの距離を変化させ、次々に画像を取り込めば、３次元観察を行うことも可能である。
【００９１】
以上のように、第１，２，５の実施の形態ではディスクに減光手段を設けた共焦点顕微鏡について述べた。
【００９２】
なお、本発明は上記各実施の形態のみに限定されず、要旨を変更しない範囲で適宜変形して実施できる。
【００９３】
【発明の効果】
本発明の共焦点顕微鏡によれば、ディスクの一部の領域を一定の透過率とし、明視野像の観察時にこの領域を通して観察を行なうよう構成したので、顕微鏡を非常に安価に作製でき、観察が容易になるばかりでなく、共焦点像と明視野像とを切り換えて観察をしても、焦点の移動が無くなる。また、ディスクの一定の透過率の領域にＮＤフィルタを形成し、その透過率を、パターン部分の透過率Ｔとしたときに、
【数７】

（ｑ＝１〜０．２）とすることで、共焦点像と明視野像とを切り換えて観察したときに観察像の明るさがほぼ同じになり、光量を調節する必要が無くなる。
【００９４】
以上のように本発明によれば、簡単な構成により良好な状態で観察を行なえる共焦点顕微鏡を提供できる。
【００９５】
また本発明によれば、共焦点像と明視野像の観察切換えを容易に行え、かつ切換え時に焦点の調整を必要としない共焦点顕微鏡を提供できる。
【図面の簡単な説明】
【図１】本発明の第１の実施の形態に係る共焦点顕微鏡の構成を示す図。
【図２】本発明の第１の実施の形態に係るディスクの構成を示す図。
【図３】本発明の第２の実施の形態に係るディスクの構成を示す図。
【図４】本発明の第３の実施の形態に係る移動部材とディスクの構成を示す上面図。
【図５】本発明の第３の実施の形態に係る移動部材とディスクの構成を示す側面図。
【図６】本発明の第４の実施の形態に係る共焦点顕微鏡の構成を示す図。
【図７】本発明の第４の実施の形態に係るディスクの構成を示す図。
【図８】本発明の第５の実施の形態に係る共焦点顕微鏡の構成を示す図。
【図９】本発明の第５の実施の形態に係るディスクの構成を示す図。
【図１０】本発明の第５の実施の形態に係る透過特性と反射特性を示す図。
【図１１】従来例に係る共焦点顕微鏡の構成を示す図。
【図１２】従来例に係るＮｉｐｋｏｗディスクの構成を示す図。
【符号の説明】
１…光源
２…コレクタレンズ
３…ビームスプリッタ
４…モータ
４ａ…軸
５…ディスク
６…移動部材
６ａ、６ｂ、６ｃ…Ｖ溝
７…位置決め機構
８…レバー
９…結像レンズ
１０…対物レンズ
１１…試料
１２…結像レンズ
１３…接眼レンズ
１４…観察者
１５…光軸
２５…ディスク
３５…ディスク
３６…移動部材
３６ａ、３６ｂ、３６ｃ…Ｖ溝
３６ｌ、３６ｍ、３６ｎ…孔
３０１…減光部材
３６ｓ、３６ｔ、３６ｕ…当て付け
１０１…光源
１０２…コンデンサーレンズ
１０３…偏光板
１０４…ＰＢＳ
１０５…ディスク
１０６…回転軸
１０７…第１の結像レンズ
１０８…１／４波長板
１０９…対物レンズ
１１０…試料
１１１…第２の結像レンズ
１１２…ＣＣＤカメラ
１１３…モニター
３００…光源
３０２…コリメータレンズ
３０３…励起フィルター
３０４…ダイクロイックミラー
３０５…ディスク
３０５１…回転軸
３０６…モータ
３０７…第１の結像レンズ
３０８…対物レンズ
３０９…試料
３１０…吸収フィルタ
３１１…第２の結像レンズ
３１２…ＣＣＤ[0001]
BACKGROUND OF THE INVENTION
The present invention provides a confocal microscope that obtains a confocal image using light transmitted through a pinhole or the like formed on a rotating disk. For example, the microstructure and three-dimensional shape of a sample can be observed at high speed using light. The present invention relates to a disk scanning confocal microscope used for measurement.
[0002]
[Prior art]
(First conventional technology)
In recent years, disc rotating confocal microscopes that can obtain observation images with high resolution in real time have been commercialized. This confocal microscope inserts and rotates a disk provided with a light passage such as a pinhole in a normal microscope optical path, removes the image of the out-of-focus surface, Only images can be extracted. Thereby, a confocal image having a very high contrast can be obtained.
[0003]
Such a confocal microscope is usually used for the purpose of obtaining an image that is further emphasized during high-magnification observation because of its nature. In general, it is necessary to align and focus a sample before observing an image. However, with the above confocal microscope, only the image of the in-focus surface can be obtained, so that it is very difficult to perform alignment and focusing operations.
[0004]
Therefore, in general, in a confocal microscope, the disk is retracted from the optical path so that a non-confocal image that does not pass through the disk can be obtained, particularly for alignment and focusing. In general, this disk is a Nipkow disk in which pinholes are arranged in a spiral, or a disk in which pinholes are randomly arranged or a line pattern is used. Since the reflected light from the available sample is as low as 0.5 to 1%, the resulting confocal image is very dark.
[0005]
In order to improve this, mercury or xenon is used as the light source. However, when the disk is retracted and the non-confocal image is viewed as described above, the light is felt dazzling because it is very bright. In such a case, since the light source of mercury or xenon cannot be dimmed, it cannot be adjusted by reducing the amount of light.
[0006]
In order to solve this problem, in the microscope apparatus as described above, one polarizing plate is rotated using a dimming means such as an ND filter or two polarizing plates that can be arbitrarily inserted into and removed from the optical path. A mechanism to reduce the amount of light is installed.
[0007]
Japanese Utility Model Laid-Open No. 5-96816 and Japanese Patent Laid-Open No. 9-325279 do not retract the disk from the optical path as described above, but provide optical paths for confocal and non-confocal, respectively. And a branching optical system member using a beam splitter is inserted into and removed from the optical path to selectively branch the optical path and switch between a confocal image and a non-confocal image. . In this configuration, separate illumination is provided for confocal and non-confocal illumination, so a high-intensity light source such as mercury or xenon is used for confocal illumination, and a low-luminance light source such as halogen is used for non-confocal illumination. If it is used, it will not be too bright during non-confocal observation.
[0008]
Unlike Japanese Utility Model Laid-Open No. 5-96816 and Japanese Patent Laid-Open No. 9-325279, JP 2001-91842 A uses a polarizing plate and a polarizing beam splitter instead of inserting and removing the optical system member. In this document, the optical path is selectively branched by rotating the polarizing plate to switch between the confocal image and the non-confocal image. The structure described in this publication uses the technique described in “Efficient rea1-time confocal microscopy with white light sources” by R. Jackaitis, T. Wilson et al., Nature, Vo 1.383 0ct. 1996 p804-806. , So-called T.A. A Wilson type disk is used. When this disk is used, the reflected light that can be used with respect to the incident light is as high as 25 to 50%, and a bright observation image can be obtained even during confocal observation, so that a halogen light source can be used. For this reason, even during non-confocal observation, it is not too bright and feels dazzling.
[0009]
Japanese Patent Laid-Open No. 2001-83426 also discloses T.W. In a confocal microscope apparatus using a Wilson type disk, a non-confocal image is obtained by electrically controlling a light beam so as to pass through an aperture and stopping the disk. Even in this configuration, since the same disk as described above is used, it is not too bright and dazzling even during non-confocal observation.
[0010]
(Second conventional technology)
As a conventional typical disk scanning confocal microscope, a confocal microscope using a Nipkow disk having a large number of pinholes is known.
[0011]
FIG. 11 is a diagram showing a configuration of a confocal microscope using the above-mentioned Nippon disc. A condenser lens 602, a polarizing plate 603, and a PBS (deflection beam splitter) 604 are disposed on the optical path of light emitted from the light source 601. On the reflection optical path of the PBS 604, a Nipkow disk (rotating disk) 605, a first imaging lens 607, a quarter-wave plate 608, an objective lens 609, and a sample 610 placed on a stage (not shown) are arranged. . A second imaging lens 611 and a CCD camera 612 are arranged on the transmission optical path for the sample 610 of the PBS 604. A monitor 613 is connected to the image output terminal of the CCD camera 612. The monitor 613 displays an image.
[0012]
FIG. 12 is a diagram showing the structure of a Nipkow disk. The Nippon disk 605 has a disk shape and is divided into three regions A, B, and C by concentric circles as shown in FIG. A large number of pinholes are spirally provided in the three regions A, B, and C. The diameter of the pinhole between each area | region A, B, and C differs, and becomes small in order of area | region A, B, and C. FIG. The pinholes are arranged so that the interval between them is about ten times the diameter of the pinholes.
[0013]
The Nipkow disk 605 can be moved in the direction of arrow A in FIG. Further, the Nippon disk 605 is connected to a motor shaft (not shown) via a rotating shaft 606, and rotates at a constant rotational speed by driving the motor.
[0014]
Next, the operation of the confocal microscope configured as described above will be described.
[0015]
The light emitted from the light source 601 passes through the condenser lens 602 and the polarizing plate 603, and only the polarized component in a certain direction is reflected by the PBS 604 and is incident on the Nippon disk 605 that rotates at a constant speed. The light that has passed through the pinhole of the Nippon disk 605 passes through the first imaging lens 607, becomes circularly polarized light by the quarter wavelength plate 608, forms an image by the objective lens 609, and enters the sample 610.
[0016]
The light reflected from the sample 610 becomes a polarization direction orthogonal to the polarization direction at the time of incidence by the quarter wavelength plate 608 again through the objective lens 609, and the first imaging lens 607 makes a Nippon disk. A sample image is projected onto 605. The in-focus portion of the projected sample image passes through the pinhole, further passes through the PBS 604 in the direction of the second imaging lens 611, and passes through the second imaging lens 611, the CCD camera 612. The image is taken with. A confocal image captured by the CCD camera 612 is displayed on the monitor 613.
[0017]
The pinhole diameter R for obtaining an appropriate confocal effect is expressed as follows: λ is the wavelength of light, M is the projection magnification of the sample 610 to be observed on the disk 605, and NA is the numerical aperture of the objective lens 609.
R = kλM / NA
It is set in relation to k is a coefficient, and k = 0.5 to 1 is often used. Therefore, when the objective lens 609 is replaced, the projection magnification M onto the disk 605 and the numerical aperture NA change, and therefore the pinhole diameter R for confocal observation needs to be changed.
[0018]
When changing the magnification required for observation, the objective lens 609 is replaced. When using an objective lens with a high magnification, the Nippon disk 605 is moved in the direction of arrow A in FIG. 11 to use the region A with a large pinhole diameter. In such a confocal microscope, only a focused image is observed.
[0019]
In order to perform normal observation, it is necessary to first focus and search for an observation position, but this operation is very difficult with a confocal microscope. Therefore, in general, in the confocal microscope, the disk 605 is moved in the direction of arrow A in FIG. Confocal images are also obtained.
[0020]
In addition, as disclosed in Japanese Utility Model Laid-Open No. 5-96816 and Japanese Patent Laid-Open No. 9-325279, an optical path is provided for confocal use and non-confocal use instead of moving the disk, and mirrors and beam splitters are provided. A device is disclosed in which a confocal image and a bright field image are obtained by switching the optical path by inserting and removing the branching optical system member to and from the optical path.
[0021]
[Problems to be solved by the invention]
(Problems of the first conventional technique)
As described above, the conventional configuration is such that the observation can be arbitrarily switched to a confocal image or a non-confocal image, and it is configured so as not to be too bright and dazzling at the time of non-confocal observation. A confocal microscope has been proposed that can be inserted into and removed from the optical path.
[0022]
However, in a configuration in which the disk is retracted from the optical path so that a non-confocal image that does not pass through the disk can be obtained, after the operation of retracting the disk is performed, a new dimming means is inserted on the optical path. And rotating the polarizing plate, the operation is very troublesome. In addition, the observer feels very dazzling and discomfort from the time the disk is evacuated to the next operation.
[0023]
In addition, in the configuration described in Japanese Utility Model Publication No. 5-96816 and Japanese Patent Application Laid-Open No. 9-325279, two optical paths for confocal and non-confocal are necessary, and there are also two types of illumination. A very large space is required.
[0024]
Further, in the configuration described in Japanese Patent Application Laid-Open No. 2001-91842, two optical paths for confocal and non-confocal are necessary, and a very large space is required. In addition, T.W. Since a Wilson disc is used, it is necessary to calculate the difference between the confocal image including the non-confocal component obtained from the line pattern area in the disc and the non-confocal image obtained from the aperture, and the confocal image is visually observed. I can't.
[0025]
In the configuration described in Japanese Patent Laid-Open No. 2001-83426, T. Since a Wilson disc is used, it is necessary to calculate the difference between the confocal image including the non-confocal component obtained from the line pattern area in the disc and the non-confocal image obtained from the aperture, and the confocal image is visually observed. In addition, since electrical control for stopping the disk is performed, a circuit for that purpose is required and the mechanism is complicated.
[0026]
(Problems of the second conventional technique)
The conventional disk scanning confocal microscope has the following problems.
[0027]
In a configuration in which the disc is retracted from the optical path and a non-confocal image that does not pass through the disc can be obtained, the air-converted length of light differs depending on whether the disc is in the optical path or retracted. This type of disk is usually made of optical glass such as BK7 and has a refractive index of about 1.5. On the other hand, when the disk is retracted, the thickness of the disk in the optical path becomes air, so the refractive index becomes 1. As a result, a phenomenon occurs in which the focal point moves. Therefore, it is necessary to refocus between the observation of the bright field image and the observation of the confocal image.
[0028]
In the configuration described in Japanese Utility Model Laid-Open No. 5-96816 and Japanese Patent Laid-Open No. 9-325279, two optical paths for confocal and bright fields are required, and there are also two types of illumination. The space becomes large and it is necessary to adjust the optical axes of the two optical paths.
[0029]
A first object of the present invention is to provide a confocal microscope capable of observing in a good state with a simple configuration.
[0030]
A second object of the present invention is to provide a confocal microscope that can easily switch between observation of a confocal image and a bright-field image and does not require adjustment of the focus at the time of switching.
[0031]
[Means for Solving the Problems]
In order to solve the above problems and achieve the object, the confocal microscope of the present invention is configured as follows.
[0032]
(1) The confocal microscope of the present invention illuminates a confocal disk arranged at or near the image position of the objective lens, a position conjugate with the image position with the light from the light source, and passes the passing light. The objective lens forms an image on the specimen and scans it. The light from the specimen is again imaged on the confocal disk or its vicinity via the objective lens, and the light passing through the confocal disk is re-applied. In a confocal microscope that forms an image by obtaining an image of the specimen, a moving unit that moves a surface of the confocal disk in a direction substantially perpendicular to an optical axis; The locking unit is configured to lock the moving unit so as to be positioned on the axis, and the dimming unit is detachable on the optical axis in conjunction with the movement of the moving unit.
[0033]
(2) The confocal microscope of the present invention is the microscope described in (1) above, and the dimming means is provided on the confocal disk.
[0034]
(3) The confocal microscope of the present invention is the microscope described in (1) above, and the dimming means is provided in the moving means.
[0035]
(4) The confocal microscope of the present invention rotates a disk on which a predetermined pattern including a light transmitting portion and a shielding portion is formed, scans the sample with the light transmitted through the pattern, and In a confocal microscope for obtaining a confocal image of a sample, the disk includes a plurality of regions divided concentrically, and at least one of the plurality of regions includes a region that transmits light uniformly.
[0036]
(5) The confocal microscope of the present invention is the microscope described in the above (4), and uniformly transmits the light when T is a light transmittance in the pattern formed on the disk. The transmittance of the area is
[Expression 2]

(Q = 1 to 0.2).
[0037]
As a result of taking the above-mentioned means, the following effects are obtained.
[0038]
(1) According to the confocal microscope of the present invention, by providing a plurality of regions having a pattern on the disk, the best suited to the conditions can be obtained only by a simple operation of moving the disk in a direction substantially perpendicular to the optical axis. The confocal observation can be performed in the state. Further, by making the dimming means detachable on the optical axis, the observer does not feel dazzling during both confocal observation and non-confocal observation. This eliminates the need for insertion and removal of other optical members and multiple optical paths, and eliminates the need for complicated optical systems and electrical control, eliminating the need for large spaces and making the entire microscope compact and simple. And can be observed in good condition.
[0039]
(2) According to the confocal microscope of the present invention, the dimming means is inserted / removed onto / from the optical axis simultaneously with the insertion / removal of the disc, so that a new mechanism such as an insertion / removal mechanism for the dimming means is not required. Can be easy.
[0040]
(3) According to the confocal microscope of the present invention, it is not necessary to increase the diameter of the disk in order to provide the dimming means, so that further space is reduced, the entire microscope can be configured compactly, and in good condition. Can observe.
[0041]
(4) According to the confocal microscope of the present invention, when observing a bright field image, the observation is made through a portion where there is no disc pattern and uniformly transmits light. Light can pass through the same material when observing the image.
[0042]
(5) According to the confocal microscope of the present invention, light is transmitted during observation of a bright field image.
[Equation 3]

Since the light passes through the region (q = 1 to 0.2), the brightness is almost the same as that during confocal image observation.
[0043]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[0044]
(First embodiment)
FIG. 1 is a diagram showing a configuration of a confocal microscope according to the first embodiment of the present invention. In FIG. 1, for example, light emitted from a light source 1 of mercury or xenon becomes parallel light via a collector lens 2 and enters a beam splitter 3. The light incident on the beam splitter 3 is bent by 90 ° and irradiates the disk 5. The disk 5 is fitted on the shaft 4 a of the motor 4 and is always rotated by driving of the motor 4.
[0045]
FIG. 2A is a diagram showing a configuration of a disk 5 which is a line pattern disk (confocal disk). The disk 5 is provided with a plurality of line patterns having a transmission part 5x and a reflection part 5y as shown in FIG. 2B, and the width of the transmission part 5x is about 1/20 of the width of the reflection part 5y. Degree. The disk 5 includes a region 5a having a small width of the transmissive portion, a region 5b having a large transmissive portion, and a region 5c having no line pattern and having, for example, ND6 (a dimming member having a transmittance of 6%). Has two areas. The disk 5 is disposed at the image position of the objective lens 10, a position conjugate with the image position, or the vicinity thereof.
[0046]
In the present embodiment, as shown in FIG. 1, the motor 4 is supported by the moving member 6 so that the surface of the disk 5 is inclined by 5 ° with respect to the surface perpendicular to the optical axis 15. As the moving member 6 moves, the motor 4 and the disk 5 are in three stages in the inclined state as described above so that one of the three regions 5a, 5b, and 5c shown in FIG. Moving. The moving member 6 is provided with V grooves 6a, 6b, 6c and a lever 8, and a positioning mechanism 7 is fixed to a microscope body (not shown). The moving member 6 is moved when the observer operates the lever 8. Along with this movement, any one of the V grooves 6a, 6b, 6c is engaged with the positioning mechanism 7, whereby the moving member 6 is positioned so that one of the three regions 5a, 5b, 5c passes through the optical axis 15. The
[0047]
The light irradiated on the disk 5 passes through the imaging lens 9 and irradiates the sample 11 via the objective lens 10. The reflected light from the sample 11 is imaged on the disk 5 by the imaging lens 9 via the objective lens 10, and the confocal image formed by passing through the disk 5 is imaged via the beam splitter 3. The image is formed by the lens 12 and is observed by the observer 14 through the eyepiece lens 13.
[0048]
Next, the operation of the confocal microscope configured as described above will be described. First, when confocal observation is performed using, for example, a low-magnification objective lens, the observer moves the moving member 6 with the lever 8 and positions the disk 5 so that the optical axis 15 passes through the region 5a. In this case, the positioning mechanism 7 is engaged with the V groove 6a.
[0049]
For example, when confocal observation is performed using an objective lens with a high magnification, the observer moves the moving member 6 with the lever 8 and positions the disk 5 so that the optical axis 15 passes through the region 5b. In this case, the positioning mechanism 7 is engaged with the V groove 6b.
[0050]
Next, when performing non-confocal observation, the observer moves the moving member 6 with the lever 8 and positions the disk 5 so that the optical axis 15 passes through the region 5c. In this case, the positioning mechanism 7 is engaged with the V groove 6c.
[0051]
After any of these operations, the light emitted from the light source 1 irradiates the disk 5 through the collector lens 2 and the beam splitter 3, and then irradiates the sample 11 through the imaging lens 9 and the objective lens 10. To do. At this time, the disk 5 rotates at high speed, and light is scanned on the sample 11 at high speed. Reflected light from the sample 11 passes through the objective lens 10 and is imaged on or near the disk 5 by the imaging lens 9, and an image formed by passing through the area of the disk 5 is re-coupled by the imaging lens 12. And is observed by the observer 14 through the eyepiece 13.
[0052]
As described above, in the first embodiment, one of the regions 5a and 5b where the line pattern of the disk 5 is arranged moves so as to be positioned on the optical axis 15 according to the magnification of the objective lens to be used. Since the member 6 moves, confocal observation can be performed in the best state. In addition, since the reflected light from the sample 11 that can be used for the incident light from the light source 1 is as low as about 5% due to the area where the line pattern is arranged, a high-intensity light source such as a mercury or xenon light source is used. ing. However, when the observer intends to perform non-confocal observation, a dimming member such as ND6 or 25 is installed in the area 5c without the line pattern of the disk 5, so that the area 5c is positioned on the optical axis 15. If the moving member 6 is moved in this manner, the observer can perform confocal observation without feeling dazzling.
[0053]
(Second Embodiment)
Since the second embodiment is different from the first embodiment only in the pattern on the disk, the pattern of the disk will be described with reference to FIGS. Description of the portion is omitted.
[0054]
FIG. 3A is a diagram showing a configuration of a disk 25 which is a line pattern disk (confocal disk). The disk 25 is provided with a plurality of line patterns having a transmission part 25x and a reflection part 25y as shown in FIG. 3B, and the width of the transmission part 25x is about 1/20 of the reflection part 25y. is there. The disk 25 has a small transmissive part width, for example, an area 25a where ND25 (a dimming member having a transmittance of 25%) is installed in the transmissive part, a wide transmissive part width area 25b, and no line pattern. There are three areas 25c where the ND6 is installed.
[0055]
The operation of the confocal microscope provided with the disk 25 configured as described above is the same as that of the first embodiment, and thus description thereof is omitted.
[0056]
For example, when the difference in magnification between the low-magnification objective lens and the high-magnification objective lens used is large, the low-magnification objective lens has a significantly larger pupil diameter than the high-magnification objective lens. For this reason, when the type of light source and the amount of light are set in accordance with observation using a high-magnification objective lens, it is dazzling when confocal observation is performed using a low-magnification objective lens.
[0057]
On the other hand, in the second embodiment, unlike the first embodiment, the light reducing member is also installed in the region where the width of the transmission portion is small. Therefore, the low magnification objective lens is used for transmission. The observer does not feel dazzling when observing the region 25a having a small width on the optical axis.
[0058]
(Third embodiment)
The third embodiment differs from the first embodiment only in the shape and pattern on the disk and the shape of the moving member, and these portions will be described with reference to FIGS. Explanation of other parts is omitted.
[0059]
FIG. 4A is a top view showing a configuration of the moving member 36 and a disk 35 which is a line pattern disk (confocal disk). The disk 35 is provided with a plurality of line patterns having a transmission part 35x and a reflection part 35y as shown in FIG. 4B. The width of the transmission part 35x is about 1/20 of the reflection part 35y. is there. The disc 35 has two regions, a region 35a where the width of the transmission part is small and a region 35b where the width of the transmission part is large.
[0060]
FIG. 5 is a side view showing the configuration of the moving member 36 and the disk 35. The moving member 36 is provided with V grooves 36a, 36b, 36c and holes 36l, 36m, 36n. As the moving member 36 moves, one of the V grooves 36a, 36b, 36c engages with the positioning mechanism 7 so that the moving member 36 is positioned so that the holes 36l, 36m, 36n pass through the optical axis 15, respectively. . In addition, abutting 36s, 36t, and 36u are installed at the entrances of the holes 36l, 36m, and 36n, respectively. The light reducing member 301 made of ND6 is inserted into the hole 36l while being held by the contact 36s.
[0061]
The operation of the confocal microscope configured as described above is the first in the case of performing confocal observation using a low-magnification objective lens and in the case of performing confocal observation using a high-magnification objective lens. This is the same as the above embodiment, but is different in the case of non-confocal observation, so that case will be described below, and description of other observations will be omitted.
[0062]
When performing non-confocal observation, the observer moves the moving member 36 with the lever 8 and positions the disk 35 so that the optical axis 15 passes through the hole 36l. In this case, the positioning mechanism 7 is engaged with the V groove 36a. After performing this operation, the observer 14 observes through the eyepiece lens 13 as in the first embodiment.
[0063]
As described above, in the third embodiment, one of the regions 35a and 35b where the line pattern of the disk 35 is arranged is positioned on the optical axis 15 according to the magnification of the objective lens used and the observation method. Since the moving member 6 moves as described above, confocal observation can be performed in the best state. Further, if the optical axis 15 is positioned so as to pass through the hole 36l outside the disk 35, the light reducing member 301 is installed in the hole 36l. Therefore, as in the first embodiment, the observer does not feel dazzling. Can perform non-confocal observation. For example, a dimming member made of ND25 may be inserted into the hole 36n. In this case, as in the second embodiment, the observer will not feel dazzled when using a low-magnification objective lens. .
[0064]
In the above-described embodiment, ND6 and ND25 are installed as the dimming means. However, a dimming member having a transmittance different from these may be disposed at each location. In the above embodiment, for example, the ND 6 is installed on the disk or the moving member. However, the ND 6 is installed so that it moves in a direction perpendicular to the optical axis and can be inserted / removed at an arbitrary position on the optical axis. Also good. Further, in the above embodiment, two types of line patterns are arranged on the disk, but this may be one type or three or more types.
[0065]
In the above embodiment, the observer observes the image through the eyepiece, but it may be picked up and observed by an imaging means such as a CCD camera. In the above embodiment, the beam splitter is installed to irradiate the sample from the light source. However, the polarizing beam splitter is installed, and the polarizing beam splitter is arranged with the polarizing plate on the three directions side where light enters and exits. You may comprise. In the above embodiment, a mercury or xenon light source is used as the light source, but other light sources such as halogen may be used.
[0066]
In the above-described embodiment, the disk on which the line pattern is installed is used. However, for example, a disk such as a Nipkow disk in which pinholes are spirally arranged may be used. In the above embodiment, the surface of the disk 5 is inclined by 5 ° with respect to the surface perpendicular to the optical axis 15 so that unnecessary light reflected from the disk surface, so-called ghost, does not return to the observer's field of view. It was. The angle of inclination is not limited to 5 °, and may be 0 °, for example, but is preferably between 0 ° and 5 °. Moreover, in the said embodiment, although the observer moved the moving member manually, you may make it move electrically.
[0067]
(Fourth embodiment)
FIG. 6 is a diagram showing a configuration of a confocal microscope according to the fourth embodiment of the present invention. This confocal microscope performs sectioning image observation. In FIG. 6, a condenser lens 102, a polarizing plate 103, and a PBS (polarizing beam splitter) 104 are arranged on the optical path of light emitted from a light source 101 of mercury or xenon, for example. On the reflection optical path of the PBS 104, a disk 105, a first imaging lens 107, a quarter-wave plate 108, an objective lens 109, and a sample 110 placed on a stage (not shown) are arranged. A second imaging lens 111 and a CCD camera 112 are arranged on the transmission optical path of the PBS 104 with respect to the sample 110. A monitor 113 is connected to the image output terminal of the CCD camera 112. The monitor 113 displays an image.
[0068]
FIG. 7 is a diagram showing the configuration of the disk 105. The disk 105 has a disk shape, and a pattern as shown in FIG. 7 is formed on an optical glass substrate, and is divided into three regions A ′, B ′, and C ′ by concentric circles. Among these, the innermost region A is transparent so that light can be transmitted uniformly. In the other regions B ′ and C ′, a large number of pinholes are provided in a spiral shape. The diameters of the pinholes between the regions B ′ and C ′ are different and increase in the order of the regions B ′ and C ′. Each pinhole is arranged so that the distance between each pinhole is about 4 to 10 times the diameter of the pinhole. In addition, the part shown with the net | network in FIG. 7 is a shielding part.
[0069]
The disk 105 can be moved in the direction of the arrow a or the arrow b in FIG. The disk 105 is connected to a motor shaft (not shown) via a rotating shaft 106, and rotates at a constant rotational speed by driving the motor.
[0070]
Next, the operation of the confocal microscope configured as described above will be described.
[0071]
The light emitted from the light source 101 passes through the condenser lens 102 and the polarizing plate 103, and only the polarization component in a certain direction is reflected by the PBS 104 and is incident on the disk 105 rotating at a constant speed. The light that has passed through the pinhole of the disk 105 passes through the first imaging lens 107, becomes circularly polarized light by the quarter-wave plate 108, forms an image by the objective lens 109, and enters the sample 110.
[0072]
The light reflected from the sample 110 passes through the objective lens 109 and becomes a polarization direction orthogonal to the polarization direction at the time of incidence again by the quarter wavelength plate 108, and the first imaging lens 107 causes the disc 105 to be reflected. A sample image is projected on top. The in-focus portion of the projected sample image passes through the pinhole, further passes through the PBS 104 in the direction of the second imaging lens 111, and passes through the second imaging lens 111 to the CCD camera 112. The image is taken with. The confocal image captured by the CCD camera 112 is displayed on the monitor 113.
[0073]
In order to obtain an appropriate confocal effect, the pinhole diameter R is set such that the wavelength of light is λ, the projection magnification of the sample 110 to be observed onto the disk 105 is M, and the numerical aperture of the objective lens 109 is NA.
R = kλM / NA
It is set in relation to k is a coefficient, and k = 0.5 to 1 is often used. Therefore, when the objective lens 109 is replaced, the projection magnification M and the numerical aperture NA on the disk 105 change, and therefore the pinhole diameter R for confocal observation needs to be changed.
[0074]
When the magnification required for observation is changed, the objective lens 109 is replaced. At this time, it is necessary to change the pinhole diameter on the disk 105 as described above. Normally, the NA often increases as the magnification of the objective lens 109 increases. However, the rate at which the NA increases is smaller than the rate at which the magnification increases. Therefore, when the objective lens 109 having a large magnification is used, the disk 105 is moved in the direction of arrow a in FIG. 6 so that the region C ′ having a large pinhole diameter is used.
[0075]
Further, when a bright field image (non-confocal image) is to be observed for focusing or searching for an observation position, the disk 105 is moved in the direction of arrow b in FIG. The (transparent) area A ′ is used. At this time, since the optical path passes through the disk 105 formed of the glass substrate, there is no change in the optical path length, and the focus does not move between the observation of the confocal image and the observation of the bright field image. .
[0076]
Note that when observing the bright field image, the portion without the pattern of the disk 105 is used, and therefore it is not always necessary to rotate the disk 105. However, the disk 105 is on the image plane, and when it is stationary, dust or the like is deposited on the disk 5 and is very conspicuous. Therefore, it is desirable to rotate the disk 105 when observing a bright field image.
[0077]
(Fifth embodiment)
FIG. 8 is a diagram showing a configuration of a confocal microscope according to the fifth embodiment of the present invention. For example, a collimator lens 302, an excitation filter 303, and a dichroic mirror 304 are arranged on the optical path of light emitted from a mercury or xenon light source 300. On the reflected light path of the dichroic mirror 304, a disk 305, a first imaging lens 307, an objective lens 308, and a sample 309 placed on a stage (not shown) are arranged. An absorption filter 310, a second imaging lens 311, and a CCD camera 312 are disposed on the transmission light path with respect to the sample 309 of the dichroic mirror 304.
[0078]
The disk 305 can be moved together with the motor 306 in the direction of the arrow a or the arrow b in FIG. Further, the disk 305 is connected to the shaft of the motor 306 via the rotating shaft 3051, and rotates at a constant rotational speed by driving the motor 306.
[0079]
9A is an overall view showing the configuration of the disk 305, and FIG. 9B is a partially enlarged view of the disk 305. As shown in FIG. The disk 305 has a disk shape and is divided into regions A ″ and B ″ by concentric circles as shown in FIG. In the region A ″ (pattern portion), slit-shaped transmission portions are arranged at equal intervals, and a region 401 having a constant width D in which a plurality of linear patterns are arranged is provided so as to be orthogonal to the other portions. FIG. 9B is an enlarged view of a part of the area A ″ in FIG. In the pattern of the area A ″, as shown in FIG. 9B, slit portions 405 through which linear light passes and shielding portions 404 that shield light are alternately arranged. The width of this line is wider than the width of the slit portion 405, for example, in a ratio of 19: 1. Also, the region B ″ is an ND filter that transmits light with a uniform transmittance, The transmittance is as follows when the transmittance of the region A ″ is T:
[Expression 4]

(Q = 1 to 0.2). For example, as described above, the transmittance T of the region B ″ is 5%, that is, 0.05. If q = 0.8 is selected, the transmittance of the ND filter is
[Equation 5]

Therefore, an ND filter having a transmittance of about 18% is formed in the region B ″. Note that when q = 0.2, the focus is in the range of 1/25 (4%) in the field of view. In the case of q = 1, the focus is in the range of all (100%) in the field of view, and almost all the samples observed with the confocal microscope have q = 1 to 0.2. 9 (a) and 9 (b) is a shielding part.
[0080]
Next, the operation of the confocal microscope configured as described above will be described.
[0081]
The light emitted from the light source 300 passes through the collimator lens 302, is selected by the excitation filter 303, and enters the dichroic mirror 304. The excitation filter 303 has transmission characteristics as shown in FIG. 10A, selectively transmits light having a wavelength that excites fluorescence from light from the light source 300, and blocks light having other wavelengths. Further, the dichroic mirror 304 reflects the light having the wavelength transmitted through the excitation filter 303 as shown in FIG. 10B, and changes the wavelength of the fluorescence from the sample 309 as shown in FIG. 10C. It has the property of transmitting. Further, the absorption filter 310 described later has a transmission characteristic as shown in FIG. 10C, which blocks the excitation wavelength and transmits the fluorescence wavelength.
[0082]
These wavelength characteristics differ depending on the fluorescent dye used. For example, when observing FITC, the excitation maximum wavelength is 490 nm and the fluorescence maximum wavelength is 520 nm. For this reason, the excitation filter 303 has a wavelength of transmitted light of 460 to 490 nm, the dichroic mirror 304 has a reflection wavelength of 460 to 490 nm, and the dichroic mirror 304 and the absorption filter have a transmission wavelength of 510 nm. Often used.
[0083]
The excitation light reflected by the dichroic mirror 304 is incident on a disk 305 that rotates at a constant speed. When observing the confocal image, the disk 5 is moved so that the area A ″ in FIG. 9A where the linear pattern is formed is positioned on the optical path. The linear shape of the disk 305 is moved. The light transmitted through the slit portion 405 passes through the first imaging lens 307, is imaged by the objective lens 308, and is incident on the sample 309.
[0084]
The sample 309 emits fluorescence by the excitation light. The fluorescence from the sample 309 passes through the objective lens 308 and enters the disk 305 via the first imaging lens 307, and an image of the sample 309 is formed on the disk 305. Of the formed image, the focused component passes through the slit portion 405 on the disk 305, and the light having the fluorescence wavelength passes through the dichroic mirror 304. Even the absorption filter 310 blocks light having an excitation wavelength. The light having the fluorescence wavelength passes through the absorption filter 310 and enters the CCD camera 312 via the second imaging lens 311, and a sample fluorescence image is formed by the CCD camera 312.
[0085]
When it is desired to observe a bright field image (non-confocal image) for focusing or searching for an observation position, the disk 305 is moved in the direction of arrow a in FIG. The transmission region B ″ is used. Here, when the region A ″ of the linear pattern is switched to the region B ″, the region B ″ is a transparent glass substrate having a transmittance of almost 100%. In some cases, the observation image becomes too bright, and it is necessary to reduce the light by some means. When the confocal image is observed, when the transmittance of the disk 305 is T, the sample 309 is irradiated with light T times the light from the light source 300. Fluorescence from sample 309 passes through disk 305. At this time, if it is in focus, about 90% of the fluorescence is transmitted, but if it is not in focus, the transmitted fluorescence is reduced depending on the degree of out-of-focus. If the focus is several hundred μm or more, the fluorescence is also T times by the disk 305. However, since the thickness of the sample usually observed with a microscope is several μm to several tens μm, the T times do not become dark.
[0086]
The transmittance of the uniform transmission region B ″ is
[Formula 6]

When (q = 1 to 0.2), the brightness of the observed image is q for 100% transmission. ² T times. In the present embodiment, the brightness of the bright field image is 0.18. ² = 0.03 times, and the brightness of the confocal image is 0.05 times only in the in-focus portion, and the non-focus image is much darker than this, so the brightness of the bright field image and the confocal image is It will be almost the same.
[0087]
In addition, as in the fourth embodiment, the optical path passes through the disk 305 formed of the glass substrate even when observing the bright field image, so that there is no change in the optical path length, and the confocal image is observed. The focus does not move.
[0088]
In the fifth embodiment, the ratio of the width of the linear transmission portion and the width of the shielding portion in the disk 305 is 1:19, but this ratio may be smaller or larger. For example, in the case of very dark fluorescence, the out-of-focus component increases, but when the ratio is about 1: 5, the image becomes brighter. In the case of very bright fluorescence, when the ratio is about 1:50, there is almost no out-of-focus component, and a sectioned image of only an in-focus image can be obtained. In this case, however, the image becomes dark.
[0089]
In the fourth embodiment, the reflection image of the sample is observed with a Nipkow disk on which pinholes are formed. In the fifth embodiment, the fluorescent sample is observed with a disk having a linear pattern. The disk pattern may be exchanged between the fourth embodiment and the fifth embodiment.
[0090]
Also in the fifth embodiment, the ND filter portion may be made transparent as in the fourth embodiment so that only the effect of defocusing is eliminated. Further, in the fifth embodiment as well, as in the fourth embodiment, the disk is divided into concentric areas, and different line patterns are formed for each area so as to cope with different objective lenses. Is also possible. In this case, a region that uniformly transmits light may be disposed in the outer concentric region. If a Z stage is added, the distance between the sample and the objective lens is changed, and images are taken one after another, three-dimensional observation can be performed.
[0091]
As described above, the first, second, and fifth embodiments have described the confocal microscope in which the disk is provided with the dimming means.
[0092]
In addition, this invention is not limited only to said each embodiment, In the range which does not change a summary, it can deform | transform suitably and can implement.
[0093]
【The invention's effect】
According to the confocal microscope of the present invention, since a part of the disk has a constant transmittance and is configured to observe through this area when observing the bright field image, the microscope can be manufactured very inexpensively and observed. In addition to facilitating the observation, even if the observation is performed by switching between the confocal image and the bright field image, the focus is not moved. Further, when an ND filter is formed in a certain transmittance region of the disk and the transmittance is the transmittance T of the pattern portion,
[Expression 7]

By setting (q = 1 to 0.2), the brightness of the observation image becomes almost the same when switching between the confocal image and the bright field image, and it is not necessary to adjust the amount of light.
[0094]
As described above, according to the present invention, it is possible to provide a confocal microscope capable of performing observation in a good state with a simple configuration.
[0095]
Further, according to the present invention, it is possible to provide a confocal microscope that can easily switch between observation of a confocal image and a bright field image and does not require adjustment of the focus at the time of switching.
[Brief description of the drawings]
FIG. 1 is a diagram showing a configuration of a confocal microscope according to a first embodiment of the present invention.
FIG. 2 is a diagram showing a configuration of a disk according to the first embodiment of the present invention.
FIG. 3 is a diagram showing a configuration of a disk according to a second embodiment of the present invention.
FIG. 4 is a top view showing a configuration of a moving member and a disc according to a third embodiment of the present invention.
FIG. 5 is a side view showing a configuration of a moving member and a disc according to a third embodiment of the present invention.
FIG. 6 is a diagram showing a configuration of a confocal microscope according to a fourth embodiment of the present invention.
FIG. 7 is a diagram showing a configuration of a disk according to a fourth embodiment of the present invention.
FIG. 8 is a diagram showing a configuration of a confocal microscope according to a fifth embodiment of the present invention.
FIG. 9 is a diagram showing a configuration of a disc according to a fifth embodiment of the invention.
FIG. 10 is a diagram showing transmission characteristics and reflection characteristics according to a fifth embodiment of the present invention.
FIG. 11 is a diagram showing a configuration of a confocal microscope according to a conventional example.
FIG. 12 is a diagram showing a configuration of a Nippon disc according to a conventional example.
[Explanation of symbols]
1 ... Light source
2 ... Collector lens
3. Beam splitter
4 ... Motor
4a ... axis
5 ... Disc
6 ... Moving member
6a, 6b, 6c ... V groove
7 ... Positioning mechanism
8 ... Lever
9 ... imaging lens
10 ... Objective lens
11 ... Sample
12 ... Imaging lens
13 ... Eyepiece
14 ... Observer
15: Optical axis
25 ... Disc
35 ... Disc
36 ... Moving member
36a, 36b, 36c ... V groove
36l, 36m, 36n ... hole
301 ... Light-reducing member
36s, 36t, 36u ... application
101: Light source
102 ... Condenser lens
103 ... Polarizing plate
104 ... PBS
105: Disc
106: Rotating shaft
107: First imaging lens
108: 1/4 wavelength plate
109 ... Objective lens
110 ... Sample
111 ... second imaging lens
112 ... CCD camera
113 ... Monitor
300: Light source
302 ... Collimator lens
303 ... Excitation filter
304 ... Dichroic mirror
305 ... Disc
3051: Rotating shaft
306 ... Motor
307: First imaging lens
308 ... Objective lens
309 ... Sample
310 ... Absorption filter
311 ... Second imaging lens
312 ... CCD

Claims (5)

The light from the light source illuminates the image position of the objective lens, a confocal disk located at or near the image position of the objective lens, and the passing light is imaged on the sample by the objective lens. Scanning, the light from the specimen is imaged again on the confocal disk or its vicinity through the objective lens, and the light passing through the confocal disk is re-imaged to obtain an image of the specimen. In the focus microscope,
Moving means for moving the surface of the confocal disc in a direction substantially perpendicular to the optical axis;
Locking means for locking the moving means to position a predetermined area on the confocal disc on the optical axis;
Dimming means detachable on the optical axis in conjunction with movement of the moving means;
A confocal microscope characterized by comprising: 2. The confocal microscope according to claim 1, wherein the dimming means is provided on the confocal disk. The confocal microscope according to claim 1, wherein the dimming unit is provided in the moving unit. In a confocal microscope that rotates a disk on which a predetermined pattern composed of a light transmitting part and a shielding part is formed, scans the sample with light passing through the pattern, and obtains a confocal image of the sample. ,
The disc includes a plurality of regions concentrically divided, and at least one of the plurality of regions includes a region that transmits light uniformly. When the light transmittance in the pattern formed on the disk is T, the transmittance of the region that uniformly transmits the light is:

The confocal microscope according to claim 5, wherein (q = 1 to 0.2).

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