JP4827335B2 - Scanning laser microscope - Google Patents

Description

として演算するわけである。そして、画面全体の構成ピクセルそれぞれについての当該演算を行い、その結果を反映させた画像を生成する。
【００８９】
３９０[ｎｍ]から４２０[ｎｍ]のスペクトルに対応する画像をＡ、４６５[ｎｍ]から４８５[ｎｍ]のスペクトルに対応する画像をＢとする。カルシウムイオン濃度の上昇とともに試料１０からの蛍光スペクトルはそれまでの図３（ａ）のような特性から図３（ｂ）に示すように、短波長側に移動したとすると、これにより、画像Ａは明るさを増し、画像Ｂは暗くなることが認められる。
【００９０】
そして、カルシウムイオン濃度を求めるために、
【数２】

但し、αは検査者が定めることのできる適宜な定数である。
を演算する。
【００９１】
例えば、“ＩＮＤＯ−１”で染色した試料（標本）は、ＵＶ光（紫外線）を照射することにより、当該試料から励起される蛍光を２つの異なる波長域（中心波長４０５[ｎｍ]と４８０[ｎｍ]）で検出し、検出光量の比（中心波長４０５[ｎｍ]の蛍光／中心波長４８０[ｎｍ]の蛍光）を演算して求めることにより、カルシウムイオン濃度を測定できる。
【００９２】
従って、このことを利用して[数２]の演算をコンピュータ３２にて行うことで、カルシウムイオン濃度を求めることができる。
【００９３】
各ピクセル別にこのようなカルシウムイオン濃度を求め、１画面全てのピクセルについてこのような演算を終えたならば、次にコンピュータ３２はピクセル毎に求めたカルシウムイオン濃度を反映させた画像を生成する。
【００９４】
そして、得られたカルシウムイオン濃度を表わす画像表示のためのデータをＣとすれば、１画像毎に図１（ｂ）に示すように、画像Ａ，Ｂ，Ｃが得られるので、上記の例では５０枚分それぞれにおけるＡ，Ｂ，Ｃの画像が得られることになる。得られたこれらの画像はモニタ装置３３に表示させ、検査者はこの表示画像を観察することになる。
【００９５】
尚、上述の例では５０枚分の画像を得ているので、これら５０枚の画像を順次表示すれば、細胞内カルシウムイオン濃度の変化を動画として表示することができる。
【００９６】
本実施形態に示した走査型レーザ顕微鏡装置は、もとになる全てのスペクトル成分のデータを、コンピュータ３２のメモリ上に保持させてあるので、選択するスペクトル範囲はコンピュータ３２に対して指定することにより、何度でも再設定できる。そのため、選択するスペクトルの範囲を種々変更して画像の状態を比較することにより、カルシウムイオン濃度の変化を表わす最善の画像を得ることができる。
【００９７】
以上、この実施形態に示したものは、レーザ光源からのレーザ光を観察対象の試料に集束させて照射させると共に試料に対して前記レーザ光を移動走査させ、試料から得られた光を分光手段により分光させて検出器で検出することにより、スペクトルのデータを得るようにした走査型レーザ顕微鏡において、前記検出器は入射光量対応に電気信号を発生する複数の微小受光素子を密に直線的に配列させた１次元光検出手段を用い、前記分光手段の分光出力をこの１次元光検出手段の微小受光素子配列範囲にその入射位置と波長域とが所定の関係を以て入射される配置関係とするようにしたものである。
【００９８】
また、前記分光手段の分光出力をこの１次元光検出手段の微小受光素子配列範囲にその入射位置と波長域とが所定の関係を持つよう集光させる光学系を設けて構成した。
【００９９】
また、前記１次元光検出手段の各微小受光素子の出力電気信号をディジタル信号化して微小受光素子配列順のデータを得る信号処理手段（信号処理装置３０）と、この得られたデータを保持する保持手段とを設けた。
【０１００】
更には、保持手段に保持したデータを用いて指定のスペクトル範囲のデータを抽出しその指定範囲のスペクトル成分による画像を生成する構成とした。
【０１０１】
レーザ光源からのレーザ光を観察対象の試料に集束させて照射させると共に試料に対して前記レーザ光を移動走査させ、試料から得られた光を分光手段により分光させて検出器で検出することにより、試料における１地点毎のスペクトルデータを得るが、スペクトルデータは例えばプリズム等による分光手段を用い、この分光手段により分光して１次元光検出手段に入射させることで得る。１次元光検出手段は、複数の微小受光素子（受光セル）を密に直線的（１次元的）に配列させた構成であり、前記分光手段の分光出力をこの１次元光検出手段の微小受光素子配列範囲にその入射位置と波長域とが関係を以て入射される配置関係としてあることから、微小受光素子はその位置がどこであるかにより、入射される波長域が定まる。従って、特定位置の微小受光素子にはスペクトルの特定波長域の光が入射する構成となる。そのため、この各微小受光素子よりその入射光量対応の信号を得てこれを受光素子個別にディジタルデータ化し、受光素子の並び順に収集すると波長成分が特定できるデータ、すなわち、全てのスペクトル分布がわかるデータとして収集されると言う効果がある。
【０１０２】
しかも、試料に照射するレーザ光は、試料上をＸ−Ｙ走査させるべく移動させるので、その時々で収集される上記データは試料に対するレーザ光の照射点における放出光のデータであり、Ｘ−Ｙ走査範囲で定まる画面を構成する画素単位でのスペクトル分布データとなる。そのため、画素毎に全てのスペクトル分布が把握できるデータとして収集できる。このデータを画面構成分収集し、記憶保存し、この保存データを用いて画像を生成するが、画素毎に全てのスペクトル分布が把握できるデータとして収集されていることから、所望にスペクトル範囲を種々指定してその指定範囲のスペクトルによる画像を表示でき、従って、１度データを収集してしまえば、そのデータを利用して種々に画像を生成することで、最適な画像を見付けることができる。
【０１０３】
蛍光試薬による試料染色した試料（蛍光染色試料）の蛍光観察においては、単染色のみならず多重染色が多用されており、特に、蛍光染色は細胞、組織内の特定対象を視認可能にするために行われることが多い。このため、多重染色観察では、各染色部位が明確な色の差、すなわち、蛍光波長の違いとして検出されなければならない。しかも、蛍光波長の部分的な重なり（クロスオーバ部分）を除去して検出する必要がある。
【０１０４】
本発明では画素毎に全てのスペクトル分布が把握できるデータとして収集でき、このデータを画面構成分収集して記憶保存し、この保存データを用いて所望のスペクトル範囲の画像を生成するが、データが保存されているので、スペクトル範囲を繰り返し何度でも種々指定し直して画像を表示できることから、１度データを収集してしまえば、そのデータを利用して種々に画像を生成することで、最適な状態の画像を見付けることができるようになる。
【０１０５】
また、スペクトル領域の範囲指定は同時に複数可能であり、しかも、指定したスペクトル領域内のスペクトル成分のデータを加算してその指定領域のスペクトル成分による画像を生成し、さらにそれを利用してイオン濃度などを演算にて求めることができ、その求めたイオン濃度の画像を表示したり他の画像と合成したりして表示できるので、検査者に試験結果についての詳細な情報を提供できる効果もある。
【０１０６】
なお、上記例では２次元画像を対象としたが、ライン走査（１次元）であってもよいことは言うまでもない。この場合には画像の横方向には走査の空間軸が、縦軸には時間（または走査回数）がとられる。また、分光にはプリズムを用いた例を示したが、これは回折格子を利用しても良く、分光可能な手段であれば利用することができる。
【０１０７】
以上は、ピクセル毎のスペクトル分布のデータを収集して保持しておき、その保持データを用いて所望のスペクトル領域を定めてその領域の成分による画像を求める方式であったが、試験を実施しながらその状況を把握できるようにし、状況如何により試験継続か中止かを判断できるようにして、もし、試験が上手くいっていないようであるならば、そこで試験を中止する等の対応ができて、時間の浪費をせずに済むようにした実施形態を次に第２の実施形態として説明する。
【０１０８】
（第２の実施形態）
この実施形態では、スペクトルデータの収集時に、あらかじめレーザ光源の出力レーザ波長成分を除く、これよりも長い二つのスペクトル域のデータの合計値を演算するとともに、ピクセル毎の比を演算し、これらの演算結果をもとに画像表示するように構成するとともに、試料に対する１画面分のレーザ走査終了のたびに画像表示を更新するようにして経過を逐次、把握できるようにするものである。
【０１０９】
図４（ａ）は、本発明の第２の実施の形態に係わる共焦点走査型レーザ顕微鏡の構成を示す図である。この実施形態においても、基本構成は第１の実施形態で説明したものと同じで良い。但し、第２の実施形態では、レーザ光源１として４４２[ｎｍ]の発振線を有するヘリウムカドミウムレーザ光源を用いている点、Ｘ−Ｙ走査光学系６は５１２×５１２ピクセルに亙り、走査するようにした点、１次元ＣＣＤ １７は例えば、１０２４素子相当のラインセンサであり、最短波長４３０[ｎｍ]、１画素当たり約０．２５[ｎｍ]に相当するように、プリズム１５と集光レンズ１６の焦点距離が光学的に設定されていて、１次元ＣＣＤ １７は４００[ｎｍ]から６８５[ｎｍ]のスペクトルが検出可能に構成されている点、コンピュータ３２は予めレーザ波長（４４２[ｎｍ]）成分を除く、これよりも長い二つのスペクトル域のデータの合計値を求めるとともに、ピクセル毎の比を演算し、これらの演算結果をもとに画像を生成し、モニタ装置３３に画像表示するように設定されており、Ｘ−Ｙ走査光学系６による１画面分の画素範囲のレーザ光走査終了のたびにモニタ装置３３に対する画像表示の更新をするようにした点が第１の実施形態と異なる。尚、３４はコンピュータ３２に接続されたハードディスクなどの大容量記憶装置である。
【０１１０】
このような本実施形態で開示した構成の装置の作用を説明する。ここで観察対象とする試料１０は２種類の蛍光蛋白（例えば、ＣＦＰとＹＦＰの２種類の蛍光蛋白）を遺伝子発現させた細胞であるとする。
【０１１１】
４４２[ｎｍ]の発振線を有するヘリウムカドミウムレーザ光源１から出射されたレーザ光束２は、ビームエクスパンダ３により対物レンズ瞳径に略一致するようにビーム径が拡大されレーザラインフィルタ４に導かれる。レーザラインフィルタ４は４４２[ｎｍ]の発振線を選択的に透過させる。
【０１１２】
レーザラインフィルタ４を透過した４４２[ｎｍ]の波長を有するレーザ光束はダイクロイックミラー５で反射され、Ｘ−Ｙ走査光学系６、瞳投影レンズ７、結像レンズ８、対物レンズ９を経て試料１０に達する。
【０１１３】
試料１０は、ＣＦＰとＹＦＰの２種類の蛍光蛋白を遺伝子発現させた細胞であり、細胞内カルシウムイオン濃度の変化により、ＣＦＰからＹＦＰへのエネルギ移動の変化が生じ、これにより蛍光量が変化するもので、この光量の比を測定することで細胞内カルシウムイオン濃度を測定することができる。
【０１１４】
試料１０に到達したレーザ光によってその到達位置での蛍光蛋白を励起し、または反射光を生じる。
【０１１５】
ここで、試料１０に対するレーザ光集束点は対物レンズ９で定まり、試料１０上でのレーザ光集束位置はレーザ光をＸ−Ｙ走査するためのＸ−Ｙ走査光学系６によるＸ−Ｙ走査位置により定まる。
【０１１６】
試料１０からの反射光および蛍光からなる光束２０は、対物レンズ９、結像レンズ８、瞳投影レンズ７、Ｘ−Ｙ走査光学系６、を経てダイクロイックミラー５に戻る。そして光束２０はダイクロイックミラー５、共焦点レンズ１１を透過し、共焦点絞り１２、コリメートレンズ１３を介して分光器１４に入る。
【０１１７】
分光器１４はプリズム１５、集光レンズ１６、および１次元ＣＣＤ１７からなり、ダイクロイックミラー５を透過した光束はプリズム１５でスペクトル分解され、集光レンズ１６を介して１次元ＣＣＤ １７上に集光される。なお１次元ＣＣＤ １７は集光レンズ１６の焦点位置に配置されている。
【０１１８】
Ｘ−Ｙ走査光学系６を駆動制御する制御装置３１のサンプリング信号に同期して、信号処理装置３０は１次元ＣＣＤ １７で検出された光強度信号をディジタル信号に変換し、ＰＣＩバス経由でコンピュータ３２に転送する。
【０１１９】
１次元ＣＣＤ １７は例えば、受光セルが１０２４素子相当のラインセンサであり、最短波長４３０[ｎｍ]、１画素当たり約０．２５[ｎｍ]に相当するように、プリズム１５と集光レンズ１６の焦点距離が光学的に設定されている。したがって、受光セル１０２４素子は、順に４００[ｎｍ]から６８５[ｎｍ]のスペクトル範囲をカバーしている。そして、その各受光セルは、自己の対応する波長成分の光の強度に対応する電気信号を発生することになる。すなわち、１次元ＣＣＤ１７は４００[ｎｍ]から６８５[ｎｍ]のスペクトルが検出可能に構成されているわけである。
【０１２０】
信号処理装置３０は１次元ＣＣＤ １７の出力を各素子毎に８ビット分解能で処理し、その並び順に並ぶデータとして出力する。
【０１２１】
したがって、この信号処理装置３０の出力するデータは、制御装置３１の一つのサンプリング信号に対して（つまり画像上の１ピクセルに対して）、１[ＫＢyte]（キロバイト）のデータ容量を有する。
【０１２２】
Ｘ−Ｙ走査光学系６によるＸ−Ｙ走査の範囲は、１画像当たり５１２×５１２ピクセル相当であるから、制御装置３１はこれに対応するサンプリング信号を出力することとなるので、画像１枚（５１２×５１２ピクセル）につき２６８[ＭＢyte]のデータ容量となる。
【０１２３】
１枚の画像を６０秒おきに取得する場合、信号処理装置３０からコンピュータ３２に必要な転送レートは、一般的なコンピュータに搭載されているＰＣＩバス経由で十分に対応可能である。また、このデータをコンピュータ３２に接続されたハードディスクなどの大容量記憶装置３４に、試料に対する１画面分のレーザ走査終了ごとに書き込むことにより、少なくとも画像５０枚分のデータを記録することが可能である。信号処理装置３０からコンピュータ３２へのデータ転送時間とハードディスク（大容量記憶装置３４）ヘの書き込み時間の合計が画面毎のレーザ走査間隔時間に対して小さければ良いことになる。
【０１２４】
この実施形態では、コンピュータ３１での処理は、あらかじめレーザ波長（４４２[ｎｍ]）成分を除く、これよりも長い二つのスペクトル域のデータの合計値を演算するとともに、ピクセル毎の比を演算し、これらの演算結果をもとに画像を生成して画像表示するように設定されており、レーザ走査終了のたびに画像表示を更新するようになっている。
【０１２５】
すなわち、二つのスペクトル範囲をあらかじめ指定しておく。一つは４６５[ｎｍ]から４９５[ｎｍ]までを、他方は５２０[ｎｍ]から５５０[ｎｍ]までである。
【０１２６】
この指定情報を受けてコンピュータ３２は、その指定範囲のスペクトルデータ積算値ΣＩ（Ｋ）ｎを、それぞれピクセル単位で求める。上述の例の場合、
【数３】

として演算するわけである。そして、画面全体の構成ピクセルそれぞれについての当該演算を行い、その結果を反映させた画像を生成する。
【０１２７】
４６５[ｎｍ]から４９５[ｎｍ]のスペクトルに対応する画像をＡ、５２０[ｎｍ]から５５０[ｎｍ]のスペクトルに対応する画像をＢとする。カルシウムイオン濃度の上昇とともに試料からの蛍光スペクトルはエネルギ移動により、画像Ｂは明るさを増し、画像Ａは暗くなる。
【０１２８】
そしてカルシウムイオン濃度を求めるために、
【数４】

但し、αは検査者が定めることのできる適宜な定数である。
を演算する。
【０１２９】
そして、演算により求めたカルシウムイオン濃度を表わす画像表示のためのデータによる画像をＣとすれば、１画像毎に図４（ｂ）に示すように、画像Ａ，Ｂ，Ｃが得られるので、例えば、ｎ＝１の画像のための走査が終了段階でそのｎ＝１の画像は生成が可能であるから、その生成画像を表示するようにする。
得られたこれらの画像は大容量記憶装置３４に保存させ、また、モニタ装置３３に表示させるので、検査者はこの表示画像を観察することになる。
【０１３０】
ｎ＝２の画像のためのレーザ走査が終了した段階で、ｎ＝２の画像を生成し、大容量記憶装置３４に保存し、また、ｎ＝１の画像に代えてそのｎ＝２の画像を表示し、ｎ＝３の画像のためのレーザ走査が終了した段階でｎ＝３の画像を生成し、大容量記憶装置３４に保存し、また、ｎ＝２の画像に代えてそのｎ＝３の画像を表示し、…と云う具合に、画像Ａ，Ｂ，Ｃを走査終了の度に画像の生成とその生成した画像のモニタ装置３３への更新表示を実施する。これにより試験の状況を把握しながら、時間経過観察ができることになる。
【０１３１】
また、生成画像は大容量記憶装置３４に保持させてあるので、所定回（前記例では５０画面に亘って）のレーザ走査終了後に当該保持させてある所定枚（前記例では５０枚）の画像を順次表示すれば細胞内カルシウムイオン濃度の変化を動画として表示して時間経過観察することができる。
【０１３２】
すなわち、この実施形態においては時間経過観察のために試料走査は所定回数だけ間欠的に行われ、前記画像表示は各レーザ走査終了毎にあるいは全枚数分の画像取得のためのレーザ走査全てが完了した後に所定回数行われるようにした。
【０１３３】
また、レーザ走査によって収集された各ピクセル毎のスペクトル分布のデータは保存されているので、選択するスペクトル範囲は何度でも再設定し直して画像表示できることから、カルシウムイオン濃度の変化を表わす最善の画像を得ることができる。
【０１３４】
このようにこの実施形態に示したものは、レーザ光源からのレーザ光を観察対象の試料に集束させて照射させると共に試料に対して前記レーザ光を移動走査させ、試料から得られた光を分光手段により分光させて検出器で検出することにより、スペクトルのデータを得るようにした走査型レーザ顕微鏡において、スペクトルデータの収集時に、あらかじめレーザ光源の出力レーザ波長成分を除く、これよりも長い二つのスペクトル域のデータの合計値を演算するとともに、ピクセル毎の比を演算し、これらの演算結果をもとにした画像を表示するように構成したものであり、レーザ光による試料の走査終了のたびに画像表示を更新するようにして経過を逐次、把握できるようにしたものである。
【０１３５】
そのため、試験を実施しながらその状況を把握できるので、状況如何により試験継続か中止かを判断できるようになり、もし、試験が上手くいっていないようであるならば、そこで試験を中止する等の対応ができて、時間の浪費をせずに済むようになる等の効果が得られる。
【０１３６】
従って、この実施形態においては、１画面分の走査を終えると時間をおいて再びレーザ光による試料の１画面分の走査を実施する間欠走査において、一つの走査が終了するたびに画像表示されるので、検査者は試験途中でＣＦＰ，ＹＦＰそれぞれの画像と、カルシウムイオン濃度の変化を確認できる。そのため、もし、試験がうまくいっていないようならば、そこで試験を中止する等の対応をとることができ、時間の浪費をせずに済むようになる。
【０１３７】
尚、上記例ではレーザ光源としてヘリウムカドミウムレーザ光源を用いて１光子励起で観察を行っているが、モードロックチタンサファイヤレーザ等の赤外域の波長を有する超短パルスレーザ光源を用いて、２光子励起観察を行い、これよりも短い２つのスペクトル域のデータの合計値を演算するとともに、ピクセル毎の比を演算し、これらの演算結果をもとに画像表示するように設定し、走査終了のたびに演算結果の画像表示を更新するようにしてもよい。ここで、２光子励起（２光子励起法）とは吸収波長の何倍化の波長を有する長波長のレーザ光を高密度で照射することにより、焦点面で局所的に蛍光分子を励起する方式で多光子励起法とも呼ばれるものであり、ピンホールなしで共焦点観察法と同等なセクショニング画像を得ることができる手法である。
【０１３８】
そして、このような超短パルスの長波長レーザ光を用いて、２光子励起によりデータを取得する方法は細胞に対する光毒性が少なく、より長時間に亙る観察が可能になる。
【０１３９】
また、この例では１次元のＣＣＤを用いたが、試料からの光束が集光レンズ１６により集光され、ここで形成される回折スポット径がＣＣＤの画素サイズよりも大きい場合は２次元ＣＣＤを用いる構成としてもよい。これにより、スポット径が大きい場合に、試料からの光を効率良く検出でき、Ｓ／Ｎの良いデータを取得することができるようになる。
【０１４０】
なお、本発明は上述した実施形態に示す例に限定されるものではなく、種々変形して実施可能である。
【０１４１】
また、本発明において、上記実施形態には種々の段階の発明が含まれており、開示される複数の構成要件における適宜な組み合わせにより種々の発明が抽出され得るものである。例えば、実施形態に示される全構成要件から幾つかの構成要件が削除されても、発明が解決しようとする課題の欄で述べた課題の少なくとも１つが解決でき、発明の効果の欄で述べられている効果の少なくとも１つが得られる場合には、当該実施形態での構成要件が一部削除された構成についても発明として成立し得るものである。
【０１４２】
【発明の効果】
本発明によれば、走査ピクセル毎にスペクトルデータを有しており、画像表示のためのスペクトル域を自由に選択することができ、しかも何度でもスペクトル域の指定を変更できるので、細胞内カルシウムイオン濃度を測定するような、試料からの蛍光スペクトルを正確に知ることができない試験や撮像を行う場合でも、最適な解析結果を得ることのできる、自由度の高い走査型レーザ顕微鏡を提供することができる。
【０１４３】
さらに、多重染色観察において、試料の退色を防止するために、少ない走査回数で蛍光波長の部分的な重なり（クロスオーバ部分）を効率良く除去して画像表示することのできる走査型レーザ顕微鏡を提供することができる。
【図面の簡単な説明】
【図１】本発明の第１の実施形態を説明するための図である。
【図２】本発明を説明するための図であって、本発明の走査型レーザ顕微鏡における分光器の構成例を説明するための図である。
【図３】得られたスペクトルの例とスペクトル範囲指定の例およびその遷移状況の例を説明するための図である。
【図４】本発明の第２の実施形態を説明するための図である。
【図５】従来技術を説明するための図である。
【図６】従来技術を説明するための図である。
【図７】従来技術を説明するための図である。
【図８】従来技術を説明するための図である。
【符号の説明】
１…レーザ光源、 ２…レーザ光束
３…ビームエクスパンダ、
４…レーザラインフィルタ
５…ダイクロイックミラー、６…Ｘ−Ｙ走査光学系
７…瞳投影レンズ、 ８…結像レンズ
９…対物レンズ、 １０…試料
１１…共焦点レンズ、 １２…共焦点絞り
１３…コリメートレンズ、１４…分光器
１５…プリズム、 １６…集光レンズ
１７…１次元ＣＣＤ、 ２０…試料からの光束
３０…信号処理装置、 ３１…制御装置
３２…コンピュータ、 ３３…モニタ装置
３４…ハードディスク装置（ＨＤＤ）[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a scanning laser microscope capable of obtaining spectral data by spectrally scanning light from a specimen obtained by scanning a stained specimen with laser light.
[0002]
[Prior art]
In order to facilitate observation of cells and tissues, staining with a staining reagent is performed. There are various types of reagents for staining, and there are a reagent that is easily dyed and a reagent that is difficult to be dyed depending on components. Utilizing this, a sample (specimen) is stained with a desired reagent according to the purpose and used for microscopic observation. Now, one step further, we irradiate a sample stained with multiple types of fluorescent reagents with laser light, and collect the spectral data obtained by spectroscopically analyzing the fluorescent radiation excited from the reagents by this laser light irradiation. It is used for analysis.
[0003]
For this purpose, a scanning laser microscope is used, which condenses laser light through an objective lens, scans the sample with a scanning device, applies light from the sample to a spectroscope, and performs spectroscopy. By obtaining spectral data in synchronization with the scanning signal from the scanning device, spectral information of light from the laser irradiation position on the sample is obtained.
[0004]
(Prior art 1)
As an example of a scanning laser microscope, there is a technique as shown in JP-T-9-502269. The technique disclosed here splits the fluorescent light beam by spectral decomposition means such as a prism, narrows the first spectral range on the one hand, and reflects at least a part of the spectral range that does not pass through the diaphragm to form the second spectral range. This is a confocal scanning laser microscope configured with two optical paths and a photodetector provided for each of the optical paths.
[0005]
FIG. 5 is a diagram showing a configuration of the confocal scanning laser microscope. The laser beam 203 emitted from the laser light source 202 is guided to the dichroic mirror 208 through the direction changing mirror 204, the laser line filter 205, the lens 206, and the diaphragm 207, and is reflected there, and the lens 209, XY scanning. The sample 213 is reached through the optical system 210, the pupil projection lens 211, and the objective lens 212.
[0006]
A light beam 214 composed of reflected light and fluorescence from the sample 213 returns to the dichroic mirror 208 via the objective lens 212, pupil projection lens 211, XY scanning optical system 210, and lens 209. Of the light beam 214, the fluorescence component passes through the dichroic mirror 208 and becomes a fluorescent light beam 217, and among these, the light beam that has passed through the confocal stop 215 enters the spectroscope 216.
[0007]
FIG. 6 is a diagram showing the configuration of the spectroscope 216. The selection device 225 has spectral decomposition means 227 for resolving the light beam 217 and means 228 for narrowing the first spectral range 229 on the one hand and reflecting at least a portion 230 of the spectral range that does not pass through the stop. The photodetector 226 includes a first photodetector 231 arranged in the narrowed optical path of the first spectral range 229 and a second photodetector 232 installed in the optical path of the reflected spectral range. Yes.
[0008]
Furthermore, the selection device 225 has means 233 that is installed in the optical path of the reflected spectral range 230 and narrows down the second spectral range 234. The second photodetector 232 is installed in the narrowed optical path of the second spectral range 234.
[0009]
(Prior art 2)
As another example, Japanese Patent Application Laid-Open No. 2000-56244 discloses wavelength selection for both or one of the illumination light and the light from the object that are divided while being dispersed, such as reflected light and fluorescence. A scanning laser microscope is disclosed having at least one micromirror array (DMD) that can be selectively switched for illumination and / or detection step.
[0010]
The configuration of the scanning laser microscope disclosed in this publication is as shown in FIG. That is, in this configuration, the laser light LB from the laser light source Ls is scanned in an XY manner on the sample Smp by being scanned by the scanning means Scn so as to move the position intermittently, and the sample Smp at the scanning position is scanned. Emits fluorescence excited by the laser beam LB.
[0011]
The (fluorescent) radiation emitted from the sample Smp is focused onto the confocal pinhole Ph located in a plane conjugated with the object. The pinhole Ph is also an opening of the spectroscopic device, and the spectroscope using the prism P separates the sample radiation into its spectral components by the dispersion action of the prism P. There is a one-dimensional DMD at the focal plane of the sample radiation, where the sample spectrum is optically imaged.
[0012]
A one-dimensional DMD is composed of a number of switching mirrors that can be individually driven. Then, while the laser beam LB from the laser light source Ls stays on the sample Smp, the mirrors are sequentially driven one by one (and switched by this), so that the light that has been split and reached at that position To the detector Dtc. In this way, the individual spectral components of the sample radiation are sequentially detected by the detector Dtc and the total spectral data of the (fluorescence) radiation emitted from the sample Smp is obtained.
[0013]
As another method, a plurality of mirrors can be simultaneously driven to selectively guide light in a desired spectral range from the sample to the detector.
[0014]
[Problems to be solved by the invention]
In recent years, not only single staining but also multiple staining is frequently used in fluorescence observation. Naturally, fluorescent staining is performed to make it possible to visually recognize a specific target in a cell or tissue. For this reason, in the multiple staining observation, each stained part must be detected as a clear color difference, that is, a difference in fluorescence wavelength. Moreover, it is necessary to efficiently remove and detect a partial overlap (crossover portion) of the fluorescence wavelengths.
[0015]
Furthermore, fluorescent probes for detecting intracellular calcium ion concentration have been widely used for studying cell functions. This is because calcium ions are closely related to cell activity. For staining calcium ions, “INDO-1” is well known as a fluorescent reagent. By using “INDO-1”, the intracellular calcium ion concentration can be quantitatively measured.
[0016]
A sample (specimen) stained with “Indo-1” emits fluorescence when irradiated with UV light (ultraviolet rays). Then, the generated fluorescence is detected in two different wavelength ranges (center wavelength 405 [nm] and 480 [nm]), and the ratio of detected light quantity (fluorescence of center wavelength 405 [nm] / center wavelength 480 [nm] By calculating (fluorescence), the calcium ion concentration can be measured.
[0017]
The sample is repeatedly scanned with laser light using a scanning laser microscope, and the time change of the intracellular calcium ion concentration is measured by the above calculation. In general, fluorescence from a sample is branched into two optical paths at 450 [nm] by a dichroic mirror having an optical characteristic that reflects light in a specific wavelength band and transmits others, and one has a center wavelength of 405 [ nm], a band width of about 20 [nm], and the other is a bandpass filter BPF1, BPF2 having a center wavelength of 480 [nm] and a band width of about 20 [nm], thereby narrowing the detection wavelength region.
[0018]
However, the central wavelength of the fluorescence shifts depending on the state of the cell, for example, pH (pH; hydrogen ion concentration). For example, if the central wavelengths of the Ca2 + max and Ca2 + min fluorescence are shifted to O1 and O2 as shown in FIG. 8, the center wavelengths BPF1o and BPF2o of the bandpass filters BPF1 and BPF2 and the fluorescent Ca2 are used in this case. Deviation occurs between the central wavelengths O1 and O2 of + max and Ca2 + min. As a result, the amount of light that should be originally obtained cannot be obtained, and not only the S / N (signal / noise) of the data deteriorates. The calculation result represented by the light quantity ratio may be different from the value that should be originally obtained.
[0019]
That is, the filter set as described above in the related art 1 is not always the best.
[0020]
On the other hand, since the apparatus of Japanese Patent Publication No. 9-502269 can freely change the wavelength range of fluorescence from the sample guided to each detector, it is effective in solving the above-mentioned problems in the prior art 1. Conceivable. In fact, for the purpose of efficiently removing and detecting partial overlap (crossover part) of fluorescence wavelengths in multiple staining observation, the fluorescence from the sample can be controlled by controlling the slit while checking the image repeatedly. Since the spectrum can be freely selected, it is far better than the method using an optical bandpass filter. However, it is desirable that laser scanning can be reduced as much as possible because the sample fades due to repeated laser scanning.
[0021]
However, in the above-described example of measuring the intracellular calcium ion concentration, the state of the cell, for example, pH is unknown to the examiner, and where the central wavelength of the fluorescence is located, a partial overlap of the fluorescence wavelengths (crossover portion) ) Has no way of knowing in advance exactly what level it is, there is a problem of not knowing whether the results obtained are the best.
[0022]
That is, even if the change in calcium ion concentration can be captured, the inspector never knows whether the result is the best, and there is no guarantee that the best result will be obtained. is there.
[0023]
If it is possible to repeat the test by trial and error, it is possible to bring the obtained result closer to the optimum one. However, in this type of test, once the data is acquired, the sample must basically be exchanged (generally, the test is started by applying a stimulus with a chemical. Stimulation of the second time is ineffective.) Therefore, enormous labor and cost are required for cell preparation and the like, and it must be said that it is difficult in practice.
[0024]
Thus, the fundamental drawback of the above-described prior art is that only a preset spectrum, that is, the average intensity of fluorescence passing through the slit, can be used to form an image of the sample.
[0025]
On the other hand, in the technique disclosed in Japanese Patent Laid-Open No. 2000-56244, when collecting all spectral data when obtaining fluorescence spectral data from a sample, the DMD mirror elements are driven one by one and the sample is applied to the photodetector. Therefore, it is necessary to derive the spectral spectrum of the fluorescence from the light source, so that it takes a long time to obtain the spectral data of the entire spectral region.
Therefore, it is an unsuitable device for observing and measuring a phenomenon that changes rapidly such as intracellular calcium ion concentration. Therefore, in the prior art, it is impossible to observe and measure a phenomenon that changes rapidly such as intracellular calcium ion concentration. Therefore, the rapid development of technology to overcome this is eagerly desired.
[0026]
Therefore, the object of the present invention is to obtain an optimal analysis result when performing a test or imaging in which the fluorescence spectrum from the sample cannot be accurately known as in the case of measuring the intracellular calcium ion concentration. An object of the present invention is to provide a scanning laser microscope that can be obtained reliably.
[0027]
Another object of the present invention is to provide a scanning laser microscope that can collect data at once for the entire spectrum of light emitted from a stained sample.
[0028]
In addition, an object of the present invention is to provide a scanning laser microscope capable of displaying an image reflected on a desired spectrum with a small number of scans in order to prevent fading of a sample in multiple staining observation. There is to do.
[0029]
In addition, the purpose of the present invention is to prevent partial discoloration of the fluorescence wavelength by a small number of scans, preferably one scan, in order to prevent discoloration of the sample in multiple staining observation. An object of the present invention is to provide a scanning laser microscope capable of efficiently removing images and displaying images.
[0030]
[Means for Solving the Problems]
In order to solve the above problems and achieve the object, the scanning laser microscope of the present invention is configured as follows.
[0031]
[1] The laser light from the laser light source is focused on the sample to be observed and irradiated, and the laser light is moved and scanned with respect to the sample, and the light obtained from the sample is dispersed by the spectroscopic means.InspectionIn a scanning laser microscope that obtains spectral data by
  EnterUsing a one-dimensional light detection means in which a plurality of minute light receiving elements that generate an electrical signal corresponding to the amount of incident light are linearly arranged, the spectral output of the spectroscopic means is placed in the minute light receiving element arrangement range of the one-dimensional light detection means. Spectral data is obtained in units of pixels by making the incident relationship between the incident position and the wavelength range incident with a predetermined relationship.A detector;
  Means for collecting and storing electrical signals obtained from each micro light receiving element of the detector as spectral distribution data in pixel units;
  Means for receiving an instruction of a spectral range for image display;
  Means for extracting and adding data corresponding to the designated spectral range from the stored data, and generating an image of the spectral range;
  Means for displaying the generated imageWith,
  The instruction input of the spectral range and the image generation and display of the corresponding spectral range can be repeatedly performed based on the same stored and stored data.It is characterized by that.
[2] In addition, in the configuration of the item [1], the means for receiving an instruction for the spectral range includes an interface for specifying the desired spectral range from the spectral distribution displayed on the screen. Features.
[0032]
By focusing and irradiating a laser beam from a laser light source on a sample to be observed, moving and scanning the laser beam with respect to the sample, spectrally separating the light obtained from the sample by a spectroscopic unit, and detecting it by a detecting unit Spectral data for each point in the sample is obtained. For example, spectral data is obtained by using a spectroscopic means such as a prism, and spectrally splitting the spectroscopic means so as to enter the one-dimensional light detecting means. The one-dimensional light detecting means has a configuration in which a plurality of minute light receiving elements are arranged linearly (one-dimensionally), and the spectral output of the spectroscopic means is incident on the minute light receiving element array range of the one-dimensional light detecting means. Since the position and the wavelength range are arranged so as to be incident with a corresponding relationship, the incident wavelength range is determined depending on where the minute light receiving element is located. Therefore, since the light of a specific wavelength region of the spectrum is incident on the minute light receiving element at the specific position, a signal corresponding to the incident light amount is obtained from each minute light receiving element, and this is converted into digital data for each light receiving element, If the light receiving elements are collected in the order of arrangement, there is an effect that the wavelength components can be specified, that is, the data can be collected as data in which all the spectral distributions are known.
[0033]
In addition, since the laser light applied to the sample is moved and scanned over the sample, the data collected at that time is the data of the emitted light at the laser light irradiation point on the sample, and constitutes a screen determined by the scanning range. It becomes spectral distribution data in pixel units. Therefore, it can be collected as data from which all spectral distributions can be grasped for each pixel.
[0034]
Therefore, according to the present invention, a technique is established that can acquire all fluorescence spectral data from a sample at once in a short time. In addition, according to the present invention, an optimal analysis result is reliably obtained when performing a test or imaging in which the fluorescence spectrum from a sample cannot be accurately known, as in the case of measuring intracellular calcium ion concentration. It is possible to provide a scanning laser microscope.
[0035]
Further, in the multiple staining observation, it is possible to provide a scanning laser microscope capable of collecting necessary fluorescence spectrum data with a small number of scans in order to prevent discoloration of the sample.
[0037]
According to this configuration, the data obtained by collecting the electrical signals obtained from the micro light receiving elements of the detector are collected for the screen configuration, stored and stored, and an image is generated using the stored data. Since all spectrum distributions are collected as data that can be grasped every time, various spectrum ranges can be specified as desired, and images based on the spectra in the specified ranges can be displayed. Therefore, once data is collected, An optimal image can be found by generating various images using the data.
[0038]
Therefore, according to the present invention, it is possible to provide a scanning laser microscope capable of displaying an image reflected on a desired spectrum with a small number of scans in order to prevent fading of a sample in multiple staining observation.
[0039]
[3] In the configuration of the item [1],The means for accepting an indication of the spectral range accepts an indication of at least two types of spectral ranges that are longer than the output laser wavelength of the laser light source,
  The means for generating an image of the spectral range extracts and adds data corresponding to the two specified spectral ranges from the held data, and calculates a ratio between the two to obtain the ratio. An image based on the generated image, and output the generated image until the next image is generated,
  The display means displays the output image.
[0040]
In this configuration, when scanning for one screen is completed, an image is displayed every time one scanning is completed in intermittent scanning in which scanning for one screen of the sample by laser light is performed again after a while. The person can check how the image changes. For this reason, if the test does not go well, it is possible to take measures such as stopping the test there, so that time is not wasted.
[0041]
In addition, the present invention may include the following aspects.
[0042]
(1) The scanning laser microscope of the present invention condenses laser light through an objective lens, scans the sample with a scanning device, projects the light from the sample onto the spectroscope, and emits light from the scanning device. A scanning laser microscope capable of obtaining spectroscopic data corresponding to a predetermined spectral band in synchronization with a scanning signal, wherein in the spectroscopic data, only data in a desired spectral region is extracted and calculated, and the calculation result Based on the above, an image is displayed after the scanning is completed.
[0043]
(2) The scanning laser microscope of the present invention is the scanning laser microscope described in (1) above, and again performs data computation by changing a desired spectral range, and again based on the computation result. An image is displayed, and this sequence is repeated.
[0044]
(3) The scanning laser microscope of the present invention is the scanning laser microscope according to (1) or (2) above, wherein a plurality of desired spectral regions are designated in the spectral data, and Only data is extracted and calculated, and an image is displayed on at least one image plane based on the calculation result.
[0045]
(4) The scanning laser microscope of the present invention is the scanning laser microscope according to the above (1) or (2), wherein a plurality of desired spectral regions are designated in the spectral data, and each of these spectral regions is specified. Only data is extracted and calculated, and based on the calculation result, images corresponding to the plurality of designated spectral regions are displayed on each image plane.
[0046]
(5) The scanning laser microscope of the present invention is the scanning laser microscope according to any one of (1) to (4) above, and the image display shows a total value of data (light intensity data) in a desired spectral region. This is what we do.
[0047]
(6) The scanning laser microscope of the present invention is the scanning laser microscope described in (3) above, wherein two desired spectral regions are designated, and only data in each spectral region is extracted, A value is calculated, a ratio of these total values is calculated, and an image is displayed based on the calculation result.
[0048]
(7) The scanning laser microscope of the present invention is the scanning laser microscope according to the above (1) to (6), and the image display includes data (light intensity data) of the entire spectral region excluding the laser wavelength. It is set in advance to perform based on the total value of data (light intensity data) in a longer spectral range excluding the total value or laser wavelength.
[0049]
(8) The scanning laser microscope of the present invention is the scanning laser microscope according to the above (1) to (6), and the image display is data (light intensity data) of the entire spectral region excluding the laser wavelength. It is set in advance to perform based on a total value of data (light intensity data) in a shorter spectral range excluding the total value or laser wavelength.
[0050]
(9) The scanning laser microscope of the present invention is the scanning laser microscope described in (1) above, wherein the spectroscope includes a one-dimensional or two-dimensional CCD array.
[0051]
(10) The scanning laser microscope of the present invention is the scanning laser microscope described in (1) above, in which the sample scan is intermittently performed a predetermined number of times for time-lapse observation, and the image display is performed after scanning. It is performed a predetermined number of times.
[0052]
DETAILED DESCRIPTION OF THE INVENTION
The present invention allows spectral data of fluorescence from a sample stained with a reagent to be acquired over the entire spectral range in a short time, and from the acquired spectral data of the entire spectral range, a desired value can be obtained. Analysis and image display can be performed using spectral data in the spectral region, and details of embodiments of the present invention will be described below with reference to the drawings.
[0053]
(First embodiment)
FIG. 1 is a diagram showing a configuration of a confocal scanning laser microscope according to the first embodiment of the present invention. In the figure, reference numeral 1 denotes a laser light source that oscillates and outputs a laser beam having a desired wavelength. When the measurement target is an intracellular calcium ion concentration labeled with Indo-1, for example, 351 [nm] An argon laser light source having an oscillation line is used. Reference numeral 2 denotes a laser beam (laser beam) emitted from the laser light source 1.
[0054]
A beam expander 3 is an optical system for enlarging the beam diameter of the laser beam 2 emitted from the laser light source 1 so as to substantially match the objective lens pupil diameter. A laser line filter 4 is a filter that selectively transmits light having a preset desired wavelength. When the measurement object is the intracellular calcium ion concentration labeled with Indo-1, an optical filter that selectively transmits a wavelength of 351 [nm] is used. Reference numeral 5 denotes a dichroic mirror, which is a half mirror having optical characteristics that reflects light of a specific wavelength band and transmits the other light. When the measurement object has an intracellular calcium ion concentration, a mirror having an optical characteristic of selectively reflecting the wavelength of 351 [nm] and transmitting the others is used.
[0055]
6 is an XY scanning optical system, 7 is a pupil projection lens, 8 is an imaging lens, 9 is an objective lens, and 10 is a sample. The sample 10 is stained with a reagent according to the purpose. The objective lens 9 can be adjusted forward and backward close to the sample 10 and is a lens for focusing the optical system with respect to the sample 10. The imaging lens 8 and the pupil projection lens 7 have their optical axes aligned with those of the objective lens 9. It is arranged to match the optical axis.
[0056]
Further, 11 is a confocal lens, 12 is a confocal stop, 13 is a collimating lens, and 15 is a prism. The confocal lens 11, the confocal stop 12, and the collimating lens 13 transmit light from the sample 10 to the prism 15. The optical axis is arranged on the optical path to be guided.
[0057]
The prism 15 is for splitting incident light of each wavelength.
[0058]
The dichroic mirror 5 is disposed on an optical path that guides light from the sample 10 to the prism 15 and is inclined on the optical path between the confocal lens 11 and the XY scanning optical system 6. The laser beam 2 guided from the laser light source 1 through 4 is reflected here and guided to the sample 10 side, and the optical axis of the laser light source 1 is guided to the confocal lens 11 side. It is arranged at the intersection with the top.
[0059]
The confocal lens 11 is a lens having a focal position conjugate with the focal position of the objective lens 9 with respect to the sample 10, and the confocal stop 12 is a stop disposed at the confocal position of the confocal lens 11. The confocal stop 12 plays the role of a pinhole, and since the ideal pinhole diameter varies depending on the wavelength of the light to be extracted, the confocal stop 12 also has a function as a stop so that it can be adjusted.
[0060]
Since the fluorescence emitted from the sample 10 is focused on the confocal stop 12 located in a plane conjugate with the observation target portion of the sample, the light passing through the confocal stop 12 enters the spectroscope 14. In this mechanism, the light is incident on the prism 15 of the spectroscope 14 and split.
[0061]
The XY scanning optical system 6 is disposed in the optical path between the dichroic mirror 5 and the pupil projection lens 7 and can optically move the optical path within a predetermined range. 16 is a condensing lens, 17 is a one-dimensional CCD (solid-state image sensor in which pixels are arranged in a line), and the condensing lens 16 is a lens for collecting the light dispersed by the prism 15 in a predetermined range, This is for condensing light on the one-dimensional CCD 17. The one-dimensional CCD 17 is disposed at the focal position of the condenser lens 16.
[0062]
As shown in FIG. 2, the one-dimensional CCD 17 is a line image sensor in which a plurality of light receiving cells corresponding to pixels are densely arranged in a line, and the light dispersed by the prism 15 is one-dimensionally collected by the condenser lens 16. By collecting within the light receiving cell array range of the CCD 17, light having a specific spectrum is incident on a specific cell position and detected. The CCD 17 is configured to output pixel signals (luminance signals) due to charges generated in the cells by the incidence of light as serial data in the order in which the cells are arranged at the time of reading. Each of them has a correspondence relationship that becomes a detection signal of a specific spectrum.
[0063]
The spectroscope 14 is constituted by the prism 15, the condenser lens 16, and the one-dimensional CCD 17. The laser light is XY scanned with respect to the sample 10 by the XY scanning optical system 6, and the one-dimensional CCD 17 output after the spectroscopy by the fluorescent prism 15 excited by the laser light at a certain moment is This is a detection pixel signal that individually includes all the spectra included in the excitation fluorescence (or reflected light) at the position irradiated with the laser light.
[0064]
31 is a control device for driving and controlling the XY scanning optical system 6, and 30 is a signal processing device, and a signal corresponding to the incident light intensity detected by the one-dimensional CCD 17 is supplied to the control device. The digital signal is converted into a digital signal and output in synchronization with the sampling signal 31. The computer 32 receives data output from the signal processing device 30 and stores it in a storage means as a file. It has functions such as generating a spectrum image using these stored files, displaying a graph of the spectrum, and generating an image of the specified spectrum range by specifying the spectrum range. Reference numeral 33 denotes a monitor device, which is an image display device that displays an output screen of the computer 32.
[0065]
The computer 32 is a graphical user interface (GUI) that is an interface that supports an image generation application and an inspector's operations and instructions so that graphical elements such as icons and windows displayed on the screen can be used as a mouse. Interface of a computer using a pointing device) and display all the spectral distributions collected for each image on the screen, or at least the desired spectral range from the displayed spectral distributions It has a function of individually specifying one or more, extracting data about the specified spectral region from the storage means, generating an image, and displaying the image.
[0066]
Next, the operation of this apparatus having such a configuration will be described. Let us consider a case in which a measurement relating to intracellular calcium ion concentration is to be carried out. In this case, “INDO-1” is used as a fluorescent reagent for staining the sample 10. In this case, an argon laser light source is used as the laser light source 1. The sample 10 stained with “INDO-1” is placed under the objective lens 9 and the objective lens 9 is focused.
[0067]
Thereafter, laser light is emitted from the laser light source 1. The laser light source 1 is an argon laser light source having an oscillation line (laser light) of 351 [nm], and the laser beam 2 of 351 [nm] emitted from the laser light source 1 is reflected by the beam expander 3 on the objective lens pupil diameter. The beam diameter is enlarged so as to substantially coincide with. Then, the light is guided to the dichroic mirror 5 through the laser line filter 4.
[0068]
The laser line filter 4 selectively transmits an oscillation line of 351 [nm]. The laser beam having a wavelength of 351 [nm] that has passed through the laser line filter 4 is reflected by the dichroic mirror 5, passes through the XY scanning optical system 6, the pupil projection lens 7, the imaging lens 8, and the objective lens 9, and then the sample 10. To reach.
[0069]
Here, the laser beam focusing point with respect to the sample 10 is determined by the objective lens 9, and the laser beam focusing position on the sample 10 is the XY scanning position by the XY scanning optical system 6 for XY scanning the laser beam. It depends on.
[0070]
The sample 10 is labeled with a fluorescent reagent “INDO-1”. When the sample 10 is irradiated with laser light having a wavelength of 351 [nm], reflected light or laser is emitted from the sample 10 serving as the irradiation point. Fluorescence is generated by excitation with light. A light beam 20 composed of reflected light and fluorescence from the sample 10 returns to the dichroic mirror 5 through the objective lens 9, the imaging lens 8, the pupil projection lens 7, and the XY scanning optical system 6. Then, the light beam 20 passes through the dichroic mirror 5, where the light of the original laser light wavelength component is removed and becomes only the light of the fluorescent component, which further passes through the confocal lens 11, The light from the position that becomes the irradiation point is focused and focused on the confocal stop 12 side. The confocal stop 12 at the focal point takes out light from the position of the sample 10 that is the irradiation point, and the rest is removed from the stop opening (pinhole) of the confocal stop 12. Removed. The light that has passed through the confocal stop 12 is collimated (collimated) by the collimating lens 13 and enters the spectroscope 14.
[0071]
The spectroscope 14 includes a prism 15, a condensing lens 16, and a one-dimensional CCD 17, and the light beam that has passed through the confocal stop 12 and converted into parallel rays by the collimating lens 13 is spectrally resolved by the prism 15, and the condensing lens 16. Is condensed on the one-dimensional CCD 17. The one-dimensional CCD 17 is arranged at the focal position of the condensing lens 16, and a plurality of light receiving cells of minute size are arranged linearly and densely, The light is condensed by the condensing lens 16 so that the distribution range matches the arrangement range of the light receiving cells in the one-dimensional CCD 17.
[0072]
Therefore, the arrangement position of each light receiving cell and the wavelength range of the spectrum to be dispersed have a specific relationship. The wavelength of the spectrum obtained by the spectrum and the position of the light receiving cell for detecting the spectrum of that wavelength in the one-dimensional CCD 17 are determined.
[0073]
Therefore, the light separated for each wavelength by being spectrally resolved by the prism 15 is incident on the light receiving cell at the position corresponding to the spectral light path on the one-dimensional CCD 17 arranged one-dimensionally. In the one-dimensional CCD 17, the spectral component of the output light at the laser beam irradiation position at that time in the sample 10 enters the light receiving cell corresponding to the component, and as a result, the light receiving cell generates a charge corresponding to the incident light amount. To do.
[0074]
The electric charges generated in this way are read in order according to the arrangement order of the light receiving cells, and are given to the signal processing device 30 as a light intensity signal continuous in light receiving cell units. The signal processing device 30 converts the light intensity signal detected by the one-dimensional CCD 17 into a digital signal in synchronism with the sampling signal from the control device 31 and, for example, passes through a PCI bus which is a standard bus of a personal computer. To the computer 32.
[0075]
The computer 32 receives this and stores it in the storage means. The stored digital signal of each light receiving cell for one line is component-specific data (spectral distribution characteristic data) of all the spectral components of the output light at the specific scanning position in the sample 10. Therefore, component-specific data of all spectral components of the output light at the specific scanning position in the sample 10 can be collected at a time.
[0076]
When the data collection at one point is finished, the control device 31 drives and controls the XY scanning optical system 6 in synchronization with the sampling signal generated by itself to acquire the spectral data of the next pixel. The position of the laser beam 2 with respect to 10 is moved. Then, as described above, the light from the position is split by the prism 15 to collect all data (spectral distribution characteristic data) of the spectral components of the sample 10 at the position.
[0077]
The control device 31 drives and controls the XY scanning optical system 6 in synchronization with the sampling signal to move the position of the laser light 2 with respect to the sample 10, thereby performing XY scanning so as to cover each position of the sample 10. Then, all data of the spectral components of the sample 10 at each position are collected.
[0078]
The above-described one-dimensional CCD 17 is, for example, a line image sensor having 256 light receiving cells corresponding to 256 elements, and the prism 15 and the condensing lens so as to correspond to the shortest wavelength 350 [nm] and about 1 [nm] per element. If 16 focal lengths are set optically, in this case, the one-dimensional CCD 17 has a configuration capable of detecting a spectrum in a range from 350 [nm] to 605 [nm].
[0079]
The signal processing device 30 is assumed to be configured to digitize the output of the one-dimensional CCD 17 for each element, for example, with 8-bit resolution. In this case, for each sampling signal of the control device 31 (that is, for one pixel (one pixel) on the image), data with a capacity of 256 [Byte] (bytes) (data of spectral distribution characteristics) Will be collected. If the XY scanning optical system 6 covers 256 × 256 pixels and is to be scanned, 256 × 256 sampling signals are output per screen, so one image (256 × 256 pixels) is output. ) Has a data capacity of 17 [MByte].
[0080]
When one image having a 256 × 256 pixel configuration is acquired in one second, the transfer rate for transferring data from the signal processing device 30 to the computer 32 is about 17 [MByte / second]. Can be sufficiently transferred via the PCI bus installed in the PC.
[0081]
Further, by mounting a 1 [GByte] memory in the computer 32, it is possible to transfer the spectral distribution characteristic data for each pixel of at least 50 images to the computer and record it in the computer memory. .
[0082]
From the above, for the sample to be observed, for every pixel, it covers the entire spectrum, and includes light intensity data for each component (data of spectral distribution characteristics for each pixel) for 50 seconds (50 images). Time-lapse observation data can be obtained.
[0083]
Here, as shown below, the entire spectrum data sequence for each image pixel (Xi, Yj) n (where (Xi, Yj) is the pixel position and n is the nth scanned image) For each spectral component, I (K1) n, I (K2) n, I (K3) n,..., I (Km) n, for example, I (350) n, I (351) n, I (352 ) N,...,..., I (605) n (n = 1 to 50, and K1, K2,... Indicate wavelengths. If I (350) n, n. The incident light amount value at the wavelength 350 [nm] of the first image, and I (351) n indicates the incident light amount value at the wavelength 351 [nm] of the nth image).
[0084]
The above is the data collection of spectral components in the scanning laser microscope of the present invention. Next, an image display method will be described.
[0085]
After acquiring the data, the examiner selects a spectral range for displaying an image. This is performed by software of the computer 32.
[0086]
The computer 32 includes an image generation application, a GUI that is an interface that supports easy operation and instructions by the inspector, operation means, and the like. Then, a menu screen based on these software is displayed on the monitor device 33 to instruct the examiner what processing the user wants to perform. Now, assuming that the inspector gives an instruction to generate an image of a desired spectral component from the collected spectrum data, the computer 32 receives this instruction and generates a screen for specifying the spectrum range. Send and display. The inspector then indicates the desired spectral range.
[0087]
For example, a distribution chart of spectral characteristics is displayed in a graph from collected spectrum data, and a desired spectral range is designated on the displayed distribution chart. As a result, it is assumed that two spectral ranges are designated as shown by hatched areas denoted by symbols p and q in FIG. That is, one is a range (p) from a wavelength of 390 [nm] to 420 [nm], and the other is a range (q) from 465 [nm] to 485 [nm].
[0088]
Receiving this designation information, the computer 32 obtains the spectrum data integrated value ΣI (K) n in the designated range in units of pixels. In the above example,
[Expression 1]

Is calculated as follows. Then, the calculation is performed for each pixel constituting the entire screen, and an image reflecting the result is generated.
[0089]
An image corresponding to a spectrum from 390 [nm] to 420 [nm] is A, and an image corresponding to a spectrum from 465 [nm] to 485 [nm] is B. Assuming that the fluorescence spectrum from the sample 10 has shifted to the short wavelength side as shown in FIG. 3B from the characteristics shown in FIG. Increases the brightness, and it is recognized that the image B becomes darker.
[0090]
And to find the calcium ion concentration,
[Expression 2]

Here, α is an appropriate constant that can be determined by the examiner.
Is calculated.
[0091]
For example, a sample (specimen) stained with “INDO-1” irradiates UV light (ultraviolet rays) to emit fluorescence excited from the sample in two different wavelength ranges (central wavelengths 405 [nm] and 480 [ nm]) and calculating the ratio of the detected light quantity (fluorescence with central wavelength 405 [nm] / fluorescence with central wavelength 480 [nm]), the calcium ion concentration can be measured.
[0092]
Therefore, the calcium ion concentration can be obtained by performing the calculation of [Equation 2] using the computer 32 by the computer 32.
[0093]
Such a calcium ion concentration is obtained for each pixel, and when such calculation is completed for all pixels on one screen, the computer 32 then generates an image reflecting the calcium ion concentration obtained for each pixel.
[0094]
Then, assuming that the data for image display representing the obtained calcium ion concentration is C, images A, B, and C are obtained for each image as shown in FIG. Then, images of A, B, and C for 50 sheets are obtained. These obtained images are displayed on the monitor device 33, and the inspector observes this display image.
[0095]
In the above example, since 50 images are obtained, if these 50 images are sequentially displayed, a change in intracellular calcium ion concentration can be displayed as a moving image.
[0096]
In the scanning laser microscope apparatus shown in the present embodiment, since all the data of the spectrum components that are the base are stored in the memory of the computer 32, the spectrum range to be selected is designated to the computer 32. You can reset it again and again. Therefore, the best image representing the change in the calcium ion concentration can be obtained by variously changing the range of the selected spectrum and comparing the image states.
[0097]
As described above, in this embodiment, the laser light from the laser light source is focused on the sample to be observed and irradiated, and the laser light is moved and scanned with respect to the sample, and the light obtained from the sample is spectroscopic means. In the scanning laser microscope in which spectrum data is obtained by performing spectroscopy with a detector and detecting with a detector, the detector densely and linearly arranges a plurality of minute light receiving elements that generate an electrical signal corresponding to the amount of incident light. The arrayed one-dimensional light detecting means is used, and the spectral output of the spectroscopic means is arranged so that the incident position and the wavelength region are incident on the minute light receiving element array range of the one-dimensional light detecting means with a predetermined relationship. It is what I did.
[0098]
Further, an optical system for condensing the spectral output of the spectroscopic means in the micro light receiving element array range of the one-dimensional photodetecting means so that the incident position and the wavelength range have a predetermined relationship is provided.
[0099]
Further, the signal processing means (signal processing device 30) for obtaining the data in the order of the arrangement of the micro light receiving elements by converting the output electric signal of each micro light receiving element of the one-dimensional light detecting means into a digital signal, and holding the obtained data And holding means.
[0100]
Further, the data held in the holding means is used to extract data in a designated spectral range, and an image is generated using spectral components in the designated range.
[0101]
By focusing and irradiating laser light from a laser light source on the sample to be observed, moving and scanning the laser light on the sample, and separating the light obtained from the sample with a spectroscopic means and detecting it with a detector Spectral data for each point in the sample is obtained. For example, spectral data is obtained by using a spectroscopic means such as a prism, and spectrally splitting the spectroscopic means so as to enter the one-dimensional light detecting means. The one-dimensional light detecting means has a configuration in which a plurality of minute light receiving elements (light receiving cells) are arranged densely and linearly (one-dimensionally), and the spectral output of the spectroscopic means is received by the minute light receiving of the one-dimensional light detecting means. Since the incident position and the wavelength range are related to each other in the element arrangement range, the incident wavelength range is determined depending on where the minute light receiving element is located. Accordingly, the light receiving element at a specific position is configured to receive light in a specific wavelength region of the spectrum. Therefore, the data corresponding to the amount of incident light is obtained from each minute light receiving element, converted into digital data for each light receiving element, and collected in the order in which the light receiving elements are arranged, that is, data in which the wavelength component can be specified, that is, data in which all spectral distributions are known. It has the effect of being collected as.
[0102]
Moreover, since the laser light applied to the sample is moved so as to perform XY scanning on the sample, the data collected at that time is data of emitted light at the irradiation point of the laser light on the sample, and XY This is spectral distribution data in units of pixels constituting a screen determined by the scanning range. Therefore, it can be collected as data from which all spectral distributions can be grasped for each pixel. This data is collected for each screen configuration, stored and saved, and an image is generated using this saved data. Since all the spectral distribution is collected for each pixel, it is collected in various desired spectral ranges. It is possible to designate and display an image with a spectrum in the designated range. Therefore, once data is collected, an optimum image can be found by generating various images using the data.
[0103]
In fluorescence observation of a sample stained with a fluorescent reagent (fluorescent stained sample), not only single staining but also multiple staining is frequently used. In particular, fluorescent staining is used to make it possible to visually recognize specific objects in cells and tissues. Often done. For this reason, in the multiple staining observation, each stained part must be detected as a clear color difference, that is, a difference in fluorescence wavelength. In addition, it is necessary to detect by removing a partial overlap (crossover portion) of the fluorescence wavelength.
[0104]
In the present invention, it is possible to collect all the spectral distribution for each pixel as data that can be grasped. This data is collected and stored for the screen configuration, and an image in a desired spectral range is generated using the stored data. Since it is saved, the spectrum range can be repeatedly specified and displayed again and again, so the image can be displayed. Once data has been collected, it is optimal to generate various images using that data. It becomes possible to find an image in a proper state.
[0105]
Multiple spectral region ranges can be specified at the same time. In addition, the spectral component data in the specified spectral region is added to generate an image based on the spectral component in the specified region. Etc. can be obtained by calculation, and the image of the obtained ion concentration can be displayed or synthesized with other images, so that it is possible to provide detailed information about the test result to the inspector. .
[0106]
In the above example, a two-dimensional image is targeted, but it goes without saying that line scanning (one-dimensional) may be used. In this case, a horizontal axis of scanning is taken in the horizontal direction of the image, and time (or the number of scans) is taken in the vertical axis. Moreover, although the example which used the prism for the spectroscopy was shown, this may use a diffraction grating and can be used if it is a means which can carry out spectroscopy.
[0107]
The above is a method that collects and holds spectral distribution data for each pixel, determines the desired spectral region using the stored data, and obtains an image based on the components of that region. However, the situation can be grasped, it can be judged whether the test is continued or stopped depending on the situation, and if the test does not seem to be successful, the test can be stopped there, etc. Next, an embodiment that eliminates the need for wasting will be described as a second embodiment.
[0108]
(Second Embodiment)
In this embodiment, when spectral data is collected, the total value of data in two spectral regions longer than this, excluding the output laser wavelength component of the laser light source, is calculated, and the ratio for each pixel is calculated. An image is displayed based on the calculation result, and the image display is updated every time one screen of the sample is scanned with the laser so that the progress can be grasped sequentially.
[0109]
FIG. 4A is a diagram showing a configuration of a confocal scanning laser microscope according to the second embodiment of the present invention. Also in this embodiment, the basic configuration may be the same as that described in the first embodiment. However, in the second embodiment, a helium cadmium laser light source having an oscillation line of 442 [nm] is used as the laser light source 1, and the XY scanning optical system 6 is scanned over 512 × 512 pixels. The one-dimensional CCD 17 is, for example, a line sensor corresponding to 1024 elements, and the prism 15 and the condenser lens 16 so as to correspond to the shortest wavelength 430 [nm] and about 0.25 [nm] per pixel. The one-dimensional CCD 17 is configured to detect a spectrum from 400 [nm] to 685 [nm], and the computer 32 has a laser wavelength (442 [nm]) in advance. The total value of data in two spectral regions longer than this, excluding components, is calculated, the ratio for each pixel is calculated, and an image is generated based on these calculation results. The image display on the monitor device 33 is updated every time the scanning of the laser beam in the pixel range for one screen by the XY scanning optical system 6 is completed. Different from the first embodiment. Reference numeral 34 denotes a mass storage device such as a hard disk connected to the computer 32.
[0110]
The operation of the apparatus having the configuration disclosed in this embodiment will be described. Here, it is assumed that the sample 10 to be observed is a cell in which two types of fluorescent proteins (for example, two types of fluorescent proteins of CFP and YFP) are expressed.
[0111]
A laser beam 2 emitted from a helium cadmium laser light source 1 having an oscillation line of 442 [nm] is expanded by a beam expander 3 so as to substantially coincide with the objective lens pupil diameter and guided to the laser line filter 4. . The laser line filter 4 selectively transmits an oscillation line of 442 [nm].
[0112]
The laser beam having a wavelength of 442 [nm] that has passed through the laser line filter 4 is reflected by the dichroic mirror 5, passes through the XY scanning optical system 6, the pupil projection lens 7, the imaging lens 8, and the objective lens 9, and then the sample 10. To reach.
[0113]
Sample 10 is a cell in which two types of fluorescent proteins, CFP and YFP, are expressed. A change in intracellular calcium ion concentration causes a change in energy transfer from CFP to YFP, which changes the amount of fluorescence. Therefore, the intracellular calcium ion concentration can be measured by measuring the ratio of the light amounts.
[0114]
The fluorescent protein at the arrival position is excited by the laser beam reaching the sample 10 or reflected light is generated.
[0115]
Here, the laser beam focusing point with respect to the sample 10 is determined by the objective lens 9, and the laser beam focusing position on the sample 10 is the XY scanning position by the XY scanning optical system 6 for XY scanning the laser beam. It depends on.
[0116]
A light beam 20 composed of reflected light and fluorescence from the sample 10 returns to the dichroic mirror 5 through the objective lens 9, the imaging lens 8, the pupil projection lens 7, and the XY scanning optical system 6. The light beam 20 passes through the dichroic mirror 5 and the confocal lens 11 and enters the spectroscope 14 via the confocal stop 12 and the collimator lens 13.
[0117]
The spectroscope 14 includes a prism 15, a condensing lens 16, and a one-dimensional CCD 17. A light beam that has passed through the dichroic mirror 5 is spectrally resolved by the prism 15, and is condensed on the one-dimensional CCD 17 through the condensing lens 16. The The one-dimensional CCD 17 is arranged at the focal position of the condenser lens 16.
[0118]
In synchronism with the sampling signal of the control device 31 that drives and controls the XY scanning optical system 6, the signal processing device 30 converts the light intensity signal detected by the one-dimensional CCD 17 into a digital signal, and the computer via the PCI bus. 32.
[0119]
For example, the one-dimensional CCD 17 is a line sensor having a light receiving cell equivalent to 1024 elements, and the prism 15 and the condenser lens 16 are arranged so as to correspond to the shortest wavelength 430 [nm] and about 0.25 [nm] per pixel. The focal length is set optically. Therefore, the light receiving cell 1024 element covers the spectral range from 400 [nm] to 685 [nm] in order. Each of the light receiving cells generates an electric signal corresponding to the intensity of light of the corresponding wavelength component. That is, the one-dimensional CCD 17 is configured to be able to detect a spectrum from 400 [nm] to 685 [nm].
[0120]
The signal processing device 30 processes the output of the one-dimensional CCD 17 with 8-bit resolution for each element, and outputs it as data arranged in the order of arrangement.
[0121]
Therefore, the data output from the signal processing device 30 has a data capacity of 1 [KByte] (kilobytes) for one sampling signal of the control device 31 (that is, for one pixel on the image).
[0122]
Since the range of XY scanning by the XY scanning optical system 6 is equivalent to 512 × 512 pixels per image, the control device 31 outputs a sampling signal corresponding to this, so that one image ( The data capacity is 268 [MByte] per 512 × 512 pixels.
[0123]
When one image is acquired every 60 seconds, the transfer rate required from the signal processing device 30 to the computer 32 can be sufficiently handled via a PCI bus mounted on a general computer. In addition, by writing this data in a large-capacity storage device 34 such as a hard disk connected to the computer 32 at the end of one screen of laser scanning for the sample, it is possible to record data for at least 50 images. is there. The sum of the data transfer time from the signal processing device 30 to the computer 32 and the writing time to the hard disk (mass storage device 34) should be smaller than the laser scanning interval time for each screen.
[0124]
In this embodiment, the processing in the computer 31 calculates the total value of data in two longer spectral ranges excluding the laser wavelength (442 [nm]) component in advance and calculates the ratio for each pixel. Based on these calculation results, an image is generated and displayed, and the image display is updated every time laser scanning is completed.
[0125]
That is, two spectrum ranges are designated in advance. One is from 465 [nm] to 495 [nm], and the other is from 520 [nm] to 550 [nm].
[0126]
Receiving this designation information, the computer 32 obtains the spectrum data integrated value ΣI (K) n in the designated range in units of pixels. In the above example,
[Equation 3]

Is calculated as follows. Then, the calculation is performed for each pixel constituting the entire screen, and an image reflecting the result is generated.
[0127]
An image corresponding to a spectrum from 465 [nm] to 495 [nm] is A, and an image corresponding to a spectrum from 520 [nm] to 550 [nm] is B. As the calcium ion concentration increases, the fluorescence spectrum from the sample increases in brightness due to energy transfer, and the image A becomes darker.
[0128]
And to find the calcium ion concentration,
[Expression 4]

Here, α is an appropriate constant that can be determined by the examiner.
Is calculated.
[0129]
Then, if an image based on data for image display representing the calcium ion concentration obtained by calculation is C, images A, B, and C are obtained for each image as shown in FIG. For example, since the n = 1 image can be generated at the end of scanning for the n = 1 image, the generated image is displayed.
These obtained images are stored in the large-capacity storage device 34 and displayed on the monitor device 33, so that the inspector observes this display image.
[0130]
When the laser scanning for the image of n = 2 is completed, an image of n = 2 is generated and stored in the mass storage device 34, and the image of n = 2 is substituted for the image of n = 1. When the laser scanning for the image of n = 3 is completed, an image of n = 3 is generated and stored in the mass storage device 34, and the n = 3 is replaced with the n = 2 image. 3 images are displayed, and so on, every time images A, B, and C are scanned, an image is generated and the generated image is updated and displayed on the monitor device 33. This makes it possible to observe the passage of time while grasping the status of the test.
[0131]
Further, since the generated image is held in the large-capacity storage device 34, the predetermined number of images (50 images in the above example) that are held after completion of laser scanning a predetermined number of times (in the above example, over 50 screens). Can be displayed in a moving image, and the time course can be observed.
[0132]
That is, in this embodiment, the sample scan is intermittently performed a predetermined number of times for the time lapse observation, and the image display is completed at the end of each laser scan or all of the laser scans for acquiring all the images. After a certain number of times.
[0133]
In addition, since the spectral distribution data for each pixel collected by laser scanning is stored, the selected spectral range can be reset and displayed again and again, so that the best representation of changes in calcium ion concentration is possible. An image can be obtained.
[0134]
As described above, in this embodiment, the laser light from the laser light source is focused on the sample to be observed and irradiated, and the laser light is moved and scanned with respect to the sample, and the light obtained from the sample is dispersed. In a scanning laser microscope in which spectral data is obtained by spectroscopically detecting and detecting with a detector, when collecting spectral data, the output laser wavelength component of the laser light source is removed in advance. It is configured to calculate the total value of data in the spectral range, calculate the ratio for each pixel, and display an image based on these calculation results. As the image display is updated, the progress can be grasped sequentially.
[0135]
Therefore, since the situation can be grasped while conducting the test, it becomes possible to judge whether the test is continued or stopped depending on the situation, and if the test does not seem to be successful, the test is stopped there. Can be obtained, and the effect of not wasting time can be obtained.
[0136]
Therefore, in this embodiment, when scanning for one screen is completed, an image is displayed every time one scanning is completed in intermittent scanning in which scanning for one screen of the sample by laser light is performed again after a while. Therefore, the inspector can check the images of CFP and YFP and the change of the calcium ion concentration during the test. For this reason, if the test is not successful, it is possible to take measures such as stopping the test there, so that time is not wasted.
[0137]
In the above example, a helium cadmium laser light source is used as a laser light source for observation by one-photon excitation. However, an ultrashort pulse laser light source having an infrared wavelength such as a mode-locked titanium sapphire laser is used for two-photon excitation. Excitation observation is performed, the total value of data in two shorter spectral ranges is calculated, the ratio for each pixel is calculated, and an image is displayed based on these calculation results. You may make it update the image display of a calculation result each time. Here, the two-photon excitation (two-photon excitation method) is a method in which fluorescent molecules are excited locally at the focal plane by irradiating a long-wavelength laser beam having a wavelength multiple of the absorption wavelength at a high density. It is also called the multiphoton excitation method, and is a technique that can obtain a sectioning image equivalent to the confocal observation method without a pinhole.
[0138]
A method of acquiring data by two-photon excitation using such an ultrashort pulsed long-wavelength laser beam has less phototoxicity to cells and enables observation over a longer time.
[0139]
In this example, a one-dimensional CCD is used. However, when the light beam from the sample is collected by the condenser lens 16 and the diffraction spot diameter formed here is larger than the pixel size of the CCD, the two-dimensional CCD is used. It is good also as a structure to use. Thereby, when the spot diameter is large, light from the sample can be detected efficiently, and data with good S / N can be acquired.
[0140]
The present invention is not limited to the examples shown in the above-described embodiments, and can be implemented with various modifications.
[0141]
In the present invention, the above embodiment includes inventions at various stages, and various inventions can be extracted by appropriately combining a plurality of disclosed constituent elements. For example, even if some constituent elements are deleted from all the constituent elements shown in the embodiment, at least one of the problems described in the column of the problem to be solved by the invention can be solved, and is described in the column of the effect of the invention. When at least one of the effects is obtained, a configuration in which the configuration requirements in the embodiment are partially deleted can also be realized as an invention.
[0142]
【The invention's effect】
According to the present invention, the spectral data is provided for each scanning pixel, the spectral range for image display can be freely selected, and the designation of the spectral range can be changed any number of times. To provide a scanning laser microscope with a high degree of freedom that can obtain optimal analysis results even when performing tests or imaging in which the fluorescence spectrum from a sample cannot be accurately known, such as measuring the ion concentration Can do.
[0143]
In addition, in order to prevent fading of the sample during multiple staining observation, a scanning laser microscope that can efficiently remove partial overlaps (crossover portions) of fluorescence wavelengths and display images with a small number of scans is provided. can do.
[Brief description of the drawings]
FIG. 1 is a diagram for explaining a first embodiment of the present invention.
FIG. 2 is a diagram for explaining the present invention and a diagram for explaining a configuration example of a spectroscope in a scanning laser microscope of the present invention.
FIG. 3 is a diagram for explaining an example of an obtained spectrum, an example of spectrum range designation, and an example of a transition state thereof.
FIG. 4 is a diagram for explaining a second embodiment of the present invention.
FIG. 5 is a diagram for explaining a conventional technique.
FIG. 6 is a diagram for explaining a conventional technique.
FIG. 7 is a diagram for explaining a conventional technique.
FIG. 8 is a diagram for explaining a conventional technique.
[Explanation of symbols]
1 ... laser light source, 2 ... laser beam
3 ... Beam expander,
4 ... Laser line filter
5 ... Dichroic mirror, 6 ... XY scanning optical system
7 ... Pupil projection lens, 8 ... Imaging lens
9 ... Objective lens, 10 ... Sample
11 ... Confocal lens, 12 ... Confocal stop
13 ... Collimating lens, 14 ... Spectroscope
15 ... Prism, 16 ... Condensing lens
17 ... one-dimensional CCD, 20 ... luminous flux from sample
30 ... Signal processing device, 31 ... Control device
32 ... Computer, 33 ... Monitor device
34. Hard disk drive (HDD)

Claims (3)

Moving to scan the laser beam to the sample with is irradiated by focusing the laser beam to a sample to be observed from the laser light source, by detect by divided by the spectral means the light obtained from a sample, the spectrum In a scanning laser microscope that can obtain the data of
Using a one-dimensional light detection means linearly are arranged a plurality of micro-light-receiving element for generating an electric signal to the input Shako amount corresponding the spectral output of the spectroscopic unit to the micro light-receiving element array range of the one-dimensional light detection means A detector that obtains spectral data in pixel units by adopting an arrangement relationship in which the incident position and the wavelength region are incident with a predetermined relationship ;
Means for collecting and storing electrical signals obtained from each micro light receiving element of the detector as spectral distribution data in pixel units;
Means for receiving an instruction of a spectral range for image display;
Means for extracting and adding data corresponding to the designated spectral range from the stored data, and generating an image of the spectral range;
Means for displaying the generated image ;
A scanning laser microscope characterized in that the instruction input of a spectral range and image generation and display of the corresponding spectral range can be repeatedly performed based on the same stored and stored data . 2. The scanning laser microscope according to claim 1, wherein the means for receiving an instruction for the spectral range includes an interface for indicating the desired spectral range from a spectral distribution displayed on a screen. The means for accepting an indication of the spectral range accepts an indication of at least two types of spectral ranges that are longer than the output laser wavelength of the laser light source,
The means for generating an image of the spectral range extracts and adds data corresponding to the two specified spectral ranges from the held data, and calculates a ratio between the two to obtain the ratio. An image based on the generated image, and output the generated image until the next image is generated,
2. The scanning laser microscope according to claim 1, wherein the display means displays the output image.

Images