JP4148392B2

JP4148392B2 - Method and apparatus for observing fine three-dimensional localization of gene expressed in vivo

Info

Publication number: JP4148392B2
Application number: JP2002057963A
Authority: JP
Inventors: 秀夫横田; 佐紀子中村; 昭武牧野内; 俊郎樋口; 龍太郎姫野
Original assignee: RIKEN Institute of Physical and Chemical Research
Current assignee: RIKEN Institute of Physical and Chemical Research
Priority date: 2002-03-04
Filing date: 2002-03-04
Publication date: 2008-09-10
Anticipated expiration: 2022-03-04
Also published as: JP2003254964A

Description

【０００１】
【発明の属する技術分野】
本発明は、生体内発現遺伝子の微細３次元局在の観察方法およびその装置に関し、さらに詳細には、動物や植物などの生体内において各種遺伝子の発現状況を観察する際に用いて好適な生体内発現遺伝子の微細３次元局在の観察方法およびその装置に関する。
【０００２】
【発明の背景】
従来、生体内部で発現した遺伝子、即ち、生体内発現遺伝子の局在を観察するにあたっては、試料となる生体（本明細書においては、「試料となる生体」を「生体試料」と適宜に称することとする。）を切片化し、切片化した生体試料を観察することにより、生体試料内における発現遺伝子の２次元配置を観察するという手法がとられていた。
【０００３】
また、上記したような観察手法を行う際に、観察対象となる遺伝子に対して発現時に検出が可能となるマーカー（標識）を組み込み、このマーカーを組み込んだ遺伝子を有する遺伝子組み換え生体試料を観察するようにして、生体試料内部における特定の遺伝子の発現部位を分別することが可能になってきている。
【０００４】
なお、マーカーとしては、蛍光物質、発光物質あるいは色素などを用いることができ、具体的には、ＧＦＰ、ＥＧＦＰ、ＹＦＰ、ＲＦＰ、ルシフェリン、メラニン色素などがある。
【０００５】
こうした観察を行う際において、微細な領域を観察するミクロ観察を行う場合には、共焦点レーザー顕微鏡を用いた観察手法が用いられているが、その観察可能な範囲は、生体試料の厚さ方向では当該生体試料の表面から１００μｍ程度までと小さいものであった。
【０００６】
このため、生体一個体全体における生体内発現遺伝子の３次元局在を観察することが極めて困難であるという問題点があった。
【０００７】
【発明が解決しようとする課題】
本発明は、上記したような従来の技術の有する問題点に鑑みてなされたものであり、その目的とするところは、生体一個体全体を観察の対象領域とし、かつ、その観察の対象領域内で細胞１つレベルでの微細な領域における生体内発現遺伝子の局在を観察することを可能にした生体内発現遺伝子の微細３次元局在の観察方法およびその装置を提供しようとするものである。
【０００８】
【課題を解決するための手段】
上記目的を達成するために、本発明による生体内発現遺伝子の微細３次元局在の観察方法あるいはその装置においては、特定の遺伝子の発現時に検出が可能となるマーカーを有する遺伝子組み換え生体試料を連続して切断し、当該切断毎に共焦点撮像により得られた断面の画像たる断面画像を観察することを繰り返して、生体試料内部の３次元構造を再構築することにより生体試料体内での発現遺伝子の微細３次元局在を観察するようにしたものである。
【０００９】
即ち、本発明のうち請求項１に記載の発明は、観察対象となる特定の遺伝子に対して発現時に検出が可能となるマーカーを予め組み込み、該マーカーを組み込んだ遺伝子を有する遺伝子組み換え体よりなるマーカー組み込み遺伝子組み換え生体試料の生体一個体全体をμｍの分解能で一定の方向に順次押し出す第１のステップと、上記第１のステップにおいて押し出された上記マーカー組み込み遺伝子組み換え生体試料を、上記第１のステップの押し出し毎に順次に連続して切断する第２のステップと、共焦点レーザー顕微鏡を用い、上記第２のステップの切断毎に、上記第２のステップにおいて切断された上記マーカー組み込み遺伝子組み換え生体試料の切断面に光を集光させ、上記光を集光させた切断面からの反射光によって上記切断面の２次元画像を連続して撮像し、該撮像した上記切断面の２次元画像のそれぞれを順次記録手段に記録させる第３のステップと、上記第１のステップ、上記第２のステップ、上記第３のステップを順次繰り返す第４のステップと、上記第４のステップの後に、上記第３のステップにおいて記録された２次元画像データを上記記録手段から取り出して２次元画像群から立体画像の構築を行い、上記マーカーにより上記観察対象となる特定の遺伝子の発現を検出可能で、上記マーカー組み込み遺伝子組み換え生体試料の生体一個体全体における生体内発現遺伝子の微細３次元局在を観察可能な３次元的な上記マーカー組み込み遺伝子組み換え生体試料の観察を行う第５のステップとを有するようにしたものである。
また、本発明のうち請求項２に記載の発明は、観察対象となる特定の遺伝子に対して発現時に検出が可能となるマーカーを予め組み込み、該マーカーを組み込んだ遺伝子を有する遺伝子組み換え体よりなるマーカー組み込み遺伝子組み換え生体試料の生体一個体全体をμｍの分解能で一定の方向に順次押し出す押し出し手段と、上記押し出し手段によって押し出された上記マーカー組み込み遺伝子組み換え生体試料を、上記押し出し手段の押し出し毎に順次に連続して切断する切断手段と、共焦点レーザー顕微鏡を用い、上記切断手段による切断毎に、上記切断手段によって切断された上記マーカー組み込み遺伝子組み換え生体試料の切断面に光を集光させ、上記光を集光させた切断面からの反射光によって上記切断面の２次元画像を連続して撮像する共焦点撮像手段と、上記共焦点撮像手段によって撮像された２次元画像に基づいて立体画像の構築を行い、上記マーカーにより上記観察対象となる特定の遺伝子の発現を検出可能で、上記マーカー組み込み遺伝子組み換え生体試料の生体一個体全体における生体内発現遺伝子の微細３次元局在を観察可能な３次元的な上記マーカー組み込み遺伝子組み換え生体試料の観察の処理を行う画像処理手段とを有するようにしたものである。
また、本発明のうち請求項３に記載の発明は、請求項２に記載の発明において、上記共焦点撮像手段は、複数のピンホールを有するニポウディスクと、上記ニポウディスクのピンホールに対応する位置に複数のマイクロレンズアレイを有するマイクロレンズアレイディスクと、上記ニポウディスクと上記マイクロレンズアレイディスクとを一体的に回転させる回転装置とを有し、レーザー光を複数の上記マイクロレンズアレイにより対応するそれぞれの上記ピンホールに集光するようにしたものである。
【００１０】
また、本発明のうち請求項４に記載の発明は、観察対象となる特定の遺伝子に対して発現時に検出が可能となるマーカーを予め組み込み、該マーカーを組み込んだ遺伝子を有する遺伝子組み換え体よりなるマーカー組み込み遺伝子組み換え生体試料の生体一個体全体をμｍの分解能で一定の方向に順次押し出す第１のステップと、上記第１のステップにおいて押し出された上記マーカー組み込み遺伝子組み換え生体試料を、上記第１のステップの押し出し毎に順次に連続して切断する第２のステップと、上記第２のステップの切断毎に、マイクロレンズアレイディスクの複数のマイクロレンズアレイによりニポウディスクの対応するそれぞれのピンホールに集光し、上記ピンホールを通過した光を対物レンズで上記第２のステップにおいて切断された上記マーカー組み込み遺伝子組み換え生体試料の切断面に集光させ、上記マーカー組み込み遺伝子組み換え生体試料の切断面から反射された光をＣＣＤカメラで撮像することにより、上記マーカー組み込み遺伝子組み換え生体試料の切断面の複数の点からの反射光を同時に検出して、上記マーカー組み込み遺伝子組み換え生体試料の切断面の２次元画像を連続して撮像する第３のステップと、上記第３のステップにおいて撮像した２次元画像に基づいて立体画像の構築を行い、上記マーカーにより上記観察対象となる特定の遺伝子の発現を検出可能で、上記マーカー組み込み遺伝子組み換え生体試料の生体一個体全体における生体内発現遺伝子の微細３次元局在を観察可能な３次元的な上記マーカー組み込み遺伝子組み換え生体試料の観察を行う第４のステップとを有し、上記ニポウディスクと上記マイクロレンズアレイディスクとを一体的に高速回転して、光の焦点を上記マーカー組み込み遺伝子組み換え生体試料の切断面全体に走査するようにしたものである。
【００１１】
また、本発明のうち請求項５に記載の発明は、請求項４に記載の発明において、上記マーカー組み込み遺伝子組み換え生体試料のマーカーとして光により励起されて発光または発色する物質を用いる場合には、上記マーカー組み込み遺伝子組み換え生体試料のマーカーへ光を照射するための光源と、上記第３のステップにおいて上記マーカー組み込み遺伝子組み換え生体試料の切断面の２次元画像を撮像するために上記マーカー組み込み遺伝子組み換え生体試料の切断面に集光させる光の光源とを共用化するようにしたものである。
【００１２】
また、本発明のうち請求項６に記載の発明は、観察対象となる特定の遺伝子に対して発現時に検出が可能となるマーカーとして蛍光物質を予め組み込み、該マーカーを組み込んだ遺伝子を有する遺伝子組み換え体よりなるマーカー組み込み遺伝子組み換え生体試料の生体一個体全体をμｍの分解能で一定の方向に順次押し出す押し出し手段と、上記押し出し手段によって押し出された上記マーカー組み込み遺伝子組み換え生体試料を、上記押し出し手段の押し出し毎に順次に連続して切断する切断手段と、上記切断手段によって切断された上記マーカー組み込み遺伝子組み換え生体試料の切断面に対して、上記マーカー組み込み遺伝子組み換え生体試料のマーカーたる上記蛍光物質を励起するための特異的な波長の光を出射する光源と、複数のピンホールを有するニポウディスクと、上記ニポウディスクの対応するそれぞれの上記ピンホ−ルに上記光源から出射された光を集光する複数のマイクロレンズアレイを有するマイクロレンズアレイディスクと、上記ニポウディスクと上記マイクロレンズアレイディスクを一体的に回転させ上記光源から出射された光の焦点を上記マーカー組み込み遺伝子組み換え生体試料の切断面全体に走査可能とする回転装置と、上記ニポウディスクの上記ピンホールを通過した光を上記マーカー組み込み遺伝子組み換え生体試料の切断面に集光させる対物レンズと、上記対物レンズによって光を集光させた上記マーカー組み込み遺伝子組み換え生体試料の切断面から反射された光が入射し上記マーカー組み込み遺伝子組み換え生体試料の切断面の複数の点からの反射光を同時に検出して上記マーカー組み込み遺伝子組み換え生体試料の切断面の２次元画像を連続して撮像するＣＣＤカメラとを有する共焦点撮像手段と、上記共焦点撮像手段により撮像された上記マーカー組み込み遺伝子組み換え生体試料の切断面の２次元画像を記憶する画像記録手段と、上記画像記録手段に記録された上記マーカー組み込み遺伝子組み換え生体試料の切断面の２次元画像に対する画像処理を行って立体画像の構築を行い、上記マーカーたる上記蛍光物質の発光により上記観察対象となる特定の遺伝子の発現を検出可能で、上記マーカー組み込み遺伝子組み換え生体試料の生体一個体全体における生体内発現遺伝子の微細３次元局在を観察可能な３次元的な上記マーカー組み込み遺伝子組み換え生体試料の観察の処理を行う画像処理手段と、上記画像処理手段により処理された処理画像を表示する表示手段とを有し、上記光源を、上記共焦点撮像手段の光源として共用化するようにしたものである。
【００１３】
また、本発明のうち請求項７に記載の発明は、請求項６に記載の発明において、上記光源は、ハロゲン光源と光学フィルターを用いたキセノン水銀光源とであり、上記共焦点撮像手段は、上記マーカー組み込み遺伝子組み換え生体試料の切断面の上記ハロゲン光源による白色光観察と、上記キセノン水銀光源および上記光学フィルターによるＶ・Ｂ・Ｇ励起の蛍光観察との同時観察が可能な構成を有し、上記画像記録手段は、上記マーカー組み込み遺伝子組み換え生体試料の同一の切断面に関して、白色光における観察を示す上記切断面の２次元画像をはじめに記録し、次ぎに蛍光における観察を示す上記切断面の２次元画像を記録し、上記画像処理手段は、上記画像記録手段に記録された上記マーカー組み込み遺伝子組み換え生体試料の切断面の２次元画像を画像処理することにより、白色光の観察時における３次元的な上記マーカー組み込み遺伝子組み換え生体試料の観察の処理と蛍光の観察時における３次元的な上記マーカー組み込み遺伝子組み換え生体試料の観察の処理とを行なうようにしたものである。
また、本発明のうち請求項８に記載の発明は、観察対象となる特定の遺伝子に対して発現時に検出が可能となるマーカーとして発光する物質を予め組み込み、該マーカーを組み込んだ遺伝子を有する遺伝子組み換え体よりなるマーカー組み込み遺伝子組み換え生体試料の生体一個体全体をμｍの分解能で一定の方向に順次押し出す押し出し手段と、上記押し出し手段によって押し出された上記マーカー組み込み遺伝子組み換え生体試料を、上記押し出し手段の押し出し毎に順次に連続して切断する切断手段と、上記切断手段によって切断された上記マーカー組み込み遺伝子組み換え生体試料の切断面に光を集光させ、上記光を集光させた切断面からの反射光によって上記切断面の２次元画像を連続して撮像する共焦点撮像手段と、上記共焦点撮像手段によって撮像された２次元画像に基づいて立体画像の構築を行い、上記マーカーたる上記発光する物質の発光により上記観察対象となる特定の遺伝子の発現を検出可能で、上記マーカー組み込み遺伝子組み換え生体試料の生体一個体全体における生体内発現遺伝子の微細３次元局在を観察可能な３次元的な上記マーカー組み込み遺伝子組み換え生体試料の観察の処理を行う画像処理手段とを有し、上記共焦点撮像手段は、複数のピンホールを有するニポウディスクと、上記ニポウディスクのピンホールに対応する位置に複数のマイクロレンズアレイを有するマイクロレンズアレイディスクと、上記ニポウディスクと上記マイクロレンズアレイディスクとを一体的に回転させる回転装置とを有し、レーザー光を複数の上記マイクロレンズアレイにより対応するそれぞれの上記ピンホールに集光し、上記回転装置により上記ニポウディスクと上記マイクロレンズアレイディスクとを一体的に高速回転して、レーザー光の焦点を上記マーカー組み込み遺伝子組み換え生体試料の切断面全体に走査するようにしたものである。
【００１４】
【発明の実施の形態】
以下、添付の図面に基づいて、本発明による生体内発現遺伝子の微細３次元局在の観察方法およびその装置の実施の形態の一例を詳細に説明するものとする。
【００１５】
図１には、本発明による生体内発現遺伝子の微細３次元局在の観察装置の実施の形態の一例を示す概念構成説明図が示されている。
【００１６】
この本発明による生体内発現遺伝子の微細３次元局在の観察装置（以下、「本装置」と適宜に称する。）は、観察対象の試料たる生体試料として、特定の遺伝子の発現時に検出が可能となるマーカーを組み込んだ遺伝子を有する遺伝子組み換え体を用いる。
【００１７】
ここで、上記した特定の遺伝子の発現時に検出が可能となるマーカーを有する遺伝子組み換え体を作出するにあたっては、例えば、卵細胞に組み換えＤＮＡをインジェクションする手法を用いてもよいし、また、集合キメラにより組み換え遺伝子体を作出する手法を用いてもよい。
【００１８】
即ち、本発明において生体試料として用いられる遺伝子組み換え体は、どのような手法により作出されたものでもよく、本発明はその作出の手法に制限されるものではない。
【００１９】
また、マーカーとしては、従来より公知の蛍光物質、発光物質あるいは色素などを用いることができ、具体的には、ＧＦＰ、ＥＧＦＰ、ＹＦＰ、ＲＦＰ、ルシフェリン、メラニン色素などを用いればよい。しかしながら、マーカーについても、上記したものに限定されるものではなく、特定の遺伝子の発現時に検出が可能となるものであるならば、上記以外の物質を適宜に用いることができる。
【００２０】
なお、マーカーの検出にあたっては、例えば、電磁波を用いるようにすればよい。具体的には、電磁波として白色光を用いる場合には、幅広い波長の光を当てて反射してきた特定の波長を検出するものであるため、マーカーとして色素を用いる場合には白色光での観察が可能となる。即ち、マーカーとして色素を用いる場合には、非特異的な波長の電磁波を照射して、特異的な波長の電磁波を受けて検出する。
【００２１】
また、マーカーとして発光する物質を用いる場合には、電磁波を照射する必要はなく、特異的な波長の電磁波を受けて検出する。
【００２２】
さらに、マーカーとして蛍光色素を用いる場合には、特異的な波長の電磁波を照射して、特異的な波長の電磁波を受けて検出する。
【００２３】
図１に示す本装置は、生体内で発現したときに所定の波長の光を照射すると励起されることにより発光したり発色したりして他の遺伝子と異なる振る舞いをするマーカーを組み込んだ遺伝子を有する遺伝子組み換え体よりなる生体試料（以下、「マーカー組み込み遺伝子組み換え生体試料」と適宜に称する。）を切断手段としてのナイフにより切断して、当該マーカー組み込み遺伝子組み換え生体試料の断面を作成する断面作成装置（スライサ）１０と、断面作成装置１０により作成されたマーカー組み込み遺伝子組み換え生体試料の断面に対して、当該マーカー組み込み遺伝子組み換え生体試料のマーカーを励起するための所定の波長の光を照射する光源としてのレーザー１２と、断面作成装置１０に作成されるとともにレーザー１２によって所定の波長の光を照射されたマーカー組み込み遺伝子組み換え生体試料の断面の画像を撮像するＣＣＤカメラ１４を有する共焦点撮像手段としての共焦点撮像装置などにより構成される撮像手段としての断面画像観察部と、断面画像観察部により撮像されたマーカー組み込み遺伝子組み換え生体試料の断面の画像を記憶する画像記録装置１６と、断面作成装置１０ならびに画像記録装置１６の動作を制御するための制御信号を出力するパーソナルコンピュータなどにより構成される制御装置１８と、画像記録装置１６に記録されたマーカー組み込み遺伝子組み換え生体試料の断面の画像に対する画像処理を行って任意の断面や立体画像（３次元画像）などの処理画像を生成する画像処理手段としての画像処理装置（グラフィックワークステーション）２０と、画像記録装置１６に記録されたマーカー組み込み遺伝子組み換え生体試料の断面の画像や画像処理装置２０により処理された任意の断面や立体像（３次元像）などの処理画像を表示するモニタ２２とを有し、観察者２４はモニタ２２を監視することにより、マーカー組み込み遺伝子組み換え生体試料におけるマーカー組み込み遺伝子の発現状態を観察する。
【００２４】
なお、マーカーとして、発光物質などを用いる場合には、マーカーを励起するための光を照射する光源としては、本装置に光源１２を設ける必要はない。
【００２５】
ここで、断面作成装置１０、断面画像観察部、画像記録装置１６、制御装置１８、画像処理装置２０ならびにモニタ２２としては、例えば、本願発明者らが特願２０００−３４７３９８号「３次元内部構造の解析方法及び装置」として提案した装置を用いることができる。
【００２６】
上記出願に開示された装置は、観察対象の試料を切断して、その断面の断面画像を観察することを繰り返して試料内部の３次元構造を再構築する装置である。
【００２７】
より詳細には、断面作成装置１０は、マーカー組み込み遺伝子組み換え生体試料を切削希望量だけ上方へ送り、当該マーカー組み込み遺伝子組み換え生体試料の切削希望量だけ、当該マーカー組み込み遺伝子組み換え生体試料の上方を回転するナイフで切削するという処理を自動的に繰り返すものである。
【００２８】
ここで、マーカー組み込み遺伝子組み換え生体試料を切削希望量だけ上方へ送る処理を実行する押し出し手段としての装置は、例えば、ステッピングモータとボールネジとを用いた直動装置により構成し、マーカー組み込み遺伝子組み換え生体試料を一定の方向（図２おいては上向きである。）に順次押し出すようにすればよい。なお、マーカー組み込み遺伝子組み換え生体試料の押し出しの分解能は、例えば、最小０．５μｍとすることができる。
【００２９】
また、マーカー組み込み遺伝子組み換え生体試料を切断する切断手段としては、ナイフを駆動装置などにより水平回転させ、押し出されたマーカー組み込み遺伝子組み換え生体試料を順次切断するように構成すればよい。
【００３０】
図２には、断面画像観察部の共焦点撮像装置を中心に示す概略構成説明図が示されている。
【００３１】
ここで、共焦点撮像装置は、複数のピンホール１００ａを有するニポウディスク１００と、ニポウディスク１００のピンホ−ル１００ａに対応する位置に複数のマイクロアレイレンズ１０２ａを有するマイクロアレイレンズディスク１０２と、ニポウディスク１００とマイクロアレイレンズディスク１０２を一体に回転させる回転装置１０４と、レーザー１２から出射されたレーザー光をマーカー組み込み遺伝子組み換え生体試料の断面に集光させる対物レンズ１０６と、マーカー組み込み遺伝子組み換え生体試料からの反射光を水平に反射するダイクロイックミラー１０８と、ダイクロイックミラー１０８により水平に反射された光を集光してＣＣＤカメラ１４へ入射する接眼レンズ１１０と有している。
【００３２】
この構成により、共焦点撮像装置においては、レーザー１２から出射されたレーザー光を複数のマイクロアレイレンズ１０２ａにより対応するそれぞれのピンホール１００ａに集光し、複数のピンホール１００ａを通過したレーザー光を対物レンズ１０６でマーカー組み込み遺伝子組み換え生体試料のナイフで切断した断面に集光させる。そして、マーカー組み込み遺伝子組み換え生体試料の断面から反射された光をダイクロイックミラー１０８で水平に反射し、接眼レンズ１１０を通してＣＣＤカメラ１４で撮像するすることにより、断面の複数の点からの反射光を同時に検出する。
【００３３】
さらに、回転装置１０４によりニポウディスク１００とマイクロアレイレンズディスク１０２とを高速回転させるだけで、レーザー光の焦点を切断した断面全体に走査することができ、断面全体の２次元画像を短時間で得ることができる。
【００３４】
以上の構成において、本装置においては、マーカー組み込み遺伝子組み換え生体試料を凍結などで固定して包埋しする。
【００３５】
次いで、マーカー組み込み遺伝子組み換え生体試料を一定の方向に順次押し出す押し出しステップと、押し出されたマーカー組み込み遺伝子組み換え生体試料を順次切断する切断ステップと、切断したマーカー組み込み遺伝子組み換え生体試料の断面にレーザー１２から出射されたレーザー光を集光させ、その反射光から断面の２次元画像を撮像する共焦点撮像ステップとを順次行う。
【００３６】
この結果、マーカー組み込み遺伝子組み換え生体試料の断面の連続断面画像が得られる。さらに、この連続断面画像を画像処理装置２０で処理して、マーカー組み込み遺伝子組み換え生体試料の３次元内部構造をモニタ２２により表示する。
【００３７】
こうした本装置によれば、共焦点撮像装置によりマーカー組み込み遺伝子組み換え生体試料の切断した断面にのみにレーザー光を集光させてその反射光を観察するので、例えば、マーカー組み込み遺伝子組み換え生体試料の透けなどような、その焦点位置以外の光の影響をほとんど排除することができ、撮像した２次元画像の解像度を高め、マーカー組み込み遺伝子組み換え生体試料の内部構造を高精密に再構築できる。
【００３８】
また、マーカーとして光により励起されて発光したり発色したりする物質を用いる場合には、レーザー１２をマーカーへ光を照射するための光源と共焦点撮像装置の光源として共用化することができる。
【００３９】
なお、マーカー組み込み遺伝子組み換え生体試料の切断した断面に照射してマーカーを励起する光源は、上記したようなレーザーに限られるものではなく、例えば、ハロゲン光源やキセノン水銀光源などを、適宜にフィルターなどを組み合わせて用いることができる。即ち、上記した光源から照射される光としては、白色光や蛍光などを適宜に設定するようにしてもよい。
【００４０】
即ち、光源としてハロゲン光源と光学フィルターを用いたキセノン水銀光源とを用いるようにすると、ハロゲン光源による白色光観察とキセノン水銀光源および光学フィルターによるＶ・Ｂ・Ｇ励起の蛍光観察との同時観察が可能となる。
【００４１】
ここで、断面作成装置１０は、例えば、「１５ｍｍ×１２ｍｍ×１２０ｍｍ〜１８０ｍｍ×１５０ｍｍ×２００ｍｍ」の大きさのマーカー組み込み遺伝子組み換え生体試料を、最小１０μｍの厚さで切断することができる。
【００４２】
断面画像観察部は、断面作成装置１０がマーカー組み込み遺伝子組み換え生体試料を切断する毎に、その断面に光源１２から照射された光を照射することにより観察する。この断面画像観察部は、例えば、マーカー組み込み遺伝子組み換え生体試料の断面はハロゲン光源による白色光観察と、キセノン水銀光源および光学フィルターによるＶ・Ｂ・Ｇ励起の蛍光観察との同時観察が可能であるように構成することができる。
【００４３】
画像記録装置１６においては、観察した断面の断面画像を記録する際に、同一の断面に関して、白色光における観察を示す断面画像をはじめに記録し、次ぎに蛍光における観察を示す断面画像を記録するように、画像処理装置２０により制御することができる。
【００４４】
画像処理装置２０は、画像記録装置１６に記録した断面画像を画像処理することにより、白色光の観察時における任意の断面の画像や立体画像あるいは蛍光の観察時における任意の断面の画像や立体画像を示すデータをモニタ２２に出力し、モニタ２２は出力されたデータに基づいて画像を映し出すことができる。
【００４５】
次ぎに、上記した本装置によって行った実験結果について説明すると、マーカー組み込み遺伝子組み換え生体試料として、維管束に特異的に発現する遺伝子ＯｓＴｒｘｈのプロモーターの制御下でＧＦＰを発現する遺伝子組み換えイネを用い、その発現部位の３次元構造をＧＦＰの蛍光により観察した。
【００４６】
マーカー組み込み遺伝子組み換え生体試料は、人工光条件下にて栽培し、種子ができた時点でその茎および根と茎の移行部をナイフにて切り出し、凍結包埋剤を用いて凍結包埋した。
【００４７】
観察条件は、マーカー組み込み遺伝子組み換え生体試料の切断の厚さを２μｍとし、切断に用いる断面作成装置１０のナイフの回転数９０ｒｐｍとし、マーカー組み込み遺伝子組み換え生体試料の温度を「−４５℃」とした。
【００４８】
なお、断面作成装置１０のナイフとしては、ミクロトーム用ナイフを用い、レーザー１２の出力を１５０ｍｗで照射した断面を観察した。
【００４９】
図３乃至図４には、共焦点撮像装置を用いない従来の断面画像観察部による観察結果を示し、図３は落射蛍光で５倍の対物レンズ１０６を使用して観察したイネの茎の断面画像であり、図４は落射蛍光で２０倍の対物レンズ１０６で観察したイネの茎の断面画像である。
【００５０】
図３乃至図４において、やや色の薄い部位（実際には、青色に発色している。）が自家蛍光を示し、最も色の薄い部位（実際には、緑色に発色している。）がＧＦＰによる発現遺伝子を示している。
【００５１】
落射蛍光では、下層の透けがあるために、ＧＦＰ発現部位がぼやけており、大まかな局在はわかるが、詳細な局在を観察することはできない。
【００５２】
また、図５乃至図６には、共焦点撮像装置を用いた本発明における断面画像観察部による観察結果を示し、図５は共焦点撮像装置で５倍の対物レンズ１０６を使用して観察したイネの茎の断面画像であり、図６は共焦点撮像装置で２０倍の対物レンズ１０６で観察したイネの茎の断面画像である。
【００５３】
上記した図５乃至図６により、維管束の周囲でＧＦＰが特異的に発現していることがわかる。また、維管束を構成しているいくつかの細胞で強く発現していることがわかる。
【００５４】
以上のことより、共焦点撮像装置を用いた本発明における断面画像観察部を用いることにより、マーカー組み込み遺伝子組み換え生体試料内における発現遺伝子の３次元局在を観察することができた。
【００５５】
その際に、後から染色するのではなく、生体の発現遺伝子が発色することから、試料内部での染色のムラがなく、試料の大きさの影響を受けない。
【００５６】
即ち、本発明は、共焦点撮像装置による下層の透けのない観察と、断面作成装置１０を用いた深さ方向に制限のない観察との利点を併せ持つものである。
【００５７】
また、本発明による観察手法は、対象とするマーカー組み込み遺伝子組み換え生体試料に依存するものではないので、幅広い対象の対象のマーカー組み込み遺伝子組み換え生体試料の微細３次元局在を得ることができる。
【００５８】
なお、共焦点撮像装置は、上記した構成に限られるものではなく、例えば、共焦点顕微鏡を用いるようにしてもよい。
【００５９】
【発明の効果】
本発明は、以上説明したように構成されているので、生体一個体全体を観察の対象領域とし、かつ、その観察の対象領域内で細胞１つレベルでの微細な領域における生体内発現遺伝子の局在を観察することができるようになるという優れた効果を奏する。
【図面の簡単な説明】
【図１】本発明による生体内発現遺伝子の微細３次元局在の観察装置の実施の形態の一例を示す概念構成説明図である。
【図２】断面画像観察部の共焦点撮像装置を中心に示す概略構成説明図である。
【図３】共焦点撮像装置を用いない従来の断面画像観察部による観察結果を示し、落射蛍光で５倍の対物レンズを使用して観察したイネの茎の断面画像である。
【図４】共焦点撮像装置を用いない従来の断面画像観察部による観察結果を示し、落射蛍光で２０倍の対物レンズを使用して観察したイネの茎の断面画像である。
【図５】共焦点撮像装置を用いた本発明によるの断面画像観察部による観察結果を示し、落射蛍光で５倍の対物レンズを使用して観察したイネの茎の断面画像である。
【図６】共焦点撮像装置を用いた本発明によるの断面画像観察部による観察結果を示し、落射蛍光で２０倍の対物レンズを使用して観察したイネの茎の断面画像である。
【符号の説明】
１０断面作成装置（スライサ）
１２レーザー
１４ＣＣＤカメラ
１６画像記録装置
１８制御装置
２０画像処理装置（グラフィックワークステーション）
２２モニタ
２４観察者
１００ニポウディスク
１００ａピンホール
１０２マイクロアレイディスク
１０２ａマイクロアレイレンズ
１０４回転装置
１０６対物レンズ
１０８ダイクロイックミラー
１１０接眼レンズ[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method and apparatus for observing fine three-dimensional localization of genes expressed in vivo, and more particularly, suitable for use in observing the expression status of various genes in living bodies such as animals and plants. The present invention relates to a method and apparatus for observing fine three-dimensional localization of a gene expressed in the body.
[0002]
BACKGROUND OF THE INVENTION
Conventionally, in observing the localization of a gene expressed in a living body, that is, a gene expressed in the living body, a living body serving as a sample (in this specification, “a living body serving as a sample” is appropriately referred to as a “biological sample”) The method of observing the two-dimensional arrangement of the expressed gene in the biological sample by sectioning and observing the sectioned biological sample was taken.
[0003]
In addition, when performing the observation technique as described above, a marker (label) that can be detected at the time of expression is incorporated into a gene to be observed, and a genetically modified biological sample having a gene incorporating this marker is observed. In this way, it has become possible to distinguish the expression site of a specific gene within a biological sample.
[0004]
As the marker, a fluorescent substance, a luminescent substance, a dye, or the like can be used. Specific examples include GFP, EGFP, YFP, RFP, luciferin, and melanin dye.
[0005]
When performing such observation, when performing micro observation to observe a fine region, an observation method using a confocal laser microscope is used, but the observable range is the thickness direction of the biological sample. Then, it was as small as about 100 μm from the surface of the biological sample.
[0006]
For this reason, there has been a problem that it is extremely difficult to observe the three-dimensional localization of the in-vivo-expressed gene in one whole living organism.
[0007]
[Problems to be solved by the invention]
The present invention has been made in view of the problems of the prior art as described above, and the object of the present invention is to make an entire living body an observation target region and within the observation target region. It is an object of the present invention to provide a method and apparatus for observing a fine three-dimensional localization of a gene expressed in vivo that makes it possible to observe the localization of the gene expressed in vivo in a minute region at a single cell level. .
[0008]
[Means for Solving the Problems]
In order to achieve the above object, in the method or apparatus for observing fine three-dimensional localization of a gene expressed in vivo according to the present invention, a genetically modified biological sample having a marker that can be detected when a specific gene is expressed is continuously used. And observing a cross-sectional image, which is a cross-sectional image obtained by confocal imaging for each cutting, and reconstructing the three-dimensional structure inside the biological sample, thereby expressing the gene expressed in the biological sample. The three-dimensional localization is observed.
[0009]
That is, the invention described in claim 1 of the present invention is To be observed Specific genes Against Markers that can be detected during expression Pre-integrate the gene with the marker Have Incorporating genetically modified markers Genetically modified biological sample A whole living body with a resolution of μm A first step of sequentially extruding in a fixed direction, and the above-mentioned extruding in the first step Built-in marker Genetically modified biological samples , For each extrusion of the first step Sequentially Consecutively Using a second step of cutting and a confocal laser microscope, Every time the second step is cut, The above cut in the second step Built-in marker Of genetically modified biological samples Cut off The light was collected on the cross section and the light was collected. Cut off Above by reflected light from the cross section Cut off A two-dimensional image of the cross section Continuously Image Each of the captured two-dimensional images of the cut surface A third step that causes the sequential recording means to record, a fourth step that sequentially repeats the first step, the second step, the third step, and the third step after the fourth step. The two-dimensional image data recorded in the step is taken out from the recording means and taken out from the two-dimensional image group. A three-dimensional image is constructed, the expression of a specific gene to be observed can be detected by the marker, and the micro three-dimensional localization of the in vivo expressed gene in one whole individual organism of the marker-embedded genetically modified biological sample is observed Possible three-dimensional marker incorporation And a fifth step of observing the genetically modified biological sample.
Moreover, invention of Claim 2 among this invention is the following. To be observed Specific genes Against Markers that can be detected during expression Pre-integrate the gene with the marker Have Incorporating genetically modified markers Genetically modified biological sample A whole living body with a resolution of μm Extruding means that sequentially extrudes in a certain direction, and the above-mentioned extruded by the extruding means Built-in marker Genetically modified biological samples , For each extrusion of the extrusion means Sequentially Consecutively Using a cutting means for cutting and a confocal laser microscope, For each cutting by the cutting means, The above cut by the cutting means Built-in marker Of genetically modified biological samples Cut off The light was collected on the cross section and the light was collected. Cut off Above by reflected light from the cross section Cut off A two-dimensional image of the cross section Continuously Based on the confocal imaging means for imaging, and the two-dimensional image captured by the confocal imaging means A three-dimensional image is constructed, the expression of a specific gene to be observed can be detected by the marker, and the micro three-dimensional localization of the in vivo expressed gene in one whole individual organism of the marker-embedded genetically modified biological sample is observed Possible three-dimensional marker incorporation And an image processing means for observing the genetically modified biological sample.
According to a third aspect of the present invention, in the second aspect of the present invention, the confocal imaging means is provided at a position corresponding to a tipping disc having a plurality of pinholes and a pinhole of the tipping disc. A microlens array disk having a plurality of microlens arrays, and a rotating device that integrally rotates the nipou disk and the microlens array disk, each of which corresponds to the laser beam by the plurality of microlens arrays. It concentrates on the pinhole.
[0010]
Moreover, invention of Claim 4 among this invention is the following. To be observed Specific genes Against Markers that can be detected during expression Pre-integrate the gene with the marker Have Incorporating genetically modified markers Genetically modified biological sample A whole living body with a resolution of μm A first step of sequentially extruding in a certain direction, and the above-mentioned extruding in the first step Built-in marker Genetically modified biological samples , For each extrusion of the first step Sequentially Consecutively A second step of cutting; Every time the second step is cut, The plurality of microlens arrays of the microlens array disk are focused on the corresponding pinholes of the Niipou disk, and the light passing through the pinholes is cut by the objective lens in the second step. Built-in marker Of genetically modified biological samples Cut off Focus on the cross section Built-in marker Of genetically modified biological samples Cut off By imaging the light reflected from the cross section with a CCD camera, Built-in marker Of genetically modified biological samples Cut off Detecting reflected light from multiple points on the cross section at the same time, Built-in marker Of genetically modified biological samples Cut off A two-dimensional image of the cross section Continuously Based on the third step of imaging and the two-dimensional image captured in the third step A three-dimensional image is constructed, the expression of a specific gene to be observed can be detected by the marker, and the three-dimensional localization of the in vivo expression gene in the whole living organism of the marker-incorporated genetically modified biological sample is observed. Possible three-dimensional marker incorporation A fourth step of observing the genetically modified biological sample, wherein the Nipo disk and the microlens array disk are integrally rotated at high speed to focus the light on the Built-in marker Of genetically modified biological samples Cut off The entire section is scanned.
[0011]
The invention according to claim 5 is the invention according to claim 4, wherein Built-in marker When using a substance that emits light or develops color when excited by light as a marker for a genetically modified biological sample, Built-in marker A light source for irradiating the marker of the genetically modified biological sample with light, and in the third step, Built-in marker Of genetically modified biological samples Cut off Above to capture a two-dimensional image of the cross section Built-in marker Of genetically modified biological samples Cut off The light source for condensing light on the cross section is shared.
[0012]
Moreover, invention of Claim 6 among this invention is the following. To be observed Specific genes Against Markers that can be detected during expression As a gene incorporating a fluorescent substance in advance and incorporating the marker Have Incorporating genetically modified markers Genetically modified biological sample A whole living body with a resolution of μm Extruding means that sequentially extrudes in a certain direction, and the above-mentioned extruded by the extruding means Built-in marker Genetically modified biological samples , For each extrusion of the extrusion means Sequentially Consecutively Cutting means for cutting, and the above-mentioned cutting means by the cutting means Built-in marker Of genetically modified biological samples Cut off Above for the cross section Built-in marker Markers for genetically modified biological samples The above fluorescent substance For exciting Specific A microlens having a light source that emits light of a wavelength, a nipou disk having a plurality of pinholes, and a plurality of microlens arrays that collect the light emitted from the light source on each of the pinholes corresponding to the nipou disk The focus of the light emitted from the light source is rotated by integrally rotating the array disk, the Nipkow disk and the microlens array disk. Built-in marker Of genetically modified biological samples Cut off Rotating device capable of scanning the entire cross section and the light passing through the pinhole of the Niipou disc Built-in marker Of genetically modified biological samples Cut off The objective lens that focuses light on the cross section, and the light that is condensed by the objective lens Built-in marker Of genetically modified biological samples Cut off The light reflected from the cross section is incident Built-in marker Of genetically modified biological samples Cut off Detect the reflected light from multiple points of the cross section at the same time Built-in marker Of genetically modified biological samples Cut off A two-dimensional image of the cross section Continuously Confocal imaging means having a CCD camera for imaging, and the above-mentioned image captured by the confocal imaging means Built-in marker Of genetically modified biological samples Cut off An image recording means for storing a two-dimensional image of the cross section, and the above-mentioned image recorded in the image recording means Built-in marker Of genetically modified biological samples Cut off Perform image processing on 2D cross-sectional images A three-dimensional image is constructed, and the expression of a specific gene to be observed can be detected by light emission of the fluorescent substance as the marker. Incorporation of the above three-dimensional markers that can observe three-dimensional localization An image processing means for observing the genetically modified biological sample and a display means for displaying the processed image processed by the image processing means, and sharing the light source as the light source of the confocal imaging means It is what I did.
[0013]
The invention according to claim 7 of the present invention is the invention according to claim 6, wherein the light source is a halogen light source and a xenon mercury light source using an optical filter, and the confocal imaging means includes: the above Built-in marker Of genetically modified biological samples Cut off The cross-section has a configuration capable of simultaneous observation of white light observation with the halogen light source and V, B, G excitation fluorescence observation with the xenon mercury light source and the optical filter. Built-in marker The same in a genetically modified biological sample Cut off Above, showing observation in white light with respect to cross section Cut off A two-dimensional image of the cross section is recorded first, followed by the fluorescence observation above Cut off A two-dimensional image of the cross section is recorded, and the image processing means is recorded on the image recording means. Built-in marker Of genetically modified biological samples Cut off By processing the two-dimensional image of the cross section, the above three-dimensional image is obtained when white light is observed. Built-in marker Three-dimensional above-mentioned observation process and fluorescence observation of genetically modified biological samples Built-in marker And processing for observing the genetically modified biological sample.
Moreover, invention of Claim 8 among this invention is To be observed Specific genes Against Markers that can be detected during expression As a gene that has previously incorporated a substance that emits light as Have Incorporating genetically modified markers Genetically modified biological sample A whole living body with a resolution of μm Extruding means that sequentially extrudes in a certain direction, and the above-mentioned extruded by the extruding means Built-in marker Genetically modified biological samples , For each extrusion of the extrusion means Sequentially Consecutively Cutting means for cutting, and the above-mentioned cutting means by the cutting means Built-in marker Of genetically modified biological samples Cut off The light was collected on the cross section and the light was collected. Cut off Above by reflected light from the cross section Cut off A two-dimensional image of the cross section Continuously Based on the confocal imaging means for imaging, and the two-dimensional image captured by the confocal imaging means A three-dimensional image is constructed, the expression of the specific gene to be observed can be detected by luminescence of the luminescent substance as the marker, and the in vivo expressed gene in the whole living individual of the marker-incorporated genetically modified biological sample Incorporation of the above three-dimensional markers that can observe fine three-dimensional localization Image processing means for observing the genetically modified biological sample, and the confocal imaging means includes a nippo disk having a plurality of pinholes and a plurality of microlens arrays at positions corresponding to the pinholes of the nipou disk. Having a microlens array disk and a rotating device that integrally rotates the nipou disk and the microlens array disk, and condensing the laser light into the corresponding pinholes by the plurality of microlens arrays. The nip disk and the microlens array disk are integrally rotated at a high speed by the rotating device to focus the laser beam on the Built-in marker Of genetically modified biological samples Cut off The entire section is scanned.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, an example of an embodiment of an observation method and apparatus for fine three-dimensional localization of a gene expressed in vivo according to the present invention will be described in detail with reference to the accompanying drawings.
[0015]
FIG. 1 is a conceptual configuration explanatory diagram showing an example of an embodiment of an observation apparatus for fine three-dimensional localization of a gene expressed in vivo according to the present invention.
[0016]
The observation apparatus for fine three-dimensional localization of a gene expressed in vivo according to the present invention (hereinafter referred to as “this apparatus” as appropriate) can detect a specific gene as a biological sample to be observed. A gene recombinant having a gene incorporating a marker is used.
[0017]
Here, in order to produce a genetic recombinant having a marker that can be detected during the expression of the specific gene described above, for example, a technique of injecting a recombinant DNA into an egg cell may be used, You may use the method of producing a recombinant gene body.
[0018]
That is, the gene recombinant used as a biological sample in the present invention may be produced by any technique, and the present invention is not limited to the production technique.
[0019]
As the marker, conventionally known fluorescent substances, luminescent substances, dyes, and the like can be used, and specifically, GFP, EGFP, YFP, RFP, luciferin, melanin dye, and the like may be used. However, the markers are not limited to those described above, and substances other than those described above can be appropriately used as long as they can be detected during expression of a specific gene.
[0020]
In detecting the marker, for example, an electromagnetic wave may be used. Specifically, when white light is used as an electromagnetic wave, it is intended to detect a specific wavelength that has been reflected by applying light of a wide wavelength, so when using a dye as a marker, observation with white light is possible. It becomes possible. That is, when a dye is used as a marker, an electromagnetic wave with a non-specific wavelength is irradiated and an electromagnetic wave with a specific wavelength is received and detected.
[0021]
In addition, when a substance that emits light is used as a marker, it is not necessary to irradiate an electromagnetic wave, and an electromagnetic wave having a specific wavelength is received and detected.
[0022]
Further, when a fluorescent dye is used as a marker, an electromagnetic wave having a specific wavelength is irradiated and an electromagnetic wave having a specific wavelength is received and detected.
[0023]
The device shown in FIG. 1 is a gene incorporating a marker that emits light or develops a color when excited when irradiated with light of a predetermined wavelength when expressed in a living body and behaves differently from other genes. Creating a cross-section by cutting a biological sample made of a genetically modified product (hereinafter referred to as “marker-embedded genetically modified biological sample” as appropriate) with a knife as a cutting means to create a cross-section of the marker-embedded genetically modified biological sample A light source for irradiating a cross-section of a marker-embedded genetically modified biological sample created by the apparatus (slicer) 10 and the cross-section creating device 10 with light of a predetermined wavelength for exciting the marker of the marker-embedded genetically modified biological sample As a laser 12 and a cross-section creating apparatus 10 and a laser 12 Cross-sectional image observation as an imaging means composed of a confocal imaging device as a confocal imaging means having a CCD camera 14 for taking an image of a cross-section of a marker-incorporated genetically modified biological sample irradiated with light of a predetermined wavelength. And an image recording device 16 for storing cross-sectional images of the marker-embedded genetically modified biological sample imaged by the cross-sectional image observation unit, and a control signal for controlling the operations of the cross-section creating device 10 and the image recording device 16 are output. The image processing is performed on the cross-sectional image of the genetically-incorporated genetically modified biological sample recorded in the control device 18 constituted by a personal computer or the like and the marker-recording biologic sample recorded in the image recording device 16, so Image processing apparatus (graphic works) as image processing means for generating processed images 20), and a processed image such as a cross-sectional image of the marker-embedded genetically modified biological sample recorded in the image recording device 16 and an arbitrary cross-section or a three-dimensional image (three-dimensional image) processed by the image processing device 20 are displayed. The observer 24 observes the expression state of the marker-integrated gene in the marker-integrated genetically modified biological sample by monitoring the monitor 22.
[0024]
In addition, when using a luminescent substance etc. as a marker, it is not necessary to provide the light source 12 in this apparatus as a light source which irradiates the light for exciting a marker.
[0025]
Here, as the cross-section creating device 10, the cross-sectional image observation unit, the image recording device 16, the control device 18, the image processing device 20, and the monitor 22, for example, the inventors of the present application disclosed Japanese Patent Application No. 2000-347398 “three-dimensional internal structure”. The apparatus proposed as “Analysis Method and Apparatus” can be used.
[0026]
The apparatus disclosed in the above application is an apparatus for reconstructing a three-dimensional structure inside a sample by cutting a sample to be observed and observing a cross-sectional image of the cross section.
[0027]
More specifically, the cross-section creating device 10 sends the marker-incorporated genetically modified biological sample upward by a desired amount of cutting, and rotates the marker-incorporated genetically modified biological sample above the marker-incorporated genetically modified biological sample by the desired amount of cutting. The process of cutting with a cutting knife is automatically repeated.
[0028]
Here, the device as the push-out means for executing the process of sending the marker-incorporated genetically modified biological sample upward by the desired cutting amount is constituted by, for example, a linear motion device using a stepping motor and a ball screw. The sample may be sequentially extruded in a certain direction (upward in FIG. 2). Note that the resolution of extruding the marker-incorporated genetically modified biological sample can be, for example, a minimum of 0.5 μm.
[0029]
In addition, the cutting means for cutting the marker-incorporated genetically modified biological sample may be configured to horizontally cut the extruded marker-incorporated genetically modified biological sample in order by horizontally rotating the knife with a driving device or the like.
[0030]
FIG. 2 is a schematic configuration explanatory diagram mainly showing the confocal imaging device of the cross-sectional image observation unit.
[0031]
Here, the confocal imaging apparatus includes a Nipo disk 100 having a plurality of pinholes 100a, a microarray lens disk 102 having a plurality of microarray lenses 102a at positions corresponding to the pinholes 100a of the Nipo disk 100, the Nipo disk 100 and the microarray lens. A rotating device 104 that rotates the disk 102 integrally, an objective lens 106 that focuses the laser light emitted from the laser 12 on the cross section of the marker-modified genetically modified biological sample, and horizontal reflected light from the marker-modified genetically modified biological sample The dichroic mirror 108 that reflects the light and the eyepiece 110 that collects the light horizontally reflected by the dichroic mirror 108 and enters the CCD camera 14.
[0032]
With this configuration, in the confocal imaging device, the laser light emitted from the laser 12 is condensed to the corresponding pinholes 100a by the plurality of microarray lenses 102a, and the laser light that has passed through the plurality of pinholes 100a is used as the objective. The lens 106 is focused on a cross-section cut with a knife of a genetically modified biological sample incorporating a marker. Then, the light reflected from the cross section of the genetically modified biological sample incorporating the marker is reflected horizontally by the dichroic mirror 108 and imaged by the CCD camera 14 through the eyepiece 110, thereby simultaneously reflecting the reflected light from a plurality of points on the cross section. To detect.
[0033]
Furthermore, by simply rotating the Nipo disk 100 and the microarray lens disk 102 at a high speed by the rotating device 104, the entire cross section obtained by cutting the focal point of the laser beam can be scanned, and a two-dimensional image of the entire cross section can be obtained in a short time. it can.
[0034]
In the above configuration, in the present apparatus, the marker-incorporated genetically modified biological sample is fixed and embedded by freezing or the like.
[0035]
Next, an extruding step for sequentially extruding the marker-incorporated genetically modified biological sample in a predetermined direction, a cutting step for sequentially cutting the extruded marker-incorporating genetically modified biological sample, and a cross section of the cut marker-incorporating genetically modified biological sample from the laser 12 A confocal imaging step of condensing the emitted laser light and sequentially capturing a two-dimensional image of the cross section from the reflected light is sequentially performed.
[0036]
As a result, a continuous cross-sectional image of the cross-section of the marker-incorporated genetically modified biological sample is obtained. Further, this continuous cross-sectional image is processed by the image processing device 20, and the three-dimensional internal structure of the marker-incorporated genetically modified biological sample is displayed on the monitor 22.
[0037]
According to such an apparatus, since the laser beam is focused only on the cut section of the marker-modified genetically modified biological sample by the confocal imaging device and the reflected light is observed, for example, the marker-modified genetically-modified biological sample is transparent. Thus, the influence of light other than the focal position can be almost eliminated, the resolution of the captured two-dimensional image can be increased, and the internal structure of the genetically modified biological sample incorporating a marker can be reconstructed with high precision.
[0038]
Further, when a substance that emits light or develops color when excited by light is used as the marker, the laser 12 can be shared with the light source for irradiating the marker with light and the light source of the confocal imaging device.
[0039]
The light source that excites the marker by irradiating the cut cross-section of the genetically modified biological sample with the marker incorporated therein is not limited to the laser as described above. For example, a halogen light source, a xenon mercury light source, or the like can be appropriately filtered. Can be used in combination. That is, as the light emitted from the light source described above, white light, fluorescence, or the like may be set as appropriate.
[0040]
That is, if a halogen light source and a xenon mercury light source using an optical filter are used as the light source, simultaneous observation of white light observation using a halogen light source and fluorescence observation of V, B, G excitation using a xenon mercury light source and an optical filter is possible. It becomes possible.
[0041]
Here, for example, the cross-section creating apparatus 10 can cut a marker-incorporated genetically modified biological sample having a size of “15 mm × 12 mm × 120 mm to 180 mm × 150 mm × 200 mm” with a thickness of a minimum of 10 μm.
[0042]
The cross-sectional image observation unit observes the cross-section creating apparatus 10 by irradiating the cross-section with the light emitted from the light source 12 every time the marker-embedded genetically modified biological sample is cut. For example, the cross-sectional image observing unit can simultaneously observe white light observation using a halogen light source and V, B, G excitation fluorescence observation using a xenon mercury light source and an optical filter on the cross section of the genetically modified biological sample incorporating a marker. It can be constituted as follows.
[0043]
In the image recording device 16, when recording a cross-sectional image of the observed cross-section, a cross-sectional image showing observation in white light is recorded first for the same cross-section, and then a cross-sectional image showing observation in fluorescence is recorded next. Further, it can be controlled by the image processing apparatus 20.
[0044]
The image processing device 20 performs image processing on the cross-sectional image recorded in the image recording device 16 so that an arbitrary cross-sectional image or stereoscopic image at the time of white light observation or an arbitrary cross-sectional image or stereoscopic image at the time of fluorescence observation is obtained. Is output to the monitor 22, and the monitor 22 can project an image based on the output data.
[0045]
Next, the experimental results performed by the above-described apparatus will be described. Using genetically modified rice that expresses GFP under the control of the promoter of the gene OsTrxh that is specifically expressed in the vascular bundle, The three-dimensional structure of the expression site was observed by GFP fluorescence.
[0046]
The marker-incorporated genetically modified biological sample was cultivated under artificial light conditions, and when seeds were formed, the stem and root-to-stem transition portion were cut out with a knife and frozen and embedded using a frozen embedding agent.
[0047]
The observation conditions were that the cutting thickness of the marker-incorporated genetically modified biological sample was 2 μm, the rotation speed of the knife of the cross-section creating apparatus 10 used for cutting was 90 rpm, and the temperature of the marker-incorporating genetically modified biological sample was “−45 ° C.” .
[0048]
In addition, as a knife of the cross-section creating apparatus 10, a microtome knife was used, and a cross section irradiated with an output of the laser 12 at 150 mw was observed.
[0049]
FIGS. 3 to 4 show the observation results of a conventional cross-sectional image observation unit that does not use a confocal imaging device, and FIG. 3 shows a cross-section of rice stems observed by epi-fluorescence using the objective lens 106 of 5 times. FIG. 4 is a cross-sectional image of a rice stalk observed with an objective lens 106 of 20 times by epifluorescence.
[0050]
In FIG. 3 to FIG. 4, the slightly light-colored portion (actually colored blue) shows autofluorescence, and the lightest-colored portion (actually colored green). The gene expressed by GFP is shown.
[0051]
In epifluorescence, since there is transparency in the lower layer, the GFP expression site is blurred, and rough localization can be seen, but detailed localization cannot be observed.
[0052]
5 to 6 show the observation results by the cross-sectional image observation unit in the present invention using the confocal imaging device, and FIG. 5 is an observation using the confocal imaging device using the objective lens 106 of 5 times. 6 is a cross-sectional image of a rice stem, and FIG. 6 is a cross-sectional image of a rice stem observed with a 20 × objective lens 106 using a confocal imaging device.
[0053]
5 to 6 described above, it can be seen that GFP is specifically expressed around the vascular bundle. It can also be seen that it is strongly expressed in several cells that make up the vascular bundle.
[0054]
From the above, it was possible to observe the three-dimensional localization of the expressed gene in the marker-embedded genetically modified biological sample by using the cross-sectional image observation unit in the present invention using a confocal imaging device.
[0055]
At this time, since the expression gene in the living body is colored instead of staining later, there is no uneven staining inside the sample and it is not affected by the size of the sample.
[0056]
In other words, the present invention has the advantages of the observation of the lower layer with the confocal imaging device without see-through and the observation with the cross-section creating device 10 without limitation in the depth direction.
[0057]
In addition, since the observation technique according to the present invention does not depend on the target marker-incorporated genetically modified biological sample, it is possible to obtain a fine three-dimensional localization of a wide range of target marker-incorporated genetically modified biological samples.
[0058]
Note that the confocal imaging device is not limited to the above-described configuration, and for example, a confocal microscope may be used.
[0059]
【The invention's effect】
Since the present invention is configured as described above, an entire living body is set as an observation target region, and an in vivo expression gene in a minute region at one cell level in the observation target region. There is an excellent effect that the localization can be observed.
[Brief description of the drawings]
FIG. 1 is an explanatory diagram of a conceptual configuration showing an example of an embodiment of an observation apparatus for fine three-dimensional localization of a gene expressed in vivo according to the present invention.
FIG. 2 is a schematic configuration explanatory diagram mainly showing a confocal imaging device of a cross-sectional image observation unit.
FIG. 3 is a cross-sectional image of a rice stalk observed using a 5 × objective lens with epi-fluorescence, showing an observation result by a conventional cross-sectional image observation unit that does not use a confocal imaging device.
FIG. 4 is a cross-sectional image of a rice stalk observed using a 20-times objective lens with epi-fluorescence, showing an observation result by a conventional cross-sectional image observation unit that does not use a confocal imaging device.
FIG. 5 is a cross-sectional image of a rice stalk observed using a cross-sectional image observation unit according to the present invention using a confocal imaging apparatus, and observed using epi-fluorescence with a 5 × objective lens.
FIG. 6 is a cross-sectional image of a rice stalk observed using a 20-times objective lens with epi-fluorescence, showing an observation result by a cross-sectional image observation unit according to the present invention using a confocal imaging device.
[Explanation of symbols]
10 Cross-section creation device (slicer)
12 Laser
14 CCD camera
16 Image recording device
18 Control device
20 Image processing device (graphic workstation)
22 Monitor
24 observers
100 Nipou Disc
100a pinhole
102 Microarray disk
102a microarray lens
104 Rotating device
106 Objective lens
108 dichroic mirror
110 Eyepiece

Claims

A marker that can be detected at the time of expression of a specific gene to be observed is pre-incorporated, and the entire bio- organism of a genetically-modified genetically-modified biological sample made of a genetically modified recombinant having a gene into which the marker is incorporated has a resolution of μm. A first step of sequentially extruding in a certain direction with
A second step of sequentially cutting the marker-incorporated genetically modified biological sample extruded in the first step sequentially for each extrusion in the first step;
Using a confocal laser microscope, for each cut of the second step, the to focus the light to the switching section of the marker embedded recombinant biological sample is cut in the second step was condensed to the light and continuously captured two-dimensional images of the switching section by reflected light from the switching section, and a third step of sequentially recorded in the recording means the respective two-dimensional image of the cut surface was imaging,
A fourth step of sequentially repeating the first step, the second step, and the third step;
After the fourth step, the two-dimensional image data recorded in the third step is taken out from the recording means and a three-dimensional image is constructed from a group of two-dimensional images . 3D observation of the marker-incorporated genetically modified biological sample capable of detecting gene expression and observing the fine three-dimensional localization of the in vivo-expressed gene in the whole living organism of the marker-incorporated genetically modified biological sample A method for observing a fine three-dimensional localization of a gene expressed in vivo, comprising: a fifth step.

A marker that can be detected at the time of expression of a specific gene to be observed is pre-incorporated, and the entire bio- organism of a genetically-modified genetically-modified biological sample made of a genetically modified recombinant having a gene into which the marker is incorporated has a resolution of μm. Extruding means for sequentially extruding in a certain direction with,
Cutting means for sequentially cutting the marker-incorporated genetically modified biological sample pushed out by the push-out means sequentially for each push-out of the push-out means ;
Using a confocal laser microscope, for each cut by said cutting means, to collect the light to the switching section of the marker embedded recombinant biological sample is cut by said cutting means, from the switching section obtained by focusing the light confocal imaging means for imaging by continuous two-dimensional images of the switching section by reflected light,
A three-dimensional image is constructed based on the two-dimensional image captured by the confocal imaging means, the expression of the specific gene to be observed can be detected by the marker, in vivo expressed genes characterized by having an image processing means for processing the observable three-dimensional the marker embedded recombinant biological sample observation fine 3-dimensional localization of in vivo expressed genes in the entire population Fine three-dimensional observation device.

In the observation apparatus for fine three-dimensional localization of the in-vivo expressed gene according to claim 2,
The confocal imaging means includes
Nipkow disc having a plurality of pinholes;
A microlens array disk having a plurality of microlens arrays at positions corresponding to the pinholes of the nipou disk;
A rotating device that integrally rotates the nipou disk and the microlens array disk;
An apparatus for observing a fine three-dimensional localization of a gene expressed in a living body, characterized in that laser light is condensed on each corresponding pinhole by a plurality of microlens arrays.

A marker that can be detected at the time of expression of a specific gene to be observed is pre-incorporated, and the entire bio- organism of a genetically-modified genetically-modified biological sample made of a genetically modified recombinant having a gene into which the marker is incorporated has a resolution of μm. A first step of sequentially extruding in a certain direction with
A second step of sequentially cutting the marker-incorporated genetically modified biological sample extruded in the first step sequentially for each extrusion in the first step;
Every time the second step is cut, a plurality of microlens arrays of microlens array discs collect light in corresponding pinholes of the Niipou disc, and light that has passed through the pinholes is collected by the objective lens in the second step. It is converged on the switching section of the cut the marker embedded recombinant biological sample in, by imaging the light reflected from the switching section of the marker embedded recombinant biological sample with a CCD camera, the marker embedded recombinant biological sample simultaneously detected reflected light from a plurality of points of the switching section of a third step for capturing a two-dimensional image of the switching section of the marker embedded recombinant biological sample sequentially,
A three- dimensional image is constructed on the basis of the two-dimensional image captured in the third step, the expression of the specific gene to be observed can be detected by the marker, and one living organism of the marker-incorporated genetically modified biological sample And a fourth step of observing the three-dimensional marker-incorporated genetically modified biological sample capable of observing the fine three-dimensional localization of the gene expressed in vivo in the whole ,
And integrally high speed rotation and the said Nipkow disk microlens array disk, 3D station fine in vivo expressed genes, characterized by scanning the focal point of the light to the switching entire cross-section of the marker embedded recombinant biological sample Current observation method.

In the observation method of the fine three-dimensional localization of the in-vivo expressed gene according to claim 4,
When using a material which emits light or color are excited by light as a marker of the marker embedded recombinant biological sample includes a light source for irradiating light to the markers of said marker embedded recombinant biological sample, said third step the marker incorporation recombinant biological sample of the switching section 2D image the marker incorporation recombinant biological sample cut cross section of the light for focusing the light source and in vivo expression, characterized by sharing the to image the at A method for observing fine three-dimensional localization of genes.

Fluorescent substance is pre-incorporated as a marker that can be detected at the time of expression with respect to a specific gene to be observed , and a whole living organism of a marker-incorporated genetically modified biological sample comprising a gene recombinant having a gene into which the marker is incorporated Extruding means that sequentially extrudes in a certain direction with a resolution of μm ,
Cutting means for sequentially cutting the marker-incorporated genetically modified biological sample pushed out by the push-out means sequentially for each push-out of the push-out means ;
A light source for emitting against switching section of the marker embedded recombinant biological sample is cut by said cutting means, specific wavelengths of light to excite the marker serving the fluorescent substance of said marker embedded recombinant biological sample ,
A Nipo disk having a plurality of pinholes, a microlens array disk having a plurality of microlens arrays for condensing light emitted from the light source on the corresponding pinholes of the Nipo disk, the Nipo disk and the micro a rotating device for the focus of the light emitted from the light source integrally rotating the lens array disc scannable the switching entire cross-section of the marker embedded recombinant biological sample, the light passed through the pinhole of the Nipkow disk said marker embedded recombinant an objective lens for converging to the switching section of the biological sample, wherein the objective lens is light reflected from the switching section of the marker embedded recombinant biological sample is condensed light is incident by the marker integrated gene switching section recombinant biological sample Confocal imaging means and a CCD camera for imaging by detecting the reflected light from the number of points at the same time continuously 2D image switching section of the marker embedded recombinant biological sample,
An image recording means for storing the 2-dimensional image of a switching section of the marker embedded recombinant biological samples imaged by the confocal imaging means,
Performed the construction of three-dimensional image by performing image processing on the two-dimensional image of the switching section of the marker embedded recombinant biological samples recorded on said image recording means, certain to be the observation target by light emission of the marker serving as the fluorescent substance The three-dimensional observation of the genetically-incorporated genetically modified biological sample can detect the expression of the gene and can observe the fine three-dimensional localization of the in vivo-expressed gene in one whole individual organism. Image processing means for performing processing;
Display means for displaying the processed image processed by the image processing means,
An apparatus for observing fine three-dimensional localization of a gene expressed in vivo, wherein the light source is shared as a light source of the confocal imaging means.

In the observation apparatus for fine three-dimensional localization of the in-vivo expressed gene according to claim 6,
The light source is a halogen light source and a xenon mercury light source using an optical filter,
The confocal imaging means, and the white light observation with the halogen light source of the switching section of the marker embedded recombinant biological sample, simultaneous observation of the fluorescence observation of V · B · G excitation by said xenon mercury light source and the optical filter Has a possible configuration,
It said image recording means, for the same switching section of the marker embedded recombinant biological sample, recorded at the beginning of the two-dimensional image of the switching section showing the observation in the white light, the switching section showing the observed in fluorescence following 2 Record dimensional images,
Wherein the image processing means, said by the two-dimensional image switching section of the recorded said marker embedded recombinant biological sample to the image processing to the image recording means, 3-dimensional the marker embedded recombinant during the white light observation An apparatus for observing a fine three-dimensional localization of a gene expressed in a living body, characterized by performing a process of observing a biological sample and a process of observing a three-dimensional marker-incorporated genetically modified biological sample during fluorescence observation.

An entire living organism of a genetically-modified genetically-modified biological sample comprising a genetically-recombinant that has previously incorporated a luminescent substance as a marker that can be detected at the time of expression for a specific gene to be observed , and that has a gene incorporating the marker Extrusion means for sequentially extruding the material in a fixed direction with a resolution of μm ,
Cutting means for sequentially cutting the marker-incorporated genetically modified biological sample pushed out by the push-out means sequentially for each push-out of the push-out means ;
The cut by the cutting means and the marker is condensed light to the switching section of the integrated gene recombination biological samples, are continuously captured two-dimensional images of the switching section by reflected light from the cut cross-section is focusing the light Confocal imaging means,
A three-dimensional image is constructed based on the two-dimensional image imaged by the confocal imaging means, the expression of the specific gene to be observed can be detected by luminescence of the luminescent substance as the marker, and the marker is incorporated Image processing means for observing the three-dimensional marker-incorporated genetically modified biological sample capable of observing the fine three-dimensional localization of the in vivo expressed gene in one whole individual of the genetically modified biological sample,
The confocal imaging means includes
Nipkow disc having a plurality of pinholes;
A microlens array disk having a plurality of microlens arrays at positions corresponding to the pinholes of the nipou disk;
A rotating device that integrally rotates the nipou disk and the microlens array disk;
The laser light is condensed on each of the corresponding pinholes by a plurality of the microlens arrays, and the Nipkow disk and the microlens array disk are integrally rotated at a high speed by the rotating device to focus the laser light on the observation apparatus microscopic three-dimensional localization of in vivo expressed genes is to scan the switching entire cross-section of the marker embedded recombinant biological sample.