JP3635381B2 - X-ray microscope - Google Patents

Description

【００１２】
ここで、χｉ（ｒ）はｉ番目の分子構成原子の２ｐ軌道の波動関数、Ｃｉは規格化定数を示す。
【００１３】
原子の２ｐ軌道は、光励起により１ｓ軌道の電子と強い相互作用をもつ。すなわち、量子力学によれば１ｓ軌道の電子は、光励起を行う場合２ｐ軌道へ高い遷移確率を持つ。そこで、炭素原子において１ｓ電子が各ｐ軌道へ遷移する場合と、１ｓ電子が電離する場合の吸収断面積を計算した結果、図５のようになった（Y.Iketaki et.al:Rev.Sci.Instrum.66(1995)982参照)。
図５によれば、１ｓ電子が２ｐ軌道へ遷移する場合には１５０Ｍｂ（１５０×１０~¹⁸ｃｍ²）程度の値を持ち、「水の窓」の励起波長を用いた１ｓ電子が電離する場合と比較すると１００倍以上の強い吸収をもつことが分かる。
【００１４】
一般に、生体分子内では原子軌道を存在しないが、式（１）の様に原子軌道が線形結合した分子軌道が存在する。従って、分子構成原子の１ｓ電子が該分子軌道に遷移する内殻励起過程による共鳴吸収が可能となる。式（１）によれば、π軌道やπ＊軌道の波動関数は分子の構成原子の２ｐ軌道線形結合であり、構成原子である炭素、窒素、酸素の１ｓ電子が電子により占有されていないπ＊の分子軌道へ遷移する場合の吸収断面積も大きな値を持つ。
【００１５】
ベンゼン分子を初めとする６員環分子において、１ｓ電子が各ｐ軌道へ遷移する場合の計算結果を図６に示す。図６によれば、「水の窓」の励起波長を用いた１ｓ電子が電離する場合と比較すると光子エネルギーが２８５ｅＶ前後で６倍程度大きな吸収断面積を持つ。この他、６員環分子以外にも２重結合をもつ分子であれば、「水の窓」のよりも光子エネルギーが若干小さい領域（「水の窓」の励起波長よりも若干長い）において同様に非常に強い内殻励起過程による非常に強い共鳴吸収がある。
【００１６】
以上の議論から、蛍光分析で用いるラベラーは豊富に二重結合が含まれるので内殻励起過程を起こす波長を持つ軟Ｘ線に対する吸収ラベラーとしても機能する。すなわち、生体分子内のアミノ基を初めとする特定の化学基をラベリングして、内殻励起過程を生じさせる共鳴する波長のＸ線を用いて、生体試料の透過像を撮影すれば特定の化学基のみの分布像が得られる。しかも、該共鳴による吸収は非常に強いので微量な量の化学基を検出できるので、この様なラベリング又は染料技術を導入することにより従来のＸ線顕微法と比較すると格段に高機能のＸ線顕微法が実現する。
【００１７】
本原理を実現する際には、２つの必要条件を満たす必要がある。
１）ラベラーとしては二重結合に含む分子を用いる。
２）ラベラーの持つ内殻励起過程を生じさせる共鳴ラインよりも波長幅（あるいは光子エネルギー幅）の狭いバンド幅のＸ線を用いること。
また、生体用顕微鏡に限定した場合、機能を上げる付帯条件として、
３）ウェットな試料を撮影できるために、Ｘ線が水分子に吸収されないことも挙げられる。
以下、１）、２）、３）について具体的に考察をしてみる。
【００１８】
１）について
一般にラベラーは、Ｈ，Ｃ，Ｎ，Ｏを主成分として、その他若干のＳ，Ｂｒ，Ｐｂ等を含む有機分子である。したがって、Ｃ，Ｎ，Ｏの１ｓ電子がラベラーのπ軌道又はπ＊軌道に光励起される場合の吸収ラインを利用するのが有利である。その場合の、共鳴吸収線の光子エネルギー（又は波長）は、各種有機分子の例をとると、過去の理論又は実験の論文より調べることができ、おおよそ推測がつく。以下、列挙する。
ｉ）Ｃ：１ｓ→π＊ ２８０〜３００ｅＶ（４４Ａ〜４１Ａ）
a)M.N.Piancastelli et.al, J.Chem.Phys.90(1989),1987〜
ii）Ｎ：１ｓ→π＊ ３９０〜４１０ｅＶ（３２Ａ〜３０Ａ）
b)J.A.Horsley et.al, J.Chem.Phys.83(1985),6099〜
iii）Ｏ：１ｓ→π＊ ５２０〜５５０ｅＶ（２４Ａ〜２２Ａ）
c)I.Ishii et.al, J.Chem.Phys.87(1987),830〜
d)N.Kosugi et.al, J.Chem.Phys.97(1992),8842
以上の領域で１ｓ→π＊を含む、様々な内殻励起過程による吸収ピーク群が観測されている。この他のＳ，Ｂｒ，Ｐｂ等の重元素の場合、１ｓ→π＊に限らず２ｐ，２ｓ，３ｓ，３ｐ，３ｄ……を始めとして、様々な内殻軌道よりの吸収ピークが２０００ｅＶ以下で観測されている。従って、以上にあげた波長帯域のＸ線を利用すれば、本Ｘ線顕微法を効果的に適応できる。
【００１９】
２）について
また、ａ），ｂ），ｃ）の文献より、１ｓ→π＊遷移に伴う最も強い吸収ピークに着目すると大体、１ｅＶ程度のライン幅を持っている。従って、少なくとも光子エネルギー幅１ｅＶ以下のバンド幅のＸ線を用いることが必要条件となる。それ以上のバンド幅で、透過像を撮影すると、吸収に関与しない波長が混入するために著しくコントラストが低下する。従って、必然的に上記のバンド幅以下の高い分解能をもつ波長分散性のある光学素子や単色化した光源を用いることを意味する。
【００２０】
３）について
ウェットな試料を撮影できるために、Ｘ線が水分子に吸収されないことが必要条件である。これは即ち、水分子中の酸素原子のＫ吸収端より長い波長（２３．６Ａより波長領域）の軟Ｘ線を用いることを意味する。
この様に、本原理を用いれば特定の化学基を分別できる従来法を上回る高機能の軟Ｘ線顕微法が実現できる。
【００２１】
本発明を詳細に述べる。
本発明のＸ線顕微法では使用するＸ線の光子エネルギーは２０００ｅＶ以下で、波長幅が１ｅＶ以下の単色化されたＸ線である。Ｘ線の光子エネルギーは２０００ｅＶ以下とした理由は先に述べたように、１ｓ電子を初めとして、２ｐ，２ｓ，３ｓ，３ｐ，３ｄ……の様々な内殻軌道よりの吸収ピークが２０００ｅＶ以下で観測されることによる。そして、特にＸ線の光子エネルギーが２８０ｅＶ〜５５０ｅＶであり、しかも波長可変であることが好ましい。
【００２２】
本発明のＸ−線顕微法で観測できる生体分子をより広く適用するため試料となる生体分子を２重結合を含む分子でラベリングを行う。ラベリングを行うことによってこれらの分子のＣ，Ｎ，Ｏの１ｓ電子を励起、π＊に吸収するのを利用するのである。Ｃ，Ｎ，Ｏの１ｓ→π＊吸収として２８０〜５５０ｅＶのＸ線の光子エネルギーを利用するのである。特に、２８０〜２９０ｅＶの光子エネルギーのＸ線を用いれば、炭素原子の１ｓ電子がπ＊軌道に遷移するときの吸収像を得ることになる。この際、Ｘ線の光子エネルギーは炭素の１ｓ電子の電離エネルギーより小さくなるので、純粋にπ＊軌道のつくる影のみ良いＳ／Ｎで撮影できる。具体的には染色する分子が５員環または６員環を含む化合物、特にベンゼン環を含むものが好ましい。更に本発明で使用される染色する分子としては先に述べたように蛍光分析化学の分野でラベラ−として使用されている化合物は何れでもよく、その二、三を列挙すると、次の通りである。
１）チオール基のラベラー（Ｒ−ＳＨ）
ジスルフィードの交換反応を起こす分子やリアールハライドなどがあり、ＳＨ基の特異的反応を利用するマレイミド基を持つ分子。
例えば、４−ｆｌｕｏｒｏ−７−ｓｕｌｆａｍｏｙｌｂｅｎｚｏｆｕｒａｚａｎ，２，２−Ｄｉｈｙｄｒｏｘｙ−６，６’−ｄｉｎａｐｈｔｈｙｌ ｄｉｓｕｌｆｉｄｅ，Ｎ−（９−Ａｃｒｉｄｉｎｙｌ）ｍａｌｅｉｍｉｄｅ
２）カルボキシ基のラベラー（Ｒ−ＣＯＯＨ）
プロメチル基を反応活性基とするもの。
例えば、４−Ｂｒｏｍｏｍｅｔｈｙｌ−７−ｍｅｔｈｏｘｙｃｏｕｍａｒｉｎ，３−Ｂｒｏｍｏｍｅｔｈｙｌ−６，７−ｄｉｍｅｔｈｏｘｙ−１−ｍｅｔｈｙｌ−１，２−ｄｉｔｙｄｒｏｑｕｉｎｏｘａｌｉｎｅ−２−ｏｎｅ
３）アミノ基のラベラー（Ｒ−ＮＨ，Ｒ−ＮＨ２）
イソチオシアネート基、フェロセニルイソチオシアネート基、ニトロアリールハライド、酸クロリド基を活性基とするもの。
例えば、Ｆｌｕｏｒｅｓｃｅｉｎｉｓｏｔｈｉｏｃｙａｎａｔｅ，ｉｓｏｍｅｒ−１，４−〔４−（Ｄｉｍｅｔｈｙｌａ）ｍｉｎｏ）ｐｈｅｎｙｌａｚｏ〕ｐｈｅｎｙｌｉｓｏｔｈｉｏｃｙａｎａｔｅ，４−Ｃｈｌｏｒｏ−７−ｎｉｔｒｏｂｅｎｚｏｆｕｒａｚａｎ，４−Ｆｌｕｒｏｒｏ−７−ｎｉｔｒｏｂｅｎｚｏｆｕｒａｚａｎ，３−Ｃｈｌｏｒｏｃａｒｂｏｎｙｌｅ−６，７−ｄｉｍｅｔｈｏｘｙ−１−ｍｅｔｈｙｌ−２（１Ｈ）−ｑｕｉｎｏｘａｌｉｎｏｎｅ，Ｎ−Ｓｕｃｃｉｎｉｍｉｄｙｌ−４−ｎｉｔｒｏｐｈｅｎｙｌａｃｅｔａｔｅ，Ｓｕｌｆｏｒｒｈｏｄａｍｉｎｅ １０１ ａｃｉｄ ｃｈｌｏｒｉｄｅ
４）アルデヒド基のラベラー
例えば、１，２−Ｄｉａｍｉｎｏ−４，５−ｄｉｍｅｔｈｏｘｙｂｅｎｚｅｎ，ｄｉｈｙｄｒｏｃｈｌｏｒｉｄｅ，２，２’−Ｄｉｔｈｉｏｂｉｓ（１−ａｍｉｎｏｎａｐｈｔｈａｌｅｎｅ）
５）水酸基のラベラ
酸クロリド等が利用される。
例えば、
３−Ｃｈｌｏｒｏｃａｒｂｏｎｙｌｅ−６，７−ｄｉｍｅｔｈｏｘｙ−１−ｍｅｔｈｙｌ−２（１Ｈ）−ｑｕｉｎｏｘａｌｉｎｏｎ，
６）蛋白質の疎水ポケットのラベラー
例えば、３，６−Ｂｉｓ（ｄｉｍｅｔｈｙｌａｍｉｎｏ）−１０−ｄｏｄｅｃｙｌａｃｒｉｄｉｎｉｕｍ ｂｒｏｍｉｄｅ，４−Ｂｅｎｚｙｌａｍｉｎｏ−７−ｎｉｔｒｏｂｅｎｚｏｆｕｒａｚａｎ，４−（４−Ｍｅｔｈｏｘｙｂｅｎｚｙｌａｍｉｎｏ）−７−ｎｉｔｒｏｂｅｎｚｏｆｕｒａｚａｎ
７）細胞内カルシウムイオンのラベラー
例えば、・Ｏ，Ｏ’−Ｂｉｓ（２−ａｍｉｎｏｐｈｅｎｙｌ）ｅｔｈｙｌｅｎｅｇｌｙｃｏｌ−Ｎ，Ｎ，Ｎ’，Ｎ’，−ｔｅｔｒａａｃｅｔｉｃ ａｃｉｄｅ，ｔｅｔｒａｐｏｔａｓｓｉｕｍ ｓａｌｔ，ｈｙｄｒａｔｅ・Ｎ，Ｎ，Ｎ’，Ｎ’，−Ｔｅｔｒａｋｉｓ（２−ｐｈｒｉｄｙｌｍｅｔｈｙｌ）ｅｔｈｙｌｅｎｅｄｉａｍｉ・１−〔２−Ａｍｉｄｏ−５−（２，７−ｄｉｃｈｌｏｒｏ−６−ｈｙｄｒｏｚｙ−３−ｏｘｙ−９−ｘａｎｔｈｅｎｙｌ〕−２−（２−ａｍｉｎｏ−５−ｍｅｔｈｙｌｐｈｅｎｏｘｙ）ｅｔｈａｎｅ−Ｎ，Ｎ，Ｎ’，Ｎ’，−ｔｅｔｒａａｃｅｔｉｃ ａｃｉｄｅ
８）細胞内のｐｐＨのラベラー
例えば、２’，７’−Ｂｉｓ（ｃａｒｂｏｘｙｅｔｈｙｌ）−４ｏｒ５−ｃａｒｂｏｘｙｆｌｕｏｒｅｓｃｅｉｎ，３’−Ｏ−Ａｃｅｔｙｌ−２’，７’−ｂｉｓ（（ｃａｒｂｏｘｙｅｔｈｙｌ）−４ｏｒ５−ｃａｒｂｏｘｙｆｌｕｏｒｅｓｃｅｉｎ，ｄｉａｃｅｔｏｘｙｍｅｔｈｙｌｅｓｔｅｒ
９）細胞膜のラベラー（リン脂質、生体膜）
例えば、１，３−Ｂｉｓ（１−ｐｈｒｅｎｙｌ）ｐｒｏｐａｎｅ，１−（４−Ｔｒｉｍｅｔｈｙｌａｍｍｏｎｉｕｍｐｈｅｎｙｌ）−６−ｐｈｅｎｙｌ−１，３，５−ｈｅｘａｔｒｉｅｎｅ ｉｏｄｉｄｅ
１０）ＤＮＡをはじめとする核酸のラベラー
例えば、４’，６−ｄｉａｍｉｎｏ−２−ｐｈｅｎｙｌｉｎｄｏｌｅ（ＤＡＰＩ）
また、本発明の顕微鏡法においては、試料容器の窓材として試料との相互作用を生じないように、π＊軌道を持たない有機分子又は炭素化物を利用する。
更に本願発明を実施例を用いて具体的に説明する。
【００２３】
【実施例及び比較例】
実施例１
本実施例では、Ｏ−（４−Ｎｉｔｒｏｂｅｎｚｙｌ）ｈｙｄｒｏｘｙｌａｍｉｎｅ塩酸塩を用いた染色法を示す。この化合物が次のような構造式を有し、ケトン基またはアルデヒド基と結合し、ラベル化を行う。
【００２４】
【化２】

【００２５】
この化合物はベンゼン環を持ち、光子エネルギー２８５ｅＶ前後で炭素１ｓ→π＊遷移に伴う強いＸ線の吸収ピークをもつ。本実施例では、代表的なタバコモザイクウイルスを例にとり、このウイルスの蛋白質分子中のケトン基またはアルデヒド基をラベリングする場合を考える。タバコモザイクウイルスの形状は、１６×３００ｎｍ棒状であり、ウイルスの蛋白質は１５８個のアミノ酸分子で構成される２１３０個の小集団の蛋白質からなる。アミノ酸分子１個に対して、ケトン基またはアルデヒド基を有するペプチド結合が１個存在する。従って、タバコモザイクウイルスは１５８×２１３０のケトン基またはカルボニル基をもつ。そこで、Ｏ−（４−Ｎｉｔｒｏｂｅｎｚｙｌ）ｈｙｄｒｏｘｙｌａｍｉｎｅでタバコモザイクウイルスのケトン基またはアルデヒド基をラベリングしたときの１ｓ→π＊遷移に伴う最も強い吸収を見積もる。
本発明者らが先に発表した論文（Y.Iketaki et.al:Rev.Sci.Instrum.66(1995)982）によれば、ベンゼン分子１個あたりの吸収断面積σは２５Ｍｂ（２５×１０~¹⁸ｃｍ²程度であり、内殻励起による線吸収係数μは、σと単位体積あたりのケトン基またはアルデヒド基の密度の積で与えられる。染色されたタバコモザイクウイルスの共鳴波長における線吸収係数μは１３／μｍとなる。この値は通常の蛋白質の「水の窓」領域の波長の線吸収係数の倍以上の値をもつ。
顕微鏡のコントラストＣは、色々なコントラストの領域をもつ試料の最大透過率Ｉｍａｘと対象とする領域の透過率Ｉが分かると、式（２）で与えられる。
【００２６】
【化３】

【００２７】
Ｘ＝１６ｎｍのサイズのタバコモザイクウイルスの透過率Ｉは式（３）で与えられるので、
【００２８】
【化４】

【００２９】
タバコモザイクウイルスのコントラストは０．１程度となる。この値は、「水の窓」領域の波長を用いる従来技術と比較すると、２倍以上となる。この事実は、本技術により非常に高い感度で蛋白質を検出できることを示している。
【００３０】
実施例２
実施例１で使用したＯ−（４−Ｎｉｔｒｏｂｅｎｚｙｌ）ｈｙｄｒｏｘｙｌａｍｉｎｅにかえて、次に列挙した各種のラベラー分子を適宜選択使用することによって、生体細胞の特定の化学基、分子、構造を選別できる。
基本的には、先に列挙した各種のラベラー分子の様な複雑な分子を利用しなくても、官能基と少なくとも１つの２重結合をもつ分子であれば、本発明のＸ線顕微法のラベラーとして機能することができる。
【００３１】
【表１】

【００３２】
【表２】

【００３３】
【表３】

【００３４】
【表４】

【００３５】
【表５】

【００３６】
【表６】

【００３７】
【表７】

【００３８】
【表８】

【００３９】
【表９】

【００４０】
【表１０】

【００４１】
【表１１】

【００４２】
【表１２】

【００４３】
【表１３】

【００４４】
【表１４】

【００４５】
【表１５】

【００４６】
【表１６】

【００４７】
【表１７】

【００４８】
【表１８】

【００４９】
【表１９】

【００５０】
【表２０】

【００５１】
【表２１】

【００５２】
【表２２】

【００５３】
【表２３】

【００５４】
【表２４】

【００５５】
【表２５】

【００５６】
【表２６】

【００５７】
【表２７】

【００５８】
【表２８】

【００５９】
【表２９】

【００６０】
【表３０】

【００６１】
【表３１】

【００６２】
【表３２】

【００６３】
【表３３】

【００６４】
【表３４】

【００６５】
【表３５】

【００６６】
【表３６】

【００６７】
【表３７】

【００６８】
【表３８】

【００６９】
【表３９】

【００７０】
【表４０】

【００７１】
実施例３
本発明は化学状態の選別の他に、Ｘ線顕微鏡像のコントラストの制御も可能とする。図６によれば、ベンゼンの１ｓ→π＊遷移に伴う吸収断面積の光子エネルギーの依存性であるが、吸収ピークは約１ｅＶ程のバンド幅を持つ。もし、共鳴ラインのバンド幅より一桁程度狭いバンド幅で単色化されてしかも波長可変のＸ線光源を用いて透過画像を撮影すると、容易にコントラストが調整できる。本例において、実施例１と同じ様にタバコモザイクウイルスの蛋白質をＯ−（４−Ｎｉｔｒｏｂｅｎｚｙｌ）ｈｙｄｒｏｘｙｌａｍｉｎｅでラベリングする場合を想定する。
例えば、文部省高エネルギー物理学研究所フォトンファクトリーのビームーラインＢＦ−ＢＬ２は、光子エネルギー２８５ｅＶ前後でバンド幅５０ｍｅＶ程度の分光されたＸ線を取り出すことができる。この様なＸ線光源を用いて、１ｓ→π＊遷移の共鳴ラインピークの先頭値の光子エネルギーでラベリングしたタバコモザイクウイルスを撮影を行うと、実施例１で示した様に０．１程度のコントラストが得られる。しかし、共鳴ラインピークの先頭値よりも２５０ｍｅＶ程度低い光子エネルギーで撮影すると、吸収断面積が半減するのでコントラストが半減する。この様に、撮影するＸ線の光子エネルギー（波長）を選択することで、コントラストが変化する。タバコモザイクウイルスよりも大きいサイズでしかも蛋白質を多量に含む細胞では必要以上にコントラストがつきすぎ、微細構造の観察が不可能となる。この様な場合、本発明を用いれば、Ｘ線の光子エネルギーの選択によりコントラストが任意に調整できる。これに対して、「水の窓」と呼ばれる波長４２．７〜２３．６Ａの波長領で撮影する内殻電離型の吸収過程を用いた方法では図（８）に示すように線吸収係数の変化が穏やかな為、本発明の様な技術を用いることができず、コントラストは繁雑で高度の技術を要する試料の厚み調整に頼っていた。
【００７２】
実施例４
本発明では、微小な構造の吸収像を得るための吸収効果を増幅する技術をも可能にする。観察しようとする化学基の密度をＮ、ラベラーのＸ線吸収断面積をσχとすると線吸収係数μは（４）で与えられる。
【００７３】
【化５】

【００７４】
従来法では、観察しようとする化学基の密度Ｎが少なければ、式（４）より吸収像を得るのが大変困難になる。しかし、本発明ではラベラーの分子設計で解決できる。
基本的に、内殻電子がπ＊軌道へ遷移する場合の吸収断面積はラベラー分子の持っている二重結合の数に比例する。したがって、ラベラー分子に二重結合をもつ側鎖を必要な数だけ付加すれば、ラベラー分子のＸ線の吸収断面積を比例して高めることができる。付加する側鎖の数をｍ、Ｘ線の吸収断面積をσ_sとすると
、
【００７５】
【化６】

【００７６】
となり、従って線吸収係数は式（６）の様になる。
【００７７】
【化７】

【００７８】
例えばＯ−（４−Ｎｉｔｒｏｂｅｎｚｙｌ）ｈｙｄｒｏｘｙｌａｍｉｎｅは二重結合を有するベンゼン環を一つしか含まないが、さらにｍ個のベンゼン環を側鎖を付加すると、タバコモザイクウイルスに関しては線吸収係数は式（７）の様になる。
【００７９】
【化８】

【００８０】
従って、ベンゼン環を５つ付加すればコントラスト０．５まで増加し、タバコモザイクウイルスの像を強調できる。
【００８１】
実施例５
本技術は、生体試料の試料容器の設計も容易にする。一般に、生体用Ｘ線顕微鏡で用いられる波長領域の光は大気中で減衰が激しく、一般に全システムが真空容器内に収容される。従って、濡れたままの生体試料は超薄膜の窓でできた試料容器内に封入され、真空中で試料は乾燥しない工夫がなされている。一般に、用いられる薄膜の材料は、「水の窓」領域及びその近傍でＸ線の透過率が高く、同時に薄膜にしやすいものを選択する。その際、２重結合を持たない有機材料より選択すると、炭素の１ｓ→π＊遷移を利用して撮影すると透過率が高く、強度のある有機薄膜を薄膜材料として利用できる。例えば、ポリエチレンは２重結合を持たない代表的な飽和した高分子であり、したがってπ＊の分子軌道を有しない。従って、４４〜４３。７Ａ（２９０〜２８０ｅＶ）の波長領域で非常に高い透過率を持つことが期待できる。
図９は、実際にＪ．Ｒ．Ｒａｂｅ等によって測定されたポリエチレンフィルムのＸ線透過特性である（Ｊ．Ｒ．Ｒａｂｅ ｅｔ．ａｌ．：Ｔｈｉｎ Ｓｏｌｉｄ Ｆｉｌｍｓ １５９（１９８８）２７５）。図８によればポリエチレンフィルムは、２８７ｅＶ以下で非常に高い透過率を持つことが分かる。内殻励起過程を用いた顕微鏡法において、Ｘ線の波長が４３．５Ａ（２８５ｅＶ）前後で強い吸収線をもつラベラーのベンゼン環の吸収像を撮影する場合には、該フィルムは最適な試料容器の窓材である。
【００８２】
【発明の効果】
以上述べたように、本発明においては光子エネルギー２０００ｅＶ以下で、波長幅が１ｅＶ以下の単色化されたＸ線を用い、特に試料を２重結合を含む分子で染色することることによって、従来の内殻電離過程によるＸ線顕微法の場合に比してコントラストがつき易く、Ｘ線の光子エネルギーの選択によりコントラストが任意に調整で、高機能のＸ線顕微法を提供することができる。
【図面の簡単な説明】
【図１】 従来のウォルタ−型Ｘ線顕微鏡の光学系を示す説明図
【図２】 フレネルゾーンプレートの正面図
【図３】 シュワルツシルド光学系を示す説明図
【図４】 内殻電離過程と内殻励起過程との説明図
【図５】 炭素原子１ｓ電子が各ｐ軌道に励起した場合の光子エネルギーと断面積との関係図
【図６】 ベンゼン１ｓ電子がπ＊の分子軌道へ遷移する場合の光子エネルギーと断面積との関係図
【図７】 水及び蛋白の波長に対する吸収係数との関係図
【図８】 ポリエチレンの光子エネルギーに対する強度との関係図[0001]
[Industrial application fields]
The present invention provides a new high-performance X-ray microscope About.
[0002]
[Prior art]
In recent years, technological development of an X-ray light source and an X-ray optical system has progressed, and an X-ray microscope has been proposed as an applied technology thereof. There are various types of such microscopes. For example, as shown in FIGS. 1 to 3, an oblique oblique optical system represented by the Walter type, a full-nel zone plate using a diffraction effect, a direct oblique oblique type Schwartz in which an X-ray multilayer mirror is deposited on two spherical mirrors. Microscope systems using various imaging elements such as a schild optical system have been developed. In addition, a direct contact photographing method in which a sample image is directly recorded on an X-ray film or a photoresist without using an imaging element has also been used for a long time.
In particular, X-rays are useful because they are less damaging to biological samples like electron beams, and are attracting attention as new biological microscopes that can be observed with high resolution and no staining while the biological samples remain wet. In particular, in the wavelength range of 42.7 to 23.6 mm, called the “water window”, X-rays are absorbed most by carbon or nitrogen, and have a high transmittance for water molecules consisting of oxygen and hydrogen. The transmission image of a protein having carbon as the main constituent atom can be observed with good contrast even in water. The wavelength 42.7． corresponds to the K absorption edge of carbon, and the wavelength 23.6Å corresponds to the oxygen K absorption edge.
[0003]
The imaging principle of the X-ray absorption image is based on the inner-shell ionization process of carbon and nitrogen 1s electrons. Until recently, this imaging principle attracted attention, but an X-ray microscopic method using a core excitation process to a molecular orbital of a biomolecule different from the core ionization process has been proposed. The wavelength of the X-ray used at this time is only slightly longer than the K absorption edge of carbon. The absorption process by the inner shell excitation process is resonance absorption, and this X-ray microscopic method is more sensitive than that using the inner shell ionization process.
[0004]
FIG. 4 shows the principle diagram of the X-ray microscopic method using the inner-shell ionization process and the inner-shell excitation process to the molecular orbitals of biomolecules. In other words, FIG. 4-1 shows an X-ray microscopic method using an inner shell ionization process, in which intracellular carbon or carbon by X-rays in a wavelength range of 42.7 to 23.6 mm called a “water window”. The 1s electrons of nitrogen are ionized, and an X-ray absorption image at this time can be obtained, and an X-ray absorption image is obtained using this. On the other hand, FIG. 4-2 is a principle diagram of the X-ray microscopic method using the inner shell excitation process, which is a method of exciting carbon 1s electrons to molecular orbitals not occupied by biomolecule electrons by X-rays. This method is a kind of resonance absorption process, and strong X-ray absorption can be expected (reference document Y. Iketakiet.al: Rev. Sci. Instrum. 66 (1995) 982). Moreover, since the molecular orbital has an energy level unique to the molecule, it is possible to see an absorption image of only a specific molecule by selecting the X-ray resonance wavelength. It can be said that the method is superior to the method.
[0005]
Molecular orbitals that play an important role in the inner shell excitation process are antibonding molecular orbitals called π * orbitals, and are usually empty orbitals. This π * orbit is often observed when the 2p orbit of carbon constituting the biomolecule is double-bonded with the orbit of another atom. For example, a benzene ring or a nitrogen base has a strong absorption line at an X-ray wavelength of around 43.5 mm (285 eV). The energy position of these absorption lines is about 1 mm longer than the limit on the long wavelength side of the “water window” region (on the low energy side about 5 eV in terms of photon energy).
However, it has been found that such an excellent X-ray microscopic method has a limit of adaptation. That is, the biomolecules that can be observed are limited to some molecules having a benzene ring or a nitrogen base.
[0006]
[Means for Solving the Problems]
Therefore, the present inventor has further expanded the application limit of the conventional X-ray microscopic method, and as a result of various studies to observe an absorption image of a selected chemical group in a living cell, as a result of using a specific X-ray photon energy. In addition, the inventors have found that the intended purpose can be achieved by labeling or staining a nuclear chemical group with a molecule having a π * orbital that causes specific strong soft X-ray absorption, and the present invention has been completed. An object of the present invention is to provide a novel high-performance X-ray microscopic method.
[0007]
[Means for Solving the Problems]
The gist of the present invention is that an X-ray light source emitting monochromatic X-rays having a photon energy of 280 eV to 550 eV and a wavelength width of 1 eV or less, and a double in which 1s electrons are excited by X-rays from the X-ray light source. An X-ray microscope having a sample container containing a sample stained with molecules containing bonds and having a window through which X-rays from the X-ray light source pass Microscopy The window material constituting the window of the sample container uses an organic molecule or carbonized material having no π * orbit, and the 1s electrons of the stained sample by X-rays transmitted through the window An X-ray microscope characterized by observing absorption or fluorescence of the X-ray generated by excitation Law It is.
[0008]
Hereinafter, the principle of the present invention will be described.
In fluorescence analytical chemistry or biology, a specific chemical group of a biological cell molecule is modified (labeled) with a molecule having a fluorophore (hereinafter, such a molecule is called a labeler), and fluorescence from the labeled molecule is emitted. Techniques for analyzing the structure and composition of living cells through observation have been developed. For example, N-succinimidyl-4-nitrophenylacetate and 5- (4-dimethylaminophenyl) -2,4-pentadienal bind to the amino group (N-terminal) of the protein and emit strong ultraviolet fluorescence. O- (4-Nitrobenzyl-N, N′-diisopropylisourea binds to the carbonyl group (C-terminal) of the protein and emits strong ultraviolet fluorescence. Furthermore, N, N, N ′, N′-Tetrakis (2- Phenylethyl) ethylenediamin labels the signaling substance Ca 2+ which plays an important role in the cell, and 4-Fluoro-7-sulfobenzofurazan, ammonium salt binds to the SH group of cysteine, which is a kind of amino acid, for example, at 385 nm. Emits strong 515 nm fluorescence upon excitation.
[0009]
All of these molecules are characterized by strong fluorescence, which is due to the structure of the molecular orbitals of these molecules. That is, these molecules surely have a chemical group having a double bond. If there is a double bond, π electrons are present. These electrons usually exist in molecular orbitals called π orbitals, and are easily excited by higher-order unoccupied π orbitals or antibonding π * orbitals by ultraviolet or visible light. Then, stronger fluorescence is emitted from these excited states when they are deexcited to the ground state.
[0010]
By the way, when the π orbital or π * orbital is considered from a quantum chemical viewpoint, these molecular orbitals are generally formed by linear combinations of 2p orbitals of atoms constituting the molecule. That is,
[0011]
[Chemical 1]

[0012]
Here, χ i (r) is a wave function of the 2p orbit of the i-th molecular constituent atom, and Ci is a normalization constant.
[0013]
The 2p orbit of the atom has a strong interaction with the 1s orbital electron by photoexcitation. That is, according to quantum mechanics, electrons in the 1s orbital have a high transition probability to the 2p orbital when photoexcitation is performed. Therefore, as a result of calculating the absorption cross section when the 1s electron transitions to each p orbit in the carbon atom and when the 1s electron ionizes, the result is as shown in FIG. 5 (Y.Iketaki et.al:Rev.Sci .Instrum.66 (1995) 982).
According to FIG. 5, when the 1s electron transits to the 2p orbit, 150 Mb (150 × 10˜ ¹⁸ cm ² ) And has a strong absorption of 100 times or more compared to the case where 1s electrons using the excitation wavelength of the “water window” are ionized.
[0014]
In general, there is no atomic orbital in a biomolecule, but there is a molecular orbital in which atomic orbitals are linearly coupled as in equation (1). Therefore, resonance absorption by the inner shell excitation process in which the 1s electrons of the molecular constituent atoms transition to the molecular orbitals becomes possible. According to Equation (1), the wave function of the π orbit or π * orbit is a 2p orbital linear combination of the constituent atoms of the molecule, and the 1s electrons of carbon, nitrogen, and oxygen as constituent atoms are not occupied by electrons. The absorption cross section when transitioning to the * molecular orbital also has a large value.
[0015]
FIG. 6 shows the calculation result when 1s electrons transition to each p orbital in a 6-membered ring molecule such as a benzene molecule. According to FIG. 6, the photon energy has an absorption cross section about 6 times larger at around 285 eV compared to the case where 1s electrons using the excitation wavelength of the “water window” are ionized. In addition, in the case of a molecule having a double bond in addition to the six-membered ring molecule, the same applies in a region where the photon energy is slightly smaller than that of the “water window” (slightly longer than the excitation wavelength of the “water window”). There is a very strong resonance absorption due to the very strong inner-shell excitation process.
[0016]
From the above discussion, since the labeler used in the fluorescence analysis contains abundant double bonds, it also functions as an absorption labeler for soft X-rays having a wavelength causing the inner shell excitation process. That is, if a specific chemical group such as an amino group in a biomolecule is labeled and a transmission image of a biological sample is photographed using X-rays having a resonating wavelength that causes an inner shell excitation process, a specific chemistry is obtained. A distribution image of only the group is obtained. In addition, since the absorption due to the resonance is very strong, a trace amount of chemical groups can be detected. By introducing such labeling or dye technology, X-rays with much higher function than conventional X-ray microscopic methods are obtained. A microscopic method is realized.
[0017]
In order to realize this principle, it is necessary to satisfy two requirements.
1) As a labeler, a molecule contained in a double bond is used.
2) Use X-rays with a narrower wavelength width (or photon energy width) than the resonance line that causes the inner shell excitation process of the labeler.
In addition, when it is limited to a biological microscope, as an incidental condition to increase the function,
3) Since a wet sample can be imaged, X-rays are not absorbed by water molecules.
Hereinafter, 1), 2), and 3) will be specifically considered.
[0018]
About 1)
In general, a labeler is an organic molecule containing H, C, N, and O as main components and some other S, Br, Pb, and the like. Therefore, it is advantageous to use an absorption line when C, N, O 1s electrons are photoexcited in the π orbit of the labeler. In this case, the photon energy (or wavelength) of the resonance absorption line can be examined from past theoretical or experimental papers by taking examples of various organic molecules, and can be roughly estimated. Listed below.
i) C: 1s → π * 280-300 eV (44A-41A)
a) MNPiancastelli et.al, J. Chem. Phys. 90 (1989), 1987-
ii) N: 1s → π * 390-410 eV (32A-30A)
b) JAHorsley et.al, J. Chem. Phys. 83 (1985), 6099-
iii) O: 1s → π * 520-550 eV (24A-22A)
c) I. Ishii et.al, J. Chem. Phys. 87 (1987), 830-
d) N. Kosugi et.al, J. Chem. Phys. 97 (1992), 8842
In the above region, absorption peak groups due to various core excitation processes including 1s → π * are observed. In the case of other heavy elements such as S, Br, and Pb, absorption peaks from various inner orbitals are 2000 eV or less, including not only 1s → π * but 2p, 2s, 3s, 3p, 3d. Observed. Therefore, the present X-ray microscopic method can be effectively applied by using X-rays in the above-mentioned wavelength bands.
[0019]
About 2)
Further, from the documents a), b), and c), when attention is paid to the strongest absorption peak associated with the 1s → π * transition, the line width is about 1 eV. Therefore, it is necessary to use X-rays having a bandwidth of at least a photon energy width of 1 eV or less. When a transmission image is photographed with a bandwidth larger than that, the contrast is remarkably lowered due to the inclusion of wavelengths that are not involved in absorption. Therefore, it means that a wavelength-dispersible optical element having a high resolution equal to or less than the above-described bandwidth and a monochromatic light source are used.
[0020]
About 3)
In order to be able to photograph a wet sample, it is a necessary condition that X-rays are not absorbed by water molecules. This means that soft X-rays having a wavelength longer than the K absorption edge of oxygen atoms in water molecules (wavelength region from 23.6 A) are used.
In this way, by using this principle, it is possible to realize a high-functional soft X-ray microscopic method that exceeds the conventional method capable of separating a specific chemical group.
[0021]
The present invention will be described in detail.
In the X-ray microscopic method of the present invention, the photon energy of X-rays to be used is monochromatic X-rays having a wavelength width of 2000 eV or less and a wavelength width of 1 eV or less. The reason why the photon energy of X-rays is 2000 eV or less is that, as described above, absorption peaks from various inner orbitals of 2p, 2s, 3s, 3p, 3d, etc. including 1s electrons are 2000 eV or less. Depending on what is observed. In particular, the photon energy of the X-ray is preferably 280 eV to 550 eV, and the wavelength is preferably variable.
[0022]
In order to more widely apply biomolecules that can be observed by the X-ray microscopic method of the present invention, the biomolecules that are samples are labeled with molecules containing double bonds. By performing labeling, the Cs, N, and O 1s electrons of these molecules are excited and utilized to absorb π *. X-ray photon energy of 280 to 550 eV is used as 1s → π * absorption of C, N, and O. In particular, if X-rays having a photon energy of 280 to 290 eV are used, an absorption image when a 1s electron of a carbon atom transitions to a π * orbit is obtained. At this time, since the photon energy of the X-ray is smaller than the ionization energy of the 1s electron of carbon, only the shadow created by the π * orbit can be photographed with a good S / N. Specifically, compounds in which the molecule to be dyed contains a 5-membered ring or a 6-membered ring, particularly those containing a benzene ring are preferred. Furthermore, as described above, any compound used as a labeler in the field of fluorescence analysis chemistry may be used as a staining molecule used in the present invention, and a few of them are listed as follows. .
1) Labeler of thiol group (R-SH)
Molecules that have a maleimide group that uses a specific reaction of the SH group, such as a molecule that causes a disulfide exchange reaction or a rearal halide.
For example, 4-fluoro-7-sulfamoylbenzufurazan, 2,2-Dihydroxy-6,6'-dinaphthyl disulphide, N- (9-Acridinyl) maleimide
2) Labeler of carboxy group (R-COOH)
A promethyl group is a reactive group.
For example, 4-Bromomethyl-7-methoxycoumarin, 3-Bromomethyl-6,7-dimethyl-1-methyl-1,2-dioxyquinoxaline-2-one
3) Labeler of amino group (R-NH, R-NH2)
An active group consisting of an isothiocyanate group, a ferrocenyl isothiocyanate group, a nitroaryl halide, and an acid chloride group.
For example, Fluoresceinisothiocyanate, isomer-1,4- [4- (Dimethyla) mino) phenylyazothiocyanate, 4-Chloro-7-nitrobenzofuranzan, 4-Fluoro-7-nitrobroziofurazolan-6, methyl-2 (1H) -quinoxalinone, N-Succinimidyl-4-nitrophenylate, Sulforrhodamine 101 acid chloride
4) Labeler of aldehyde group
For example, 1,2-Diamino-4,5-dimethylbenzen, dihydrochloride, 2,2′-Dithiobis (1-aminophathalene)
5) Hydroxyl label
Acid chloride or the like is used.
For example,
3-Chlorocarbon-6,7-dimethyl-1-methyl-2 (1H) -quinoxalinon,
6) Hydrophobic pocket labeler for proteins
For example, 3,6-Bis (dimethylamino) -10-dedecylacridinium bromide, 4-Benzylamino-7-nitrobenzofurazan, 4- (4-Methoxybenzylamino) -7-nitrobenzofurazazan
7) Intracellular calcium ion labeler
For example, • O, O′-Bis (2-aminophenyl) ethyleneglycol-N, N, N ′, N ′, -tetraacetic acid, tetrapotassium salt, hydrate, N, N, N ′, N ′, —Tetrakis (2- phenylmethyl) ethyleneami · 1- [2-amido-5- (2,7-dichloro-6-hydroxy-3-oxy-9-xanthenyl] -2- (2-amino-5-methylphenoxy) ethane-N, N, N ′, N ′, -tetraacetic acid
8) Intracellular ppH labeler
For example, 2 ′, 7′-Bis (carboxyethyl) -4or5-carboxyfluorescein, 3′-O-Acetyl-2 ′, 7′-bis ((carboxyethyl) -4or5-carboxycecein, diacetoxymethylester
9) Cell membrane labelers (phospholipids, biological membranes)
For example, 1,3-Bis (1-phenylyl) propane, 1- (4-Trimethylammoniumphenyl) -6-phenyl-1,3,5-hexatriene iodide
10) Labelers for nucleic acids including DNA
For example, 4 ′, 6-diamino-2-phenyllinole (DAPI)
In the microscope method of the present invention, an organic molecule or carbonized material having no π * orbital is used as a window material for the sample container so as not to cause an interaction with the sample.
Further, the present invention will be specifically described using examples.
[0023]
[Examples and Comparative Examples]
Example 1
In this example, a staining method using O- (4-Nitrobenzoyl) hydroxylamine hydrochloride is shown. This compound has the following structural formula and binds to a ketone group or an aldehyde group for labeling.
[0024]
[Chemical 2]

[0025]
This compound has a benzene ring, and has a strong X-ray absorption peak associated with a carbon 1s → π * transition around a photon energy of 285 eV. In this example, a typical tobacco mosaic virus is taken as an example, and a case where a ketone group or an aldehyde group in a protein molecule of this virus is labeled is considered. The shape of the tobacco mosaic virus is a 16 × 300 nm rod, and the viral protein is composed of 2130 small groups of proteins composed of 158 amino acid molecules. There is one peptide bond having a ketone group or an aldehyde group for one amino acid molecule. Thus, tobacco mosaic virus has 158 × 2130 ketone or carbonyl groups. Therefore, the strongest absorption associated with the 1s → π * transition when the ketone group or aldehyde group of tobacco mosaic virus is labeled with O- (4-Nitrobenzoyl) hydroxylamine is estimated.
According to a paper previously published by the present inventors (Y. Iketaki et.al: Rev. Sci. Instrum. 66 (1995) 982), the absorption cross section σ per benzene molecule is 25 Mb (25 × 10 ~ ¹⁸ cm ² The linear absorption coefficient μ due to inner shell excitation is given by the product of σ and the density of ketone groups or aldehyde groups per unit volume. The linear absorption coefficient μ at the resonance wavelength of the stained tobacco mosaic virus is 13 / μm. This value is more than double the linear absorption coefficient of the wavelength of the “water window” region of normal protein.
The contrast C of the microscope is given by Equation (2) when the maximum transmittance Imax of a sample having various contrast regions and the transmittance I of a target region are known.
[0026]
[Chemical 3]

[0027]
Since the transmittance I of tobacco mosaic virus having a size of X = 16 nm is given by equation (3),
[0028]
[Formula 4]

[0029]
The contrast of tobacco mosaic virus is about 0.1. This value is more than doubled compared to the prior art using wavelengths in the “water window” region. This fact indicates that this technique can detect proteins with very high sensitivity.
[0030]
Example 2
A specific chemical group, molecule, or structure of a living cell can be selected by appropriately selecting and using the various kinds of labeler molecules listed below instead of O- (4-Nitrobenzoyl) hydroxylamine used in Example 1.
Basically, it is possible to use the X-ray microscopic method of the present invention as long as it is a molecule having at least one double bond with a functional group without using complex molecules such as the various labeler molecules listed above. Can function as a labeler.
[0031]
[Table 1]

[0032]
[Table 2]

[0033]
[Table 3]

[0034]
[Table 4]

[0035]
[Table 5]

[0036]
[Table 6]

[0037]
[Table 7]

[0038]
[Table 8]

[0039]
[Table 9]

[0040]
[Table 10]

[0041]
[Table 11]

[0042]
[Table 12]

[0043]
[Table 13]

[0044]
[Table 14]

[0045]
[Table 15]

[0046]
[Table 16]

[0047]
[Table 17]

[0048]
[Table 18]

[0049]
[Table 19]

[0050]
[Table 20]

[0051]
[Table 21]

[0052]
[Table 22]

[0053]
[Table 23]

[0054]
[Table 24]

[0055]
[Table 25]

[0056]
[Table 26]

[0057]
[Table 27]

[0058]
[Table 28]

[0059]
[Table 29]

[0060]
[Table 30]

[0061]
[Table 31]

[0062]
[Table 32]

[0063]
[Table 33]

[0064]
[Table 34]

[0065]
[Table 35]

[0066]
[Table 36]

[0067]
[Table 37]

[0068]
[Table 38]

[0069]
[Table 39]

[0070]
[Table 40]

[0071]
Example 3
The present invention makes it possible to control the contrast of the X-ray microscope image in addition to the selection of the chemical state. According to FIG. 6, although it is dependence of the photon energy of the absorption cross section accompanying the 1 s → π * transition of benzene, the absorption peak has a bandwidth of about 1 eV. If a transmission image is captured using an X-ray light source that is monochromatic and has a wavelength that is narrower by an order of magnitude than the resonance line bandwidth, the contrast can be easily adjusted. In this example, it is assumed that the tobacco mosaic virus protein is labeled with O- (4-Nitrobenzoyl) hydroxylamine as in Example 1.
For example, the Ministry of Education High Energy Physics Laboratory Photon Factory's beam line BF-BL2 can take out a spectral X-ray having a photon energy of about 285 eV and a bandwidth of about 50 meV. When such a X-ray light source is used to photograph the tobacco mosaic virus labeled with the photon energy of the first value of the resonance line peak of the 1s → π * transition, as shown in Example 1, it is about 0.1. Contrast is obtained. However, if the photon energy is about 250 meV lower than the starting value of the resonance line peak, the absorption cross section is halved, so the contrast is halved. In this way, the contrast changes by selecting the photon energy (wavelength) of the X-ray to be imaged. Cells that are larger in size than tobacco mosaic virus and contain a large amount of protein have too much contrast, making it impossible to observe the fine structure. In such a case, if the present invention is used, the contrast can be arbitrarily adjusted by selecting the photon energy of X-rays. On the other hand, in the method using the inner shell ionization type absorption process, which is called “the window of water” and is photographed in the wavelength range of 42.7 to 23.6 A, the linear absorption coefficient is changed as shown in FIG. Since the change is gentle, the technique as in the present invention cannot be used, and the contrast is complicated, and it relies on the adjustment of the thickness of the sample which requires a high technique.
[0072]
Example 4
The present invention also enables a technique for amplifying the absorption effect for obtaining an absorption image having a minute structure. When the density of the chemical group to be observed is N and the X-ray absorption cross section of the labeler is σχ, the linear absorption coefficient μ is given by (4).
[0073]
[Chemical formula 5]

[0074]
In the conventional method, if the density N of the chemical group to be observed is small, it is very difficult to obtain an absorption image from the equation (4). However, in the present invention, it can be solved by labeler molecular design.
Basically, the absorption cross section when the inner-shell electron transitions to the π * orbit is proportional to the number of double bonds of the labeler molecule. Therefore, if a necessary number of side chains having double bonds are added to the labeler molecule, the X-ray absorption cross section of the labeler molecule can be increased in proportion. The number of side chains to be added is m, and the X-ray absorption cross section is σ. _s If
,
[0075]
[Chemical 6]

[0076]
Therefore, the linear absorption coefficient is as shown in Equation (6).
[0077]
[Chemical 7]

[0078]
For example, O- (4-Nitrobenzoyl) hydroxylamine contains only one benzene ring having a double bond, but when m side-chains are added to m benzene rings, the linear absorption coefficient is expressed by the formula (7 )
[0079]
[Chemical 8]

[0080]
Therefore, if five benzene rings are added, the contrast is increased to 0.5, and the image of tobacco mosaic virus can be enhanced.
[0081]
Example 5
The present technology also facilitates the design of sample containers for biological samples. In general, light in the wavelength region used in the X-ray microscope for living organisms is strongly attenuated in the atmosphere, and the entire system is generally housed in a vacuum vessel. Therefore, the biological sample as it is wet is enclosed in a sample container made of an ultra-thin window, and the sample is not dried in a vacuum. In general, the material of the thin film to be used is selected so as to have a high X-ray transmittance in the “water window” region and the vicinity thereof, and at the same time easy to form a thin film. At this time, when an organic material having no double bond is selected, an organic thin film having high transmittance and high strength can be used as the thin film material when photographing using the 1s → π * transition of carbon. For example, polyethylene is a typical saturated polymer that has no double bonds and therefore does not have a π * molecular orbital. Therefore, it can be expected to have a very high transmittance in the wavelength region of 44 to 43.7 A (290 to 280 eV).
FIG. R. It is the X-ray transmission characteristic of the polyethylene film measured by Rabbe et al. (JR Rabet et.al .: Thin Solid Films 159 (1988) 275). According to FIG. 8, it can be seen that the polyethylene film has a very high transmittance at 287 eV or less. In the microscope method using the inner shell excitation process, when taking an absorption image of a labeler's benzene ring having a strong absorption line at an X-ray wavelength of around 43.5 A (285 eV), the film is an optimum sample container. Window material.
[0082]
【The invention's effect】
As described above, in the present invention, conventional X-rays having a photon energy of 2000 eV or less and a wavelength width of 1 eV or less are used, particularly by staining a sample with molecules containing double bonds. Compared with the case of the X-ray microscopic method using the inner shell ionization process, a contrast can be easily obtained, and a high-functional X-ray microscopic method can be provided by arbitrarily adjusting the contrast by selecting the photon energy of the X-ray.
[Brief description of the drawings]
FIG. 1 is an explanatory view showing an optical system of a conventional Walter X-ray microscope.
[Figure 2] Front view of Fresnel zone plate
FIG. 3 is an explanatory diagram showing a Schwarzschild optical system.
[Fig. 4] Explanatory diagram of inner-shell ionization process and inner-shell excitation process
FIG. 5 is a relationship diagram of photon energy and cross-sectional area when 1s electrons of carbon atoms are excited in each p-orbital.
FIG. 6 is a diagram showing the relationship between photon energy and cross-sectional area when benzene 1s electrons transition to a π * molecular orbital.
FIG. 7 is a graph showing the relationship between water and protein wavelength and absorption coefficient.
FIG. 8 is a relationship diagram of the intensity of polyethylene with respect to photon energy.

Claims (8)

Staining with an X-ray light source that emits monochromatic X-rays having a photon energy of 280 eV to 550 eV and a wavelength width of 1 eV or less, and molecules containing double bonds in which 1s electrons are excited by X-rays from the X-ray light source A microscope using an X-ray microscope containing a sample container and a sample container having a window through which X-rays from the X-ray light source pass.
The window material constituting the window of the sample container uses an organic molecule or carbonized material having no π * orbital, and is generated by excitation of 1s electrons of the stained sample by X-rays transmitted through the window. , X-rays microscopy, characterized by observing the absorption or fluorescence of the X-ray. X-ray microscopy of 請 Motomeko 1 wherein shall be the variable wavelength of the X-ray. The X-ray microscopy according to claim 1, wherein the molecule for staining the sample is a compound containing a 5-membered ring or a 6-membered ring. 2. The X-ray microscopy method according to claim 1, wherein the molecule for staining the sample is a compound containing a benzene ring. The X-ray microscopy according to claim 1, wherein the molecule for staining the sample is a fluorescent dye containing a double bond. 2. The X-ray microscope according to claim 1, wherein 1s electrons of C, N, and O of the stained sample are excited to absorb the X-ray in a π * orbit. The X-ray microscope according to claim 6, wherein the Cs, N, and O 1s electrons of the stained sample are excited and absorbed in a π * orbit. 8. X-ray microscopy according to claim 7, wherein the 1s electron of C of the stained sample is excited by π * to absorb X-rays.

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