JP2005114450A - Fluorescence analyzing method due to single-ion irradiation and fluorescence analyzer - Google Patents
Fluorescence analyzing method due to single-ion irradiation and fluorescence analyzer Download PDFInfo
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Abstract
Description
本発明は、イオン加速器や放射性同位元素（ＲＩ）等からの高エネルギーイオンを試料に照射して、試料より発せられる蛍光の分布とその寿命を測定する技術に関するものである。本発明によれば、生物などの生きた試料の分析も可能であり、特に医学・生物学分野での利用が期待される。 The present invention relates to a technique for irradiating a sample with high-energy ions from an ion accelerator, a radioisotope (RI) or the like, and measuring the distribution of fluorescence emitted from the sample and its lifetime. According to the present invention, it is possible to analyze a living sample such as a living organism, and it is expected to be used particularly in the medical / biological field.
従来、医学・生物学分野で、よく利用される蛍光顕微鏡は、試料に蛍光色素を投与し、励起光（可視〜紫外光）の照射により発せられる蛍光を光学顕微鏡で観察することにより、試料観察を行うものである。このような蛍光顕微鏡による観察は、透過／反射光では見ることのできない、試料中の分子の動きや化学状態を可視化できるため、特に医学・生物分野でよく利用されている（例えば、非特許文献１参照のこと）。 Conventionally, a fluorescence microscope that is often used in the medical and biological fields is to observe a sample by administering a fluorescent dye to the sample and observing the fluorescence emitted by irradiation with excitation light (visible to ultraviolet light) with an optical microscope. Is to do. Such observation with a fluorescence microscope is often used particularly in the medical and biological fields because it can visualize the movement and chemical state of molecules in a sample, which cannot be seen with transmitted / reflected light (for example, non-patent documents). 1).
しかしながら、この手法には、蛍光色素投与による生体試料に対する二次的影響、連続的な励起光照射による蛍光の減衰、光の回折限界による空間分解能の制限、試料の密度を含む厚さを測定することが困難、等の問題点がある。
本発明は、上記従来技術の、蛍光色素投与による生体試料に対する二次的影響、連続的な励起光照射による蛍光の減衰、光の回折限界による空間分解能の制限、試料の密度を含む厚さを測定することが困難、等の問題点を解消することをその課題とする。 The present invention relates to the above-mentioned prior art, the secondary effects on biological samples due to administration of fluorescent dyes, fluorescence decay due to continuous excitation light irradiation, spatial resolution limitation due to light diffraction limit, and thickness including sample density. The problem is to eliminate problems such as difficulty in measurement.
上記従来技術の課題を解決するため本発明者らは鋭意研究した結果、本発明を完成させた。
要するに、本発明は、シングルイオン照射による蛍光分析方法であって、予め蛍光色素を添加した試料や自らが蛍光を発する試料に、シングルイオンを照射し、前記試料から発せられる蛍光の光量と前記試料を透過する前記シングルイオンのエネルギーとを測定することを特徴とするものである。
In order to solve the above-mentioned problems of the prior art, the present inventors have intensively studied, and as a result, completed the present invention.
In short, the present invention is a fluorescence analysis method using single ion irradiation, in which a sample to which a fluorescent dye has been added in advance or a sample that emits fluorescence in advance is irradiated with single ions, and the amount of fluorescence emitted from the sample and the sample And measuring the energy of the single ions that pass through.
また、本発明は、上記の方法において、シングルイオンが、イオン加速によるイオンマイクロビーム又は放射性同位元素からのα粒子であることを特徴とするものである。
また、本発明は、上記の方法において、試料から発せられる蛍光及びその発光寿命、並びに試料を透過するシングルイオンのエネルギー損失を同時に測定できることを特徴とするものである。
In the above method, the present invention is characterized in that the single ion is an α particle from an ion microbeam or a radioisotope by ion acceleration.
Further, the present invention is characterized in that, in the above method, the fluorescence emitted from the sample, its emission lifetime, and the energy loss of single ions passing through the sample can be measured simultaneously.
更に、本発明は、シングルイオン照射による蛍光分析装置であって、試料に照射するためのシングルイオン発生源と、前記シングルイオン発生源からのシングルイオン照射により試料から発せられる蛍光の光量を測定するための光検出器と、前記試料への前記シングルイオン照射により前記試料を透過するイオンエネルギーを測定するための粒子検出器と、場合により、光学顕微鏡及び高感度カメラを含む蛍光分布測定系とを含むことを特徴とするものである。 Furthermore, the present invention is a fluorescence analyzer using single ion irradiation, which measures a single ion generation source for irradiating a sample and the amount of fluorescence emitted from the sample by single ion irradiation from the single ion generation source. And a particle detector for measuring the ion energy transmitted through the sample by the single ion irradiation to the sample, and optionally a fluorescence distribution measurement system including an optical microscope and a high-sensitivity camera. It is characterized by including.
本発明のシングルイオン照射による蛍光分析によれば、極微量の蛍光色素の投与で試料の観察を行えることから、蛍光色素の生体試料への影響を最小限に抑え、より自然な状態での観察が可能になる。また、０．１μｍ以下の高い空間分解能で試料観察を行うこともできることから、光学顕微鏡では観察できなかった微細な構造を可視化することが可能になる。 According to the fluorescence analysis by single ion irradiation of the present invention, the sample can be observed by administration of a very small amount of the fluorescent dye, so that the influence of the fluorescent dye on the biological sample is minimized and the observation is performed in a more natural state. Is possible. In addition, since the sample can be observed with a high spatial resolution of 0.1 μm or less, it is possible to visualize a fine structure that could not be observed with an optical microscope.
本発明は、予め蛍光色素を添加した試料や自らが蛍光を発する試料に、にシングルイオンを照射し、前記試料から発せられる蛍光の光量と前記試料を透過する前記シングルイオンのエネルギーとを測定することを特徴とする、シングルイオン照射による蛍光分析方法である。 The present invention irradiates a sample to which a fluorescent dye has been added in advance or a sample that fluoresces itself with a single ion, and measures the amount of fluorescence emitted from the sample and the energy of the single ion transmitted through the sample. This is a fluorescence analysis method using single ion irradiation.
本発明にしたがえば、蛍光色素の励起に光を用いないためバックグランドが少なく、本質的に高感度であり、かつ高エネルギーのシングルイオンを用いるため、試料の局所に大きなエネルギーを与えることができ、極微量の蛍光色素の投与でも感度よく試料の蛍光観察を行うことが可能である。更に、シングルイオンの照射によって蛍光を逐次観察していくため、蛍光色素の減衰は非常に少ないという特徴を有する。また、イオンマイクロビームを用いるため、光の回折限界に制限されることなく、非常に高い空間分解能で試料の観察を行うことができる。更に、試料を透過したイオンのエネルギー損失を同時測定することにより、試料の密度を含む厚さを正確に観測できるなどの利点を有する。 According to the present invention, no light is used to excite the fluorescent dye, so there is little background, which is inherently highly sensitive, and high energy single ions are used. It is possible to observe the fluorescence of the sample with high sensitivity even by administration of a very small amount of fluorescent dye. Furthermore, since fluorescence is sequentially observed by irradiation with a single ion, the fluorescent dye has a very small attenuation. In addition, since an ion microbeam is used, the sample can be observed with very high spatial resolution without being limited by the diffraction limit of light. Furthermore, by simultaneously measuring the energy loss of ions transmitted through the sample, there is an advantage that the thickness including the density of the sample can be accurately observed.
本発明において用いることができるイオンの種類は、分析項目に依存して選択することができる。試料を透過するイオンのエネルギーを測定する場合は、水素、ヘリウムなどの軽イオンを選択することができる。蛍光のみを測定する（イオンが試料中で停止する）場合は、例えば、鉄などの重イオンを選択することができる。 The kind of ion that can be used in the present invention can be selected depending on the analysis item. When measuring the energy of ions passing through the sample, light ions such as hydrogen and helium can be selected. When only fluorescence is measured (the ions stop in the sample), for example, heavy ions such as iron can be selected.
本発明の一実施形態においては、シングルイオンとして、イオン加速器によるイオンマイクロビームを用いることができる。この実施形態の蛍光分析装置の構成概略図を図１に示す。図１において、シングルイオン照射による蛍光分析装置は、試料に照射するためのシングルイオン発生源と、前記シングルイオン発生源からのシングルイオン照射により試料から発せられる蛍光の光量を測定するための光検出器と、前記試料への前記シングルイオン照射により前記試料を透過するイオンエネルギーを測定するための粒子検出器とを含むことを特徴とする。
イオンマイクロビームは、イオン加速器からの連続的なイオンビームをシングルイオンにまびいた後、磁気レンズで集束して大気中に取り出し、走査電極により偏向して試料に走査照射する。シングルイオンの照射により、試料自らや予め試料に添加した蛍光色素からの蛍光が発せられる。光検出器により励起光の光量を測定し、粒子検出器により試料を透過するイオンのエネルギーを測定することにより、蛍光分布、蛍光寿命、エネルギー損失分布を分析することができる。この実施形態においては、蛍光の分布をイオンマイクロビームの照射位置で知ることができるため、光学顕微鏡の使用を必要としない。
In one embodiment of the present invention, an ion microbeam by an ion accelerator can be used as a single ion. A schematic diagram of the configuration of the fluorescence analyzer of this embodiment is shown in FIG. In FIG. 1, a fluorescence analyzer using single ion irradiation includes a single ion generation source for irradiating a sample, and light detection for measuring the amount of fluorescence emitted from the sample by single ion irradiation from the single ion generation source. And a particle detector for measuring ion energy transmitted through the sample by the single ion irradiation to the sample.
In the ion microbeam, a continuous ion beam from an ion accelerator is spread into single ions, then focused by a magnetic lens, taken out into the atmosphere, deflected by a scanning electrode, and scanned onto a sample. By irradiation with single ions, fluorescence is emitted from the sample itself or a fluorescent dye previously added to the sample. The fluorescence distribution, fluorescence lifetime, and energy loss distribution can be analyzed by measuring the amount of excitation light with a photodetector and measuring the energy of ions that pass through the sample with a particle detector. In this embodiment, since the fluorescence distribution can be known from the irradiation position of the ion microbeam, the use of an optical microscope is not required.
また、本発明の別の実施形態においては、シングルイオンとして、ＲＩからのα線を用いることができる。この実施形態の蛍光分析装置の構成概略図を図２に示す。図２において、シングルイオン照射による蛍光分析装置は、試料に照射するためのシングルイオン発生源と、前記シングルイオン発生源からのシングルイオン照射により試料から発せられる蛍光の光量を測定するための光検出器と、前記試料への前記シングルイオン照射により前記試料を透過するイオンエネルギーを測定するための粒子検出器と、光学顕微鏡及び高感度カメラを含む蛍光分布測定系とを含むことを特徴とする。 In another embodiment of the present invention, α rays from RI can be used as single ions. A schematic diagram of the configuration of the fluorescence analyzer of this embodiment is shown in FIG. In FIG. 2, a fluorescence analyzer using single ion irradiation includes a single ion generation source for irradiating a sample, and light detection for measuring the amount of fluorescence emitted from the sample by single ion irradiation from the single ion generation source. A particle detector for measuring ion energy transmitted through the sample by the single ion irradiation to the sample, and a fluorescence distribution measurement system including an optical microscope and a high sensitivity camera.
α粒子の衝撃により、試料自らや予め試料に添加した蛍光色素からの蛍光が発せられる。発光した蛍光を光学顕微鏡と高感度カメラとを組み合わせて測定することにより、その分布を計測することができる。また、光検出器により蛍光の発光タイミングを測定し、粒子検出器により試料を透過するイオンのエネルギーとタイミングを測定することにより、エネルギー損失、蛍光寿命を分析することができる。 Due to the impact of the α particles, fluorescence is emitted from the sample itself or from a fluorescent dye previously added to the sample. The distribution can be measured by measuring the emitted fluorescence in combination with an optical microscope and a high sensitivity camera. Further, energy loss and fluorescence lifetime can be analyzed by measuring the emission timing of fluorescence with a photodetector and measuring the energy and timing of ions passing through the sample with a particle detector.
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