JP6104703B2 - Staining agent for electron microscope observation and staining method for sample for electron microscope observation - Google Patents
Staining agent for electron microscope observation and staining method for sample for electron microscope observation Download PDFInfo
- Publication number
- JP6104703B2 JP6104703B2 JP2013105150A JP2013105150A JP6104703B2 JP 6104703 B2 JP6104703 B2 JP 6104703B2 JP 2013105150 A JP2013105150 A JP 2013105150A JP 2013105150 A JP2013105150 A JP 2013105150A JP 6104703 B2 JP6104703 B2 JP 6104703B2
- Authority
- JP
- Japan
- Prior art keywords
- electron microscope
- sample
- staining
- microscope observation
- ytterbium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Analysing Materials By The Use Of Radiation (AREA)
- Sampling And Sample Adjustment (AREA)
Description
本発明は、電子顕微鏡観察用染色剤および電子顕微鏡観察用試料の染色方法に関する。 The present invention relates to a staining agent for electron microscope observation and a staining method for a sample for electron microscope observation.
生体試料の微細な構造等を観察する装置として、光学顕微鏡や電子顕微鏡が知られている。特に、電子顕微鏡は光学顕微鏡と比べて分解能が高いため、より微細な構造を観察する際に有効である。しかしながら、生体試料は、炭素、酸素、窒素、水素などの軽元素を含んで構成されているため、電子線を十分に散乱させることができない。したがって、電子顕微鏡で観察しても、電子顕微鏡像に十分なコントラストが得られない場合がある。そのため、電子顕微鏡で生体試料を観察する際には、一般的に、試料を重金属等で電子染色する。試料を電子染色することにより、電子線の散乱を促し、電子顕微鏡像にコントラストをつけることができる。 As an apparatus for observing the fine structure of a biological sample, an optical microscope and an electron microscope are known. In particular, since the electron microscope has a higher resolution than the optical microscope, it is effective for observing a finer structure. However, since the biological sample is configured to include light elements such as carbon, oxygen, nitrogen, and hydrogen, the electron beam cannot be sufficiently scattered. Therefore, even when observed with an electron microscope, sufficient contrast may not be obtained in the electron microscope image. Therefore, when observing a biological sample with an electron microscope, the sample is generally electronically stained with heavy metal or the like. By electron-staining the sample, scattering of the electron beam can be promoted, and contrast can be given to the electron microscope image.
従来、電子染色剤（電子顕微鏡観察用染色剤）として、酢酸ウラニルが用いられていた。酢酸ウラニルは、高い染色効果を有しており、酢酸ウラニルで生体試料を染色することにより、高いコントラストの電子顕微鏡像を得ることができる。 Conventionally, uranyl acetate has been used as an electron stain (stain for electron microscope observation). Uranyl acetate has a high staining effect, and a high contrast electron microscope image can be obtained by staining a biological sample with uranyl acetate.
また、特許文献１には、電子染色剤として、白金ブルー（［Ｐｔ４（ＮＨ３）８（Ｃ６Ｈ１３Ｏ５）４］＋５）が開示されている。 Patent Document 1 discloses platinum blue ([Pt 4 (NH 3 ) 8 (C 6 H 13 O 5 ) 4 ] +5 ) as an electron stain.
上述したように、酢酸ウラニルは高い染色効果を有しているが、放射性物質のため、入手や使用に厳しい規制がある。そのため、酢酸ウラニルに代替する電子染色剤が求められている。上述した特許文献１に開示された白金ブルーは、酢酸ウラニルに代替する電子染色剤の１つとして知られている。 As described above, uranyl acetate has a high staining effect, but because of the radioactive substance, there are strict regulations on its availability and use. Therefore, there is a need for an electronic stain that replaces uranyl acetate. Platinum blue disclosed in Patent Document 1 described above is known as one of electron staining agents that substitute for uranyl acetate.
白金ブルーは、時間が経過すると変質する場合があるため、使用にあわせて合成することが望ましい。しかしながら、白金ブルーの合成には、通常５〜７日程度の時間が必要であり、かつ高度な化学的知識を必要とする。したがって、白金ブルーを用いた電子染色では、白金ブルーの合成に時間や手間がかかってしまい、簡便に電子染色を行うことができないという問題があった。 Since platinum blue may change in quality over time, it is desirable to synthesize it according to use. However, the synthesis of platinum blue usually requires about 5-7 days and requires advanced chemical knowledge. Therefore, in the electron staining using platinum blue, there is a problem that the synthesis of platinum blue takes time and labor, and the electron staining cannot be easily performed.
本発明は、以上のような問題点に鑑みてなされたものであり、本発明のいくつかの態様によれば、簡便に電子染色を行うことができ、かつ高い染色効果を有する電子顕微鏡観察用染色剤および電子顕微鏡観察用試料の染色方法を提供することができる。 The present invention has been made in view of the above-described problems, and according to some embodiments of the present invention, it is possible to easily perform electron staining and for electron microscope observation having a high staining effect. A staining method and a staining method of a sample for electron microscope observation can be provided.
（１）本発明に係る電子顕微鏡観察用染色剤は、
ネガティブ染色法に用いられる電子顕微鏡観察用染色剤であって、
三酢酸イッテルビウムと、
メタノールを含む溶媒と、
を含有する。
(1) The stain for electron microscope observation according to the present invention is:
An electron microscope observation stain used for negative staining,
Ytterbium triacetate,
A solvent comprising methanol;
Containing.
このような電子顕微鏡観察用染色剤によれば、高い染色効果を有することができる。さらに、このような電子顕微鏡観察用染色剤によれば、三酢酸イッテルビウムを、メタノールを含む溶媒に溶解させるだけで電子染色剤として用いることができる。したがって、簡便に電子染色を行うことができる。また、このような電子顕微鏡観察用染色剤は、分散性が高く、試料粒子間に染色剤を分散させることができるため、試料が凝集している場合でも、染色剤を凝集させないことができる。 Such a staining agent for electron microscope observation can have a high staining effect. Furthermore, according to such an electron microscope observation stain, ytterbium triacetate can be used as an electron stain simply by dissolving it in a solvent containing methanol. Therefore, it is possible to easily perform electron staining. In addition, since such a staining agent for electron microscope observation has high dispersibility and can disperse the staining agent between the sample particles, the staining agent can be prevented from aggregating even when the sample is aggregated.
（２）本発明に係る電子顕微鏡観察用試料の染色方法は、
本発明に係る電子顕微鏡観察用染色剤に試料を接触させる工程を含む。
(2) The staining method of the sample for electron microscope observation according to the present invention is as follows:
A step of bringing the sample into contact with the electron microscope observation stain according to the present invention.
このような電子顕微鏡観察用試料の染色方法によれば、試料を、簡便かつ良好に電子染色することができる。また、このような電子顕微鏡観察用試料の染色方法によれば、試料が凝集している場合でも、染色剤を凝集させないことができる。 According to such a method for staining an electron microscope observation sample, the sample can be easily and satisfactorily electron-stained. In addition, according to such a method for staining an electron microscope observation sample, even when the sample is aggregated, the staining agent can not be aggregated.
以下、本発明の好適な実施形態について図面を用いて詳細に説明する。なお、以下に説明する実施形態は、特許請求の範囲に記載された本発明の内容を不当に限定するものではない。また、以下で説明される構成の全てが本発明の必須構成要件であるとは限らない。 DESCRIPTION OF EMBODIMENTS Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. The embodiments described below do not unduly limit the contents of the present invention described in the claims. In addition, not all of the configurations described below are essential constituent requirements of the present invention.
１． 電子顕微鏡観察用染色剤
まず、本実施形態に係る電子顕微鏡観察用染色剤について説明する。
1. Electron Microscope Observation Staining Agent First, the electron microscope observation staining agent according to this embodiment will be described.
本実施形態に係る電子顕微鏡観察用染色剤は、三酢酸イッテルビウム（Ｙｂ（ＣＨ３ＣＯＯ）３）と、メタノールを含む溶媒と、を含有する。なお、三酢酸イッテルビウムを含有するとは、三酢酸イッテルビウムの水和物を含有している場合も含むものとする。三酢酸イッテルビウムの水和物としては、例えば、酢酸イッテルビウム四水和物（Ｙｔｔｅｒｂｉｕｍ（ＩＩＩ） ａｃｅｔａｔｅ ｔｅｔｒａｈｙｄｒａｔｅ，Ｙｂ（ＣＨ３ＣＯＯ）３・４Ｈ２Ｏ）が挙げられる。 The stain for electron microscope observation according to the present embodiment contains ytterbium triacetate (Yb (CH 3 COO) 3 ) and a solvent containing methanol. It should be noted that “containing ytterbium triacetate” includes the case of containing hydrate of ytterbium triacetate. Examples of the hydrate of ytterbium triacetate include ytterbium acetate tetrahydrate (Yterbium (III) acetate tetrahydrate, Yb (CH 3 COO) 3 .4H 2 O).
また、本実施形態に係る電子顕微鏡観察用染色剤の溶媒は、例えば、１００体積％メタノールである。なお、該溶媒は、メタノールと、水と、を含んでいてもよい。このとき、該溶媒のメタノールの濃度は、特に限定されない。なお、該溶媒は、メタノールおよび水以外の物質を含んでいてもよい。 Moreover, the solvent of the staining agent for electron microscope observation which concerns on this embodiment is 100 volume% methanol, for example. The solvent may contain methanol and water. At this time, the concentration of methanol in the solvent is not particularly limited. In addition, this solvent may contain substances other than methanol and water.
本実施形態に係る電子顕微鏡観察用染色剤において、三酢酸イッテルビウムの濃度は特に限定されず、例えば１〜１０質量％である。本実施形態に係る電子顕微鏡観察用染色剤において、三酢酸イッテルビウムの濃度は、例えば飽和濃度である。 In the electron microscope observation stain according to this embodiment, the concentration of ytterbium triacetate is not particularly limited, and is, for example, 1 to 10% by mass. In the electron microscope observation stain according to this embodiment, the concentration of ytterbium triacetate is, for example, a saturated concentration.
本実施形態に係る電子顕微鏡観察用染色剤は、三酢酸イッテルビウムに加えて、さらに、三酢酸イッテルビウム以外の物質を含有していてもよい。 In addition to ytterbium triacetate, the stain for electron microscope observation according to the present embodiment may further contain a substance other than ytterbium triacetate.
ここで、電子顕微鏡観察用染色剤とは、電子顕微鏡観察の対象となる試料を染色するための染色剤（電子染色剤）をいう。なお、染色とは、いわゆる電子染色をいい、試料の特定の部位や、試料の周囲（グリッドの支持膜と試料との間、試料の凹凸の凹部等）に電子の散乱を促す物質（重金属等）を吸着または結合させることをいう。電子顕微鏡観察用染色剤を用いて試料を染色することにより、電子顕微鏡像にコントラストをつけることができる。 Here, the electron microscope observation staining agent refers to a staining agent (electron staining agent) for staining a sample to be observed with an electron microscope. Staining is a so-called electron staining, which is a substance (heavy metal, etc.) that promotes scattering of electrons around a specific part of the sample or around the sample (between the support film of the grid and the sample, the concave and convex portions of the sample). ) Is adsorbed or bound. By staining the sample with a staining agent for electron microscope observation, contrast can be given to the electron microscope image.
また、染色された試料の観察に用いられる電子顕微鏡としては、例えば、走査電子顕微鏡（Ｓｃａｎｎｉｎｇ Ｅｌｅｃｔｒｏｎ Ｍｉｃｒｏｓｃｏｐｅ、ＳＥＭ）、透過電子顕微鏡（Ｔｒａｎｓｍｉｓｓｉｏｎ Ｅｌｅｃｔｒｏｎ Ｍｉｃｒｏｓｃｏｐｅ、ＴＥＭ）、走査透過電子顕微鏡（Ｓｃａｎｎｉｎｇ Ｔｒａｎｓｍｉｓｓｉｏｎ Ｅｌｅｃｔｒｏｎ Ｍｉｃｒｏｓｃｏｐｅ、ＳＴＥＭ）などが挙げられる。 Examples of the electron microscope used for observing the stained sample include a scanning electron microscope (SEM), a transmission electron microscope (Transmission Electron Microscope, TEM), a scanning transmission electron microscope (Scanning Transmission Electron Microscope, TEM), and the like. STEM).
本実施形態に係る電子顕微鏡観察用染色剤は、例えば、タンパク質などの生体高分子を含んで構成される生体試料、炭素、酸素、窒素、水素などの軽元素を含んで構成される試料、ウイルス、リポソーム等の微粒子等を染色することができる。 The stain for electron microscope observation according to the present embodiment includes, for example, a biological sample including a biopolymer such as a protein, a sample including a light element such as carbon, oxygen, nitrogen, and hydrogen, a virus In addition, fine particles such as liposomes can be stained.
２． 電子顕微鏡観察用試料の作製方法
次に、本実施形態に係る電子顕微鏡観察用試料の作製方法について説明する。本実施形態に係る電子顕微鏡観察用試料の作製方法は、本実施形態に係る電子顕微鏡観察用試料の染色方法を含む。以下に示す本実施形態に係る電子顕微鏡観察用試料の作製方法では、本発明に係る電子顕微鏡観察用染色剤を、ネガティブ染色法に適用した場合について説明する。
2. Next, a method for producing an electron microscope observation sample according to this embodiment will be described. The method for producing an electron microscope observation sample according to the present embodiment includes the electron microscope observation sample staining method according to the present embodiment. In the following method for producing an electron microscope observation sample according to this embodiment, a case where the electron microscope observation stain according to the present invention is applied to a negative staining method will be described.
本実施形態に係る電子顕微鏡観察用試料の作製方法は、本発明に係る電子顕微鏡観察用染色剤にウイルスやタンパク質等の試料を接触させる工程を含む。具体的には、例えば、カーボン支持膜等を備えたグリッド上に、ウイルス等を含む試料を載せ、本実施形態に係る電子顕微鏡観察用染色剤を加える。これにより、染色剤の一部が、グリッドの支持膜と試料との間、試料の凹凸の凹部等に残留し、透過電子顕微鏡像にコントラストがつく（ネガティブ染色法）。 The method for producing an electron microscope observation sample according to this embodiment includes a step of bringing a sample such as a virus or protein into contact with the electron microscope observation stain according to the present invention. Specifically, for example, a sample containing a virus or the like is placed on a grid provided with a carbon support film or the like, and the staining for electron microscope observation according to this embodiment is added. As a result, a part of the staining agent remains between the grid support film and the sample, in the concave and convex portions of the sample, and the transmission electron microscope image is contrasted (negative staining method).
以上の工程により、電子顕微鏡観察用試料を作製することができる。 Through the above steps, an electron microscope observation sample can be produced.
なお、ここでは、本実施形態に係る電子顕微鏡観察用染色剤をネガティブ染色法に適用した場合について説明したが、本実施形態に係る電子顕微鏡観察用染色剤をその他の染色法にも適用することができる。例えば、本実施形態に係る電子顕微鏡観察用染色剤をポジティブ染色法に適用することができる。ここでポジティブ染色法とは、試料の特定部位に重金属等を結合させて、電子顕微鏡像にコントラストをつける手法である。例えば、エポキシ樹脂等に包埋された生体試料を、ミクロトーム等で薄片化し、薄片化された試料を、本実施形態に係る電子顕微鏡観察用染色剤に浸漬させることにより、試料をポジティブ染色することができる。 In addition, although the case where the stain for electron microscope observation according to the present embodiment is applied to the negative staining method has been described here, the stain for electron microscope observation according to the present embodiment is also applied to other staining methods. Can do. For example, the electron microscope observation stain according to the present embodiment can be applied to the positive staining method. Here, the positive staining method is a method of adding contrast to an electron microscope image by binding heavy metal or the like to a specific part of a sample. For example, a biological sample embedded in an epoxy resin or the like is sliced with a microtome or the like, and the sliced sample is immersed in the staining for electron microscope observation according to the present embodiment, thereby positively staining the sample. Can do.
また、本実施形態に係る電子顕微鏡観察用染色剤で染色された試料を、さらに、鉛化合物を含有する染色剤に接触（浸漬）させてもよい。すなわち、試料を、本実施形態に係る電子顕微鏡観察用染色剤と鉛化合物を含有する染色剤とによって、二重染色してもよい。ここで、上記鉛化合物としては、例えば、クエン酸鉛が挙げられる。 Moreover, you may make the sample dye | stained with the stain | dye for electron microscope observation which concerns on this embodiment further contact (immerse) the stain | dye containing a lead compound. That is, the sample may be double-stained with the electron microscope observation stain according to this embodiment and the stain containing a lead compound. Here, examples of the lead compound include lead citrate.
３． 実施例
以下、実施例を挙げて本実施形態をさらに詳細に説明するが、本発明はこれによって制限されるものではない。
3. Examples Hereinafter, the present embodiment will be described in more detail with reference to examples, but the present invention is not limited thereto.
３．１． 実施例１
（１）試料作製
試料は、液体培地で培養したＴ４ファージおよび大腸菌を用いた。染色剤は、三酢酸イッテルビウム四水和物（和光純薬工業株式会社製）を、１００体積％メタノールで溶解させることによって作製した。該染色剤において、三酢酸イッテルビウムの濃度は、１質量％とした。
3.1. Example 1
(1) Sample preparation T4 phage and E. coli cultured in a liquid medium were used as samples. The staining agent was prepared by dissolving ytterbium triacetate tetrahydrate (manufactured by Wako Pure Chemical Industries, Ltd.) with 100% by volume methanol. In the staining agent, the concentration of ytterbium triacetate was 1% by mass.
本実施例の電子顕微鏡観察用試料の作製方法を説明する。まず、カーボン膜を張った銅製グリットに上記試料を滴下し濾紙で余剰の液を吸い取る。次に、上記染色剤をグリットに滴下しすぐに濾紙で余剰の液を吸い取ることでネガティブ染色し、電子顕微鏡観察用試料を作製した。 A method for manufacturing the electron microscope observation sample of this example will be described. First, the sample is dropped onto a copper grit covered with a carbon film, and excess liquid is sucked off with a filter paper. Next, the dyeing agent was dropped onto the grit, and the excess liquid was immediately sucked off with filter paper to perform negative staining, thereby preparing an electron microscope observation sample.
なお、比較例として、同様の手順により、上記試料を１質量％の三酢酸イッテルビウム水溶液で染色（ネガティブ染色）した。三酢酸イッテルビウム水溶液は、三酢酸イッテルビウム四水和物（和光純薬工業株式会社製）を、蒸留水で溶解させることによって作製した。 As a comparative example, the sample was stained with 1% by mass of an aqueous ytterbium triacetate solution (negative staining) by the same procedure. The ytterbium triacetate aqueous solution was prepared by dissolving ytterbium triacetate tetrahydrate (manufactured by Wako Pure Chemical Industries, Ltd.) with distilled water.
（２）観察結果
このようにして作製された電子顕微鏡観察用試料を、透過電子顕微鏡（日本電子株式会社製ＪＥＭ−１４００）で観察した。図１〜図４は、三酢酸イッテルビウム水溶液で染色されたＴ４ファージの透過電子顕微鏡像である。図５および図６は、１００体積％メタノールに溶解した１質量％の三酢酸イッテルビウムで染色されたＴ４ファージの透過電子顕微鏡像である。
(2) Observation result The sample for electron microscope observation produced in this way was observed with the transmission electron microscope (JEM-1400 by JEOL Ltd.). 1 to 4 are transmission electron microscope images of T4 phage stained with an aqueous ytterbium triacetate solution. 5 and 6 are transmission electron microscopic images of T4 phage stained with 1% by mass of ytterbium triacetate dissolved in 100% by volume of methanol.
図１および図２に示すように、三酢酸イッテルビウム水溶液を用いてネガティブ染色を行うと、染色剤が試料周囲に存在し、試料の輪郭がはっきりと見て取れた。しかしながら、図３および図４に示すように、試料が凝集している場合、染色剤も凝集し、試料の微細な構造を確認することができないことがあった。 As shown in FIG. 1 and FIG. 2, when negative staining was performed using an aqueous ytterbium triacetate solution, the staining agent was present around the sample, and the outline of the sample was clearly visible. However, as shown in FIGS. 3 and 4, when the sample is aggregated, the staining agent also aggregates, and the fine structure of the sample may not be confirmed.
これに対して、１００体積％メタノールに溶解した１質量％の三酢酸イッテルビウムを用いてネガティブ染色を行うと、図５および図６に示すように、試料が凝集している場合でも、染色剤は凝集せずに、１つ１つのＴ４ファージやその他の構造物を明瞭に見て取れた。なお、図５および図６に示す以外の視野でも、染色剤の目立った凝集は見られなかった。これは、三酢酸イッテルビウムをメタノールに溶解することで、水に溶解した場合と比べて、分散性が増し、試料粒子間により良く染色剤が分散するためであると考えられる。 On the other hand, when negative staining is performed using 1% by mass of ytterbium triacetate dissolved in 100% by volume of methanol, as shown in FIG. 5 and FIG. Each T4 phage and other structures could be clearly seen without aggregation. In addition, in the field of view other than those shown in FIGS. 5 and 6, no conspicuous aggregation of the staining agent was observed. This is considered to be because by dissolving ytterbium triacetate in methanol, the dispersibility increases as compared with the case where it is dissolved in water, and the staining agent is better dispersed between the sample particles.
３．２． 実施例２
次に、三酢酸イッテルビウム水溶液にメタノールを添加することによる染色効果の変化について実験を行った。
3.2. Example 2
Next, an experiment was conducted on the change in staining effect by adding methanol to an aqueous ytterbium triacetate solution.
（１）試料作製
試料は、液体培地で培養したＴ４ファージおよび大腸菌を用いた。
(1) Sample preparation T4 phage and E. coli cultured in a liquid medium were used as samples.
染色剤は、メタノールの濃度を変えた３種類を準備した。 Three types of staining agents were prepared with different concentrations of methanol.
第１の染色剤は、三酢酸イッテルビウム四水和物（和光純薬工業株式会社製）を、メタノールの体積と水の体積が１：１、すなわち、５０体積％（ｖ／ｖ）メタノールに溶解させることによって作製した。第１の染色剤において、三酢酸イッテルビウムの濃度は、１質量％とした。 The first staining agent is ytterbium triacetate tetrahydrate (manufactured by Wako Pure Chemical Industries, Ltd.), in which the volume of methanol and the volume of water are 1: 1, that is, 50% by volume (v / v) dissolved in methanol. It was produced by making it. In the first staining agent, the concentration of ytterbium triacetate was 1% by mass.
第２の染色剤は、三酢酸イッテルビウム四水和物（和光純薬工業株式会社製）を、２５体積％メタノールで溶解させることによって作製した。第２の染色剤において、三酢酸イッテルビウムの濃度は、１質量％とした。 The second staining agent was prepared by dissolving ytterbium triacetate tetrahydrate (manufactured by Wako Pure Chemical Industries, Ltd.) with 25% by volume methanol. In the second staining agent, the concentration of ytterbium triacetate was 1% by mass.
第３の染色剤は、三酢酸イッテルビウム四水和物（和光純薬工業株式会社製）を、１０体積％メタノールで溶解させることによって作製した。第３の染色剤において、三酢酸イッテルビウムの濃度は、１質量％とした。 The third staining agent was prepared by dissolving ytterbium triacetate tetrahydrate (manufactured by Wako Pure Chemical Industries, Ltd.) with 10% by volume methanol. In the third staining agent, the concentration of ytterbium triacetate was 1% by mass.
本実施例の電子顕微鏡観察用試料の作製方法を説明する。まず、カーボン膜を張った銅製グリットに上記試料を滴下し濾紙で余剰の液を吸い取る。次に、第１の染色剤をグリットに滴下しすぐに濾紙で余剰の液を吸い取ることでネガティブ染色し、電子顕微鏡観察用試料を作製した。同様の手順により、上記試料を第２の染色剤でネガティブ染色して、電子顕微鏡観察用試料を作製した。また、同様の手順により、上記試料を第３の染色剤でネガティブ染色して、電子顕微鏡観察用試料を作製した。 A method for manufacturing the electron microscope observation sample of this example will be described. First, the sample is dropped onto a copper grit covered with a carbon film, and excess liquid is sucked off with a filter paper. Next, the first dyeing agent was dropped on the grit, and the excess liquid was immediately sucked off with a filter paper to perform negative staining, thereby preparing an electron microscope observation sample. According to the same procedure, the sample was negatively stained with the second staining agent to prepare an electron microscope observation sample. Further, according to the same procedure, the sample was negatively stained with a third stain, and a sample for observation with an electron microscope was prepared.
（２）観察結果
このようにして作製された電子顕微鏡観察用試料を、透過電子顕微鏡（日本電子株式会社製ＪＥＭ−１４００）で観察した。図７〜図１０は、第１の染色剤（５０体積％メタノールに溶解した１質量％の三酢酸イッテルビウム）で染色されたＴ４ファージの透過電子顕微鏡像である。図１１〜図１４は、第２の染色剤（２５体積％メタノールに溶解した１質量％の三酢酸イッテルビウム）で染色されたＴ４ファージの透過電子顕微鏡像である。図１５〜図２０は、第３の染色剤（１０体積％メタノールに溶解した１質量％の三酢酸イッテルビウム）で染色されたＴ４ファージの透過電子顕微鏡像である。
(2) Observation result The sample for electron microscope observation produced in this way was observed with the transmission electron microscope (JEM-1400 by JEOL Ltd.). 7 to 10 are transmission electron microscopic images of T4 phage stained with the first staining agent (1% by mass of ytterbium triacetate dissolved in 50% by volume of methanol). FIGS. 11 to 14 are transmission electron microscopic images of T4 phage stained with a second staining agent (1% by mass of ytterbium triacetate dissolved in 25% by volume of methanol). 15 to 20 are transmission electron microscopic images of T4 phage stained with a third staining agent (1% by mass of ytterbium triacetate dissolved in 10% by volume of methanol).
第１の染色剤でネガティブ染色を行った試料では、図７および図８に示すように、試料が凝集している場合でも、染色剤は凝集せずに、１つ１つのＴ４ファージやその他の構造物を明瞭に見て取れた。しかしながら、図９および図１０に示すように、一部に、染色剤が凝集し、試料の微細な構造を確認することができない箇所があった。 As shown in FIG. 7 and FIG. 8, in the sample subjected to negative staining with the first staining agent, even when the sample is aggregated, the staining agent does not aggregate, and each T4 phage or other The structure was clearly visible. However, as shown in FIG. 9 and FIG. 10, there was a part where the staining agent aggregated and the fine structure of the sample could not be confirmed.
第２の染色剤でネガティブ染色を行った試料では、図１１および図１２に示すように、試料が凝集している場合でも、染色剤は凝集せずに、１つ１つのＴ４ファージやその他の構造物を明瞭に見て取れた。しかしながら、図１３および図１４に示すように、第１の染色剤でネガティブ染色を行った場合と比べて、染色剤が凝集し、試料の微細な構造を確認することができない箇所が多くみられた。 In the sample that was negatively stained with the second staining agent, as shown in FIGS. 11 and 12, even when the sample is aggregated, the staining agent does not aggregate and each T4 phage or other The structure was clearly visible. However, as shown in FIG. 13 and FIG. 14, compared to the case where negative staining is performed with the first staining agent, there are many places where the staining agent aggregates and the fine structure of the sample cannot be confirmed. It was.
第３の染色剤でネガティブ染色を行った試料では、図１５および図１６に示すように、試料が凝集している場合でも、三酢酸イッテルビウム水溶液で染色を行った場合と比べて（図３および図４参照）、染色剤は凝集せずに、１つ１つのＴ４ファージやその他の構造物を明瞭に見て取れた。しかしながら、図１７および図１８に示すように、第１の染色剤および第２の染色剤でネガティブ染色を行った場合と比べて、染色剤が凝集し、試料の微細な構造を確認することができない箇所が多くみられた。また、ごく稀に、図１９および図２０に示すような、結晶状の構造物が見られた。 As shown in FIGS. 15 and 16, in the sample that was negatively stained with the third staining agent, even when the sample was aggregated, compared to the case where the sample was stained with the aqueous ytterbium triacetate solution (see FIGS. 3 and 3). The staining agent did not aggregate, and each T4 phage and other structures could be clearly seen. However, as shown in FIGS. 17 and 18, compared with the case where negative staining is performed with the first staining agent and the second staining agent, the staining agent aggregates, and the fine structure of the sample can be confirmed. There were many places that could not be done. Very rarely, crystalline structures as shown in FIGS. 19 and 20 were observed.
以上のことから、三酢酸イッテルビウムとメタノールを含む溶媒とを含む染色剤は、高い染色効果を有することがわかった。さらに、三酢酸イッテルビウム水溶液にメタノールを添加することで、メタノールを添加しない場合と比べて、試料が凝集している場合でも、染色剤が凝集せずに、構造物を明瞭に観察できることがわかった。そして、メタノールの濃度が高いほど、その効果が高いことがわかった。三酢酸イッテルビウム水溶液にメタノールを添加することで、分散性が増し、試料粒子間に染色剤がより分散するためであると考えられる。 From the above, it was found that a staining agent containing ytterbium triacetate and a solvent containing methanol has a high staining effect. Furthermore, it was found that by adding methanol to the ytterbium triacetate aqueous solution, the structure can be clearly observed without staining, even when the sample is aggregated, compared to the case where methanol is not added. . And it turned out that the effect is so high that the concentration of methanol is high. It is considered that the addition of methanol to the ytterbium triacetate aqueous solution increases the dispersibility and further disperses the stain between the sample particles.
本実施形態に係る電子顕微鏡観察用染色剤では、三酢酸イッテルビウムと、メタノールを含む溶媒と、を含有している。そのため、上述のように、高い染色効果を有することができる。さらに、三酢酸イッテルビウムは、合成等の必要がなく、例えば、メタノールを含む溶媒に溶解させるだけで電子染色剤として用いることができる。したがって、簡便に電子染色を行うことができる。さらに、本実施形態に係る電子顕微鏡観察用染色剤は、分散性が高く、試料粒子間に染色剤を分散させることができるため、試料が凝集している場合でも、染色剤を凝集させないことができる。 The electron microscope observation stain according to this embodiment contains ytterbium triacetate and a solvent containing methanol. Therefore, as described above, it can have a high dyeing effect. Furthermore, ytterbium triacetate does not require synthesis or the like, and can be used as an electron stain only by dissolving it in a solvent containing methanol, for example. Therefore, it is possible to easily perform electron staining. Furthermore, the electron microscope observation stain according to this embodiment has high dispersibility and can disperse the stain between the sample particles. Therefore, even when the sample is aggregated, the stain may not be aggregated. it can.
本実施形態に係る電子顕微鏡観察用試料の染色方法によれば、上述したように、簡便に電子染色を行うことができ、かつ高い染色効果を有する本実施形態に係る電子顕微鏡観察用染色剤を用いて染色するため、試料を、簡便かつ良好に染色することができる。さらに、試料が凝集している場合でも、染色剤を凝集させないことができる。 According to the method for staining an electron microscope observation sample according to this embodiment, as described above, the electron microscope observation staining agent according to this embodiment can be easily dyed and has a high staining effect. Since it is used and dye | stained, a sample can be dye | stained simply and favorably. Furthermore, even when the sample is aggregated, the staining agent can be prevented from aggregating.
本発明は、実施の形態で説明した構成と実質的に同一の構成（例えば、機能、方法およ
び結果が同一の構成、あるいは目的及び効果が同一の構成）を含む。また、本発明は、実施の形態で説明した構成の本質的でない部分を置き換えた構成を含む。また、本発明は、実施の形態で説明した構成と同一の作用効果を奏する構成又は同一の目的を達成することができる構成を含む。また、本発明は、実施の形態で説明した構成に公知技術を付加した構成を含む。
The present invention includes configurations that are substantially the same as the configurations described in the embodiments (for example, configurations that have the same functions, methods, and results, or configurations that have the same objects and effects). In addition, the invention includes a configuration in which a non-essential part of the configuration described in the embodiment is replaced. In addition, the present invention includes a configuration that exhibits the same operational effects as the configuration described in the embodiment or a configuration that can achieve the same object. Further, the invention includes a configuration in which a known technique is added to the configuration described in the embodiment.
Claims (2)
三酢酸イッテルビウムと、
メタノールを含む溶媒と、
を含有する、電子顕微鏡観察用染色剤。 An electron microscope observation stain used for negative staining,
Ytterbium triacetate,
A solvent comprising methanol;
A stain for electron microscope observation, comprising
A method for staining a sample for electron microscope observation, comprising the step of bringing the sample into contact with the staining agent for electron microscope observation according to claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2013105150A JP6104703B2 (en) | 2013-05-17 | 2013-05-17 | Staining agent for electron microscope observation and staining method for sample for electron microscope observation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2013105150A JP6104703B2 (en) | 2013-05-17 | 2013-05-17 | Staining agent for electron microscope observation and staining method for sample for electron microscope observation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2014224793A JP2014224793A (en) | 2014-12-04 |
| JP6104703B2 true JP6104703B2 (en) | 2017-03-29 |
Family
ID=52123551
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2013105150A Active JP6104703B2 (en) | 2013-05-17 | 2013-05-17 | Staining agent for electron microscope observation and staining method for sample for electron microscope observation |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP6104703B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20240201113A1 (en) * | 2021-05-26 | 2024-06-20 | Hitachi High-Tech Corporation | Particle analyzing device, and particle analyzing method |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3088861B2 (en) * | 1992-10-28 | 2000-09-18 | ナカライテスク株式会社 | Dye aqueous solution for protein staining, staining kit and staining method |
| JP5487636B2 (en) * | 2008-02-18 | 2014-05-07 | 株式会社豊田中央研究所 | Monodispersed spherical inorganic phosphor, method for producing the same, and ordered array |
| JP5199220B2 (en) * | 2009-11-26 | 2013-05-15 | 国立大学法人 東京大学 | Electrostaining agent, staining solution, and electrostaining method |
| US10809167B2 (en) * | 2010-08-30 | 2020-10-20 | Konica Minolta, Inc. | Tissue staining method with staining agent containing particle holding plural phosphors |
| JP6077735B2 (en) * | 2011-06-08 | 2017-02-08 | 日本電子株式会社 | Staining agent for electron microscope observation and staining method for sample for electron microscope observation |
-
2013
- 2013-05-17 JP JP2013105150A patent/JP6104703B2/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| JP2014224793A (en) | 2014-12-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chen et al. | Three-dimensional imaging of dislocations in a nanoparticle at atomic resolution | |
| Arán‐Ais et al. | Electrochemical characterization of clean shape‐controlled Pt nanoparticles prepared in presence of oleylamine/oleic acid | |
| Balci et al. | Electroless synthesis of 3 nm wide alloy nanowires inside tobacco mosaic virus | |
| Menumerov et al. | Identifying the true catalyst in the reduction of 4-nitrophenol: a case study showing the effect of leaching and oxidative etching using ag catalysts | |
| CN106233394A (en) | Ferromagnetism metal nanometer line dispersion liquid and manufacture method thereof | |
| Munusamy et al. | Comparison of 20 nm silver nanoparticles synthesized with and without a gold core: Structure, dissolution in cell culture media, and biological impact on macrophages | |
| Duester et al. | Can cloud point-based enrichment, preservation, and detection methods help to bridge gaps in aquatic nanometrology? | |
| Liu et al. | Precision synthesis: designing hot spots over hot spots via selective gold deposition on silver octahedra edges | |
| Su et al. | In situ observations of shell growth and oxidative etching behaviors of Pd nanoparticles in solutions by liquid cell transmission electron microscopy | |
| Ahrens et al. | Electrochemical formation of gold nanoparticles on polycrystalline gold electrodes during prolonged potential cycling | |
| Asselin et al. | Decoration of plasmonic Mg nanoparticles by partial galvanic replacement | |
| Bonggotgetsakul et al. | A method for coating a polymer inclusion membrane with palladium nanoparticles | |
| JP6104703B2 (en) | Staining agent for electron microscope observation and staining method for sample for electron microscope observation | |
| CN106872438A (en) | A kind of self assembled three-dimensional copper Raman enhancing substrate and preparation method and application | |
| Peng et al. | Identification of a quasi-liquid phase at solid–liquid interface | |
| JP6357354B2 (en) | Staining agent for electron microscope observation and staining method for sample for electron microscope observation | |
| Loget et al. | Direct visualization of symmetry breaking during janus nanoparticle formation | |
| JP6077757B2 (en) | Staining agent for electron microscope observation and staining method for sample for electron microscope observation | |
| Ogawa et al. | Formation and stability of cellulose–copper–NaOH crystalline complex | |
| JP6077735B2 (en) | Staining agent for electron microscope observation and staining method for sample for electron microscope observation | |
| Bailey et al. | Microswimmers from Scalable Galvanic Displacement | |
| CN104607656B (en) | A kind of ultra-long silver nanowire and preparation method thereof | |
| JP2009172574A (en) | Metal particle carrying catalyst and its manufacturing method | |
| Semenova et al. | A new route for SERS analysis of intact erythrocytes using polydisperse silver nanoplatelets on biocompatible scaffolds | |
| JP6325009B2 (en) | Method for producing nano colloidal particle carrier and carrier |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20151117 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20160810 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20160812 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20161011 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20170222 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20170301 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 6104703 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |