JP5631513B1 - Method and apparatus for manufacturing replica thin film for specimen of electron microscope - Google Patents

Abstract

【課題】生物試料の断面組織のレプリカを正確に形成することができる電子顕微鏡の検体用レプリカ薄膜の作製方法およびその装置を提供する。【解決手段】金属ガスの直流グロー放電による薄膜を堆積するガス反応容器１０の内部に、外形が半球状の凸型形状に形成される陽極１１と、椀状の凹面形状に形成される陰極１２とが、一定間隔をもって対向して設けられ、陰極１２側に試料４を保持する試料ホルダー２０が設けられている。この試料ホルダー２０の間隙部にカッターの刃３１を挿入して試料４の外周のみにカッターの刃３１を突き当てると同時に、試料ホルダー２０を展開することにより試料４を割断して表面に露出させ、その表面に薄膜を直流グロー放電により形成する。【選択図】図１An object of the present invention is to provide a method and apparatus for producing a replica thin film for a specimen of an electron microscope capable of accurately forming a replica of a cross-sectional tissue of a biological sample. A gas reaction vessel 10 for depositing a thin film by direct current glow discharge of a metal gas has an anode 11 formed in a convex shape having a hemispherical shape and a cathode 12 formed in a bowl-like concave shape. And a sample holder 20 that holds the sample 4 on the cathode 12 side. The cutter blade 31 is inserted into the gap of the sample holder 20 and the cutter blade 31 is abutted only on the outer periphery of the sample 4, and at the same time, the sample holder 20 is expanded to cleave the sample 4 and expose it to the surface. A thin film is formed on the surface by direct current glow discharge. [Selection] Figure 1

Description

本発明は、電子顕微鏡の検体用凍結試料割断面のレプリカ薄膜作製方法およびその作製装置に関する。さらに詳しくは、直流グロー放電法により四酸化オスミウムガスのプラズマ化オスミウム金属を堆積してレプリカ薄膜を作製するレプリカ薄膜の作製方法およびその作製装置に関する。   The present invention relates to a method for manufacturing a replica thin film of a frozen sample fracture section for a specimen of an electron microscope and a manufacturing apparatus therefor. More specifically, the present invention relates to a replica thin film manufacturing method and a manufacturing apparatus therefor, in which a replica thin film is manufactured by depositing an osmium tetroxide gas plasma metal by a direct current glow discharge method.

透過型電子顕微鏡用の高分解能レプリカ膜作製方法は、本発明者が提案した直流グロー放電法による有機化合物ガスのプラズマ重合被膜法が知られている（例えば特許文献１、２参照）。即ち、図５に直流グロー放電法の電極部位の概略図が示されるように、２枚の円板からなる陽極５１と陰極５２とが、４５ｍｍ程度の間隔で配置され、被膜される試料６０は、陰極５２の上に固定され、図示しないガス反応容器内を排気してから、ハイドロカーボンガスを規定のガス圧まで導入し、１〜１.５ｋＶ程度の直流電圧を両電極５１、５２間に印加することにより、グロー放電が発生し、例えばエチレンガスのプラズマ重合薄膜が陰極５２に置かれた試料６０の表面に付着して精密なレプリカ被膜を作製する。その被膜だけを透過型電子顕微鏡で検鏡すると、試料６０の表面微細構造を立体像で観察することができる。なお、図５で、５３は陽光柱、５４は負グロー相領域、５５はファラデー暗部である。   As a method for producing a high-resolution replica film for a transmission electron microscope, a plasma polymerization coating method of an organic compound gas by a direct current glow discharge method proposed by the present inventor is known (see, for example, Patent Documents 1 and 2). That is, as shown in the schematic diagram of the electrode part of the direct current glow discharge method in FIG. 5, the anode 51 and the cathode 52 made of two discs are arranged at an interval of about 45 mm, and the sample 60 to be coated is Then, after being fixed on the cathode 52 and evacuating a gas reaction vessel (not shown), a hydrocarbon gas is introduced to a specified gas pressure, and a DC voltage of about 1 to 1.5 kV is applied between the electrodes 51 and 52. When applied, glow discharge is generated, and for example, a plasma polymerized thin film of ethylene gas adheres to the surface of the sample 60 placed on the cathode 52 to produce a precise replica film. When only the coating film is examined with a transmission electron microscope, the surface fine structure of the sample 60 can be observed in a three-dimensional image. In FIG. 5, 53 is a positive column, 54 is a negative glow phase region, and 55 is a Faraday dark part.

一方、走査型電子顕微鏡の検体レプリカ膜もグロー放電で形成することを検討したが、検体の殆どは嵩張る試料が多くて負グロー相の浅い領域からはみ出すために、均一な超薄膜を得るのが難しい。   On the other hand, we examined that the specimen replica film of the scanning electron microscope is also formed by glow discharge. However, since most specimens are bulky and protrude from the shallow area of the negative glow phase, a uniform ultrathin film can be obtained. difficult.

また、生物試料の凍結割断面レプリカ法の初期は、２枚の対向円板型電極を用いて有機化合物ガスのプラズマ重合膜レプリカ法を用いていたが、装置が充分でなかったのと、負グロー相領域が浅くて全面均一被膜に無理があった。一方、常温試料装置は、極端に簡易化した卓上型のオスミウム被膜法の採用で急速に普及してきた。しかし、凍結割断面レプリカ法は取り残されている。   In the initial stage of the frozen section replica method for biological samples, the plasma polymerized film replica method of organic compound gas was used using two opposing disk-type electrodes. The glow phase region was shallow and the uniform coating on the entire surface was impossible. On the other hand, the room temperature sample apparatus has been rapidly spread by adopting an extremely simplified desktop osmium coating method. However, the frozen section replica method is left behind.

特許第２６９７７５３号公報Japanese Patent No. 2,697,753 特公昭６２−５５０９５号公報Japanese Examined Patent Publication No. 62-55095

前述のように、有機金属ガスの膜厚に限界がある以上、一桁も薄くなったオスミウムのプラズマ化金属被膜法であれば、硬度、密度、導電性において問題はなく、後は試料の処理法、凍結割断法、被膜法、検鏡法の過程をより合理的に行えば利用価値は充分あると思われる。   As mentioned above, there is no problem in hardness, density, and conductivity with the osmium plasma metal coating method, which has been reduced by an order of magnitude as long as the film thickness of the organometallic gas is limited. If the process of the method, freezing cleaving method, coating method, and microscopic method is performed more rationally, it seems that the utility value is sufficient.

本発明は、このような状況に鑑みてなされたもので、オスミウムのプラズマ化金属被膜を用いて、走査型電子顕微鏡用にも用い得ると共に、試料の凍結割断面の正確なレプリカ薄膜の作製方法および作製装置を提供することを目的とする。   The present invention has been made in view of such a situation, and can be used for a scanning electron microscope using a plasma metal film of osmium, and a method for producing a replica thin film with an accurate frozen section of a sample. Another object is to provide a manufacturing apparatus.

本発明の他の目的は、凍結試料の割断面を正確に露出させると共に、割断面の両側の断面を得ることができる試料ホルダーおよびその試料ホルダーに固定された試料を衝撃で割断することができる試料割断装置を提供することにある。   Another object of the present invention is to accurately expose a fractured surface of a frozen sample and to cleave a sample holder capable of obtaining both sides of the fractured surface and a sample fixed to the sample holder by impact. The object is to provide a sample cleaving apparatus.

本発明者は、嵩張る検体でも直流グロー放電法によりレプリカ薄膜を形成するために鋭意検討を重ねた結果、負グロー相領域を拡張する以外にはなく、専ら負グロー相領域を広げるための放電システムの検討を繰り返し重ねた。その結果、弱い発光の広領域を占める陽光柱領域に注目し、この領域を減少させる方法があれば、負グロー相領域は必ず広がる筈だと考えて実験を重ねた。その結果、放電電圧は、ガスの種類により陽光柱領域の電位分布、発光強度、定位置からのずれ等に変化があることを見出し、陽極面を小さくし、陰極面を大きくして対向させることにより、安定した均等な電界にするための電極形状と配置および電極間隔等を任意選択できる方法について種々考慮した結果、同心で、半球形状の大小の陰極と陽極を対向させることにより、極めて珍しい凹凸形の陰、陽両電極を作製した。そして、これにより、数年間苦しんだ末の問題を解決した。   The present inventor has conducted extensive studies to form a replica thin film by a direct current glow discharge method even for a bulky specimen, and as a result, the discharge system not only expands the negative glow phase region but also exclusively expands the negative glow phase region. The examination was repeated repeatedly. As a result, we focused on the positive column region that occupies a wide area of weak light emission, and if there was a way to reduce this region, we thought that the negative glow phase region would surely spread and repeated experiments. As a result, the discharge voltage is found to change in the potential distribution in the positive column region, emission intensity, deviation from a fixed position, etc. depending on the type of gas, and the anode surface is made smaller and the cathode surface made larger to face each other. As a result of various considerations regarding the electrode shape and arrangement for achieving a stable and uniform electric field, as well as methods for arbitrarily selecting the electrode spacing, concentric, hemispherical large and small cathodes are made to face each other and extremely uneven Both negative and positive electrodes were produced. And this solved the problem that suffered for several years.

本発明の電子顕微鏡の検体用レプリカ薄膜の作製方法は、外形が半球状の凸型形状の陽極と該陽極と同心で椀状の凹面形状に形成された陰極とを一定間隔で対向させ、検体用の試料を棒状に採取し、棒状に採取した前記試料を凍結して蝶番式で閉じた状態の試料ホルダーにセットし、または前記棒状の試料を蝶番式の試料ホルダーにセットしてから凍結して前記陰極の凹面の底部に載置し、前記試料ホルダーを展開すると共に、前記試料を割断し、該割断により露出した断面にグロー放電法によりレプリカ用金属薄膜を形成することを特徴とする。 The method for producing a replica thin film for a specimen of an electron microscope according to the present invention is such that a convex anode having a hemispherical outer shape and a cathode formed in a bowl-like concave shape concentrically with the anode face each other at a constant interval. samples of use were taken into a rod, and freeze the samples taken in a rod shape was set on the sample holder in the closed position by hinged, or a sample of the bar was frozen after setting the sample holder hinged was placed on the bottom portion of the concave surface of the cathode Te, while developing the sample holder, fractured the sample, to characterized in that that form the metal thin replica by glow discharge method to a cross section exposed by該割sectional that.

前記試料ホルダーのセッティングを、液体窒素に浸るようにして行い、前記棒状の試料の割断を、前記試料ホルダーの温度が−１２０℃〜−８０℃になった時点で行うことができる。   The sample holder can be set so as to be immersed in liquid nitrogen, and the rod-shaped sample can be cleaved when the temperature of the sample holder reaches −120 ° C. to −80 ° C.

前記試料の割断は、カッターの刃先を前記試料に突き当てると共に、前記試料ホルダーの閉じた状態の拘束力を開放して展開することにより行うことができる。また、さらに具体的には、前記試料ホルダーの割断を、前記カッターの刃を押出しバネにより突出させることにより行い、該カッターの刃を前記棒状の試料の表面に突き当てて前記試料の表面に切り込みを入れると同時に該カッターの刃を後退させることにより行い、かつ、該カッターの刃を前記試料の表面に突き当てる際に、前記試料ホルダーの２枚の板状部材を閉じた状態に保持する拘束糸を切断することにより前記試料ホルダーを展開させることが、簡単に、かつ、確実に試料を切断することができるため好ましい。 The cleaving of the sample can be performed by abutting the cutting edge of the cutter against the sample and opening the sample holder in a closed state and opening the sample holder. More specifically, the sample holder is cleaved by causing the blade of the cutter to protrude by an extrusion spring, and the blade of the cutter is abutted against the surface of the rod-shaped sample and cut into the surface of the sample. The restraint that holds the two plate-like members of the sample holder in a closed state when the cutter blade is abutted against the surface of the sample when the cutter blade is retreated. It is preferable to unfold the sample holder by cutting the yarn because the sample can be cut easily and reliably.

本発明の検体用レプリカ薄膜の作製装置は、ガス反応容器と、該ガス反応容器の内部に設けられ、外形が半球状の凸型の陽極と、該陽極と一定間隔をおいて対向し、前記陽極と同心に設けられた半球状の凹型内面を有する陰極と、前記陰極の凹面の底部に設けられ、蝶番式に固定された２枚の板状部材が閉じた状態で該２枚の板状部材に連通して挿入された試料が、前記２枚の板状部材が展開することにより割断される試料ホルダーと、前記陰極に着脱自在に取り付けられ、前記試料ホルダーの前記２枚の板状部材の間隙部からカッターの刃を挿入して前記試料を割断すると共に前記試料ホルダーを展開する試料割断具と、前記ガス反応容器内を真空にし得る排気装置と、前記ガス反応容器内に反応ガスを導入するガス導入パイプと、を有している。 The specimen replica thin film preparation apparatus of the present invention is provided with a gas reaction vessel, a convex anode having a hemispherical outer shape, facing the anode at a predetermined interval, and provided in the gas reaction vessel, A cathode having a hemispherical concave inner surface provided concentrically with the anode, and two plate-like members provided at the bottom of the concave surface of the cathode and fixed in a hinged manner in a closed state sample inserted in communication with the member, the the sample holder to be fractured by the two plate-like member is deployed, removably attached to the cathode, the two sheet members of the sample holder A cutter blade is inserted from the gap of the sample to cleave the sample, and the sample cleaving tool that expands the sample holder, an exhaust device that can evacuate the gas reaction vessel, and a reaction gas into the gas reaction vessel A gas introduction pipe to be introduced; There.

前記試料ホルダーが、一端縁側で蝶番状に軸心周りに回転自在に保持される２枚の板状部材と、前記２枚の板状部材が閉じられて重ね合せられるときその間に一定の間隙部を有すると共に、前記２枚の板状部材が閉じた状態に保持するように、前記間隙部を通って設けられる拘束糸と、拘束力がない場合に前記２枚の板状部材が展開して開くように設けられるバネ部材と、を有し、前記２枚の板状部材が閉じられた状態で該２枚の板状部材を貫通して試料を挿入し得る貫通孔が形成されていることが好ましい。The sample holder has two plate-like members that are rotatably held around the axis in a hinge shape on one edge side, and when the two plate-like members are closed and overlapped with each other, a fixed gap portion is provided between them. And the two plate-like members are unfolded when there is no restraining force, and a restraining thread provided through the gap so that the two plate-like members are held closed. A spring member provided so as to open, and a through hole is formed through which the sample can be inserted through the two plate-like members in a state where the two plate-like members are closed. Is preferred.

本発明の検体用レプリカ薄膜の作製装置に用いられる蝶番式の試料ホルダーは、一端縁側で蝶番状に軸心周りに回転自在に保持される２枚の板状部材と、前記２枚の板状部材が閉じられて重ね合せられるときその間に一定の間隙部を有すると共に、前記２枚の板状部材が閉じた状態に保持するように、前記間隙部を通って設けられる拘束糸と、拘束力がない場合に前記２枚の板状部材が展開して開くように設けられるバネ部材と、を有し、前記２枚の板状部材が閉じられた状態で該２枚の板状部材を貫通して試料を挿入し得る貫通孔が形成される構造になっている。   The hinge-type sample holder used in the specimen replica thin film manufacturing apparatus of the present invention has two plate-like members that are rotatably held around an axis in a hinge shape on one end edge side, and the two plate-like members. When the members are closed and overlapped with each other, there is a certain gap portion therebetween, and a restraining thread provided through the gap portion so as to hold the two plate-like members in a closed state, and a restraining force A spring member provided so that the two plate-like members are unfolded and opened when the two plate-like members are open, and the two plate-like members penetrate through the two plate-like members in a closed state. Thus, a through hole into which a sample can be inserted is formed.

本発明の検体用レプリカ薄膜の作製方法によれば、試料の外周部を叩くように切り込んで衝撃を与え、同時に試料ホルダーの２枚の板状部材を閉じた状態にしている拘束糸を切断することにより、２枚の板状部材が強い回転バネの力による展開力で試料が割断される。即ち、ナイフ（カッター）の刃は試料の周囲を叩きつけることによる０.５ｍｍ程度以下の切り込みだけで、後は試料を叩きつけることによる衝撃と、試料ホルダーが展開する際の引っ張り力とで割断されるため、割断面には、カッターの刃の傷などが入ることなく、非常にきれいな割断面となる。そして、その割断面に四酸化オスミウムのプラズマ化金属を堆積するため、非常に薄くて強靭なアモルファスのレプリカ被膜を得ることができる。   According to the method for producing a specimen replica thin film of the present invention, the sample is cut so as to strike the outer periphery of the sample, and an impact is applied. At the same time, the constraining yarn that closes the two plate-like members of the sample holder is cut. As a result, the two plate-like members are cleaved by the deployment force due to the force of the strong rotating spring. That is, the blade of the knife (cutter) is only cut by about 0.5 mm or less by hitting the periphery of the sample, and thereafter it is cleaved by the impact by hitting the sample and the pulling force when the sample holder is deployed. For this reason, the split section has a very clean split section without any scratches on the cutter blade. Then, since the plasma metal of osmium tetroxide is deposited on the fractured surface, a very thin and strong amorphous replica film can be obtained.

また、本発明の検体用レプリカ薄膜の作製装置によれば、グロー放電をさせる両電極を半球状の凸面とその凸面とほぼ同心の凹面とを対向させて形成しており、その凹面を陰極面としているため、負グロー相領域を陰極の表面上に陰極と陽極の間隔の８０％程度の高さの広い範囲で形成することができる。その結果、試料部分の厚さが厚くなっても全体に均一な薄膜を形成することができ、試料を試料ホルダーに挿入して、陰極面から試料の成膜面の位置が離れても均一な厚さの薄膜を形成することができる。そのため、試料ホルダーの貫通孔に試料を挿入して陰極上に載置した状態で試料ホルダーを展開すると共に試料を割断して、そのままの状態で薄膜を形成し、検鏡することができる。   Further, according to the specimen replica thin film manufacturing apparatus of the present invention, both electrodes for glow discharge are formed so that a hemispherical convex surface and a concave surface substantially concentric with the convex surface are opposed to each other, and the concave surface is formed on the cathode surface. Therefore, the negative glow phase region can be formed on the surface of the cathode in a wide range having a height of about 80% of the distance between the cathode and the anode. As a result, even if the thickness of the sample portion is increased, a uniform thin film can be formed on the entire surface. Even when the sample is inserted into the sample holder and the position of the film formation surface of the sample is separated from the cathode surface, it is uniform. A thin film can be formed. Therefore, the sample holder can be unfolded while the sample is inserted into the through hole of the sample holder and placed on the cathode, and the sample can be cleaved to form a thin film as it is, and can be microscopically examined.

本発明の試料ホルダーによれば、２枚の板状部材が一端縁側で回転自在に保持され、蝶番のような構造になっており、しかも、２枚の板状部材は拘束力がない場合に展開して開放するようにバネ部材（回転バネ）を介して取り付けられているため、拘束力となる固定手段（拘束糸）を解除することにより、容易に試料ホルダーを展開することができる。そのため、例えば試料ホルダーを閉じた（折り畳んだ）状態で、２枚の板状部材に貫通する貫通孔内に試料を挿入しておけば、試料ホルダーの固定手段を解除することにより、自動的に試料ホルダーが展開しようとし、その際に試料に若干の切込みを入れることにより試料が割断されて展開した２枚の板状部材の露出面に試料の割断面の両面が露出する。この貫通孔を２個（２組）形成しておくことにより、２つの試料を同時に割断して４個の断面にレプリカ被膜を形成することができる。   According to the sample holder of the present invention, when the two plate-like members are rotatably held at one edge side and have a hinge-like structure, and the two plate-like members have no binding force. Since it is attached via a spring member (rotating spring) so as to be unfolded when it is unfolded, the sample holder can be easily unfolded by releasing the fixing means (restraint thread) that becomes a restraining force. Therefore, for example, if the sample holder is closed (folded) and the sample is inserted into the through hole that penetrates the two plate-like members, the sample holder is automatically released by releasing the fixing means. The sample holder tries to expand, and at that time, the sample is cleaved by making a slight cut in the sample, and both surfaces of the divided section of the sample are exposed on the exposed surfaces of the two plate-shaped members that have been expanded. By forming two (two sets) of these through holes, two samples can be cleaved simultaneously to form a replica film on four cross sections.

本発明の検体用レプリカ薄膜の形成装置を用いて薄膜を形成する装置の概略図で、（ａ）は試料ホルダーを閉じた状態を示す図、（ｂ）は試料を割断して試料ホルダーを展開した状態を示す図である。BRIEF DESCRIPTION OF THE DRAWINGS It is the schematic of the apparatus which forms a thin film using the formation apparatus of the replica thin film for specimens of this invention, (a) is a figure which shows the state which closed the sample holder, (b) expands a sample holder by cleaving a sample. It is a figure which shows the state which carried out. 本発明のレプリカ薄膜の形成に用いる試料ホルダーの概略図で、（ａ）が２枚の板状部材を閉じた状態の図、（ｂ）が２枚の板状部材を展開した状態の図である。FIG. 2 is a schematic view of a sample holder used for forming a replica thin film of the present invention, in which (a) shows a state in which two plate-like members are closed, and (b) shows a state in which two plate-like members are developed. is there. 本発明のレプリカ薄膜形成方法に用いる試料割断装置の一例を示す斜視説明図である。It is perspective explanatory drawing which shows an example of the sample cleaving apparatus used for the replica thin film formation method of this invention. 図１の陽極および陰極の部分の構造を主体的に示した断面説明図である。FIG. 2 is a cross-sectional explanatory view mainly showing the structure of the anode and cathode portions of FIG. 1. 従来のグロー放電の状態を説明する図である。It is a figure explaining the state of the conventional glow discharge.

つぎに、図面を参照しながら本発明の電子顕微鏡の検体用レプリカ薄膜の作製方法、作製装置およびその作製に用いられる試料ホルダーと試料割断装置について説明をする。   Next, a method for manufacturing a replica thin film for a specimen of an electron microscope according to the present invention, a manufacturing apparatus, and a sample holder and a sample cleaving apparatus used for the manufacturing will be described with reference to the drawings.

本発明の作製装置は、図１に一実施形態の概略図が示されるように、ガス反応容器１０内の上方に小型の半球型陽極１１を配置し、例えば４０ｍｍ程度の間隔をおいて下方に、陽極１１と同心で凹状の陰極１２を対向させて配置されている。この状態で直流グロー放電をさせると、図４に断面説明図が示されるように、均一な負グロー相領域４１が、陽極１１と陰極１２との間隔の８０％程度の広い領域で形成された。なお、図４において、４２は陽光柱である。   The production apparatus of the present invention has a small hemispherical anode 11 arranged in the upper part of the gas reaction vessel 10 as shown in a schematic diagram of one embodiment in FIG. The concave cathode 12 concentric with the anode 11 is arranged opposite to the anode 11. When a direct current glow discharge is performed in this state, a uniform negative glow phase region 41 is formed in a wide region of about 80% of the distance between the anode 11 and the cathode 12 as shown in the cross-sectional explanatory diagram of FIG. . In FIG. 4, reference numeral 42 denotes a positive column.

図１において、１１ａ、１２ａは、それぞれ陽極１１、陰極１２に電圧を印加するためのリード線、１３は試料ステージ（試料載置台）、１４は四酸化オスミウムなどの反応ガスを導入するガス導入パイプ、１５はガス反応容器１０内を低圧にするため、図示しないロータリーポンプなどの排気装置と接続する排気管、１９は絶縁板である。この陰極１２の底面に凹部１６（図４参照）が形成され、その凹部１６内に試料ステージ１３が置かれ、その試料ステージ１３に、試料４を挿入した蝶番式の試料ホルダー２０が載置され、割断装置３０により、試料４を割断できる構成になっている。   In FIG. 1, 11a and 12a are lead wires for applying a voltage to the anode 11 and the cathode 12, respectively, 13 is a sample stage (sample mounting table), and 14 is a gas introduction pipe for introducing a reaction gas such as osmium tetroxide. , 15 is an exhaust pipe connected to an exhaust device such as a rotary pump (not shown) in order to make the gas reaction vessel 10 low in pressure, and 19 is an insulating plate. A recess 16 (see FIG. 4) is formed on the bottom surface of the cathode 12, a sample stage 13 is placed in the recess 16, and a hinged sample holder 20 into which the sample 4 is inserted is placed on the sample stage 13. The sample 4 can be cleaved by the cleaving device 30.

蝶番式の試料ホルダー２０は、図２に、２つの板状部材２１、２２を閉じた状態の図（ａ）と、２つの板状部材２１、２２が開いて展開した状態の図（ｂ）がそれぞれ示されるように、例えば厚さが８ｍｍ程度で、２０ｍｍ角程度の大きさの２つの板状部材２１、２２の一端縁部で蝶番のように、支軸２３の周りに回転自在に固定されると共に、強い回転バネ２８により、拘束力がなければ展開して開く（図２（ｂ）参照）ようになっている。そのため、２枚の板状部材２１、２２が閉じた状態（図２（ａ）参照）を維持できるように、図示しないクランプで挟むための溝２１ａ、２２ａが形成されている。さらに、２枚の板状部材２１、２２が閉じた状態のときに、２枚の板状部材２１、２２の間に、例えば２ｍｍ程度の間隙部２７が形成されるように、ストッパ２６が形成されている。   The hinge type sample holder 20 is shown in FIG. 2 (a) in a state in which the two plate-like members 21 and 22 are closed, and in a state in which the two plate-like members 21 and 22 are opened and unfolded (b). As shown, each of the two plate-like members 21 and 22 having a thickness of about 8 mm and a size of about 20 mm square, for example, is fixed to be rotatable around the support shaft 23 like a hinge. At the same time, the strong rotating spring 28 opens and opens when there is no restraining force (see FIG. 2B). Therefore, grooves 21a and 22a are formed to be sandwiched by clamps (not shown) so that the two plate-like members 21 and 22 can be kept closed (see FIG. 2A). Further, when the two plate-like members 21 and 22 are closed, a stopper 26 is formed so that a gap portion 27 of about 2 mm, for example, is formed between the two plate-like members 21 and 22. Has been.

また、図２（ａ）に示されるように、２枚の板状部材２１、２２が閉じた状態で、２枚の板状部材２１、２２を貫通する貫通孔２４が、例えば２ｍｍ程度の直径で形成され、その貫通孔２４に棒状の試料４を挿入できるように形成されている。さらに、試料４を挿入する作業時などでは、前述のように、閉じた状態を、図示しないクランプで保持することができるが、後述するように、遠隔操作によりこの２枚の板状部材２１、２２を展開するように拘束力を解除するために、図２（ａ）に示されるように、拘束糸２５を通す溝２５ａが形成されている。即ち、作業中は、図示しないクランプにより閉じた状態を保持しながら、最終的に陰極１２の底部にセッティングする際には、この拘束糸２５により閉じた状態を保持し、クランプは除去する。   Further, as shown in FIG. 2 (a), when the two plate-like members 21 and 22 are closed, the through-hole 24 that penetrates the two plate-like members 21 and 22 has a diameter of about 2 mm, for example. The rod-shaped sample 4 can be inserted into the through hole 24. Further, when the sample 4 is inserted, the closed state can be held by a clamp (not shown) as described above. However, as will be described later, the two plate-like members 21, In order to release the restraining force so as to unfold 22, as shown in FIG. 2A, a groove 25 a through which the restraining thread 25 is passed is formed. In other words, during the operation, while holding the closed state by a clamp (not shown), when finally setting the bottom of the cathode 12, the closed state is held by the restraining thread 25, and the clamp is removed.

この試料ホルダー２０に挿入する試料４は、例えば直径が２ｍｍ程度の棒状に採取したり、外径が２ｍｍ程度で、厚さが０.２ｍｍ程度の細いプラスティック細管に試料を詰め込んだりして形成され、この試料を予め凍結して試料ホルダー２０の貫通孔２４に挿入しても良いし、試料ホルダー２０の貫通孔２４に挿入してから、試料ホルダー２０ごと凍結しても良い。   The sample 4 to be inserted into the sample holder 20 is formed, for example, by collecting it in a rod shape having a diameter of about 2 mm, or packing the sample into a thin plastic tube having an outer diameter of about 2 mm and a thickness of about 0.2 mm. The sample may be frozen in advance and inserted into the through hole 24 of the sample holder 20, or the sample holder 20 may be frozen after being inserted into the through hole 24 of the sample holder 20.

試料割断装置３０は、例えば図１および図３に示されるように、例えば全長が約１０ｃｍで、カッターカバー３４の直径が約１２ｍｍの大きさで、カッターの刃３１が水平面に対して約５０度に傾斜した位置で、カッター支持枠３６により、陰極１２の上部に取り付けられるようになっている。カッター支持棒３７の支持筒３７ａを押して、カッターカバー３４を水平にすることにより、カッター部分を陰極１２の外側に回転させることができ、試料４の割断後に試料割断装置３０を、図１（ｂ）に示されるように陰極１２の外側の絶縁板１９上に移動させることができる。   For example, as shown in FIGS. 1 and 3, the sample cleaving apparatus 30 has a total length of about 10 cm, a diameter of the cutter cover 34 of about 12 mm, and the cutter blade 31 is about 50 degrees with respect to the horizontal plane. It is attached to the upper part of the cathode 12 by a cutter support frame 36 at a position inclined to the upper side. By pressing the support cylinder 37a of the cutter support rod 37 and leveling the cutter cover 34, the cutter portion can be rotated to the outside of the cathode 12, and the sample cleaving device 30 can be operated as shown in FIG. As shown in FIG. 4, the light can be moved onto the insulating plate 19 outside the cathode 12.

カッターの外部操作用の細い操作棒（図示せず）が中心部にある。また、カッターの刃３１は、試料ホルダー２０に近接しており、５ｍｍ程度突出させて試料４に０.２〜０.５ｍｍ程度切り込むようになっている。このカッターの刃３１は、ハンマーリング３２の先端部に固定されている。ハンマーリング３２は、強い押出しバネ３３により一定距離だけ押し出される構造になっている。通常は、このハンマーリング３２は、押出しバネ操作板３３ａにより、押出しバネ３３の付勢力に抗して引っ込んだ状態になっている。即ち、押出しバネ操作板３３ａを外部から解除することにより、カッターの刃３１が押し出されて、試料ホルダー２０の拘束糸２５を切断すると共に、試料４に強い衝撃と僅かな切り込みが入り、拘束糸２５の切断による試料ホルダー２０の２枚の板状部材２１、２２の展開と相俟って試料４が割断される。この浅い切り込みが入れられるのと同時に、カッターの刃３１は、図示しない細い引きバネにより、元の位置に引き戻される。従って、試料４の割断面には、カッターの刃３１の傷跡が入らず、しかも、割断した両面が２枚の板状部材２１、２２の両方に現れる。なお、３５は回転軸、３７は支持棒、３８は上下移動バネである。このバネ３８などは図１では簡略化のため省略してある。   A thin operation rod (not shown) for external operation of the cutter is at the center. The cutter blade 31 is close to the sample holder 20 and protrudes about 5 mm so as to cut about 0.2 to 0.5 mm into the sample 4. The cutter blade 31 is fixed to the tip of the hammer ring 32. The hammer ring 32 is structured to be pushed out by a strong distance by a strong pushing spring 33. Normally, the hammer ring 32 is retracted against the biasing force of the push spring 33 by the push spring operating plate 33a. That is, by releasing the push-out spring operation plate 33a from the outside, the cutter blade 31 is pushed out to cut the restraint thread 25 of the sample holder 20, and a strong impact and a slight notch are applied to the sample 4, and the restraint thread In combination with the development of the two plate-like members 21 and 22 of the sample holder 20 by cutting 25, the sample 4 is cleaved. At the same time that the shallow cut is made, the cutter blade 31 is pulled back to its original position by a thin pulling spring (not shown). Accordingly, the scar of the cutter blade 31 does not enter the fractured surface of the sample 4, and the both sides that have been cleaved appear on both of the two plate-like members 21 and 22. Reference numeral 35 denotes a rotating shaft, 37 denotes a support rod, and 38 denotes a vertical movement spring. The spring 38 and the like are omitted in FIG. 1 for simplification.

また、陰極１２の底部の凹部１６内には、試料ステージ１３が設けられ、その内部に試料ホルダー２０と接するように設けられた温度センサ１７および筒型の加熱用ヒータ１８が設けられている。この凹部１６は、例えば直径が３０ｍｍ程度、深さが８ｍｍ程度に形成され、後述する試料ホルダー２０が展開した状態で入る大きさに形成されている。試料ホルダー２０のセッティングが完了しても液体窒素が蒸発しないで残っていたり、試料ホルダー２０の温度が所定の温度まで上昇していなかったりした場合には、加熱用ヒータ１８を点灯して温度を上昇させることができるようになっている。このような冷却および加熱手段が形成されていることにより、試料ホルダー２０の温度を正確に制御することができる。例えば、試料４を割断する際の試料ホルダー２０の温度が、凍結した−１９０℃程度から例えば−１００℃±２０℃になるように温度が調整される。この温度は、試料４の種類や密度などに応じて選択される。   A sample stage 13 is provided in the recess 16 at the bottom of the cathode 12, and a temperature sensor 17 and a cylindrical heating heater 18 provided in contact with the sample holder 20 are provided therein. The recess 16 is formed to have a diameter of about 30 mm and a depth of about 8 mm, for example, and is formed to have a size that allows the sample holder 20 to be described later to be opened. Even if the setting of the sample holder 20 is completed, if liquid nitrogen does not evaporate or remains, or if the temperature of the sample holder 20 has not risen to a predetermined temperature, the heater 18 is turned on to adjust the temperature. It can be raised. By forming such cooling and heating means, the temperature of the sample holder 20 can be accurately controlled. For example, the temperature of the sample holder 20 when cleaving the sample 4 is adjusted such that the temperature is about −100 ° C. ± 20 ° C. from the frozen −190 ° C. This temperature is selected according to the type and density of the sample 4.

この装置でレプリカを作製する方法について説明する。生体の凍結用試料採取は、直径が約２ｍｍの棒状にして液体窒素に投入する。試料ホルダー２０の溝２１ａ、２２ａをクランプで挟んで２枚の板状部材２１、２２を閉じた状態にし、その貫通孔２４（図２参照）に、凍結した試料４を挿入する。なお、この状態で、拘束糸２５でクランプを外しても閉じた状態を維持できるようにする。この試料ホルダー２０を、予め陰極１２の底部の凹部１６内に液体窒素と共に配設された試料ステージ１３にセッティングする。そして、クランプを外して、試料ホルダー２０の間隙部２７内にその拘束糸２５が現れるようにし、カッターの刃３１がその間隙部２７に一定の距離をおいて入るよう試料割断装置３０をセッティングする。凹部（冷却ホール）１３内の液体窒素がなくなるのを待って、ガス反応容器１０のベルジャーを閉じ、真空排気を始める。   A method for producing a replica with this apparatus will be described. In order to collect a sample for freezing a living body, a rod having a diameter of about 2 mm is put into liquid nitrogen. The two plate-like members 21 and 22 are closed with the grooves 21a and 22a of the sample holder 20 held between clamps, and the frozen sample 4 is inserted into the through hole 24 (see FIG. 2). In this state, the closed state can be maintained even when the clamp is removed with the restraining thread 25. This sample holder 20 is set in advance on a sample stage 13 disposed with liquid nitrogen in the recess 16 at the bottom of the cathode 12. Then, the clamp is removed so that the restraining thread 25 appears in the gap 27 of the sample holder 20, and the sample cleaving device 30 is set so that the cutter blade 31 enters the gap 27 with a certain distance. . After the liquid nitrogen in the recess (cooling hole) 13 disappears, the bell jar of the gas reaction vessel 10 is closed and evacuation is started.

この状態で、温度センサにより試料ホルダー２０の温度を測定し、温度が低すぎる場合はヒータ１８を点滅して温度を上昇させ、−１００℃±２０℃になれば、試料割断装置３０を作動させて試料ホルダー２０の２枚の板状部材２１、２２を展開すると共に試料４を割断する。その後、試料割断装置３０を回転させて、陰極１２の外側に降し、絶縁板１９の定位置に移す。この絶縁板１９には、角溝が形成されており、カッター支持枠３６が嵌め込まれるように形成されている。また、カッター支持枠３６は、操作棒３７で半回転するだけで簡単に陰極１２から外れて着脱自在に保持されているため操作は容易である。試料割断の際に飛散した微細な氷晶は、排気により蒸発しながら除去される。これらの操作と同時に重要なことは、生体の凍結割断面の氷を除去して試料構造を露出させる操作であり、数ｎｍの浅いものから、検鏡目的によって、数十μｍの深いエッチングをした後、グロー放電による四酸化オスミウムガスのプラズマ化金属堆積レプリカ被膜の形成を行うことである。   In this state, the temperature of the sample holder 20 is measured by the temperature sensor. If the temperature is too low, the heater 18 is flashed to increase the temperature, and when it reaches −100 ° C. ± 20 ° C., the sample cleaving device 30 is operated. Then, the two plate-like members 21 and 22 of the sample holder 20 are developed and the sample 4 is cleaved. Thereafter, the sample cleaving device 30 is rotated and lowered to the outside of the cathode 12 and moved to a fixed position of the insulating plate 19. The insulating plate 19 is formed with a square groove so that the cutter support frame 36 is fitted therein. Further, the cutter support frame 36 is easily detached from the cathode 12 by being only half-turned by the operation rod 37 and is easily detachably held. The fine ice crystals scattered during the cleaving of the sample are removed while being evaporated by the exhaust. What is important at the same time with these operations is the operation to remove the ice on the frozen section of the living body to expose the sample structure. From a shallow one of several nm, deep etching of several tens of μm was performed depending on the speculum purpose. Thereafter, formation of a plasma deposition metal deposition replica film of osmium tetroxide gas by glow discharge is performed.

その後、ガス反応容器１０内の圧力が１Ｐａになれば、主バルブを絞って４Ｐａに安定させ、昇華筒内で昇華した四酸化オスミウムを一定量、ガス導入パイプ１４の調整によりガス反応容器１０内に導入し、７Ｐａで安定したらグロー放電をオンにする。その結果、１〜１０秒（膜厚０.５ｎｍ／秒）で所望の厚さの超薄膜が得られる。この場合のグロー放電電圧は、約１ｋＶである。所定時間を経てグロー放電がオフになれば、直ちに昇華筒のノズルを閉じ、主排気バルブを全開にして、ガス反応容器１０内の残留オスミウムガスを完全に排気してから、主排気バルブを閉じてガス反応容器１０内を開放する。２枚の板状部材２１、２２が展開した試料ホルダー２０をそのまま取り出し、走査型電子顕微鏡の試料台にセットして検鏡すれば、双面レプリカの立体像および超高倍率像を観察することができる。   Thereafter, when the pressure in the gas reaction vessel 10 becomes 1 Pa, the main valve is throttled to stabilize to 4 Pa, and a certain amount of osmium tetroxide sublimated in the sublimation cylinder is adjusted in the gas reaction vessel 10 by adjusting the gas introduction pipe 14. When it is stabilized at 7 Pa, the glow discharge is turned on. As a result, an ultrathin film having a desired thickness can be obtained in 1 to 10 seconds (film thickness 0.5 nm / second). In this case, the glow discharge voltage is about 1 kV. When the glow discharge is turned off after a predetermined time, the nozzle of the sublimation tube is immediately closed, the main exhaust valve is fully opened, the residual osmium gas in the gas reaction vessel 10 is exhausted completely, and then the main exhaust valve is closed. The gas reaction vessel 10 is opened. If the sample holder 20 on which the two plate-like members 21 and 22 are spread out is taken out as it is and set on the sample stage of the scanning electron microscope and examined, a stereoscopic image and an ultrahigh magnification image of the double-faced replica can be observed. Can do.

この四酸化オスミウムは、アンプルに封入された四酸化オスミウム結晶（ＯｓＯ₄）の０.５ｇを用いるが、大気中に放出した場合は、強烈な刺激臭を発散するので、慎重に取り扱う必要がある。ガス導入パイプ１４はオスミウム昇華筒室にあり、アンプルをセットし、筒内を真空にしてからアンプルを割断して昇華筒のノズルによって一定量をガス反応容器１０内に導入する。また、この前後に流す生ガスの大半は排気用ロータリーポンプのオイルに付着して酸化し、オイルを黒く汚染する。素通りした生ガスはポンプの排気口から放出して強い匂いを出すのでポンプの排気口は必ずドラフトに差し込むことになっている。それができなければオスミウムガス専用の濾過装置を通す必要がある。要は、ガス反応容器の排気口にオスミウムガス専用の濾過装置を直結して、その後にロータリーポンプを接続する必要がある。そうすれば、オイルの汚染も、生ガスの放出も確実になくすることができる。この濾過装置で排気効率が落ちることはない。オスミウム専用の濾過装置は小型の筒状（直径７０ｍｍ、長さ１３０ｍｍ）で実用化されている。 This osmium tetroxide uses 0.5 g of osmium tetroxide crystals (OsO ₄ ) enclosed in ampoules, but when released into the atmosphere, it emits a strong irritating odor and must be handled with care. . The gas introduction pipe 14 is in the osmium sublimation cylinder chamber. An ampule is set, the inside of the cylinder is evacuated, the ampule is cleaved, and a predetermined amount is introduced into the gas reaction vessel 10 by the nozzle of the sublimation cylinder. In addition, most of the raw gas flowing before and after this adheres to the oil of the exhaust rotary pump and oxidizes, contaminating the oil black. The raw gas passed through is discharged from the exhaust port of the pump and gives off a strong odor, so the exhaust port of the pump must be inserted into the draft. If this is not possible, it is necessary to pass a filtration device dedicated to osmium gas. In short, it is necessary to connect a filtration device dedicated to osmium gas directly to the exhaust port of the gas reaction vessel and then connect a rotary pump. By doing so, it is possible to reliably eliminate oil contamination and release of raw gas. Exhaust efficiency is not reduced by this filtering device. An osmium-only filtering device is practically used in a small cylindrical shape (diameter 70 mm, length 130 mm).

有機化合物ガスに関しては、従来は主にエチレン（ＣＨ₂＝ＣＨ₂）とナフタリン（Ｃ₁₀Ｈ₈）が用いられていたが、その他のガスの各々の特徴を調べているうちに、有機ガスのプラズマ重合膜は約数十ｎｍの膜厚になると生体膜と被膜のコントラストがなくなり、それ以下に薄くすると被膜の強度が無くなるという欠点があり、これが有機ガスを用いる場合の限界だと考えられ、これ以下に薄くするにはオスミウム薄膜の領域だと考えたことに基づく。そのオスミウム膜の威力は、文献でも証明されている。最近のオスミウム超薄膜を用いた研究によると、シリカナノ粒子を１５万倍の立体像観察に成功した例、オージェ分析（ＡＥＳ）に２ｎｍの被膜を帯電防止膜にして成功した例、電子後方散乱回折法（ＥＢＳＤ）でチタン酸ジルコン酸鉛の多結晶体には０.２５ｎｍの被膜で分析可能にした例、などの研究成果を知って充分納得することができる。 As for organic compound gas, ethylene (CH ₂ ═CH ₂ ) and naphthalene (C ₁₀ H ₈ ) have been mainly used in the past. However, while investigating the characteristics of other gases, When the plasma polymerized film has a film thickness of about several tens of nanometers, the contrast between the biological film and the film disappears, and when it is reduced below that, the film strength is lost, which is considered to be the limit when using organic gas, To make it thinner than this is based on the idea that it is an osmium thin film region. The power of the osmium film has been proven in the literature. According to recent research using ultra-thin osmium films, examples of successful 150,000-fold stereoscopic image observation of silica nanoparticles, examples of successful Auger analysis (AES) with a 2 nm coating as an antistatic film, electron backscatter diffraction We can fully understand the results of research such as an example in which a polycrystal of lead titanate zirconate can be analyzed with a 0.25 nm film by the method (EBSD).

４ 試料
１０ ガス反応容器
１１ 陽極
１２ 陰極
１３ 試料載置台
１４ ガス導入パイプ
１５ 排気管
１６ 凹部
１７ 温度センサ
１８ ヒータ
１９ 絶縁板
２０ 試料ホルダー
２１、２２ 板状部材
２３ 回転軸部
２４ 試料挿入孔
２５ 固定手段（拘束糸）
２５ａ 固定手段挿入孔
２６ ストッパ
２７ 間隙部
２８ バネ部材
３０ 試料割断具
３１ カッターの刃
３２ ハンマーリング（押出し部材）
３３ 押出しバネ（押出し手段）
３４ カッターカバー
３５ 回転軸
３６ カッター支持枠
３７ カッター操作軸
３７ａ カッター層軸カバー兼支持筒
３８ バネ 4 Sample 10 Gas reaction vessel 11 Anode 12 Cathode 13 Sample mounting table 14 Gas introduction pipe 15 Exhaust pipe 16 Recess 17 Temperature sensor 18 Heater 19 Insulating plate 20 Sample holder 21, 22 Plate member 23 Rotating shaft portion 24 Sample insertion hole 25 Fixed Means (restraint thread)
25a Fixing means insertion hole 26 Stopper 27 Gap 28 Spring member 30 Sample cleaving tool 31 Cutter blade 32 Hammer ring (extrusion member)
33 Extrusion spring (extrusion means)
34 Cutter cover 35 Rotating shaft 36 Cutter support frame 37 Cutter operation shaft 37a Cutter layer shaft cover / support cylinder 38 Spring

Claims (8)

外形が半球状の凸型形状の陽極と該陽極と同心で椀状の凹面形状に形成された陰極とを一定間隔で対向させ、
検体用の試料を棒状に採取し、棒状に採取した前記試料を凍結して蝶番式で閉じた状態の試料ホルダーにセットし、または前記棒状の試料を蝶番式の試料ホルダーにセットしてから凍結して前記陰極の凹面の底部に載置し、
前記試料ホルダーを展開すると共に、前記試料を割断し、
該割断により露出した断面にグロー放電法によりレプリカ用金属薄膜を形成する
ことを特徴とする電子顕微鏡の検体用レプリカ薄膜の作製方法。 A convex-shaped anode having a hemispherical outer shape and a cathode formed in a bowl-like concave shape concentrically with the anode are made to face each other at regular intervals.
Samples were taken for the specimen into bars, frozen and freeze the samples taken in a rod shape was set on the sample holder in the closed position by hinged, or a sample of the bar from the set in the sample holder of the hinged And placed on the bottom of the concave surface of the cathode,
While unfolding the sample holder, cleaving the sample ,
The method for manufacturing a sample replica thin film of the electron microscope, characterized in that that form the metal thin replica by glow discharge method to a cross section exposed by該割cross. 前記陰極の凹面底部に形成した凹部内に液体窒素を注入し、該凹部の上に前記試料ホルダーを載置し、液体窒素を蒸発させて所定の温度にした後に、前記凍結した試料を割断する請求項１記載の電子顕微鏡の検体用レプリカ薄膜の作製方法。 Liquid nitrogen is injected into a recess formed in the bottom of the concave surface of the cathode, the sample holder is placed on the recess, the liquid nitrogen is evaporated to a predetermined temperature, and then the frozen sample is cleaved. A method for producing a replica thin film for a specimen of an electron microscope according to claim 1 . 前記試料ホルダーのセッティングを、液体窒素に浸るようにして行い、前記棒状の試料の割断を、前記試料ホルダーの温度が−１２０℃〜−８０℃になった時点で行う請求項１または２記載の電子顕微鏡の検体用レプリカ薄膜の作製方法。 3. The sample holder according to claim 1 or 2 , wherein the setting of the sample holder is performed so as to be immersed in liquid nitrogen, and the cutting of the rod-shaped sample is performed when the temperature of the sample holder reaches -120 ° C to -80 ° C. A method for producing a replica thin film for an electron microscope specimen. 前記試料の割断を、カッターの刃先を前記試料に突き当てると共に、前記試料ホルダーの閉じた状態の拘束力を開放して展開することにより行う請求項１〜３のいずれか１項に記載の検体用レプリカ薄膜の作製方法。 The specimen according to any one of claims 1 to 3, wherein the specimen is cleaved by abutting a cutting edge of a cutter against the specimen and releasing the specimen holder in a closed state to expand the specimen. Method for manufacturing a replica thin film. 前記試料ホルダーの展開および前記試料の割断を、カッターの刃を押出しバネにより突出させることにより該カッターの刃を前記棒状の試料の表面に突き当てて前記試料の表面に切り込みを入れると同時に該カッターの刃を後退させることにより行い、かつ、該カッターの刃を前記試料の表面に突き当てる際に、前記試料ホルダーの２枚の板状部材を閉じた状態に保持する拘束糸を切断することにより前記試料ホルダーを展開させることにより行う請求項４記載の電子顕微鏡の検体用レプリカ薄膜の作製方法。 The development and the cleaving of the sample in the sample holder, the incision blades Ri該 cutter by the blade mosquito Potter to be protruded by extruding a spring on the surface of the abutment in the sample on the surface of the sample of the bar At the same time, the cutter blade is retracted, and when the cutter blade is abutted against the surface of the sample, the restraining yarn that holds the two plate-like members of the sample holder closed is cut. The method for producing a replica thin film for a specimen of an electron microscope according to claim 4, wherein the specimen holder is developed by doing so . ガス反応容器と、該ガス反応容器の内部に設けられ、外形が半球状の凸型の陽極と、該陽極と一定間隔をおいて対向し、前記陽極と同心に設けられた半球状の凹型内面を有する陰極と、前記陰極の凹面の底部に設けられ、蝶番式に固定された２枚の板状部材が閉じた状態で該２枚の板状部材に連通して挿入された試料が、前記２枚の板状部材が展開することにより割断される試料ホルダーと、前記陰極に着脱自在に取り付けられ、前記試料ホルダーの前記２枚の板状部材の間隙部からカッターの刃を挿入して前記試料を割断すると共に前記試料ホルダーを展開する試料割断具と、前記ガス反応容器内を真空にし得る排気装置と、前記ガス反応容器内に反応ガスを導入するガス導入パイプと、を有する検体用レプリカ薄膜の作製装置。 A gas reaction vessel; a hemispherical convex anode provided inside the gas reaction vessel; and a hemispherical concave inner surface facing the anode at a predetermined interval and concentrically with the anode And a sample inserted in communication with the two plate-like members in a state in which the two plate-like members provided at the bottom of the concave surface of the cathode and fixed in a hinged manner are closed, A sample holder that is cleaved when the two plate-shaped members are developed , and a detachable attachment to the cathode, and a cutter blade is inserted through a gap between the two plate-shaped members of the sample holder, A specimen replica having a sample cleaving tool for cleaving a sample and developing the sample holder, an exhaust device capable of evacuating the gas reaction vessel, and a gas introduction pipe for introducing a reaction gas into the gas reaction vessel Thin film production equipment. 前記試料ホルダーが、一端縁側で蝶番状に軸心周りに回転自在に保持される２枚の板状部材と、前記２枚の板状部材が閉じられて重ね合せられるときその間に一定の間隙部を有すると共に、前記２枚の板状部材が閉じた状態に保持するように、前記間隙部を通って設けられる拘束糸と、拘束力がない場合に前記２枚の板状部材が展開して開くように設けられるバネ部材と、を有し、前記２枚の板状部材が閉じられた状態で該２枚の板状部材を貫通して試料を挿入し得る貫通孔が形成されてなる請求項６記載の検体用レプリカ薄膜の作製装置。 The sample holder has two plate-like members that are rotatably held around the axis in a hinge shape on one edge side, and when the two plate-like members are closed and overlapped with each other, a fixed gap portion is provided between them. And the two plate-like members are unfolded when there is no restraining force, and a restraining thread provided through the gap so that the two plate-like members are held closed. And a spring member provided so as to open, and a through hole is formed through which the sample can be inserted through the two plate-like members while the two plate-like members are closed. Item 7. An apparatus for producing a replica thin film for specimen according to Item 6 . 検体用レプリカ薄膜の作製装置に用いられる蝶番式の試料ホルダーであって、一端縁側で蝶番状に軸心周りに回転自在に保持される２枚の板状部材と、前記２枚の板状部材が閉じられて重ね合せられるときその間に一定の間隙部を有すると共に、前記２枚の板状部材が閉じた状態に保持するように、前記間隙部を通って設けられる拘束糸と、拘束力がない場合に前記２枚の板状部材が展開して開くように設けられるバネ部材と、を有し、前記２枚の板状部材が閉じられた状態で該２枚の板状部材を貫通して試料を挿入し得る貫通孔が形成されてなる試料ホルダー。 A hinge-type sample holder used in a replica thin film manufacturing apparatus for a specimen, comprising two plate-like members rotatably held around an axis in a hinge shape on one edge side, and the two plate-like members And a constraining force provided through the gap so that the two plate-like members are held in a closed state while the two sheets are closed. A spring member provided so that the two plate-like members are unfolded and opened when there is not, and the two plate-like members pass through the two plate-like members in a closed state. A sample holder in which a through hole into which a sample can be inserted is formed.

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