JPH075375A - Living body sample vessel for x-ray observation - Google Patents

Abstract

PURPOSE:To replace a sample and to add a reagent simply and to facilitate the observation of the degree of damage of a living body sample by irradiation with X-rays and to lessen the attenuation of X-rays in mid-way by providing a part of a housing with a hole communicating with a space for insertion of a sample vessel and a cap closing this hole. CONSTITUTION:Spacers 4 are discontinuous and do not act as a partition wall. The space 3' is also filled with a sample soln. The living body sample chamber is installed in an X-ray microscope, etc., in the state of hermetically closing the space 3' with a threaded cap 10. The living body sample in the soln. is irradiated with X-rays through the window 1 under a vacuum. The sample chamber is thereafter taken out into the space and the cap 10 is exchanged with the threaded cap with a pipe so that the replacement of the sample soln. and the addition of a reagent are executed. Since the living body sample is directly put into a vacuum vessel, the attenuation of the X-rays is lessened and further, the living body sample reacts through the threaded cap with the pipe under the optical microscope and the degree of the reaction of the living body sample is observed, by which the degree of the damage of the sample by the irradiation with the X-rays is known.

Description

【発明の詳細な説明】Detailed Description of the Invention

【０００１】[0001]

【産業上の利用分野】この発明は、Ｘ線観察用の生体試
料槽に関するものである。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biological sample tank for X-ray observation.

【０００２】[0002]

【従来の技術】近年の生物工学技術の発展は、光学顕微
鏡や電子顕微鏡などの観察手段に負うところが大きい。
ところが、光学顕微鏡は生きた生体試料を扱えるのが特
長であるが、可視光の波長に空間分解能が制限される欠
点がある。一方、電子顕微鏡は空間分解能は高いが、真
空中に試料を置かなければならず、電子線が透過する窓
材が存在しないために生きたままでは生体試料を観察で
きなかった。2. Description of the Related Art Recent developments in biotechnological technology largely depend on observation means such as optical microscopes and electron microscopes.
However, although the optical microscope has a feature that it can handle a living biological sample, it has a drawback that the spatial resolution is limited to the wavelength of visible light. On the other hand, the electron microscope has a high spatial resolution, but the sample must be placed in a vacuum, and the biological sample cannot be observed alive because there is no window material through which the electron beam passes.

【０００３】そこで、生きたままの生体試料を高分解能
で観察できる可能性を持つＸ線顕微鏡が注目され、その
開発が進められており、微細精密工学の発展によってＸ
線顕微鏡用のＸ線光学素子の性能が向上し、Ｘ線顕微鏡
の試験機が作られるまでになっている。このＸ線顕微鏡
に使用する波長２．４〜４．４ｎｍのＸ線は、水に余り
吸収されないので、Ｘ線顕微鏡で生体試料を観察するの
に適しているが、空気には吸収される。そのため、生体
試料を真空中に置かなければならないので、生体試料を
真空雰囲気から隔離する生体試料槽が必要になる。Therefore, an X-ray microscope, which has the possibility of observing a living biological sample as it is at a high resolution, has been attracting attention, and its development is being advanced.
The performance of X-ray optical elements for X-ray microscopes has improved, and testing machines for X-ray microscopes have been manufactured. X-rays having a wavelength of 2.4 to 4.4 nm used in this X-ray microscope are not so much absorbed by water, and are suitable for observing a biological sample with an X-ray microscope, but are absorbed by air. Therefore, since the biological sample must be placed in a vacuum, a biological sample tank for isolating the biological sample from the vacuum atmosphere is required.

【０００４】幸い、電子線と違ってＸ線の場合には、窒
化珪素のようなＸ線を透過する窓材が存在するため、生
体試料槽に窓を設ければ、生体試料を生体試料槽内に置
いて観察できる。生体試料へのＸ線照射位置を確認する
ためや、照射（観察）後の生体試料のＸ線損傷を光学的
に観察するために、窓は可視光を透過するものが好まし
い。生体試料は生きたままとするため、生体試料を水に
入れた試料溶液の形で、生体試料槽に入れる。こうし
て、水中での生体試料の観察が試みられている。Fortunately, in the case of X-rays, unlike electron beams, there is a window material such as silicon nitride that allows X-rays to pass therethrough. You can place it inside and observe. In order to confirm the X-ray irradiation position on the biological sample and to optically observe the X-ray damage of the biological sample after irradiation (observation), the window preferably transmits visible light. In order to keep the biological sample alive, the biological sample is placed in the biological sample tank in the form of a sample solution in water. In this way, attempts have been made to observe biological samples in water.

【０００５】生体試料槽は、通常、主に生体試料を含む
試料溶液を入れる空間と、この空間を真空雰囲気から気
密に隔離するハウジング及び該ハウジングの一部に取り
つけられたＸ線および可視光を透過する窓から構成され
ている。図５は従来の生体試料槽の断面を示したもので
ある。すなわち、この一対の窓５１は、シリコンウエハ
からなる支持板５２の上に形成された窒化珪素膜であ
る。この一対の窓５１に挟まれた空間５３の厚さはスペ
ーサ５４によって５〜１０μｍに規定されている。スペ
ーサ５４は、空間５３に置かれる試料溶液を封じ込める
隔壁も兼ねている。支持板５２は枠５５に装着され、Ｏ
リング５７、５９を介して蓋５６で押さえられる。蓋５
６はネジ５８で枠５５に固定される。固定圧はネジ５８
でＯリング５９を押しつぶして調節される。ハウジング
は支持板５２とスペーサ５４からなるが、ハウジングの
組立は極めて面倒であり、時間がかかる。The biological sample tank usually contains a space for containing a sample solution mainly containing a biological sample, a housing for hermetically isolating this space from a vacuum atmosphere, and X-rays and visible light attached to a part of the housing. It consists of a transparent window. FIG. 5 shows a cross section of a conventional biological sample tank. That is, the pair of windows 51 is a silicon nitride film formed on the support plate 52 made of a silicon wafer. The thickness of the space 53 sandwiched by the pair of windows 51 is defined by the spacer 54 to be 5 to 10 μm. The spacer 54 also serves as a partition for containing the sample solution placed in the space 53. The support plate 52 is attached to the frame 55 and
It is pressed by the lid 56 via the rings 57 and 59. Lid 5
6 is fixed to the frame 55 with screws 58. Fixing pressure is screw 58
It is adjusted by crushing the O-ring 59 with. Although the housing is composed of the support plate 52 and the spacer 54, the assembly of the housing is extremely troublesome and time consuming.

【０００６】従って、別の生体試料を観察する場合に
は、その入れ換えが面倒であり、時間がかかることにな
る。特に一度観察してＸ線損傷があったか否か調べる場
合、特別な試薬が必要になり、これを試料溶液に添加す
る必要がある。添加する場合にはハウジングを分解し、
その後組立なければならない。そこで、これまでも、試
料溶液を入れ換え、また生体試料の位置を調整できる等
の機能を備えた生体試料槽がいくつか提案された（特開
平３−２８２２３０号、特願平３−２９０７６６）。し
かしながら、これまでに提案されたものは、いずれも大
気中で使用するものであった。従って、ハウジングは真
空雰囲気に対して気密性がない。Therefore, when observing another biological sample, the replacement is troublesome and time-consuming. In particular, when observing once and checking whether or not there is X-ray damage, a special reagent is required and it is necessary to add this to the sample solution. When adding, disassemble the housing,
Then you have to assemble. Therefore, up to now, some biological sample tanks having functions such as replacing the sample solution and adjusting the position of the biological sample have been proposed (Japanese Patent Laid-Open No. 3-228230 and Japanese Patent Application No. 3-290766). However, all the proposals so far have been used in the atmosphere. Therefore, the housing is not airtight to the vacuum atmosphere.

【０００７】また、Ｘ線顕微鏡は大きく分けて２つの直
列につないだ真空容器で構成されており、一方の真空容
器に第１の窓があり、第１の窓を通して、第１の窓の外
に置かれた生体試料槽にＸ線が照射される。他方の真空
容器には第２の窓があり、生体試料槽に照射されたＸ線
は、第２の窓を通じて真空容器内に入射し、結像する。The X-ray microscope is roughly divided into two vacuum vessels connected in series. One of the vacuum vessels has a first window, and the first window passes through the outside of the first window. The biological sample tank placed at is irradiated with X-rays. The other vacuum container has a second window, and the X-rays applied to the biological sample tank enter the vacuum container through the second window and form an image.

【０００８】[0008]

【発明が解決しようとする課題】以上の通りの従来技術
では、様々に工夫された構成に差異はあるものの、いず
れも共通して線源を出たＸ線は、少なくとも第１の窓、
生体試料槽の窓（通常は一対）及び第２の窓を通るよう
になっている。しかしながら、この窓材は、Ｘ線を透過
するとは言うものの、従来の技術ではその透過率は比較
的低く、また、強力なＸ線源が現在のところ存在しない
ことから、途中でＸ線の減衰が激しく、鮮明なＸ線観察
像が得られないという問題点があった。In the prior art as described above, although there are differences in various devised configurations, all of the X-rays emitted from the radiation source in common have at least the first window,
It is adapted to pass through the window (usually a pair) of the biological sample tank and the second window. However, although this window material is said to transmit X-rays, its transmittance is relatively low in the prior art, and since there is no strong X-ray source at present, attenuation of X-rays is in progress. However, there is a problem that a clear X-ray observation image cannot be obtained.

【０００９】このため、簡単に試料の入れ換えや試薬添
加が可能で、Ｘ線照射による生体試料の損傷程度の観察
が容易で、しかも観察操作過程でのＸ線の減衰を少なく
することのできる改善された方策の実現が求められてい
た。この発明はこのような問題点を解決することを目的
としている。Therefore, it is possible to easily replace the sample and add the reagent, to easily observe the degree of damage to the biological sample due to X-ray irradiation, and to reduce the attenuation of X-ray during the observation operation process. It was required to realize the measures taken. The present invention aims to solve such problems.

【００１０】[0010]

【課題を解決するための手段】すなわち、この発明の発
明者は、上記の問題点に鑑みて、直接に真空容器内に入
れられ、しかも簡単に試料溶液の入れ換えや試薬の添加
が可能な生体試料槽を得るべく研究の結果、生体試料を
含む試料溶液を入れる空間と、この空間を真空雰囲気か
ら気密に隔離するハウジング並びに該ハウジングの一部
に取りつけられたＸ線及び可視光を透過する窓からなる
Ｘ線観察用の生体試料槽であって、ハウジングの一部に
試料溶液の装入空間に連通する穴と、その穴を閉じる蓋
とを備えた生体試料槽を提供するものである。In view of the above-mentioned problems, the inventor of the present invention provides a living body that can be directly placed in a vacuum container and that can easily exchange sample solutions and add reagents. As a result of research to obtain a sample tank, a space for containing a sample solution containing a biological sample, a housing for hermetically isolating this space from a vacuum atmosphere, and a window for transmitting X-rays and visible light attached to a part of the housing The present invention provides a biological sample tank for X-ray observation, which is provided with a hole that communicates with a sample solution loading space in a part of a housing and a lid that closes the hole.

【００１１】[0011]

【作用】図１および図２に沿ってこの発明の構成に基づ
く作用を説明すると、まず図１および図２のスペーサ３
は不連続で隔壁とはなっておらず、空間３’も試料溶液
で満たされている。図１のように、空間３’をネジ蓋１
０で密閉した状態で、生体試料槽を図３で一例を示した
Ｘ線顕微鏡あるいは図４で一例を示したＸ線照射装置に
設置し、真空下で窓１を通して溶液中の生体試料にＸ線
を照射する。その後、生体試料槽を空気中に取り出し、
ネジ蓋１０を図２のように管付きのネジ蓋１０’に交換
し、試料溶液の入れ換えや試薬の添加ができるようにす
る。The operation based on the structure of the present invention will be described with reference to FIGS. 1 and 2. First, the spacer 3 shown in FIGS.
Is discontinuous and does not form a partition, and the space 3 ′ is filled with the sample solution. As shown in Fig. 1, the space 3'is set in the screw lid 1
In a state of being sealed with 0, the biological sample tank is installed in the X-ray microscope of which an example is shown in FIG. 3 or the X-ray irradiation device of which an example is shown in FIG. Irradiate a line. After that, remove the biological sample tank into the air,
The screw lid 10 is replaced with a screw lid 10 'with a tube as shown in FIG. 2 so that the sample solution can be exchanged and the reagent can be added.

【００１２】従って、簡単に試料の入れ換えや試薬添加
が可能となる。また、直接に真空容器内に入れられるの
で、Ｘ線の減衰も少ない。光学顕微鏡下では、管付きの
ネジ蓋１０’を通して、生体試料が反応するような試薬
を添加し、生体試料の反応の程度を観察することによっ
て、Ｘ線照射による生体試料の損傷の程度を知ることが
できる。Therefore, it is possible to easily replace the sample and add the reagent. Further, since it is directly placed in the vacuum container, there is little attenuation of X-rays. Under an optical microscope, a reagent that allows a biological sample to react is added through a screw cap 10 'with a tube, and the degree of reaction of the biological sample is observed to know the degree of damage to the biological sample due to X-ray irradiation. be able to.

【００１３】[0013]

【実施例】図１および図２にこの発明の一実施例を示
す。一対の窓１は、シリコンウエハからなる円形の支持
板２の上に形成された窒化珪素膜である。開口部は、シ
リコンウエハ上に窒化珪素膜を化学気相堆積法（ＣＶ
Ｄ）によって形成した後に、シリコンウエハのバックエ
ッチングによって形成した。一対の窓１に挟まれた空間
３の厚さはスペーサ４によって５〜１０μｍに規定され
ている。1 and 2 show an embodiment of the present invention. The pair of windows 1 are silicon nitride films formed on a circular support plate 2 made of a silicon wafer. The opening is formed by depositing a silicon nitride film on a silicon wafer by chemical vapor deposition (CV).
After forming by D), it was formed by back etching of a silicon wafer. The thickness of the space 3 sandwiched by the pair of windows 1 is defined by the spacer 4 to be 5 to 10 μm.

【００１４】スペーサ４はＸ線入射方向からみて不連続
（つまり、切れ目がある）で、空間３は空間３’とつな
がっており、試料溶液は空間３と空間３’とを満たす。
支持板２は下枠５に装着され、Ｏリング９を介して上蓋
６で押さえられる。上蓋６はネジ８で下枠５に固定され
る。固定圧はネジ８でＯリング９を押しつぶして調節さ
れる。Ｏリング７と９が、試料溶液と外界（真空雰囲
気）との間の気密を保つ要である。The spacer 4 is discontinuous (that is, has a break) when viewed from the X-ray incident direction, the space 3 is connected to the space 3 ', and the sample solution fills the space 3 and the space 3'.
The support plate 2 is attached to the lower frame 5, and is pressed by the upper lid 6 via the O-ring 9. The upper lid 6 is fixed to the lower frame 5 with screws 8. The fixing pressure is adjusted by crushing the O-ring 9 with the screw 8. It is essential that the O-rings 7 and 9 maintain airtightness between the sample solution and the outside (vacuum atmosphere).

【００１５】ここでは、主に支持板２と枠５と蓋６がハ
ウジングを構成する。空間３’は必ずしも必要ではな
く、溶液交換用の穴は、たとえば試料槽の側面に設けて
空間３’が最小限になるような構成にすることも可能で
ある。このようにして作製した生体試料槽に試料溶液を
入れ、図１のようにＯリング１１を介して２個のネジ蓋
１０（この発明で言う「蓋」に相当）で密封する。試料
はツリガネムシストークの収縮要素であるスパスモネー
ムで、溶液はスパスモネーム収縮を引き起こすカルシウ
ムイオンを取り除くキレート剤ＥＧＴＡの水溶液とし
た。光学顕微鏡下で、スパスモネームが視野内にあるこ
とを確認してから、以上の構成からなる試料槽４０を図
３のような構成のＸ線顕微鏡にセットし、試料観察を行
った。Here, the support plate 2, the frame 5 and the lid 6 mainly form a housing. The space 3'is not always necessary, and the solution exchange hole may be provided on the side surface of the sample tank, for example, so that the space 3'is minimized. The sample solution is put into the biological sample tank manufactured in this way, and is sealed with two screw lids 10 (corresponding to the “lid” in the present invention) via the O-ring 11 as shown in FIG. The sample was spasmoname, which is a contraction element of Tristomane Cistoke, and the solution was an aqueous solution of a chelating agent EGTA that removes calcium ions causing the spasmoname contraction. After confirming that Spasmoname was in the visual field under the optical microscope, the sample tank 40 having the above-described configuration was set in the X-ray microscope having the configuration as shown in FIG. 3 to observe the sample.

【００１６】このＸ線顕微鏡は、用いる波長のＸ線が数
ｍｍ程度の空気層で吸収されてしまうため、Ｘ線光学系
のすべてが真空容器３４の中にあり、真空下で作動す
る。高輝度パルスレーザ光３１を集光レンズ３２で集光
して、レーザ光取り込み窓３３を通して標的板３５の表
面に当て、プラズマ３６を生成させる。プラズマから発
生したＸ線３７の内、所望の波長のＸ線だけをフィルタ
３８で取り出し、集光用Ｘ線光学素子ウォルタ鏡３９で
生体試料槽４０の窓の部分に集光し、生体試料槽内の試
料を照射する。生体試料槽を透過したＸ線は、結像用Ｘ
線光学素子ウォルタ鏡４１で撮像器４２の受光面上に結
像する。In this X-ray microscope, X-rays of the wavelength used are absorbed by the air layer of about several millimeters, so that the entire X-ray optical system is inside the vacuum container 34 and operates under vacuum. The high-intensity pulsed laser light 31 is condensed by the condenser lens 32 and is applied to the surface of the target plate 35 through the laser light intake window 33 to generate plasma 36. Of the X-rays 37 generated from the plasma, only the X-rays having a desired wavelength are taken out by the filter 38, condensed by the Wolter mirror 39 of the X-ray optical element for condensing on the window portion of the biological sample tank 40, and the biological sample tank is obtained. Irradiate the sample inside. The X-rays that have passed through the biological sample tank are X-rays for imaging.
An image is formed on the light receiving surface of the image pickup device 42 by the linear optical element Walter mirror 41.

【００１７】生体試料に過剰のＸ線を照射してから、生
体試料槽４０を真空容器から取り出した。光学顕微鏡下
では、生体試料の形態に異常は見られなかった。しか
し、ネジ蓋１０を図２のように管付きネジ蓋１０’に替
えて、一方から吸い出しながらもう一方からカルシウム
溶液（試薬）を注入したところ、カルシウムイオンが生
体試料に到達した時点で、Ｘ線を照射してない部分は収
縮したが、Ｘ線を照射した部分は収縮しなかった。After irradiating the biological sample with excess X-rays, the biological sample tank 40 was taken out from the vacuum container. No abnormality was observed in the morphology of the biological sample under the light microscope. However, when the screw lid 10 is replaced with the screw lid 10 'with a tube as shown in FIG. 2 and a calcium solution (reagent) is injected from one side while sucking out from the other side, when calcium ions reach the biological sample, X The part that was not irradiated with radiation shrank, but the part that was irradiated with X-rays did not shrink.

【００１８】ここでは、Ｘ線顕微鏡を用いて試料にＸ線
を照射したが、図４に示すように、結像系のない装置で
も同様の実験は行える。ただし、照射Ｘ線量を知るため
に、Ｘ線検出器４３は備えていたほうがよい。また、Ｘ
線源はここで用いたレーザプラズマＸ線源に限らず、ど
のようなＸ線源でも使える。Ｘ線光学素子もウォルタ鏡
に限らない。Here, the X-ray microscope was used to irradiate the sample with X-rays. However, as shown in FIG. 4, the same experiment can be performed with an apparatus having no imaging system. However, the X-ray detector 43 should be provided in order to know the irradiation X-ray dose. Also, X
The X-ray source is not limited to the laser plasma X-ray source used here, and any X-ray source can be used. The X-ray optical element is not limited to the Walter mirror.

【００１９】[0019]

【発明の効果】この発明の試料槽は、直接に真空容器内
に入れられるので、途中でのＸ線の減衰が少なく、鮮明
なＸ線観察像が得られる。しかも簡単に試料溶液の入れ
換えや試薬の添加が可能である。そのため、Ｘ線照射に
よる生体試料の損傷の程度を大気下で光学顕微鏡で容易
に調べることができる。また、光学顕微鏡を真空容器内
に組み込み、図２のように管付きネジ蓋１１’を試料槽
に装着し、管を真空容器外に通じされば、生体試料を真
空容器から出さなくても同様の試験ができる。Since the sample tank of the present invention is directly placed in the vacuum container, a clear X-ray observation image can be obtained with little attenuation of X-rays on the way. Moreover, it is possible to easily replace the sample solution and add the reagent. Therefore, the degree of damage to the biological sample due to X-ray irradiation can be easily examined under an atmosphere with an optical microscope. In addition, the optical microscope is incorporated into the vacuum container, the screw cap 11 'with a tube is attached to the sample tank as shown in FIG. 2, and the tube is passed outside the vacuum container, the biological sample does not have to be taken out from the vacuum container. Can be tested.

【図面の簡単な説明】[Brief description of drawings]

【図１】この発明の一実施例に係わる生体試料槽の概略
垂直断面である。FIG. 1 is a schematic vertical cross section of a biological sample tank according to an embodiment of the present invention.

【図２】別の実施例に係わる生体試料槽の概略垂直断面
である。FIG. 2 is a schematic vertical cross section of a biological sample tank according to another embodiment.

【図３】この発明の試料槽を組み込んだＸ線顕微鏡の構
成を示す概念図である。FIG. 3 is a conceptual diagram showing the configuration of an X-ray microscope incorporating the sample tank of the present invention.

【図４】この発明の試料槽を組み込んだＸ線照射装置の
構成を示す概念図である。FIG. 4 is a conceptual diagram showing the configuration of an X-ray irradiation device incorporating the sample tank of the present invention.

【図５】従来の生体試料槽の概略垂直断面である。FIG. 5 is a schematic vertical cross section of a conventional biological sample tank.

【符号の説明】 １，５１ Ｘ線透過窓 ２，５２ 支持板（窓基板） ３，３’，５３ 試料溶液が入る空間 ４，５４ スペーサ ５，５５ 下枠 ６，５６ 上蓋 ７，９，１１，５７，５９ Ｏリング ８，５８ 上蓋の留めネジ １０ 蓋ネジ １０’管付き蓋ネジ ３１ レーザ光 ３２ レーザ光集光用レンズ ３３ レーザ光取り込み窓 ３４ 真空容器 ３５ 標的板 ３６ プラズマ ３７ Ｘ線 ３８ フィルタ ３９ 集光用Ｘ線光学素子 ４０ 試料槽 ４１ 結像用Ｘ線光学素子 ４２ Ｘ線撮像器 ４３ Ｘ線検出器[Explanation of reference symbols] 1,51 X-ray transmission window 2,52 Support plate (window substrate) 3,3 ', 53 Space for containing sample solution 4,54 Spacer 5,55 Lower frame 6,56 Upper lid 7,9,11 , 57, 59 O-ring 8, 58 Upper lid retaining screw 10 Lid screw 10 'Lid screw with tube 31 Laser light 32 Lens for condensing laser light 33 Laser light capturing window 34 Vacuum container 35 Target plate 36 Plasma 37 X-ray 38 Filter 39 Condensing X-ray optical element 40 Sample tank 41 Imaging X-ray optical element 42 X-ray imager 43 X-ray detector

Claims (1)

【特許請求の範囲】[Claims] 【請求項１】 生体試料を含む試料溶液を入れる空間
と、この空間を真空雰囲気から気密に隔離するハウジン
グ並びにこのハウジングの一部に取りつけられたＸ線及
び可視光を透過する窓からなるＸ線観察用の生体試料槽
において、 ハウジングの一部に試料溶液装入空間に連通する穴と、
その穴を閉じる蓋とを備えたことを特徴とするＸ線観察
用の生体試料槽。1. An X-ray comprising a space for containing a sample solution containing a biological sample, a housing for hermetically isolating this space from a vacuum atmosphere, and an X-ray and a window for transmitting visible light attached to a part of this housing. In the biological sample tank for observation, a hole that communicates with the sample solution charging space in a part of the housing,
A biological sample tank for X-ray observation, comprising a lid for closing the hole.