JP5207300B2 - High pressure sample container for optical microscopy - Google Patents
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- 238000000399 optical microscopy Methods 0.000 title 1
- 239000000463 material Substances 0.000 claims description 39
- 230000003287 optical effect Effects 0.000 claims description 28
- 229910052594 sapphire Inorganic materials 0.000 claims description 15
- 239000010980 sapphire Substances 0.000 claims description 15
- 239000007788 liquid Substances 0.000 description 12
- 238000000034 method Methods 0.000 description 7
- 230000000052 comparative effect Effects 0.000 description 6
- 239000000853 adhesive Substances 0.000 description 5
- 230000001070 adhesive effect Effects 0.000 description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 239000011324 bead Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000004005 microsphere Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical class [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 2
- 229910045601 alloy Inorganic materials 0.000 description 2
- 239000000956 alloy Substances 0.000 description 2
- 239000003822 epoxy resin Substances 0.000 description 2
- 238000002073 fluorescence micrograph Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 229910000856 hastalloy Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 229920000647 polyepoxide Polymers 0.000 description 2
- 239000010453 quartz Substances 0.000 description 2
- 239000005294 BK7 Substances 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
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- 239000010432 diamond Substances 0.000 description 1
- 229910003460 diamond Inorganic materials 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 239000005304 optical glass Substances 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
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- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002834 transmittance Methods 0.000 description 1
Description
本発明は、高圧力下での試料の光学顕微観察に用いられる光学顕微観察用高圧力試料容器に関する。 The present invention relates to a high-pressure sample container for optical microscopic observation used for optical microscopic observation of a sample under high pressure.
高圧力技術は、地球深部をはじめとする高圧力環境の再現、新規物質の合成、食品加工並びに殺菌手法の開発等、幅広い分野で用いられている技術である。このような高圧力技術に光学顕微鏡を組み合わせた高圧力光学顕微観察は、高圧力環境下における物質の物理的特性、化学的特性の変化等を観察する手法として用いられている(非特許文献１）。 High pressure technology is a technology that is used in a wide range of fields, including reproduction of high pressure environments such as deep in the earth, synthesis of new substances, food processing, and development of sterilization techniques. High pressure optical microscopic observation in which an optical microscope is combined with such high pressure technology is used as a technique for observing changes in physical properties and chemical properties of substances in a high pressure environment (Non-patent Document 1). ).
高圧力光学顕微観察では、例えば金属製の高圧力試料容器が観察用セルとして用いられる。この高圧力試料容器は、内部に収容された液体試料に光を照射したり、液体試料からの光（透過光や反射光、蛍光等）を取り出すための窓を有している。前記窓は、高圧力試料容器に形成された透孔を石英ガラス等の窓材で塞いだ構成になっており、液体試料からの光は窓材を通して放出され、対物レンズによって結像される。 In high-pressure optical microscopic observation, for example, a metal high-pressure sample container is used as an observation cell. This high-pressure sample container has a window for irradiating the liquid sample accommodated in the light and taking out light (transmitted light, reflected light, fluorescence, etc.) from the liquid sample. The window has a structure in which a through hole formed in a high-pressure sample container is closed with a window material such as quartz glass, and light from the liquid sample is emitted through the window material and imaged by an objective lens.
ところで、従来の高圧力試料容器では、高耐圧性を確保するために厚みが数mmの窓材を高圧力試料容器の内側から貼り付けて透孔を塞いでいた。このため、対物レンズを試料に近づけることができず、高開口数の対物レンズを用いることができなかった。
本発明が解決しようとする課題は、試料の近くに対物レンズを配置することができる光学顕微観察用高圧力試料容器を提供することである。
By the way, in the conventional high pressure sample container, in order to ensure high pressure resistance, a window material having a thickness of several millimeters was attached from the inside of the high pressure sample container to close the through hole. For this reason, the objective lens cannot be brought close to the sample, and an objective lens having a high numerical aperture cannot be used.
The problem to be solved by the present invention is to provide a high-pressure sample container for optical microscopic observation in which an objective lens can be disposed near the sample.
上記課題を解決するために成された本発明の光学顕微観察用高圧力試料容器は、高圧力下における試料を光学顕微観察するための、容器本体と蓋体とから成る高圧力試料容器であって、
前記容器本体は、試料導入路を内部に有すると共に、高圧に加圧された試料を前記試料導入路に取り込むための高圧ライン取付口と、前記蓋体が嵌合可能な凹部とを有し、
前記蓋体は、前記凹部に嵌合されたときに前記試料導入路側で開口し、外部に向かって先細となる円錐部と、試料から放射される光を放出する観察窓とを有し、
前記観察窓は、前記円錐部の先端に形成された透孔と、前記蓋体の外側面に接着固定された、前記透孔を閉塞する窓材とから成ることを特徴とする。
ここで、本発明の光学顕微観察用高圧力試料容器は光学顕微観察の際に観察窓と対物レンズとが対向するように配置される。
The high pressure sample container for optical microscopic observation of the present invention, which has been made to solve the above problems, is a high pressure sample container composed of a container body and a lid for optical microscopic observation of a sample under high pressure. And
The container body has a sample introduction path therein, a high pressure line attachment port for taking a sample pressurized to a high pressure into the sample introduction path, and a recess into which the lid body can be fitted,
The lid body has a conical portion that opens on the sample introduction path side when being fitted into the concave portion and tapers outward, and an observation window that emits light emitted from the sample,
The observation window is a through hole formed in said tip of the cone, is adhered and fixed to the outer surface of the front Symbol lid, characterized in that it consists of a window material for closing said through hole.
Here, the high pressure sample container for optical microscopic observation of the present invention is arranged so that the observation window and the objective lens face each other during the optical microscopic observation.
サファイアガラスは薄くても高強度であることから、窓材をサファイア製の薄板から構成すると良い。サファイアガラスは、可視領域を含む幅広い波長領域にわたって光の透過率が高いことから、この点においても窓材の材料として優れている。 Support from that fire glass is a high strength even thinner, may constitute a window material from sapphire made of a thin plate. Since sapphire glass has high light transmittance over a wide wavelength region including the visible region, it is also excellent as a window material in this respect.
本発明の光学顕微観察用高圧力試料容器によれば、対物レンズを試料の観察点に近づけることができるため、作動距離が短い開口数が大きな対物レンズを用いて光学顕微鏡観察を行うことができる。このため、高圧力環境下においても、通常圧力下と変わらぬ高解像度、高感度の光学顕微鏡観察を実施できる。 According to the high pressure sample container for optical microscopic observation of the present invention, since the objective lens can be brought close to the observation point of the sample, the optical microscope observation can be performed using the objective lens having a short working distance and a large numerical aperture. . For this reason, even in a high pressure environment, observation with an optical microscope with high resolution and high sensitivity that is the same as under normal pressure can be performed.
以下、本発明の実施の形態について図面を参照しながら説明する。
図１は本実施の形態に係る光学顕微観察用高圧力試料容器（以下、高圧力試料容器という）の構造を示している。図１において、ほぼ直方体状の高圧力試料容器１０は、金属製の容器本体１２及び蓋体１４から構成されている。容器本体１２及び蓋体１４は、十分な耐圧性を確保するために、ハステロイＣ２７６等のニッケル基合金、或いはステンレス鋼等から構成されている。
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
FIG. 1 shows the structure of a high pressure sample container for optical microscope observation (hereinafter referred to as a high pressure sample container) according to the present embodiment. In FIG. 1, a substantially rectangular parallelepiped high-pressure sample container 10 includes a metal container body 12 and a lid body 14. The container body 12 and the lid body 14 are made of nickel-base alloy such as Hastelloy C276, stainless steel or the like in order to ensure sufficient pressure resistance.
容器本体１２の内部には液体試料の導入路１６が形成されていると共に、側部には圧力ポンプからの圧力ライン（図示せず）を取り付けるための２個の取付口１８ａ、１８ｂが設けられている。前記圧力ラインは、その接続形状に応じて２個の取付口１８ａ、１８ｂのうちの一方に接続され、他方の取付口は栓で閉塞される。以上の構成により、圧力ポンプによって高圧に加圧された液体試料が導入路１６を通って高圧力試料容器１０内に導入される。
なお、図示しないが、容器本体１２内には流水路が形成されている。この流水路には恒温槽からの流水管が接続されるようになっている。恒温槽からの水が流水路を流通することにより高圧力試料容器１０内の温度が調節される。
また、容器本体１２の下部中央には、光照射窓２０が設けられている。光照射窓２０は、透孔２１と透孔２１を閉塞する窓材２２から構成されている。この窓材２２は例えば石英ガラスから成り、接着剤によって容器本体１２の内側面に貼り付けられている。高圧力試料容器１０内の液体試料には、光照射窓２０を通して光が照射される。
A liquid sample introduction path 16 is formed inside the container body 12, and two attachment ports 18a and 18b for attaching a pressure line (not shown) from a pressure pump are provided on the side portion. ing. The pressure line is connected to one of the two attachment ports 18a and 18b according to the connection shape, and the other attachment port is closed with a stopper. With the above configuration, the liquid sample pressurized to a high pressure by the pressure pump is introduced into the high-pressure sample container 10 through the introduction path 16.
Although not shown, a flow channel is formed in the container body 12. A water pipe from a thermostatic bath is connected to the water channel. The temperature in the high-pressure sample container 10 is adjusted by the water from the thermostat flowing through the flowing water channel.
A light irradiation window 20 is provided at the lower center of the container body 12. The light irradiation window 20 includes a through hole 21 and a window member 22 that closes the through hole 21. The window material 22 is made of, for example, quartz glass, and is attached to the inner surface of the container body 12 with an adhesive. The liquid body sample in a high pressure sample container 10, light is irradiated through the light irradiation window 20.
一方、蓋体１４は円柱状をなし、容器本体１２の上部中央のねじ穴２４に螺挿されるようになっている。蓋体１４の底面にはＯ-リング２６が取り付けられている。蓋体１４をねじ穴２４に装着したとき、Ｏ-リング２６は当該ねじ穴２４の底面に当接する。このような構成により、蓋体１４と容器本体１２との間が気密に封止される。
ねじ穴２４の底部には光照射窓２０と対向する開口２８が形成されている。また、蓋体１４の下面のほぼ中央には光照射窓２０と対向する凹部３０が形成されており、この凹部３０の上面部（以下、上底部ともいう）に観察窓３２が設けられている。観察窓３２は、蓋体１４に形成された透孔３３と、この透孔３３を閉塞する窓材３４から構成されている。窓材３４は例えばサファイア製の薄板から成り、蓋体１４の外側面に接着剤で貼り付けられている。ここで、「外側面」とは蓋体１４を容器本体１２に取り付けたときに高圧力試料容器１０の外面となる面をいう。液体試料から発せられた光は観察窓３２を通して放出され、対物レンズ１００（図２参照）で結像される。
On the other hand, the lid body 14 has a cylindrical shape and is screwed into a screw hole 24 at the upper center of the container body 12. An O-ring 26 is attached to the bottom surface of the lid body 14. When the lid body 14 is attached to the screw hole 24, the O-ring 26 comes into contact with the bottom surface of the screw hole 24. With such a configuration, the space between the lid 14 and the container body 12 is hermetically sealed.
An opening 28 facing the light irradiation window 20 is formed at the bottom of the screw hole 24. In addition, a concave portion 30 that faces the light irradiation window 20 is formed substantially at the center of the lower surface of the lid body 14, and an observation window 32 is provided on the upper surface portion (hereinafter also referred to as an upper bottom portion) of the concave portion 30. . The observation window 32 includes a through hole 33 formed in the lid body 14 and a window member 34 that closes the through hole 33. The window member 34 is made of, for example, a thin plate made of sapphire, and is attached to the outer surface of the lid body 14 with an adhesive. Here, the “outer surface” refers to a surface that becomes the outer surface of the high-pressure sample container 10 when the lid 14 is attached to the container body 12. Light emitted from the liquid sample is emitted through the observation window 32 and imaged by the objective lens 100 (see FIG. 2).
図２は蓋体１４の観察窓３２周辺部分を対物レンズ１００と共に示す拡大図である。図２に示すように、凹部３０の上面には上方に向かって先細となる円錐部３０ａが形成されている。この円錐部３０ａの傾斜面の角度は４５度に設定されており、その先端に直径が0.4mmの透孔３３が形成されている。このような円錐部３０ａを設けたことにより、液体試料中の観察対象物を窓材３４の近くに導入することができる。
また、蓋体１４の上面のうち前記透孔３３を中心とする部分には円形状の凹部３６が形成されており、この凹部３６に窓材３４が接着剤により貼り付けられている。凹部３６は、当該凹部３６に窓材３４を固定したときに窓材３４の外面が蓋体１４の外面とほぼ面一になるような寸法に設計されている。
FIG. 2 is an enlarged view showing the periphery of the observation window 32 of the lid 14 together with the objective lens 100. As shown in FIG. 2, a conical portion 30 a that tapers upward is formed on the upper surface of the recess 30. The angle of the inclined surface of the conical portion 30a is set to 45 degrees, and a through hole 33 having a diameter of 0.4 mm is formed at the tip thereof. By providing such a conical portion 30a, the observation object in the liquid sample can be introduced near the window member 34.
In addition, a circular recess 36 is formed in a portion of the upper surface of the lid 14 centering on the through hole 33, and a window member 34 is attached to the recess 36 with an adhesive. The recess 36 is designed to have such a dimension that the outer surface of the window member 34 is substantially flush with the outer surface of the lid 14 when the window member 34 is fixed to the recess 36.
本実施の形態では、観察窓３２の窓材３４として直径寸法が7mm、厚さ寸法が0.2mmの非常に薄いサファイア製の薄板を用いた。このような薄い窓材３４でも十分な耐圧性を確保するために、窓材３４の直径を透孔３３の直径よりも非常に大きく設定し、窓材３４と蓋体１４との接着面を広くしている。
また、本実施の形態では、蓋体１４に対して窓材３４を強固に接着するために窓材３４に所定の前処理を施してから蓋体１４に貼り付けている。以下に、窓材３４の貼り付け手順１〜５を示す。以下の手順は、ニッケル基合金（ハステロイＣ２７６）から成る蓋体１４に一液性エポキシ樹脂接着剤（ナガセケムテックス株式会社製、EPOXY RESIN XD911）を用いて窓材３４（サファイア製の薄板）を接着する手順の例である。
In the present embodiment, a very thin sapphire plate having a diameter of 7 mm and a thickness of 0.2 mm is used as the window member 34 of the observation window 32. In order to ensure sufficient pressure resistance even with such a thin window member 34, the diameter of the window member 34 is set to be much larger than the diameter of the through hole 33, and the bonding surface between the window member 34 and the lid 14 is widened. doing.
Further, in the present embodiment, in order to firmly bond the window material 34 to the lid body 14, the window material 34 is applied to the lid body 14 after being subjected to a predetermined pretreatment. Below, the sticking procedures 1-5 of the window material 34 are shown. In the following procedure, a window material 34 (a thin plate made of sapphire) is applied to a lid 14 made of a nickel-based alloy (Hastelloy C276) using a one-component epoxy resin adhesive (manufactured by Nagase ChemteX Corporation, EPOXY RESIN XD911). It is an example of the procedure to adhere | attach.
１．サファイア製の窓材（以下、サファイア窓材）３４をアセトン中で３０分間超音波洗浄する。
２．アセトンからサファイア窓材３４を取り出し、飽和ＮａＯＨ溶液で３０分間超音波洗浄する。
３．飽和ＮａＯＨ溶液からサファイア窓材３４を取り出し、蒸留水で洗浄する。
４．サファイア窓材を乾燥させる。
５．乾燥後、サファイア窓材３４に接着剤を塗布し、当該窓材を蓋体１４の凹部３６に押し当てて１２０度で２時間加熱する。
1. A sapphire window material (hereinafter, sapphire window material) 34 is ultrasonically cleaned in acetone for 30 minutes.
2. The sapphire window material 34 is taken out from acetone and ultrasonically washed with a saturated NaOH solution for 30 minutes.
3. The sapphire window material 34 is taken out from the saturated NaOH solution and washed with distilled water.
4). Dry the sapphire window material.
5. After drying, an adhesive is applied to the sapphire window material 34, the window material is pressed against the recess 36 of the lid body 14, and heated at 120 degrees for 2 hours.
以上の手順でサファイア窓材３４を固定した蓋体１４を容器本体１２に取付け、50MPaの耐圧試験を行ったところ、12時間経過しても、サファイア窓材３４に割れが発生したり、サファイア窓材３４が凹部から剥がれたりしなかった。２時間あれば高圧下での光学顕微鏡観察を十分に行えることから、前記高圧力試料容器１０は高圧下での試料の光学顕微鏡観察に使用可能であるといえる。 The lid 14 with the sapphire window member 34 fixed thereto was attached to the container body 12 by the above procedure, and a 50 MPa pressure resistance test was performed. The material 34 did not peel from the recess. It can be said that the high-pressure sample container 10 can be used for optical microscope observation of a sample under high pressure because the optical microscope observation under high pressure can be sufficiently performed in two hours.
また、本実施の形態の高圧力試料容器１０では、非常に薄いサファイア窓材３４を蓋体１４の外側の面に取り付けて観察窓３２を構成した。このため、対物レンズ１００を液体試料の近くに配置することができる。
図３は、高圧力試料容器１０の蓋体として従来の蓋体１４’を用いた場合の対物レンズ１００から液体試料までの距離を説明するための図である。上述の図２との比較から明らかなように、従来の蓋体では内側面に窓材３４が貼り付けられている。試料Ｓの観察ポイントは窓材３４の内面であるため、窓材３４の厚さ寸法が同じであっても、本実施の形態の蓋体１４を用いた場合の対物レンズ１００から液体試料までの距離ｄ１は、従来の蓋体１４’を用いた場合の距離ｄ２よりも蓋体の厚みの分だけ短くすることができる。このため、本実施の形態の高圧力試料容器は、開口数が大きく、作動距離が小さな対物レンズを用いて光学顕微鏡観察することができる。
なお、開口数が大きい対物レンズ１００は使用の際にレンズと観察窓との間にオイル１０１を満たす必要があるが、このことを考慮しても、本実施の形態の蓋体１４を用いた場合の距離ｄ１は、従来の蓋体を用いた場合の距離ｄ２よりも短くなる。
Further, in the high pressure sample container 10 of the present embodiment, the observation window 32 is configured by attaching a very thin sapphire window member 34 to the outer surface of the lid 14. For this reason, the objective lens 100 can be disposed near the liquid sample.
FIG. 3 is a diagram for explaining the distance from the objective lens 100 to the liquid sample when the conventional lid 14 ′ is used as the lid of the high-pressure sample container 10. As is clear from the comparison with FIG. 2 described above, the window material 34 is attached to the inner surface of the conventional lid. Since the observation point of the sample S is the inner surface of the window member 34, even if the thickness dimension of the window member 34 is the same, the distance from the objective lens 100 to the liquid sample when the lid body 14 of the present embodiment is used. The distance d1 can be made shorter by the thickness of the lid than the distance d2 when the conventional lid 14 ′ is used. For this reason, the high pressure sample container of the present embodiment can be observed with an optical microscope using an objective lens having a large numerical aperture and a small working distance.
Note that the objective lens 100 having a large numerical aperture needs to be filled with oil 101 between the lens and the observation window when used, but the lid body 14 of the present embodiment is used in consideration of this. The distance d1 in this case is shorter than the distance d2 when the conventional lid is used.
図４に、本実施の形態の高圧力試料容器１０を用いて直径１μmの蛍光ビーズの光学顕微観察を行った結果を比較例の光学顕微観察結果と共に示す。比較例としては、厚さ1.5mmのＢＫ７製窓材を蓋体の透孔に内側から貼り付けて成る高圧力試料容器を用いた。蛍光ビーズは窓材３４に吸着させて観察した。
図４の（ａ）及び（ｃ）は比較例及び本実施の形態の高圧力試料容器を用いたときの明視野像を、図（ｂ）及び（ｄ）は比較例及び本実施の形態の高圧力試料容器を用いたときの蛍光像をそれぞれ示す。
本実施の形態の高圧力試料容器１０内の試料は、開口数NAが1.65の超高開口数対物レンズ（オリンパス株式会社製、Apo 100×OHR)を用いて観察した。この対物レンズは、レンズと窓材との間にオイルを満たして用いる油浸レンズである。一方、比較例の高圧力試料容器内の試料は開口数NAが0.6の対物レンズ（オリンパス株式会社製、LUCPLFLN40Ｘ）を用いて観察した。
図４の（ａ）と(ｂ）及び(ｃ）と(ｄ）から、蛍光像はいずれも蛍光ビーズの発する蛍光であることを確認した。また、図４の（ｂ）及び（ｄ）から、従来の高圧力試料容器を用いた場合に比べて、本実施の形態の高圧力試料容器を用いた場合の方が、明るい蛍光像を観察できることが分かった。
FIG. 4 shows the results of optical microscopic observation of fluorescent beads having a diameter of 1 μm using the high-pressure sample container 10 of the present embodiment, together with the optical microscopic observation results of the comparative example. As a comparative example, a high pressure sample container in which a BK7 window material having a thickness of 1.5 mm was attached to the through hole of the lid from the inside was used. The fluorescent beads were observed by adsorbing to the window material 34.
4 (a) and 4 (c) show bright field images when the high pressure sample container of the comparative example and this embodiment is used, and FIGS. 4 (b) and (d) show the comparative example and this embodiment. Fluorescent images when using a high pressure sample container are shown.
The sample in the high-pressure sample container 10 of the present embodiment was observed using an ultra-high numerical aperture objective lens having a numerical aperture NA of 1.65 (manufactured by Olympus Corporation, Apo 100 × OHR). This objective lens is an oil immersion lens used by filling oil between a lens and a window material. On the other hand, the sample in the high-pressure sample container of the comparative example was observed using an objective lens (LUCPLFLN40X, manufactured by Olympus Corporation) having a numerical aperture NA of 0.6.
From (a) and (b) and (c) and (d) of FIG. 4, it was confirmed that all the fluorescence images were fluorescence emitted by the fluorescent beads. Also, from FIGS. 4B and 4D, a brighter fluorescent image is observed when the high-pressure sample container of the present embodiment is used than when a conventional high-pressure sample container is used. I understood that I could do it.
なお、本発明は上記した実施の形態に限らず、例えば次のような変形が可能である。
本発明者は、本発明の高圧力試料容器の観察窓の窓材と液体試料との界面で全反射するようにレーザー光を入射させは、窓材近傍の試料のみをエバネッセント照明により励起させることができることを確認した。このことから、本発明の高圧力試料容器を用いることにより、例えば生体分子に蛍光分子を修飾することで高圧力によって引き起こされる生体分子の構造変化や酵素活性の変化などを１分子ずつ検出することが可能になる。
The present invention is not limited to the above-described embodiment, and for example, the following modifications are possible.
The inventor makes laser light incident so as to be totally reflected at the interface between the window material of the observation window of the high pressure sample container of the present invention and the liquid sample, and excites only the sample near the window material by evanescent illumination. I confirmed that I was able to. From this, by using the high-pressure sample container of the present invention, for example, by modifying a fluorescent molecule to a biomolecule, a change in the structure of a biomolecule or a change in enzyme activity caused by a high pressure can be detected one molecule at a time. Is possible.
また、本発明者は、近赤外レーザー光を本発明の高圧力試料容器内部で集光させることにより、当該試料容器内に封入した微小球を集光点近傍に捕捉し三次元的に操作できることを確認した。これにより、微小球に生体分子を吸着させることで、高圧力環境下の溶液中で生体分子の位置を三次元的に操作可能となる。従って、微小球の光学像をカメラやフォトダイオードに投影し、その位置を検出することで、ナノメートルの精度で生体分子の重心位置を検出することができる。 In addition, the inventor condenses near-infrared laser light inside the high-pressure sample container of the present invention, thereby capturing the microsphere enclosed in the sample container in the vicinity of the condensing point and operating it three-dimensionally. I confirmed that I can do it. Thereby, the position of the biomolecule can be manipulated three-dimensionally in the solution under a high pressure environment by adsorbing the biomolecule to the microsphere. Therefore, by projecting an optical image of a microsphere onto a camera or a photodiode and detecting the position, the position of the center of gravity of the biomolecule can be detected with nanometer accuracy.
窓材は、光学的性質が均質な光学ガラスであれば良く、サファイア製の窓材の他、石英製、合成石英製、ＢＫ７製、ダイアモンド製の窓材等でも良い。
要求される耐圧性能や対物レンズの規格等を満たすのであれば、窓材は0.2mmよりも薄くても良く、また、0.2mmよりも厚くても良い。窓材を厚くした場合であっても蓋体の内側の面に固定する場合に比べて、対物レンズを試料の観察点に近づけることができる。
The window material may be any optical glass having a uniform optical property, and may be a window material made of quartz, synthetic quartz, BK7, diamond, or the like in addition to a window material made of sapphire.
The window material may be thinner than 0.2 mm or thicker than 0.2 mm as long as the required pressure resistance performance and objective lens standards are satisfied. Even when the window material is thick, the objective lens can be brought closer to the observation point of the sample as compared with the case where the window material is fixed to the inner surface of the lid.
観察窓の透孔の直径は0.4mmに限らず、観察対象の大きさに応じて適宜の直径に設定することができる。この場合、透孔の大きさに対する窓材の大きさの比が大きいほど窓材の接着面が大きくなり、窓材の耐圧性能が向上すると考えられることから、透孔の大きさに応じて、また、要求される耐圧性能に応じて、窓材を適宜の大きさにする必要がある。 The diameter of the through hole of the observation window is not limited to 0.4 mm, and can be set to an appropriate diameter according to the size of the observation target. In this case, the larger the ratio of the size of the window material to the size of the through hole, the larger the bonding surface of the window material, and it is considered that the pressure resistance performance of the window material is improved. Moreover, it is necessary to make a window material into a suitable magnitude | size according to the pressure | voltage resistant performance requested | required.
容器本体に観察窓を設け、蓋体に照射窓を設けても良い。
容器本体及び蓋体のいずれか一方に窓を設け、この窓が観察窓と照射窓を兼用するようにしても良い。
An observation window may be provided on the container body, and an irradiation window may be provided on the lid.
A window may be provided on one of the container main body and the lid, and this window may be used as an observation window and an irradiation window.
１０…光学顕微観察用高圧力試料容器
１２…容器本体
１４…蓋体
１６…導入路
１８…取付口
２０…光照射窓
２１…透孔
２２…窓材
２６…Ｏ−リング
３２…観察窓
３３…透孔
３４…窓材
DESCRIPTION OF SYMBOLS 10 ... High pressure sample container 12 for optical microscope observation ... Container main body 14 ... Cover body 16 ... Introduction path 18 ... Installation port 20 ... Light irradiation window 21 ... Through-hole 22 ... Window material 26 ... O-ring 32 ... Observation window 33 ... Through hole 34 ... Window material
Claims (2)
前記容器本体は、試料導入路を内部に有すると共に、高圧に加圧された試料を前記試料導入路に取り込むための高圧ライン取付口と、前記蓋体が嵌合可能な凹部とを有し、
前記蓋体は、前記凹部に嵌合されたときに前記試料導入路側で開口し、外部に向かって先細となる円錐部と、試料から放射される光を放出する観察窓とを有し、
前記観察窓は、前記円錐部の先端に形成された透孔と、前記蓋体の外側面に接着固定された、前記透孔を閉塞する窓材とから成ることを特徴とする光学顕微観察用高圧力試料容器。 In a high pressure sample container consisting of a container main body and a lid for optical microscopic observation of a sample under high pressure,
The container body has a sample introduction path therein, a high pressure line attachment port for taking a sample pressurized to a high pressure into the sample introduction path, and a recess into which the lid body can be fitted,
The lid body has a conical portion that opens on the sample introduction path side when being fitted into the concave portion and tapers outward, and an observation window that emits light emitted from the sample,
The observation window has a through hole formed at the tip of the conical portion, which is bonded to the outer surface of the front Symbol lid, optical microscopic observation, characterized in that it consists of a window material for closing said through hole High pressure sample container.
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