JP5930364B2 - Biological sample fixator - Google Patents
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- JP5930364B2 JP5930364B2 JP2011259007A JP2011259007A JP5930364B2 JP 5930364 B2 JP5930364 B2 JP 5930364B2 JP 2011259007 A JP2011259007 A JP 2011259007A JP 2011259007 A JP2011259007 A JP 2011259007A JP 5930364 B2 JP5930364 B2 JP 5930364B2
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Description
本発明は、顕微鏡のステージに載せた生体試料の動きを拘束するための生体試料固定器に関する。 The present invention relates to a biological sample fixing device for restraining movement of a biological sample placed on a stage of a microscope.
近年、細胞等を観察する観察装置及び標識法の開発が進められており、リアルタイムイメージング及び各種細胞内イベントの可視化が可能となっている。特に培養プレート上の細胞を生きたまま観察できる生細胞リアルタイムイメージング技術により、これまでは観察することのできなかった細胞内の分子シグナル等の観察が可能となっている。
しかし、例えば小動物の臓器における細胞等の挙動は、周囲の細胞を含む三次元構造、及び、周囲の細胞から分泌されるタンパク質によるシグナル伝達等、周囲の細胞からの影響を受けることから、培養細胞を用いた細胞の挙動を観察するのみでは足らず、生きた状態にある小動物の臓器の細胞を可視化する必要がある。
In recent years, development of observation devices and labeling methods for observing cells and the like has been promoted, and real-time imaging and visualization of various intracellular events are possible. In particular, a live cell real-time imaging technique that allows observation of cells on a culture plate while they are alive enables observation of intracellular molecular signals that could not be observed so far.
However, for example, the behavior of cells in small animal organs is affected by surrounding cells such as the three-dimensional structure including surrounding cells and signal transduction by proteins secreted from surrounding cells. It is not only necessary to observe the behavior of cells using, but it is necessary to visualize the cells of small animal organs in a living state.
小動物の臓器又は組織の細胞を観察する場合、例えば心臓及び肺の動きに影響を受けにくい、脂肪、固形腫瘍及び皮膚等は、特に固定しなくても、共焦点レーザ顕微鏡を用いて観察することが可能である。しかし、麻酔下であっても生命維持に不可欠である、心臓、肺、腸及び横隔膜直下に存在しその動きの影響を受ける肝臓等に関しては、固定して観察を行うことが必要となる。 When observing cells in organs or tissues of small animals, for example, fat, solid tumors, and skin that are not easily affected by heart and lung movements should be observed using a confocal laser microscope, even if they are not fixed. Is possible. However, it is necessary to fix and observe the liver, which is indispensable for life support even under anesthesia, and which is directly under the heart, lungs, intestines and diaphragm and is affected by the movement.
生きた状態にある小動物（例えばマウス）の心臓、肝臓等の生体試料を観察するためにその生体試料を固定する技術として、例えば特許文献１に、生体観察用のスタビライザが開示されている。このスタビライザは、図１３に示すように、観察領域９４よりも大きな貫通孔９８を有し臓器に接触させる環状の接触部９１を備えている。この接触部９１のうち、臓器の表面９０との接触面に凹部９２が設けられ、この凹部９２には、凹部９２と表面９０との間に形成される空間を減圧する減圧手段９３が接続されている。 As a technique for fixing a biological sample such as the heart and liver of a small animal (for example, mouse) in a living state, for example, Patent Literature 1 discloses a stabilizer for observing the living body. As shown in FIG. 13, the stabilizer includes an annular contact portion 91 having a through hole 98 larger than the observation region 94 and making contact with an organ. Of the contact portion 91, a concave portion 92 is provided on the contact surface with the organ surface 90, and a decompression means 93 is connected to the concave portion 92 for decompressing a space formed between the concave portion 92 and the surface 90. ing.
このスタビライザによれば、顕微鏡のステージ上に載せた小動物の臓器の表面９０に対して環状の接触部９１を上から押し付け、減圧手段９２によって凹部９２（空間）を減圧する。すると、この接触部９１は臓器の表面９０を吸着して固定することができ、これにより顕微鏡のステージに対して臓器を固定することが可能となる。
そして、環状である接触部９１の径方向内側を観察領域９４とし、この観察領域９４の中央にある観察部位９５を対物レンズ９６の視野内に収め、顕微鏡観察が行われる。
According to this stabilizer, the annular contact portion 91 is pressed from above against the surface 90 of the organ of a small animal placed on the stage of the microscope, and the recess 92 (space) is decompressed by the decompression means 92. Then, the contact portion 91 can adsorb and fix the surface 90 of the organ, thereby fixing the organ to the microscope stage.
Then, the radially inner side of the annular contact portion 91 is set as an observation region 94, and the observation region 95 at the center of the observation region 94 is placed in the field of view of the objective lens 96, and microscope observation is performed.
小動物等の生体の臓器を顕微鏡で観察する場合、麻酔下であっても、その小動物の生命活動に不可欠である呼吸及び消化運動等により、臓器の運動が継続的に行われていることから、小動物及びその臓器に対する負担をできるだけ抑える必要がある。
しかし、従来のスタビライザの場合、観察部位９５の周囲に、環状の接触部９１を設置する必要があることから、小動物の皮膚の切開範囲９７は、この接触部９１よりも充分に大きな範囲が必要となり、切開範囲９７が観察部位９５に比べてはるかに広くなってしまい、小動物に対する侵襲性が問題となる。
つまり、観察部位９５が例えば２ｍｍ程度の領域であっても、その周囲に存在している環状の接触部９５の外径が３ｃｍであるならば、この３ｃｍよりも充分に大きな切開範囲９７が必要となる。さらに、小動物の臓器における観察部位９５を変更するためには、臓器に対して接触部９１を移動させる必要があり、この場合、さらに大きな切開範囲９７が必要となる。
When observing living organs such as small animals with a microscope, even under anesthesia, the organs are continuously exercised by breathing and digestion movements that are essential for the life activity of the small animals. It is necessary to minimize the burden on small animals and their organs.
However, in the case of the conventional stabilizer, since it is necessary to install the annular contact portion 91 around the observation site 95, the incision range 97 of the skin of the small animal needs to be sufficiently larger than the contact portion 91. Thus, the incision range 97 becomes much wider than the observation site 95, and the invasiveness to small animals becomes a problem.
That is, even if the observation site 95 is an area of about 2 mm, for example, if the outer diameter of the annular contact portion 95 existing around the observation site 95 is 3 cm, an incision range 97 sufficiently larger than this 3 cm is necessary. It becomes. Furthermore, in order to change the observation site 95 in the organ of a small animal, it is necessary to move the contact portion 91 with respect to the organ, and in this case, a larger incision range 97 is required.
そこで、本発明は、侵襲性を抑えつつ生体試料を固定することが可能となる生体試料固定器を提供することを目的とする。 Then, an object of this invention is to provide the biological sample fixing device which can fix a biological sample, suppressing invasiveness.
本発明は、顕微鏡のステージに設置され、生体試料を当該ステージに対して固定するための生体試料固定器であって、前記ステージに設置される透明の板部と、前記板部に重ねて設けられ当該板部との間に流路を形成するカバー部とを備え、前記カバー部又は前記板部には、前記流路の一部と繋がり当該流路内の流体を吸引するための吸引部が設けられ、前記カバー部には、前記流路の他部と繋がっていると共に前記生体試料の一部に臨ませる開口が設けられていることを特徴とする。 The present invention is a biological sample fixing device that is installed on a stage of a microscope and fixes a biological sample to the stage, and is provided so as to overlap with a transparent plate portion installed on the stage. And a cover part that forms a flow path between the plate part and the plate part, and the cover part or the plate part is connected to a part of the flow path and sucks the fluid in the flow path The cover part is provided with an opening that is connected to the other part of the flow path and faces a part of the biological sample.
本発明によれば、カバー部に形成されている開口に生体試料を臨ませ、流路内の流体を吸引すれば、開口において生体試料の一部を陰圧（負圧）により保持することができる。このため、開口に保持されている生体試料の一部を観察部位として、透明の板部を通じて顕微鏡観察することが可能となる。すなわち、開口に保持されている生体試料の一部は、顕微鏡による観察部位となることから、この生体試料の一部が、例えば小動物等の生体の皮膚から露出していれば足りる。したがって、小動物の皮膚の切開範囲は、生体試料の一部及びその周囲の僅かな部分が露出する範囲で済み、切開範囲を従来と比較して小さくすることができ、小動物等の生体に対する侵襲性を抑えることが可能となる。 According to the present invention, when a biological sample is exposed to the opening formed in the cover portion and the fluid in the flow path is sucked, a part of the biological sample can be held by the negative pressure (negative pressure) in the opening. it can. For this reason, it becomes possible to make a microscopic observation through a transparent plate part by using a part of the biological sample held in the opening as an observation site. That is, since a part of the biological sample held in the opening serves as an observation site by a microscope, it is sufficient that a part of the biological sample is exposed from the skin of a living body such as a small animal. Therefore, the incision range of the skin of the small animal is a range in which a part of the biological sample and a small portion around the biological sample are exposed, and the incision range can be made smaller than before, and is invasive to a living body such as a small animal. Can be suppressed.
また、前記開口は、前記板部を間に挟んで、前記顕微鏡の対物レンズに対向する位置に設けられることにより、流路内の流体を吸引することで開口に保持される生体試料の一部を、顕微鏡の対物レンズの視野内に収めることができる。 In addition, the opening is provided at a position facing the objective lens of the microscope with the plate portion interposed therebetween, and thereby a part of the biological sample held in the opening by sucking the fluid in the flow path In the field of view of the objective lens of the microscope.
また、前記カバー部は、前記流路を構成するための凹溝部と、この凹溝部を囲み前記板部の面に当接する当接面を有する本体部とを有し、前記当接面は、前記流路内の流体を吸引すると前記板部の前記面に密着し、当該吸引を解除した状態では前記板部の前記面と分離可能である非接着面であるのが好ましい。
この場合、流路内の流体を吸引すれば、カバー部の当接面は板部の面に密着し、カバー部を板部に固定することができる。そして、流路内の流体の吸引を解除すれば、カバー部は板部から分離可能となり、カバー部と板部との内の一方を交換することができる。
Further, the cover portion includes a groove portion for forming the flow path, and a main body portion having a contact surface that surrounds the groove portion and contacts the surface of the plate portion, When the fluid in the flow path is sucked, it is preferably a non-adhesive surface that is in close contact with the surface of the plate portion and is separable from the surface of the plate portion when the suction is released.
In this case, if the fluid in the flow path is sucked, the contact surface of the cover portion comes into close contact with the surface of the plate portion, and the cover portion can be fixed to the plate portion. If the suction of the fluid in the flow path is released, the cover portion can be separated from the plate portion, and one of the cover portion and the plate portion can be exchanged.
また、流路内の流体を吸引することによって、生体試料の一部はカバー部（開口）に密着することから、生体試料を傷めないように、前記カバー部は、前記板部よりも弾性係数の小さい部材からなるのが好ましい。これにより、カバー部は板部より柔軟性を有し、生体試料を傷めるのを防ぐことができる。 In addition, by sucking the fluid in the flow path, a part of the biological sample comes into close contact with the cover part (opening), so that the cover part has an elastic coefficient more than that of the plate part so as not to damage the biological sample. It is preferable that it consists of a small member. Thereby, a cover part has a softness | flexibility rather than a board part, and can prevent damaging a biological sample.
また、前記板部は、前記流路内の流体の吸引により前記開口に保持されかつ当該開口から前記流路内に侵入させる前記生体試料の一部を接触可能とする接触面を有しているのが好ましい。
この場合、開口に保持されかつこの開口から流路内に侵入させた生体試料の一部が、板部のステージと反対側の面（接触面）に接触可能となる。そして、この接触面に接触している生体試料の一部を顕微鏡により観察することができる。
In addition, the plate portion has a contact surface that is held in the opening by suction of fluid in the flow path and allows a part of the biological sample to enter the flow path from the opening. Is preferred.
In this case, a part of the biological sample held in the opening and intruded into the flow channel from the opening can come into contact with the surface (contact surface) on the side opposite to the stage of the plate portion. A part of the biological sample in contact with the contact surface can be observed with a microscope.
また、前記流路は、前記吸引部と繋がっている主流路と、この主流路から分岐し前記開口とそれぞれが繋がっている複数の枝流路とを有しているのが好ましい。
この場合、開口に保持される生体試料の一部の周囲を複数方向から吸引し、その周囲の空間を減圧することができ、生体試料の一部を開口に保持させることができる。
Moreover, it is preferable that the said flow path has the main flow path connected with the said suction part, and the several branch flow path branched from this main flow path and each connected with the said opening.
In this case, the periphery of a part of the biological sample held in the opening can be sucked from a plurality of directions, the surrounding space can be decompressed, and a part of the biological sample can be held in the opening.
また、主流路から複数の枝流路が分岐している場合において、前記カバー部の前記枝流路間には、前記流路内の減圧によって当該枝流路が潰れるのを防ぐ補剛部が形成されているのが好ましい。
枝流路が形成されている領域ではカバー部の剛性が、主流路が形成されている領域の剛性に比べて低くなる場合があるが、補剛部によってカバー部の剛性が低下するのを抑えることができる。
In addition, when a plurality of branch channels are branched from the main channel, a stiffening portion that prevents the branch channels from being crushed by decompression in the channel is provided between the branch channels of the cover portion. Preferably it is formed.
In the area where the branch flow path is formed, the rigidity of the cover part may be lower than the rigidity of the area where the main flow path is formed, but the stiffening part prevents the rigidity of the cover part from being lowered. be able to.
また、本発明は、顕微鏡のステージに設置され、生体試料を当該ステージに対して固定するための生体試料固定器であって、前記ステージに設置される透明の板部材に重ねて設けられ当該板部材との間に流路を形成するカバー部材を備え、前記カバー部材には、前記流路の一部と繋がり当該流路内の流体を吸引するための吸引部と、前記流路の他部と繋がっていると共に前記生体試料の一部に臨ませる開口とが形成されていることを特徴とする。
さらに本発明は、顕微鏡のステージに設置され、生体試料を当該ステージに対して固定するための生体試料固定器であって、前記ステージに設置される透明の板部材に重ねて設けられ当該板部材との間に流路を形成するカバー部材を備え、前記カバー部材には、前記流路の一部と繋がっていると共に前記生体試料の一部に臨ませる開口が形成され、前記カバー部材は、前記流路の他部と繋がり当該流路内の流体を吸引するための吸引部が形成されている前記板部材に重ねて設けられることを特徴とする。
Further, the present invention is a biological sample fixing device that is installed on a stage of a microscope and fixes a biological sample to the stage, and is provided so as to overlap with a transparent plate member installed on the stage. A cover member that forms a channel with the member, the cover member being connected to a part of the channel and a suction part for sucking the fluid in the channel, and the other part of the channel And an opening that faces a part of the biological sample is formed.
Furthermore, the present invention is a biological sample fixing device that is installed on a stage of a microscope and fixes a biological sample to the stage, and is provided so as to overlap with a transparent plate member installed on the stage. A cover member that forms a flow path between the cover member, the cover member is connected to a part of the flow path and has an opening that faces a part of the biological sample. It is connected to the other part of the said flow path, It is provided so that it may overlap with the said plate member in which the suction part for attracting | sucking the fluid in the said flow path is formed.
これら本発明によれば、カバー部材に形成されている開口に生体試料を臨ませ、流路内の流体を吸引すれば、開口において生体試料の一部を陰圧（負圧）により固定することができる。このため、開口に保持されている生体試料の一部を観察部位として、透明の板部材を通じて顕微鏡観察することが可能となる。すなわち、開口に保持されている生体試料の一部は、顕微鏡による観察部位となることから、この生体試料の一部が、例えば小動物等の生体の皮膚から露出していれば足りる。したがって、小動物の皮膚の切開範囲は、生体試料の一部及びその周囲の僅かな部分が露出する範囲で済み、切開範囲を従来と比較して小さくすることができ、小動物等の生体に対する侵襲性を抑えることが可能となる。 According to these aspects of the present invention, when a biological sample is exposed to the opening formed in the cover member and the fluid in the flow path is sucked, a part of the biological sample is fixed by the negative pressure (negative pressure) in the opening. Can do. For this reason, it becomes possible to perform microscopic observation through a transparent plate member using a part of the biological sample held in the opening as an observation site. That is, since a part of the biological sample held in the opening serves as an observation site by a microscope, it is sufficient that a part of the biological sample is exposed from the skin of a living body such as a small animal. Therefore, the incision range of the skin of the small animal is a range in which a part of the biological sample and a small portion around the biological sample are exposed, and the incision range can be made smaller than before, and is invasive to a living body such as a small animal. Can be suppressed.
本発明の生体試料固定器によれば、小動物等の皮膚の切開範囲は、生体試料の一部及びその周囲の僅かな部分が露出する範囲で済み、切開範囲を従来と比較して小さくすることができ、小動物等の生体に対する侵襲性を抑えることが可能となる。 According to the biological sample fixing device of the present invention, the incision range of the skin of a small animal or the like is a range in which a part of the biological sample and a small portion around the biological sample are exposed, and the incision range is made smaller than the conventional one. It is possible to suppress invasiveness to a living body such as a small animal.
〔生体試料固定器の全体構成〕
図１は、本発明の生体試料固定器１をステージ６に設置した顕微鏡５の模式図である。この生体試料固定器１は、生きた状態にある小動物等の生体の臓器又は組織（生体試料）を、ステージ６に対して固定し、その動きを拘束するための装置であり、しかも、その臓器等を、生体の体外へ取り出すことなく体内に収められた状態で、その生体の皮膚の一部を切開して露出させ、顕微鏡５によって観察するために用いられる機器である。なお、本実施形態では生体をマウスＭとして説明する。
[Overall configuration of biological sample fixture]
FIG. 1 is a schematic diagram of a microscope 5 in which a biological sample fixing device 1 of the present invention is installed on a stage 6. The biological sample fixing device 1 is a device for fixing a living organ or tissue (biological sample) such as a small animal in a living state with respect to the stage 6 and restraining the movement thereof. Is a device used for observing with a microscope 5 by incising and exposing a part of the skin of the living body in a state in which the body is housed without being taken out of the living body. In the present embodiment, the living body is described as a mouse M.
顕微鏡５は、ステージ６と、対物レンズ７を有しステージ６の下側に設けられている対物ユニット８とを備えた倒立顕微鏡である。ステージ６の上に生体試料固定器１が載せられ、この生体試料固定器１の上にマウスＭが載せられ、ステージ６に形成されている貫通孔６ａを通じて、マウスＭの臓器Ｐが観察される。なお、マウスＭには麻酔が適用されるが、このマウスＭの全身をステージ６に固定する必要はない。
対物ユニット８は、対物レンズ７の他に、レーザ光源８ａ、マイクロレンズディスク８ｂ、ピンホールディスク８ｃ、ダイクロイックミラー８ｄ及びＣＣＤカメラ８ｅを有しており、臓器Ｐの高速撮影を行う。なお、観察対象となるマウスＭは、うつ伏せ姿勢であり、その腹部の皮膚が切開され、臓器Ｐを体内に残しながら体外へ露出させた状態とする。
The microscope 5 is an inverted microscope including a stage 6 and an objective unit 8 having an objective lens 7 and provided on the lower side of the stage 6. The biological sample fixing device 1 is mounted on the stage 6, the mouse M is mounted on the biological sample fixing device 1, and the organ P of the mouse M is observed through the through hole 6 a formed in the stage 6. . Although anesthesia is applied to the mouse M, it is not necessary to fix the whole body of the mouse M to the stage 6.
The objective unit 8 includes a laser light source 8a, a microlens disk 8b, a pinhole disk 8c, a dichroic mirror 8d, and a CCD camera 8e in addition to the objective lens 7, and performs high-speed imaging of the organ P. Note that the mouse M to be observed is in a prone posture, the abdominal skin is incised, and the organ P is exposed outside the body while remaining in the body.
図２は、生体試料固定器１の斜視図である。図３は、生体試料固定器１の断面図（図２のＢ矢視の断面図）であり、臓器Ｐを保持した状態にある。図４は、図２のＡ矢視における断面図である。生体試料固定器１は、ステージ６上に設置される透明の板部（１０）と、この板部（１０）の上面１１（ステージ６と反対側の面）に重ねて設けられているカバー部（２０）とを備えている。なお、板部（１０）とカバー部（２０）とは、一体不可分の構造であってもよいが、本実施形態では別の部材からなり、板部は板部材（ガラス板１０）からなり、カバー部はカバー部材２０からなる。また、カバー部材２０の内部を説明するために、その内部構造を、図２では細線で示している。 FIG. 2 is a perspective view of the biological sample fixing device 1. 3 is a cross-sectional view of the biological sample fixing device 1 (a cross-sectional view taken in the direction of arrow B in FIG. 2), in which the organ P is held. 4 is a cross-sectional view taken along arrow A in FIG. The biological sample fixing device 1 includes a transparent plate portion (10) installed on the stage 6 and a cover portion provided so as to overlap the upper surface 11 (surface opposite to the stage 6) of the plate portion (10). (20). The plate portion (10) and the cover portion (20) may have an integral inseparable structure, but in this embodiment, the plate portion is composed of another member, and the plate portion is composed of a plate member (glass plate 10). The cover part is composed of a cover member 20. Moreover, in order to explain the inside of the cover member 20, the internal structure is shown by a thin line in FIG.
本実施形態では、前記板部材は透明の薄いガラス板１０であって、光に対して透明であり、一般的に光学顕微鏡観察において用いられるカバーガラスである。ガラス板１０はステージ６に固定される。
カバー部材２０は、ガラス板１０と同様に薄い板部材であって、本実施形態では、シリコンゴム製（ポリジメチルシロキサン（ＰＤＭＳ）製）である。カバー部材２０は、ガラス板１０の上に載って積層状態にある。なお、カバー部材２０及び板部材は他の材質であってもよい。
In this embodiment, the said plate member is the transparent thin glass plate 10, Comprising: It is transparent with respect to light, and is a cover glass generally used in an optical microscope observation. The glass plate 10 is fixed to the stage 6.
The cover member 20 is a thin plate member similar to the glass plate 10, and is made of silicon rubber (made of polydimethylsiloxane (PDMS)) in this embodiment. The cover member 20 is placed on the glass plate 10 and in a laminated state. The cover member 20 and the plate member may be made of other materials.
〔カバー部材２０及びガラス板１０について〕
カバー部材２０の一面（下面）側であってその中央部には、凹溝部２２が形成されており、カバー部材２０がガラス板１０に重ねられると、この凹溝部２２によってガラス板１０との間に流路３０が形成される。本実施形態では、顕微鏡５及び生体試料固定器１は大気中に設置されることから、この流路３０には空気（大気）が流れる。
[About the cover member 20 and the glass plate 10]
A concave groove portion 22 is formed on the one surface (lower surface) side of the cover member 20 and in the center thereof. When the cover member 20 is stacked on the glass plate 10, the concave groove portion 22 forms a gap with the glass plate 10. A flow path 30 is formed in In this embodiment, since the microscope 5 and the biological sample fixing device 1 are installed in the atmosphere, air (atmosphere) flows through the flow path 30.
図４において、ガラス板１０の上にカバー部材２０が重ねられた状態で、凹溝部２２の溝底面（上面）２２ａと、左右両側の溝側面２２ｂ，２２ｃと、ガラス板１０の上面１１とで囲まれた空間によって、流路３０が形成される。
そして、カバー部材２０に凹溝部２２が形成されることにより、その溝底面２２ａを下面として有する薄肉部２５と、それ以外の厚肉部２６とが形成される。厚肉部２６は凹溝部２２が形成されていない部分である。
In FIG. 4, with the cover member 20 overlaid on the glass plate 10, the groove bottom surface (upper surface) 22 a of the concave groove portion 22, the left and right groove side surfaces 22 b and 22 c, and the upper surface 11 of the glass plate 10. A flow path 30 is formed by the enclosed space.
Then, by forming the concave groove portion 22 in the cover member 20, a thin portion 25 having the groove bottom surface 22a as a lower surface and the other thick portion 26 are formed. The thick portion 26 is a portion where the concave groove portion 22 is not formed.
また、図２と図３とにおいて、カバー部材２０には、吸引部３１と、開口３２とが設けられている。吸引部３１は、薄肉部２５上に設けられており、流路３０の一部３０ａ（図３参照）と繋がっており、流路３０内の空気を吸引するためのパイプ３３が接続されている接続ポートである。
開口３２は、流路３０の他部３０ｂ（図３参照）と繋がっていると共にマウスＭの臓器Ｐの一部Ｐａに臨ませる貫通孔からなる。つまり、カバー部材２０の薄肉部２５をその厚さ方向に貫通して形成した貫通孔によって、開口３２が構成されており、開口３２は流路３０内の空間とマウスＭが存在する外部空間とを連通している。
2 and 3, the cover member 20 is provided with a suction portion 31 and an opening 32. The suction part 31 is provided on the thin-walled part 25, is connected to a part 30a (see FIG. 3) of the flow path 30, and is connected to a pipe 33 for sucking air in the flow path 30. It is a connection port.
The opening 32 is connected to the other portion 30 b (see FIG. 3) of the flow path 30 and includes a through hole that faces a part Pa of the organ P of the mouse M. That is, the opening 32 is constituted by a through hole formed by penetrating the thin portion 25 of the cover member 20 in the thickness direction, and the opening 32 is formed between the space in the flow path 30 and the external space in which the mouse M exists. Is communicated.
そして、パイプ３３に減圧手段３４が接続されており、減圧手段３４がパイプ３３を通じて流路３０内の空気を吸引することで、流路３０内が減圧される。これにより、開口３２に臨ませたマウスＭの臓器Ｐの一部Ｐａが、開口３２に吸着して保持され、臓器Ｐの動きが拘束される（図３参照）。減圧手段３４は、図示しないが、ポンプ又はシリンジ等とすることができる。 The pressure reducing means 34 is connected to the pipe 33, and the pressure reducing means 34 sucks air in the flow path 30 through the pipe 33, whereby the pressure in the flow path 30 is reduced. Thereby, a part Pa of the organ P of the mouse M facing the opening 32 is adsorbed and held in the opening 32, and the movement of the organ P is restrained (see FIG. 3). Although not shown, the decompression means 34 can be a pump or a syringe.
そして、開口３２は、ガラス板１０を間に挟んで、対物レンズ７に対向する位置に設けられており（図３参照）、吸引部３１を通じて流路３０内の空気が吸引されることで、流路３０内が減圧され、臓器Ｐの一部Ｐａが、開口３２に保持されると共に開口３２から流路３０内に侵入する。そして、ガラス板１０の上面１１（ステージ６と反対側の面）の一部１２は、開口３２から流路３０内に侵入した臓器Ｐの一部Ｐａが接触可能となる接触面となる。
この接触面（１２）に接触している臓器Ｐの一部Ｐａを、対物レンズ７の視野内に収め、この接触している一部Ｐａに焦点を合わせることにより、顕微鏡５により観察することができる。また、ガラス板１０の接触面（１２）に臓器Ｐの一部Ｐａが接触することで、この一部Ｐａを観察部位とした場合に、この観察部位とガラス板１０との間に空気の層が介在しないため良好な観察状態が得られる。
And the opening 32 is provided in the position which opposes the objective lens 7 on both sides of the glass plate 10 (refer FIG. 3), and the air in the flow path 30 is attracted | sucked through the suction part 31, The pressure in the flow path 30 is reduced, and a part Pa of the organ P is held in the opening 32 and enters the flow path 30 from the opening 32. A part 12 of the upper surface 11 (surface opposite to the stage 6) of the glass plate 10 serves as a contact surface on which part Pa of the organ P that has entered the flow path 30 through the opening 32 can come into contact.
A part Pa of the organ P in contact with the contact surface (12) is accommodated in the field of view of the objective lens 7, and the part Pa in contact with the focus can be focused and observed with the microscope 5. it can. Further, when a part Pa of the organ P comes into contact with the contact surface (12) of the glass plate 10 and this part Pa is set as an observation site, an air layer is formed between the observation site and the glass plate 10. Good observation state can be obtained because no intervening.
また、図４において、カバー部材２０は、流路３０を構成するための凹溝部２２と、この凹溝部２２を囲む本体部２３とを有している。本体部２３のうち凹溝部２２を前後左右四方から囲む部分が前記厚肉部２６となり、凹溝部２２を上から覆う部分が前記薄肉部２５となる。
そして、本体部２３は、ガラス板１０の上面１１に当接する当接面２４を有しており、この当接面２４は、ガラス板１０の上面１１と分離可能である平滑な非接着面である。つまり、カバー部材２０の下面２１とガラス板１０の上面１１とは、面接触しているが、接着剤等が用いられて接着されておらず、分離可能である。
Further, in FIG. 4, the cover member 20 includes a concave groove portion 22 for constituting the flow path 30 and a main body portion 23 surrounding the concave groove portion 22. A portion of the main body portion 23 surrounding the concave groove portion 22 from the front, rear, left and right sides is the thick portion 26, and a portion covering the concave groove portion 22 from above is the thin portion 25.
And the main-body part 23 has the contact surface 24 contact | abutted to the upper surface 11 of the glass plate 10, This contact surface 24 is a smooth non-adhesive surface which can be separated from the upper surface 11 of the glass plate 10. is there. That is, the lower surface 21 of the cover member 20 and the upper surface 11 of the glass plate 10 are in surface contact, but are not bonded using an adhesive or the like and can be separated.
凹溝部２２は、カバー部材２０の外縁を除く中央にのみ形成されており、当接面２４は、凹溝部２２の開口縁（下縁）を全周にわたって囲む範囲に設けられている。したがって、吸引部３１を通じて流路３０内の空気を吸引すれば、流路３０内は減圧され、カバー部材２０の当接面２４はガラス板１０の上面１１に密着し、カバー部材２０をガラス板１０に固定することができる。そして、流路３０内の空気の吸引を解除（停止）すれば、カバー部材２０はガラス板１０から分離可能となり、カバー部材２０とガラス板１０との内の一方を残して他方のみを交換することができる。 The concave groove portion 22 is formed only in the center excluding the outer edge of the cover member 20, and the contact surface 24 is provided in a range surrounding the opening edge (lower edge) of the concave groove portion 22 over the entire circumference. Therefore, if the air in the flow path 30 is sucked through the suction portion 31, the pressure in the flow path 30 is reduced, the contact surface 24 of the cover member 20 is in close contact with the upper surface 11 of the glass plate 10, and the cover member 20 is attached to the glass plate. 10 can be fixed. When the suction of the air in the flow path 30 is released (stopped), the cover member 20 can be separated from the glass plate 10, and only one of the cover member 20 and the glass plate 10 is exchanged while leaving the other. be able to.
〔流路３０について〕
図２に示すように、流路３０は、吸引部３１と繋がっている主流路３６と、この主流路３６から分岐している複数の枝流路３７とを有している。枝流路３７それぞれは、開口３２と繋がっている。
主流路３６は、直線的に一条形成されており、その端部に設けられている分岐部３８ａから、３つの枝流路３７ａ，３７ｂ，３７ｃが分岐している。枝流路３７ａは、開口３２と繋がっているが、他の枝流路３７ｂ，３７ｃの端部には、第２と第３の分岐部３８ｂ，３８ｃが設けられており、第２の分岐部３８ｂから２つの枝流路３７ｄ，３７ｅが分岐し、第３の分岐部３８ｃから２つの枝流路３７ｆ，３７ｇが分岐している。枝流路３７ｄ，３７ｆは、開口３２と繋がっているが、他の枝流路３７ｅ，３７ｇは、合流部３９で合流し、合流した枝流路３７ｈが開口３２と繋がっている。なお、第１〜第８の枝流路３７ａ〜３７ｈは、カバー部材２０に形成されている凹溝によって構成されることから、この凹溝の溝底面を下面として有する部分は、薄肉部２５となる（図５参照）。図５は、図２のＣ矢視の断面図である。
[About the channel 30]
As shown in FIG. 2, the flow path 30 includes a main flow path 36 connected to the suction portion 31 and a plurality of branch flow paths 37 branched from the main flow path 36. Each branch channel 37 is connected to the opening 32.
The main flow path 36 is formed in a straight line, and three branch flow paths 37a, 37b, and 37c are branched from a branch portion 38a provided at an end thereof. The branch channel 37a is connected to the opening 32, but the second and third branch parts 38b and 38c are provided at the ends of the other branch channels 37b and 37c. Two branch channels 37d and 37e branch from 38b, and two branch channels 37f and 37g branch from the third branch portion 38c. The branch flow paths 37 d and 37 f are connected to the opening 32, but the other branch flow paths 37 e and 37 g are joined at the joining portion 39, and the joined branch flow path 37 h is connected to the opening 32. In addition, since the 1st-8th branch flow paths 37a-37h are comprised by the concave groove formed in the cover member 20, the part which has the groove bottom face of this concave groove as a lower surface is the thin part 25. (See FIG. 5). FIG. 5 is a cross-sectional view taken along arrow C in FIG.
このように複数の枝流路３７が形成されていることによって、流路３０内の減圧によって枝流路３７が潰れるのを防ぐための補剛部として隔壁４０が、カバー部材２０に形成されている。つまり、例えば枝流路３７ａ，３７ｃ間には、隔壁４０が形成されている。この隔壁４０の下面は、ガラス板１０の上面１１と面で接触する（図５参照）。
カバー部材２０のうち、枝流路３７が複数形成されている領域では、空間が多くなってカバー部材２０の剛性が、主流路３６が形成されている領域の剛性に比べて低くなる場合があるが、隔壁４０によって、この剛性の低下を抑えることができる。つまり、隔壁４０は補剛部（柱部材）として機能することができる。
By forming the plurality of branch flow paths 37 in this way, the partition wall 40 is formed in the cover member 20 as a stiffening portion for preventing the branch flow paths 37 from being crushed by the reduced pressure in the flow path 30. Yes. That is, for example, the partition wall 40 is formed between the branch channels 37a and 37c. The lower surface of the partition wall 40 is in surface contact with the upper surface 11 of the glass plate 10 (see FIG. 5).
Of the cover member 20, in a region where a plurality of branch flow paths 37 are formed, the space increases and the rigidity of the cover member 20 may be lower than the rigidity of the region where the main flow path 36 is formed. However, the partition wall 40 can suppress the decrease in rigidity. That is, the partition wall 40 can function as a stiffening portion (column member).
〔その他について〕
カバー部材２０は、上記のとおりＰＤＭＳ製であり、ガラス板１０よりも弾性係数（ヤング率）の小さい部材からなるが、領域に応じてその特性（弾性係数）を変化させてもよい。つまり、凹溝部２２が形成されている同じ薄肉部２５であっても、その薄肉部２５は、開口３２から離れている第１領域４１（図３参照）と、開口３２を取り囲む領域である第２領域４２（図３参照）とを有しており、第２領域４２は、第１領域４１よりも弾性係数が小さく構成されている。本実施形態では、第２領域４２は、枝流路３７が形成されている領域である。
なお、弾性係数の差は、第１領域４１と第２領域４２とで、ＰＤＭＳの特性（材料の混合比）を変化させることにより生じさせてもよく、（薄肉部２５ではあるが）さらにその厚さを相違させることにより生じさせてもよい。
このように、カバー部材２０を弾性係数の小さい部材とすることで、カバー部材２０は柔軟性を有することができる。また、カバー部材２０はガラス板１０よりも硬さが低い。
[Others]
The cover member 20 is made of PDMS as described above, and is made of a member having a smaller elastic coefficient (Young's modulus) than the glass plate 10, but the characteristic (elastic coefficient) may be changed depending on the region. That is, even if it is the same thin part 25 in which the recessed groove part 22 is formed, the thin part 25 is the 1st area | region 41 (refer FIG. 3) which is separated from the opening 32, and the area | region which surrounds the opening 32. 2 region 42 (refer to FIG. 3), and the second region 42 is configured to have a smaller elastic coefficient than the first region 41. In the present embodiment, the second region 42 is a region where the branch channel 37 is formed.
The difference in elastic modulus may be caused by changing the characteristics (mixing ratio of materials) of the PDMS between the first region 41 and the second region 42 (even though it is the thin portion 25). It may be caused by making the thicknesses different.
Thus, the cover member 20 can have flexibility by making the cover member 20 a member having a small elastic coefficient. Further, the cover member 20 is lower in hardness than the glass plate 10.
このように、開口３２を取り囲む領域である第２領域４２の弾性係数を小さくすることで、吸引部３１を通じて流路３０内の空気を吸引した際に、この第２領域４２に含まれる開口３２は、臓器Ｐの一部Ｐａに追従して変形可能となり、開口３２と臓器Ｐとの間に隙間が生じるのを防ぎ密封性を高め、流路３０内の陰圧による臓器Ｐの固定性能を高めることができる。そして、剛性の高い第１領域４１では、流路３０内が陰圧となって流路３０が潰れるのを防ぐことができる。 In this way, by reducing the elastic coefficient of the second region 42 that is a region surrounding the opening 32, when the air in the flow path 30 is sucked through the suction portion 31, the opening 32 included in the second region 42. Can be deformed following a part Pa of the organ P, prevents a gap from being formed between the opening 32 and the organ P, improves sealing performance, and improves the performance of fixing the organ P by negative pressure in the flow path 30. Can be increased. And in the 1st area | region 41 with high rigidity, it can prevent that the inside of the flow path 30 becomes a negative pressure and the flow path 30 is crushed.
生体試料固定器１の各部の寸法について説明する。ガラス板１０は、例えば、短辺２５ｍｍ、長辺７５ｍｍ、厚さ０．１７ｍｍ程度である。カバー部材２０は、外周輪郭形状がガラス板１０よりも小さく設定されている。カバー部材２０（本体部２３）の厚さは、２ｍｍ以下であり、この厚さが厚肉部２６の厚さとなる。そして、このカバー部材２０に形成されている凹溝部２２の溝深さは、例えば５０〜２００μｍである。つまり、流路３０の厚さが５０〜２００μｍとなる。なお、流路３０（主流路３６）の幅は、０．１ｍｍ〜１ｍｍ程度である。カバー部材２０の厚さから凹溝部２２の溝深さを除いた厚さが、薄肉部２５の厚さとなる。
そして、開口３２の開口寸法、つまり本実施形態では円形である開口３２の直径は２ｍｍ〜４ｍｍ程度であり、本実施形態では３ｍｍである。
このように、カバー部材２０及び凹溝部２２（流路３０）は非常に薄く、カバー部材２０及びマイクロ流路構造である凹溝部２２は、例えば鋳造により成型される。
開口３２の開口寸法（直径）は、流路３０（主流路３６）の幅よりも大きいことから、開口３２が形成される領域の周囲には、主流路３６から流体通過断面が拡大される拡大空間が形成され、この拡大空間に隔壁４０が設けられている。
The dimension of each part of the biological sample fixing device 1 will be described. The glass plate 10 has, for example, a short side of 25 mm, a long side of 75 mm, and a thickness of about 0.17 mm. The cover member 20 is set to have an outer peripheral contour shape smaller than the glass plate 10. The thickness of the cover member 20 (main body portion 23) is 2 mm or less, and this thickness is the thickness of the thick portion 26. And the groove depth of the recessed groove part 22 currently formed in this cover member 20 is 50-200 micrometers, for example. That is, the thickness of the flow path 30 is 50 to 200 μm. The width of the flow path 30 (main flow path 36) is about 0.1 mm to 1 mm. The thickness obtained by removing the groove depth of the concave groove portion 22 from the thickness of the cover member 20 is the thickness of the thin portion 25.
The opening size of the opening 32, that is, the diameter of the opening 32 which is circular in this embodiment is about 2 mm to 4 mm, and is 3 mm in this embodiment.
Thus, the cover member 20 and the groove portion 22 (flow channel 30) are very thin, and the cover member 20 and the groove portion 22 having the micro flow channel structure are formed by casting, for example.
Since the opening dimension (diameter) of the opening 32 is larger than the width of the flow path 30 (main flow path 36), the fluid passage cross section is enlarged from the main flow path 36 around the area where the opening 32 is formed. A space is formed, and a partition wall 40 is provided in the enlarged space.
また、開口３２が形成される部分も薄肉部２５であり、薄肉部２５の厚さ及び流路３０の厚さを薄くすることによって、開口３２から侵入させた臓器Ｐの一部Ｐａは、ガラス板１０の一部（接触面）１２に接触可能となる。
つまり、薄肉部２５の厚さ及び流路３０の厚さが大きくなると、臓器Ｐの一部Ｐａをガラス板１０に接触させるためには、その厚さに応じて臓器Ｐの一部Ｐａを大きく流路３０内に引き込む必要があり、この場合、臓器Ｐの一部Ｐａが例えば鬱血して観察部位として不適切な状態になるおそれがある。また、薄肉部２５の厚さ及び流路３０の厚さが大きすぎて、臓器Ｐの一部Ｐａをガラス板１０に接触させることができない場合、一部Ｐａとガラス板１０との間に空気の層が介在して、顕微鏡５による良好な観察状態が得られない。しかし、本実施形態のように、カバー部材２０等を薄く構成することによって、このような不具合の発生を防ぐことが可能となる。
なお、開口３２の開口寸法（本実施形態では直径３ｍｍ）の１／３以上（つまり１ｍｍ以上）の寸法の広さを有する範囲で、臓器Ｐの一部Ｐａがガラス板１０に接触できるような値に、カバー部材２０の厚さ（及び流路３０の厚さ）は設定されている。
Further, the portion where the opening 32 is formed is also the thin portion 25, and by reducing the thickness of the thin portion 25 and the thickness of the flow path 30, a part Pa of the organ P invaded from the opening 32 is made of glass. A part (contact surface) 12 of the plate 10 can be contacted.
That is, when the thickness of the thin portion 25 and the thickness of the flow path 30 are increased, in order to bring a part Pa of the organ P into contact with the glass plate 10, the part Pa of the organ P is increased according to the thickness. In this case, a part Pa of the organ P may be congested and become in an inappropriate state as an observation site. Further, when the thickness of the thin portion 25 and the thickness of the flow path 30 are too large to allow a part Pa of the organ P to contact the glass plate 10, the air between the part Pa and the glass plate 10 Thus, a good observation state with the microscope 5 cannot be obtained. However, it is possible to prevent such a problem from occurring by forming the cover member 20 and the like thinly as in the present embodiment.
It should be noted that a part Pa of the organ P can come into contact with the glass plate 10 in a range having a size of 1/3 or more (that is, 1 mm or more) of the opening size of the opening 32 (diameter 3 mm in this embodiment). In the value, the thickness of the cover member 20 (and the thickness of the flow path 30) is set.
〔実施例〕
図６（Ａ）は、上記実施形態（図３）の生体試料固定器１を用いて、マウスＭの腸を固定して観察した結果を示している。マウスＭの腹部に、切開範囲として５ｍｍ四方程度の切り口を作成し、高速撮影（３３ｍｓ／ｆｒａｍｅ）を行った。また、その比較として図６（Ｂ）は、生体試料固定器１を用いることなく、つまり、マウスＭの腸を固定しないで観察した結果を示している。図６（Ａ）（Ｂ）では、蛍光色素をＰＢＳ、生理食塩水等に分散させ、これをマウスＭの尾静脈より投与している。図６（Ａ）に示すように、生体試料固定器１を用いた場合、生体試料の一部（観察部位）の動きを拘束することができ、明確な観察が可能となる。これに対して、図６（Ｂ）に示すように、固定しない場合は、時間が変化すると焦点が合わず、また、観察部位がずれてしまう。
なお、本実施形態の生体試料固定器１によれば、マウスＭの肝臓、心臓及び肺についても同様の観察が可能である。
〔Example〕
FIG. 6A shows the result of observation with the intestine of mouse M fixed using the biological sample fixing device 1 of the above embodiment (FIG. 3). An incision range of about 5 mm square was created in the abdomen of mouse M, and high-speed imaging (33 ms / frame) was performed. As a comparison, FIG. 6B shows the result of observation without using the biological sample fixing device 1, that is, without fixing the intestine of the mouse M. 6 (A) and 6 (B), the fluorescent dye is dispersed in PBS, physiological saline or the like and administered from the tail vein of mouse M. As shown in FIG. 6A, when the biological sample fixing device 1 is used, the movement of a part (observation site) of the biological sample can be restricted, and clear observation is possible. On the other hand, as shown in FIG. 6B, when it is not fixed, when time changes, the focus is not achieved and the observation site is shifted.
In addition, according to the biological sample fixing device 1 of the present embodiment, the same observation can be performed on the liver, heart, and lung of the mouse M.
図７は、上記実施形態（図３）の生体試料固定器１を用いて、マウスＭの肝臓を固定して観察した結果を示しており、図７の左側から順に、流路３０内の空気を吸引した状態（ＯＮ状態）、流路３０内の空気の吸引を停止した状態（ＯＦＦ状態）、再びＯＮ状態とした場合の観察結果を示している。
一旦、吸引をオフ状態にすると、肝臓に対する顕微鏡５の焦点がずれるが、再びオン状態にすると、肝臓の別の場所を保持することができ、観察を再開することができる。つまり、吸引のＯＮ／ＯＦＦの切り替えにより観察部位を変更することが可能となる。
FIG. 7 shows the result of observation of the liver of the mouse M fixed using the biological sample fixing device 1 of the above embodiment (FIG. 3). The air in the flow path 30 is sequentially shown from the left side of FIG. The observation results are shown when the air is sucked (ON state), the suction of the air in the flow path 30 is stopped (OFF state), and turned on again.
Once the suction is turned off, the microscope 5 is out of focus with respect to the liver, but when it is turned on again, another part of the liver can be held and observation can be resumed. That is, the observation site can be changed by switching the suction ON / OFF.
図８は、上記実施形態（図３）の生体試料固定器１を用いて、マウスＭの肝臓における幹細胞の分布及び移動を観察した結果を示している。まず、マウスＭの大腿骨より採取した骨髄細胞から得られた間葉系幹細胞（ＭＳＣ）を、培養プレートに播種し、量子ドット含有培地で培養することにより、細胞の蛍光標識を行った。次に、蛍光標識されたＭＳＣを、マウスＭの尾静脈より投与した。そして、マウス腹部に、切開範囲として５ｍｍ四方程度の切り口を作成し、生体試料固定器１で肝臓を固定し、高速撮影（３３ｍｓ／ｆｒａｍｅ）を行った。図８に示すように、ＭＳＣが肝臓に流入し、分布する様子、及び、ローリング、接着等の挙動も可視化することができる。
また、この応用として、内在性の細胞の挙動を可視化することを目的として、白血球に特異的に認識される蛍光修飾抗体をマウスの尾静脈から投与し、生きたマウスの腸における白血球の挙動の撮影を行うことが可能である。
FIG. 8 shows the result of observing the distribution and migration of stem cells in the liver of mouse M using the biological sample fixing device 1 of the above embodiment (FIG. 3). First, mesenchymal stem cells (MSC) obtained from bone marrow cells collected from the femur of mouse M were seeded on a culture plate and cultured in a quantum dot-containing medium to label the cells with fluorescence. Next, fluorescently labeled MSC was administered from the tail vein of mouse M. Then, an incision range of about 5 mm square was created in the mouse abdomen, the liver was fixed with the biological sample fixing device 1, and high-speed imaging (33 ms / frame) was performed. As shown in FIG. 8, MSC flows into the liver and is distributed, and behaviors such as rolling and adhesion can be visualized.
In addition, as an application of this, for the purpose of visualizing the behavior of endogenous cells, a fluorescently modified antibody that is specifically recognized by leukocytes is administered from the tail vein of mice, and the behavior of leukocytes in the intestine of living mice is examined. It is possible to take a picture.
図９は、上記実施形態（図３）の生体試料固定器１を用いて、マウスＭの肝臓を長時間観察した結果を示している。蛍光標識されたリポソームを、マウスの尾静脈より投与し、マウス腹部に、切開範囲として５ｍｍ四方程度の切り口を作成し、生体試料固定器１で肝臓を固定し、高速撮影（５００ｍｓ／ｆｒａｍｅ）を行った。蛍光標識リポソームが肝臓に流入し、分布する様子を、長時間にわたって撮影することができている。しかも、長時間にわたって観察部位が対物レンズの視野内でずれることがなく、撮影されている。
なお、図９では、７０分観察した結果を示しているが、１２０分、観察部位が対物レンズの視野内でずれることがなく撮影することができたことも確認されている。
FIG. 9 shows a result of observing the liver of mouse M for a long time using the biological sample fixing device 1 of the above embodiment (FIG. 3). Fluorescently labeled liposomes are administered from the tail vein of the mouse, a cut is made about 5 mm square as an incision range in the abdomen of the mouse, the liver is fixed with the biological sample fixing device 1, and high-speed imaging (500 ms / frame) is performed. went. The state where the fluorescently labeled liposome flows into the liver and is distributed can be photographed over a long period of time. Moreover, the observation site is photographed without being shifted in the field of view of the objective lens for a long time.
Note that FIG. 9 shows the result of observation for 70 minutes, but it was also confirmed that an image could be taken for 120 minutes without the observation site being displaced within the field of view of the objective lens.
以上のように、本実施形態に係る生体試料固定器１によれば、カバー部材２０に形成されている開口３２に臓器Ｐを臨ませ、吸引部３１を通じて流路３０内の空気を吸引すれば、開口３２において臓器Ｐの一部Ｐａを陰圧（負圧）により保持することができる（図３参照）。このため、臓器Ｐをステージ６に対して固定することができ、開口３２に保持されている臓器Ｐの一部Ｐａを観察部位（観察対象部位）として、ガラス板１０を通じて顕微鏡観察することが可能となる。
そして、開口３２に保持されている臓器Ｐの一部Ｐａは、顕微鏡５による観察部位となることから、この臓器Ｐの一部Ｐａが、マウスＭの皮膚Ｓから露出していれば足りる。したがって、マウスＭの皮膚Ｓの切開範囲は、臓器Ｐの一部Ｐａ及びその周囲の僅かな部分が露出する範囲で済み、切開範囲を従来（図１３参照）と比較して小さくすることができ、マウスＭに対する負担（侵襲性）を抑えることが可能となる。
As described above, according to the biological sample fixing device 1 according to the present embodiment, if the organ P faces the opening 32 formed in the cover member 20 and the air in the flow path 30 is sucked through the suction part 31. In the opening 32, a part Pa of the organ P can be held by negative pressure (negative pressure) (see FIG. 3). For this reason, the organ P can be fixed to the stage 6, and a part Pa of the organ P held in the opening 32 can be observed with a microscope through the glass plate 10 as an observation site (observation site). It becomes.
Since a part Pa of the organ P held in the opening 32 becomes an observation site by the microscope 5, it is sufficient that the part Pa of the organ P is exposed from the skin S of the mouse M. Therefore, the incision range of the skin S of the mouse M only needs to be a range in which a part Pa of the organ P and a slight portion around the organ P are exposed, and the incision range can be made smaller than the conventional one (see FIG. 13). It becomes possible to reduce the burden (invasiveness) on the mouse M.
具体的に説明すると、本実施形態では、開口３２は直径が３ｍｍであり、ガラス板１０の一部１２に接触させる臓器Ｐの一部Ｐａが１ｍｍ程度でよく、この場合、マウスＭの皮膚Ｓの切り口寸法は、開口３２の開口寸法（３ｍｍ）よりも僅かに大きい５ｍｍ程度で済み、マウスＭへの負担は小さい。つまり、本実施形態に係る生体試料固定器１では、観察部位である臓器Ｐの一部Ｐａが、この一部Ｐａを陰圧によって固定する固定部位と同じであるため、この部位及びその周囲の小領域のみを皮膚Ｓから露出可能となる切開で済み、低侵襲的である。
以上より、麻酔、人工呼吸器、体温維持装置等によってマウスＭを生かした状態とし、対物レンズ７の視野程度の小さな切り口をマウスＭの皮膚Ｓに設ければよいので、マウスＭへの影響を最小限にして臓器Ｐの観察が可能となる。また、マウスＭ及び臓器Ｐを自然な状態として観察することができる。
Specifically, in the present embodiment, the opening 32 has a diameter of 3 mm, and the part Pa of the organ P to be brought into contact with the part 12 of the glass plate 10 may be about 1 mm. In this case, the skin S of the mouse M The cut-out dimension is about 5 mm, which is slightly larger than the opening dimension (3 mm) of the opening 32, and the burden on the mouse M is small. That is, in the biological sample fixing device 1 according to the present embodiment, the part Pa of the organ P that is the observation part is the same as the fixing part that fixes the part Pa by negative pressure. Only a small region needs to be incised so as to be exposed from the skin S, and is minimally invasive.
From the above, since the mouse M can be kept alive by anesthesia, a ventilator, a body temperature maintenance device, etc., and a small incision about the visual field of the objective lens 7 is provided on the skin S of the mouse M, the influence on the mouse M is affected. The organ P can be observed with a minimum. In addition, the mouse M and the organ P can be observed in a natural state.
開口３２において行われる臓器Ｐの一部Ｐａの動きの拘束についてさらに説明する。図３に示すように、生体試料固定器１の上にマウスＭをうつ伏せの姿勢で載せることにより、開口３２にその臓器Ｐの一部Ｐａを載せることができ、この開口３２に載せた臓器Ｐの一部Ｐａは、マウスＭの自重によって、開口３２に接触すると共に、開口３２に僅かに入り込むことができる。そして、吸引部３１を通じて流路３０内の空気が吸引されることによって、流路３０内が減圧され、臓器Ｐの一部Ｐａは、更に流路３０内に引き込まれ、ガラス板１０の一部１２に接触する。この際、臓器Ｐの一部Ｐａと開口３２との間には、微小隙間δが形成され、この微小隙間δ、つまり、一部Ｐａの周囲が減圧される。
この減圧状態では、空気の吸引方向は、開口３２の中心線に平行な方向（本実施形態では鉛直方向）ではなく、この中心線に直交する方向の成分を有した方向（本実施形態では水平方向）であり、特に流路３０内であって、開口３２から離れた部分では、中心線に直交する方向となる。このように、開口３２に保持した臓器Ｐに対する空気の吸引方向は、従来（図１３）の吸引方向とは異なる。
The restraint on the movement of a part Pa of the organ P performed in the opening 32 will be further described. As shown in FIG. 3, by placing the mouse M in a prone posture on the biological sample fixing device 1, a part Pa of the organ P can be placed on the opening 32, and the organ P placed on the opening 32. A part of the Pa can contact the opening 32 by the weight of the mouse M and can enter the opening 32 slightly. Then, the air in the flow path 30 is sucked through the suction unit 31, whereby the pressure in the flow path 30 is reduced, and a part Pa of the organ P is further drawn into the flow path 30, and a part of the glass plate 10. 12 is contacted. At this time, a minute gap δ is formed between a part Pa of the organ P and the opening 32, and the area around the minute gap δ, that is, part Pa is decompressed.
In this reduced pressure state, the air suction direction is not a direction parallel to the center line of the opening 32 (vertical direction in the present embodiment), but a direction having a component in a direction perpendicular to the center line (horizontal in the present embodiment). Direction), and particularly in the flow path 30 and away from the opening 32, the direction is perpendicular to the center line. Thus, the air suction direction with respect to the organ P held in the opening 32 is different from the conventional suction direction (FIG. 13).
また、本実施形態では、枝流路３７を複数形成することによって、開口３２に保持される臓器Ｐの一部Ｐａの周囲を、複数方向から減圧することができ、臓器Ｐの一部Ｐａを安定して開口３２に保持することが可能となる。
そして、本実施形態に係る生体試料固定器１によれば、高倍率（２０倍、４０倍）となっても、長時間にわたって観察部位が対物レンズ７の視野内でずれることなく観察可能となる。また、本実施形態によれば、臓器を体外へ出して行う観察とは異なることから、長時間の観察を行っても臓器の乾燥が問題とならない。しかも、生体試料固定器１をステージ６に載せ、その上に生体（生体試料）を載せればよいことから、観察の準備が簡単である。なお、従来のスタビライザ（図１３）の場合、生体試料に対するスタビライザの位置調整が必要となり、観察の準備に手間を要する。
また、上記実施例で説明したように、流路３０内の空気の吸引を、ＯＮ／ＯＦＦ切り替えることによって、何度でも、観察部位を変更することが可能となる（図７参照）。
Further, in the present embodiment, by forming a plurality of branch channels 37, the periphery of a part Pa of the organ P held in the opening 32 can be decompressed from a plurality of directions, and the part Pa of the organ P is reduced. The opening 32 can be stably held.
Then, according to the biological sample fixing device 1 according to the present embodiment, even when the magnification is high (20 times, 40 times), the observation site can be observed without being shifted in the field of view of the objective lens 7 for a long time. . In addition, according to the present embodiment, since it is different from the observation performed by taking the organ out of the body, drying of the organ does not become a problem even if observation is performed for a long time. In addition, since the biological sample fixing device 1 is placed on the stage 6 and the living body (biological sample) is placed thereon, preparation for observation is simple. In the case of the conventional stabilizer (FIG. 13), it is necessary to adjust the position of the stabilizer with respect to the biological sample, and it takes time to prepare for observation.
Further, as described in the above embodiment, the observation site can be changed any number of times by switching ON / OFF the suction of the air in the flow path 30 (see FIG. 7).
図１０は、生体試料固定器の他の実施形態を示す斜視図である。この生体試料固定器５１と、図２に示している生体試料固定器１とは、枝流路３７及び隔壁４０の構成が異なるが、その他の構成は同じである。図１０の実施形態の場合、開口３２に対して三方向から空気が吸引される。 FIG. 10 is a perspective view showing another embodiment of the biological sample fixing device. The biological sample fixing device 51 and the biological sample fixing device 1 shown in FIG. 2 are different in the configuration of the branch channel 37 and the partition wall 40, but the other configuration is the same. In the case of the embodiment of FIG. 10, air is sucked into the opening 32 from three directions.
図１１は、生体試料固定器のさらに別の実施形態を示す断面図である。図１１（Ａ）に示すように、カバー部材２０は、開口３２の周囲に環状の突起部４５を有している。突起部４５も、本体部２３と同じ素材（ＰＤＭＳ）からなり、弾性変形が可能である。突起部４５が形成されている点以外は、図２に示す生体試料固定器１と同じである。
そして、吸引部３１を通じて流路３０内の空気を吸引すると、図１１（Ｂ）に示すように、臓器Ｐの一部Ｐａが突起部４５に吸着すると共に、この突起部４５が一部Ｐａに沿って拡径方向に弾性変形する。これにより、開口３２と臓器Ｐとは密着し、密封性を高めることができる。
FIG. 11 is a cross-sectional view showing still another embodiment of the biological sample fixing device. As shown in FIG. 11A, the cover member 20 has an annular protrusion 45 around the opening 32. The protrusion 45 is also made of the same material (PDMS) as the main body 23 and can be elastically deformed. The biological sample fixing device 1 is the same as the biological sample fixing device 1 shown in FIG. 2 except that the protrusion 45 is formed.
Then, when the air in the flow path 30 is sucked through the suction portion 31, as shown in FIG. 11B, a part Pa of the organ P is adsorbed to the protrusion 45, and the protrusion 45 becomes a part Pa. It is elastically deformed along the diameter increasing direction. Thereby, the opening 32 and the organ P can be in close contact with each other, and the sealing performance can be improved.
また、前記各実施形態では、ガラス板１０とカバー部材２０とにより生体試料固定器が構成される場合を説明したが、本発明の生体試料固定器は、ガラス板１０に重ねて載せるためのカバー部材２０のみを備えた構成であってもよい。 In each of the above-described embodiments, the case where the biological sample fixing device is configured by the glass plate 10 and the cover member 20 has been described. However, the biological sample fixing device of the present invention is a cover for stacking and mounting on the glass plate 10. The structure provided only with the member 20 may be sufficient.
また、本発明の生体試料固定器は、図示する形態に限らず本発明の範囲内において他の形態のものであってもよい。例えば、吸引部３１の形状は図示した以外の形状であってもよい。また、枝流路３７を省略して、一条の流路の一端部側に吸引部３１を設け、他端部側に開口３２を形成した簡単な構成であってもよい。なお、枝流路３７を設ける場合、その分岐の構成も図示した構成以外であってもよい。
また、吸引部３１を通じて流路３０内の吸引を行う対象は、上記実施形態では空気として説明したが、流路３０内に液体を充填しこれを吸引してもよい。
In addition, the biological sample fixing device of the present invention is not limited to the illustrated form, and may be in other forms within the scope of the present invention. For example, the shape of the suction part 31 may be a shape other than that illustrated. Alternatively, a simple configuration in which the branch channel 37 is omitted, the suction part 31 is provided on one end of the single channel, and the opening 32 is formed on the other end may be employed. In addition, when providing the branch flow path 37, the structure of the branch may be other than the illustrated structure.
In addition, in the above-described embodiment, the object to be sucked in the flow path 30 through the suction unit 31 has been described as air. However, the flow path 30 may be filled with a liquid and sucked.
さらに、上記実施形態では、ガラス板１０とカバー部材２０とは分離可能として説明したが、ガラス板１０とカバー部材２０とは接着等によって固定されていてもよく、さらには、上記実施形態では、ガラス板１０とカバー部材２０とは異なる材質であり、別部材からなる場合を説明したが、ガラス板１０に相当する板部と、カバー部材２０に相当するカバー部とは、ＰＤＭＳ等により一体成型品であってもよい。
また、上記実施形態では、吸引部３１と開口３２との双方がカバー部材（カバー部）２０に設けられている場合を説明したが、図１２に示すように、吸引部３１については、ガラス板（板部）１０に設けられていてもよい。
Furthermore, in the said embodiment, although demonstrated that the glass plate 10 and the cover member 20 were separable, the glass plate 10 and the cover member 20 may be fixed by adhesion | attachment etc. Furthermore, in the said embodiment, The case where the glass plate 10 and the cover member 20 are made of different materials and are made of different members has been described. However, the plate portion corresponding to the glass plate 10 and the cover portion corresponding to the cover member 20 are integrally formed by PDMS or the like. It may be a product.
Moreover, although the said embodiment demonstrated the case where both the suction part 31 and the opening 32 were provided in the cover member (cover part) 20, as shown in FIG. (Plate part) 10 may be provided.
さらに、図示しないが、カバー部材２０は二層構造であってもよい。この場合、図１２と同様に、吸引部３１については、ガラス板（板部）１０に設けられていてもよく、図３のように開口３２と同層である上側のカバー部材（上側のカバー部）に設けられていてもよく、又は、その下層である下側のカバー部材（下側のカバー部）に設けられていてもよい。この二層構造の場合、下側のカバー部材がガラス板１０に重ねて設けられる。また、この場合、上側のカバー部材と下側のカバー部材とは、同じ材質であってもよく、異なっていてもよい。また、各層は接着されていてもよく、分離可能であってもよい。
また、上記実施形態では、生体試料固定器を倒立顕微鏡に設置した場合として説明したが、正立顕微鏡に設置してもよい。この場合、生体試料固定器を、図３の場合と上下反対にして設置する。
Further, although not shown, the cover member 20 may have a two-layer structure. In this case, similarly to FIG. 12, the suction part 31 may be provided on the glass plate (plate part) 10, and the upper cover member (upper cover) which is the same layer as the opening 32 as shown in FIG. Part) or a lower cover member (lower cover part) which is a lower layer thereof. In the case of this two-layer structure, the lower cover member is provided so as to overlap the glass plate 10. In this case, the upper cover member and the lower cover member may be made of the same material or different from each other. Moreover, each layer may be adhere | attached and may be separable.
In the above embodiment, the biological sample fixing device is described as being installed on an inverted microscope, but it may be installed on an upright microscope. In this case, the biological sample fixing device is installed upside down as in FIG.
１：生体試料固定器 ５：顕微鏡 ６：ステージ ７：対物レンズ １０：ガラス板（板部） １１：上面 １２：一部（接触面） ２０：カバー部材（カバー部） ２２：凹溝部 ２３：本体部 ２４：当接面 ３０：流路 ３０ａ：一部 ３０ｂ：他部 ３１：吸引部 ３２：開口 ３６：主流路 ３７：枝流路 ４０：隔壁（補剛部） ５１：生体試料固定器 Ｐ：臓器（生体試料） Ｐａ：一部（生体試料の一部） DESCRIPTION OF SYMBOLS 1: Biological sample fixing device 5: Microscope 6: Stage 7: Objective lens 10: Glass plate (plate part) 11: Upper surface 12: Part (contact surface) 20: Cover member (cover part) 22: Groove part 23: Main body Part 24: Contact surface 30: Channel 30a: Part 30b: Other part 31: Suction part 32: Opening 36: Main channel 37: Branch channel 40: Bulkhead (stiffening part) 51: Biological sample fixing device P: Organ (biological sample) Pa: Part (part of biological sample)
Claims (10)
前記ステージに設置される透明の板部と、前記板部に重ねて設けられ当該板部との間に流路を形成するカバー部と、を備え、
前記カバー部又は前記板部には、前記流路の一部と繋がり当該流路内の流体を吸引するための吸引部が設けられ、前記カバー部の、前記板部を間に挟んで前記顕微鏡の対物レンズに対向する位置には、前記流路の他部と繋がっていると共に前記生体試料の一部を前記流路内に侵入させる開口が設けられていることを特徴とする生体試料固定器。 A biological sample fixing device installed on a microscope stage for fixing a biological sample to the stage,
A transparent plate portion installed on the stage, and a cover portion that is provided to overlap the plate portion and forms a flow path between the plate portion, and
The cover part or the plate part is provided with a suction part that is connected to a part of the flow path and sucks the fluid in the flow path, and the microscope is sandwiched between the plate part and the microscope. The biological sample fixing device is characterized in that an opening is provided at a position facing the objective lens of the other of the flow paths and is connected to the other part of the flow path and allows a part of the biological sample to enter the flow path . .
前記ステージに設置される透明の板部と、前記板部に重ねて設けられ当該板部との間に流路を形成するカバー部と、を備え、
前記カバー部又は前記板部には、前記流路の一部と繋がり当該流路内の流体を吸引するための吸引部が設けられ、前記カバー部には、前記流路の他部と繋がっていると共に前記生体試料の一部を前記流路内に侵入させる開口が設けられ、
前記板部は、前記流路内の流体の吸引により前記開口に保持されかつ当該開口から前記流路内に侵入させる前記生体試料の一部を接触可能とする接触面を有している、生体試料固定器。 A biological sample fixing device installed on a microscope stage for fixing a biological sample to the stage,
A transparent plate portion installed on the stage, and a cover portion that is provided to overlap the plate portion and forms a flow path between the plate portion, and
The cover part or the plate part is provided with a suction part that is connected to a part of the flow path and sucks the fluid in the flow path, and the cover part is connected to the other part of the flow path. And an opening for allowing a part of the biological sample to enter the flow path is provided ,
The plate portion has a contact surface that is held in the opening by suction of fluid in the flow path and allows a part of the biological sample to enter the flow path from the opening to be in contact with the living body. Sample fixture.
前記当接面は、前記流路内の流体を吸引すると前記板部の前記面に密着し、当該吸引を解除した状態では前記板部の前記面と分離可能である非接着面である請求項１又は２に記載の生体試料固定器。 The cover part includes a groove part for configuring the flow path, and a main body part having a contact surface that surrounds the groove part and contacts the surface of the plate part,
The contact surface is a non-adhesive surface that comes into close contact with the surface of the plate portion when the fluid in the flow path is sucked and is separable from the surface of the plate portion in a state where the suction is released. The biological sample fixing device according to 1 or 2.
前記ステージに設置される透明の板部材に重ねて設けられ当該板部材との間に流路を形成するカバー部材を備え、
前記カバー部材には、前記流路の一部と繋がり当該流路内の流体を吸引するための吸引部と、前記流路の他部と繋がっていると共に前記生体試料の一部を前記流路内に侵入させる開口と、が形成され、
前記開口は、前記カバー部材の、前記板部材を間に挟んで前記顕微鏡の対物レンズに対向する位置に設けられる、生体試料固定器。 A biological sample fixing device installed on a microscope stage for fixing a biological sample to the stage,
Provided with a cover member that is provided over the transparent plate member installed on the stage and forms a flow path between the plate member,
The cover member is connected to a part of the flow path and is connected to the suction part for sucking the fluid in the flow path and the other part of the flow path, and a part of the biological sample is transferred to the flow path. And an opening for intruding into ,
The said opening is a biological sample fixing device provided in the position which opposes the objective lens of the said microscope on both sides of the said plate member of the said cover member .
前記ステージに設置される透明の板部材に重ねて設けられ当該板部材との間に流路を形成するカバー部材を備え、
前記カバー部材には、前記流路の一部と繋がり当該流路内の流体を吸引するための吸引部と、前記流路の他部と繋がっていると共に前記生体試料の一部を前記流路内に侵入させる開口と、が形成され、
前記板部材は、前記流路内の流体の吸引により前記開口に保持されかつ当該開口から前記流路内に侵入させる前記生体試料の一部を接触可能とする接触面を有している、生体試料固定器。 A biological sample fixing device installed on a microscope stage for fixing a biological sample to the stage,
Provided with a cover member that is provided over the transparent plate member installed on the stage and forms a flow path between the plate member,
The cover member is connected to a part of the flow path and is connected to the suction part for sucking the fluid in the flow path and the other part of the flow path, and a part of the biological sample is transferred to the flow path. And an opening for intruding into ,
The plate member has a contact surface that is held in the opening by suction of fluid in the flow path and allows a part of the biological sample to enter the flow path from the opening to be in contact with the living body. Sample fixture.
前記ステージに設置される透明の板部材に重ねて設けられ当該板部材との間に流路を形成するカバー部材を備え、
前記カバー部材の、前記板部材を間に挟んで前記顕微鏡の対物レンズに対向する位置には、前記流路の一部と繋がっていると共に前記生体試料の一部を前記流路内に侵入させる開口が形成され、
前記カバー部材は、前記流路の他部と繋がり当該流路内の流体を吸引するための吸引部が形成されている前記板部材に重ねて設けられることを特徴とする生体試料固定器。 A biological sample fixing device installed on a microscope stage for fixing a biological sample to the stage,
Provided with a cover member that is provided over the transparent plate member installed on the stage and forms a flow path between the plate member,
The cover member is connected to a part of the flow path at a position facing the objective lens of the microscope with the plate member interposed therebetween, and a part of the biological sample is allowed to enter the flow path. An opening is formed,
The biological sample fixing device according to claim 1, wherein the cover member is provided to overlap with the plate member that is connected to the other part of the flow path and has a suction part for sucking the fluid in the flow path.
前記ステージに設置される透明の板部材に重ねて設けられ当該板部材との間に流路を形成するカバー部材を備え、
前記カバー部材には、前記流路の一部と繋がっていると共に前記生体試料の一部を前記流路内に侵入させる開口が形成され、
前記カバー部材は、前記流路の他部と繋がり当該流路内の流体を吸引するための吸引部が形成されている前記板部材に重ねて設けられ、
前記板部材は、前記流路内の流体の吸引により前記開口に保持されかつ当該開口から前記流路内に侵入させる前記生体試料の一部を接触可能とする接触面を有している、生体試料固定器。 A biological sample fixing device installed on a microscope stage for fixing a biological sample to the stage,
Provided with a cover member that is provided over the transparent plate member installed on the stage and forms a flow path between the plate member,
The cover member is connected to a part of the flow path and has an opening for allowing a part of the biological sample to enter the flow path .
The cover member is connected to the other part of the flow path and is provided to overlap the plate member on which a suction part for sucking the fluid in the flow path is formed ,
The plate member has a contact surface that is held in the opening by suction of fluid in the flow path and allows a part of the biological sample to enter the flow path from the opening to be in contact with the living body. Sample fixture.
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