JPH03134527A - Apparatus for measuring temperature of minute amount of sample - Google Patents
Apparatus for measuring temperature of minute amount of sampleInfo
- Publication number
- JPH03134527A JPH03134527A JP1273392A JP27339289A JPH03134527A JP H03134527 A JPH03134527 A JP H03134527A JP 1273392 A JP1273392 A JP 1273392A JP 27339289 A JP27339289 A JP 27339289A JP H03134527 A JPH03134527 A JP H03134527A
- Authority
- JP
- Japan
- Prior art keywords
- sample
- sheet
- temperature
- cooling
- thermocouple
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000001816 cooling Methods 0.000 claims abstract description 40
- 239000007788 liquid Substances 0.000 claims abstract description 24
- 238000010438 heat treatment Methods 0.000 claims abstract description 6
- 239000002470 thermal conductor Substances 0.000 claims description 6
- 229910052751 metal Inorganic materials 0.000 claims description 5
- 239000002184 metal Substances 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 4
- 150000002739 metals Chemical class 0.000 claims description 3
- 125000006850 spacer group Chemical group 0.000 abstract description 12
- 230000004083 survival effect Effects 0.000 abstract description 11
- 239000007789 gas Substances 0.000 abstract description 8
- 239000001307 helium Substances 0.000 abstract description 7
- 229910052734 helium Inorganic materials 0.000 abstract description 7
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 abstract description 7
- 239000011810 insulating material Substances 0.000 abstract description 3
- 229910052782 aluminium Inorganic materials 0.000 abstract description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 abstract description 2
- 239000011888 foil Substances 0.000 abstract description 2
- 239000004020 conductor Substances 0.000 abstract 2
- 239000007767 bonding agent Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 25
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 14
- 238000007710 freezing Methods 0.000 description 8
- 230000008014 freezing Effects 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 7
- 229910052757 nitrogen Inorganic materials 0.000 description 7
- 229910052802 copper Inorganic materials 0.000 description 6
- 239000010949 copper Substances 0.000 description 6
- 239000012790 adhesive layer Substances 0.000 description 5
- 239000013078 crystal Substances 0.000 description 5
- SWQJXJOGLNCZEY-RNFDNDRNSA-N helium-8 atom Chemical compound [8He] SWQJXJOGLNCZEY-RNFDNDRNSA-N 0.000 description 4
- 239000010410 layer Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 229920003002 synthetic resin Polymers 0.000 description 4
- 239000000057 synthetic resin Substances 0.000 description 4
- 229910001006 Constantan Inorganic materials 0.000 description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 230000001070 adhesive effect Effects 0.000 description 2
- 210000000601 blood cell Anatomy 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000853 adhesive Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000001772 blood platelet Anatomy 0.000 description 1
- 238000009529 body temperature measurement Methods 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 239000002577 cryoprotective agent Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003302 ferromagnetic material Substances 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 229910052594 sapphire Inorganic materials 0.000 description 1
- 239000010980 sapphire Substances 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000004544 sputter deposition Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 239000010409 thin film Substances 0.000 description 1
- 238000007740 vapor deposition Methods 0.000 description 1
Landscapes
- Measuring Temperature Or Quantity Of Heat (AREA)
- Sampling And Sample Adjustment (AREA)
- Radiation Pyrometers (AREA)
Abstract
Description
【発明の詳細な説明】
産業上の利用分野
本発明は、生体細胞などの極微小量の試料の温度を測定
するための装置に関する。DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a device for measuring the temperature of an extremely small amount of sample such as a living cell.
従来の技術
生体細胞を、高い生存率で凍結保存するためには、その
生体細胞を高速度で冷却する必要があり、その冷凍時の
生体細胞の温度変化を観察するために、温度測定装置が
必要になる。Conventional technology In order to cryopreserve living cells with a high survival rate, it is necessary to cool the living cells at a high rate, and in order to observe the temperature change of the living cells during freezing, a temperature measuring device is used. It becomes necessary.
本件発明者は、生体細胞である試料を高速度で冷却する
ために、液体ヘリウムによって純銅から成る冷却部材の
表面に、極微小量の試料を当接して密着する構成を提案
している。この構成において、試料の温度を測定するた
めには、単純には、その試料に小形の熱電対を浸漬ない
しは埋設することが考えられるであろう、このような構
成とすると、熱電対が前記冷却部材に接触する恐れがあ
り、したがって試料の正確な温度を測定することができ
ないという問題がある６また、熱電対をさらに小形化し
て、その熱電対の熱容量に起因した誤差を抑制する必要
がある。The present inventor has proposed a configuration in which a very small amount of sample is brought into close contact with the surface of a cooling member made of pure copper using liquid helium in order to cool the sample, which is a biological cell, at a high rate. In this configuration, in order to measure the temperature of a sample, it would be conceivable to simply immerse or bury a small thermocouple in the sample. There is a problem that there is a risk of contact with other members, and therefore it is not possible to accurately measure the temperature of the sample6.Also, it is necessary to further reduce the size of the thermocouple to suppress errors caused by the heat capacity of the thermocouple. .
このような問題を解決するために、前記冷却部材に接触
される試料の静電容量を測定し、この静電容量が、生体
細胞である試料に含まれている水分の液体から固体への
相変化に伴って変化することを観察し、これによって試
料の温度を測定することが考えられる。このような構成
では、生体細胞である試料の急速な過冷却を行う際の温
度は、試料の水分の相変化をする温度よりももつと低い
温度であるので、そのような低い温度の測定を行うこと
ができない。In order to solve this problem, the capacitance of the sample that is brought into contact with the cooling member is measured, and this capacitance is determined by the phase change of water contained in the sample, which is biological cells, from liquid to solid. It is conceivable to observe the change as the temperature changes and measure the temperature of the sample based on this. In such a configuration, the temperature at which the sample, which is a living cell, is rapidly supercooled is lower than the temperature at which the phase change of water in the sample occurs, so it is difficult to measure such low temperatures. can't do it.
発明が解決しようとする課題
本発明の目的は、極微小量の試料の温度を高精度で、し
かも広い温度範囲に亘って測定することができるように
した極微小量の試料の温度測定装置を提供することであ
る。Problems to be Solved by the Invention An object of the present invention is to provide a temperature measuring device for extremely small amounts of samples, which is capable of measuring the temperature of extremely small amounts of samples with high precision and over a wide temperature range. It is to provide.
課題を解決するための手段
本発明は、液体状の試料が載置されて保持する領域を有
し、熱容量の小さい熱良導体から成るシートと、
このシートに接触して試料とは反対側に設けられる温度
検出素子と、
試料を、シートとは反対側から冷却または加熱する手段
とを含むことを特徴とする極微小量の試料の温度測定装
置である。Means for Solving the Problems The present invention includes a sheet having a region for placing and holding a liquid sample and made of a good thermal conductor with a small heat capacity; This is an apparatus for measuring the temperature of an extremely small amount of sample, characterized in that it includes: a temperature detecting element that can be used as a sample; and means for cooling or heating the sample from the side opposite to the sheet.
また本発明は、異種金属の接続部分が偏平に形成され、
その偏平な接続部分の一表面に、液体状の試料を載置し
て保持する領域を有する、そのような熱電対と、
試料を、熱電対の前記接続部分とは反対側から冷却また
は加熱する手段とを含むことを特徴とする極微小量の試
料の温度測定装置である。Further, in the present invention, the connecting portion of dissimilar metals is formed flat,
Such a thermocouple has a region on one surface of its flat connecting portion for placing and holding a liquid sample, and the sample is cooled or heated from the side opposite to the connecting portion of the thermocouple. 1. An apparatus for measuring the temperature of an extremely small amount of sample, characterized by comprising means.
作 用
本発明に従えば、熱容量の小さい熱良導体から成るシー
トの予め定める領域に、液体状の試料を載置して保持し
、このシートに接触して試料とは反対側に温度検出素子
を設け、試料を、シートとは反対側から冷却または加熱
するようにしたので、試料が冷却または加熱される際に
、温度検出素子がその冷却または加熱する手段に接触す
ることはなく、試料の温度を、シートを介して間接的に
、しかも高精度で測定することができる。According to the present invention, a liquid sample is placed and held in a predetermined area of a sheet made of a good thermal conductor with a small heat capacity, and a temperature detection element is placed on the opposite side of the sample in contact with the sheet. Since the sample is cooled or heated from the side opposite to the sheet, the temperature detection element does not come into contact with the cooling or heating means when the sample is cooled or heated, and the temperature of the sample is can be measured indirectly and with high precision through the sheet.
また本発明に従えば、熱電対の異種金属接続部分を偏平
にし、この接続部分の予め定める領域に、液体状の試料
を載置して保持し、その試料を、熱電対の前記接続部分
とは反対側から冷却または加熱するようにしたので、熱
電対の熱容量をできるだけ小さくし、これによって高精
度で極微小量の試料の温度を測定することが可能になる
。またこのような熱電対を用いることによって、広範囲
に亘って、試料の温度を測定することができる。Further, according to the present invention, the dissimilar metal connecting portion of the thermocouple is flattened, a liquid sample is placed and held in a predetermined area of the connecting portion, and the sample is connected to the connecting portion of the thermocouple. Since the thermocouple is cooled or heated from the opposite side, the heat capacity of the thermocouple is made as small as possible, making it possible to measure the temperature of an extremely small amount of sample with high precision. Furthermore, by using such a thermocouple, the temperature of the sample can be measured over a wide range.
この試料として、たとえば生体細胞が挙げられ、この生
体細胞は、たとえば赤血球および血小板などの血液細胞
、骨髄細胞、***、卵子ならびに培養細胞などがある。Examples of this sample include living cells, such as blood cells such as red blood cells and platelets, bone marrow cells, sperm, eggs, and cultured cells.
極微小量というのは、たとえばマイクロピペットを用い
て滴下することができる程度の１未満であり得る。A microscopic amount may be less than 1, for example, such that it can be dropped using a micropipette.
実施例
第１図は、本発明の一実施例の全体の構成を示す断面図
である。基台１上に立設された支柱２の上部には、案内
ローラ３が設けられ、案内棒４が鉛直方向に昇降可能と
なっている。この案内棒４の下部には取付体５が固定さ
れており、この取付体５の下端部には試料台である取付
部材６が着脱可能に取付けられる。取付体５にはまた、
強磁性材料、たとえば鉄などから成る環状の吸着部材７
が固定される。Embodiment FIG. 1 is a sectional view showing the overall structure of an embodiment of the present invention. A guide roller 3 is provided on the upper part of a support column 2 erected on a base 1, and a guide rod 4 can be moved up and down in the vertical direction. A mounting body 5 is fixed to the lower part of the guide rod 4, and a mounting member 6, which is a sample stage, is removably mounted to the lower end of the mounting body 5. The mounting body 5 also has
An annular adsorption member 7 made of a ferromagnetic material, such as iron.
is fixed.
基台１上には、寒剤である極低温液化ガス、たとえば液
体ヘリウム８が貯留された容器９が設けられており、こ
の容器９には接続口１０からヘリウムガスが供給される
。容器９内に設けられた筒体１１の下端部は液体ヘリウ
ム８に浸漬されており、その上端部は、断熱材から成る
コンテナ１２の下部に開口している。このコンテナ１２
内には冷却部材１３が配置され、この冷却部材１３の外
周面とコンテナ１２の内周面との間の空間１４から気化
したヘリウムガスが放散される。A container 9 in which cryogenic liquefied gas, such as liquid helium 8, as a cryogen is stored is provided on the base 1, and helium gas is supplied to the container 9 from a connection port 10. The lower end of the cylindrical body 11 provided in the container 9 is immersed in liquid helium 8, and the upper end thereof is open to the lower part of the container 12 made of a heat insulating material. This container 12
A cooling member 13 is disposed inside, and vaporized helium gas is diffused from a space 14 between the outer peripheral surface of the cooling member 13 and the inner peripheral surface of the container 12.
冷却部材１３は、熱良導体である高純度の銅、銀などの
金属であってもよく、あるいはまたサファイアなどの材
料から成ってもよく、その熱容量は生体細胞である試料
２５（次に述べる第２図参照）などよりも充分に大きく
選ばれ、したがって冷却部材１３は常に極低温、たとえ
ば−２６９℃付近に保たれる。液体ヘリウムに代えて、
液体窒素を用いることもでき、そのとき冷却部材１３は
一１９６℃付近に冷却されて保たれる。The cooling member 13 may be made of a metal such as high-purity copper or silver, which is a good thermal conductor, or may be made of a material such as sapphire, and its heat capacity is higher than that of the sample 25 (described below), which is a biological cell. 2), etc., so that the cooling member 13 is always kept at an extremely low temperature, for example, around -269°C. Instead of liquid helium,
Liquid nitrogen can also be used, in which case the cooling member 13 is cooled and maintained at around -196°C.
冷却部材１３の上端面１６は、水平であり、鏡面仕上げ
が施される。The upper end surface 16 of the cooling member 13 is horizontal and has a mirror finish.
コンテナ１２は取付片１７によって支柱２に固定されて
おり、この取付片１７には永久磁石片１８が固定されて
おり、これによって吸着部材７を磁気吸着することがで
きる。コンテナ１２の上部は、ｆｉ１９によって開閉可
能とされる。The container 12 is fixed to the column 2 by a mounting piece 17, and a permanent magnet piece 18 is fixed to the mounting piece 17, so that the attraction member 7 can be magnetically attracted. The upper part of the container 12 can be opened and closed by fi19.
第２図は、取付部材６付近の分解斜視図である。FIG. 2 is an exploded perspective view of the vicinity of the mounting member 6.
取付部材６の下部は環状にくびれな係止部２０となって
おり、その下端面２１は水平である。この下端面２１上
には、生体細胞である試料２５を保持する保持部材２２
が着脱可能に取付けられる。The lower part of the mounting member 6 is an annularly constricted locking part 20, and the lower end surface 21 of the locking part 20 is horizontal. On this lower end surface 21 is a holding member 22 that holds a sample 25 that is a biological cell.
is removably attached.
第３図は、保持部材２２付近の拡大断面図である。保持
部材２２は、環状の合成樹脂材料などの断熱材から成る
スペーサ２３と、熱良導体、たとえばアルミニウムフォ
イルなどから成るシート２４とを有し、スペーサ２３と
シート２４とは接着剤によ′つて固定される。シート２
４の厚みは、たとえば１０μｍとし、その熱容量を充分
に小さく選ぶ、このシート２４のスペーサ２３によって
囲まれた下面は、試料２５がたとえばマイクロピペット
などによって載置されて、保持するための領域２４ａと
なっており、この試料は第３図において参照符２５で示
される。シート２４の外周部には、係止片２６が周方向
に間隔をあけて形成されており、この係止片２６を仮想
線２７で示す状態から矢符２８のように折曲げて、取付
部材６の係止部２０に係止し、こうして保持部材２２を
取付部材６に取付けることができ、またこの係止片２６
を矢符２８の逆方向に開いて、保持部材２２を取付部材
６から取外すことができる。FIG. 3 is an enlarged sectional view of the vicinity of the holding member 22. The holding member 22 has an annular spacer 23 made of a heat insulating material such as a synthetic resin material, and a sheet 24 made of a good thermal conductor such as aluminum foil, and the spacer 23 and the sheet 24 are fixed with adhesive. be done. sheet 2
The thickness of the sheet 24 is, for example, 10 μm, and its heat capacity is selected to be sufficiently small.The lower surface of the sheet 24 surrounded by the spacer 23 is a region 24a on which the sample 25 is placed and held by, for example, a micropipette. This sample is indicated by reference numeral 25 in FIG. Locking pieces 26 are formed on the outer periphery of the seat 24 at intervals in the circumferential direction, and the locking pieces 26 are bent as shown by the arrow 28 from the state shown by the imaginary line 27 to attach the mounting member. 6, the holding member 22 can be attached to the mounting member 6, and this locking piece 26
The holding member 22 can be removed from the mounting member 6 by opening in the direction opposite to the arrow 28.
シート２４と取付部材６の下端面２１との間には、温度
検出素子としての熱電対２９が介在される。このような
熱電対２９は、接着層４３に部分的に埋込まれて取付部
材６の下端面２１に取付けられる。接着層４３は、たと
えば粘着性を有する、いわゆる合成樹脂などから成るい
わゆる両面テープであってもよく、熱電対２９の接続部
分２１は接着層４３から露出して、シート２４に当接す
る。A thermocouple 29 as a temperature detection element is interposed between the sheet 24 and the lower end surface 21 of the mounting member 6. Such a thermocouple 29 is partially embedded in the adhesive layer 43 and attached to the lower end surface 21 of the attachment member 6. The adhesive layer 43 may be, for example, a so-called double-sided tape made of a so-called synthetic resin having adhesive properties, and the connecting portion 21 of the thermocouple 29 is exposed from the adhesive layer 43 and comes into contact with the sheet 24 .
接着層４３は、断熱層としての働きを兼ねており、試料
２５およびシート２４の高速度の冷却を確実にする。こ
の熱電対２９は、熱容量が充分に小さくなるようにする
ために、異種金属から成る細線３０から成り、その接続
部分３１はシート２４の前記領域２４ａとは反対側の表
面２４ｂに接触する。こうして領域２４ａ、したがって
試料２５の温度を高精度で検出することができる。細線
３０は、たとえばコンスタンタンと銅とからそれぞれ成
ってもよい、４ａＩ線３線間０熱起電力を電圧計たとえ
ばオシロスコープ３２によって測定することによって、
試料２５の温度を測定することができる。The adhesive layer 43 also serves as a heat insulating layer and ensures rapid cooling of the sample 25 and sheet 24. This thermocouple 29 is made of a thin wire 30 made of different metals in order to have a sufficiently small heat capacity, and its connecting portion 31 contacts the surface 24b of the sheet 24 on the opposite side from the region 24a. In this way, the temperature of the region 24a, and therefore the sample 25, can be detected with high precision. The thin wire 30 may be made of, for example, constantan and copper, respectively, by measuring the zero thermoelectromotive force between the three 4aI wires with a voltmeter, for example, an oscilloscope 32.
The temperature of the sample 25 can be measured.
案内棒４には、突起３４が形成されており、電磁プラン
ジャ３５によって駆動されるストッパ３６が突起３４に
当接している状態では、案内棒４は上方位置で保たれ、
電磁ソレノイド３５が励磁されることによって、突起３
４とストッパ３６とが外れて、案内棒４がその自重で、
取付体５および取付部材６などとともに、落下すること
ができる。A protrusion 34 is formed on the guide rod 4, and when a stopper 36 driven by an electromagnetic plunger 35 is in contact with the protrusion 34, the guide rod 4 is maintained at an upper position.
By energizing the electromagnetic solenoid 35, the protrusion 3
4 and the stopper 36 come off, and the guide rod 4 is moved by its own weight.
It can fall together with the mounting body 5, mounting member 6, and the like.
試料２５の凍結保存の手順を述べると、先ず取付部材６
を取付体５から取外した状態で、取付部材６には保持部
材２２を固定しておき、領域２４ａを上方に向けた姿勢
で、前述のようにマイクロピペットによって、生体細胞
である試料２５を領域２４ａに付着して保持する。To describe the procedure for cryopreservation of sample 25, first, attaching member 6
The holding member 22 is fixed to the mounting member 6 with the sample 25 removed from the mounting body 5, and with the region 24a facing upward, the sample 25, which is a living cell, is placed in the region using a micropipette as described above. It is attached to and held on 24a.
コンテナ１２は、蓋１９によって閉じたままとしておき
、これによって冷却部材１３は一１３０℃未満の極低温
に保っておく。The container 12 is kept closed by the lid 19, which keeps the cooling member 13 at an extremely low temperature below -130°C.
そこで、取付部材６を取付体５に固定する。蓋１９は、
コンテナ１２を閉じているので、冷却部材１３の上端面
１６に空気中の酸素、窒素および水分等（たとえばＣＯ
□）が結露して付着することを防ぐことができるととも
に、液体ヘリウムの気化したガスが試料２５に接触して
、その試料２５が不所望に冷却されるのを防ぐ。Therefore, the mounting member 6 is fixed to the mounting body 5. The lid 19 is
Since the container 12 is closed, the upper end surface 16 of the cooling member 13 is exposed to oxygen, nitrogen, moisture, etc. (for example, CO
□) can be prevented from condensing and adhering to the sample 25, and the sample 25 is also prevented from being undesirably cooled due to the vaporized gas of liquid helium coming into contact with the sample 25.
そこで次に、１１１９を移動してコンテナ１２の上部を
開き、その状態で電磁ソレノイド３５を励磁して、突起
３４からストッパ３６を外す、これによって、案内棒４
はローラ３によって案内されて取付体５、取付部材６お
よび保持部材２２などが自重で落下する。これによって
、スペーサ２３の下面２３ａは、第４図に明らかに示さ
れるように、冷却部材１３の上端面１６に衝突し、試料
２５は鏡面仕上げされている上端面１６に当接して圧着
される。スペーサ２３は、試料２５が過大な圧力で圧縮
されることを防いで、試料２５を保護する。吸着片７は
、永久磁石片１８によって磁気吸着され、こうして保持
部材２２のスペーサ２３が冷却部材１３の上端面１６に
衝突したときにおける上下の変動を防ぎ、試料２５を上
端面１６に圧着させる。これによって、試料２５は１０
３℃／分以上の冷却速度で、好ましくは１０４℃／分以
上の冷却速度で急速に冷却される。そのため、試ｆ１２
５の生存率を高くすることができる。Therefore, next, 1119 is moved to open the upper part of the container 12, and in this state, the electromagnetic solenoid 35 is energized to remove the stopper 36 from the protrusion 34. As a result, the guide rod 4
is guided by the rollers 3, and the mounting body 5, the mounting member 6, the holding member 22, etc. fall under their own weight. As a result, the lower surface 23a of the spacer 23 collides with the upper end surface 16 of the cooling member 13, as clearly shown in FIG. 4, and the sample 25 is pressed against the mirror-finished upper end surface 16. . The spacer 23 protects the sample 25 by preventing it from being compressed with excessive pressure. The attraction piece 7 is magnetically attracted by the permanent magnet piece 18, thereby preventing vertical fluctuation when the spacer 23 of the holding member 22 collides with the upper end surface 16 of the cooling member 13, and presses the sample 25 onto the upper end surface 16. As a result, sample 25 becomes 10
It is rapidly cooled at a cooling rate of 3° C./min or more, preferably 104° C./min or more. Therefore, test f12
5 survival rate can be increased.
第５図は、試料２５の冷却速度と生存率との関係を示す
本件発明者の実験結果を示すグラフである。生存率は、
ライン１１．１２間の範囲内にある０本件発明に従って
、試料２５の冷却速度を１０’、”Ｃ／分以上とするこ
とによって、生存率を高めることができ、特にその冷却
速度１０４℃／分以上とすることによって生存率をほぼ
１００％とすることができることが判る。FIG. 5 is a graph showing the experimental results of the present inventor showing the relationship between the cooling rate and survival rate of sample 25. The survival rate is
According to the present invention, the survival rate can be increased by cooling the sample 25 to 10'C/min or more, especially at a cooling rate of 104C/min. It can be seen that by doing the above, the survival rate can be made almost 100%.
冷却部材１３の上端面１６は、前述のように鏡面仕上げ
されているので、試料２５の熱伝導が極めて良好であり
、しかもこの冷却部材１３の熱容量は、試料２５、シー
ト２４および温度検出素子２９の各熱容量に比べて充分
に大きいので、その上端面１６の温度はほぼ一定であり
、このことによって、試料２５を確実に、高速度で冷却
することができ、その試料２５に含まれている水分は非
晶質で凍結され、たとえ氷晶が生成されても、それは極
めて微細であって、生体細胞が凍害を受けることはない
。試料２５と冷却部材１３の上端面１６との接触状態は
、たとえば２〜３秒間保ってもよい。Since the upper end surface 16 of the cooling member 13 is mirror-finished as described above, the heat conduction of the sample 25 is extremely good. Since the heat capacity of the sample 25 is sufficiently large compared to the respective heat capacities of the sample 25, the temperature of the upper end surface 16 is almost constant. Water is frozen in an amorphous state, and even if ice crystals are formed, they are extremely fine and do not cause freezing damage to living cells. The state of contact between the sample 25 and the upper end surface 16 of the cooling member 13 may be maintained for, for example, 2 to 3 seconds.
そこで次に、案内棒４を上昇して試料２５と冷却部材１
３の上端面１６とを離間し、ソレノイド３５を消磁して
ストッパ３６によって突起３４を支えた状態とし、この
状態で、次回の試料の凍結のために、取付体５から取付
部材６を取外し、第６図で示されるように、デユワ−ビ
ンまたは合成樹脂製容器などの槽３−８内に貯留されて
いる液体窒素３９内に浸漬する。この液体窒素３つ内で
、ビンセットを用いてシート２４の係止片２６を前述の
第３図の仮想線２７で示すように開いて、試料２５と一
体的となっている保持部材２２を取付部材６から取外す
、そこで、試料２５および保持部材２２を、合成樹脂製
容器４０内に液体窒素３つ内で、収納し、第７図に示さ
れるようにして断熱保存容器４１に貯留されている液体
窒素４２内に貯留する。こうして生体細胞を高い生存率
で、−１３０℃未満の温度範囲で、その生体細胞の水分
が結晶に転移しない状態で、保存することができる。Therefore, next, the guide rod 4 is raised to remove the sample 25 and the cooling member 1.
3, the solenoid 35 is demagnetized, and the protrusion 34 is supported by the stopper 36. In this state, the mounting member 6 is removed from the mounting body 5 in order to freeze the sample next time. As shown in FIG. 6, it is immersed in liquid nitrogen 39 stored in a tank 3-8 such as a dewar bottle or a container made of synthetic resin. In these three liquid nitrogen bottles, use a bottle set to open the locking piece 26 of the sheet 24 as shown by the imaginary line 27 in FIG. The sample 25 and the holding member 22 are removed from the mounting member 6, and then stored in a synthetic resin container 40 in three volumes of liquid nitrogen, and stored in an insulated storage container 41 as shown in FIG. It is stored in liquid nitrogen 42. In this way, biological cells can be preserved with a high survival rate in a temperature range below -130° C. without the water content of the biological cells converting to crystals.
試料２５の温度は熱電対２つによって測定され、この試
料２５は、熱容量の小さい熱良導体であるシート２４の
領域２４ａに広い面積で接触し、したがって試料２５の
温度を間接的に、高精度で測定することができる。その
ため、生体細胞である試料２５の急速凍結時における温
度変化を観察することができる。The temperature of the sample 25 is measured by two thermocouples, and the sample 25 has a large area in contact with the region 24a of the sheet 24, which is a good thermal conductor with a small heat capacity, so that the temperature of the sample 25 can be measured indirectly and with high precision. can be measured. Therefore, it is possible to observe temperature changes during rapid freezing of the sample 25, which is a living cell.
保持部材２２のスペーサ２３の厚み１（前述の第３図参
照）を、たとえば１５．２５および５０μｍに変化して
、熱電対２つを用いてその試Ｔ’−１２５の温度の時間
経過を観察することによって、試料２５の厚み方向の各
部位の組織が、どの程度の冷却速度で冷却されているか
を正確に知ることができ、これによって液体ヘリウムな
どの寒剤すなわち冷媒の違い、冷却部材１３の材質の違
い、冷却部材１３の上端面１６からの試料２５の深さの
違いによって冷却速度に及ぼす影響を知ることができる
。これによって、最適な冷却方法を見つけることが可能
になる。The thickness 1 (see FIG. 3 above) of the spacer 23 of the holding member 22 was changed to, for example, 15.25 and 50 μm, and the temperature of the sample T'-125 was observed over time using two thermocouples. By doing this, it is possible to accurately know at what cooling rate the structure of each part of the sample 25 in the thickness direction is being cooled. The influence on the cooling rate can be determined by the difference in the material and the difference in the depth of the sample 25 from the upper end surface 16 of the cooling member 13. This makes it possible to find the optimal cooling method.
水を始めとする液体は、冷却速度を上げることによって
、生成する結晶の大きさが小さくなり、やがては非晶質
、すなわちアモルファス状となる。By increasing the cooling rate of liquids such as water, the size of crystals formed becomes smaller and eventually becomes amorphous.
生体細胞は、生成する氷晶の大きさが１０ｎｍ未満、ま
たは非晶質状であれば、凍結解凍後も生存していると考
えられており、そういった状態を確実に造りだすために
本発明を実施することができる。Living cells are thought to survive even after freezing and thawing if the size of ice crystals produced is less than 10 nm or in an amorphous state, and the present invention was developed to reliably create such a state. It can be implemented.
本件発明者の実験によれば、液体ヘリウム８を用いて、
試料２５を一２６９℃付近まで冷却したとき、その試料
２５は１００μｍ以上の厚みに亘って、６Ｘ１０”Ｃ／
分以上の冷却速度が達成された。また、液体ヘリウム８
の代りに液体窒素を用いて、試料２５を一１９０℃付近
まで冷却した場合、５０μｍ以上の厚みに亘って、６Ｘ
１０’℃／分以上の冷却速度が達成された。このときの
試料２５は、たとえば赤血球細胞である。このようにし
て、生体細胞を凍結することによって、含水率が低く、
容易に非晶質で凍結可能な細胞はもとより、血球細胞の
ような高含水率の細胞においても、凍害保護剤を用いる
ことなしに、凍結保存可能となり、熱電対２９によって
その温度を観察することによって、生存率の予測を行う
ことができる。According to the inventor's experiments, using liquid helium 8,
When the sample 25 is cooled to around -269°C, the sample 25 has a 6X10"C/
Cooling rates in excess of minutes were achieved. Also, liquid helium 8
When sample 25 is cooled to around -190°C using liquid nitrogen instead, 6X
Cooling rates of greater than 10'°C/min were achieved. The sample 25 at this time is, for example, red blood cells. In this way, by freezing living cells, the water content is low,
Not only cells that are amorphous and can be frozen easily, but also cells with high water content such as blood cells can be cryopreserved without using a cryoprotectant, and the temperature can be observed using a thermocouple 29. The survival rate can be predicted by
第８図は、本発明の他の実施例の断面図である。FIG. 8 is a sectional view of another embodiment of the invention.
この実施例では、前述の熱電対２９に代えて、熱電対４
４が用いられる。この熱電対４４は、コンスタンタンの
薄Ｍ４５の表面に銅の［４６を、蒸着またはスパッタリ
ングなどの薄膜技術を用いて形成し、こうしてコンスタ
ンタンと銅との接続部分の全体の形状を扁平にし、コン
スタンクンおよび銅の素線４７，４８を電圧計４９に接
続する。In this embodiment, the thermocouple 4 is replaced with the thermocouple 29 described above.
4 is used. This thermocouple 44 is made by forming copper [46] on the surface of a thin M45 of constantan using a thin film technique such as vapor deposition or sputtering, thereby flattening the overall shape of the connection part between constantan and copper. And copper wires 47 and 48 are connected to a voltmeter 49.
層４６にはスペーサ２３が介在され、試料２５が層４６
の領域４６ａに保持される。この層４６とスペーサ２３
とは、試料２５を保持するための保持部材としての働き
を兼ねる。このような構成によれば、熱電対４４の熱容
量をさらに小さくして試料２５が冷却部材１３の上端面
１６に接触したときにおける温度計測における精度の向
上を、さらに図ることができる。The spacer 23 is interposed in the layer 46, and the sample 25 is placed in the layer 46.
is held in the area 46a. This layer 46 and spacer 23
Also serves as a holding member for holding the sample 25. According to such a configuration, it is possible to further reduce the heat capacity of the thermocouple 44 and further improve accuracy in temperature measurement when the sample 25 contacts the upper end surface 16 of the cooling member 13.
第９図は、第１図〜第７図の実施例の本件発明者による
実験結果を示すグラフである。熱電対２９の熱起電力は
電圧計３２としてのオシロスコープによって観測され、
スペーサ２３の厚みｔは５０μｍとし、試料２５は血液
３０μ！であり、第９図（１）によって得られた温度の
時間変化は、時間Ｗ１が拡大されて第９図（２）で示さ
れている。この実験によれば、冷却速度２．ｌＸｌ０’
℃／分が得られたことが確認された。FIG. 9 is a graph showing the experimental results of the present inventor for the embodiments shown in FIGS. 1 to 7. The thermoelectromotive force of the thermocouple 29 is observed by an oscilloscope as a voltmeter 32,
The thickness t of the spacer 23 is 50 μm, and the sample 25 is blood 30 μm! The time change in temperature obtained from FIG. 9(1) is shown in FIG. 9(2) with time W1 enlarged. According to this experiment, the cooling rate was 2. lXl0'
It was confirmed that ℃/min was obtained.
本発明の他の実施例として、熱電対２９．４４に代えて
、他の構造を有する温度検出素子、たとえばサーミスタ
などを用いることもまた可能である０本発明では、生体
細胞の他に、食品などの液体状の試料の温度測定を行う
ことができる６本発明では、生体細胞などのような含水
試料を電子類ｖ１１鏡で観察するための前処理として、
試料を急速凍結する際に、その試料の温度を測定するた
めにもまた、実施することができる６本発明は冷却だけ
でなく、加熱のためにも、実践することができる。As another embodiment of the present invention, instead of the thermocouple 29.44, it is also possible to use a temperature sensing element having another structure, such as a thermistor. 6 In the present invention, as a pretreatment for observing a water-containing sample such as a biological cell using an electronic V11 mirror,
It can also be practiced to measure the temperature of a sample when it is rapidly frozen.6 The invention can be practiced not only for cooling but also for heating.
試料としてマイクロとペラＩ・による液体状のものの付
着保持以外に、固体試料（たとえば肉片、細胞塊）をビ
ンセットで１寸着保持することもありうる。生体細胞の
高速冷却後、それを極低温液化ガスの気化した極低温ガ
ス雲囲気中に保持してもよい。In addition to adhering and holding a liquid sample as a sample using a micro and pelletizer, it is also possible to hold a solid sample (for example, a piece of meat, a cell mass) by one inch using a bottle set. After rapid cooling of the biological cell, it may be kept in a vaporized cryogenic gas cloud surround of cryogenic liquefied gas.
発明の効果
以上のように本発明によれば、極微小量の温度を、高精
度で、しかも、広い温度範囲に亘って、温度の測定を行
うことが可能になる。また本発明では、試料としてたと
えば生体細胞を凍結するに当たり、その試料の冷却速度
を確認することができ、これによって生体細胞が凍結障
害がないと考えられている大きさ以下の氷晶しか生成し
ておらず、したがって高い生存率で凍結されたか、また
は非晶質の状態では凍結していないかなどを確認するこ
とができる。Effects of the Invention As described above, according to the present invention, it is possible to measure extremely small amounts of temperature with high precision over a wide temperature range. Furthermore, according to the present invention, when freezing living cells as a sample, it is possible to check the cooling rate of the sample, thereby ensuring that living cells only generate ice crystals of a size smaller than that considered to be free from freezing damage. Therefore, it can be confirmed whether the frozen material has a high survival rate or whether it has not been frozen in an amorphous state.
第１図は本発明の一実施例の全体の構成を示す断面図、
第２図は取付部材６付近の分解斜視図、第３図は保持部
材２２付近の拡大断面図、第４図は試料２５を冷却部材
１３の上端面１６に圧着している状態を示す断面図、第
５図は生体細胞である試料２５とその生存率との関係を
示すグラフ、第６図は試料２５の急速冷凍後に保持部材
２２を取付部材６から取外す作業を示す斜視図、第７図
は試料２５を保存凍結した状態で保存するときの構造を
示す断面図、第８図は本発明の他の実施例の断面図、第
９図は第１図〜第７図の実施例の本件発明者による実験
結果を示すグラフである。
５・・・取付体、６・・・取付部材、８・・・液体ヘリ
ウム、１２・・・コンテナ、１３・・・冷却部材、１６
・・・上端面、２２・・・保持部材、２３・・・スペー
サ、２４・・・シート。
２５・・・試料、２９．４４・・・熱電対、３２．４９
・・・電圧計、４３・・・接着層FIG. 1 is a sectional view showing the overall configuration of an embodiment of the present invention;
2 is an exploded perspective view of the vicinity of the mounting member 6, FIG. 3 is an enlarged sectional view of the vicinity of the holding member 22, and FIG. 4 is a sectional view showing the sample 25 being crimped onto the upper end surface 16 of the cooling member 13. , FIG. 5 is a graph showing the relationship between the sample 25, which is a living cell, and its survival rate, FIG. 6 is a perspective view showing the operation of removing the holding member 22 from the mounting member 6 after rapid freezing of the sample 25, and FIG. 7 is a cross-sectional view showing the structure when the sample 25 is stored in a frozen state, FIG. 8 is a cross-sectional view of another embodiment of the present invention, and FIG. 9 is a main part of the embodiment of FIGS. 1 to 7. It is a graph showing experimental results by the inventor. 5... Mounting body, 6... Mounting member, 8... Liquid helium, 12... Container, 13... Cooling member, 16
... Upper end surface, 22 ... Holding member, 23 ... Spacer, 24 ... Sheet. 25...Sample, 29.44...Thermocouple, 32.49
...Voltmeter, 43...Adhesive layer
Claims (2)
熱容量の小さい熱良導体から成るシートと、このシート
に接触して試料とは反対側に設けられる温度検出素子と
、 試料を、シートとは反対側から冷却または加熱する手段
とを含むことを特徴とする極微小量の試料の温度測定装
置。(1) having an area where a liquid sample is placed and held;
It is characterized by comprising a sheet made of a good thermal conductor with a small heat capacity, a temperature detection element provided in contact with the sheet on the side opposite to the sample, and means for cooling or heating the sample from the side opposite to the sheet. A device for measuring the temperature of extremely small amounts of samples.
な接続部分の一表面に、液体状の試料を載置して保持す
る領域を有する、そのような熱電対と、試料を、熱電対
の前記接続部分とは反対側から冷却または加熱する手段
とを含むことを特徴とする極微小量の試料の温度測定装
置。(2) A thermocouple in which a connecting part of dissimilar metals is formed flat and has an area for placing and holding a liquid sample on one surface of the flat connecting part, and a sample. An apparatus for measuring the temperature of an extremely small amount of sample, characterized in that it includes means for cooling or heating from the opposite side of the pair of connecting parts.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1273392A JPH03134527A (en) | 1989-10-19 | 1989-10-19 | Apparatus for measuring temperature of minute amount of sample |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1273392A JPH03134527A (en) | 1989-10-19 | 1989-10-19 | Apparatus for measuring temperature of minute amount of sample |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH03134527A true JPH03134527A (en) | 1991-06-07 |
Family
ID=17527261
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1273392A Pending JPH03134527A (en) | 1989-10-19 | 1989-10-19 | Apparatus for measuring temperature of minute amount of sample |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH03134527A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1423501A2 (en) * | 2001-08-06 | 2004-06-02 | Vanderbilt University | Device and methods for measuring the response of at least one cell to a medium |
| WO2007142061A1 (en) * | 2006-06-06 | 2007-12-13 | Konica Minolta Medical & Graphic, Inc. | Microchip inspection device |
| CN103398883A (en) * | 2013-07-29 | 2013-11-20 | 佳木斯大学 | Piece pressing machine for manufacturing pieces in cell genetics |
-
1989
- 1989-10-19 JP JP1273392A patent/JPH03134527A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1423501A2 (en) * | 2001-08-06 | 2004-06-02 | Vanderbilt University | Device and methods for measuring the response of at least one cell to a medium |
| EP1423501A4 (en) * | 2001-08-06 | 2008-08-06 | Univ Vanderbilt | Device and methods for measuring the response of at least one cell to a medium |
| US7704745B2 (en) | 2001-08-06 | 2010-04-27 | Vanderbilt University | Apparatus and methods for monitoring the status of a metabolically active cell |
| US7713733B2 (en) | 2001-08-06 | 2010-05-11 | Vanderbilt University | Device and methods for detecting the response of a plurality of cells to at least one analyte of interest |
| US7981649B2 (en) | 2001-08-06 | 2011-07-19 | Vanderbilt University | Device and methods for monitoring the status of at least one cell |
| WO2007142061A1 (en) * | 2006-06-06 | 2007-12-13 | Konica Minolta Medical & Graphic, Inc. | Microchip inspection device |
| CN103398883A (en) * | 2013-07-29 | 2013-11-20 | 佳木斯大学 | Piece pressing machine for manufacturing pieces in cell genetics |
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