JPH04370089A - Separation of fine particle - Google Patents
Separation of fine particleInfo
- Publication number
- JPH04370089A JPH04370089A JP16926291A JP16926291A JPH04370089A JP H04370089 A JPH04370089 A JP H04370089A JP 16926291 A JP16926291 A JP 16926291A JP 16926291 A JP16926291 A JP 16926291A JP H04370089 A JPH04370089 A JP H04370089A
- Authority
- JP
- Japan
- Prior art keywords
- particles
- fine particles
- laser beam
- particle
- flow
- 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.)
- Withdrawn
Links
- 239000010419 fine particle Substances 0.000 title claims abstract description 64
- 238000000926 separation method Methods 0.000 title claims abstract description 19
- 239000002245 particle Substances 0.000 claims abstract description 64
- 238000000034 method Methods 0.000 claims abstract description 17
- 230000003287 optical effect Effects 0.000 claims abstract description 10
- 210000004027 cell Anatomy 0.000 description 17
- 239000011521 glass Substances 0.000 description 5
- 230000001678 irradiating effect Effects 0.000 description 3
- 239000011859 microparticle Substances 0.000 description 3
- 238000005192 partition Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 239000004065 semiconductor Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
Abstract
Description
【０００１】0001
【産業上の利用分野】本発明は、非接触で細胞、高分子
等の微粒子を分離、選別する方法に関するものである。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for separating and sorting fine particles such as cells and polymers without contact.
【０００２】0002
【従来の技術】従来、微粒子を分離する方法として、フ
ローサイトメトリーによる方法がある。この方法では、
微粒子を含む溶液をノズルから振動させながら押し出す
ことによって液滴を飛ばし、この液滴にレーザ光を照射
し液滴の蛍光、散乱光強度を計測することによって微粒
子の大きさあるいは種類を判別する。そして、この情報
をもとに必要とする微粒子が含まれる液滴を帯電させ、
この液滴が分離容器前の高電圧に印加された電極板間を
通過するときに液滴の飛ぶ方向を変え、液滴を所定の分
離容器に入れることにより、微粒子を分離するものであ
る。2. Description of the Related Art Conventionally, as a method for separating fine particles, there is a method using flow cytometry. in this way,
A solution containing fine particles is extruded through a nozzle while vibrating to eject droplets, and the size or type of the fine particles is determined by irradiating the droplets with laser light and measuring the fluorescence and scattered light intensity of the droplets. Then, based on this information, the droplets containing the necessary particles are charged,
When the droplets pass between electrode plates applied to a high voltage in front of the separation container, the flying direction of the droplets is changed and the droplets are placed in a predetermined separation container, thereby separating fine particles.
【０００３】0003
【発明が解決しようとする課題】しかしながら、上記の
ようなフローサイトメトリーによる微粒子の分離方法に
おいては、微粒子を含む液滴が、ノズルから高速に押し
出され、高速で分離容器に入るため、壊れ易い微粒子（
たとえば、植物細胞のプロトプラスト等）の場合は、損
傷を受ける可能性がある（鷲津、他：応用物理，第５８
卷，第３号，ｐ．３８３（１９８９））。また上記手法
では、微粒子が含まれる液滴が外気にふれるため完全な
非接触で微粒子を分離することはできない。それゆえ、
微粒子を壊さずに、非接触で分離する方法が望まれてい
た。[Problems to be Solved by the Invention] However, in the method for separating microparticles by flow cytometry as described above, droplets containing microparticles are pushed out from the nozzle at high speed and enter the separation container at high speed, so they are easily broken. Fine particles (
For example, protoplasts of plant cells) may be damaged (Washizu et al.: Applied Physics, No. 58).
Volume 3, p. 383 (1989)). Further, in the above method, the droplets containing the particles come into contact with the outside air, so it is not possible to separate the particles in a completely non-contact manner. therefore,
There was a desire for a method for separating fine particles without breaking them and without contact.
【０００４】本発明は上記事情に基づいてなされたもの
であり、微粒子を壊さずに、非接触で分離することがで
きる微粒子の分離方法を提供することを目的とするもの
である。The present invention has been made based on the above-mentioned circumstances, and it is an object of the present invention to provide a method for separating fine particles that can be separated in a non-contact manner without destroying the fine particles.
【０００５】[0005]
【課題を解決するための手段】上記の目的を達成するた
めの本発明に係る微粒子の分離方法は、異なる微粒子あ
るいは細胞（以下、細胞等も含めて微粒子と称す。）を
含む溶液が一定流量で流れている流れ系において、集光
したレーザビームを微粒子に照射し、微粒子をレーザの
出射方向の光軸上に一列に並ぶように移動させ、ビーム
ウエスト付近で散乱光強度を計測することにより、流れ
てきた個々の微粒子の違いを判別し、その結果、所望の
微粒子である場合には、該微粒子が分離容器近くに流れ
てきたときに、別の集光されたレーザビームを該微粒子
に照射することにより、該微粒子を特定の分離容器の方
向に流れ込ませ、異なる微粒子から所望の微粒子を分離
することを特徴とするものである。[Means for Solving the Problems] In order to achieve the above object, a method for separating particles according to the present invention is such that a solution containing different particles or cells (hereinafter referred to as particles including cells) is supplied at a constant flow rate. In a flowing flow system, a focused laser beam is irradiated onto fine particles, the particles are moved in a line on the optical axis in the laser emission direction, and the scattered light intensity is measured near the beam waist. , determines the difference between the individual flowing particles, and if the particle is the desired particle, another focused laser beam is applied to the particle when it flows near the separation container. The method is characterized in that by irradiating the particles, the particles flow in the direction of a specific separation container, and a desired particle is separated from different particles.
【０００６】[0006]
【作用】本発明は上記の構成によって、集光したレーザ
ビームを微粒子に照射して微粒子を一列に整列して移動
させ、各微粒子の散乱光強度を計測することにより、た
とえば微粒子の粒径を判断し、その判断に応じて、所望
の微粒子の移動方向を変えることができる。したがって
、完全な非接触で所望の微粒子を確実に分離することが
できる。[Operation] With the above configuration, the present invention irradiates fine particles with a focused laser beam, aligns and moves the fine particles in a line, and measures the intensity of scattered light of each fine particle, thereby determining, for example, the particle size of the fine particles. The desired movement direction of the fine particles can be changed according to the judgment. Therefore, desired fine particles can be reliably separated completely without contact.
【０００７】[0007]
【実施例】以下、本発明の一実施例を図面を参照して説
明する。図１は本発明の一実施例である微粒子の分離方
法を説明するための図である。DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will be described below with reference to the drawings. FIG. 1 is a diagram for explaining a method for separating fine particles, which is an embodiment of the present invention.
【０００８】本実施例の微粒子の分離方法に使用される
装置は、図１に示すように、セル１と、三個のレーザと
、集光レンズ５，６，８と、分離容器Ａ，Ｂとを備える
ものである。セル１は上下面及び側面すべてガラス製の
流路形状をもつ。微粒子３を含む溶液は左上に設けた一
つの入口２からセル１内に流し込まれ、粒径が判断され
た後、仕切り板１０で分けられた流路を通じて二つの分
離容器Ａ，Ｂのいずれかに流入する。分離容器Ａは所望
の粒径を有する微粒子を入れるものであり、分離容器Ｂ
はその粒径と異なる粒径の微粒子を入れるものである。As shown in FIG. 1, the apparatus used in the particle separation method of this embodiment includes a cell 1, three lasers, condensing lenses 5, 6, and 8, and separation containers A and B. It is equipped with the following. The cell 1 has a channel shape in which the upper, lower and side surfaces are all made of glass. A solution containing fine particles 3 is poured into the cell 1 from one inlet 2 provided at the upper left, and after the particle size is determined, it is sent to one of two separation containers A and B through a flow path separated by a partition plate 10. flows into. Separation container A contains fine particles having a desired particle size, and separation container B
contains fine particles with a particle size different from that particle size.
【０００９】一般に、微粒子を非接触で分離するために
は、分離する微粒子を含んだ溶液がガラス等で覆われた
セル内を流れている状態で微粒子の粒径や質量等を識別
し、分離することが望ましい。この微粒子を分離するた
めには、微粒子を押して微粒子の流れの方向を変えて分
けることが考えられ、微粒子を押す手段としては、水流
あるいは空気圧や光の圧力が挙げられる。微粒子が光の
圧力によって押される現象については、分献（Ａ．Ａｓ
ｈｋｉｎ：Ｐｈｙｓ．Ｒｅｖ．Ｌｅｔｔ．，２４，１５
６（１９７０））に報告されており、光が微粒子内を屈
折して通過する際の運動量変化によって生じるものであ
る。微粒子を押す手段として、水流を利用する場合には
渦の発生による流れの乱れの問題があり、また空気圧を
利用する場合には、泡の発生及び泡の混入の影響がある
ため、光を用いて分離することが望ましい。Generally, in order to separate fine particles without contact, a solution containing the fine particles to be separated is flowing through a cell covered with glass, etc., and the particle size and mass of the fine particles are identified and separated. It is desirable to do so. In order to separate these fine particles, it is possible to push the fine particles and change the direction of flow of the fine particles to separate them. Examples of means for pushing the fine particles include water flow, air pressure, and light pressure. Regarding the phenomenon in which fine particles are pushed by the pressure of light, see the section (A.As
hkin:Phys. Rev. Lett. ,24,15
6 (1970)), and is caused by a change in momentum when light is refracted and passes through a fine particle. When using a water stream as a means to push particles, there is a problem with flow disturbance due to the generation of eddies, and when using air pressure, there is an effect of bubble generation and bubble inclusion, so it is difficult to use light. It is desirable to separate the
【００１０】このため、本実施例では、微粒子３を直進
させたり、微粒子３の流れの方向を変えるために、レー
ザビーム４，７，９を使用している。Therefore, in this embodiment, the laser beams 4, 7, and 9 are used to make the particles 3 move straight or to change the direction of flow of the particles 3.
【００１１】次に、本実施例において、所望の粒径を有
する微粒子を分離する方法を説明する。まず、入口２よ
り粒径等の異なる微粒子３を一定粒量あるいは一定流速
で流し込む。微粒子３が位置３ａに来ると、左側から放
射されたレーザビーム４はレンズ５で集光され、セル１
のガラス側面を通過して微粒子３に照射する。微粒子３
は集光されたレーザビーム４による光の圧力を受けて、
レーザビーム４の光軸Ｌ上に引き寄せられて一列状にな
って光軸Ｌ上をビームウエスト３ｂの方向に移動する。Next, in this example, a method for separating fine particles having a desired particle size will be explained. First, fine particles 3 having different particle sizes, etc. are poured into the inlet 2 at a constant particle amount or at a constant flow rate. When the particle 3 reaches the position 3a, the laser beam 4 emitted from the left side is focused by the lens 5, and the cell 1
The light passes through the glass side surface of the glass and irradiates the particles 3. Fine particles 3
receives the pressure of light from the focused laser beam 4,
The beams are drawn onto the optical axis L of the laser beam 4 and move in a line along the optical axis L in the direction of the beam waist 3b.
【００１２】また、ビームウエスト３ｂ付近では、微粒
子３は特に明確にミー散乱（Ｍｉｅ ｓｃａｔｔｅｒｉ
ｎｇ）を起こすことがわかる。ミー散乱光強度は、微粒
子３の粒径等で決まるものであるため、レーザビーム４
の光軸Ｌに垂直な方向に設置したフォトダイオード等で
、上記ミー散乱光強度を計測し、微粒子３の粒径の情報
を得る。Further, near the beam waist 3b, the fine particles 3 are particularly clearly affected by Mie scattering (Mie scattering).
ng). Since the Mie scattered light intensity is determined by the particle size of the fine particles 3, the laser beam 4
The intensity of the Mie scattered light is measured by a photodiode or the like installed in a direction perpendicular to the optical axis L of the microparticles 3, and information on the particle size of the fine particles 3 is obtained.
【００１３】必要とする粒径を持った微粒子である場合
には、微粒子３がビームウエスト３ｂの位置より下流に
あるレーザビーム４の光軸Ｌ上の位置３ｃへ流れてきた
ときに、別のレーザビーム７を集光レンズ６で集光した
ビームを微粒子３の流れ方向と垂直な方向（図１では下
方向）から微粒子３に照射する。集光されたレーザビー
ム４，７による光の圧力によって微粒子３の流れる方向
は変わり、微粒子３は位置３ｅの方向に流れる。セル１
においては仕切り板１０があるため、微粒子３は、分離
容器Ａに通じる矢印１１の方向へ流れ込む。[0013] When the fine particles have the required particle size, when the fine particles 3 flow to a position 3c on the optical axis L of the laser beam 4 downstream from the position of the beam waist 3b, another The laser beam 7 is focused by a condenser lens 6 and is irradiated onto the particles 3 from a direction perpendicular to the flow direction of the particles 3 (downward in FIG. 1). The flow direction of the particles 3 changes due to the pressure of the light from the focused laser beams 4 and 7, and the particles 3 flow in the direction of the position 3e. cell 1
Since there is a partition plate 10 in the case, the fine particles 3 flow in the direction of the arrow 11 leading to the separation container A.
【００１４】また、上記計測して得た粒径の情報から必
要とする微粒子でない場合には、レーザビーム７と集光
レイズ６による集光ビームを照射させずに、位置３ｃよ
りも下流にあるレーザビーム４の光軸Ｌ上の位置３ｄに
微粒子３が達したときに、レーザビーム９と集光レンズ
８による集光ビームを上方向から照射して位置３ｆの方
向に微粒子３を流す。この場合、微粒子３は分離容器Ｂ
に通じる矢印１２の方向に流れ込む。Further, if the particle size information obtained by the above measurement indicates that the particle is not the required particle, the laser beam 7 and the condensing beam 6 are not irradiated with the condensed beam, and the particle located downstream of the position 3c is When the fine particles 3 reach the position 3d on the optical axis L of the laser beam 4, the laser beam 9 and the condensed beam from the condenser lens 8 are irradiated from above to cause the fine particles 3 to flow in the direction of the position 3f. In this case, the fine particles 3 are in the separation container B.
It flows in the direction of arrow 12 leading to.
【００１５】以上のようにレーザビーム４の光軸Ｌに対
して図１のように互いに反対の位置にあるレーザビーム
７及び集光レンズ６とレーザビーム９及び集光レンズ８
のうち一方の集光したレーザビームのみを微粒子３に照
射することにより、微粒子３の流れ方向を変え、所望の
粒径を有する微粒子を確実に分離することができる。As described above, the laser beam 7 and the condenser lens 6 and the laser beam 9 and the condenser lens 8 are located at opposite positions as shown in FIG. 1 with respect to the optical axis L of the laser beam 4.
By irradiating the fine particles 3 with only one of the focused laser beams, the flow direction of the fine particles 3 can be changed, and fine particles having a desired particle size can be reliably separated.
【００１６】また、このような流れ系において集光され
た光の圧力を利用した分離方法では、微粒子３が浮遊し
た状態で分離するため、たとえ壊れ易い細胞等でも、損
傷なく容易に分離することができる。[0016] In addition, in a separation method that utilizes the pressure of light focused in such a flow system, since the particles 3 are separated in a suspended state, even fragile cells can be easily separated without damage. Can be done.
【００１７】微粒子として壊れ易い細胞等を用いる場合
、使用するレーザの波長としては、細胞の光の波長に対
する吸収特性を考慮した範囲内であればよい。また、光
出力は細胞を死滅させない範囲の出力を与える必要があ
り、約数ｍＷ〜数十ｍＷである。この場合にはレーザビ
ームには、高効率、長寿命、安定である半導体レーザを
用いてもかまわない。本発明者等は、波長７９０ｎｍ，
光出力２５ｍＷの半導体レーザを使用し、ＮＡ（開口角
）＝０．４程度の集光レンズ５，６，８を用いて集光し
たレーザビームを、粒径数μサイズのポリスチレンラテ
ックス粒子に照射することにより、この粒子をレーザビ
ームの圧力で押し、本方法で所望の粒径を有する微粒子
だけを容易に且つ精確に分離することができた。When fragile cells or the like are used as the fine particles, the wavelength of the laser to be used may be within a range that takes into consideration the absorption characteristics of the cells with respect to the wavelength of light. Further, the light output must be within a range that does not kill cells, and is approximately several mW to several tens of mW. In this case, a semiconductor laser with high efficiency, long life, and stability may be used as the laser beam. The present inventors have a wavelength of 790 nm,
Using a semiconductor laser with an optical output of 25 mW, a laser beam focused using condensing lenses 5, 6, and 8 with an NA (aperture angle) of about 0.4 is irradiated onto polystyrene latex particles with a particle diameter of several microns. By pressing the particles with the pressure of the laser beam, it was possible to easily and accurately separate only fine particles having a desired particle size using this method.
【００１８】[0018]
【発明の効果】以上説明したように本発明によれば、微
粒子を含んだ流れ系においてガラス越しに集光されたレ
ーザビームを用い、溶液に浮遊した状態の微粒子を分離
するため、完全な非接触で微粒子を確実に分離すること
ができ、しかも壊れ易い細胞等の微粒子であっても壊す
ことなく分離することができる微粒子の分離方法を提供
することができる。Effects of the Invention As explained above, according to the present invention, a laser beam focused through glass in a flow system containing fine particles is used to separate fine particles suspended in a solution. It is possible to provide a method for separating fine particles that can reliably separate fine particles by contact, and can also separate fine particles such as fragile cells without breaking them.
【図１】本発明の一実施例である微粒子の分離方法を説
明するための図である。FIG. 1 is a diagram for explaining a method for separating fine particles, which is an embodiment of the present invention.
１ セル ２ セルの入口 ３ 微粒子 ４，７，９ レーザビーム ５，６，８ 集光レンズ １０ 仕切り板 1 Cell 2 Cell entrance 3. Fine particles 4, 7, 9 Laser beam 5, 6, 8 Condensing lens 10 Partition plate
Claims (1)
胞等も含めて微粒子と称す。）を含む溶液が一定流量で
流れている流れ系において、集光したレーザビームを微
粒子に照射し、微粒子をレーザの出射方向の光軸上に一
列に並ぶように移動させ、ビームウエスト付近で散乱光
強度を計測することにより、流れてきた個々の微粒子の
違いを判別し、その結果、所望の微粒子である場合には
、該微粒子が分離容器近くに流れてきたときに、別の集
光されたレーザビームを該微粒子に照射することにより
、該微粒子を特定の分離容器の方向に流れ込ませ、異な
る微粒子から所望の微粒子を分離することを特徴とする
微粒子の分離方法。Claim 1: In a flow system in which a solution containing different particles or cells (hereinafter referred to as particles including cells) is flowing at a constant flow rate, the particles are irradiated with a focused laser beam, and the particles are exposed to the laser beam. By moving the particles so that they are lined up in a line on the optical axis in the emission direction and measuring the intensity of scattered light near the beam waist, it is possible to determine the differences between individual particles that have flown in. As a result, if the particles are the desired particles, When the particles flow near the separation vessel, another focused laser beam is irradiated onto the particles to cause the particles to flow in the direction of a specific separation vessel, and to separate the particles from different particles into the desired one. A method for separating fine particles, characterized by separating fine particles.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP16926291A JPH04370089A (en) | 1991-06-14 | 1991-06-14 | Separation of fine particle |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP16926291A JPH04370089A (en) | 1991-06-14 | 1991-06-14 | Separation of fine particle |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH04370089A true JPH04370089A (en) | 1992-12-22 |
Family
ID=15883252
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP16926291A Withdrawn JPH04370089A (en) | 1991-06-14 | 1991-06-14 | Separation of fine particle |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH04370089A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2335737A (en) * | 1998-03-24 | 1999-09-29 | Pittway Corp | Smoke detector with particle sensor |
| WO2004039501A1 (en) * | 2002-11-01 | 2004-05-13 | Techno Network Shikoku Co., Ltd. | Method for sorting and recovering fine particle and apparatus for recovery |
| JP2007175581A (en) * | 2005-12-27 | 2007-07-12 | National Institute Of Advanced Industrial & Technology | Particulate recovering apparatus |
| KR100938927B1 (en) * | 2007-12-31 | 2010-01-27 | 재단법인서울대학교산학협력재단 | Microfluidic device for sorting cells using laser ablation |
-
1991
- 1991-06-14 JP JP16926291A patent/JPH04370089A/en not_active Withdrawn
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2335737A (en) * | 1998-03-24 | 1999-09-29 | Pittway Corp | Smoke detector with particle sensor |
| WO2004039501A1 (en) * | 2002-11-01 | 2004-05-13 | Techno Network Shikoku Co., Ltd. | Method for sorting and recovering fine particle and apparatus for recovery |
| US7428971B2 (en) | 2002-11-01 | 2008-09-30 | Techno Network Shikoku Co., Ltd. | Method for sorting and recovering fine particle and apparatus for recovery |
| JP2007175581A (en) * | 2005-12-27 | 2007-07-12 | National Institute Of Advanced Industrial & Technology | Particulate recovering apparatus |
| KR100938927B1 (en) * | 2007-12-31 | 2010-01-27 | 재단법인서울대학교산학협력재단 | Microfluidic device for sorting cells using laser ablation |
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| A300 | Application deemed to be withdrawn because no request for examination was validly filed |
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