JPH01302138A - Data correction method of particle analyzer - Google Patents
Data correction method of particle analyzerInfo
- Publication number
- JPH01302138A JPH01302138A JP63133149A JP13314988A JPH01302138A JP H01302138 A JPH01302138 A JP H01302138A JP 63133149 A JP63133149 A JP 63133149A JP 13314988 A JP13314988 A JP 13314988A JP H01302138 A JPH01302138 A JP H01302138A
- Authority
- JP
- Japan
- Prior art keywords
- fluorescence
- correction
- detectors
- particle
- fluorescent agents
- 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
- 239000002245 particle Substances 0.000 title claims abstract description 37
- 238000012937 correction Methods 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims description 12
- 238000005259 measurement Methods 0.000 claims abstract description 7
- 238000001514 detection method Methods 0.000 claims description 2
- 238000012545 processing Methods 0.000 abstract description 12
- 239000007850 fluorescent dye Substances 0.000 abstract description 11
- 239000003795 chemical substances by application Substances 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 101150104680 MCH1 gene Proteins 0.000 description 1
- 101100382379 Rattus norvegicus Cap1 gene Proteins 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
【発明の詳細な説明】
[産業上の利用分野]
本発明は、例えばフローサイトメータ等のように、検体
粒子からの散乱光及び蛍光を検出することにより検体粒
子の性質を解析する粒子解析装置のデータ補正方法に関
するものである。Detailed Description of the Invention [Industrial Application Field] The present invention relates to a particle analysis device, such as a flow cytometer, which analyzes the properties of sample particles by detecting scattered light and fluorescence from the sample particles. The present invention relates to a data correction method.
[従来の技術]
フローサイトメータ等に用いられる従来の粒子解析装置
では、フローセルの中央部の例えば200 JLmX
2004mの微小な断面を有する流通部内を、シース液
に包まれて通過する検体粒子に光ビームを照射し、その
結果生ずる散乱光及び蛍光により、検体粒子の形状、大
きさ、屈折率等の粒子的性質を解明することが可能であ
る。[Prior Art] In a conventional particle analysis device used in a flow cytometer or the like, a particle size of 200 JLmX in the center of a flow cell, for example, is
A light beam is irradiated onto the specimen particles passing through the flow section, which has a minute cross section of 2004 m, wrapped in sheath liquid, and the resulting scattered light and fluorescence are used to determine the shape, size, refractive index, etc. of the specimen particles. It is possible to elucidate the properties of
発生した蛍光は分光され、赤色蛍光及び緑色蛍光として
検出されるが、赤色蛍光と緑色蛍光の波長領域には重な
り合う部分があるため、互いの検出器に他の蛍光が混入
することが避けられない。The generated fluorescence is spectrally analyzed and detected as red fluorescence and green fluorescence, but since the wavelength regions of red fluorescence and green fluorescence overlap, it is inevitable that other fluorescence will mix into each other's detectors. .
これは例えば白血球中のリンパ球を赤色及び緑色の蛍光
剤を用いて同時に染色し、それぞれの蛍光強度により2
種類の免疫機能を検査する場合に、一方の蛍光色だけが
発生しても双方の検出器に出力が生じてしまうという問
題となる。For example, lymphocytes in white blood cells are simultaneously stained using red and green fluorescent agents, and the fluorescence intensity of each is used to stain the lymphocytes at the same time.
When testing different types of immune function, a problem arises in that even if only one fluorescent color is generated, outputs are generated in both detectors.
そこで、混入した信号を除去する補正を必要とするが、
従来の補正方法では測定者が検体粒子から発生する赤色
及び緑色蛍光の信号強度を見ながら補正の程度を手動で
決定しているために、個人差が生じて客観性に欠け、ま
た時間が掛かる等の問題点がある。Therefore, correction is required to remove the mixed signal, but
In conventional correction methods, the measurer manually determines the degree of correction while observing the signal intensity of red and green fluorescence generated from sample particles, which results in individual differences, lacks objectivity, and is time-consuming. There are other problems.
[発明の目的]
本発明の目的は、上述の問題点を解決し、測定者個人に
よる信号の補正誤差を無くして正確な補正を可能とし、
更に測定時間が短縮できる粒子解析装置のデータ補正方
法を提供することにある。[Object of the Invention] The object of the present invention is to solve the above-mentioned problems, eliminate signal correction errors caused by individual measurers, and enable accurate correction.
Another object of the present invention is to provide a data correction method for a particle analyzer that can shorten measurement time.
[発明の概要]
上述の目的を達成するための本発明の要旨は、蛍光剤に
より染色した検体粒子に光ビームを照射し、検体粒子か
ら発生する蛍光を分光して、分光された蛍光強度を測定
する検出波長範囲が異なる複数個の検出器を備えた粒子
解析装置において、予め特定の蛍光剤で染色した検体粒
子に対して光ビームを照射し、前記各検出器が測定した
蛍光強度から前記特定の蛍光剤に対する前記各検出器間
の補正係数を算出し、測定時における前記各検出器の出
力から他の検出器が測定すべき蛍光成分の影響を除去す
るように補正することを特徴とする粒子解析装置のデー
タ補正方法である。[Summary of the Invention] The gist of the present invention for achieving the above-mentioned object is to irradiate specimen particles stained with a fluorescent agent with a light beam, to separate the fluorescence generated from the specimen particles, and to calculate the spectroscopic fluorescence intensity. In a particle analyzer equipped with a plurality of detectors measuring different detection wavelength ranges, a light beam is irradiated onto sample particles that have been stained with a specific fluorescent agent in advance, and the fluorescence intensity measured by each of the detectors is used to determine the A correction coefficient between each of the detectors for a specific fluorescent agent is calculated, and correction is made to remove the influence of the fluorescent component to be measured by other detectors from the output of each of the detectors at the time of measurement. This is a data correction method for particle analysis equipment.
[発明の実施例]
本発明に係る方法を図示の実施例に基づいて詳細に説明
する。[Embodiments of the Invention] The method according to the present invention will be explained in detail based on illustrated embodiments.
第1図は光学系と信号処理系の構成図である。FIG. 1 is a block diagram of an optical system and a signal processing system.
フローセルlの流通部la内を高速暦法となったシース
液に包まれて、赤色及び緑色の蛍光剤で染色された検体
粒子が通過し、この流れと直交する方向にレーザー光源
2が配置されている。このレーザー光源2から照射され
たレーザービームLを流通部1aに導くために、光軸G
1上に結像レンズ3が配置され、更に検体粒子からの9
0°散乱光及び蛍光を測定するために、検体粒子の流れ
の方向と光軸01にそれぞれ直交する方向である光軸0
2上に、フローセル1側から集光レンズ4、緑色光を反
射するグイクロイックミラー5、赤色光を反射するグイ
クロイックミラー6が順次に配列されている。グイクロ
イックミラー5の反射方向には集光レンズ7、光検出器
8が、グイクロイックミラー6の反射方向には集光レン
ズ9、光検出器10が配列されている。Specimen particles stained with red and green fluorescent agents pass through the flow section la of the flow cell l while being wrapped in a sheath liquid using a high-speed calendar method, and a laser light source 2 is arranged in a direction perpendicular to this flow. There is. In order to guide the laser beam L irradiated from this laser light source 2 to the circulation part 1a, the optical axis G
An imaging lens 3 is disposed on 1, and furthermore, 9
In order to measure the 0° scattered light and fluorescence, the optical axis 0, which is a direction perpendicular to the flow direction of the sample particles and the optical axis 01, respectively.
2, a condensing lens 4, a guichroic mirror 5 that reflects green light, and a guichroic mirror 6 that reflects red light are sequentially arranged from the flow cell 1 side. A condenser lens 7 and a photodetector 8 are arranged in the direction of reflection of the guichroic mirror 5, and a condenser lens 9 and a photodetector 10 are arranged in the direction of reflection of the guichroic mirror 6.
信号処理系としては、光検出器8,10の出力は信号処
理部11に接続され、この信号処理部11の出力はA/
D変換部12を経てインタフェイス13に接続されてい
る。更に、インタフェイス13は解析部14と接続され
、この解析部14の出力が再び信号処理部11に入力さ
れるようになっている。As a signal processing system, the outputs of the photodetectors 8 and 10 are connected to a signal processing section 11, and the output of this signal processing section 11 is connected to an A/
It is connected to an interface 13 via a D conversion section 12. Furthermore, the interface 13 is connected to an analysis section 14 so that the output of this analysis section 14 is inputted to the signal processing section 11 again.
ここで、結像レンズ3を介してフローセルlの流通部1
aに集光されたレーザービームLは検体粒子によって散
乱され、90°蛍光は集光レンズ4を経て、緑色蛍光は
グイクロイックミラー5で、赤色蛍光はグイクロイック
ミラー6で反射され、それぞれ集光レンズ7.9を介し
て光検出器8.10に入射し電気信号に変換される。光
検出器8.10からの電気信号は後述する信号処理部1
1へ入力され1次にA/D変換部12でA/D変換され
、インタフェイス13を介して解析部14に送られる。Here, the flow section 1 of the flow cell l is
The laser beam L focused on a is scattered by the specimen particles, the 90° fluorescence passes through the condenser lens 4, the green fluorescence is reflected by the guichroic mirror 5, and the red fluorescence is reflected by the guichroic mirror 6, respectively. The light enters a photodetector 8.10 via a condensing lens 7.9 and is converted into an electrical signal. The electrical signal from the photodetector 8.10 is sent to the signal processing unit 1, which will be described later.
1, the signal is first A/D converted by the A/D converter 12, and sent to the analyzer 14 via the interface 13.
第2図は緑色及び赤色蛍光剤とグイクロイックミラー5
.6の波長特性図を示し、曲線G及びRは緑色及び赤色
の蛍光剤波長特性であり、グイクロイックミラー5は波
長がD1以下の光、グイクロイックミラー6は波長がD
2以上の光を反射する。Figure 2 shows the green and red fluorescent agents and the glycoic mirror 5.
.. 6, curves G and R are the wavelength characteristics of green and red fluorescent agents, the guichroic mirror 5 is for light with a wavelength of D1 or less, and the guichroic mirror 6 is for light with a wavelength of D1.
Reflects 2 or more lights.
このため、01以下の波長は緑色蛍光として、 02以
上の波長は赤色蛍光として検出されることになる。一方
1曲線G及びRは波長特性の重なりが大きいため、波長
がD2以上の斜線で示す緑色蛍光部分G°は赤色蛍光に
混入し、逆に波長が旧以下の赤色蛍光部分R′は緑色蛍
光に混入する。Therefore, wavelengths of 01 or less are detected as green fluorescence, and wavelengths of 02 or more are detected as red fluorescence. On the other hand, curves G and R have a large overlap in wavelength characteristics, so the green fluorescent part G° shown by diagonal lines with a wavelength of D2 or more is mixed with red fluorescence, and conversely, the red fluorescent part R' with a wavelength of D2 or less is green fluorescent. be mixed into.
第3図はこれらの波長部分が混入した電気信号を補正す
るために設けられた信号処理部内の補正系ブロック図で
あり、Gl、 G2は信号処理系のゲイン、11.12
はそれぞれ緑色、赤色蛍光による光電流、R1,R2は
電流電圧変換用抵抗、)[1,R2は補正係数である。Figure 3 is a block diagram of the correction system in the signal processing unit provided to correct the electrical signal mixed with these wavelength parts, where Gl and G2 are the gains of the signal processing system, 11.12
are photocurrents caused by green and red fluorescence, respectively, R1 and R2 are current-voltage conversion resistors, )[1 and R2 are correction coefficients.
第2図からも理解し得るように、入力信号の補正を行う
には一方の信号の混入比分の信号を他方の信号から差し
引けばよい、そこで、赤色蛍光剤の波長特性曲線Rの内
部において、波長がD2以上の領域に対する領域R°の
割合をα、同様に緑色蛍光の曲線Gの内部において、波
長がO1以下の領域に対する領域G′の割合をβとし、
また補正前の電圧をVl’ 、 V2°、補正後の電圧
をvl、v2とすると、次式が成立する。As can be understood from Fig. 2, in order to correct the input signal, the signal corresponding to the mixing ratio of one signal can be subtracted from the other signal. Therefore, within the wavelength characteristic curve R of the red fluorescent agent, , α is the ratio of the region R° to the region whose wavelength is D2 or more, and β is the ratio of the region G′ to the region whose wavelength is O1 or less within the green fluorescence curve G.
Further, if the voltages before correction are Vl' and V2°, and the voltages after correction are vl and v2, the following equation holds true.
vl=vt′−に2V2’ −(t)
= GIRI(It◆α12) −に2G2R2(12
◆βII)= (GIRI−βに2G2R2)11+
(αGIRI−に2G2R2)12・・・(2)
ここで、光電流I2の影響を無くすには、αGIRI−
K2G2R2= 0
−”、に2=αGIRI/ G2R2・・・(3)同様
にして、
V2= V2’−KIVI’ ・(
4)が成立し、
K1=βG2R2/ GIRI ・・
・(5)ここで、 (3) 、 (5)式から、Vl
= GIRI (1−(X 73 )11
・・・(6)V2= G2R2(1−(X 13 )
f2 ・・・(7)αβ= KIK
2 ・・・(8)(
8) 、 (7) 、 (8)式から、Vl= GIR
I (1−KIK2)11 ・ (
9)V2= G2R2(1−KIK2)12
・ (10)(9) 、 (10)式から、
補正後の値Vl、 V2を真の値V1= GIRIIl
、 V2= G2R2V2ト等シくスルタメニハ、第4
図に示すようにVl、・v2を更に次式で書き換えれば
よい。vl=vt'- to 2V2'-(t)
= GIRI(It◆α12) −2G2R2(12
◆βII) = (GIRI-β2G2R2)11+
(2G2R2 for αGIRI−) 12...(2) Here, in order to eliminate the influence of photocurrent I2, αGIRI−
K2G2R2= 0 -'', 2=αGIRI/G2R2... (3) Similarly, V2= V2'-KIVI' ・(
4) holds, K1=βG2R2/GIRI...
・(5) Here, from equations (3) and (5), Vl
= GIRI (1-(X73)11
...(6)V2=G2R2(1-(X13)
f2...(7) αβ= KIK
2...(8)(
8) From equations (7) and (8), Vl=GIR
I (1-KIK2)11 ・ (
9) V2=G2R2(1-KIK2)12
・From equations (10)(9) and (10),
The corrected value Vl, V2 is the true value V1 = GIRIIl
, V2= G2R2V2, etc. Sultameniha, 4th
As shown in the figure, Vl and v2 can be further rewritten as the following equations.
V1=(Vlo−に2V2’ )/(1−KIK2)
−(11)V2 = (V2’−KIVI’ )
/(1−KIK2) ・(12)次に、補正係
数Kl、 K2の算出方法について述べる。第5図は赤
色蛍光剤のみにより染色された検体粒子の測定データで
あり、縦軸を赤色蛍光強度、横軸を緑色蛍光強度とした
二次元サイトグラム上に1つのまとまった粒子群として
表示される。この粒子群をウィンドウWで囲み、ウィン
ドウW内の粒子群の緑色及び赤色蛍光強度の平均値をM
C)l 1及びMeO2とすると、MCll0CVI’
MCH2oc V2’
・ (13)となる、ここで、赤色蛍光の緑色
側への混入成分を除去するためには、(11)式でv1
=0として、K2=V1’ /V2° =MCH1/M
GH2・(14)となるように補正係数に2を定めれば
よい、同様に、補正係数Klについては、線色蛍光剤の
みにより染色された粒子を測定して求めることができる
。V1=(Vlo-2V2')/(1-KIK2)
-(11)V2 = (V2'-KIVI')
/(1-KIK2) (12) Next, a method for calculating the correction coefficients Kl and K2 will be described. Figure 5 shows measurement data of specimen particles stained only with a red fluorescent agent, and is displayed as a group of particles on a two-dimensional cytogram with red fluorescence intensity on the vertical axis and green fluorescence intensity on the horizontal axis. Ru. This particle group is surrounded by a window W, and the average value of the green and red fluorescence intensities of the particle group within the window W is M
C) If l 1 and MeO2, MCll0CVI' MCH2oc V2'
・ (13) Here, in order to remove the mixed component of red fluorescence to the green side, v1 in equation (11) is
=0, K2=V1' /V2° =MCH1/M
It is sufficient to set the correction coefficient to 2 so that GH2·(14).Similarly, the correction coefficient Kl can be determined by measuring particles dyed only with the line color fluorescent agent.
このように、2種類の蛍光剤を用いる粒子解析の蛍光補
正を行うには、予め一方の蛍光剤だけが発光する二次元
サイトグラム上の粒子群にウィンドウをかけ、解析部1
4において各蛍光強度の平均値を算出して補正係数に1
、K2を求め、それらの値を信号処理部11に入力した
後に測定を開始すればよい。In this way, in order to perform fluorescence correction in particle analysis using two types of fluorescent agents, a window is placed in advance on the particle group on the two-dimensional cytogram where only one of the fluorescent agents emits light, and the analysis unit 1
In step 4, calculate the average value of each fluorescence intensity and set the correction factor to 1.
, K2 and input these values to the signal processing section 11 before starting the measurement.
なお、補正係数Kl、 K2を求めるに当り、蛍当強度
の平均値MCHI、MeO2の代りに、最大頻度の蛍当
強度を使用することもできる。また、補正係数の算出に
ついては、補正前のアナログ信号をA/D変換して解析
部14に送り、解析部14において行うことも可能であ
る。なお、蛍光剤の種類が更に増加しても、同様な方法
で校正が可能である。Incidentally, in determining the correction coefficients Kl and K2, the maximum frequency of the fluorescent light intensity can be used instead of the average value MCHI and MeO2 of the fluorescent light intensity. Further, the calculation of the correction coefficient can also be performed in the analysis section 14 by A/D converting the analog signal before correction and sending it to the analysis section 14 . Note that even if the number of types of fluorescent agents increases further, calibration can be performed using the same method.
また、蛍光剤が1種類でも、結合する検体粒子によって
は発生する波長領域が異なる場合にも応用可能である。Furthermore, even if there is only one type of fluorescent agent, the present invention can be applied to cases where different wavelength ranges are generated depending on the analyte particles to which the fluorescent agent is bound.
[発明の効果]
以上説明したように本発明に係る粒子解析装置のデータ
補正方法は、単一又は複数の蛍光剤を用いた場合の信号
強度から予め補正係数を算出して補正することにより、
補正値の客観性及び信頼性が向上し、また測定時間の短
縮や検体粒子の節約が可能となる。[Effects of the Invention] As explained above, the data correction method for a particle analyzer according to the present invention calculates and corrects a correction coefficient in advance from the signal intensity when a single or multiple fluorescent agents are used.
The objectivity and reliability of correction values are improved, and measurement time and specimen particles can be saved.
図面は本発明に係る粒子解析装置のデータ補正方法の実
施例を示し、第1図は構成図、第2図は蛍光剤及び分光
系の波長特性図、第3図、第4図は補正系の構成図、第
5図は二次元ヒストグラム図である。
符号1はフローセル、2はレーザー光源、5.6はグイ
クロイックミラー、8,10は光検出器、11は信号処
理部、12はA/D変換部、13はインタフェイス、1
4は解析部である。
特許出願人 キャノン株式会社
、5
第1図The drawings show an embodiment of the data correction method for a particle analyzer according to the present invention, in which Fig. 1 is a block diagram, Fig. 2 is a wavelength characteristic diagram of the fluorescent agent and spectroscopic system, and Figs. 3 and 4 are the correction system. FIG. 5 is a two-dimensional histogram diagram. 1 is a flow cell, 2 is a laser light source, 5.6 is a gicroic mirror, 8 and 10 are photodetectors, 11 is a signal processing section, 12 is an A/D conversion section, 13 is an interface, 1
4 is an analysis section. Patent applicant: Canon Co., Ltd., 5 Figure 1
Claims (1)
、検体粒子から発生する蛍光を分光して、分光された蛍
光強度を測定する検出波長範囲が異なる複数個の検出器
を備えた粒子解析装置において、予め特定の蛍光剤で染
色した検体粒子に対して光ビームを照射し、前記各検出
器が測定した蛍光強度から前記特定の蛍光剤に対する前
記各検出器間の補正係数を算出し、測定時における前記
各検出器の出力から他の検出器が測定すべき蛍光成分の
影響を除去するように補正することを特徴とする粒子解
析装置のデータ補正方法。1. Particle analysis equipped with multiple detectors with different detection wavelength ranges that irradiate sample particles stained with a fluorescent agent with a light beam, separate the fluorescence generated from the sample particles, and measure the intensity of the separated fluorescence. In the apparatus, a light beam is irradiated onto sample particles that have been stained with a specific fluorescent agent in advance, and a correction coefficient between the respective detectors for the specific fluorescent agent is calculated from the fluorescence intensity measured by each of the detectors; A data correction method for a particle analyzer, characterized in that the output of each of the detectors during measurement is corrected so as to remove the influence of fluorescence components to be measured by other detectors.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP63133149A JPH01302138A (en) | 1988-05-31 | 1988-05-31 | Data correction method of particle analyzer |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP63133149A JPH01302138A (en) | 1988-05-31 | 1988-05-31 | Data correction method of particle analyzer |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH01302138A true JPH01302138A (en) | 1989-12-06 |
Family
ID=15097862
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP63133149A Pending JPH01302138A (en) | 1988-05-31 | 1988-05-31 | Data correction method of particle analyzer |
Country Status (1)
Country | Link |
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JP (1) | JPH01302138A (en) |
-
1988
- 1988-05-31 JP JP63133149A patent/JPH01302138A/en active Pending
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