JPH01302138A - Data correction method of particle analyzer - Google Patents

Abstract

PURPOSE:To enhance the objectivity and reliability of a correction value and to shorten a measuring time, by preliminarily calculating a correction factor from the signal intensity obtained when a single or a plurality of fluorescent agents are used to perform correction. CONSTITUTION:The laser beam L condensed to the flowing part 1a of a flow cell 1 through an image forming lens 3 is scattered by a sample particle and 90 deg. fluorescence passes through a condensing lens 4 to reach dichroic mirrors 5, 6 and green and red fluorescences are respectively reflected by said mirrors 5, 6 to enter beam detectors 8, 10 through condensing lenses 7, 9 while the electric signals from said detectors 8, 10 are inputted to a signal processing part 11 and converted by an A/D converting part 12 to be sent to an analytical part 14 through an interface 13. In performing the fluorescence correction of particle analysis using two kins of fluorescent agents, a window is preliminarily applied to the particle group on a two-dimensional sightgram emitted only by one of both fluorescent agents and the average value of respective fluorescence intensities in the analytical part 14 to calculate correction factors K1, K2 which are, in turn, inputted to the signal processing part 11 to start measurement.

Description

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.

[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

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. .

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.

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.

[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.

[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.

[Embodiments of the Invention] The method according to the present invention will be explained in detail based on illustrated embodiments.

FIG. 1 is a block diagram of an optical system and a signal processing system.

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.

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.

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.

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.

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.

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.

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'- 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-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.

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.

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.

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.

[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.

[Brief explanation of the drawing]

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)

[Claims] 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.