JPH0277636A - Particle measuring device - Google Patents

Abstract

PURPOSE:To measure correct size of a particle by providing an aperture equipped with an opening portion of a predetermined shape in front of a measuring unit to be detected. CONSTITUTION:When a measuring particle passes a measuring unit to be detected, the light projected to the measuring particle is radiated to be scattered. Among the scattering light, components of a predetermined angle radiated by an aperture 6 in the forward direction of an optical path pass the aperture and the remainder is shut off. The strength of the light passing the aperture 6 and condensed by a condensing lens 7 is detected by a photodetector 8. The particle size is calculated in an operation circuit based on an output of the detector 8. There is provided a photometric optical system for lateral scattering light and fluorescent light in a lateral direction of a flow cell 1. An output of the flow cell 1 is also inputted to the operation circuit where an analysis of the particle is operated. The strength of the front scattering light detected by the photodetector 8 is linearly related to the particle size of the measuring particle, thereby achieving a correct measurement of the particle size.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention is directed to, for example, irradiating sample particles passing through a flow cell with a laser beam or the like, and detecting scattered light, transmitted light, or fluorescence emitted by the sample particles. The present invention relates to a particle measuring device for analyzing the properties, structure, etc. of sample particles, and for performing immunological tests.

[Conventional technology] Conventional particle analysis devices, such as flow cytometers,
Irradiation light is irradiated onto sample liquids such as blood cells and sensitized latex particles that are wrapped in sheath liquid and pass through a flow section with a minute rectangular cross section of about 200 μm x 200 μm in the center of the flow cell, and as a result, forward scattered light is bundled. Particle analysis is performed using side scattered light and fluorescence. Information representing the particle size can be obtained from the forward scattered light, structural information about the particle can be obtained from the side scattered light, and chemical properties of the particle such as the amount of DNA and RNA can be obtained from the fluorescence. Conventionally, it has been thought that the detected intensity of forward scattered light has a one-to-one correspondence with the particle diameter of sample particles, and the particle diameter has been calculated based on the detected intensity of forward scattered light.

[Problem that the invention seeks to solve] However,
In conventional methods that use monochromatic light such as a laser beam as the irradiation light, if the sample particles are translucent, the function of the detected scattered light intensity and particle diameter becomes a monotonically increasing function, as shown in Figure 6. figure,
There is a problem in that the linearity collapses around a certain particle size, making it impossible to calculate the particle size around that area.

As a means to solve this problem, in Japanese Patent Application No. 62-58301 previously filed by the applicant, optical filters with different transmittances are arranged concentrically in the optical path of the forward scattered light receiving optical system. discloses an example in which the relationship between the detected intensity of scattered light and the particle diameter is made linear by adjusting the transmittance for each scattering angle, and accurate particle diameters are determined.

An object of the present invention is to provide a particle analyzer that can accurately determine particle diameters regardless of the size of sample particles using a simpler method.

[Means for Achieving the Objective] In order to achieve the above-mentioned objective, a particle measuring device is provided that includes an irradiation system that irradiates a light beam onto sample particles, and a detection system that detects light emitted by the sample particle from the light beam. In this method, an aperture is provided in the optical path of the detection system, and the aperture has an opening shape with an opening area ratio that varies depending on the scattering angle so that the detected intensity of the scattered light becomes a monotonous function of the particle diameter.

[Example] Embodiments of the present invention will be described in detail below with reference to the drawings.

FIG. 1(a) is a configuration diagram of an embodiment, in which laser light emitted from a laser light source 3 is imaged onto a test part of a flow section 2 in a flow cell 1 by an imaging lens 4 such as a cylindrical lens. Ru. In the flow section 2, sample particles S such as blood cells or latex aggregates are flowed one by one or one lump at a time in a direction perpendicular to the drawing using a sheath flow system.

A predetermined angular component of the forward scattered light emitted in the forward direction of the optical path passes through the aperture 6 made of a light-absorbing member among the scattered light emitted by the analyte particles passing through the test part and irradiated with light to the sample particles. However, everything else is blocked. The light passing through the aperture 6 is focused by a condensing lens 7, and the light intensity is detected by a photodetector 8. Based on the output of the photodetector 8, a calculation circuit (not shown) calculates the particle diameter. , will be. Further, although not shown, a photometric optical system for side scattered light and fluorescence is arranged in the lateral direction of the flow cell 1, and the photometric output of these lights is also input to an arithmetic circuit to perform calculations for particle analysis. Here, the forward scattered light intensity detected by the photodetector 8 has a linear relationship with the particle diameter of the sample particles, as shown in FIG. 5, so that the accurate particle diameter can be determined. The reason will be explained below.

As stated in the previous Japanese Patent Application No. 62-58301, the applicant of the present invention has found that the relationship between the detected intensity of scattered light (2) and the particle diameter differs depending on the scattering angle component, as shown in FIG. Based on this assumption, by using concentric filters with different light transmittances, the light intensity I (01 to θ2) is halved in the range of angles θ to θ2, and the light intensity is 100% in the range of angles 02 to θ! (
02 to θ3). Therefore, the amount of light detected by the photodetector is 34I (θl ~ θ2) + I (02 ~ θ,), which is a monotonically increasing function as shown in Figure 5, making it possible to determine the accurate particle diameter. Instead of using a filter, the present invention uses concentric apertures having different aperture size ratios for each angular component to obtain the same effect as a filter.

FIG. 1(b) shows an example of the shape of the aperture 6, in which an inner light shielding part and an outer light shielding part are connected by a thin beam. This aperture 6 is placed at a predetermined distance from the test area through which the sample particles S pass, and the scattered light emitted from the sample particles S is 100% blocked in the range from angle O to θ by the circular light shielding part. be done. Note that this portion also serves as a stopper that blocks direct light from the irradiated laser beam. In the surrounding angle range of 01 to θ, 50% of the light passes through the semicircular light shielding portion. Further, in the range of angles 02 to θ3 around it, the projected area of the beam is very small, so almost 100% of the light passes through. 0
All angles greater than or equal to 3 are blocked from passing. In this example, θ is 8 degrees, θ2 is 13 degrees, and θ3 is 32 degrees, and these numbers are values determined by simulation.

It goes without saying that the shape of the aperture is not limited to the shape shown in FIG. 1(b). Angle θ.

As long as 50% of the scattered light in the range of ~θ2 can pass through, the shape of the light shielding part is acceptable. For example, modifications as shown in FIG. 2(a) and FIG. 2(b) are also possible. In FIG. 2(a), 50% of the light is blocked by providing two 1/4 circular light blocking portions in the range of angles 01 to θ2. Further, in the second F (c), a mesh-like member having a transmittance of 50% is used to block light in the range of angle θ to θ2.

Furthermore, theoretically, the amount of light passing through the angle range of 01 to θ2 is 5
Even if the angles θ and ˜θ are not 100%, the same effect can be obtained as long as the amount of passing light is at a ratio of 1:2. Therefore, an opening shape as shown in FIG. 2(C) is also possible. In this example, in the range of angle 01 to θ, 33% transmission, 0
In the range of 2 to θ3, the transmission is 66%. however,
In order to improve the measurement sensitivity of the particle analyzer, it is better to use a configuration that can detect as large a scattered light intensity as possible, and the former example with less light loss is preferable.

As another modification, a light-absorbing light-shielding pattern having the same shape as described above is attached to a transparent member such as a glass plate, thereby eliminating the need for support using a beam. As a more extended variation, flow cell 1 can be constructed without using plate glass.
It is also possible to attach a light shielding pattern directly to the forward scattered light exit surface or the condenser lens 7. In these cases, the size of the light shielding pattern is determined according to the distance from the sample particle S so that the radiation angle θ11θ2, θ from the sample particle S does not change.

Furthermore, the light shielding portions of each angular component of the aperture do not need to be in the same plane, and may be placed separately as long as they are in the optical path between the subject and the photodetector 8.

In this case as well, each aperture size is determined in consideration of the radiation angle component.

[Other Embodiments] FIG. 3(a) is a block diagram of another embodiment of the present invention, and the same reference numerals as in FIG. 1 represent the same members.

Further, FIG. 3(b) shows the shape of the aperture used.

This embodiment is characterized in that the aperture 9 is arranged obliquely with respect to the straight direction of the optical path. The material of the aperture 9 used does not have to be light absorbing. Further, the shape is an ellipse so that the projected shape when tilted obliquely is the same as the open pattern of the previous embodiment. As a result, the opening shape of the aperture when viewed from the direction in which the optical path advances becomes a perfect circle.

The forward scattered light emitted from the sample particles S travels straight along the optical path at the aperture portion of the aperture 9, which is arranged obliquely, and is reflected in a different direction from the optical path at the light-shielding portion of the aperture. A light absorbing means (not shown) is provided at the reflected end. Therefore, unlike the previous embodiment, there is no need to use a light-absorbing member for the aperture, and the same effect can be obtained.

It goes without saying that this embodiment can also be modified in many ways similar to the previous embodiments.

In the embodiments of the present invention described above, 50% and 100%
Although the angular component of the % transmittance was corrected in two stages, it is also possible to further improve the accuracy by using an aperture shaped to correct the angular component in multiple stages. Also,
Depending on the type of sample particles to be measured, for example, a method of rotating the turret, selecting an optimally shaped aperture according to the characteristics of the sample particles, and arranging it in the optical path may also be effective in improving accuracy. The design of the shape of these apertures can be determined by computer simulation or the like.

[Effects of the Invention] According to the present invention, by arranging an aperture having an opening of a predetermined shape in front of the test portion, it is possible to determine the accurate particle diameter of the sample particles.

[Brief explanation of the drawing]

FIG. 1(a) is a configuration diagram of an embodiment of the present invention, FIG. 1(b)
Figure 2 (a), (b), and (c) are examples of modified aperture shapes, Figure 3 (a) is a configuration diagram of another embodiment, and Figure 3 (b) is a different example. Figures 4 and 5 are diagrams showing the function of scattered light intensity ■ and particle diameter according to the present invention, and Figure 6 is a diagram showing the function of scattered light detection intensity ■ and particle diameter according to the conventional example. The main symbols in the figure are: 1...flow cell, 2...tb il part, 3
... Laser light source, 4... Imaging lens, 6.9.
...Aperture, 7...Condensing lens, 8...
Photodetector, phantom 1 wild goose

Claims (1)

[Scope of Claims] 1. In a particle measuring device comprising an irradiation system that irradiates a light beam onto sample particles, and a detection system that detects light emitted by the sample particles, the light beam is located within the optical path of the detection system. A particle measuring device comprising an aperture, the aperture having an aperture shape having an aperture area ratio that varies depending on the scattering angle so that the detected intensity of the scattered light becomes a monotonous function of the particle diameter. 2. The particle measuring device according to claim 1, wherein the center portion of the aperture is a light shielding portion for direct light removal.