JPH02138851A - Particle measuring apparatus - Google Patents

Abstract

PURPOSE:To obtain highly accurate information concerning a shape by a method wherein first and second photometry means to measure scattered light due to sample particles arranged at a position to which first and second light irradiation means correspond and output values of both the photometry means are compared with a comparison means and an identification is performed to determine whether the sample particles are spherical or not spherical from the results of the comparison. CONSTITUTION:A first light irradiation means is composed of a laser light source 1 with a wavelength lambda1 of a particle measuring device and an image forming optical system 3 while a second light irradiation means is composed of a laser light source 11 with a wavelength lambda2 and an image forming optical system 13. Irradiation light from both the irradiation means is made to irradiate the sample particles flowing sequentially through a passage section 2 within a flowcell 4 and scattered light from the sample particles is detected selectively first and second photo detectors 10 and 18 arranged in respective optical axes. Detection outputs thereof are stored into first and second memory sections of a memory means 19 in such a manner that a larger data and a smaller data correspond to the first and second memory sections among detection data from one sample particle and values thereof are computed by a computing means 20 to determine a shape of the sample particle.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a particle measuring device that performs particle measurement by irradiating light onto sample particles and measuring light from the sample particles, such as transmitted light or scattered light. .

[Prior Art] In conventional particle measuring devices, such as flow cytometers,
By irradiating light from one direction onto specimen particles such as biological cells that are flowing one by one at high speed, and measuring the scattered light and fluorescence generated by this, information about the particle size and properties of the specimen particles can be obtained, and a large number of specimen particles can be analyzed. Based on this information about cells, we analyzed the sample particles. Recently, a test sample is added to latex particles sensitized with antibodies, and the latex particles aggregate due to the antigen-antibody reaction, and the size of the latex aggregates is detected using a flow cytometer. It has also been used to detect the presence of specific antigens in test samples.

[The problem that the invention is trying to solve] However,
Conventional particle measuring devices that emit light from one direction cannot distinguish between spherical and non-spherical sample particles, making it difficult to accurately analyze the shape of sample particles.

An object of the present invention is to provide an apparatus that can obtain particle shape information of sample particles, such as information on whether they are spherical or non-spherical, by irradiating light from a plurality of directions and obtaining a plurality of pieces of information.

[Means to solve the problem] In order to solve the above-mentioned problem, the sample particles can be measured by irradiating light onto the test area through which the sample particle passes and measuring the light from the test part. In the particle measuring device that performs
a first light irradiation unit that irradiates the test area with irradiation light from a direction; and a second light irradiation unit that irradiates the test unit with irradiation light from a second direction different from the first direction. Said first and second
comprising first and second photometric means disposed at positions corresponding to the light irradiation means and respectively photometrically measuring the light scattered by the sample particles;
It has a comparing means for comparing the output values of the first and second photometric means, and an arithmetic means for identifying the shape of the sample particle from the comparison result of the comparing means.

[Example] Hereinafter, an example of the particle measuring device of the present invention will be described in detail with reference to the drawings.

FIG. 1 is a block diagram of an embodiment of the present invention, where 1 is the wavelength λ1
3 is an imaging optical system consisting of a cylindrical lens and the like, forming a first light irradiation means. Wavelength ^1 emitted from laser light source 1
The laser beam is imaged by the imaging optical system 3 onto the flow section 2 in the flow cell 4 made of transparent glass. At this time, the imaging optical system 3
The shape of the beam spot imaged on the test area by
It has an elliptical shape with respect to the flow of sample particles.

The purpose of this is to ensure that the sample particles are irradiated with light at a nearly uniform intensity even if the flow position of the sample particles in the test area is slightly shifted downward or to the left or right. It is.

Further, as a second light irradiation means, a laser beam having a wavelength λ2 different from the previous wavelength λ1 is directed to the laser light source 11 from a direction perpendicular to the optical path of the laser beam from the laser light source 1 and the flow direction of the sample particles. Or fired. This laser light is
- Similar to the light irradiation means of the memory 1, the imaging lens system 13 forms an image of the test portion in the flow cell from a direction perpendicular to the first light irradiation means.

Sample particles such as blood cells and latex aggregates are sequentially flowed one by one or one lump at a time in the direction perpendicular to the page of the paper into the flow section 2 in the flow cell 4 using a sheath flow system, and are exposed to a laser beam with a wavelength λ1 and a laser beam with a wavelength λ2. The laser beam sequentially passes through the test portions in the flow cell 4 where it is imaged. Here, when there are no sample particles 5 in the test area, the laser light from the laser light source 1 travels straight through the flow cell 4 and is blocked by the stopper 6.
Similarly, the laser light from the laser light source 11 is blocked by the stopper 14. However, when sample particles approach the test area and are irradiated with a laser beam, light scattering occurs due to the sample particles, and scattered light with wavelengths λ1 and λ2 is generated. Condensing lenses 7 and 15 are arranged in the straight direction of the optical paths of the laser light sources and 11, respectively, and the scattered light at a predetermined angle is condensed. Here, the scattered light collected by the lens 7 is selectively transmitted by the barrier filter 8, in which only the light having the wavelength λ1 is transmitted. That is, only the scattered light generated by irradiation by the first light irradiation means is selected. The light having the wavelength λ that has passed through the barrier filter 8 passes through the diaphragm 9 and is detected by the photodetector 10 as a light pulse intensity. Further, from the scattered light collected by the lens 15, only the light having the wavelength λ2 is selected by the barrier filter 16. That is, only the scattered light from the second light irradiation means is selected, and the light pulse intensity of wavelength λ2 is detected by the aperture 17 and photodetector 18. The pulse outputs of the photodetectors 10 and 18 are time-integrated by respective integration circuits (not shown) to obtain an integral value for each pulse. The two integral values obtained each time the sample particles pass through the test area are stored separately in the storage means 19. Thus 1
Measurement data from different angles are obtained for each sample particle, and based on the contents of the storage means 19, calculations for particle measurement are performed by the calculation means 20, and the calculation results are displayed on the display means 21.

Next, a calculation method for identifying the shape of sample particles from detection data will be described.

The storage means 19 is provided with a first storage section and a second storage section.
Among the two pieces of scattered light detection data, the larger data is stored, and the smaller data is stored in the second storage section. In the case of the same value, the same value is stored respectively. The histograms of each data stored in the 11th second storage section are shown in FIGS. 2(a) and 2(b).
).

Here, the horizontal axis is the scattered light detection intensity, and the vertical axis is the number of sample particles. It is generally known that the detected intensity of scattered light increases as the size of the sample particle increases.If the sample particle is perfectly spherical, the size of the sample particle seen from two sides is the same, and the two obtained The detected data also has almost the same value. On the other hand, when the sample particles are non-spherical, the size changes depending on the direction from which the sample particles are viewed. Therefore, first,
The values stored in the second storage section differ. The more non-spherical the specimen particle is, the more different this becomes. That is, the degree of difference between the data stored in the first and second storage units represents the non-sphericity of the sample particles. The degree of difference may be expressed by the difference between two data, or may be expressed as a ratio.

Figures 3 (a) and (b) are histograms obtained by taking the difference between the data in the first storage section and the second storage section, and Figure 3 (a) shows when the sample particle is completely spherical. As a result,
FIG. 3(b) shows the results when the sample particles are non-spherical. The horizontal axis represents the difference between the data in the first and second storage units, that is, the non-sphericity of the sample particles, and the vertical axis represents the number of sample particles. Note that the horizontal axis may use a ratio instead of a difference. As is clear from this, data for spherical sample particles is concentrated near the left end of the histogram as shown in Figure 3 (a), whereas for non-spherical sample particles the data is concentrated near the left end as shown in Figure 3 (b). It shows a tendency to widen. In this way, it is possible to distinguish between spherical and non-spherical shapes from the histogram pattern, and further to determine the degree of non-spherical shape. In this embodiment, the discrimination of the histogram pattern is automatically performed by the discrimination circuit of the calculation means 20 using pattern recognition technology, or it may be determined whether it is a human by looking at the displayed image.

However, although the non-sphericity of the sample particles can be determined from this histogram, the size of the sample particles cannot be determined at the same time. Therefore, Figure 4 (a) and (b)
Use a site duram like . The horizontal axis is the first one as before.
, the difference between the data in the second storage section, that is, the non-sphericity of the sample particles, and the vertical axis is the average value of the data in the first and second storage sections, that is, the average scattered light intensity, which represents the average particle diameter. represent. The data of each sample particle is plotted as one point on the cytogram, and the tendency of the plot set differs depending on the shape of the sample particle. For example, in Figure 4 (a), completely spherical sample particles of different sizes are distinguished. has been done. 2
Specimen particles of different sizes are each plotted in a range approximately surrounded by a dotted line. Further, FIG. 4(b) shows that the range of the plot expands horizontally depending on the degree of non-sphericity of the sample particles, and is separated in the vertical direction depending on the size of the sample particles. By displaying the sample particles in such a cytogram, the shape (non-sphericity, size) of the sample particle can be determined based on how it is displayed on the cytogram. Further, even when specimen particles having a plurality of shapes are mixed, the shapes can be distinguished and the M number can be determined. As before, the determination of the site duram pattern may be performed automatically by pattern recognition, or may be determined by a human.

As described above, particle analysis can be performed by determining and counting the shape of sample particles, such as determining and counting the type of blood cells, determining and counting the type of microorganisms, and detecting antigen-antibody reactions using sensitized latex. I'll come. In addition, in addition to the scattered light from two directions, more detailed analysis can be performed by measuring the fluorescence from fluorescently stained specimen particles and adding the biochemical properties of the specimen particles obtained from the fluorescence intensity as parameters for particle analysis. can be done. Furthermore, it is also possible to use transmitted light instead of scattered light, although the accuracy is slightly lower.

In addition, in the examples described in "--, information was obtained from two directions by irradiating the sample particles with light from two directions," but the information is not limited to this, but by irradiating light from three to ten directions. ,
It is also possible to perform a more detailed analysis.

Further, as in the embodiment, a plurality of laser beams may be irradiated at the same position in the flow section, or the positions may be shifted in the flow direction and irradiated separately.

Furthermore, in the example, as a preferred embodiment, different laser light sources are used to irradiate irradiation light of different wavelengths, and the light receiving system selects the wavelength of the scattered light and performs photometry for each, or multiple laser light sources with the same wavelength are used. It is also possible.

This is because the side scattered light intensity is generally very weak compared to the forward scattered light intensity, and even if one forward scattered light output is mixed with the other side scattered light, there is a noise component that will cause a big problem. This is because it does not become. In this case, instead of preparing a plurality of identical laser light sources, a modification may be considered in which the laser beam from a single laser light source is divided using a heam splitter or the like and irradiated from multiple directions simultaneously.

``Effects of the Invention'' - According to the invention, it is possible to obtain information from multiple directions of each specimen particle to determine the shape, etc. of the specimen particle.

[Brief explanation of the drawing]

Figure 1 is a configuration diagram of an uninvented embodiment, Figures 2 (a) and (b) are histograms of scattered light measurement data, and Figures 3 (a) and (b) are graphs showing the non-spherical shape and shape of the sample particles. The histograms in Figures 4(a) and 4(b) are cytograms representing the shape of the sample particles. Laser light source with wavelength λ2, 8... barrier filter that transmits light with wavelength λ1, 16... barrier filter that transmits light with wavelength λ2]
1 Ruta, 6.14...stopper, 19...memory means, 20...arithmetic means 21...display display ganglia 9 HashiP Kobe Hirabe's choice made me cry. Renshu

Claims (1)

[Claims] 1. In a particle measuring device that measures sample particles by irradiating light onto a test area through which sample particles pass and measuring the light from the test area, the irradiated light is emitted from a first direction. a first light irradiation means that irradiates the test area with irradiation light; a second light irradiation unit that irradiates the test area with irradiation light from a second direction different from the first direction; A first and a second light emitting means arranged at positions corresponding to the light irradiation means of No. 2 and measuring the light from the sample particles, respectively.
A particle measurement method comprising: a photometric means, a comparing means for comparing the output values of the first and second photometric means, and a determining means for determining the shape of the sample particle from the comparison result of the comparing means. Device. 2. The particles according to claim 1, wherein the discrimination means performs statistical processing to discriminate the shape of the sample particles using a large number of comparison data obtained by comparing the output values of each sample particle by the comparison means. measuring device.