JPH0274845A - Particle measuring apparatus - Google Patents

Abstract

PURPOSE:To make it possible to perform highly accurate analysis by scanning specimen particles with light beams having the different wavelengths in the plurality of directions, and obtaining the data of the specimen particles in a plurality of the directions. CONSTITUTION:Laser light having a wavelength of lambda1 is emitted from a laser light source 1, and the image is formed on a flowing part 2 in a flow cell 4 comprising transparent glass through an image forming optical system 3. Laser light having the wavelength lambda2 is emitted from a laser light source 11, and the image is formed at the detecting part of the flow cell 4 through an image forming lens system 13 in the direction perpendicular to the above described direction. When the laser light is projected on specimen particles, the light is scattered. The light is condensed through a lens 7. Only the light having the wavelength lambda1 is selectively transmitted through a barrier filter 8. The light passes through a stop 9, and the light intensity is detected with a photodetector 10. The light is also condensed through a lens 15, and only the light having the wavelength lambda2 is selected through a barrier filter 16. The light intensity is detected with a stop 17 and a photodetector 18.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is a method for measuring particles by irradiating light onto specimen particles and measuring light such as transmitted light, scattered light, or fluorescence by the specimen particles. Concerning a measuring device.

[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 thereby, information about the particle size and properties of the specimen particles can be obtained, and a large number of specimen particles can be collected. The sample particles were quantitatively analyzed from this information about the cells. 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 identify specific antigens inside.

In addition to a flow cytometer, particle measurement based on image information of sample particles is generally performed using a device such as an optical microscope or an electron microscope. Particularly recently, devices have come into use that obtain high-contrast images that reflect the internal structure of cells by two-dimensionally scanning stationary cells with a minute laser spot.

The applicant of this application previously filed patent application No. 63-100572.
proposed a particle measuring device that combines the functions of both a flow cytometer and a scanning microscope.

[Problem that the invention seeks to solve] However,
Conventional flow cytometer and patent application No. 63-100572
In this device, each sample particle is irradiated with light from one direction, so information can only be extracted from one direction, making it impossible to understand the sample particle three-dimensionally.

An object of the present invention is to provide a particle measuring device that can obtain information about each sample particle from multiple directions and perform highly accurate analysis.

[Means for solving the problem] In order to solve the above-mentioned problem, the sample particles are measured by irradiating light onto the sample particles passing through the sample part and measuring the light from the sample part. In the particle measuring device that performs
A first beam that irradiates the test area with a first wavelength O light from a first direction.
a second light irradiation means for irradiating the test area with light of a second wavelength from a second direction different from the first direction; A photometer is provided for measuring light of two wavelengths.

[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 configuration diagram of an embodiment of the present invention, where 1 is the wavelength λ1
A laser light source emits a laser beam, and 3 is an imaging optical system consisting of a cylindrical lens and the like, forming a first light irradiation means. Laser light with a wavelength λ1 emitted from a laser light source 1 is imaged by an imaging optical system 3 onto a flow section 2 within a flow cell 4 made of transparent glass. At this time, the shape of the beam spot imaged onto the test area by the imaging optical system 3 is an ellipse that is horizontally elongated with respect to the flow of the sample particles.

This is so that even if the flow position of the sample particles in the test area is slightly shifted in the vertical or horizontal direction of the page, the sample particles can be irradiated with light at a substantially uniform intensity.

Further, as a second light irradiation means, the laser light source 11 emits a laser light having a wavelength λ2 different from the previous wavelength λ1 from a direction perpendicular to the optical path of the laser light from the laser light source 1 and the flow direction of the sample particles. be done. This laser light is transmitted by the imaging lens system 13 similarly to the first light irradiation means.
An image is formed on the test area in the flow cell from a direction perpendicular to the first light irradiation means.

Sample particles, such as blood cells or latex aggregates, are sequentially flowed one by one or in chunks in a direction perpendicular to the plane of the paper into the flow section 2 in the flow cell 4 using a sheath flow system, and are exposed to laser light with a wavelength λ1 and laser light with a wavelength λ2. The light 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 specimen particles approach the test area and are irradiated with laser light, light scattering occurs due to the specimen 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 1 and 11, respectively, to condense scattered light at a predetermined angle. Here, from the scattered light collected by the lens 7, only the light having the wavelength λ1 is selectively transmitted by the barrier filter 8. That is, only the scattered light generated by irradiation by the first light irradiation means is selected. The light having the wavelength λ1 that has passed through the barrier filter 8 passes through the aperture 9, and the light intensity is detected by the photodetector 10.

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 by the second light irradiation means is selected, and the light intensity of the wavelength λ2 is detected by the aperture 17 and the photodetector 18. The outputs of the photodetectors 10 and 18 are connected to storage means 19, and each detection data is stored separately.

In this way, measurement data from different angles are obtained for each sample particle, and based on the contents of the storage means 19, the calculation means 20 performs calculations for particle measurement.

[Other Embodiments Next, another embodiment of the present invention will be described in which more detailed particle information can be obtained by optically scanning sample particles from a plurality of directions. FIG. 2 is a block diagram of another embodiment of the present invention, and FIG. 3 is a side view of the flow cell section viewed from the laser beam irradiation direction, and the same reference numerals as in FIG. 1 represent the same members.

The wavelength λ1 is used as the first light irradiation means for irradiating the sample particles with light.
A laser beam emitted from a laser light source 21 that emits a laser beam with a wavelength of is deflected and scanned at high speed within a plane perpendicular to the flow of specimen particles by an optical deflector 22 provided in the optical path. Note that a stopper 23 is provided in the straight direction from the laser light source 1 to cut off the zero-order light. Optical deflector 22
The laser beam deflected by is telecentrically imaged by the objective lens 24 onto the test portion of the flow section 2 in the flow cell 4 . Here, the size of the imaging spot is assumed to be smaller than the size of the sample particles.

Further, from a direction perpendicular to the irradiation direction of the scanning light and the passage direction of the sample particles, a laser light source 31 serving as a second light irradiation means emits a laser light with a wavelength λ2 different from the wavelength λ of the laser light source 21. , optical deflector 32, objective lens 3
4 and a stopper 33 are arranged in the same manner as the first irradiation means, and scan the subject part from a direction orthogonal to the first irradiation means.

Here, the optical deflectors 22 and 32 are controlled by a control circuit 39 to perform deflection scanning at the same speed.

Specimen particles 5 such as biological cells are sequentially flowed one by one in the direction perpendicular to the plane of the paper in FIG. 1, that is, in the vertical direction in FIG. At this time, the scanning speed of the laser is set to be sufficiently larger than the soaking speed of the sample particles. Therefore, as shown in FIG. 3, when a sample particle approaches a test area where the laser spot 50 is scanned at high speed, the sample particle will be scanned in the lateral direction. At this time, since the optical system for laser scanning is telecentric, the irradiation beam is incident on the sample particle from the same direction at any scanning position, and uniform scanning is performed. The sample particles 5 are scanned with a laser beam of wavelength λ1 by the first light irradiation means, and the light transmitted through the sample particles 5 and scattered by the sample particles 5 is collected by a condenser provided at opposing positions with the flow cell 4 in between. The light is focused by the lens 25. The wavelength of the focused light is selected by a filter 40 that allows only light with wavelength λ1 to pass through, and only the transmitted light is selected by a diaphragm 26 located at a position conjugate with the optical deflector 22, and the intensity of the transmitted light with wavelength λ1 is selected. is detected by the photodetector 27. Note that if it is desired to detect light scattered by the sample particles instead of transmitted light, by providing a stopper with the same area as the aperture of the diaphragm 26 instead of the diaphragm 26, the transmitted light can be cut and the scattered light can be detected. .

Further, a condenser lens 35 is arranged at a position facing the second light irradiation means with the flow cell 4 in between, and a filter 41 that condenses the transmitted light and scattered light from the test area and allows only the wavelength λ2 to pass. The wavelength is selected by the aperture 36 and the photodetector 37.
The transmitted light intensity of wavelength λ2 is detected at . The case of detecting scattered light is the same as above. The photodetectors 27 and 3
The outputs of 7 are each connected to the memory 38 and stored separately in the memory in chronological order.

As described above, when the sample particles 5 pass through the inspection area of the distribution section 2 where laser beams are scanned alternately from multiple directions at high speed, the scanning speed is set sufficiently large compared to the passing speed of the particles. Therefore, the specimen particles are scanned multiple times from each direction when passing through the test section, and the specimen particles are substantially two-dimensionally scanned from multiple directions.

In this way, each time a sample particle passes through the test section, the memory 38
At the top, data representing information such as the morphology of the sample particles viewed from multiple directions is stored regarding a large number of sample particles. It is generally well known that various particle analyzes are possible by subjecting the data obtained in this way to processing such as image processing, or by representing it in a histogram or cytodrum.

This analysis is performed by the arithmetic circuit 42, and the analysis result is displayed on the CRT.
It is output by displaying it on the screen, printing it out, etc.).

In the embodiments described above, forward scattered light or transmitted light is detected by the light detection means disposed in front of the optical axis of the laser irradiation light, but side scattered light is detected at the same time by the other light detection means. It is also possible.

In addition, in the above-mentioned example, the sample particles were irradiated with light from two directions to obtain information from two directions, but the invention is not limited to this.
It is also possible to perform more detailed analysis by irradiating light from three or more directions.

[Effects of the Invention] As described above, according to the present invention, it is possible to provide a particle measuring device that can obtain information about a large number of sample particles from a plurality of directions, and can perform more accurate analysis.

[Brief explanation of the drawing]

Fig. 1 is a block diagram of an embodiment of the present invention, Fig. 2 is a block diagram of another embodiment, and Fig. 3 is a side view of a flow cell section in another embodiment. , 1.21... Laser light source with wavelength λ1, 11.32...
...Laser light source with wavelength λ2, 8.40...Wavelength λ
16.41...barrier filter that allows light of wavelength λ2 to pass through, 6.14.23.33...stopper, 19.38...
... storage means, 20.42 ... calculation means, 22.32 ... optical deflector,

Claims (1)

[Scope of Claims] 1. In a particle measuring device that measures sample particles by irradiating light onto sample particles passing through a sample part and measuring the light from the sample part, from a first direction. a first light irradiation unit that irradiates the test area with light of a first wavelength; and a second light irradiation unit that irradiates the test unit with light of a second wavelength from a second direction different from the first direction. a light irradiation means, and the first
, a particle measuring device comprising photometric means for measuring light of a second wavelength, respectively. 2. The particle measuring device according to claim 1, wherein the light irradiation means scans the test area in a direction intersecting the passing direction of the sample particles by deflecting the light beam with an optical system including a light deflector.