JPH0621859B2 - Particle measuring device - Google Patents

Description

TECHNICAL FIELD The present invention relates to a particle measuring apparatus for irradiating a flowing sample particle with a light beam to detect information on the sample particle and performing particle measurement.

[Prior Art] In a conventional particle measuring device, for example, a flow cytometer,
By irradiating sample particles such as cells flowing at high speed one by one from one direction and measuring the scattered light and fluorescence generated thereby, information about the particle size and properties of the sample particles can be obtained, and many cells can be obtained. Quantitatively analyzed the sample particles from these information about.

In this conventional flow cytometer, the shape and size of the light beam irradiating the sample particles are set to have a flow direction substantially equal to or slightly larger than the sample particles, and a flow orthogonal direction is set to a size larger than the sample particles. As a result, the light irradiation can be performed with a uniform intensity even if the flow position of the sample particles is deviated. In addition to this, a slit scan method is also used in which more detailed information of the sample particles is detected by irradiating a slit-shaped beam having a beam size in the flow direction smaller than the size of the sample particles. Recently, a test sample was added to the latex particles sensitized with an antibody, and agglutination of the latex particles occurred due to the antigen-antibody reaction, and the size of the latex aggregate was detected using a flow cytometer. It has also been used to identify specific antigens inside.

In addition to the 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. In particular, recently, an apparatus for obtaining an image that reflects the internal structure of a cell having a high contrast by two-dimensionally scanning a stationary cell with a micro laser spot has been used. The applicant of the present application filed Japanese Patent Application No. 63-100572 (Japanese Patent Application Laid-Open No. 01-
No. 270644) proposed a particle measuring device having the functions of both the flow cytometer and the scanning microscope.

[Problems to be Solved by the Invention] However, with the device of Japanese Patent Application No. 63-100572 and the flow cytometer, it is possible to extract information from only one direction for each sample particle, and the three-dimensional analysis of sample particles is not possible. I couldn't figure it out.

It is an object of the present invention to provide a particle measuring device which can obtain information from a plurality of directions for each sample particle and can perform highly accurate analysis.

[Means for Solving the Problem] The particle measuring device of the present invention for solving the above-mentioned problems includes a means for allowing a sample particle to pass through a test part, and a passage direction of the sample particle toward the test part. A first irradiation unit that irradiates the irradiation light from a crossing direction, and a second irradiation unit that crosses the passage direction of the sample particles and irradiates the irradiation light to the test portion from a direction different from the first irradiation unit. Of the first and second irradiating means, while the other of the first and second irradiating means is not irradiating so as to irradiate the same analyte particle alternately, It is characterized in that it is provided with a photometric means for measuring the light from the sample particles passing therethrough with respect to each of the first and second irradiation means.

[Examples] Hereinafter, examples of the particle measuring apparatus 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, and FIG. 2 is a side view of a flow cell portion viewed from a laser beam irradiation direction. As a first light irradiating means for irradiating the sample particles with light, the laser light emitted from the laser light source 1 is moved at a high speed in a plane orthogonal to the flow of the sample particles by an optical deflector 2 provided in the optical path. Is deflected and scanned. A stopper 3 is provided in the direction straight from the laser light source 1 to cut the 0th order light. The laser light deflected by the optical deflector 2 is transferred to the objective lens 4
At, a telecentric image is formed on the test portion of the flow section 6 in the flow cell 5. Here, it is assumed that the size of the imaging spot is smaller than the sample particle size.

In addition, from the direction orthogonal to the irradiation direction of the scanning light and the passing direction of the sample particles, the laser light source 11, the optical deflector 12, the objective lens 25, and the stopper 14 serve as the second light irradiation means. It is arranged in the same manner as the irradiating means, and the subject is telecentrically scanned from the direction orthogonal to the first irradiating means.

Here, the optical deflectors 2 and 12 are controlled by the control circuit 15 so that when one of the optical deflectors 2 and 12 scans a portion to be inspected, the other becomes blanking. That is, the sample particles are not simultaneously irradiated with laser light from a plurality of directions, and only one of them is irradiated with laser light at a certain time.

In the flow section 6 in the flow cell 5, sample particles such as blood cells and latex aggregates are arranged one by one in a direction perpendicular to the paper surface of FIG. 1, that is, in the vertical direction of FIG. Be washed away. Therefore, as shown in FIG. 2, when the sample particles reach the test part where the laser spot 50 is scanned at high speed, the sample particles flow and are scanned in the lateral direction. At this time, since the laser scanning optical system is a telecentric, the irradiation beam is incident on the specimen particles from the same direction at any scanning position, and uniform scanning is performed. Since the sample particles 7 pass through the test portion of the circulation unit 6 in which the laser beam is alternately scanned at high speed in a plurality of directions in this manner, the scanning speed is set sufficiently high with respect to the passage speed of the particles. The sample particles are scanned a plurality of times from each direction when passing through the test part, and the sample particles are substantially two-dimensionally scanned from a plurality of directions.

The laser light is scanned by the first light irradiating means on the sample particles 7, and the light transmitted through the sample particles 7 and scattered by the sample particles 7 or the fluorescence emitted from the sample particles passes through the flow cell 5 and is the first Only the transmitted light is condensed by the condenser lens 8 arranged at the position facing the light irradiation means, and only the transmitted light passes through the diaphragm 9 arranged at the conjugate position with the light deflector 2, and the photodetector 1
At 0, the transmitted light intensity is detected. When it is desired to detect the scattered light of the sample particles instead of the transmitted light, a stopper having the same area as the aperture of the diaphragm 9 is arranged instead of the diaphragm 9 to cut the transmitted light and detect the scattered light. Is possible.

Further, a condenser lens 16 is arranged at a position facing the second light irradiation means with the flow cell 5 interposed therebetween, and the condenser lens 16 is provided.
The transmitted light and scattered light from the test part collected by
Only the transmitted light is selected by 7, and the transmitted light intensity is detected by the photodetector 18. The same applies when detecting scattered light instead of transmitted light. The outputs of the photodetectors 10 and 18 are respectively connected to the memory 13 and stored separately in time series in the memory.

Next, a method of driving the optical deflectors 2 and 12 in the control circuit 15 will be described with reference to FIGS. 3 and 4. The drive signal generator 19 generates a rectangular wave of 50% duty as shown in FIG. 4 (1). This signal is divided into two signals, one of which is input to the sawtooth wave generator 21, and the other of which is inverted in waveform by the inverter 20 as shown in FIG. 4 (4) and input to the sawtooth wave generator 22. Actually, UB in Fig. 4 (2) and Fig. 4 (5)
The LK signal determines the effective scanning range. 4 (3) and 4 (6), respectively, by these sawtooth wave generators 21 and 22.
Signals having a sawtooth-like waveform are obtained. These signals are voltage controlled oscillators 23 and 33, amplifiers 24 and 3, respectively.
4 becomes a drive signal for the optical deflector, and the optical deflector 2, 12
At, the transducer is driven to deflect and scan. Thus, while one is scanning the effective scanning range, the other is in the blanking state, and the particle information viewed from each direction can be separately distinguished and taken out.

As described above, the data of information representing the morphology of sample particles viewed from a plurality of directions accumulated for a large number of sample particles, subjected to processing such as image processing, or represented in a histogram or cytogram, It is generally well known that various particle analyzes are possible. The analysis result is displayed on a CRT or output by a method such as printout.

[Second Embodiment] Next, a second embodiment of the present invention will be described. FIG. 5 is a configuration diagram thereof, and FIG. 6 is a side view of the flow cell portion viewed from the laser beam irradiation direction. In each drawing, the same reference numerals as those used in the previous embodiment represent the same members.

In FIG. 5, as the first light irradiation means, the laser light emitted from the laser light source 31 passes through the light restricting means 32 such as a mechanical shutter, a liquid crystal shutter, or a chip, and is composed of a plurality of cylindrical lenses. In the lens unit 33, it is shaped into a slit-shaped beam,
Irradiation is applied to the test portion of the circulation portion 6 in the flow cell 7. The beam shape is a shape that is narrow in the flow direction and large in the flow orthogonal direction, as indicated by 51 in FIG. Lens unit 3
Instead of 3, the beam spot may be formed by using a slit-shaped aperture. As a modified example, it is possible to irradiate an elliptical beam spot having a size larger than the sample particle size such as 51 in FIG. 7, but a slit-shaped beam gives a larger amount of information.

Further, as the second light irradiating means, the laser light source 34, the light restricting means 35, in the direction orthogonal to the first light irradiating means,
The lens unit 36 is arranged in the same manner as the first light irradiation means.

Here, the laser light irradiation of the first and second light irradiation means is
The control circuit 45 controls the light restricting means 32 and 35 to alternately irradiate in a short time. Therefore, the passage of the sample particles to the test portion causes the sample particles to be alternately slit-scanned from two directions.

In this way, the light that is alternately irradiated to the sample particles and transmitted and scattered through the sample particles, or the fluorescence emitted from the particles is received by the condenser lens 40 and the photodetector 41 in the first light irradiation means, and the second light is emitted. Condensing lens 4 for irradiation means
2. The light is received by the photodetector 43. Although it is not shown in the drawing whether to detect the transmitted light or the scattered light, a diaphragm is placed in front of the photodetectors 41 and 43 as in the previous embodiment,
You can choose either by placing the stopper.
The outputs of these photodetectors 41 and 43 are separately stored in time series in the memory 44, and thus the one-dimensional optical scanning results of the specimen particles viewed from a plurality of directions are stored in the memory 44 for a large number of specimen particles. It will be stored and various particle analyzes will be performed based on this data.

In the embodiment described above, the forward scattered light or the transmitted light is detected by the light detecting means arranged in front of the optical axis of the laser irradiation light, but the other light detecting means is also used for detecting the side scattered light. It is also possible to detect side scattered light at the same time.
Needless to say, a dedicated detection optical system may be provided to detect the side scattered light without using the other light detection means. Further, in the optical path, a detection system including a dichroic mirror and a barrier filter may be provided in front of the photodetector to detect the fluorescence emitted from the sample particles.
As a result, the number of measurement parameters can be increased, which leads to improvement in measurement accuracy.

It should be noted that in the above-mentioned embodiment, by controlling the blanking, irradiation light from two directions is prevented from being simultaneously irradiated to the particles to be tested, thereby preventing noise from being mixed by the other irradiation light. If only the detection is performed, the irradiation accuracy of the irradiation light from two directions at the same time without performing blanking control does not significantly affect the measurement accuracy. This is because the intensity of side scattered light or fluorescence is very weak compared to the intensity of transmitted light or forward scattered light.

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

[Effects of the Invention] According to the present invention as described above, it is possible to provide a particle measuring device capable of obtaining information from each of a plurality of directions for each sample particle and enabling highly accurate analysis.

[Brief description of drawings]

1 is a configuration diagram of an embodiment of the present invention, FIG. 2 is a side view of a flow cell portion, FIG. 3 is a driving method of an optical deflector, FIG. 4 is a driving timing chart of an optical deflector, and FIG. FIG. 6 is a configuration diagram of a second embodiment, FIG. 6 is a side view of a flow cell portion in the second embodiment, and FIG. 7 is a side view of a flow cell portion in a modified example of the second embodiment. , 1, 11, 31, 34 ... Laser light source, 2, 12 ... Optical deflector, 3, 14 ... Stopper, 5 ... Flow cell, 6
…… Distribution department, 7 …… Sample particles, 9,17 …… Aperture 13,
44 ... Memory, 15, 45 ... Control circuit, 19 ... Drive signal generator, 21, 22 ... Sawtooth wave generator, 23,
33 ... Voltage controlled oscillator, 24, 34 ... Amplifier, 3
2, 35 ...... Light restriction means.

Claims (3)

[Claims] 1. A means for passing sample particles through a test part, a first irradiating means for irradiating the test part with irradiation light in a direction intersecting a passage direction of the sample particles, and A second irradiation unit that irradiates irradiation light toward the inspected portion from a direction different from the first irradiation unit that intersects the passing direction, and one of the first and second irradiation units is irradiating. The other means does not irradiate, and the control means for controlling to irradiate the same analyte particles alternately, and the light from the analyte particles passing through the test part,
A particle measuring device comprising: a second photo-irradiating unit and a photo-measuring unit. 2. The first and second irradiating means deflects a light beam by an optical system including an optical deflector, and scans a portion to be inspected in a direction intersecting a passage direction of sample particles. The particle measurement device described. 3. The particle measuring device according to claim 1, wherein the first and second irradiating means irradiate a light beam having a flat shape in a direction intersecting with a passage direction of analyte particles.