JPH0274846A - Particle measuring apparatus - Google Patents

Abstract

PURPOSE:To make it possible to perform analysis more accurately by projecting light beams on specimen particles in a plurality of directions, and obtaining the data of the specimen particles in a plurality of the directions. CONSTITUTION:Laser light from a laser source 1 is deflected through a light deflector 2. The image is formed at a part to be checked of a flowing part 6 in a flow cell 5 through an objective lens 4. The image of light from a laser light source 11 is likewise formed through a light deflector 12 in the direction perpendicular to the above described scanning light. Only the laser light from either direction is projected by the control with a control circuit 15. The light which is transmitted through or scattered by specimen particles is condensed through a condenser lens 8. Only the transmitted light passes through a stop 9. The intensity of the transmitted light is detected with a photodetector 10. The light is condensed through a condenser lens 16. Only the transmitted light is selected through a stop 17. The intensity of the transmitted light is detected with a photodetector 18.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a particle measuring device that performs particle measurement by irradiating flowing sample particles with a light beam and detecting information regarding the sample particles.

[Prior Art] In conventional particle measuring devices, such as flow cytometers,
By irradiating light from one direction on specimen particles such as 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. The sample particles were quantitatively analyzed from this information.

In this conventional flow cytometer, the shape and size of the light beam that irradiates the sample particles is set to be approximately the same size or slightly larger than the sample particles in the flow direction, and set to be larger than the sample particles in the direction perpendicular to the flow. As a result, light irradiation can be performed with uniform intensity even if there is a shift in the flow position of the sample particles. In addition to this, a slit scan method is also used in which more detailed information about the sample particles is detected by irradiating a beam with a slit shape in which the beam size in the flow direction is smaller than the size of the sample particles. Recently, a test sample is added to latex particles sensitized with antibodies, and the antigen-antibody reaction causes aggregation of the latex particles, 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 the present application previously filed Japanese Patent Application No. 100572/1983, in which he proposed a particle measuring device having the functions of both the flow cytometer and the scanning microscope.

[The problem that the invention is trying to solve, however,
The apparatus disclosed in Japanese Patent Application No. 63-100572 and the flow cytometer could only extract information from one direction about each sample particle, and it was not possible 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 problems, there is provided a means for passing the sample particles through the test section, and a light beam is irradiated on the test section from a direction intersecting the passing direction of the test particles. a first irradiation means, and a second irradiation means that irradiates a light beam from a direction that intersects with the passing direction of the sample particles and is different from the irradiation direction of the first irradiation means.
irradiation means, and photometry means for measuring the light from the test area with respect to the first and second irradiation means, respectively.

[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, and FIG. 2 is a side view of the flow cell section viewed from the laser beam irradiation direction. As a first light irradiation means for irradiating sample particles with light, a laser beam emitted from a laser light source 1 is deflected at high speed within a plane perpendicular to the flow of sample particles by an optical deflector 2 provided in the optical path. is deflected and scanned. Note that a stopper 3 is provided in the straight direction from the laser light source 1 to cut off the zero-order light. The laser beam deflected by the optical deflector 2 passes through the objective lens 4.
The image is telecentrically formed on the test part of the flow section 6 in the flow cell 5. Here, the size of the imaging spot is assumed to be smaller than the sample particle size.

Further, from the direction orthogonal to the irradiation direction of the scanning light and the passing direction of the specimen particles, the laser light source 11, the optical deflector 12, the objective lens ←, and the stopper 14 serve as the second light irradiation means. The second irradiation means is arranged in the same manner as the first irradiation means, and telecentrically scans the test area from a direction orthogonal to the first irradiation means.

Here, the optical deflectors 2 and 12 are controlled by a control circuit 15 in a manner to be described later, so that when one is scanning the subject, the other is blanking. That is, the sample particles are not irradiated with laser light from multiple directions simultaneously, but are irradiated with laser light from only one direction at a certain time.

Sample particles, such as blood cells and latex aggregates, are introduced into the flow section 6 in the flow cell 5 one by one or in clusters in the direction perpendicular to the page of FIG. 1, that is, in the vertical direction of FIG. be swept away by Therefore, as shown in FIG. 2, when specimen particles approach a test area where the laser spot 50 is scanned at high speed, the specimen particles flow and are 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. In this way, the specimen particles 7 pass through the test portion of the flow section λ where the laser light is scanned alternately from multiple directions at high speed, and the scanning speed is set sufficiently large compared to the passing speed of the particles. The specimen particles are scanned multiple times from each direction as they pass through the test area, resulting in substantially two-dimensional scanning of the specimen particles from multiple directions.

The sample particles 7 are scanned with a laser beam by the first light irradiation means, and the light transmitted through the sample particles 7 and scattered by the sample particles 7, or the fluorescence emitted from the sample particles is transmitted to the first light source with the flow cell 5 in between. The light is condensed by a condenser lens 8 disposed at a position facing the light irradiation means, and only the transmitted light passes through an aperture 9 disposed at a conjugate position with the light deflector 2.
The transmitted light intensity is detected at 0. Note that if you want to detect the scattered light of the sample particles instead of the transmitted light, you can cut the transmitted light and detect the scattered light by placing a stopper with the same area as the aperture of the diaphragm 9 instead of the diaphragm 9. is possible.

Further, a condenser lens 16 is arranged at a position facing the second light irradiation means with the flow cell 5 in between.
The transmitted light and scattered light from the test area collected by the aperture 1
7 selects only the transmitted light, and the photodetector 18 detects the intensity of the transmitted light. Note that the case where scattered light is detected instead of transmitted light is similar to the above. The outputs of the photodetectors 10 and 18 are respectively connected to a memory 13 and stored separately in the memory in chronological order.

Next, a method of driving the optical deflector 2.12 in the control circuit 15 will be explained using FIGS. 3 and 4. The drive signal generator 19 generates a 50% duty rectangular wave as shown in FIG. 4(1). This signal is split into two branches, one of which is input to the sawtooth wave generator 21, and the other whose waveform is inverted by the inverter 20 as shown in FIG. 4 (4) and is input to the sawtooth wave generator 22. Actually, the U and BLK signals in FIGS. 4(2) and 4(5) determine the effective scanning range. These sawtooth generators 21, 22 generate a fourth
Signals with sawtooth waveforms as shown in Fig. (3) and Fig. 4 (6) are obtained, and each of these signals is
3.33, it becomes a driving signal for the optical deflector through amplifiers 24.34, and drives a transducer in the optical deflector 2.12 to deflect and scan the light. In this way, while one is scanning the effective scanning range, the other is in a blanking state, and particle information viewed from each direction can be extracted separately.

As described above, the information data representing the morphology of the sample particles as seen from multiple directions, which has been stored for a large number of sample particles, is subjected to processing such as image processing, and is expressed in a histogram or cytogram. It is generally well known that various particle analyzes are possible. This analysis result is displayed on a CRT or outputted by a method such as printing out.

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

In FIG. 5, a laser beam emitted from a laser light source 31 as a first light irradiation means passes through a light limiting means 32 such as a mechanical shutter, a liquid crystal shutter, or a chopper, and passes through a lens consisting of a plurality of siluntric lenses. It is formed into a slit-shaped beam in the unit 33,
The area to be inspected in the flow section 6 within the flow cell is irradiated. This beam shape is narrow in the flow direction and large in the direction perpendicular to the flow, as shown by 51 in FIG. Furthermore, lens unit 3
3, the beam spot may be formed using a slit-shaped aperture. Further, as a modification, it is possible to irradiate with an elliptical beam spot such as 51 in FIG. 7, which is larger than the sample particle size, but a slit-shaped beam can obtain a larger amount of information.

Further, as a second light irradiation means, a laser light source 34, a light restriction means 35,
A lens unit 36 is arranged in the same manner as the first light irradiation means.

Here, the laser light irradiation by the first and second light irradiation means is as follows:
The light limiting means 32,35 are controlled by the control circuit 45, and the light is irradiated alternately in a short period of time.

Therefore, by passing the sample particles to the test area, the sample particles
The slits are scanned alternately from each direction.

In this way, the light that is alternately irradiated onto the sample particles and transmitted and scattered by the sample particles, or the fluorescence emitted from the particles, is received by the condenser lens 40 and the photodetector 41 for the first light irradiation means, and is received by the light detector 41 for the first light irradiation means. Concerning the light irradiation means, a condensing lens 42
, the light is received by the photodetector 43. Note that whether to detect transmitted light or scattered light is determined by placing an aperture in front of the photodetector 41, 43 as in the previous embodiment or by placing a stopper, although it is not shown in the figure. can be selected. The outputs of these photodetectors 41 and 43 are stored in the memory 44.
They are stored separately in chronological order, and in this way, the memory 44 stores the one-dimensional optical scanning results of the specimen particles viewed from multiple directions for a large number of specimen particles.Based on this data, various particle An analysis is made.

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 the other light detection means is also used to detect side scattered light. , it is also possible to detect side scattered light at the same time.

It goes without saying that a dedicated detection optical system may be provided to detect the side scattered light without also serving as the other light detection means. Furthermore, it is also possible to detect fluorescence emitted from sample particles by providing a detection system including a dichroic mirror or a barrier filter in front of the photodetector in the optical path.

This allows the number of measurement parameters to be increased, leading to improved measurement accuracy.

In the above example, blanking control was performed to prevent the irradiation light from two directions from being irradiated onto the test particle at the same time, thereby preventing noise from being mixed in by the other irradiation light. If the purpose is to detect only light, irradiating light from two directions simultaneously without blanking control will not have much effect on measurement accuracy. This is because the intensity of side scattered light and fluorescence is extremely weak compared to the intensity of transmitted light or forward scattered light.

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 each sample particle from multiple directions and can perform more accurate analysis.

[Brief explanation of the drawing]

Figure 1 is a configuration diagram of an embodiment of the present invention, Figure 2 is a side view of the flow cell section, Figure 3 is a method of driving an optical deflector, Figure 4 is a timing chart for driving the optical deflector, and Figure 5 is a diagram of the drive timing chart of the optical deflector. 6 is a side view of the flow cell section in the second embodiment; FIG. 7 is a side view of the flow cell section in a modified example of the second embodiment. Main symbols in the figure 1.11.31.34... Laser light source, 2.12.
...Light deflector, 3! 4... Stopper, 5...
・Flow cell, 6... Distribution section, 7... Sample particles, 9.17... Aperture 13.44... Memory, 1
5.45... Control circuit, 19... Drive signal generator, 21.22... Sawtooth wave generator, 23.33...
...voltage controlled oscillator, 24.34... amplifier,
32.35...Light limiting means. 2nd Inju 6,3rd drawing 4th group

Claims (1)

[Scope of Claims] 1. means for passing sample particles through a test region; first irradiation means for irradiating irradiation light from a direction intersecting the passing direction of test particles in the test region; a second irradiation means that irradiates the test area with irradiation light from a direction that intersects with the passing direction and is different from the irradiation direction of the first irradiation unit; A particle measuring device characterized by comprising photometric means for measuring light with respect to each of the irradiation means. 2. The particle measuring device according to claim 1, further comprising a control means for controlling one of the first and second irradiation means to perform blanking while the other is irradiating. 3. The particle measuring device according to claim 1, wherein the irradiation means deflects the light beam with an optical system including a light deflector to scan the test area in a direction intersecting the passing direction of the sample particles. 4. The particle measuring device according to claim 1, wherein the irradiation means irradiates a flat light beam in a direction intersecting the passing direction of the sample particles.