JPH0213830A - Article measuring apparatus - Google Patents

Abstract

PURPOSE:To set an irradiation light with the optimum beam spot automatically depending on measuring conditions by detecting one of particle size information of a sample particle and flow diameter information of a flow thereof to alter and set a beam diameter of the irradiation light based on a detection value thereof. CONSTITUTION:A sample liquid is converged to a fine flow wrapped by a sheath liquid and passes almost through the center of a passage section within a flow cell 4. At this point, sample particles in the sample liquid are separated and flow one at a time sequentially. The sample particles passing one by one is irradiated individually with a laser light emitted from a laser light source 1. Then, a diameter of a flow of the sample liquid wrapped by the sheath liquid within the passage section of the flow cell 4 is determined by a difference between pressurized values applied to a sample liquid container 15 and a sheath liquid container 14 respectively. Then, as shown in drawings (a), (b) and (c), the larger the diameter of the sample diameter, the size of a sideway beam spot is set larger.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a particle measuring device, for example, a flow cytometer that measures particles by irradiating light onto sample particles and measuring scattered light or fluorescence.

[Prior Art] Conventionally, in a particle measuring device such as a flow cytometer, sample particles passing sequentially through a flow cell are irradiated with light such as a laser beam, and the scattered light and fluorescence generated by the irradiation are emitted. The sample particles were measured using photometric signals. At that time, it has generally been done to irradiate the sample particles with a laser beam imaged in an elliptical shape that is horizontally elongated with respect to the passing direction of the sample particles. Conventionally, an elliptical beam spot has been formed by arranging the generating lines of two cylindrical lenses orthogonal to each other. Therefore, in order to obtain various beam spots depending on the measurement conditions, it is necessary to prepare various cylindrical lenses and to replace and combine them in a turret style.

Therefore, the applicant of the present application proposed a zoom lens system with a simple structure and easy control in Japanese Patent Application No. 62-153845. This makes it possible to form a beam spot with an optimal shape according to the measurement conditions such as the particle diameter of the sample particles using a simple optical system.

[Problem that the invention seeks to solve] However,
In the conventional particle measuring device described above, the beam spot of an appropriate size according to the measurement conditions was set by humans based on the type of sample particles and measurement conditions. Even if the type of particles is known, it is difficult to set the optimal beam spot when the particle size is unknown.

An object of the present invention is to provide a particle measuring device that can automatically set an irradiation beam spot with an optimal shape according to measurement conditions and has high measurement accuracy.

[Means for Solving the Problems] In order to solve the above-mentioned problems, the analyte particles present in the flow passing through the test area are irradiated with irradiation light and the generated light is photometered. In a particle measuring device that performs measurement, a detection means for detecting at least one of particle diameter information of sample particles and flow diameter information, and a beam diameter of irradiation light is changed and set based on an output value of the detection means. It has control means.

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

FIG. 1 is a configuration diagram of a particle measuring device according to the present invention, in which a sample liquid container 15 contains a sample liquid mixed with specimen particles such as biological cells, and a sheath liquid container 14 contains distilled water as a sheath liquid. are each pressurized, and the sample liquid is wrapped in the sheath liquid and converged into a thin flow, which passes through approximately the center of the flow section in the flow cell 4. At this time, the analyte particles in the sample liquid are separated and sequentially flowed one by one. The laser light emitted from the laser light source 1 is converged into an arbitrary elliptical shape by a combination of zoom lens systems 2.3 arranged sequentially along the optical axis and irradiated onto the sample particles passing through the sample particles one by one. Ru. Here, the zoom lens system 2 has a variable power function in a direction perpendicular to the direction in which the sample particles pass, and the zoom lens system 3 has a variable power function in the direction in which the sample particles pass. Also,
The magnification of these zoom lens systems can be controlled by an external signal.

When the sample particles are irradiated with a light beam, scattered light and fluorescence are generated. Among the scattered lights, the forward scattered lights emitted in the forward direction of the optical path are photometered by a condenser lens 5 and a photodetector 6. In order to prevent the irradiated light beam from directly entering the photodetector 6, a stopper (not shown) is provided in the optical path in front of the condenser lens 5 to remove the direct light. Photodetector 6
The output is input to the arithmetic circuit 16. Among the scattered lights, the side scattered lights emitted in the lateral direction orthogonal to the irradiation light optical path are collected by the condenser lens 7, reflected by the dichroic mirror 8, and photometered by the photodetector 11. Generally, the direction in which side scattered light is photometered is often orthogonal as in this embodiment, but is not limited to orthogonal and may be, for example, a 45 degree direction or a 60 degree direction. In addition, in order to photometer the weak fluorescence generated together with the scattered light, green fluorescence is detected by the dichroic mirror 9 and photodetector 12 among the fluorescence that is focused by the condenser lens 7 and passed through the dichroic mirror 8. Red fluorescence is detected by a combination of a reflecting mirror 10 and a photodetector 13.

In front of each photodetector, there is a
Third bandpass filters 21, 22, and 23 are installed. The signals of the photodetectors 115, 12, and 13 are sent to the arithmetic circuit 1.
6, and the calculation circuit 16 performs particle measurement calculations. Further, from the arithmetic circuit 16, a zoom lens system 2.
A control signal for controlling the magnification of 3 is output.

Next, using FIG. 2, a zoom lens system 2 according to an embodiment of the present invention will be described.
．． The optical system of No. 3 will be explained. FIG. 2 is an optical configuration diagram of the zoom lens system, in which two sets of optically corrected zoom lens systems are arranged along the optical axis. However, the two zoom lens systems each have a zoom function only within the plane and within the side. FIG. 2(a) is a plan view, and FIG. 2(b) is a side view seen from a direction perpendicular to the plan view. A convex cylindrical lens 30, a concave cylindrical lens 31, and a convex cylindrical lens 32 arranged and aligned along the optical axis constitute an optically corrected zoom lens system having a zoom function only within a plane, and a convex cylindrical lens 33, The concave cylindrical lens 34 and the convex cylindrical lens 35 constitute an optically corrected zoom lens system having a zoom function only within the side surface. This optically corrected zoom lens system consists of two
The two dynamic lenses move as one, and if the power and lens spacing of each group are selected appropriately, the image position can be kept constant, but when zooming, the focal plane will shift slightly under certain conditions. There is. Therefore, when the in-plane focal length of the zoom lens system of the cylindrical lenses 30, 31, and 32 is continuously changed, the in-plane imaging beam diameter d in the flow section of the flow cell 4 is continuously changed. . Further, when the focal length of the cylindrical lenses 33, 34, and 35 is continuously changed within the side surface of the zoom lens system, the 9 imaging beam diameter d is continuously changed within the side surface of the flow cell 4. Thereby, the vertical and horizontal sizes of the beam spot in the flow section of the flow cell 4 can be freely changed separately, and a zoom spot of any vertical and horizontal size can be obtained.

Next, a method of adjusting the magnification of the zoom lens system to set a beam spot suitable for measurement conditions will be described.

The greater the intensity of the light irradiated to the sample particles, the stronger the scattered light, fluorescence, and other light generated from the sample particles.

In particular, since side scattered light and fluorescence are weak, it is preferable that the intensity of the light irradiated to the sample particles is also greater, that is, the size of the converged beam spot is smaller.

However, the individual sample particles that flow one after another through the flow cell do not always flow at a fixed position, but vary with slight positional deviations in the front and back or left and right directions when viewed from the top of the flow cell. Since the laser beam irradiated here has a Gaussian intensity distribution, if the beam spot is narrowed down too much, the fluctuation of the irradiated light intensity will increase with respect to the positional shift of the sample particles. Therefore, it is important to set the irradiation beam spot to an appropriate size so that the irradiation light intensity does not vary greatly due to positional deviation, even though the beam spot size is made as small as possible and the irradiation light intensity is increased. This is important for improving measurement accuracy.

As a first method of setting the optimum beam spot, a method of adjusting the size of the beam spot in the lateral direction perpendicular to the flow of sample particles will be described below with reference to FIG. 3.

The sample flow diameter, which is the thickness of the sample liquid flow, is used as a parameter for setting the horizontal size of the beam spot. The sample flow diameter of the sample liquid surrounded by the sheath liquid in the flow section of the flow cell 4 is determined by the difference in the pressurization values applied to the sample liquid container 15 and the sheath liquid container 14, respectively. The value of this sample flow diameter is set depending on the particle diameter of the sample particles, etc., and it is generally desirable to set the sample flow diameter to a value as close as possible to the particle diameter of the sample particles.

The sample flow diameter for setting the beam spot can be calculated from the pressure values applied to the sheath liquid container 14 and the sample liquid container 15, or alternatively, it can be calculated using a conversion table obtained through experience. It's okay.

As another method, it is also possible to directly optically measure the sample liquid flow in a flow cell using a CCD or the like, as shown in Japanese Patent Application No. 60-264158.

The horizontal size of the irradiation beam spot is set according to the flow diameter of the extracted sample. As a specific setting method, as shown in Figure 3 (a), (b), and (c),
The larger the sample flow diameter is, the larger the beam spot size in the lateral direction is set. Since the displacement of the flowing specimen particles occurs within the range of the sample flow diameter, the displacement of the flowing specimen particles may become larger as the sample flow diameter becomes larger. Therefore, in order to increase tolerance to an increase in positional deviation, this setting is made in accordance with the size of the sample flow diameter.

Regarding the beam size in the vertical direction, in addition to the size that irradiates the entire sample particle as shown in Figure 3, it is also possible to measure particles using the slit scan method by setting the beam to a slit shape that is thinner than the particle diameter of the sample particle. It is also possible to do so.

Basically, the settings are as described above, but the optimal settings vary depending on not only the sample flow diameter but also the purpose of measurement. In other words, the light intensity irradiated to the sample particles and the CV
Since the values (coefficients of variation) are contradictory to each other, the balance must be determined depending on the purpose of measurement. For example, when measuring extremely weak fluorescence, it is necessary to increase the beam intensity to increase the intensity of the generated fluorescence even if the CV value deteriorates to some extent, so the horizontal beam size is made small. Furthermore, if the main purpose is to measure particles by photometry of forward scattered light, since forward scattered light has a relatively large output, CV
It is possible to improve the value and reduce the variation in measurement data of individual sample particles.

Next, as a second method for setting the optimum beam spot, a method of adjusting the magnification of the beam spot size in the vertical direction, which is the same as the flow of sample particles, will be described below.

The particle size of the sample particles is used as a parameter for obtaining the optimum beam spot in the longitudinal direction, and the scattered light, particularly the forward scattered light intensity, that is, the output of the photodetector 6, is used as a means for obtaining the particle size.

Generally, there is a correlation between the particle diameter of the sample particles and the forward scattered light intensity. Therefore, the calculation circuit 16 calculates the particle diameter of the sample particle from the forward scattered light intensity detected by the photodetector 6.

Based on the determined particle diameter, the zoom lens system 3 is controlled and set to a size slightly larger than the particle diameter of the sample particles in the vertical direction of the beam spot. By setting in this manner, it is possible to irradiate the sample particles with light of higher intensity while ensuring the minimum beam size necessary for measuring the sample particles without worsening the CV value. Another effect is that, as shown in FIG. 4(d), the possibility of two specimen particles entering the beam spot at the same time and generating an erroneous signal is reduced.

Note that the horizontal and vertical beam spot sizes may be set independently using the first and second setting methods, but both may be set simultaneously as shown in Figures 4(a), (b), and (c). By doing so, measurement accuracy can be further improved.

Note that the beam spot setting described above is set by flowing a small amount of sample liquid at the start of measurement, and after fixing the beam spot, particle measurement is started using the remaining sample liquid.

In the embodiments of the present invention described above, an optically corrected zoom lens is used as the zoom lens system, but the invention is not limited to this.
It is also possible to use a mechanically corrected zoom lens system as shown in No. 272074. Further, the present invention is not limited to the above-mentioned flow cytometer, but is also applicable to latex immunodiagnosis devices, particle counters, and the like.

[Effects of the Invention] According to the present invention, it is possible to automatically set the optimum beam spot irradiation light depending on the measurement conditions, and it is possible to provide a particle measuring device with high measurement accuracy.

[Brief explanation of the drawing]

Figure 1 is an overall configuration diagram of the particle measuring device of the present invention, Figure 2 is an optical configuration diagram of the zoom lens system, Figure 3 is an explanatory diagram for setting the horizontal size of the beam spot, and Figure 4 is the beam spot. This is an explanatory diagram for setting the horizontal and vertical size of , and the symbols in the diagram are:

Claims (1)

[Scope of Claims] 1. In a particle measuring device that measures sample particles by photometering the light generated by irradiating sample particles existing in a flow passing through a sample part with irradiation light, at least Particle measurement characterized by having a detection means for detecting either particle diameter information of particles or flow diameter information of the flow, and control means for changing and setting the beam diameter of irradiation light based on the output value of the detection means. Device. 2. The particle measuring device according to claim 1, wherein the control means changes the beam diameter in the vertical direction of the flow of the irradiation light based on the particle diameter information. 3. The particle measuring device according to claim 1, wherein the control means changes the beam diameter in the lateral direction of the flow of the irradiated light based on the flow diameter information. 4. The particle measuring device according to claim 2, wherein the particle size information is the particle size of the sample particle calculated based on the intensity of scattered light emitted from the sample particle. 5. The particle measuring device according to claim 3, wherein the flow diameter information is a sample flow diameter calculated from respective pressurization values of a sample liquid containing specimen particles and a sheath liquid. 6. The particle measuring device according to claim 1, wherein the beam diameter of the irradiated light is changed and set before actual measurement.