JPS63182547A - Particle analyzer - Google Patents

Abstract

PURPOSE:To easily measure the diameter of a liquid flow by switching a light beam between two shapes, i.e., a shape which crosses a sample flow and a shape which is narrowed down in width to illuminate part of the sample flow when measuring the diameter of the liquid flow by projecting the light beam on particles to be inspected which flow through the distribution part of a flow cell. CONSTITUTION:The distribution part 2 which allows the sample liquid to pass is provided at the center part of the flow cell 1 and a laser light source 3 is arranged at right angles of the flow of the sample liquid; and an image forming lens 4 formed by combining cylindrical lenses is arranged on an optical axis 01 so as to guide the irradiation light from the laser light source to the distribution part 2, and the shape of the beam L is made optionally elliptic. A stopper 5, an objective 6, and a photoelectric detector 7 are provided opposite the light 3 across the distribution part 2. Then the lens 4 is rotated around the optical axis 01 by 90 deg. to switch the beam L between the normal shape and narrow-width shape, and the diameter of the liquid flow is calculated by using a dial gauge 34.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a particle analysis device that is capable of measuring the flow diameter of a sample liquid flowing through a flow section in a flow cell in a flow cytometer or the like.

[Prior art 1] In a conventional particle analysis device used in a flow cytometer, etc., a particle size of, for example, 250 m x 250 p in the center of the flow cell
A laser beam is irradiated onto the sample liquid flow surrounded by the high-speed sheath liquid flowing in a laminar flow state based on the principle of hydrodynamic focusing in a flow section with a minute cross section of m. The particle properties such as the shape, size, and refractive index of the sample particles are obtained from the forward scattered light and side scattered light scattered by the sample particles.

In order to flow the sample liquid and the sheath liquid at high speed, the sample liquid and the sheath liquid are each pressurized, and a pressure slightly higher than that of the sheath liquid is applied to the sample liquid. The flow diameter of the sample liquid flow changes depending on the difference in pressure applied to the sample liquid and the sheath liquid; as the pressure difference increases, the flow diameter of the sample liquid flow increases, and as the pressure difference decreases, the flow diameter of the sample liquid flow decreases. Become.

By the way, when the flow diameter of the sample liquid flow is larger than the size of the sample particles, variations occur in the passage positions of the sample particles in the sample liquid flow. In addition, the light intensity distribution of the laser beam irradiated to the sample particles is a Gaussian distribution, so if there is variation in the passing position of the sample particles, there will be a difference in the irradiation light intensity even for the same sample particle, and as a result, Differences in intensity occur in the photoelectric signals obtained from the sample particles, making highly accurate measurements difficult. Conversely, if the flow diameter of the sample liquid flow is small compared to the size of the sample particles, the measurement accuracy will increase, but the number of sample particles passing per unit time will decrease, and the analysis speed will decrease. In order to obtain highly accurate and efficient measurement results, it is desirable that the flow diameter of the sample liquid flow be approximately equal to the size of the sample particles.

In conventional particle analysis devices, the pressures applied to each of the sample liquid and the sheath liquid are adjusted so that the number of sample particles passing per unit time is constant. Therefore, in measurements using this method, the difference in pressure applied to the sample liquid and the sheath liquid depends on the concentration of analyte particles in the sample liquid. , the pressure applied to the sample liquid is increased to increase the flow diameter of the sample liquid flow. However, if the flow diameter of the sample liquid flow is increased, as described above, the passing positions of the sample particles will vary, making it difficult to perform highly accurate measurements.

[Objective of the Invention] The object of the present invention is to perform particle analysis efficiently and with high accuracy without being affected by the concentration of analyte particles in a sample liquid.
An object of the present invention is to provide a particle analysis device equipped with means for measuring the flow diameter of a sample liquid flow.

[Summary of the Invention] The gist of the present invention to achieve the above-mentioned object is to provide an irradiation optical system that irradiates a light beam to sample particles flowing through a flow section in a flow cell, and a photometry system that measures scattered light and fluorescence by the sample particles. a particle analysis device, wherein the flow cell is placed on a base together with the photometric optical system, and the base is movable relative to the irradiation optical axis of the light beam. In this method, the light beam has two shapes: one that crosses the sample flow in a direction perpendicular to the flow of the sample flow, and one that narrows the width in the direction perpendicular to the flow of the sample flow and irradiates a part of the sample flow. This is a particle analysis device characterized by being provided with switching means.

[Embodiments of the invention] The present invention will be explained in detail based on illustrated embodiments.

FIG. 1 is a plan view of the optical system and alignment device.

A flow section 2 through which the sample liquid passes in the vertical direction perpendicular to the plane of the paper is provided in the center of the flow cell l, and a laser light source 3 is arranged in a direction perpendicular to the flow of the sample liquid. In order to guide the irradiated light to the flow section 2, an imaging lens 4, for example, combined with a cylindrical lens, is installed on the optical axis 01 to adjust the laser beam L to an elliptical spot having arbitrary major and minor axes. It is arranged. Further, on the side of the forward scattered light from the sample particles S caused by the laser beam L, there is a stopper 5 that blocks the direct light of the laser beam L when there is no sample particle S, and an objective lens 6.
and a photoelectric detector 7 are arranged.

Further, on the optical axis 02 perpendicular to the flow axis of the sample particles S and the optical axis 01, from the flow cell l side, a photometric objective lens 8, a condensing lens 9, an aperture 10, a condensing lens 11, and a gicroic mirror are arranged. 12, 13 and mirror 14 are arranged in sequence. A barrier filter 15 and a photoelectric detector 16 are installed in the direction of reflection of the guichroic mirror 12, a barrier filter 17 and a photodetector 18 are installed in the direction of reflection of the guichroic mirror 13, a barrier filter 19 is installed in the direction of reflection of the mirror 14, and a photoelectric detection A container 20 is arranged. These photoelectric detectors 16, 18, and 20 use photomultis that enhance weak light and make it detectable.

The laser light source 3 to the photoelectric detector 7 on the optical axis 01 are fixed on the substrate 30 after axis adjustment. Further, the flow cell I and the photometric objective lens 8 to mirror 14 on the optical axis 02, barrier filters 15, 17, 19, photoelectric detectors 16, 18, .
20 is placed on a stage 31 that is movable in the Y direction parallel to the optical axis 02 after focus adjustment, and the substrate 30 and the stage 3
A stage 32 that is movable in the X direction parallel to the optical axis 01 is further interposed between the optical axis 01 and the optical axis 01. And stage 31
The amount of movement of the stage 32 in the Y direction is measured by a dial gauge 33, and the amount of movement of the stage 32 in the X direction is measured by a dial gauge 34. Further, the stage 31 is configured to be moved parallel to the substrate 30 in the Y direction by a knob 35, and the stage 32 is configured to be moved parallel to the substrate 30 in the X direction by a knob 36.

The laser beam L emitted from the laser light source 3 enters the flow section 2 of the flow cell 1 via the imaging lens 4,
Forward scattered light by the sample particles S is focused on a photoelectric detector 7 via an objective lens 6, and its light intensity is measured. Also,
Side-scattered light by the sample particles S enters the gicroic mirrors 12, 13 and 14 via the photometric objective lens 8, condensing lens 9, aperture 10, and condensing lens 11, and these gicchroic mirrors Each reflected light for each wavelength range by 12.13.14 is filtered by barrier filter 15.17.
19, the light is focused on photoelectric detectors 16, 18, and 20, respectively, and the light intensity is measured.

FIGS. 2 to 4 are cross-sectional views taken along the axis of the sample liquid flow in the flow cell 1, and show a state in which the sample liquid is surrounded by the sheath liquid and flows. Figure 2 is a diagram showing the relationship between the sample particles S and the laser beam spot B during photometry, and even if the passing position of the sample particles S flowing in the sample liquid flow varies, the sample particles S are irradiated with a sufficient laser beam. The laser beam spot B is irradiated so that the major axis direction of the laser beam spot B is perpendicular to the direction in which the sample liquid flows, that is, the direction of the arrow A.

Figures 3 and 4 are diagrams showing the relationship between the sample particles S and the laser beam spot B when measuring the flow diameter of the sample liquid flow. That is, the laser beam spot B is irradiated parallel to the direction of the arrow A.

Here, to explain the procedure for measuring the flow diameter of the sample liquid flow, first, the imaging lens 4 is
'Rotate it so that the major axis direction of the laser beam spot B becomes parallel to the direction of arrow A as shown in FIGS. 3 and 4. Next, by moving the stage 31 parallel to the substrate 30 in the Y direction using the knob 35,
The flow cell 1 is moved parallel to the optical axis O1 in the Y-axis direction, and the laser beam spot B is set at the position shown by the two-dot chain line in FIG. 3, that is, the position where the sample liquid flow is not irradiated with the laser beam spot B at all. .

Then, by using the knob 35 to move the stage 31 in parallel in the Y direction, the flow cell 1 is gradually moved in parallel in the Y-axis direction in FIG. The value indicated by the dial gauge 33 when B crosses the end face of the sample liquid flow is measured. After that, the flow cell 1 is gradually moved further in the Y-axis direction, and this time the dial gauge is at the position where the forward scattered light signal starts to be no longer detected, that is, when it crosses the other end face of the sample liquid flow as shown in FIG. Measure the value indicated by 33. Then, the difference between the latter measured value and the former measured value corresponds to the flow diameter of the sample liquid flow.

In the embodiment described above, by rotating the imaging lens 4 by 90'' about the optical axis 01, the measurement is switched from measuring the forward scattered light to measuring the diameter of the sample liquid flow. To improve the accuracy of diameter measurement,
A similar effect can be obtained by preparing an imaging lens that further shortens the minor axis of the laser beam spot B and replacing it with the imaging lens 4. Further, the laser beam spot H may be rotated by an image rotator disposed on the exit side of the imaging lens 4 instead of rotating the imaging lens 4.

Furthermore, in this example, a dial gauge is used to measure the flow diameter of the sample liquid flow, and the flow diameter of the sample liquid flow is calculated from the measured value, but it is also possible to use a magnetic linear scale or the like. It is also possible to display it digitally. Further, in this case, the flow diameter may be determined by scanning the laser beam (substrate) B without moving the stage 31 or the like.

[Advantageous Effects of the Invention 1] As explained above, the particle analyzer according to the present invention can easily measure the flow diameter of the sample liquid flow, and can make the flow diameter of the sample liquid flow almost equal to the size of the sample particles. Therefore, there is no variation in the passing position of the sample particles in the photometry section, and there is an effect that highly accurate and efficient particle analysis can be performed.

[Brief explanation of the drawing]

The drawings show an embodiment of the particle analysis device according to the present invention, and Fig. 1 shows the configuration of the optical system and alignment device, and Fig. 2 shows the positional relationship between the sample liquid flow and the laser beam spot during photometry. , 3 and 4 are diagrams showing the positional relationship between the sample liquid flow and the laser beam spot when measuring the flow diameter of the sample liquid flow. 1 is a flow cell, 2 is a flow section, 3 is a laser light source,
4 is an imaging lens, 5 is a stopper, 6 is an objective lens, 7 is a photodetector, 12.13 is a dichroic mirror, 14
is a mirror, 15.17.19 is a barrier filter, 16.
18 is a photoelectric detector, 30 is a substrate, 31.32 is a stage, and 33.34 is a dial gauge. Patent applicant Canon Co., Ltd. Figure 1 'Jy 182547 (5) Figure 2 Figure 4

Claims (1)

[Scope of Claims] 1. An irradiation optical system that irradiates a light beam to sample particles flowing through a flow section in a flow cell, and a photometric optical system that measures scattered light and fluorescence by the sample particles, and the flow cell In the particle analysis device, the light beam is placed on a base together with the photometric optical system, and the base is movable relative to the irradiation optical axis of the light beam. Particles characterized by being provided with means for switching between two shapes: one that crosses the sample flow in a direction perpendicular to the sample flow, and one that narrows the width in the direction perpendicular to the flow of the sample flow and irradiates a part of the sample flow. Analysis device. 2. The particle analysis apparatus according to claim 1, wherein the light beam switching means makes the light beam elliptical and rotates with respect to the flow direction of the sample flow. 3. The particle analysis device according to claim 1, wherein the light beam switching means rotates an imaging lens made of a cylindrical lens about an optical axis. 4. The particle analysis apparatus according to claim 1, wherein the light beam switching means includes an image rotator. The particle analysis device according to claim 1. 5. The particle analysis device according to claim 1, further comprising means for detecting the amount of movement of the base.