JPS6236542A - Particle analyzer - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides a method for easily determining the alignment state between the optical axis of a light irradiation optical system and the axis of flow of sample particles in a flow cytometer or the like. This is related to a particle analysis device that has made this possible.

[Prior Art] In a conventional particle analysis device used in a flow cytometer, etc., a particle size of 200 pm
Irradiation light is applied to a specimen such as a blood cell that passes through the flow section of the ILm, which has a minute cross section, and is wrapped in a sheath fluid.
The resulting forward and side scattered light makes it possible to obtain particulate properties such as shape, size, and refractive index of the specimen.

Further, for a specimen that can be stained with a fluorescent agent, important information for analyzing the specimen can be obtained by detecting the fluorescence of the specimen from side scattered light in a direction substantially perpendicular to the irradiation light.

Laser light is generally used as the irradiation light in such devices, and the intensity distribution of the laser light from a normal laser light source exhibits a Gaussian distribution. Therefore, if the central axis of the flow of sample particles and the optical axis of the laser beam match, the sample particles are irradiated with the most intense laser beam, allowing efficient measurement. However, when the optical axis of the laser beam and the axis of the flow of the sample particles are misaligned, the intensity of the laser beam irradiated to the sample particles becomes weaker according to a Gaussian distribution, and the resulting scattered light also becomes weaker, making it difficult to measure the same particles. However, the same photodetection signal may not be obtained.

Therefore, in order to perform accurate measurements using a flow cytometer or the like, it is necessary to precisely align the flow axis of sample particles with the optical axis. For this purpose, in conventional equipment, before measurement, air bubbles are flowed through the flow section as a standard sample and irradiated with laser light, and the state of the scattered light scattered by the air bubbles is visually observed, and the irradiation optical axis and air bubble flow are A method is adopted in which the state of coincidence with the center of is confirmed. However, this method has the disadvantage that quantitative judgment is impossible, accurate positioning is difficult, and individual differences occur among operators.

Therefore, in order to improve the above-mentioned drawbacks, the applicant has decided to
We have proposed a method that detects both the left and right edges of the cell flow area as the irradiation light intensity distribution, moves the flow cell to the center, and aligns the irradiation optical axis with the center of the flow cell. According to this method, the irradiation optical axis can be accurately and easily aligned with the center of the flow cell, and if the sample liquid containing suspended analyte particles always flows through the center of the flow cell, the flow cell If the center and the irradiation optical axis are aligned, the irradiation optical axis and the center of the sample liquid flow are aligned, so accurate measurement is possible. However, the flow position of the sample liquid is easily affected by changes in flow rate, sample pressure, etc., and it is quite difficult to always flow the sample liquid to the center of the flow section. Therefore, it is preferable to adjust the irradiation optical axis and the center of the sample flow as close to the actual measurement as possible, and we have developed a method that can be performed accurately and easily by adjusting the direction of the axis while flowing the standard sample, which was the case in the past. is desired.

In addition, the conventional device is equipped with an observation optical system to observe the flow state of the standard sample liquid and the sample liquid, which has the disadvantage of making the configuration complicated and expensive. Improvements are also needed.

[Objective of the Invention J The object of the present invention is to detect and evaluate the intensity distribution of the irradiated light when a conditioning liquid that absorbs light in the wavelength range of the irradiated light is flown, thereby determining the central axis of the flow of the sample liquid. An object of the present invention is to provide a particle analysis device that can accurately and easily adjust the optical axis of irradiation light.

[Summary of the Invention] The gist of the present invention for achieving 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 light beam scattered by the sample particles. a photometric optical system for measuring light; a detection means for detecting a change in the intensity distribution of the light beam due to a conditioning liquid that passes through the flow section and includes a substance that absorbs light in the wavelength range of the light beam; The present invention is a particle analysis device characterized by comprising an evaluation means for evaluating an output signal waveform.

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

Figure 1 shows the overall configuration of the flow cell 1, in which sample particles are hydrodynamically focused while being surrounded by a sheath liquid in a high-speed laminar flow inside the flow section 2 perpendicular to the plane of the paper in the center of the flow cell 1. passes through, and a laser light source 3 is arranged in a direction perpendicular to this flow. An imaging lens 4 is arranged on the optical axis 01 in order to guide the laser light emitted from the laser light source 3 to the flow section 2. In addition, a beam splitter 5, a condensing lens 6, and a photoelectric detector 7 are sequentially arranged on the forward scattered light side of the laser light scattered by the sample particles. The irradiated laser light is scattered by the sample particles and sent to the beam splitter 5.
The light passes through a condensing lens 6 and reaches a photoelectric detector 7, where information mainly on the size of the sample particles can be obtained. Note that 1. Normally, a light shielding plate (not shown) is placed near the condenser lens 6, which removes the straight components of the laser light that are not scattered by the sample particles, and allows the S of the information obtained by the photoelectric detector 7 to be removed.
/N ratio is improved.

Furthermore, the optical axis 02 of the luminous flux split by the beam splitter 5
A condensing lens 8 and an array photoelectric detector 9 are arranged above, and the array photoelectric detector 9 has a scale C on which a scale C is displayed.
Monitor 10 such as RT is fJF Hf.

has been done. The beam split and reflected by the beam splitter 5 is imaged by a condenser lens 8 onto an array photodetector 9, which is a one-dimensional photoelectric conversion element.

In addition, the central axis of the flow of sample particles and the optical axis 01 of the laser beam
On the optical axis 03 in directions substantially orthogonal to each other, an objective condenser lens 11 for side scattered light and an aperture stop (not shown) are arranged.
Optical systems such as a wavelength selection means and a photoelectric detector are arranged to measure the scattered light and fluorescence in the 90° direction of the sample particles.
The granularity of the sample particles can be observed using the 0° scattered light, and the biochemical properties of the sample particles can be observed using the fluorescence.

By the way, as described above, the laser light normally has a Gaussian intensity distribution, and the imaging beam in the flow section 2 also has a Gaussian intensity distribution. Here, a substance that absorbs light in the wavelength range of laser light is used as the adjustment liquid S, and the flow of this adjustment liquid surrounded by the sheath liquid inside the flow section 2 is maintained in a laminar flow state perpendicular to the plane of the paper. When it passes, a part of the laser light from the laser light source 3 is absorbed by the iA liquid regulating liquid S. The state of the light intensity distribution of the laser beam at that time is imaged on the optical array sensor 9 via the beam splitter 5 and the condensing lens 8, and the output signal waveform I is displayed as the light intensity distribution waveform P on the monitor 10. will be displayed as.

Figure 2 shows the light intensity distribution waveform P when the optical axis 01 of the laser beam and the center of the adjustment liquid flow S coincide, and the center part of the Gaussian distribution waveform is absorbed by the adjustment liquid S and becomes concave. The waveform is bilaterally symmetrical, and by observing this on the monitor 10, it can be confirmed that the optical axis O1 of the laser beam and the center of the flow of the adjustment liquid S coincide.

If the optical axis 01 of the laser beam and the center of the adjustment liquid flow do not match, the concave portion shifts to the right or left, and the light intensity distribution waveform P becomes asymmetrical. Therefore, while monitoring the light intensity distribution waveform P on the monitor 10, the flow cell 1 or the irradiation optical system can be moved until the waveform P becomes bilaterally symmetrical. In this way, the optical axis O1 of the laser beam can be adjusted. It is possible to accurately align the center of the liquid flow.

Note that the light intensity distribution waveform P may be confirmed and evaluated by calculating the output signal of the optical sensor array 9 instead of using the monitor 10.

Further, the flow diameter of the adjustment liquid S is expressed by the distance W between the two peaks of the light intensity distribution waveform P shown in FIG.

It can be read on the scale C on the monitor 10. Furthermore, when the laminar flow of the adjustment liquid flow is disturbed, the light intensity distribution waveform P is disturbed, so by monitoring the light intensity distribution waveform P on the monitor 10, the M-wave state of the adjustment liquid flow can be observed. is also possible.

In this way, while observing the monitor 10, accurately align the center of the adjustment liquid flow with the optical axis of the laser beam, measure the flow diameter of the adjustment liquid flow, and after adjusting the laminar flow of the adjustment liquid flow, perform the adjustment under the same conditions. By actually flowing the sample liquid through the flow section 2, it is possible to irradiate the center of the flow with laser light and obtain accurate measured values. Furthermore, when the sample liquid is flowed under the same conditions as the adjustment liquid flow, the flow diameter W of the adjustment liquid flow may be regarded as the flow diameter of the sample liquid flow, and the flow diameter of the sample liquid can be easily estimated. be able to.

[Effects of the Invention] As explained above, the particle analysis device according to the present invention flows a conditioning liquid that absorbs the wavelength range of the irradiated laser light into the flow cell, and the laser beam that is partially absorbed by the conditioning liquid flows. By evaluating the light intensity distribution of light via a photoelectric detector, it is possible to easily align the irradiation optical axis with the center of the adjustment liquid flow, and the sample liquid can be measured under the same conditions as the adjustment liquid flow. By flowing the laser beam, the center of the sample liquid can be accurately irradiated with the laser beam, and accurate measurement values without noise signals can be obtained. Similarly, by observing the shape of the light intensity distribution on the monitor of the laser beam irradiated to the adjustment liquid flow, it is possible to know the flow diameter and laminar flow state of the adjustment liquid flow, and the sample liquid is flowed under the same conditions. In this case, since the adjustment liquid flow and the sample liquid flow can be considered to be in the same state, it becomes possible to estimate the flow diameter of the sample liquid and the flow state of the sample liquid without providing an observation optical system.

[Brief explanation of the drawing]

The drawings show an embodiment of the particle analysis apparatus according to the present invention, and FIG. 1 is an overall configuration diagram, and FIG. 2 is a monitor display waveform diagram of laser light intensity distribution. 1 is a flow cell, 2 is a flow section, 3 is a laser light source, 5
is a beam splitter, 7 is a photoelectric detector, 9 is a photoelectric detector, and 10 is a monitor.

Claims (1)

[Scope of Claims] 1. An irradiation optical system that irradiates a light beam onto sample particles flowing through a flow section in a flow cell, a photometric optical system that measures scattered light from which the light beam is scattered by the sample particles, and a photometric optical system that measures the scattered light that is scattered by the sample particles; a detecting means for detecting a change in the intensity distribution of the light beam caused by a conditioning liquid containing a substance that absorbs light in the wavelength range of the light beam; and an evaluation means for evaluating an output signal waveform of the detecting means. A particle analysis device characterized by: 2. The light beam is a laser beam, and the detection means detects a deformation of the Gaussian intensity distribution of the laser beam due to light absorption of the adjustment liquid, and displays it on the display means, so that the irradiation optical system and the sample particle The particle analysis device according to claim 1, wherein the optical axis is adjusted with respect to the central axis of the flow. 3. The particle analysis apparatus according to item 1, wherein the evaluation means displays the output signal waveform on a monitor. 4. The particle analysis device according to claim 3, wherein the monitor is provided with a scale so that the flow diameter of the adjustment liquid can be detected.