JPH0641908B2 - Particle analyzer - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention is used in a flow cytometer or the like, and irradiates a light beam such as a laser beam to a particle to be inspected in a sample flow to determine the property of the particle to be inspected. The present invention relates to a particle analysis device for analysis.

[Prior Art] A flow cytometer is a high-speed cell suspension solution,
That is, it is a device that illuminates the sample liquid with, for example, laser light, detects the optical signal due to the scattered light and fluorescence, and elucidates the properties and structure of cells, such as cytochemistry, immunology, hematology, oncology, genetics, etc. Used in the field of.

In a conventional particle analyzer used for this flow cytometer or the like, for example, 200 μm × 2 at the center of the flow cell is used.
Irradiation light such as laser light is radiated to the test particles such as blood cells that are wrapped in the sheath liquid and pass through the flow passage having a minute rectangular cross section of 00 μm, and the resulting forward and side scattered light causes It is possible to obtain particle properties such as the shape, size, and refractive index of the test particles. Further, for the test particles that can be stained with a fluorescent agent, by detecting the fluorescence of the test particles from the side scattered light in the direction substantially perpendicular to the irradiation light, important information for analyzing the test particles. Can be asked.

However, in the conventional apparatus of this type, the alignment and focus states are determined by looking at the actual output signal of the test particles. Therefore, there is a drawback that it is difficult to determine what the alignment and focus actually look like.

[Object of the Invention] An object of the present invention is to eliminate the above-mentioned drawbacks of the conventional example and to provide a particle analysis apparatus capable of easily performing the best alignment and focus adjustment.

[Summary of the Invention] The gist of the present invention for achieving the above-mentioned object is to irradiate a test particle in a fluid with light, and to analyze the test particle from scattered light and fluorescence of the test particle, Based on the output of the photodetector, a reticle for alignment / focus confirmation that is removably provided in the optical path at a position conjugate with the test particle with respect to the objective lens, a photodetector that detects the light flux that has passed through the reticle. And a means for adjusting alignment and focus.

Embodiments of the Invention The present invention will be described in detail based on the illustrated embodiments.

In FIG. 1, reference numeral 1 is a flow cell, and a flow section 1a through which a sample flow flows is provided in the center of the flow cell. A laser light source 2 is provided on one side of the flow cell 1, an imaging optical system 3 is provided between the flow cell 1 and the flow cell 1 along the optical axis of the emitted laser light, and an objective lens 4 and light detection are provided on the opposite side of the flow cell 1. The container 5 is arranged. In addition, the objective lens 6, the reticle 7, the condenser lens 8, the dichroic mirrors 9, 10, the condenser lens 11, the barrier filter 12, and the light in the directions orthogonal to the optical axis of the laser light and the flow direction of the circulation portion 1a. The detectors 13 are arranged in sequence. Condensing lenses 14 and 15, barrier filters 16 and 17, and photodetectors 18 and 19 are arranged on the reflection sides of the dichroic mirrors 9 and 10, respectively. The reticle 7 is provided on the rotary plate 21 together with the aperture 20,
The motor 22 allows the reticle 7 and the aperture 20 to selectively move on the optical path. The reticle 7 is installed at a position conjugate with the circulation portion 1a with respect to the objective lens 6, and is provided so as to overlap two "V" -shaped slits 7a and 7b that pass light as shown in FIG. , Slits 7a and 7b appear at equal intervals along the center line B.

The light beam from the laser light source 2 is shaped by the imaging optical system 3 into a predetermined beam shape, for example, an elliptical shape, and imaged on the flow portion 1a of the flow cell 1. The forward scattered light scattered forward by the test particles mixed in the sample flow passing through the circulation unit 1 a is guided to the photodetector 5 by the objective lens 4. Further, the side scattered light or fluorescence in the 90 ° direction passes through the objective lens 6, the reticle 7 and the condenser lens 8, and is wavelength-divided by the dichroic mirrors 9, 10.
Photo-detectors 13, 18, 19 are passed through barrier filters 12, 16, 17 by condenser lenses 11, 14, 15 respectively.
Detected by.

As shown in FIG. 2, the particles to be inspected are A, B, and
When flowing through any of the parts of C, the photodetectors 13, 18,
The signal waveforms of the scattered light obtained in any one of 19 are as shown in FIGS. 3 (a), (b), and (c), respectively. Second
B in the figure is the case where the position where the particles flow is at the optical center, and as shown in (b), the intervals L1, L2, L3 of the signal waveforms are
Are equal pulse trains. When flowing through A, L1
= L3 <L2 pulse train. When flowing through C, the center 2
One pulse is overlapped to form three pulses, and the alignment can be determined from the intervals of these pulses, and the alignment can be performed while observing the intervals of these pulses so that they are equidistant as shown in (b). . Also, the focus state can be determined by determining the contrast of each pulse from the sharpness of the pulse.

Next, FIG. 4 is an explanatory diagram regarding the matching degree between the position of the irradiation light beam and the center of the aperture 20 in the flow direction of the sample flow. When the height of the laser light imaged by the imaging optical system 3 in FIG. 1 is not at the center of the aperture 20 with respect to the objective lens 6, as shown in FIG. Is a when the light beam is imaged above, b at the same height, and c when it is imaged below.

Therefore, pulse trains as shown in FIGS. 5A, 5B, and 5C are obtained according to the respective image formation states. At this time, since B in FIG. 2 is considered as the test particles are flowing, the pulse intervals are equal. As shown in FIG. 5 (b), it can be determined that the alignment is in focus when the pulse intervals are equal and the intensity distribution is symmetrical and the pulse contrast is maximum.

At the time of alignment / focus, the reticle 7 is installed on the optical axis, and when the alignment / focus adjustment is completed, the aperture 20 is inserted instead of the reticle 7 to start the measurement. The aperture 20 and the reticle 7 can be replaced manually, but in the embodiment, the rotating plate 2 is rotated by the motor 22.
1 can be rotated to be inserted selectively.

The alignment and focus can be adjusted by moving the objective lens 6. That is, for the alignment, by moving the objective lens 6 up, down, left and right, B and B of FIG.
It is possible to move the objective lens 6 in the flow direction in order to obtain the case shown in FIGS. 5 (a) and 5 (c) as shown in FIG. 5 (b). Focusing is performed by moving the objective lens 6 in the optical axis direction. The focus and the alignment shown in FIG. 3 can also be performed by moving the flow cell 1. The alignment shown in FIG. 4 is performed by tilting the flow cell 1 with respect to the optical axis of the objective lens 6. Further, when the side-scattering / fluorescence photometric optical system is arranged on the stage S, the alignment / focus can be adjusted by moving the stage S. Thus, by processing the signal waveform obtained by the photodetector, the alignment can be performed automatically or manually.
The focus can be adjusted.

EFFECTS OF THE INVENTION As described above, the particle analysis apparatus according to the present invention can determine the alignment / focus of the apparatus from the obtained signal by providing the reticle at the conjugate position of the particle to be detected in the light receiving or photometric system. Therefore, there is an advantage that the alignment and focus can be adjusted easily, and both can be performed at the same time.

[Brief description of drawings]

The drawings show an embodiment of a particle analysis apparatus according to the present invention.
FIG. 2 is its configuration diagram, FIG. 2 is a front view of the reticle, FIG.
FIG. 5 is an output waveform diagram in each alignment state, and FIG.
The figure is an illustration of the state of the laser beam on the reticle. Reference numeral 1 is a flow cell, 2 is a laser light source, 3 is an imaging optical system, 4 and 6 are objective lenses, 5, 13, 18 and 19 are photodetectors, 7 is a reticle, 9 and 10 are dichroic mirrors, and 12 and 16. , 17 is a barrier filter, 20 is an aperture, 21 is a rotating plate, and 22 is a motor.

Claims (6)

[Claims] 1. An apparatus for irradiating light to a test particle in a fluid and analyzing the test particle from scattered light and fluorescence of the test particle, wherein the device is inserted in an optical path at a position conjugate with the test particle with respect to an objective lens. A detachably provided reticle for confirmation of alignment and focus, a photodetector for detecting a light flux passing through the reticle, and means for adjusting alignment and focus based on the output of the photodetector. Particle analysis device characterized by. 2. The reticle according to claim 1, wherein the reticle has a pattern including a plurality of slits, and it can be understood that an interval between signal pulses obtained by the photodetector changes depending on an alignment state. Particle analysis device according to item. 3. The particle analysis apparatus according to claim 1, wherein the photodetector also serves as a signal detector for the particles to be detected. 4. The particle analyzer according to claim 1, wherein the focus / alignment adjusting means moves the objective lens. 5. The particle analyzer according to claim 1, wherein the focus / alignment adjusting means moves a flow cell through which the particles to be detected flow. 6. The particle analyzing apparatus according to claim 1, wherein the focus / alignment adjusting means moves a stage on which a side scattering optical system is mounted.