JPH0566980B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a particle analysis device having a focusing function for a flow cell in a flow cytometer or the like.

[Prior art] In a particle analysis device used in a flow cytometer, etc., a particle size of, for example, 200 μm×
Inside the flow section with a minute rectangular cross section of 200μm,
A laser beam is irradiated onto specimen particles such as blood cells passing through the sheath fluid, and the resulting forward and side scattered light makes it possible to obtain particle properties such as the shape, size, and refractive index of the specimen. It is possible. Furthermore, for specimens that can be stained with a fluorescent agent, important information for analyzing the specimen can be obtained by detecting fluorescence.

In order to perform accurate measurements with a flow cytometer, etc., a photometric objective lens must be used to accurately focus only the light flux from the sample particle or its immediate vicinity, so that signals from objects other than the sample particle are not mixed in. At the same time, the flow axis of the sample particles must be accurately aligned with the optical axis. for that,
It is necessary to adjust the focus using the objective lens, but
There are several inconvenient factors as described below.

(1) Since the operator manually adjusts the optical axis by visual inspection, the operation is complicated and it is difficult to perform sufficiently accurate optical axis adjustment.

(2) Even if the focal point moves during measurement, it is impossible to confirm this, so it is impossible to determine whether or not spurious signals are mixed in during measurement, and there is concern about the reliability of the data.

(3) Focus adjustment is required each time the nozzle or flow cell is replaced, making measurement time-consuming.

[Object of the Invention] An object of the present invention is to provide a particle analysis device that can automatically and accurately adjust the focus to the optical axis center of the flow cell by detecting the positional relationship of reflected light from the front and rear surfaces of the flow cell. It is about providing.

[Summary of the invention] The gist of the present invention for achieving the above object is as follows:
An irradiation optical system that irradiates a light beam onto the sample particles flowing through the flow section in the flow cell, and a photometry optical system that collects and measures light generated by the irradiation of the light beam onto the sample particles using a photometry objective lens. a projection optical system that projects an index light beam in an oblique direction from the photometric direction of the photometric optical system toward the flow section through the photometry objective lens; an optical array sensor that detects the incident position of the two reflected lights from which the index light beam is reflected and passes through the photometric objective lens; This is a particle analysis device characterized by detecting a focused state.

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

1 and 2 are configuration diagrams of the photometric optical system and the illumination system.
The figure shows the view from the cross-sectional direction of the sample liquid flow. A subject sample is injected into the flow cell 1 from the upper direction A, and at the same time, a sheath liquid flows from the B direction. Flow section 1a of flow cell 1
In this case, the sheath liquid encloses the sample liquid flow therein, flows in a laminar flow state, is hydrodynamically focused, and is discharged as a drain in the C direction. Laser light emitted from the Ar ⁺ laser light source 2 is imaged near the flow section 1a of the flow cell 1 through the imaging lens system 3. An objective lens 4 and a photodetector 5 are arranged in the straight direction of the laser beam to collect forward scattered light from the sample particles.
Also, on the optical axis perpendicular to the direction of incidence of the laser beam, a convex lens 6, a concave lens 7, a dichroic mirror 8 for emitting infrared light and transmitting visible light, an imaging lens 9, an aperture 10, and a convex lens 11 are arranged in order. , dichroic mirrors 12 and 13, and a reflection mirror 14 are arranged.

The dichroic mirror 12 has the property of transmitting light with a wavelength longer than the wavelength of 488nm of the Ar ⁺ laser beam, and an imaging lens 15, 488nm, is placed on the reflection side of the dichroic mirror 12.
A barrier filter 16 and an optical guide 17 are arranged to allow wavelengths of m to pass through. Furthermore, the dichroic mirror 13 has the characteristics of reflecting green light and transmitting red light, and an imaging lens 18 on the reflecting side.
Bandpass filter 19, optical guide 20
is provided. On the reflective side of the reflective mirror 14, an imaging lens 21, a barrier filter 22 that transmits red light, and an optical guide 23 are arranged.

In addition, on the reflection side of the dichroic mirror 8,
A focus detection section consisting of a projection optical system and a measurement optical system is provided. First, a convex lens 24, a half mirror 25, aperture masks 26, 27, and an infrared light source 28 are sequentially arranged on the optical axis as the projection optical system. It is located. Further, on the reflection side of the half mirror 25, a barrier filter 29 and an optical array sensor 30 are provided as a photometric optical system.

The forward scattered light of the laser light by the sample particles is detected by the objective lens 4 and the photodetector 5.
Since there is a correlation between the magnitude of the output signal and the size of the sample particle, it is used to determine the size of the sample particle. Further, when specimen particles dyed to emit fluorescence are irradiated with A ⁺ laser light having a wavelength of 488 nm, the reflected light exhibits green or red fluorescence. PI used to detect the amount of DNA (deoxyribonucleic acid) emits red fluorescence,
FITC, which is used to detect cell membrane surface antigens, is known to emit green fluorescence. In addition, the complex structure inside the sample particle is reflected in the side scattered light caused by irradiation with the A ⁺ laser beam.

In order to obtain information from such side-scattered light, the side-scattered light from the flow cell 1 passes through an objective lens system consisting of a convex lens 6 and a concave lens 7 and an imaging lens 9 to the focal position of the aperture 10. is imaged once. The side scattered light further passes through the convex lens 11 and reaches the dichroic mirror 12.
A ⁺ Light with a wavelength shorter than the wavelength of 488nm of laser light is
It is reflected by this dichroic mirror 12, passes through an imaging lens 15, and then passes through a barrier filter 16.
Only the 488 nm light is imaged on the end face of the optical guide 17, transmitted to a photodetector (not shown), and measured.

Of the light beams that have passed through the dichroic mirror 12, the green light is reflected by the dichroic mirror 13 and enters the optical guide 20 via the imaging lens 18 and the barrier filter 19 that allows only the green light to pass. Furthermore, the light beam that has passed through the dichroic mirror 13 is reflected by the reflection mirror 14, and enters the optical guide 23 via the imaging lens 21 and the barrier filter 22 that passes only red light.

Next, in the focus detection section, the infrared light source 28 emits light in the infrared region, and the irradiated light that has passed through the aperture mask 27 shown in FIG. The light reaches the dichroic mirror 8 via the mask 26, the half mirror 25, and the convex lens 24, is reflected to the left, and is projected onto the flow cell 1 via the concave lens 7 and the convex lens 6. Here, the width of the pattern of the mask 27 is somewhat smaller than the width of the flow section 1a of the flow cell 1, and the projection light is reflected on the front and rear surfaces of the flow cell 1. However, if reflection efficiency is ignored, the width of the pattern may extend beyond the width of the flow section 1a.

The pattern of the aperture mask 26 is such that the slit portion is eccentric from the optical axis, and is used to allow the index by the mask 27 to enter the flow cell 1 from an oblique direction. Therefore, if the flow cell 1 is in a normal position, that is, its center is optically conjugate with the photometric optical system, the light shown in FIG. A state as shown in d is obtained. FIG. 3a shows the optical conjugate positional relationship of the entire flow cell 1, b shows the positions of reflected light from the front and rear surfaces of the flow cell 1,
c represents the photoelectrically converted output waveforms of reflected light from the front and rear surfaces of the flow cell 1, respectively.
In this way, the position of the focal point is a~c,e~ as shown in a.
When shifted back and forth as shown in g, the image is formed at the position shown in b, and a signal waveform shown in c is output from the optical array sensor 30.

The signal waveform obtained in the focus detection section in this way is analyzed by the signal processing section, and if the focus position is shifted, focus adjustment is automatically performed. In addition,
The focus adjustment performed here is to adjust the focal shift with respect to the emission direction of the side scattered light in Figs. 1 and 2 and move it to the focal position of the optical system, but it also applies to the forward scattered light. Of course, a similar focus detection section may be arranged.

FIG. 4 is a block circuit configuration diagram for automatic focus adjustment, in which the output signal from the optical array sensor 30 is analyzed by a peak position detection section 31. This peak position detection section 31 obtains a peak position P1 of the signal corresponding to the light reflected from the front surface of the flow cell 1, and a peak position P2 corresponding to the reflected light from the rear surface of the flow cell 1.
The adder 32 calculates P1+P2, and the divider 33 calculates P=(P1+P2)/2. This P indicates the current focusing state of the projection optical system, that is, the focal position of the objective lens system consisting of a convex lens 6 and a concave lens 7. Therefore, the reference position setting section 34
The comparator 35 compares the output S of the optical focus position data set in advance with the output value P from the divider 33, and the cell drive unit 3
6 to control the position of flow cell 1. In the comparator 35, when P>S and when P<S
In this case, automatic focusing is performed by changing the drive direction of the cell drive unit 36 and controlling the center of the flow cell 1 to the optical focus position.

In the focus detection section of this embodiment, a CCD is used as the optical array sensor 30, and the signal waveform is extracted by electrically scanning the reflected light from the flow cell 1 by the projection light. A similar signal waveform can be obtained even if a mechanical drive mechanism is attached and scanning is performed. In addition, by introducing a microprocessor into the signal processing section and performing each calculation using software, the circuit configuration becomes simpler.
And similar performance can be obtained. It goes without saying that focus adjustment may be performed by adjusting the objective lens system instead of by moving the flow cell 1.

[Effects of the Invention] As explained above, the particle analysis device according to the present invention projects a mask pattern from the focus detection section onto the flow cell, performs focus detection based on the reflected light from the front and rear surfaces of the flow cell, and detects the flow cell. Since the center of the optical system is moved relative to the center position of the optical system, the complexity of conventional manual focus adjustment is avoided, and highly reliable data can be obtained because the focus position can be adjusted with high precision. become.

[Brief explanation of the drawing]

The drawings show an embodiment of the particle analysis device according to the present invention, and FIGS. 1 and 2 are configuration diagrams of the optical system, FIG. 3 is an explanatory diagram of the relationship between the center of the flow cell and the focal point position, and FIG.
The figure is a block circuit configuration diagram of a signal processing section and a driving section. 1 is a flow cell, 1a is a flow section, 2 is a laser light source, 3 is an imaging lens system, 4 is an objective lens,
5 is a photodetector, 6 is a convex lens, 7 is a concave lens,
8, 12, 13 are dichroic mirrors, 9 is an imaging lens, 16, 19, 22 is a barrier filter,
17, 20, 23 are optical guides, 25 is a half mirror, 26, 27 are aperture masks, 2
8 is a red light source, 30 is an optical array sensor, 31 is a peak position detector, 32 is an adder, 33 is a divider,
34 is a reference position setter, 35 is a comparator, and 36 is a cell driver.

Claims (1)

[Claims] 1. An irradiation optical system that irradiates a light beam onto the sample particles flowing through the flow section in the flow cell, and a photometric optical system that collects and measures the light generated by the irradiation of the light beam onto the sample particles with a measurement objective lens. a projection optical system that projects an index light beam in an oblique direction from the photometric direction of the photometric optical system toward the flow section through the photometry objective lens; an optical array sensor that detects the incident position of the two reflected lights from which the index light beam is reflected and passes through the photometric objective lens; A particle analysis device characterized by detecting a focused state.