JPH0552896B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a particle analysis device that is capable of determining the in-focus state of a photometric objective lens in a flow cytometer or the like.

[Prior art] In conventional particle analysis devices used in flow cytometers, etc., a particle size of 70 μm, for example,
Irradiation light is irradiated onto a specimen such as blood cells wrapped in sheath liquid and passing through a flow section with a minute rectangular cross section of m x 20 μm, and the resulting forward and side scattered light is used to determine the shape and shape of the specimen. It is possible to obtain particle properties such as size and refractive index. Also,
For specimens 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 irradiated light.

In order to perform accurate measurements with a flow cytometer, etc., the photometric objective lens must accurately focus only on the sample particles or their immediate vicinity to prevent spurious signals from objects other than the sample particles from being mixed in. Must be. For this purpose, it is necessary to adjust the focus of the objective lens, but in conventional devices, the operator manually adjusts the focus by visual inspection before measurement, which is not only complicated, but also requires the operator to manually adjust the focus. At present, it is difficult to perform sufficiently accurate focus adjustment due to individual differences.

Furthermore, if the focal point moves during measurement, it is impossible to confirm this, so it is impossible to determine whether or not a spurious signal has been mixed in during the measurement, leading to concerns about the reliability of the data.

Furthermore, it requires focus adjustment every time the nozzle, flow cell, etc. are replaced, which has the disadvantage that measurement is time-consuming. In addition, when performing fluorescence measurements, it is necessary to strengthen weak fluorescence signals, and for this purpose it is necessary to make the photoelectric detector that detects fluorescence more formal, improve the luminous efficiency of the fluorescent agent, and increase the power of the irradiation light source. Consideration has been given to increasing the amount of light and improving the light collection efficiency of the objective lens. The luminescence efficiency of fluorescent agents is currently being actively researched, and increasing the power of the irradiation light source can be considerably increased if manufacturing costs are ignored, but on the other hand, if the power is increased too much, the sample particles It is hard to say that this is a good method as it may cause damage to the person.

Improving the light collection efficiency of the objective lens can be achieved by increasing the numerical aperture of the objective lens, but this comes with the opposite effect of decreasing the depth of focus. If the depth of focus becomes shallow, even if the distance between the sample flow section and the photometric objective lens moves slightly, not only signals from the sample particles but also signals from other objects will be mixed in, making it difficult to obtain accurate Unable to perform measurements. As described above, the conventional apparatus has the disadvantage that focus adjustment is complicated, and sufficient fluorescence signal intensity cannot be obtained, so that analysis accuracy cannot be improved.

[Object of the Invention] An object of the present invention is to provide a focusing optical system that focuses a photometric objective lens, to easily and accurately adjust the focus, and to obtain sufficient fluorescence signal intensity to improve measurement accuracy. The purpose of the present invention is to provide a particle analysis device that improves particle analysis.

[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 analyte particles flowing through a flow section in the flow cell; a photometric optical system that measures light generated by irradiating the analyte particles with the light beam through a photometric objective lens; The particle analysis apparatus is characterized in that it is provided with a focus detection means for detecting a focused state of the photometric objective lens using surface reflection of the flow cell.

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

FIG. 1 is a configuration diagram of a particle analysis device, in which sample particles S pass through a flow section 2 perpendicular to the paper plane in the center of a flow cell 1, and a laser light source 3 is arranged in a direction perpendicular to this flow. . On the optical axis O of the laser light L emitted from the laser light source 3, an imaging lens 4 is formed by making two sets of cylindrical lenses perpendicular to the laser light source 3 side with respect to the detection particles S.
is located. Further, a light shielding plate 5,
A condenser lens 6 and a photoelectric detection section 7 are arranged in sequence.

Further, an autofocus unit (hereinafter referred to as AF unit) 9 including an objective lens 8 and a condenser lens 1 are arranged in a direction substantially perpendicular to the optical axis O of the laser beam L and the central direction of the flow of the sample particles S.
0, wavelength selection means 13, 14, 1 consisting of a diaphragm plate 11, a condensing lens 12, a dichroic mirror, etc.
A pariah filter 16, a photoelectric detector 17, a barrier filter 18, and a photoelectric detector are arranged on the optical axis in the direction reflected by these wavelength selection means 13, 14, and 15, which are arranged obliquely with respect to the optical axis. A detector 19, a barrier filter 20, and a photoelectric detector 21 are respectively arranged. For these photoelectric detectors 17, 19, and 21, for example, photomals that can detect weak light are used.

Therefore, the laser beam L emitted from the laser light source 3 is formed into an imaging beam with arbitrary major and minor axes by the imaging lens 4 made up of two sets of cylindrical lenses orthogonal to each other, and the laser beam L is formed into an imaging beam having arbitrary major and minor axes. Particles S are irradiated. Among the scattered light irradiated onto the sample particles S, the forward scattered light is removed by the light shielding plate 5, and the irradiated light that has passed through the position where there is no sample particle S is removed, and the forward scattered light is sent to the photoelectric detector 7 via the condenser lens 6. The properties of the sample particles S are measured.

In addition, sample particles S stained with various fluorescent agents
The light is focused as side scattered light onto the aperture plate 11 by the condenser lens 10 via the objective lens 8 in the AF unit 9. Side scattered light and fluorescence are
By passing the diaphragm plate 11 installed at a position conjugate to the sample particles S, a photometric signal with less noise can be obtained. The light beam after passing through the diaphragm plate 11 is made into a parallel light beam by a condenser lens 12, and separated into side scattered light and fluorescence by a wavelength selection means 13 having appropriate spectral characteristics, and the side scattered light is separated into side scattered light and fluorescence. Detected by filter 16 and photoelectric detector 17,
The granularity inside the sample particles S can be observed. On the other hand, the fluorescence passes through the wavelength selection means 13 and the wavelength selection means 1
4, the green fluorescence is detected by the photoelectric detector 19 via the barrier filter 18, and the red fluorescence is detected by the photoelectric detector 2 via the wavelength selection means 15 and the variable filter 20.
1, and the biochemical properties of the detected particles are observed.

In addition, wavelength selection means 14 and 15 for selecting fluorescence
Although a dichroic mirror of green and red is used as a dichroic mirror, for example, a wavelength selection means such as a spectroscopic prism or a diffraction grating that can continuously separate wavelengths may also be used. Further, a beam diameter variable means such as a beam expander or a beam compressor may be inserted between the light source 3 and the imaging lens 4.

Here, we introduced the AF unit 9, which can increase the efficiency of focusing weak light and achieve accurate focusing.
This will be explained with reference to FIGS. 2 and 3. Second
The figure is a configuration diagram of the AF unit 9 seen from the side.
FIG. 3 is a configuration diagram seen from above. An objective lens 8 is installed in the lower part of the AF unit 9, and a light source 22 is installed in the upper part to irradiate the flow cell 1 with light, and an aperture 23 and convex lenses 24 and 25 are arranged in sequence on the optical axis of the light source 22. has been done. Furthermore, a mirror 26 is provided on one side in the horizontal direction with respect to the optical axis between the aperture 23 and the convex lens 24, and an aperture 27 is provided on the other side in the horizontal direction with respect to the optical axis between the convex lenses 24 and 25. , a position detector 28 is arranged at a position where the light beam reflected by the mirror 26 is incident.

In this case, the objective lens 8 is arranged so that the focus is on the center of the flow section 2 and the condensed light beam from the flow section 2 becomes a parallel light beam by the objective lens 8. In this state, the AF unit The optical system on the upper part of the flow cell 9 is arranged to detect the surface of the flow cell 1 . That is, the convex lens 2 is
4. Aperture 23 via aperture 27 and convex lens 25
is projected onto the surface of the flow cell 1, and a slit image of the aperture 23 reflected from the surface of the flow cell 1 is formed on a position detector 28 via convex lenses 24, 25 and a mirror 26. In the states shown in FIGS. 2 and 3, the objective lens 8 is in focus, so the bit position on the position detector 28 on which the slit of the aperture 23 is imaged is the same as the one in focus of the objective lens 8. This will show that there is.

In this way, if the distance from the center to the surface of the flow cell 1 is known in advance, the opening 23
When the convex lenses 24 and 25 focus on the surface of the flow cell 1, the objective lens 8 is always focused on the center of the flow section 2, and a parallel beam of the image of the sample particle S is always obtained from the objective lens 8. . Furthermore, since the diaphragm plate 11 is arranged at the focal position of the focusing lens 10, when focusing is achieved by moving the AF unit 9, the flow section 2 is in a conjugate relationship with the diaphragm plate 11, and the sample particles S The scattered light is accurately focused on the position of the diaphragm plate 11.

Here, the objective lens 8 and the condensing lens 10 are
Since the beams between them are combined so that the beam becomes parallel, even if the dimensions from the surface of the flow cell 1 to the flow section 2 are slightly different when replacing the flow cell 1, etc., the AF unit 9 can be moved and the position detector 2
Focusing can be achieved simply by forming the slit image of the aperture 23 on the focal position 8.

In this way, in the embodiment, it is possible to focus easily and accurately, so the numerical aperture of the objective lens 8 is increased while maintaining accurate focus, the light collection efficiency of the optical system is improved, and the signal strength is increased. This means that it is possible to increase the

FIG. 4 is a perspective view of a flow cell 1 improved to be compatible with the above-mentioned AF unit 9, in which the incident surface 29 of the laser beam L is coated with a transparent film suitable for the wavelength of the laser beam L. Incidence surface 2
The photometric surface 30 below the surface perpendicular to 9 is coated with a transmission film that efficiently transmits the photometric wavelength range. Further, the focus detection surface 31 above the photometry surface 30 is coated with a reflective film that efficiently reflects the wavelength of the light emitted from the focusing light source 22.

Since it is preferable that the wavelength of the focusing light source 22 is separated from the wavelength of the laser light source 3 and the wavelength of fluorescence, it is preferable to use an infrared light source. Therefore, as the reflective film of the detection surface 31, a film that efficiently reflects infrared light may be selected. By using such a flow cell 1, a clear slit image of the aperture 23 can be obtained on the position detector 28.

A mechanism is provided that is driven by the output signal of the position detector 28, and the AF unit 9 is searched on the optical axis by the drive mechanism until the slit of the aperture 23 is imaged at a predetermined position of the position detector 28. move it,
If the drive mechanism is stopped in response to a focused signal, a focused state can be automatically obtained, further improving operability. Furthermore, if the measurement start signal of the particle analyzer is generated by the signal indicating that the drive mechanism of the AF unit 9 is stopped or the output signal of the position detector 28 at the time of focusing, the objective lens 8 can be focused. Inaccurate measurements can be avoided when the measurement is not performed. Further, a means for displaying a focusing signal indicating that the slit of the aperture 23 has been imaged at a predetermined position of the position detector 28 may be provided, and the focus signal may be manually operated.
When operating the AF unit 9, measurement may be started when the focus signal is output.

In the embodiment, a case has been described in which the AF unit 9 is installed in the photometric optical system for side scattered light, but in the photometric optical system for forward scattered light, the AF unit 9 is also installed between the light shielding plate 5 and the condenser lens 6. A similar effect can be obtained by placing an AF unit in the like this
It goes without saying that even better results can be obtained if the AF unit is installed in both the side and front photometric optical systems.

[Effects of the Invention] As explained above, the particle analyzer optical system according to the present invention can easily and accurately adjust the focus of the photometric objective lens by installing the focus detection means in the photometric optical system. This makes it possible to improve measurement accuracy and increase the numerical aperture of the objective lens, thereby improving fluorescence photometry intensity and enabling highly accurate analysis.

In addition, if desired, by providing a mechanism that drives the focus detection means, it is possible to perform focus adjustment fully automatically, and by providing a focus signal display mechanism, it is possible to easily adjust the focus manually. In addition, a mechanism that allows the device to move only when in focus can be provided to further facilitate measurement.

[Brief explanation of the drawing]

The drawings show an embodiment of the particle analysis device according to the present invention, in which Fig. 1 is a configuration diagram of the optical system, Fig. 2 is a layout diagram of the optical system as seen from the side of the AF unit, and Fig. 3 is a diagram showing the arrangement of the optical system.
Optical system layout diagram of the AF unit viewed from above, No. 4
The figure is a perspective view of a flow cell with various coatings. 1 is a flow cell, 2 is a flow section, 3 is a laser light source, 4 is an imaging lens, 5 is a light shielding plate, 6, 1
0 and 12 are condenser lenses, 7, 17, 19, and 21 are photoelectric detectors, 8 is an objective lens, 9 is an AF unit,
11 is a diaphragm plate, 13, 14, 15 are wavelength selection means, 16, 18, 20 are barrier filters, 22 is a light source, 23 is an aperture, 24, 25 are convex lenses, 26
27 is a mirror, 27 is a diaphragm, and 28 is a position detector.

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, and a light beam generated by the irradiation of the light beam onto the sample particles through a photometric objective lens. 1. A particle analysis apparatus comprising: a photometric optical system for measuring light; and a focus detection means for detecting a focused state of the photometric objective lens using surface reflection of the flow cell. 2. The particle analysis device according to claim 1, wherein a reflective film having a characteristic of reflecting light in a wavelength range used by the focus detection means is provided on the surface of the flow cell. 3. The particle analysis apparatus according to claim 2, wherein the focus detection means uses light outside the wavelength range of the light beam irradiated onto the sample particles.