JPS6236541A - Particle analyzer - Google Patents

Abstract

PURPOSE:To enable the adjustment of the attitude of a flowcell easily and accurately, by detecting the positional relationship of the reflected light on the surface of a flowcell. CONSTITUTION:Particles previously subjected to a fluidically dynamic focusing by being wrapped with a sheath liquid of a fast laminar flow passes through a passage section 2 at the center of a flowcell 1 and a laser unit 3 and photoelectric detectors 7 and 7' are arranged in the direction perpendicular to the flow. Luminous flux emitted from a light source 12 of a detection optical system I illuminated being magnified passes an opening 11 and deflected with a half- mirror 9 to project to the flowcell 1 through a condenser lens 8. Then, the reflected light on the surface of the flowcell 1 is transmitted through the half- mirror 9 again by way of the condenser lens 8 and forms an image on a split light receiving element 10 to detect the rotation A-A' and the fall C-C' of the flowcell 1. On the basis of the resulting detection signals, the attitude of the flowcell 1 is adjusted.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a particle analysis device that is capable of determining the attitude of a flow cell in a flow cytometer or the like.

[Prior Art] In a conventional particle analysis device used in a flow cytometer or the like, a particle size of, for example, 200 pm
Irradiation light is irradiated onto a specimen such as blood cells wrapped in sheath liquid and passing through a flow section with a minute cross section of 00 JLm, and the resulting forward and side scattered light is used to determine the shape, size, and shape of the specimen. It is possible to obtain particle-like properties such as refractive index.

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.

In order to perform accurate measurements with a flow cytometer, the flow cell must be held perpendicular to the irradiation optical axis and the side scattered light optical axis. FIG. 5(a) is an explanatory diagram of rotation and inclination of the flow cell, and the inclination of the flow cell 1 with respect to the optical axis 01 of the irradiation light and the optical axis 02 of the side scattered light is perpendicular to the direction of the flow F of the sample particles. The rotation shown by A-A' around the flow F in the plane, the tilting towards the forward scattered light side with respect to the direction of the flow F, and the tilting towards the side scattered light side with respect to the direction of the flow F. It is considered that the fall is cc'.

FIG. 5(b) is an explanatory diagram of the shift of the optical path when the flow cell 1 is rotated in the direction of the A-A' force in a plane perpendicular to the direction of the flow F of sample particles. The incident light is refracted because the flow cell 1 is rotating, and becomes Lo when exiting the flow cell 1, indicating that it is shifted by Δp. On the side scattered light side, the scattered light is similarly refracted, causing a shift in the optical path.

Furthermore, if there is a tilt in the B-B'' or c-c' force direction, the optical path will similarly shift, making it difficult to perform accurate measurements.

In order to hold the flow cell 1 vertically, in conventional devices, a shielding plate having small holes is arranged on the exit surface of the irradiation light source, and the operator visually observes the image of the small holes reflected by the flow cell 1. By adjusting the rotation of the flow cell 1, letting a drop of water fall from the top of the flow cell 1 through the flow section, marking that position, flowing a water stream, and repeatedly adjusting so that the water stream hits the mark, At present, the inclination of the flow cell 1 is adjusted, but the operation is complicated and there are individual differences between operators, making it difficult to perform sufficiently accurate adjustment.

[Object of the Invention] An object of the present invention is to provide a particle analysis device that can easily and accurately adjust the attitude of a flow cell by detecting the positional relationship of light reflected by the surface of the flow cell.

[Summary of the Invention] The gist of the present invention for achieving the above-mentioned object is to irradiate a light beam onto sample particles flowing through a flow section in a flow cell.
a second irradiation optical system that projects a light beam onto the flow cell surface;
The particle analysis apparatus is characterized in that it comprises a detection means including a photoelectric detector made of a split type light receiving element that detects the reflected light of the light beam by the surface of the flow cell.

[Embodiments of the Invention] The present invention will be described in detail based on embodiments illustrated in FIGS. 1 to 4.

Figure 1 is a basic configuration diagram, in which sample particles are hydrodynamically focused and surrounded by a sheath liquid in a high-speed laminar flow inside the flow section 2 in the center of the flow cell 1 perpendicular to the plane of the paper. A laser light source 3 is arranged in a direction perpendicular to this flow. An imaging optical system 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 condenser lens 5, an aperture 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 generated laser light is scattered by the specimen particles, passes through a condensing lens 5 and an aperture 6, and reaches a photoelectric detector 7, where information mainly on the size of the specimen particles can be obtained. Further, on the optical axis 02 in the direction substantially perpendicular to the central axis of the flow of sample particles and the optical axis O1 of the laser beam, there is a condensing lens for side scattered light 5゜, an aperture 6゜,
Photoelectric detectors 7'' are arranged in sequence, and the scattered light of the laser beam by the sample particles in the 90'' direction is measured, so that the granularity of the sample particles can mainly be observed.
When biochemical analysis is performed by fluorescently labeling specimen particles, measurement can be performed by installing a wavelength selection means (not shown) on the optical axis 02. Further, a condenser lens 8 is placed on the optical axis 03 on the opposite side of the optical axis 02 with the flow cell 1 in between. A half mirror 9 and a divided light receiving element 10 are arranged in sequence, and the optical axis is 0.
An aperture 11.3 is provided on the reflected optical path of the half mirror 9, which is obliquely disposed relative to the half mirror 9.3. The light sources 12 are arranged one after the other. Further, a switchable mirror (not shown) is disposed between the flow cell 1 and the imaging optical system 4 on the optical axis 01, and a switchable mirror (not shown) is provided on the reflection side of the mirror, which is similar to that from the condenser lens 8 to the light source 12. A detection optical system (2) similar to the detection optical system (2) constituted by optical members 8' to 12' (not shown) is arranged.

FIG. 2 is a perspective view of the detection optical system I, in which the light beam emitted from the light source 12 passes through the aperture 11, is deflected by the half mirror 9, and is projected onto the flow cell 1 via the condenser lens 8. There is.

Then, the reflected light on the surface of the flow cell l passes through the nof mirror 9 via the condensing lens 8 again and is imaged on the divided light receiving element 10, and the rotation A-A',
A fall cc' is detected.

As for the detection optical system (not shown), the light source 1 passes through the aperture 11' via the switching mirror in the same way as the detection optical system I.
The luminous flux from 2゛ is /＼-Fumirror 9", condenser lens 8"
The reflected light is projected onto the flow cell 1 via a switching mirror, passes through a condenser lens 8' and a half mirror 9', and is imaged on the divided light receiving element 10°, and rotates A-A'' and tilts B-Bo. is now detected.

The divided light receiving element 10.10' that detects the surface reflected light of the flow cell 1 in this way has four photoelectric detection surfaces a.
A four-segment light-receiving element composed of elements 1 to a4 is used. Figure 3 shows the apertures 11.1 on the photoelectric detection surfaces a1 to a4.
1' is a formed diagram of a reflected image in the flow cell 1, and when the flow cell 1 is held perpendicular to the laser light source 3 and the side scattered light photometry optical system, the image shown in FIG.
As shown in a), the detection surface a of the divided light-receiving element 10, lO''
1 to a4 with the same size, each detection surface a1, a
2゜Respective outputs v1, v2, v3 from a3 and a4
, v4 are equal.

FIG. 3(b) is an image formed on the divided light-receiving element lO° of the detection optical system (■) when the flow cell 1 falls in the direction of Bo, or an image of the detection optical system (■) when the flow cell 1 falls in the direction of Co. is an image formation diagram on the divided light receiving element 10, in which upper and lower detection surfaces a1, a2 and a are obtained by dividing the divided light receiving element 10, etc. horizontally.
3. Output from a4, that is (Vt◆V2) and (V3+
A difference occurs in V4), and the output relationship is m + V2) > (V3
．． V4) Tonal, *ri, Fig. 3 (C) t* Image formation diagram on the split light receiving element 10' of the detection optical system (■) when the flow cell 1 falls in the direction of B, or when the flow cell 1 falls in the direction of C. This is an image formed on the divided light-receiving element 10 of the detection optical system I when it falls down, and in this case, the relationship between the outputs of the one-divided light-receiving element 10, etc. is (Vl-V2)<(V3+V4).

FIG. 3(d) is an image formed on the divided light-receiving elements 10 and 10' of the detection optical systems 1 and 2 when the flow cell 1 rotates in the A' length direction, and the detection surface at of the 4-part light-receiving element 1O110''
- Left and right detection surfaces al, a4 and a, vertically divided into a4, etc.
2. Output from a3, i.e. (V1+V4) and (V2+V
3), and m + V4) > (V2 + V3). Figure 3 (e) shows the result on the split light receiving element 10.10° of the detection optical system (■) and (2) when the flow cell 1 rotates in the A direction. This is an image diagram, and (VL+V4) < (V2.V3).

FIG. 3(f) is an image formed on the divided light receiving element 1O110' when the flow cell 1 is tilted at the same time as it rotates, and the flow cell 1 is tilted in the direction C and rotated in the direction A''. The image formed on the divided light receiving element 10 in the case of FIG. The figure is an example. When tilting and rotation are combined in this way, the detection surface a! of the four-divided light receiving element 10.10゛!
There will be a difference in all of the outputs v1 to v4 of ~a4.

In this way, when the outputs v1 to v4 from the four-divided detection surfaces a1 to a4 of the divided light receiving element 10.10'' are equal, the flow cell 1 is directed to each of the laser optical axis 01 and the side scattered light optical axis 02. Since it is held vertically, by moving the flow cell 1 so that the four outputs of each divided light receiving element 1O110° are equal, the flow cell 1 is held perpendicular to the lights i01 and 02, respectively. I can do it.

FIG. 4 is a configuration diagram of a control mechanism for automatically maintaining the normal posture of the flow cell 1. A motor 13 is connected to the flow cell 1, and a servo circuit 14 and a motor drive are connected to the output side of the divided light receiving element 10. The circuits 15 are connected in sequence,
The drive circuit 15 is connected to the motor 13, and the motor 13 is driven by the output signal of the divided light receiving element 10. Note that the detection optical system (2) also has a completely similar configuration.

In this way, a mechanism driven by the output signal of the divided light-receiving element 10, etc. is provided, and aperture images are equally imaged on the four-divided detection surfaces a1 to a4 of the divided light-receiving element lO, etc., and the output from the detection surface al''a4 is The orientation of the flow cell 1 is searched and driven until Vl-V4 become equal, and the split light receiving element 1
If the drive mechanism is stopped by the same value signal of each output from 0, etc., the flow cell l will be automatically held perpendicular to each of the optical axes 01 and 02, making it possible to further improve operability. .

Further, it is also possible to provide means for displaying a vertical coincidence signal at a predetermined position of the split light receiving element 10 etc. to notify that an aperture image of the aperture 11 etc. has been formed, and when this vertical coincidence signal is output. If the measurement is performed on the sample particles, the operation becomes even easier.

Note that the wavelength of the light source 12 is preferably separated from the wavelength of the laser light source 3 and the wavelength of fluorescence so as not to affect the measurement of scattered light, etc. within the 70-cell 1, and an infrared light source is used. It is preferable to do so. Also, during measurement, the light source 12
By setting the switch to turn off automatically, you can obtain accurate measured values without worrying about it affecting the measurement.

[Effects of the Invention] As explained above, the particle analysis device according to the present invention projects a light beam onto the flow cell surface, images the reflected light on the divided light receiving elements, and compares the outputs on the divided light receiving element surfaces. By providing the detection means, the attitude of the blow cell can be adjusted easily and accurately, and measurement accuracy can be improved. Also, if desired, by providing a mechanism driven by the output signal of the detection means,
It is also possible to fully automatically adjust the attitude of the flow cell, and furthermore, the measurement operation can be made easier.

[Brief explanation of the drawing]

Drawings 1 to 4 show an embodiment of a particle analysis device according to the present invention, in which FIG. 1 is a configuration diagram of an optical system, FIG. 2 is a perspective view of a detection optical system, and FIGS. 3(a) to 4. (f) is an explanatory diagram of the light image distribution on the divided light-receiving element, ftIJ'j is a configuration diagram of the flow cell drive mechanism, FIG. 5 is an explanatory diagram of the rotation and tilting of the flow cell, and (a) is An explanatory diagram of the directions of rotation and inclination, and (b) is an explanatory diagram of the shift of the optical path during rotation. 1 is a flow cell, 2 is a flow section, 3 is a laser light source, 6
is an aperture, 7 is a photoelectric detector, 9 is a half mirror, 110 is a divided light receiving element, 11 is an aperture, 12 is a light source, 13 is a motor,
14 is a servo circuit, and 15 is a motor drive circuit.

Claims (1)

[Scope of Claims] 1. A first 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 from the light beam scattered by the sample particles. , comprising: a second irradiation optical system that projects a light beam onto the surface of the flow cell; and a detection means including a photoelectric detector comprising a split-type light receiving element that detects light reflected by the surface of the flow cell of the light beam. particle analysis device. 2. The second irradiation optical system is provided in two sets in order to project onto the flow cell surface from two directions, and the detection means corresponding to these second irradiation optical systems are provided.
The particle analysis device described in Section 1. 3. The attitude of the flow cell is detected based on the positional relationship of reflected images of the light beam of the second irradiation optical system on the flow cell surface obtained by a plurality of detection surfaces of the photoelectric detector. The particle analysis device according to item 1. 4. The particle analysis apparatus according to claim 1, wherein the second irradiation optical system uses light in a wavelength range outside the wavelength range used by the first irradiation optical system. 5. The particle analysis apparatus according to claim 1, further comprising means for displaying a normal state of the attitude of the flow cell based on a signal from the detection means. 6. The particle analysis apparatus according to claim 1, further comprising a drive mechanism that automatically searches and moves the flow cell until the attitude of the flow cell reaches a normal state based on the output of the detection means.