JPH0882588A - Particle analyser - Google Patents

Abstract

PURPOSE: To provide a particle analyser capable of forming two or more sample streams in one detector to simultaneously or successively measuring two or more kinds of samples treated with different reagents. CONSTITUTION: In a particle count device D1, two sample passages 12a, 12b are arranged in one flow cell 12 in close vicinity to each other. By this constitution, two sample streams A1, B1 are formed and the signals from the sample streams A1, B1 are separately detected. The flow cell 12 is constituted, for example, by parallelly arranging two passages 12a, 12b each having a square pillar shape of which one side is 0.3mm at an interval of 0.5mm.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a particle analyzer, and more particularly to a particle analyzer for measuring the size and properties of particles in a sample flow using a flow cell.

[0002]

2. Description of the Related Art Conventionally, there is known a technique for measuring the size and properties of particles in a sample flow by measuring scattered light and fluorescence from the sample flow. There are several such technologies.

For example, "Fast Imaging in Flow: A
Means of Combining Flow-Cytometry and Image Analys
is '' (J Histochemistry and Cytochemistry vol.277 N
o.11979) shows a device which allows the shape of particles to be inspected after passing them through the sensing pores. Also,
U.S. Pat. No. 3,710,933 discloses a method of combining electrical resistance measurement with light absorption detection / scattered light detection.

[0004]

However, these conventional devices and methods can measure only one type of sample flow at a time due to the limitation of the sample flow forming method in the detector. As a result, when measuring using a sample treated with two or more types of reagents with different properties, after measuring one sample, the sample flow and the discharge channel are washed once, and then the other sample is measured. There is a problem that “it takes time to measure” or “the device becomes large” because it has to be measured by one of the two methods of preparing a separate detector for each reagent used. there were.

Further, Japanese Patent Laid-Open No. 61-132841 (operating method of microscope equipment) proposes to form a sample flow in a flow cell having a flow path having a large aspect ratio and observe particles in the sample flow. It is shown. However, "simultaneous measurement of two or more types of samples treated with different reagents" was not considered.

Further, in Japanese Utility Model Laid-Open No. 58-114754 (Sheathed sample flow generator), the inner diameter of the front chamber portion of one flow cell is 2 mm, and the inner diameter of the flow passage of the measuring portion is 0.2 mm.
It is shown that two sample flows are formed in one flow path by arranging two sample outflow nozzles at 0.8 mm intervals in the front chamber.

However, in this example, since the pipe through which the sample flows has a perfect circular cross section, it can be estimated that the interval between the two sample flows is about 80 μm when the inner diameter is about 0.2 mm. It will flow in almost the same position in the measurement area. As a result, it is difficult to detect the signals from the particles in the sample stream with separate detectors. Further, when two samples having different properties are flown at the same time, there is a problem that the two samples are mixed in the detection region, so that effective measurement cannot be performed.

An object of the present invention is to solve the above problems and to form two or more sample streams in one detector so that two or more kinds of samples treated with different reagents can be treated simultaneously or By making it possible to sequentially measure, it is intended to reduce the cost of the device and speed up the measurement.

[0009]

According to the present invention, in a particle analyzer for irradiating a sample flowing in a flow cell surrounded by a sheath liquid with light and detecting light from particles in the sample, the flow cell Is configured such that a plurality of sample streams flow in parallel in the interior at a predetermined interval, and a light receiving element having a light receiving portion for receiving forward scattered light from each sample stream in the flow cell is provided via a light receiving lens, Provided is a particle analyzer, wherein the forward scattered light from each sample flow is made incident on the light receiving portion of the light receiving element independently of each other.

[0010]

The flow cell is configured such that a plurality of sample streams flow in parallel inside the flow cell at predetermined intervals. 1
The following two methods are conceivable as methods for forming two or more sample streams in parallel inside one flow cell. By providing one or a plurality of partition walls along the flow direction of the sample flow in one flow cell, two or more sample flows are formed at a predetermined interval. One flat channel is provided in one flow cell, and two or more sample streams are formed in the channel with a predetermined interval.

The number of light receiving elements that receive the forward scattered light may be one, or a plurality of light receiving elements may be provided in the same number as the sample flow.
In the case of the former, the light receiving element is provided with a plurality of light receiving portions in the same number as the sample flow, and in the case of the latter, these light receiving elements have one
Each light receiving unit is provided. Thus, in either case, the forward scattered light from each sample flow enters the light receiving portion of the light receiving element independently of each other.

A shield plate for shielding the forward scattered light emitted from the light receiving lens is arranged between the light receiving element and the light receiving lens, and the front scattered light is provided to the light receiving portion of the light receiving element on the shield plate. A slit for guiding may be provided. In such a case, the forward scattered light from each sample flow is more reliably and independently incident on the light receiving portion of the light receiving element.

In the particle analyzer according to the present invention, another light receiving element for receiving another light (for example, side scattered light) is provided corresponding to a plurality of sample streams, and the forward scattered light is used for the detection. The signal detected by another light receiving element may be gated. Here, gating refers to determining whether or not to adopt as data.

The particle analyzer according to the present invention utilizes the forward scattered light detected from each particle in a plurality of samples to discriminate which sample flow the signal is from and determine the optimum one from the plurality of light receiving elements. It may be configured to select and detect an object.

The particle analyzer according to the present invention may be adapted to detect fluorescence signals from a plurality of samples dyed with a fluorescent dye in a pretreatment step or other steps.

[0016]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, four embodiments of the present invention will be described in detail with reference to the drawings. The present invention is not limited to these.

Example 1 FIG. 1 shows the configuration of Example 1 of the particle analyzer according to the present invention. This embodiment shows a particle counter D ₁ as a particle analyzer. That is, this particle counter D
₁ has two sample channels (12a) in one flow cell (12).
・ By arranging 12b) closely, two sample streams (A ₁・ B ₁ ) are formed, and each sample stream (A ₁・ B ₁ )
It is characterized in that the signals from are separately detected.

Here, as the flow cell (12), there are two flow channels (12a.12a) having a square prism shape with one side of the flow channel being 0.3 mm.
b) is arranged in parallel with a distance of 0.5 mm from each other.

FIG. 2 shows the structure of the flow cell (12) and its surroundings. In this configuration, nozzles (22a, 22b) for sample ejection are arranged below the two flow paths (12a, 12b), respectively. Each nozzle (22a, 22b) is arranged coaxially with the central axis of each flow path (12a, 12b). A partition wall was provided between the two flow paths (12a, 12b) so that the two sample streams (A ₁ , B ₁ ) could flow in parallel at a predetermined interval. It is in the same state as.

As shown in FIG. 1, the illumination light from the laser light source (2) is condensed into an elliptical shape by the condenser lens (4) to constantly illuminate the flow cell (12). And, "When the particles to be inspected pass through the area of the illumination light, the scattered light from the particles is received
The light is condensed on the light receiving element 1 (14) by (6), and the properties of the test particles are measured. This point is similar to the conventional flow cytometer.

As the light receiving element 1 (14), the sample flow (A ₁
If there are two B ₁ ), as shown in FIG. 3, A ₂ and B ₂
A photodiode having a structure having two detection regions is used. This photodiode has a light receiving lens 1
It is placed at the focal point of (6) or at a position further away from it. When the light receiving element 1 (14) is arranged at the focal position of the light receiving lens 1 (6), the scattered light from the two sample streams (A ₁ · B ₁ ) collected by the light receiving lens 1 (6) is received. 2 of element 1 (14)
The light is focused on each of the _two detection areas (A ₂ and B ₂ ). This allows the scattered light signals from each sample flow (A ₁ · B ₁ ) to be detected separately.

Further, the light receiving element 1 (14) is replaced by the light receiving lens 1 (6)
It is placed behind the focal point of and the focus of the receiving lens 1 (6)
Shield plate with circular slit as shown in Figure 4 at the point position (24)
(For example, the hole diameter of each circular slit: 0.5 mm)
If placed, each sample flow (A ₁・ B₁From the part other than
It becomes possible to shield and remove the scattered light and fluorescence.

The light receiving lens 2 (8) is a lens for receiving the side scattered light or fluorescence from the sample flow A ₁ . A photomultiplier tube is used as the light receiving element 2 (16).
When detecting the fluorescence from the sample, an optical filter that transmits only a specific wavelength is arranged in front of the light receiving element 2 (16). This filter needs to have the property of transmitting only the wavelength region corresponding to the fluorescence wavelength region of the reagent used for dyeing the sample flow.

The light receiving lens 3 (10) is a lens for receiving the side scattered light or fluorescence from the sample flow B ₁ . As the light receiving element 3 (18), a photomultiplier tube is used like the light receiving element 2 (16). When detecting fluorescence from the sample, an optical filter is arranged in front of the light receiving element 3 (18).

The operation of the particle counter D ₁ will be described below. The sample to be measured is guided to one of the two nozzles (22a, 22b) after being subjected to pretreatment such as being dyed with a specific fluorescent dye in the pre-measurement stage. One nozzle
When the particles in the sample flow A ₁ flowing out from (22a) pass through the illumination light area from the light source (2), the scattered light from the particles is collected by the light receiving lens 1 (6) and received by the light receiving element 1 (14). Detection area A ₂
Is focused on. At the same time, the fluorescence from the particles is received by the light receiving lens 2
(8) and the light receiving lens 3 (10) collects the light, and the light receiving element 2 (1
6) and the light receiving element 3 (18).

The light receiving lens 2 (8) is set so that the signal from one flow path 12a in the flow cell (12) can be best detected, and the condenser lens 3 (10) is set in the other flow path 12a.
Set to best detect the signal from b. As a result, the signal due to the fluorescence condensed on the light receiving element 3 (18) by the light receiving lens 3 (10) is received by the light receiving element 2 (16).
The signal becomes weaker than the signal detected at.

The signal processing device (20) has a function of analyzing the detection signals of the light receiving element 1 (14), the light receiving element 2 (16) and the light receiving element 3 (18), as well as the "light receiving element 1 (14)". When a signal is detected in the detection area A ₂ , the signal detected by the light receiving element 2 (16) is used as data to clear the signal detected by the light receiving element 3 (18), and vice versa. When a signal is detected in the detection area B ₂ , the detection signal of the light receiving element 3 (18) is used as data and the detection signal of the light receiving element 2 (16) is cleared. "

This means that "one channel (for example, channel 12a
), The scattered light signal or fluorescence signal from the particle is not only detected at the light receiving portion A ₃ of the light receiving element 2 (16), but also at the light receiving portion B of the light receiving element 3 (18) at the same time. _This is for removing the signal detected in B ₃ as a false signal, in contrast to the phenomenon that “ ₃ is detected as a weak signal”.

By using such a method,
It is possible to determine whether or not the signals detected by the light receiving element 2 (16) and the light receiving element 3 (18) are signals from particles flowing through the two flow paths (12a, 12b). As a result, the signal detected in the detection area A ₂ of the light receiving element 1 (14) and the light receiving element 2 (1
The signal detected at the light receiving point A ₃ in 6) is used as one of the flow paths 12
It becomes possible to analyze it as a set of signals from particles that have passed through a. By using such a signal processing method, it was impossible in the past to measure “a sample flow is made to flow through two channels at the same time, and signals from particles flowing in each channel are simultaneously detected and analyzed”. Will be possible.

In the signal processing device (20), each light receiving element 1
The signals from (14) ・ 2 (16) ・ 3 (18) are analyzed and output as scattered light and fluorescence data from each particle. That is, when the signal from the light receiving element 1 (14), the signal from the light receiving element 2 (16) and the signal from the light receiving element 3 (18) are input to the signal processing device (20), the light receiving element 2 (16) The intensity of the signal from the light receiving element 3 (18) is compared with the intensity of the signal from the light receiving element 3 (18). Then, for example, when the intensity of the signal from the light receiving element 2 (16) is higher, the signal is adopted as the data from the particle. As a result, the light receiving element 1 (14) signal and the light receiving element 2 (16)
Signals of the two channels are processed as one set, and finally one of the channels 12
It is output as the data from the test particle that passed a.

Further, when the sample is made to flow from the other nozzle (22b) to the flow channel 12b, the signal is analyzed in the same manner and finally output as data from the test particles that have passed through the flow channel 12b. .

Further, when the sample flow is simultaneously discharged from the two nozzles (22a and 22b) to the two flow paths (12a and 12b), the signal from the sample flow A ₁ and the sample flow B ₁ are similarly obtained. Can be separately analyzed and output separately as data from the sample that has passed through each flow path. In this way, it is possible to simultaneously flow two or more sample streams (A ₁ · B ₁ ) in _one detector and detect the signals from each sample stream (A ₁ · B ₁ ). .

When the distance between the two flow paths (12a, 12b) is wide, the intensity of the signal detected by the light receiving element 2 (16) and the intensity of the signal detected by the light receiving element 3 (18) are different. Since there is a large difference, comparing the output signal of the light receiving element 2 (16) with the output signal of the light receiving element 3 (18) makes it easy to determine which of the flow paths (12a and 12b) is the signal caused by the particles. Is. However, the signal detected by the light receiving element 3 (18) becomes stronger as the distance between the two flow channels (12a and 12b) becomes narrower, and the signal is from the particles flowing through the flow channel 12a. A strong signal is detected, as if it were a signal from a particle flowing through the channel 12b.

Even at this time, no problem occurs when the sample flow is one or when two sample flows are alternately flowed. However, when two sample streams are flown at the same time, the same signal is detected from the one sample stream to the two light receiving elements 2 (16) and 3 (18) at the same time, resulting in false data. .
In this case, the signal processing by the above-mentioned method becomes impossible, and it is necessary to separate each signal by the above-mentioned method.

The signal processing device (20) includes a signal from the light receiving element 1 (14), a signal from the light receiving element 2 (16) and a light receiving element 3 (1).
When the signal from 8) is input, it is determined in which of the two detection areas (A ₂ · B ₂ ) in the light receiving element 1 (14) the scattered signal has been detected. When it is determined that the signal from the area A _{2 is} received, the signal from the light receiving element 2 (16) is held to clear the signal from the light receiving element 3 (18) and the signal from the area B _{2 is} received. When it is determined that light is being received, the signal from the light receiving element 3 (18) is held and the signal from the light receiving element 2 (16) is cleared.

Thereafter, the signal from the light receiving element 1 (14) and the signal from the light receiving element 2 (16) are combined into one set, and the signal from the light receiving element 1 (14) and the signal from the light receiving element 3 (18) are combined. Are processed as one set, and finally, the data from the test particles passing through the flow channel 12a and the data from the test particles passing through the flow channel 12b are separately output.

In this way, two flow paths (12a
Even when the sample is flowed out from 12b),
It is possible to correctly detect and measure the signal from 12b). If particles arrive at _two regions (A ₂ · B ₂ ) at the same time, neither side scattered light data (signals detected at A ₃ or B ₃ ) is used as data.

Embodiment 2 FIGS. 5 to 8 show Embodiment 2 of the particle analyzer according to the present invention.
Shows the configuration of. This embodiment shows a particle counter D ₂ as a particle analyzer in which a flat sample flow path (32a) is provided in a flow cell (32). That is, in this particle counter D ₂ , the sample channel (32a) in the flow cell (32)
The cross-sectional shape of is a rectangle with the long side having a ratio of 10 times or more that of the short side, and the direction of flow convergence in the front chamber part (32b) of the flow cell (32) is defined as It is characterized by forming a flat sheath liquid flow that is converged in only one direction by making only the side direction.

Details of the flow cell (32) are shown in FIGS.
You In this configuration, between the two nozzles (22a ・ 22b)
By setting the distance to 1 mm or more, the measurement area (32c)
Two sample streams (A₁・ B₁) Is sufficient
Sample flow (A ₁・ B₁) Formed
No sample mixing occurs and each sample flow (A₁・
B₁A) Particles flowing through can be measured accurately.

Here, by making the shape of the flow cell into a rectangle which is horizontally longer than the flow cell (32) in the particle counter D _2, it becomes easy to arrange three or more nozzles in one flow cell, Furthermore, simultaneous formation of different sample streams can easily be carried out.

Embodiment 3 FIG. 9 shows the configuration of Embodiment 3 of the particle analyzer according to the present invention. This example shows a particle counter D ₃ as a particle analyzer. That is, this particle counter D
_{In 3} , the three receiving lenses 1 for detecting the signal from the particles
・ A rod type lens (selfoc lens) is used as 2/3 (36 ・ 38 ・ 40), and these are flow cells.
The configuration is shown in the vicinity of (42).

In FIG. 9, three light receiving lenses 1, 2, 3
Although all of (36, 38, 40) are shown as rod lenses, conventional lenses can be used as the light receiving lenses 2, 3 (38, 40). In that case, as the light receiving element 1 (14), the element (see FIG. 3) having the _two detection areas (A ₂ · B ₂ ) shown in the first and second embodiments may be used. One light receiving element may be arranged in parallel. A method of directly bonding the rod lens to the flow cell (42) is also possible.

Here, as the light receiving lens 1 (36), two rod lenses having an outer diameter of 2 mm are arranged in parallel, and the intervals of the sample streams are set to 2 mm, so that the signals from the respective sample streams are separately received. The lens is arranged to receive light.

When the conventional type light receiving lens 1 (6) shown in Examples 1 and 2 is used, the size and the numerical aperture of the light receiving lens 1 (6) are set in one flow cell (12/32). The number of sample streams that can be configured is limited. For example, when using a light receiving lens 1 (6) with an aperture of 10 mm and forming a plurality of sample streams at intervals of 1 mm, theoretically, about 10 sample streams can be formed. Since the condensing ability of the lens decreases as it approaches the outside of the light receiving lens 1 (6), the limit is to form 3 to 5 sample streams.

However, if the rod lens shown in this embodiment is used, one light receiving lens 1 (36) can be used in correspondence with each sample flow. As a result, five or more sample streams can be simultaneously formed, and the difference in sensitivity between sample streams can be reduced.

Embodiment 4 FIG. 10 shows the construction of Embodiment 4 of the particle analyzer according to the present invention. This example shows a particle counter D ₄ as a particle analyzer. That is, in this particle counter D ₄ , the rod type light receiving lens 1 (36) of the third embodiment is used.
The detection light collected in (1) is transmitted by an optical fiber and guided to the light receiving elements 1 (14) (14). By transmitting the detection light through a fiber, the positions of the light-receiving lens 1 (36) and the light-receiving elements 1 (14) and (14) can be set arbitrarily, so the degree of freedom in system configuration is improved and the device can be downsized. At the same time, there is an advantage that adjustment at the time of assembly can be simplified.

[0047]

According to the particle analyzer of the first aspect of the present invention, in the flow cell, a plurality of sample streams are made to flow in parallel at predetermined intervals, and each sample stream in the flow cell is separated from each other. A light receiving element having a light receiving section for receiving the forward scattered light is provided via a light receiving lens, and the forward scattered light from each sample stream is independently incident on the light receiving section of the light receiving element. Thus, it becomes possible to simultaneously measure a plurality of samples treated with a plurality of different reagents with a single detector. Therefore, it is not necessary to provide a detector for each treatment reagent, the apparatus can be simplified, and the measurement time can be shortened as compared with the case where continuous measurement is performed with one detector.

The particle analyzer according to claim 2 or 3 of the present invention also has the same effect as the particle analyzer according to claim 1.

According to the particle analyzer of the fourth or fifth aspect of the present invention, one light-receiving element is provided with a plurality of light-receiving portions in the same number as the sample flow, or a plurality of light-receiving elements is provided with one light-receiving element.
Since the respective light receiving portions are provided, the forward scattered light from each sample flow enters the light receiving portion of the light receiving element independently of each other. Therefore, the same effect as that of the particle analyzer according to claim 1 is obtained.

According to the particle analyzer of the sixth aspect of the present invention, a shielding plate for shielding the forward scattered light emitted from the light receiving lens is arranged between the light receiving element and the light receiving lens, and the shielding plate is provided. In addition, since the slit for guiding the forward scattered light to the light receiving portion of the light receiving element is provided, the forward scattered light from each sample flow is more reliably and independently incident on the light receiving portion of the light receiving element. . Therefore, a more remarkable effect is obtained as compared with the case of the particle analyzer according to claim 1.

According to the particle analyzer of claim 7 of the present invention, in addition to the effect of the particle analyzer of claim 1, another light receiving device for receiving another light (for example, side scattered light). An element is provided corresponding to a plurality of sample streams, and it becomes possible to gate a signal detected by another light receiving element using forward scattered light, so that a particle analyzer which is easier to use can be obtained. .

According to the particle analyzer of claim 8 of the present invention, in addition to the effect of the particle analyzer of claim 1, forward scattered light detected from each particle in a plurality of samples is utilized. It is possible to discriminate which sample flow the signal is from and select and detect the optimum one from the plurality of light receiving elements, and it is possible to obtain a particle analyzer which is easier to use.

According to the particle analyzer of the ninth aspect of the present invention, in addition to the effect of the particle analyzer of the first aspect, a plurality of samples dyed with a fluorescent dye in the pretreatment step and other steps are used. It becomes possible to detect the fluorescence signal of, and a more easy-to-use particle analyzer can be obtained.

[Brief description of drawings]

FIG. 1 is a structural explanatory view showing a schematic structure of a particle analyzer according to a first embodiment of the present invention.

FIG. 2 is a structural explanatory view showing a detailed structure of a flow cell which is a constituent element of the particle analyzer.

FIG. 3 is a configuration explanatory view showing a configuration of a light receiving element that receives forward scattered light, which is a component of the particle analyzer.

FIG. 4 is a configuration explanatory view showing a configuration of a shielding plate which is a component of the particle analyzer.

FIG. 5 is a structural explanatory view showing a schematic structure of a particle analyzer according to a second embodiment of the present invention.

FIG. 6 is a configuration explanatory view showing a detailed configuration of a flow cell which is a component of the particle analyzer.

FIG. 7 is a structural explanatory view of the inside of the flow cell as viewed from the front.

FIG. 8 is a structural explanatory view of the inside of the flow cell viewed from the side.

FIG. 9 is a structural explanatory view showing a schematic structure of a particle analyzer according to a third embodiment of the present invention.

FIG. 10 is a structural explanatory view showing a schematic structure of a particle analyzer according to a fourth embodiment of the present invention.

[Explanation of symbols]

 2 light source (laser) 4 condenser lens 6 light receiving lens 1 8 light receiving lens 2 10 light receiving lens 3 12 flow cell 12a sample flow path 12b sample flow path 14 light receiving element 1 16 light receiving element 2 18 light receiving element 3 20 signal processing device 22a sample nozzle 22b Sample nozzle 24 Shielding plate 28 Beam stopper 32 Flow cell 32a Sample flow path 36 Light receiving lens 1 38 Light receiving lens 2 40 Light receiving lens 3 42 Flow cell

Claims (9)

[Claims] 1. A particle analyzer for irradiating a sample flowing in a flow cell surrounded by a sheath liquid with light to detect light from particles in the sample, wherein the flow cell has a plurality of sample streams at predetermined intervals. A light-receiving element having a light-receiving portion that receives the forward scattered light from each sample stream in the flow cell is provided via a light-receiving lens, and the light-receiving portion of the light-receiving element receives the light from each sample stream. A particle analyzer, wherein the forward scattered light is made to enter independently of each other. 2. The particle analyzer according to claim 1, wherein the flow cell is provided with partition walls along the flow direction for allowing a plurality of sample streams to flow in parallel at predetermined intervals. 3. The particle analyzer according to claim 1, wherein the flow cell is provided with one flat flow path for allowing a plurality of sample flows to flow in parallel at predetermined intervals. 4. The number of light-receiving elements is one, and the number of light-receiving parts in the light-receiving elements is the same as that of the sample flow.
The particle analyzer according to claim 1. 5. A plurality of light receiving elements are provided in the same number as the sample flow,
The particle analysis device according to claim 1, wherein each light-receiving element has one light-receiving unit. 6. A shield plate for shielding the forward scattered light emitted from the light receiving lens is disposed between the light receiving element and the light receiving lens, and the front scattered light is received by the light receiving portion of the light receiving element. The particle analyzer according to any one of claims 1 to 5, further comprising: a slit for guiding to. 7. Another light-receiving element for receiving another light is provided corresponding to a plurality of sample streams, and forward scattered light is used to gate a signal detected by the other light-receiving element. The particle analyzer according to any one of claims 1 to 6, comprising: 8. The forward scattered light detected from each particle in a plurality of samples is used to determine from which sample flow the signal is, and the optimum one is selected from a plurality of light receiving elements and detected. 8. The particle analysis device according to claim 7, wherein. 9. The particle analyzer according to claim 7, wherein fluorescence signals from a plurality of samples dyed with a fluorescent dye are detected.