JPH09166541A - Flowing grain analyzing device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To eliminate the dispersion of the measured value caused by the incident time fluctuation of irradiation light so as to be able to measure stably with accuracy by providing a correcting means or the like for correcting the position of a flow cell on the basis of a dislocation signal. SOLUTION: Sheath flow grains 40 is irradiated with laser beams 7 in a flow cell 4. Sideward scattered light or fluorescence 42 is detected by a lens 11 and a photo detector 18, and forward scattered light 43 is detected by a lens 8 and a photo detector 10. A photo detector 21 for detecting sideward scattered light 44 is provided to detect the center dislocation of the laser beams 7. When the center dislocation of the laser beams 7 is generated, dislocation is generated also to the scattered light 44. This is received by the photo detector 21, and a signal processing circuit 22 outputs a dislocation signal. On the basis of this dislocation signal, a CPU 25 controls an XY driving means 30 (correcting means) to correct the position of the flow cell 4. A quartered photodiode, a CCD, or the like is used as the photo detector 21.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fluidized particle analyzer, and particularly to a method of hydrodynamically focusing a particle suspension which flows at high speed, irradiating the spot with laser light or the like, and sequentially detecting scattered light or fluorescence. The present invention relates to a fluidized particle analyzer including a so-called flow cytometer, which analyzes properties and structures of particles.

[0002]

2. Description of the Related Art With the development of biotechnology, there has been an increasing demand for an apparatus for automatic analysis and separation of particles such as cells and DNA in fields such as medicine and biology. A flow cytometer is known as a typical fluidized particle analyzer that analyzes such particles. For example, a fluidized particle analyzer that applies flow cytometry using a laser as a light source causes particles to be analyzed such as cells to flow in a row in a flow cell that is a particle aligning means, and the laser light is applied to the flowing particles. Irradiate. Detects forward scattered light and fluorescence generated by particles and converts them into electrical signals,
Particle analysis is performed based on these electrical signals.
As a result, a large number of particles can be analyzed at high speed.

FIG. 8 shows a general flow cytometer 200.
It is a schematic diagram which shows the principal part. With reference to FIG. 8, a sample liquid 1 containing particles and contained in a container is guided to a flow cell 4 by an air pump 3 together with a sheath liquid 2 in another solution. Inside the flow cell 4, a sheath flow is formed in which the sheath liquid 2 wraps the sample liquid 1 in a cylindrical shape. This sheath flow is formed as a means for accurately flowing particles one by one along the central axis of the flow cell. The laser light 7 emitted from the laser light source 5 and narrowed down by the focusing lens 6 is applied to the lower portion of the flow cell 4. In many cases, the particles contained in the sample liquid 1 are fluorescently labeled with a fluorescent substance such as a fluorescent dye or a fluorescent label monoclonal antibody, and when the particles pass through the laser light 7, scattered light and fluorescence are generated.

The scattered light has a condenser lens 8 and a beam block 9.
Is detected by a photodetector 10 such as a photodiode. On the other hand, as for the fluorescence, the red fluorescence is collected by a condensing optical system including a condensing lens 11, a half mirror 12, a condensing lens 13, and a filter 14 and detected by a photodetector 15. The green fluorescence is emitted from the half mirror 12 on a different path from the condenser lens 16,
It is collected by the filter 17 and detected by the photodetector 18. Usually, photomultipliers capable of detecting weak light are used for the fluorescence photodetectors 15 and 18. The signals from the photodetector 10 for detecting scattered light, the photodetector 15 for detecting red fluorescent light, and the photodetector 18 for detecting green fluorescent light are respectively sent to the signal processing circuit 19, where the scattered light and the fluorescent light are detected. The particles are identified by analyzing the intensity.

In the above example, the laser light source 5 is used as the light source, but there is also an apparatus using a mercury lamp or a xenon lamp which is cheaper than this. In addition, the fluorescence measurement is performed in three colors or four.
There is also an apparatus that measures the color by subdividing it, and an apparatus that measures the 90 ° direction component of the scattered light. In addition to the particle identification function,
There is also an apparatus called a cell sorter that has a mechanism for sorting and sorting the identified particles.

[0006]

A flow cytometer as a conventional fluidized particle analyzer is constructed as described above. FIG. 9 is a diagram showing a transmission state of the sheath flow 41 in the flow cell 4 in FIG. 8 on the transmission plane of the laser light 7 and its intensity distribution. The left side of FIG. 9 shows the intensity distribution of the laser light 7 in a state (A) in which the laser light irradiates the central portion of the sheath flow 41 and a state (B) in which the laser light is displaced from the central portion as indicated by the dotted line (B).

Referring to FIG. 9, the intensity distribution of the laser light 7 normally exhibits a Gaussian distribution as shown by the characteristic A. Since the particle 40 wrapped with the sheath liquid 2 as the sample passes through the central portion of the sheath flow 41, when the particle 40 is irradiated with the laser light 7, the optical axis position of the laser light 7 is the sheath flow 41. If it coincides with the central portion of, the light with the highest intensity is incident on the particle 40, and efficient measurement can be performed.

However, when the optical axis of the laser beam 7 is relatively deviated from the center of the sheath flow 41 as shown by B,
The intensity of the laser light 7 with which the particles 40 are irradiated becomes weak according to the Gaussian distribution, and the obtained scattered light and fluorescence are also weak. As described above, in the conventional fluidized particle analyzer, if the optical axis of the laser light deviates from the center of the sheath flow, the same light detection signal cannot be obtained even if the same particle is measured, and the measurement accuracy is limited. There was a problem.

The present invention has been made in order to solve the above-mentioned problems, and it eliminates the dispersion of measured values due to fluctuations when the irradiation light is incident, and enables fluid particles to be measured accurately and stably. An analyzer is provided. In addition,
As the light source of the irradiation light, the same light source as the conventional one such as a laser light source, a mercury lamp, a xenon lamp can be used.

[0010]

A fluidized particle analyzer according to a first aspect of the present invention provides a particle liquid sending means for flowing a particle suspension liquid at high speed, and an irradiation light having a predetermined intensity distribution to the particle liquid sending means. Irradiation means for irradiating, light receiving means for receiving scattered light or fluorescence from particles by the irradiation light through the condensing optical system, signal processing means for performing light reception signal processing of the light receiving means, and signals from the signal processing means. Particle analysis means based on which particles are analyzed, position deviation detection means for detecting a position deviation between the irradiation means and the particle liquid sending means, and a position deviation signal being output, and a particle liquid flow based on the position deviation signal. Correction means for correcting the position of the feeding means.

The positional deviation detecting means detects the positional deviation between the particle liquid sending means for flowing the particle suspension and the irradiation means for irradiating the particle liquid with the irradiation light, and based on the detected positional deviation signal, the particle liquid sending means To correct the position, the position is adjusted so that the strongest light is incident on the particle. As a result, efficient analysis of particles becomes possible.

In the fluidized particle analyzer according to any one of claims 2 to 4, the detection light is taken out from the particle by interposing an optical splitter in the optical path of the forward scattering light receiving system, the back scattering light receiving system, or the side scattering light receiving system. , And it is used as a position shift signal.

Since the positional deviation detecting means extracts the detection light as the positional deviation signal through the optical splitter in the optical path of the scattering light receiving system in each direction, the positional deviation can be detected with a simple structure.

In the fluidized particle analyzer according to a fifth aspect, in the fluidized particle analyzer according to the first aspect, the misregistration detecting means is in the optical path of the side scattering light receiving system in a direction symmetrical to the direction of receiving the fluorescence from the particles. The detection light is taken out through the optical splitter and is used as a position shift signal.

Since the position shift signal is detected by the light splitter provided in the side-scattering light receiving system in the direction symmetrical to the direction of receiving the fluorescence from the particles, the position shift of the center of the irradiation light can be surely detected.

In the fluidized particle analyzer according to the sixth aspect, the positional deviation detecting means according to the first aspect uses a four-division photodiode. Since the positional deviation is detected using the four-division photodiode, the positional deviation direction can be reliably detected.

In a fluidized particle analyzer according to a seventh aspect, the positional deviation detecting means of the first aspect uses a four-divided photodiode, and the four-divided photodiodes are arranged in a matrix in a vertical direction and a horizontal direction. And the current difference in the horizontal direction are detected as the positional deviation detection signal. Since the positional deviation is detected from the current difference between the photodiodes arranged in the vertical and horizontal directions, the positional deviation can be detected with a simple configuration.

In the fluidized particle analyzer according to the eighth aspect, the area CCD is used as the positional deviation detecting means. Since the positional displacement is detected using the area CCD, the positional displacement can be detected with a simple configuration.

In the fluidized particle analyzer of the ninth aspect, the positional deviation detecting means uses a two-dimensional PSD.
Since the positional deviation is detected using the two-dimensional PSD, the positional deviation can be detected with a simple configuration.

[0020]

Embodiments of the present invention will be described below with reference to the drawings. FIG. 1 is a schematic diagram showing a main part 100 of a fluidized particle analyzer according to the present invention.

With reference to FIG. 1, a parallel laser beam 7 from a laser light source (not shown) is passed through a lens 6 to generate a sheath flow 4.
The particle 40 of No. 1 is irradiated, and side scattered light or fluorescence 42 is received and detected by the lens 11 and the photodetector 18. Also,
The forward scattered light 43 is detected by the lens 8 and the photodetector 10.
The configuration up to this point is the same as that of the conventional FIG.

Here, for the forward scattered light 43, as shown in FIG. 1, a light shielding plate 20 is inserted in front of the lens 8 in order to avoid the straight traveling light of the laser light 7.

A signal processing circuit 19 performs known signal processing such as amplification and peak hold on these signals. The signal processing circuit 19 controls the entire apparatus via the A / D converter 23 for A / D converting the input signal.
It is connected to the I / O interface 24 of U25.
The I / O interface 24 includes a ROM 26 that stores programs and the like, a RAM 27 that stores data and the like, and a CRT.
A display unit 28 for displaying the same and the like, and an alarm means 29 for issuing an alarm if necessary are connected.

The data processed by the signal processing circuit 19 and A / D converted is stored in the RAM 27, and the data is displayed on the display unit 28 by a known method.

In this embodiment, in order to detect the positional deviation of the center of the laser light 7, the photodetector 21 for detecting the light 44 reflected in the symmetrical direction of the side scattered light 42 is provided. As described above, when the position shift of the center of the laser light 7 occurs, not only the side scattered light 42 but also the side scattered light 44
There is also a displacement. This is received by the photodetector 21.

Next, the photodetector 21 will be described. The photodetector 21 has, for example, the configuration shown in FIG. Here, it is composed of four-divided photodiodes B1 to B4. The respective outputs are wired as shown in the figure, and the tracking signals Tr1 and Tr2 are output using the operational amplifiers 51 and 52, respectively.

Here, Tr1 = (IB1 + IB2)-
(IB3 + IB4) Tr2 = (IB1 + IB3)-(IB2 + IB4) where IB1 to IB4 are 4-division photodiodes B1 to
It is the output current value of B4.

Next, the photodetector 21 using the side scattered light 44.
A method of detecting the center position deviation of the laser light 7 will be described. FIG. 3 is a diagram showing a positional relationship between the photodiodes B1 to B4 and the side scattered light 44 irradiated on the photodiodes B1 to B4. Referring to FIG. 3, when the side scattered light 44 is at the center of the photodiodes B1 to B4 as shown in (A), both tracking signals Tr1 and Tr2 become almost zero. If the initial setting is made in this state, the tracking signals Tr1, Tr2
The deviation of the optical axis of the laser beam can be detected by the deviation of the output of.

That is, as shown in FIG. 3B, when the side scattered light 44 approaches the photodiodes B1 and B2, the tracking signal Tr1> 0 and the tracking signal Tr2≈0.
become. Also, the side scattered light 44 causes the photodiode B to
When approaching the B1 and B3 sides, the tracking signal Tr1≈0 and the tracking signal Tr2> 0, and the respective directions can be detected.

Referring again to FIG. 1, the fluidized particle analyzer of the present invention is connected to an I / O interface 24,
Flow cell 4 (corresponding to sheath flow 41) is shown as X, Y in the figure.
It has an XY drive means 30 for driving the flow cell 4 in the X and Y directions to correct the deviation of the flow cell 4 from the center of the laser beam 7.

FIG. 4 is a schematic diagram showing the structure of the XY drive means 30. Referring to FIG. 4, the flow cell 4 is connected to the XY drive means 30 via the flow cell support column 53.
As the XY drive means 30, for example, a precision drive device using a pulse motor or a laser actuator is used.

When the tracking signals Tr1 and Tr2 have the following relationships with respect to the predetermined values α and β, the CPU 25 stops the data collection from the signal processing circuit 19 and causes the XY drive means 30 to operate the flow cell. 4 position correction is performed.

| Tr1 | ≧ α, | Tr2 | ≧ β The position correction is performed for the flow cell 4 in the X-axis and Y-axis directions so that it falls within the range of | Tr1 | <α, | Tr2 | <β (1). Control the position. If the optimally set values for Tr1 and Tr2 are Tr10 and Tr20,
The values of α and β are, for example, α = Tr10 × 1.1 and β = T
Set to r20 × 1.1.

FIG. 5 shows detection output data FSC (forward scattering light) of the photodetector 10 which detects the forward scattered light 43.
ht) and the SSC (side scatterin) detected by the photodetector 18.
(g light) data signal waveform. As shown in the figure, the particles 40 are detected in the flow cell 4 at the portions where the concave portions or the convex portions are formed in the FSC and SSC. Therefore, it is necessary to detect and control the positional deviation of the flow cell 4 at the timing when the cells do not pass, as shown by, in FIG. These timing controls are performed by the CPU2
5 is possible.

If the CPU 25 can control the XY drive means 30 within the range of the equation (1), the signal processing circuit 19
Data is collected again and displayed on the display unit 28 as histogram data, for example. However, when it does not fall within the range of the formula (1), the alarm means 29 is used to notify the operator of the abnormality of the measurement.

In the above embodiment, the side scattered light 44
Although the positional deviation of the laser light 7 is detected by using, the present invention is not limited to this, and the backscattered light 61 of the sheath flow 41 may be detected via the half mirror 62 as shown in FIG. The forward scattered light 43 of the sheath flow 41 may be detected via the half mirror 63 as shown in FIG.

Although a four-division photodiode is used as the photodetector 3, a CCD or a PSD (Positive Sensiti) is used.
ve Device), as long as it is a photodetector that can detect the shake of the optical axis.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a main part of a fluidized particle analyzer according to the present invention.

FIG. 2 is a schematic diagram showing a configuration of a photodetector.

FIG. 3 is a diagram showing a positional relationship between a photodiode constituting a photodetector and side scattered light with which the photodetector is irradiated.

FIG. 4 is a diagram showing a relationship between an XY drive unit and a flow cell.

FIG. 5 is a diagram showing an example of output data from a front photodetector and a side photodetector.

FIG. 6 is a diagram showing another embodiment of the present invention.

FIG. 7 is a diagram showing another embodiment of the present invention.

FIG. 8 is a schematic diagram showing a main part of a conventional fluidized particle analyzer.

FIG. 9 is a diagram showing an intensity distribution of laser light in a state where the optical axis position of the laser light is deviated from the sheath flow.

[Explanation of Codes] 4 Flow Cell 6 Focusing Lens 7 Laser Light 8 Condensing Lens 10 Photodetector 11 Condensing Lens 18 Photodetector 19 Signal Processing Circuit 21 Photodetector 22 Signal Processing Circuit 23 A / D Converter 24 I / O Interface 25 CPU 26 ROM 27 RAM 28 Display 29 Alarm means 30 XY drive means 40 Particles 41 Sheath flow

Claims (9)

[Claims] 1. A particle liquid sending means for flowing a particle suspension liquid at a high speed, an irradiation means for irradiating the particle liquid sending means with light having a predetermined intensity distribution, and the light through a condensing optical system. A light receiving means for receiving scattered light or fluorescence from particles by the light receiving means, a signal processing means for processing a light receiving signal of the light receiving means, and a particle analyzing means for analyzing the particles based on a signal from the signal processing means. A position deviation detecting unit that detects a position deviation between the irradiation unit and the particle liquid sending unit and outputs a position deviation signal; and a position of the particle liquid sending unit is corrected based on the position deviation signal. A fluidized particle analyzer including a correction means. 2. The fluidized particle analyzer according to claim 1, wherein the positional deviation detecting means extracts detection light as the positional deviation signal by interposing an optical splitter in the optical path of the forward scattering light receiving system from the particles. . 3. The fluidized particle analyzer according to claim 1, wherein the positional deviation detecting means extracts detection light as the positional deviation signal through an optical splitter in the optical path of the backscattering light receiving system from the particles. . 4. The fluidized particle analyzer according to claim 1, wherein the misregistration detection means extracts detection light as the misregistration signal through an optical splitter in the optical path of the side-scattering light receiving system from the particles. apparatus. 5. The misregistration detection means extracts detection light through an optical splitter in an optical path of a side-scattering light receiving system in a direction symmetrical to a direction in which fluorescence from the particles is received, and the detection light is detected. The fluidized particle analyzer according to claim 1, wherein is the position displacement signal. 6. The fluidized particle analyzer according to claim 1, wherein the positional deviation detecting means includes a four-division photodiode. 7. The four-division photodiodes are arranged in a matrix in a vertical direction and a horizontal direction, and the current difference in the vertical direction and the current difference in the horizontal direction are used as the position shift signal. The fluidized particle analyzer according to any one of 5 above. 8. The area shift detection means is an area CCD.
The fluidized particle analyzer according to any one of claims 1 to 5, further comprising: 9. The two-dimensional PSD is used as the positional deviation detecting means.
The fluidized particle analyzer according to any one of claims 1 to 5, further comprising: