JPH05273203A - Cell or fine particle analyzing device - Google Patents

Abstract

PURPOSE:To stabilize sheath flow and to certainly measure cells or fine particles by using an array type photodetector as the photodetector of a measuring optical system. CONSTITUTION:A linear CCD sensor 24 being an array type optical sensor is used as a photodetector and the intensity of light incident on each of the windows of the sensor 24 is monitored. The image forming center position of scattered light is calculated from the intensities of light of the respective windows of the sensor 24 by a signal processing circuit 19 and, when the image forming position is shifted from the center of the sensor 24, a problem of sheath flow is judged to be present to emit a warning signal. When trouble due to the generation of air bubbles in the sheath flow is generated, in order to correct this trouble, a signal is emitted to a liquid feed control unit 25 so that a liquid is allowed to flow under high pressure at a high speed to remove air bubbles or a position correcting signal is sent to an automatic micro-movement stage 26 adjusting the position of a flow cell to take a measure to finely moving the flow cell so that cells or fine particles flow to the center of laser beam.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a device for irradiating a fluid to be measured with a light beam and measuring fluorescence or scattered light emitted from cells or fine particles contained in the fluid to identify cells or fine particles. ..

[0002]

2. Description of the Related Art With the development of biotechnology, there is an increasing demand for an apparatus for automatic analysis and sorting of cells in a wide range of fields such as medicine and biology. A flow cytometer is known as a typical one of such cell analyzers. In the semiconductor and medical fields, a laser light scattering counter is used to control fine particles in air or liquid. These devices have a common feature that cells or microparticles are caused to flow at high speed in the form of a flow to a detection unit, laser light is irradiated onto the detection section, and scattered light and fluorescence emitted from the cells or microparticles are detected for measurement.

FIG. 2 is a schematic diagram for explaining the construction of the main part of a general cell analyzer (flow cytometer) and its operation. In FIG. 2, a sample liquid 1 in a container and containing cells is guided to a flow cell 4 by an air pump 3 together with a sheath liquid 2 in another container. Inside the flow cell 4, the sheath liquid 2 collects the sample liquid 1 A sheath-shaped sheath flow that wraps in a cylindrical shape is formed. The sheath flow is formed as a means for accurately flowing cells one by one along the central axis of the flow cell 4. (The sheath flow will be described later).

A laser beam 7 emitted from a laser light source 5 and narrowed down by a focusing lens 6 is applied to the lower portion of the flow cell 4. In many cases, the cells contained in the sample liquid 1 are fluorescently labeled with a fluorescent substance such as a fluorescent dye or a fluorescently labeled monoclonal antibody, and when the cells pass through the laser light 7, scattered light and fluorescence are generated. Scattered light
It is detected by a photodetector 10 such as a photodiode through a condensing optical system including a condensing lens 8 and a beam block 9. On the other hand, with respect to fluorescence, 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 is detected by a photodetector 15, and green fluorescence is Are collected by the condenser lens 16 and the filter 17 from the half mirror 12 in another path and detected by the photodetector 18. Usually, the fluorescence detectors 15 and 18 are electron multipliers capable of detecting weak light. 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 cells are identified by analyzing the intensities.

In the above-mentioned apparatus, it is required that cell identification or particle size measurement be accurate, and for that purpose, a special flow system called a sheath flow is used.
FIG. 3A shows a schematic diagram of the flow cell 4 of FIG. 2 with only its periphery slightly enlarged. The same reference numerals are used for the parts in FIG. The controller 20 and the pressure regulating valve 21 of FIG. 3A are not shown in FIG. 2. In FIG. 3A, the liquid portion is shaded,
The sheath flow is indicated at 22. The leg portion of the flow cell 4 is formed in a rectangular shape, and the laser light 7 indicated by the arrow is passed at a right angle to reduce measurement noise. FIG. 3B is a schematic diagram showing the diameter of the main part, and the sheath flow 22
The diameter of the sample liquid column at the center of the sheath flow 22 is 100 to 200 μm.
Since it must be smaller than m, it has been shown that the thickness is 10 to 50 μm.

A feature of the sheath flow 22 is that cells and fine particles can be made to flow in a line one by one in the central portion of the laser light, and the intensity of the laser light 7 is always constant regardless of the intensity distribution in the cross-sectional direction. It has the advantage that cells and particles can be irradiated with.

[0007]

As described above, although the sheath flow system is effective, it is extremely unstable. In particular, when the sample liquid starts to flow, air bubbles are mixed and accumulated around the liquid delivery nozzle, which hinders the formation of the normal sheath flow 22 and often causes the sample liquid column to deviate from the center. FIG. 4 is a partial schematic view showing the state, FIG. 4 (a) is a view of the flow cell 4 seen from above, and FIG. 4 (b) is a sectional view. 23 represents a bubble.
If this happens, it is necessary to remove bubbles with high flow velocity and high water pressure.However, when performing automatic measurement of the device, it is possible to detect that the sample liquid column is decentered. However, if the measurement is continued as it is, cells and fine particles do not pass through the center of the laser beam, which causes a measurement error.

The present invention has been made in view of the above points, and an object thereof is to provide an analyzer (flow cytometer) capable of stabilizing the sheath flow and reliably measuring cells or fine particles. It is in.

[0009]

In order to solve the above problems, the cell or particle analyzer of the present invention uses an array of photodetectors as the photodetector of the measurement optical system.

[0010]

The scattered light or fluorescence emitted from the cells or fine particles in the sample liquid column is condensed by the measurement optical system and forms an image on the photodetector. The position where an image is formed at this time varies depending on the positions of cells and particles passing through the flow cell. When cells and particles are passing through the center of the flow cell, the optical image is formed at the center of the array of photodetectors, but the sample column is offset from the center of the flow cell, and cells and particles are in the flow cell. When passing through the end, the position where the light image is formed comes to the end of the arrayed photodetector. Therefore, by monitoring at which position of the arrayed photodetector scattered light or fluorescence is imaged, it is possible to know whether or not the sheath flow is normally formed, and if the sheath flow is defective,
You can fix this.

[0011]

EXAMPLES The present invention will be described below based on examples. FIG. 1 is a schematic diagram showing a main configuration of a cell or fine particle analyzer of the present invention. The basic structure of the entire device is
Since the device is the same as the device shown in FIG. 2, the description thereof will be omitted, but the difference from FIG. 2 is that the photodetector 10 of FIG. The linear CCD sensor 24 is used to monitor the intensity of light incident on each window of the linear CCD sensor 24. By doing so, the signal processing circuit 19 obtains the image forming center position of the scattered light from the light intensity of each window of the CCD sensor 24 which is an array-like photo detector, and the image forming position is CC.
If the D sensor 24 is displaced from the center, it can be determined that there is a problem with the sheath flow and a warning signal can be issued.

Further, when a problem occurs due to the generation of bubbles in the sheath flow, a high water pressure,
A signal is sent to the liquid feeding control device 25 or a position correction signal is sent to the automatic micro-moving stage 26 that adjusts the position of the flow cell so that the liquid is flowed at a high speed to remove the air bubbles, and cells or fine particles are centered on the laser beam. It is possible to take various measures such as finely moving the flow cell so that the flow will occur.

[0013]

[Effects of the Invention] In an analyzer for cells and particles using sheath flow, the sample liquid column is displaced from the center of the flow cell,
There was a case that measurement error occurred in the unstable state,
In the present invention, since an array-shaped photodetector is used as the photodetector of the measurement optical system, it is determined whether or not a stable sheath flow is formed depending on the position where scattered light emitted from cells or fine particles or the fluorescence image is connected. If any inconvenience occurs in the sheath flow after being detected, it can be corrected and stabilized, so that it is possible to more reliably measure cells or fine particles.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing a main configuration of a cell or fine particle analyzer of the present invention.

FIG. 2 is a schematic diagram showing the main configuration of an ordinary cell or particle analyzer.

FIG. 3A is a schematic view showing the periphery of the flow cell in a slightly enlarged manner, and FIG. 3B is a schematic view showing a diameter dimension of a main part of the flow cell.

FIG. 4A is a partial schematic view of the flow cell seen from above,
(B) is a partial sectional view

[Explanation of symbols]

 1 Sample Liquid 2 Sheath Liquid 3 Air Pump 4 Flow Cell 5 Laser Light Source 6 Focusing Lens 7 Laser Light 8 Focusing Lens 9 Beam Block 10 Photodetector 11 Focusing Lens 12 Half Mirror 13 Focusing Lens 14 Filter 15 Photodetector 16 Focusing Lens 17 Filter 18 Photodetector 19 Signal Processing Circuit 20 Controller 21 Pressure Control Valve 22 Sheath Flow 23 Bubble 24 Linear CCD Sensor 25 Liquid Transfer Control Device 26 Moving Stage

Claims (2)

[Claims] 1. A liquid feeding system for feeding a fluid containing cells or fine particles to a flow cell, a projection optical system for irradiating a light beam to the fluid passing through the flow cell, and the cell for receiving the light beam. Alternatively, a cell or fine particle analyzer that includes a measurement optical system that collects scattered light or fluorescence emitted from fine particles and measures at least one of them, and identifies the cells or fine particles from the light intensity measured by the measurement optical system. An array-type photodetector is used as the photodetector of the measurement optical system. 2. The analysis device according to claim 1, wherein the arrayed photodetector is a linear CC capable of controlling a sheath flow in a flow cell according to an imaging position output on the photodetector.
A cell or particle analysis device characterized by being a D sensor.