JPH0448245A - Particle measuring instrument - Google Patents

Abstract

PURPOSE:To improve the accuracy of particle measurement by discharging liquid drops which contain individual particles to be inspected from a nozzle opening, and sticking the discharged liquid drops from the opening on a glass plate provided opposite the opening and measuring the object particles. CONSTITUTION:The liquid drops which are discharged from the opening of a nozzle 1 strike on the glass plate 16 slantingly and stick on the glass plate 16 to move on the glass plate 16 by gushing. When a cell in the liquid passes across a measurement position irradiated with a laser beam from a laser light source 7 from the reverse surface of the glass plate 16, scattered light and fluorescent light are emitted by the cell and detectors 5 and 6 detect their intensity values. Liquid drops are discharged from the opening of the nozzle 1, one after another, the cell is measured at the measurement position, and the cell which is already measured is flushed away with following liquid drops which are discharged. Consequently, individual object particles in a particle suspension can securely be measured.

Description

Detailed Description of the Invention [Industrial Field of Application] The present invention involves separating individual test particles one by one in a suspension in which microscopic test particles such as cells are suspended, and separating them one by one using an optical method or the like. This invention relates to a particle measuring device that measures particles using.

[Prior Art] Conventionally, a sheath-flow method as shown in FIG. 4 is generally known as a method for separating a large number of particles one by one in a particle suspension. This involves applying pressure to the particle suspension liquid and the sheath liquid using pressurizing devices, wrapping the particle suspension liquid in the sheath liquid, and then hydrodynamically converging the particle suspension liquid into a thin stream to separate individual particles. This is a method of separating the particles by flowing them one by one in a line.

In addition, a flow cytometer is a device that prepares a sample such as blood, separates it using this sheath-flow method, measures the individual cells in the sample using optical methods, etc., and performs cell analysis and cell sorting. It has been put into practical use under the names of cell sorter and cell sorter.

Figure 244 shows an example of the configuration of a flow cytometer, in which individual cells that are separated by the above-mentioned sheath-flow method and flowing in a line inside the flow cell are irradiated with measurement energy, for example, a light beam from the light source 24, and the cells are exposed to light. A detector 25 detects optical reactions caused by irradiation, such as scattered light and fluorescence emitted from cells.
．． 26 for photometry. Based on the photometric output, the signal processing unit 29 performs calculations such as particle analysis type, size, etc.

Further, FIG. 345 shows an example of the configuration of a cell sorter. A cell suspension such as blood is guided into the nozzle by an external pressure device, and a laminar flow centered around the cell suspension is formed within the nozzle by the sheath flow principle, and at the orifice (70 to 100 μm) at the nozzle exit, The cell suspension is released into the air as a jet stream with an average diameter of 15-20 μm. 100 to 20 from the nozzle tip to the main jet stream in the air
Excitation light from the light source 24 is irradiated at 0 μm. When cells that have been fluorescently stained in advance are irradiated with excitation light, scattered light and fluorescence are emitted from the cells, which are detected by detectors 25 and 26 and converted into electrical signals.

On the other hand, the nozzle is operated by the vibrator 21 under the control of the excitation circuit 27.
The jet stream exiting the orifice becomes uniform droplets several mm below the nozzle tip. Depending on whether the signal detected from the cells satisfies a predetermined condition, a signal is sent from the signal processing unit 29 to the charging circuit 28, and a charging signal is applied to the nozzle in synchronization with the formation of droplets. , the droplet becomes lightly charged. While the charged droplet containing the desired cells passes between two electrodes 22 and 23 that generate a strong electric field, the droplet is deflected and distributed to the right or left depending on the type of cell etc. by static electricity. collected in separate test tubes.

In the sheath-flow type used in the above-mentioned flow cytometers and cell sorters, the cell suspension and sheath liquid are guided to the nozzle by pressurizing each liquid with a pressure system, but this requires the use of piping, pumps, etc. Since a pressurizing device is required, there are problems in that the device is large-scale and the control is complicated.

In addition, in the sheath flow type, the flow rate is determined by the pressure applied to the cell suspension and the sheath liquid, and the time interval for particle separation is determined by this flow rate and the degree of dilution of the cell suspension. It was difficult to change in range.

Therefore, the applicant of the present application has previously proposed a method of ejecting droplets by applying ejection energy into a container containing a one-particle suspended liquid, for example, applying thermal energy to generate air bubbles. We proposed a new particle measuring device that uses a method in which droplets containing individual particles to be detected are ejected from an opening by the flJ impact of expanding bubbles.

[Object of the Invention] An object of the present invention is to further improve the particle measuring device proposed above to further improve the accuracy of particle measurement.

[Means for Achieving the Object] The present invention for achieving the above object is a particle measuring device that separates test particles one by one in a particle suspension and measures the separated test particles. A storage section for storing a suspended liquid; an opening provided in the storage section for discharging the particle suspension liquid to the outside; and a liquid droplet containing individual particles to be detected by applying ejection energy to the storage section. means for discharging from the opening, and a means provided at a position opposite to the opening,
The present invention is characterized in that it includes a measuring section to which the droplets discharged from the opening are attached, and a means for measuring the particles to be detected in the measuring section.

[Example 1] Embodiments of the present invention will be described in detail below with reference to the drawings.

FIGS. 1 and 2 are configuration diagrams of an embodiment of the present invention.

In the figure, reference numeral 1 denotes a nozzle having a rectangular cross section of about 50 μm×50 μm for accommodating a cell suspension in which cells, which are test particles, are suspended, and one end is open to form an opening.

As an example of a method for creating the nozzle, a common method is to create fine grooves on a substrate by etching or a photoresist process, and then laminate a flat plate on top of the grooves, but the method is not limited to this. The size of the nozzle cross section is determined according to the size of the test particles to be measured. In this example, the measurement target is considered to be blood, and the size of various blood cells contained in the blood is about 5 μm to 30 μm. Therefore, the nozzle size was set to 50 μm×50 μm, which is slightly larger than the maximum blood cell size.

Reference numeral 2 denotes a supply port that sequentially supplies cell suspension into the nozzle 1 and is connected near the outlet of the nozzle 1. Reference numeral 3 denotes a heating section provided within the nozzle, specifically a heater whose electrodes are connected to a control circuit to be described later. Note that the heating section 3
The heating unit 3 may be heated outside the nozzle to heat the inside of the nozzle. Also, the heating unit 3 is not limited to a heater, but may be any means that generates thermal energy, such as a laser beam applied to a heat absorbing member. 4 may be provided on the outside of the nozzle 1,
This is a heat dissipation member for suppressing the temperature of the nozzle 1 from rising due to heat conduction from the heater.

Reference numeral 16 denotes a fixed transparent glass plate, which is arranged obliquely with respect to the discharge axis 1 of the nozzle 1. 7 is a laser light source which is a measurement energy generating means, and 5.6 is a pressure detector such as a photomultiplier for detecting an optical reaction from particles caused by light irradiation. The laser beam emitted from the laser light source 7 enters the back surface of the glass plate 16, and the ejection axis 1 of the nozzle 1 and the laser optical axis 0 are arranged so as to intersect near the glass plate 16. The detector 5 is placed on the laser optical axis 0, and a beam stopper (not shown) is provided on the optical axis in front of the detector 5 to prevent the laser beam from directly entering the detector. Scattered light scattered forward in the optical path by the particles S at the measurement position irradiated with the laser beam on the laser beam 16 is photometered by the detector 5 . Further, the detector 6 is arranged in a direction intersecting each of the laser optical axis 0 and the droplet ejection axis 1, and in front of the detector 6 is a bandpass filter 1 that selectively transmits only the target fluorescence wavelength.
5 is provided, and the fluorescence emitted from the particles S at the measurement position is selectively measured by the detector 6.

Next, the operation in the above configuration will be explained.

Prepare a measurement sample such as a blood sample that has been sufficiently diluted and stained with a fluorescent reagent if necessary, and supply this sample to the supply port 2 and from there into the nozzle 1.
Let the inside be filled.

Here, control by the control system shown in FIG. 3 will be specifically explained.

When the ON-OFF generation circuit 32 drives the heater 3 provided in the nozzle 1 to generate heat according to the ejection timing signal generated by the sampling synchronization circuit 31, the water in the heated cell suspension is instantly vaporized. As a result, bubbles 8 are generated as shown in Fig. 341. Then, the volume increases by the amount of vaporization, so what? # The cells S near the opening of the nozzle 1 are pushed out from the opening of the nozzle, and the cell suspension containing the cells S is discharged out of the nozzle 1. The bubbles 8, which initially continued to expand, cool and begin to contract, and due to the reduction in volume, a pulling force acts on the cell suspension discharged from the opening.In this way, the cells containing fine ms discharged from the opening. The suspended liquid turns into droplets and flies into the air. The basic principle of this discharge is disclosed in, for example, Japanese Patent Application Laid-Open No. 54-59938.
It is described in Japanese Patent Application Laid-Open No. 55-27282.

The amount of cell suspension that is discharged is transferred to the supply port 2 by capillary action.
is supplied and returns to the initial state. Since the cell suspension is supplied naturally by capillary action, there is no need for a conventional pressurized system.

The generation and disappearance of this bubble is ′! This is done in a very short time according to the sampling frequency created by the sampling synchronization circuit 31 shown in Figure J3, and it is possible to continuously eject up to several thousand droplets per second. This allows a large number of cells to be separated at high speed.

In addition, the signal generated by the sampling synchronization circuit 31 is also used as a data acquisition timing signal for the detection system 33, and the ejection timing signal of the sampling synchronization circuit 31 allows the ejected droplet to pass through the measurement position after a predetermined time. Only the detector 5.6 is configured to take in data from the detector 5.6. This makes it possible to eliminate as much as possible the intake of noise components from particles other than the test particles.

The size of the aperture and the capacity of the heater are set so that the droplet discharged from the nozzle has a diameter of approximately 50 μm to 80 μm, and the particle size is set so that only one cell particle is included in the droplet discharged. Set the dilution of the suspension.

The droplets discharged from the opening collide with the glass plate 16 from an oblique direction, adhere to the glass plate 16, and move on the glass plate 16 due to the force of the discharge. Then, when the cells in the liquid pass across the measurement position where the laser beam is irradiated from the back surface of the glass plate 16, scattered light and fluorescence are generated from the cells.
Each intensity is detected by a detection system consisting of detectors 5 and 6. Droplets are discharged one after another from the opening of the nozzle to measure cells at the measurement position, and the cells that have been measured are swept away by the droplets discharged later.

In FIG. 3, the output from the detection system 33 having a photodetector 5.6 is stored one after another in the data storage section 34, and from the obtained measurement data regarding a large number of cell particles, the signal processing section 35 generates a histogram and a site. Particle analysis, such as determining the type of cell particles and analyzing sex purchase, is performed using statistical processing such as gram analysis.

Since various methods are widely and generally known regarding specific calculation methods, detailed explanations will be omitted here. The analysis results are displayed on a TV monitor, printed out, or otherwise output.

As described above, instead of directly irradiating the flying droplets with a laser beam, the measurement is performed by attaching the cell particles to a fixed glass plate and passing it through the measuring section one after another. Effects can be obtained.

'17S1, since the cell particles are attached to a fixed glass plate, there is less displacement of the particles at the test position, and more stable measurements can be performed.

Second, if the configuration were to directly irradiate a flying droplet with a laser beam, diffuse reflection would occur on the droplet surface with an unstable surface shape, which would adversely affect the optical measurement of cell particles in the droplet. There is fear. On the other hand, this example is configured to irradiate a laser beam from the back side of a transparent glass plate and measure the m-cell particles in the liquid adhering to the glass plate, so diffuse reflection does not occur and the particles in the flow cell can be measured. It is possible to perform measurements with high reliability equivalent to that of

Although the above is applicable to devices that analyze microparticles in the biological field such as blood cells, the present invention is not limited thereto. It can be used in all devices that handle floating liquids.

In addition, in the above embodiment, in order to eject droplets containing test particles, a method is shown in which bubbles are generated using thermal energy and the droplets are ejected by the impact of the expansion, but an alternative method is to use a piezo element, etc. A method may also be used in which droplets are ejected by mechanically applying impact using a vibrator. In this case, as in the embodiment using bubbles, so-called on-demand ejection control is possible in which liquid is ejected according to an arbitrary ejection timing signal.

Furthermore, although the above embodiment shows the most basic form of an optical system for particle measurement, various improvements can be made in order to perform more accurate measurement. For example, if the irradiation optical system is used as a scanning optical system and the measuring section is optically scanned at high speed, the tolerance to the positional deviation of the particles to be measured can be further increased. Common scanning means include an acousto-optic deflection device (AOD) and a vibrating mirror. Further, if an array sensor such as a CCD is used as the light receiving optical system, image information of the particles to be detected can be captured, and the amount of analysis information can be further improved.

Furthermore, as shown in Japanese Patent Application No. 1-280707,
By arranging a plurality of ejection nozzles side by side and ejecting droplets one after another from each nozzle, the throughput can be further improved.

[Effects of the Invention] As described above, according to the present invention, individual test particles in a particle suspension can be measured more reliably with a simple configuration.

[Brief explanation of drawings]

1 and 2 are block diagrams of the embodiment of the present invention, FIG. 3 is a block diagram of the control system, and FIG. 4 and 1iS5 are block diagrams of the conventional example, and the main symbols in the figures are , 1...nozzle, 2...supply port, 3...heating section, 4...heat radiation member, 5.6...pressure detector, 7...
...Laser light source, 8...Bubble, 15...Band pass filter, 16...Transparent glass plate, S...
・Cell No. 1 ￥2

Claims (4)

[Claims] (1) A particle measuring device that separates test particles one by one in a particle suspension and measures the separated test particles, comprising: a storage section that stores the particle suspension; an opening for discharging the suspended liquid to the outside; means for applying discharging energy into the storage section to discharge droplets containing individual test particles from the opening; provided at a position opposite to the opening; A particle measuring device comprising: a measuring section to which droplets ejected from the measuring section are attached; and means for measuring test particles in the measuring section. (2) The particle measuring device according to claim 1, wherein the measuring section is a transparent plate, and the irradiation light is irradiated from the back side of the transparent plate on which the ejected liquid is attached to optically measure the particles to be detected. (3) The measuring section is disposed obliquely with respect to the opening, and the ejected droplet adheres to the measuring section from an oblique direction, and the test particles in the droplet pass through the measuring position. The particle measuring device described. (4) having means for applying thermal energy into the housing part;
The particle measuring device according to claim 1, wherein the droplets are ejected by heating to generate bubbles.