JP2749906B2 - Particle measurement device - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a particle measuring apparatus that separates and measures individual test particles in a suspension in which fine particles are suspended, one by one.

[Prior Art] Conventionally, as a method of separating many particles in a particle suspension liquid one by one, a sheath flow method as shown in FIG. 11 is generally known. In this method, the particle suspension and the sheath liquid are each pressurized by a pressurizing device, and the particle suspension is flowed so as to be wrapped in the sheath liquid. This is a method in which the components are flown in a row and separated.

In addition, a device such as a blood cytometer, which prepares a sample such as blood, measures individual cells in the sample separated by the sheath flow method using an optical method or the like, and performs cell analysis and cell sorting, is a flow cytometer. And has been put to practical use under the name of cell sorter.

FIG. 11 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 flow in a line in the flow cell are irradiated with measurement energy, for example, a light beam from a light source, and the light to the cells is irradiated. An optical reaction caused by irradiation, for example, scattered light or fluorescence emitted from cells is measured by a detector. Based on the photometric output, the signal processing unit calculates the type and size of the particle analysis.

FIG. 12 shows an example of the configuration of a cell sorter. A cell suspension such as blood is guided into the nozzle by an external pressurizing device, and a laminar flow centered on the cell suspension is formed in the nozzle by the sheath flow principle. At an orifice (70 to 100 μm) at the nozzle outlet, Average cell suspension diameter is 15
Released into the air as a jet stream of 2020 μm. Excitation light from a light source 24 is applied to the jet stream that has flown out into the air at a position 100 to 200 μm from the nozzle tip. When excitation light is applied to cells that have been previously stained with fluorescent light, scattered light and fluorescent light are emitted from the cells, and are detected by detectors 25 and 26 and converted into electrical signals. On the other hand, the nozzle is vibrated at about 40 KHz by the vibrator 21 under the control of the vibrating circuit 27, and the jet flow exiting the orifice becomes uniform droplets several mm below the nozzle tip. Depending on whether the signal detected from the cell satisfies a predetermined condition, a signal is sent from the signal processing unit 29 to the charging circuit 28, and a charging signal is added to the nozzle in synchronization with the formation of the droplet, and the liquid is discharged. The drops are lightly charged. While the charged droplet containing the desired cell passes between the two electrodes 22 and 23 that generate a strong electric field, the droplet is deflected to the right or left depending on the type of cell and the like by the static electricity, and is distributed. Collected in separate test tubes.

[Problems to be Solved by the Invention] In the sheath flow method used in the flow cytometer and the cell sorter, the cell suspension and the sheath liquid are guided to the nozzle by pressurizing each liquid with a pressurizing system. However, since a pressurizing device such as a pipe and a pump for that purpose is required, there is a problem that the device is large and the control is complicated.

In the sheath flow method, the flow velocity of the flow is determined by the pressure applied to the cell suspension and the sheath liquid, and the time interval for separating particles is determined by the flow velocity and the degree of dilution of the cell suspension. It was difficult to change in scope.

Further, it is difficult to reduce the size of the device, and it is difficult and impractical to increase the processing capacity by arranging a plurality of devices in parallel at high density.

[Object of the Invention] An object of the present invention is to provide a novel particle measuring device using a method different from the conventional sheath flow method.

[Means of Reserving Means for Solving the Problems] The present invention for solving the above-described problems is directed to an apparatus for separating and measuring individual particles in a particle suspension, wherein a storage unit for storing the particle suspension and having an opening, A particle measuring device, comprising: means for discharging liquid droplets containing particles from the opening by heating the liquid in the storage section to generate bubbles, and measuring means for measuring individual particles. .

Embodiment 1 Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings. 1 to 4 are block diagrams of a first 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 serving as test particles float, and one end thereof is opened to form an opening. As an example of a method of forming the nozzle, a method is generally used in which a fine groove is provided on a substrate by an etching or photoresist process, and a flat plate is bonded thereon, but the method is not limited thereto. The size of the cross section of the nozzle is set to a size suitable for the size of the test particles to be measured. In this example, the measurement target was blood, and the size of various blood cells contained in the blood was about 5 μm to 30 μm. Therefore, the nozzle size was set to 50 μm × 50 μm, which was slightly larger than the maximum blood cell size.

Reference numeral 2 denotes a supply port for sequentially supplying the cell suspension into the nozzle 1 and is connected near the outlet of the nozzle 1. Reference numeral 3 denotes a heating unit provided in the nozzle, specifically, a heater, whose electrodes are connected to a control circuit described later. The heating unit 3 may be located outside the nozzle to heat the inside of the nozzle. The heating unit 3 is not limited to a heater, but may be any unit that generates heat energy. For example, the heating unit 3 may be configured to apply electromagnetic wave energy such as a laser beam to a heat absorbing member to heat the heat absorbing member. Reference numeral 4 denotes a heat radiating member provided outside the nozzle 1 for suppressing the temperature of the nozzle 1 from rising due to heat conduction from the heater.

7 is a laser light source as a measurement energy generating means,
Reference numerals 5 and 6 denote photometric detectors for detecting an optical reaction from particles caused by light irradiation. The optical axis O of the laser light emitted from the laser light source 7 causes droplets ejected from the opening of the nozzle 1 to fly. The laser light source 7 is arranged so as to intersect with the axis l of the path to be moved. The detector 5 is disposed on the laser optical axis O, and a beam stopper (not shown) for preventing a laser beam from being directly input to the detector is provided on the optical axis in front of the detector 5. Only the scattered light to be scattered is measured by the detector 5. The detector 6 is arranged in a direction orthogonal to each of the laser optical axis O and the droplet discharge axis l, and an optical filter (not shown) having a property of transmitting only a fluorescence wavelength is provided in front of the detector 6. , Only the fluorescence is measured by the detector 6.

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

A sample for measurement, such as a blood sample, which has been sufficiently diluted and stained with a fluorescent reagent or the like as necessary, is prepared, and the sample is supplied to the supply port 2 and supplied to the nozzle 1 from FIG. As shown in FIG.

Here, the control by the control system of FIG. 10 will be specifically described. FIG. 10 is also used as a control system of a fourth embodiment to be described later. In this embodiment, only one heater is provided.

When the ON-OFF generating circuit drives the heater 3 provided in the nozzle 1 to generate heat, the water in the heated cell suspension is instantaneously vaporized and bubbles 8 are generated as shown in FIG. I do. Then, the volume expands by an amount corresponding to the vaporization, so that the cells S near the opening of the nozzle 1 are pushed out from the opening of the nozzle, and as shown in FIG. Is discharged to The bubbles 8 that have been expanding initially are cooled and start to be stored, and the volume of the bubbles 8 is reduced, so that a drawing force acts on the cell suspension discharged from the opening. As a result, the cell suspension containing the cells S discharged from the opening becomes droplets and flies into the air as shown in FIG. The basic principle of this ejection is described in, for example, JP-A-54-59936 and JP-A-55-27282.

The cell suspension is supplied from the supply port 2 by the capillary action as much as the discharged liquid, and returns to the state as shown in FIG. Since the supply of cell suspension is performed naturally by capillary action,
No conventional pressurizing system is required.

The generation and extinction of these bubbles are performed in a very short time according to the sampling frequency created by the sampling synchronization circuit in Fig. 10, and it is possible to continuously discharge up to several thousand droplets per second. It is. Thereby, a large number of cells can be separated at high speed.

The size of the opening and the capacity of the heater are set so that the droplet discharged from the nozzle has a diameter of about 50 μm to 80 μm, and the particle is suspended so that only one particle is contained in the discharged droplet. Set the dilution of the solution. Then, when the droplet traverses the portion to be inspected, which is irradiated with the energy for measurement, that is, the laser beam, an optical reaction by the cells previously stained with fluorescence contained in the droplet, that is, scattered light and fluorescence are generated, and the detection is performed. Detectors are detected by a detection system including the detectors 5 and 6, respectively.

Outputs from the detection system are sequentially stored in the data storage unit,
From the measurement data obtained for a large number of particles, the signal processing unit performs particle analysis such as particle type determination and property analysis using statistical processing such as histograms and cytograms. Since various methods are widely and generally known as specific calculation methods, a detailed description thereof is omitted here.
The analysis result is output on a TV monitor, printed out, or the like.

In addition, unlike the conventional sheath flow method, the apparatus of the present invention can freely set the time interval for separating particles in a wide range.Therefore, it is necessary to consider conditions such as a required processing speed, a calorific value of the nozzle, and durability. It is preferable to set the sampling frequency so as to obtain an optimum speed. In addition to driving continuously at a constant frequency, changing the frequency,
It is also possible to drive intermittently.

Embodiment 2 FIG. 5 is a configuration diagram of a second embodiment of the present invention, and the same reference numerals as those in FIG. 1 represent the same or equivalent members.

The present embodiment is characterized in that the target portion to be irradiated with the laser beam from the light source 7 is brought closer to the opening of the nozzle 1. That is, in the previous embodiment, the cells were irradiated with a laser beam in a state where the cell suspension was discharged to form droplets, and the cells were subjected to optical measurement, whereas in the present embodiment, the cells were discharged as shown in FIG. Immediately after the irradiation, the laser light is irradiated in a state before the droplets are formed.

[Embodiment 3] Figs. 6 and 7 are simplified block diagrams of a third embodiment. Note that the same reference numerals as those in the above-described explanatory drawings represent the same or equivalent members.

A cell suspension is supplied from a supply port to a nozzle 1 having a supply port and a discharge port at both ends, and droplets containing individual cells are discharged from the nozzle discharge port by heating the heater 3.

The optical measurement of the ejected droplets is the same as in the first or second embodiment, and thus the description is omitted.

Fourth Embodiment FIG. 8 is a configuration diagram of a fourth embodiment of the present invention. The same reference numerals as those in the above-described drawings denote the same or equivalent members.

Reference numeral 9 denotes a transparent material cell provided at the tip of the nozzle 1. The internal path of the cell 9 is tapered on the nozzle side, and the outlet side is narrowed down from the nozzle part so that cells flow one by one in a line. It is a thin tube.

The cell 9 is irradiated with a light beam from the light source 7, and the detectors 5 and 6 measure the scattered light and the fluorescent light from particles passing through the test portion in the thin tube in the cell 9 to which the light beam is irradiated. It has become.

When bubbles 8 are generated by the heating of the heater 3, the cell suspension in the nozzle 1 is pressed by the expanded volume, and the cells pass one by one through the narrowed inner diameter portion of the cell 9,
Discharged from the opening.

One heating may cause one or more cells to successively pass through the test portion of the cell 9, but optical measurement of individual cells is possible as long as one cell flows in a row.

In the present embodiment, instead of optically measuring the place where cells are ejected into the air, to perform optical measurement by irradiating light in a state where the cells flow through the transparent cell, the suspension containing cells is subjected to refractive index fluctuation and There is no influence of diffuse reflection, and a stable measurement value can always be obtained.

[Embodiment 5] Fig. 9 is a configuration diagram of a fifth embodiment of the present invention. The nozzle that stores the cell suspension and discharges droplets has a very small cross section and a structure that can be easily made by processes such as etching, so that it is easy to arrange a plurality of nozzles at high density in parallel. is there.

FIG. 9 is a diagram of a predetermined test portion on the laser optical axis.
A plurality of nozzles are radially arranged. In the figure, reference numeral 11 denotes a nozzle unit in which a plurality (five) of nozzles having the structure shown in FIG. 1 are arranged in parallel.
It is not limited to the individual. As an example, the nozzle unit can be formed by providing a plurality of fine grooves on a substrate by etching and laminating a flat plate.

Reference numeral 12 denotes a supply port of the cell suspension, and 13 denotes an opening of the nozzle unit 11. The structure of each nozzle may be as shown in FIG. Since the nozzles are arranged radially around the measurement point irradiated with the laser beam, the droplets sequentially discharged from the nozzles always pass through the same target portion. As a result, a single optical system for measurement is required.

The heaters in the nozzle unit configured as described above are sequentially driven in a time series by the control system shown in FIG. 10, and droplets are sequentially discharged from the openings of the nozzles. Let it. In FIG. 9, the ON-OF signal is set according to the sampling frequency generated by the sampling synchronization circuit.
The F generation circuit drives the heater of each nozzle in a time series.
The sampling frequency can be several times the critical frequency in the previous single nozzle embodiment. This is because, in order to drive a plurality of nozzles in chronological order, the cycle in which each nozzle is driven is a fraction of the cycle in the case of a single nozzle. As a result, the measurement speed can be greatly improved.

Also, when measuring cells that are extremely sensitive to temperature rise,
By setting a lower sampling frequency or increasing the number of nozzles, the driving load of each nozzle is further reduced, the amount of heat generated is reduced, and the adverse effect on the cells due to the increased temperature of the nozzle can be suppressed. Increasing the heat capacity of the heat dissipating member is also an effective means for suppressing a rise in temperature.

The output signals of the scattered light and the fluorescence measured by the detection system are detected according to the gate signal from the sampling synchronization circuit, and are separately stored in the data storage unit for each droplet discharged from each nozzle. That is, even if the cell suspension to be supplied to each nozzle is of a different type, measurement data is stored for each type in a single measurement. Of course, the same type of sample may be measured in parallel.

According to the present embodiment, a large number of nozzles are sequentially driven in time series to discharge particles one after another, so that the processing capacity can be dramatically increased.

In addition, since the nozzles are driven in time series, the driving load on each nozzle can be reduced. Thereby, the temperature rise of the nozzle can be suppressed, and the durability of the nozzle can be improved.

Further, since the nozzles can be juxtaposed at a very high density, the apparatus becomes compact, and the number of measuring optical systems is single irrespective of the number of nozzles, which is advantageous in terms of cost and space. .

Note that all the embodiments described above are examples applied to a so-called flow cytometer for analyzing particles by performing optical measurement.However, the present invention is applicable to a cell sorter provided with a sorting mechanism as shown in FIG. Is also possible. In this case, since the cell suspension can be formed into droplets without using a vibrator or a pressurizing device, the device becomes very compact and low-cost.

In addition, the measurement of individual particles in the test portion to which the energy for measurement is applied is not limited to the above-described optical method, but may be, for example, an electrical measurement using an electrical impedance or a measurement using electromagnetic energy. Or various measurement methods such as a photoacoustic method.

Further, all the embodiments described above are applied to an apparatus for analyzing fine particles in the biological field such as blood cells, but the present invention is not limited to this. The present invention can be applied to all devices that handle the suspension of test particles, such as a device for measuring.

[Effect of the Invention] According to the present invention, individual test particles in a particle suspension can be separated with a simple configuration. Therefore, a low-cost and compact particle measuring device can be obtained.

[Brief description of the drawings]

 1 to 4 are diagrams showing the configuration of a first embodiment of the present invention, FIG. 5 is a diagram showing the configuration of a second embodiment, FIGS. 6 and 7 are diagrams showing the configuration of a third embodiment, FIG. Is a block diagram of the fourth embodiment, FIG. 9 is a block diagram of the fifth embodiment, FIG. 10 is a block diagram of the control system, FIG. 11 is an explanatory diagram of the sheath flow system, and FIG. The main symbols in the drawing are: 1... Nozzle 2, supply port 3, heating unit 4, heat dissipation member 5, 6 detector, 7 laser light source, 11 ... Nozzle unit, 12 ... Supply port, 13 ... Opening

Claims (6)

(57) [Claims] An apparatus for separating and measuring individual particles in a particle suspension, wherein a storage section for storing the particle suspension and having an opening, and heating the liquid in the storage section to generate bubbles. A particle measuring apparatus, comprising: means for discharging droplets containing particles from the opening through the opening; and measuring means for measuring individual particles. 2. The apparatus according to claim 1, wherein said measuring means is an optical measuring means. 3. The apparatus according to claim 1, wherein said measuring means measures particles on a path along which a droplet discharged from the opening flies. 4. The apparatus according to claim 1, wherein said measuring means measures particles in the storage section before discharging from the opening. 5. The apparatus according to claim 1, wherein a plurality of the storage sections are provided side by side, and droplets are sequentially discharged from openings of the respective storage sections. 6. The apparatus according to claim 1, wherein the heating is performed by a heater provided in the storage section.