JPS59176674A - Separating and determining method of particulate separator - Google Patents

Abstract

PURPOSE:To improve the separation rate and separation purity of particulate by a simple device by comparing >=2 kinds of specifying signal obtained during particulate detection with the contents of a table stored with a specifying signal corresponding to the area of particulates previously, and electrifying a liquid drop so that the deflection direction of the liquid drop is determined. CONSTITUTION:A column 6 of water which flows out of the nozzle 2 of a pipe 3 and contains particulates is irradiated with light from a laser light source 14 and generated light is detected by detectors 15 and 16 to output analog specifying signals AS1 and AS2 regarding fluorescent intensity and scattered light intensity are outputted through an analog circuit 20 controlled by a timing control circuit 21. Those signals AS1 and AS2 are converted by AD converting circuits 23 and 24 into digital specifying signals DS1 and DS2, which are compared with the specifying signal table stored in an RAM26 by a muCPU25 to output a deflection signal corresponding to an area to be separated and sampled, electrifying a liquid drop to a specified code through an interface circuit 22, delay circuit 28, and liquid drop charge voltage circuit 29. Consequently, the separation rate and separation purity of particulates are improved.

Description

DETAILED DESCRIPTION OF THE INVENTION This invention is a method for handling microparticles 81 as typified by cell sorters.
! The present invention relates to a method for determining separation using an apparatus, and more specifically to a method for physically separating particles that differ in at least one of various properties such as function, size, and shape.

Recently, a device called a cell sorter that can separate and collect microparticles such as cells and lymphocytes at high speed according to their properties has been published in Japanese Patent Publication No. 46-27118 and Japanese Patent Publication No. 56-13266.
It was proposed in No. 1 and has been put into practical use.

As shown in Fig. 1, this type of device pressurizes an inert liquid 1 and sends it into a tube 3 with a nozzle 2 formed at one end of the T, so that the inert liquid flows into the tube 3 as a laminar flow. Let it pass;
On the other hand, microparticles such as sample cells are suspended in a liquid in advance, and the microparticles are suspended'? A4 is supplied into the laminar flow inside the tube 3, and an ultrasonic signal is given to the ultrasonic vibrator 5 installed at the upper end of the tube 3 to vibrate the nozzle 2, thereby causing the flow to flow down from the nozzle 2. A charging means 8 for charging the droplets 7 is connected to the tube 3, and when the water column 6 becomes a droplet 7, the droplet 7 is charged to a predetermined level. The falling droplet 7 is charged with a sign, and an electric field is applied in a predetermined direction by a deflection plate 9.10 to deflect the droplet 7 to the right or left depending on the sign of the weight of the droplet 7.
Depending on the deflection direction, the right deflection container 11 and the left deflection container 12
Alternatively, it is configured to separate and receive droplets in the non-deflecting droplet container 13. And the water column 6 flowing down from the nozzle 2
At a position above the position where the water becomes a droplet, there is a laser beam 8!14 that irradiates the water column 6 with a laser beam, and a plurality of detectors 1 for detecting fluorescence and scattered light caused by the laser beam.
5.16 is provided.

In such a device, information on the scattered light intensity and fluorescence intensity, which vary depending on the size and shape of microparticles such as cells existing within the droplet, can be obtained, and the charge sign of the droplet can be changed according to this information. This determines the right and left deflections of the droplet, and therefore, it is possible to separate microparticles such as cells with desired properties from other microparticles in accordance with the above information. In this case, separation may be performed based on only one type of information, but in cell separation, two types of information are combined, and a two-dimensional histogram based on the correlation between the two types of information, that is, a so-called cytogram, shows the cell. It is often necessary to determine C separation depending on whether it is located within a region. For example, as shown in Figure 2, a certain area A on the cytogram is determined by the scattered light intensity and fluorescence intensity.
The cell located in R is determined to be normal and the droplet containing that cell is deflected to the right, the other cells are determined to be abnormal and the droplet targeting that cell is deflected to the left, and the droplet containing that cell is deflected to the left. Often a method is required, such as dropping the drop without deflecting it.

By the way, in a conventional device of this type, the detector 15.
Two or more signals obtained from 16, and the difference, sum,
It has been usual to binarize a composite signal such as a ratio using a comparator, and then use a logic circuit such as an AND circuit or an OR circuit to determine right deflection or left deflection. Therefore, the reality is that only rectangular or similar separation areas can be set on the cytogram. A method is also known in which the signal is converted to ﾆ＼, /[1, the digitized signal is operated, and the left deflection and right deflection are determined by a calculation algorithm. However, in this case as well, the separation area set on the cytogram is limited to a separation area expressed by a relatively simple mathematical formula such as a circle, ellipse, or rectangle, and requires a computer with a high computing power of a high-speed mini combinator or higher. It was generally difficult to commercially use the space because it needed to be occupied.

However, in cell separation using this type of device,
The area on the cytogram of cells that the user needs does not necessarily have a relatively simple geometric shape such as a rectangle or an ellipse, but often has a complex shape. In this case, if you try to separate and collect the required cells in a relatively simple geometric area such as an oval or an ellipse, the originally required area Ii! It is not possible to specify an unnecessarily narrow region AROL for AR, and as a result, a considerable number of originally useful cells are discarded, resulting in a low separation rate. On the other hand, if we place emphasis on the separation rate and try to increase it, it is necessary to lower the purity of separation.

In addition, in clinical diagnosis, etc., when it is desired to separate cells based on the correlation of three or more types of information, a separation region is set on a similogram of three or more dimensions that correlates with three or more types of information. There are cases where it is desired to separate them, but such a thing is extremely difficult with devices using conventional logic circuits, and it is also difficult in practice even with conventional 1ζ loading using a measuring gun.

This invention was made in view of the above circumstances, and includes the above-mentioned particle separation device, which vibrates a liquid in which fine particles such as 1st grade are formed to form droplets, and at the same time By optically detecting the properties of the fine particles at the position and obtaining two or more types of selection signals corresponding to the properties of the particles, and by charging the droplets according to the selection signals and passing them through an electric field, a predetermined property can be obtained. In a particle separator that separates and collects droplets containing particles, the separation determination method using two or more selection signals is changed from the conventional method.
To enable separation on a cytogram of any dimension greater than or equal to two dimensions to be not limited to simple geometrical shapes such as rectangles and ellipses, but also to arbitrarily set regions with complex shapes, thereby achieving desired separation. The purpose of the present invention is to significantly improve the separation rate of microparticles such as cells compared to conventional methods, and at the same time to achieve good separation purity, and to be able to carry out the separation using simple and inexpensive equipment. It is.

That is, the method for determining separation in the particle separator of the present invention is to preset a region of microparticles to be separated and sampled on an n-dimensional (n is 2 or more) histogram that correlates with the two or more types of selection signals, and A table for each selection signal corresponding to the above is written in advance in RAM, and two or more selection signals obtained during particle detection are referred to in the table to determine the deflection direction of the droplet according to the table. This method is characterized by electrically charging the droplets.

This invention will be explained in more detail below.

FIG. 3 shows an example of an apparatus for implementing the method of the present invention, in which the outputs of detectors 15, 16 such as photomultiplier tubes are input to an analog circuit 20. The detector 15.16 is
As mentioned above, this is to irradiate the water column 6 emitted from the nozzle 2 with a laser beam at a stage before it turns into droplets and detect the resulting fluorescence intensity, scattered light intensity, etc. Multiple pieces of information can be extracted by simultaneously detecting multiple wavelengths or by changing the detection direction and angle. The analog circuit 20 performs peak hold, gating, etc. according to the signals from the detectors 15 and 16, or performs analog calculations such as sums, differences, ratios, etc. as necessary, to detect minute particles such as cells. Two or more types of analog selection signals AS+, AS2 regarding particle shape, size, strength, and other properties
Take out the analog selection signal A, for example regarding fluorescence intensity.
Analog selection signal AS2 regarding Sl and scattered light intensity
Output. Here, the timing of the analog circuit 20 is controlled by a timing signal from a timing control circuit 21. That is, the timing control circuit 21 mainly identifies passage of the detection point P of a microparticle based on the intensity of scattered light, and controls the operation of the analog circuit 20 in accordance with the timing of the passage. It also controls the timing of the R/L, E direction output interface circuit 22, etc.

The analog selection signals ASI and AS2 outputted from the analog circuit 20 are converted into digital selection signals DSI and DS2 by the A/D conversion circuits 23 and 24, and these digital selection signals DS+ and DS2 are connected to the microphone ■-J computer (or The data is taken into the microprocessor (microprocessor) 25 and compared and referenced with a table written in advance in the RA'Xl 26. Here, the RAM 26 stores in advance a region to be separated and sampled (for example, region AR in FIG. 2) on an n-dimensional (n is an integer on 2j) histogram (cytogram) that correlates with two or more digital selection signals. A table is created according to each selection signal, and the table is written. In this table, for example, addresses may be determined based on the level of the selection signal, and different values may be written depending on whether a point on the cytogram determined by the address falls within the separated region or not.

The microcomputer 25 receives an interrupt by the end A conversion (EOC) signal of the A/D conversion circuit 24 (and receives the digital selection signal DS+,
DS2 is taken in, the table value corresponding to the data is read out from the RAM 26, and R, /
'L Outputs a signal to the deflection output interface circuit 22. That is, for example, digital selection signals DS+, D
If the position on the cytogram determined by the data of S2 is within the separation area defined in the table, right deflection output data is output, and if it is outside the separation area, left deflection output data is output.

The right deflection output data or left deflection output data output in this way is output to the R, 'LB direction output interface J-
The signal is then delayed by a predetermined time period 8 in a delay circuit 28, and is applied as a right deflection control signal to a droplet charging voltage circuit 29 in synchronization with the droplet charge clock CLK. The droplet charging voltage circuit 29 applies a positive or negative voltage pulse to the droplet formation clock CLK according to the control signal in order to charge the droplet positively or negatively according to the right deflection control signal or the left deflection control signal. It is synchronously applied to a charging electrode (not shown) in the tube 3. Here, the delay circuit 28 is
This is the time required for the fine particles to reach the droplet formation point (corresponding to the charging point) Q from the detection point (measurement point) P in the water column 6. This is for delaying the Gino signal. However, since there is actually a delay time of each electric circuit and microcomputer, the delay is made by subtracting that time. By delaying the deflection ν1wJ signal in this way, the droplets are correctly formed when the liquid containing the particles is formed into droplets at the droplet formation point Q, in accordance with the data of the particles detected at the detection point P. It can be positively or negatively charged. The charged droplet is deflected by the deflection plate 9.10 as described above.
It passes through the charge between the two, is deflected to the right or left depending on the sign of the charge, and is separated and collected.

As described above, by referring to the table written in advance in the RAM 26 the selection signal obtained by particle detection, two or more types of selection signals corresponding to the properties of the particles can be separated as desired in the cytogram F. It is determined whether the particles fall within the area, and droplets containing the particles can be separated and collected accordingly. No matter how complicated the shape of the desired separation area is on the cytogram, it can be easily converted into a table and written to RAM, so that microparticles such as cells can be separated and collected using any separation area. can do.

Furthermore, the separation area is not limited to an area on a two-dimensional cytogram, but can easily be an area of any three or more dimensions. That is, it is possible to arbitrarily set a separation area based on the correlation between three or more types of selection signals.

In the example shown in FIG. 3, a pulse hide analyzer (PHA) 30 is provided separately from the analog circuit 20. This pulsed hide analyzer 30 has a detector 1
5.16, and plays the role of, for example, validating only signals with a predetermined height or higher.

FIGS. 4(A) and 4(B) show the microcomputer 2.
5 shows a flowchart in 5.

FIG. 4 (A>) shows a schematic flowchart from the start to the end of sorting, that is, particle separation operation. Once various conditions have been set in advance and a start signal is applied, the microcomputer becomes interruptible.
The data will be imported. Then, data is sequentially captured and particle separation is performed, and when it is detected that a certain amount of time has elapsed or that the number of particles has reached a certain number, an END signal is sent from another computer, counter, timer, etc. It is entered, becomes uninterruptible, and the separation operation ends.

FIG. 4(B) shows when an interrupt actually occurs, that is, when measurement is started and the EO from the A/D conversion circuits 23 and 24 is output.
This is a flowchart when an interrupt by the C signal is received, and if there is an interrupt by the EOC signal, this causes a state in which the interrupts of other signals are negative, and the data from the A/D conversion circuits 23 and 24, That is, the digital selection signals DS+ and DS2 are read. Then, the data is converted into an address of the RAM that stores the table in advance, and the value of the RAM table (map) at that address is read. Then, depending on the value in the table, right deflection data or left deflection data is outputted to the delay circuit through the RZL tfA erase interface circuit as described above. It then becomes interrupt-enabled again and returns to receive the next interrupt.

As is clear from the above explanation, according to the separation determination method of the present invention, it is possible to arbitrarily and easily divide a histogram correlated with two or more selection signals corresponding to the properties of fine particles.
One area (virtual space) can be set, and therefore, the separation area is not limited to relatively simple geometric figures such as rectangles or ellipses as in the past, but complex areas can be set. The separation rate of fine particles of different properties can be significantly improved compared to the conventional method, and even in this case, the separation purity is not reduced. Furthermore, it is possible to easily specify the separation region in a multidimensional space of three or more dimensions, and therefore the field of application can be further expanded than in the past. Furthermore, the method of the present invention can be implemented using a widely used microcomputer, and therefore can be implemented at low cost and the equipment can be miniaturized.

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram conceptually showing an example of a microparticle separation device, FIG. 2 is a diagram showing an example of a region on a two-dimensional histogram, and FIG. A block diagram showing an example, and FIG. 4 (A>, (B)) are chart diagrams showing a flowchart of a microcomputer when implementing the method of the present invention. 2... Nozzle 4... Fine particle suspension Liquid, 5...
Ultrasonic transducer, 14... Laser light source, 15.1
6...Detector, 20...Analog circuit, 25.
...Micro Combi Coater, 26...RAM,
AR, ARO...area. Applicant Nippon Bunko Kogyo Co., Ltd. Agent Patent attorney Yutaka 1) Hisashi Take (and 1 other person) Engraving of drawings (inherent (2 changes & L) (A) (B) Procedural amendment (Method) % formula % 1. Indication of the case 1982 Patent Application No. 51184 2. Name of the invention Particulate fraction IIjt Fraction determination method in gastric filling 3. Make amendments. Patent applicant address: 2967 Ishikawa-cho, Hatamako-shi, Tokyo (5 names) Japan Bunko Kogyo Co., Ltd. 4, agent address: 3-4-18-5 Mita, Minato-ku, Tokyo, amended order Date: June 28, 1982 (shipment date) 2) Submit an engraving of the drawings (no changes to the contents).

Claims (1)

[Claims] A liquid containing fine particles is vibrated to form droplets, and the properties of the particles are optically detected at a position before the droplet formation position to obtain two or more selection signals corresponding to the properties of the particles. , and in accordance with the selection signal, the droplets containing the fine particles are charged and passed through an electric field to deflect the droplets in a predetermined direction, thereby separating and collecting fine particles with predetermined properties. In the separation device, an area of microparticles to be separated and sampled is set in advance on an n-dimensional (n is 2 or more) histogram correlated with the two or more types of selection signals, and a table is provided for each selection signal corresponding to the area. is written in the RAM in advance, two or more selection signals obtained at the time of particle detection are referred to the table, and the droplet is charged in order to determine the deflection direction of the droplet according to the table. Separation determination method in a featured particle separator.