JPS5979835A - Apparatus for analysis of minute particle - Google Patents

Abstract

PURPOSE:To enable highly accurate analytical treatment due to a microcomputer, by a method wherein a specimen of a minute particle suspension is injected as a jet stream comprising liquid droplets after subjected to fluorescent staining and the optical characteristic signal of light generated by the irradiation of laser beam is separated into two signals by classification treatment. CONSTITUTION:A specimen of a minute particle suspension subjected to fluorescent staining is injected into a flow cell 2 as a jet stream comprising liquid droplets from a section 1 for selecting a liquid to be inspected and regulating pressure and irradiated with laser beam from a laser beam source 5. Generated lights are passed through lens systems 7, 7', 7'' and a dichroic mirror 8 and incident to a scattered light detector 13, a fluorescence I detector 14, a fluorescence II detector 15 and a fluorescent polarization detector 16 through a scattered light optical system 9, a fluorescent optical system I 10, a fluorescent optical system II 11 and a fluorescent polarization optical system 12. The outputs thereof are separated into a scattered light signal and a fluorescent signal through preamplifiers 17-20, a multiparameter signal treating circuit 21 and an interface 22 by classification treatment and these signals are sent to a usual microcomputer 23. The analyzed data subjected to operation treatment are displayed by a display system 24.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a microparticle analyzer, which measures the number, shape, and
Analyze sample particles based on the properties of microparticle samples by automatically determining their properties, etc. 11! This invention relates to a small particle analyzer for determining the number of particles.

The most common device based on this function is what is known as an a-cytometer, which is capable of making high-speed judgments based on the properties of cells revealed as a result of analysis, and is therefore useful for diagnosis of medical conditions and genetic biology. The current situation is that demand and demand are increasing in various fields.

A flow cytometer analyzes and measures cells at a rate of 5,000 or more cells per second and outputs the analysis results.Generally, the flow cytometer outputs the results of the analysis at a rate of 5,000 or more cells per second. The part where the test liquid such as cells @ suspension is poured into the tube, and immediately after the cover liquid flows out of this 9-capillary nozzle, it is irradiated with Rade light. Scattered light detection unit that detects scattered light, fluorescent light detection unit that detects fluorescent light, fluorescent light elimination jtt
Wipe out the fluorescent β light and detect the degree of detection, etc. /
fj part, output of photodetector Z, etc., height of 4 rus, /9
r of the circuit that detects the area of the pulse and the width of jB4 at the time of pulse.
1% of analog performance, analog performance IT section output t A/D
The frequency distribution of the interface dIS minute and the pulse height, the frequency distribution of the area of the armpit, and the frequency distribution of the time width of the arm? f-
It consists of a microcontroller part that displays the δF calculation, and the outflow velocity from the jet nozzle reaches approximately 10 mK per second. The flow cytometer, which determines the number and size of cells by detecting them with magnification, etc., determines the number of cells and the size of the cells.
In addition to simple analysis, we can also shape the Rede light using a cylindrical lens to determine the maximum diameter and reduced diameter of the cell, the position of the nucleus within the cell, or (ii) the ratio between the cell diameter and the nuclear diameter. Mli against Rede Hikari! It is also possible to add functions such as preventing erroneous judgments caused by changes in the alignment direction of the cells.Also, at a position just one point away from the tip of the Noet nozzle, the laser 11!! 1l at different angles
Detection of fluorescence from the IL cell - IA in the fluorescently stained cells is excited by the cells and undergoes a transition to the filamentous state. In order to emit fluorescent light, it can be emitted from cells; for example, the light is split into two wavelength regions, red and green, using a dichromic mirror, and the light intensity at each wavelength is multiplied by one light source. Detected on ig part 11
Wave height analysis of Ki; 1? From the fluorescence intensity, JAt of LINA in the cell is mainly measured as d1.

Fluorescence: qp, the elephant had a 1d fiber with a fluorescent flux, #+ attached. i
ll 711 Inadokoro and its surroundings 4) It also includes information about the surrounding conditions, so it's a fluorescent rtt4 light! Measuring ki light individual light dissociation, analyzing the energy transfer between molecules in the 111 cells: a, etc., etc., are detailed +! The structure of ill vacuoles, the most notable explanation 1 will be carried out.

In this way, several pieces of determination information such as If' and JL are each converted into electric lrt signals by the detector and amplified, and then
Signal processing circuit 1 with music and wave height analyzer or oscilloscope 1
1! The signal processing circuit measures the number of cells using scattered light, and the signal processing circuit measures the number of cells using scattered light. Fluorescent wavelength range signal ratio @ 舅, Temporary light polarization M
'6 Extreme Encyclopedia, cell distribution is collection control% It'+
” - 4iaj = operation control for the time lag between scattered light detection and fluorescence or fluorescence depolarization detection, which occurs in several drops, and the compatibility between scattered light and each extraction In general, analysis information from a flow cytometer such as wave height analysis is digitized rather than analog No. 16, and data processing using a microprocessor or computer is more expensive. However, data processing with a microprocessor requires multiple signals that occur at short time intervals and the time it takes to input them into the microprocessor.
:jF! It is not used in 70-cytometers because there is a discrepancy between it and E and it is difficult to directly handle the 1M number converted to /D. Conventional congi coater or mini coater
The combiator can be used for one hour, and the A/D converted signal may be collected in the data recorder and processed off-line, but the mini combi S
However, it is well known that there is a strong desire to solve this problem because it is difficult to operate, and the equipment is large and expensive.

Although it is possible to display measurement results in real time by observing Abu log information with ON0 Skog and using a multi-channel wave height analyzer, multiple types of IIT! It is also well known that there are limits to I-determination.

Therefore, it is an object of the present invention to solve these problems by providing a flow cytometer and an interface therefor that can perform advanced data processing using a commercially available combi-meter. It is. book 4
The interface by % Ming is one piece 4! - It is possible to assemble the path from the flow cytometer housing to the interior of a commercially available flow cytometer.
The micro combinator used in the present invention is the most compact Shinkai No. 1 P-belt type and is often
L makes it possible to provide a popular, small-sized, fine particle separation iron with excellent operability.

As already mentioned, the microparticle separation apparatus of the present invention is suitably used as a flow cytometer for the purpose of phase separation and separation sampling of the number, size, properties, etc. of biological cell particles, but it can also be used for other purposes. It goes without saying that it can also be effectively used as a cell sorter or as a device for separating microparticles other than various biological cells.

Hereinafter, the present invention will be described in more detail as a flow cytometer 'kM' with reference to the drawings.

FIG. 1 shows a block diaphragm of a cell sorting device with an internal interface and a micro combinator. In other words, in the drawing, 1 is the temporary structure liquid feed 0 and LE force adjustment fTbx2 is the flow
cell, 3 is a drain system, 5 is a laser light source, 6 is a light source lens system, 7, 7', 7'' are lens threads, 8a dichroic electric mirror, 9 is a scattering optical system, 1 () is a fluorescence optical system ■, 11 is a fluorescence optical system 11.12 is a fluorescence polarization optical system,
13 is a scattered light detector, 14 is a fluorescence detector 2.15 is a fluorescence 11 detector 3.16 is a fluorescence polarization detector 4.17 is a preamplifier, 18 is a preamplifier i2. ．． 19 is a preamplifier 3; 20 is a preamplifier 4; 21 is a multi-function Metai No. 8 processing circuit (analog calculation section); 22 is an interface; 23 is a microconbeater; and 24 is a display thread.

FIG. 2 shows an example of a block diagram of a portion related to the above-mentioned interface 22.

21 is the multi-parameter signal processing circuit shown in FIG. or any two of the firefly opening signals. Nire et al.'s signal is a signal output corresponding to one cell, and 21 is an analog calculation unit that converts the /4 pulse height, pulse width, and pulse area into voltage. That is, the outputs from the four detectors are subjected to one or two types of signal transmission, gain control, integration, peak hold, etc. according to the purpose of measurement.

Pulse output A% from 21 and pulse output B from A/1) & (Luuki, 31 and 3 respectively
2 to be digitized. 35 and 36 are shift registers, and when 4M No. A and 4M No. B are mutually stored in one of them, and each resistance of the shift register is discharged, the other shift register is stored with IId No. A and I. L1 No. B starts accumulating in the same way. A shift register controller 39 controls the operation of the shift register.

When the shift register is filled with the amount, the interrupt Mfl
The operation of i transfers r to the microcomputer via the input/output path interface 41. Opening/closing the flow of signals A and B to the two series of shift registers is as follows:
The circuits 33, 34, 37, and 38 perform this. The output from 41 is data processed within the microcomputer and output to a display system such as a printer or plotter. Software that requires high speed, such as interrupt processing to the computer, is written in assembly language, and other parts are programmed in RASIC. Controls the movements of the face.

Below, FIG. 3 shows a more detailed embodiment of the interface, and FIG. 4 shows a time chart of the operation.

Among the outputs from each detector, the multi-)4 shown in Figure 2
It is assumed that the scattered light signal is selected and input as the human power A in FIG. 3 and the fluorescent signal signal is selected as the human power B in FIG. 3 in the two meta signal processing circuit 21. Signals A and (MJiif8 are digitized by analog converters 51 and 52, respectively.
If the D converter is 12 pins or 5 pins, the R4 digital converters A and B pass through 12 pass lines each, then pass through one path such as a buffer amplifier, and after being processed are micro-
The output cable to the computer is sent to end 70. Third
The figure schematically represents these pass lines.

By the way, each converter outputs the signal EOC (End of Conv@rfilon) every time the A/D conversion is completed.
).

Each A/D converter IOC number is inverted by the N/D circuit 53, and the 056 input to the one-shot circuit 56 outputs an inverted output Q at the rising edge of the input signal. This signal then passes through the NOR circuit 57 and becomes one signal.

input to the cut circuit 58, 58 is the falling edge 9 of Q4M-1't.
/# /l/ x No. 414 Q is generated. Here, the /4' pulse width of the one-shot circuits 56 and 58 is determined by the time constant of each one-shot circuit, but the pulse width of the one-shot circuit 5B is τ111''1. The time relationship between them is as shown in the time chart in FIG.

When the time set by the time constant .tau.2 of the one-shot circuit 58 has elapsed, the pulse of .9 falls and this coefficient is manually applied to the counter 63, which causes the counter QA to operate 6 to count one stitch.

The QA of the counter is inverted and returns the one-shot circuit 58ρ to its original state. This results in 5
8 is τ! A pulse with a time width of is generated. Occurred in 58.
The clock operates the counter 63 and performs a carry. On the other hand, the output of counter 63 enters buffer amplifiers 54 and 55 via an inverter. Is the output of 563 mutually opening and closing buffer amplifiers 54 and 55? −) make an action. In other words, when the output of the counter 63 is positive, the buffer amplifier 5
When 0 is an inverted signal, the buffer amplifier 52 is opened. As a result, the shift register 60 receives signals A and 1 from the A/D converter.
Read h issue B alternately.

The counter in this embodiment uses a hexadecimal system, and therefore counts 8 pits for each set of A and B. The output of the maximum digit of the counter 63 switches the operations of the shift registers 59 and 60.

Furthermore, the output of the one-shot circuit 58 is branched to one-shot circuit 58.
The signal enters the shot circuit 64, which generates a predetermined pulse with a predetermined time constant τ3.

With a delay time of τ3, the output of 64 operates a one-shot circuit 65, and similarly, 65 generates a nof pulse determined by a time constant τ4. The output of the one-shot circuit 65 is sent to the shift registers 59 and 60 through a logic circuit 66 combined with AND and OR, respectively, to shift the data read into the shift registers. These shift registers 59 and 60 guide the output of the maximum digit of the counter 63 to the layer resistance circuit 66, and decide which of them is to be operated.

The data stored in the shift register is transferred to the pass line 70 after the command from the microcomputer is input from 69.
and output to the microcomputer. Depending on which of the two shift registers should be read into the micro converter, the operating order of these shift registers and the blocks are determined.
- It was added by the gogo of the tashift command, and the counter 63
Freezes depending on the state of the largest digit.

Fritsuno frogs 67' and 67'' generate a 511ill input operation command to send the data to the micro converter when the shift registers 59 and 60 operate alternately and data storage is completed.

The interrupt command is sent via the OR circuit 68 to the cutting command output terminal 72 as well as to the microconbeater. The frits and furtsnos 67' and 67'' are connected to a shift register via a logic circuit 66 and buffer amplifiers 61 and 62. In this embodiment, the shift register is used to
- Although temporary storage was performed in the evening, random access memory (RAM) can also be used.

FIG. 5 shows a block diagram using RAM. Compared to shift registers, the amount of data stored can be increased, and it is not necessary to arrange data alternately.

The signals digitized by the ADi converters 81 and 82 enter the RAM 87 via buffer amplifiers 83 and 84, respectively, and are constituted by counters. The data is read into the address specified by the command from the RAM controller 85. The RAAI has a plurality of data storage sections, and data is inserted into one of them, and the inserted data is sent from the other to the micro converter. Reading is performed by specifying the address by command.Output to the microcomputer is performed by the interrupt controller 86.
The signal appears at the 1 output terminal 42 due to the operation of .

The interface of the present invention reads analytical data from a flow cytometer into a shift register or random access memory, while transmitting previously read data to a microcomputer. Therefore, data generated at high speed can be sent to the computer in bulk, and by performing interrupt operations, the use of commercially available microcomputers becomes more efficient, and at the same time, the burden on the microcomputer is reduced. be able to.

Next, the configuration of an optical system suitable for the apparatus of the present invention will be described.

Fig. 6 mainly shows the optical system of this device, in which the sample and inert liquid are fed under pressure from the liquid to be detected/pressure vI4 adjustment section 1 to the flow cell 2, and from the capillary nozzle in the flow cell as described above. It flows out while maintaining a laminar flow state. On the other hand, the irradiated light 1 from the Rede light source 5 passes through the light source lens system 6 and is irradiated onto the sample in a laminar flow state, and the scattered light and fluorescent light from the sample are transmitted to the scattering optical system 9, the fluorescent optical systems 10, 11, respectively. In a preferred embodiment of the present invention, as shown in FIG. 171
Use. Divide one end of the bundle of Oguti fibers into multiple thin bundles, and divide the end faces into 1 to 50 areas as shown in the figure.Then, the scattered light can be divided into arbitrary angular components and photometrically measured. In this case, side torso identification processing can be performed as a five-dimensional vector. Alternatively, only essential sections among the fractionated portions may be extracted.

Figure 8 shows the condensing part in the fluorescent optical system.
181 is the optical axis of the Fi irradiation leg light, and 182 is the optical axis of the fluorescent light emitted from the sample. Generally, when the irradiated laser beam passes through the flow cell 2, the scattered light generated on the capillary wall is scattered horizontally in a donut shape. Therefore, in order to prevent this scattered light from entering the measurement optical system, the irradiated laser beam is optical axis 1
The fluorescent light is focused at an angle of 00 to the horizontal plane including 81. As for the thick field of θ, it is desirable that 0≧, where ψ is the condensing angle (half angle) of the fluorescent light.

FIG. 9(a) + <b> shows a light source lens system 6 for guiding the irradiated Radhe light to the sample. Figure 9 (a) middle 1
91 is a circular light beam from the laser beam yA5, 192 is an elliptical light beam after passing through a pair of cylindrical lenses, and 193iJ cylindrical lens. The two cylindrical lenses 192 are arranged in a cross pattern as shown in the figure. In this way, the light irradiated onto the sample can be converged into an arbitrary circular shape, so that the optimum radar irradiation light can be obtained depending on the size of the cell to be measured.

However, if the cylindrical lens 193) i is used,
As shown by a, b, e, and d in Figure (b), scattered light is generated at the entrance and exit surfaces of the cylindrical lens 1930. In order to prevent this scattered light from entering the measurement optical system, a pinpole mask 194 is placed between the cylindrical lens and the flow cell 2 in a further preferred embodiment of the invention.

Further, in a preferred embodiment of the present invention, as shown at 161 in FIG. 6, a heat exchanger lock for temperature control is attached to the tip of the capillary nozzle in the flow cell 2, and the sheath liquid is removed immediately before entering the cell. Q. Controlling the temperature of the sample liquid and the sample liquid Q. Since both liquids approach the capillary nozzle and flow out into the flow cell 2, temperature changes are likely to occur due to high flow path resistance. remains constant, increasing measurement stability.

Furthermore, in a preferred embodiment of the present invention, although not particularly shown, a particle counter is provided in the measurement system. This particle counter is a meter that monitors whether particles of interest are being measured per certain period of time. This particle monitoring allows the sample flow rate to be controlled so as not to exceed throughput limits. FIG. 10 is an example of the results obtained using an interface using a shift register, and is a forward scattering histogram of a mixed sample of mouse red blood cells and Linda's cells. FIG. 11 is an example of a fluorescence histogram, and FIG. 12 is an example of a two-dimensional distribution diagram for blood.

As described above, according to the present invention, a microparticle analyzer having an interface capable of high-speed processing by being connected to a microcomputer can be obtained.

Furthermore, a configuration including an optical system suitable for measurement with such an apparatus can be obtained.

[Brief explanation of drawings]

Figure 1 is a program diagram of this device, Figure 2 is a program diagram of an interface according to the present invention, Figure 3 is a schematic diagram of an interface circuit using a shift register, and Figure 4 is a timing diagram of the interface operation.・Chart, Figure 5 is an interface block diagram using RAM, Figure 6 is a diagram mainly showing the optical system of this device,
Figure 7 shows the scattering optical system, Figure 8 shows the condensing part of the fluorescent optical system, Figures 9 (a) and (b) respectively show the light source lens system, and Figure 10. is a diagram showing ψ1j of a forward scattering intensity histogram, FIG. 11 is a diagram showing an example of a fluorescence intensity histogram, and 9! The IJ12 diagram is a diagram showing an example of a two-dimensional distribution map output. 1... Test liquid selection/pressure cocoon adjustment section, 2... Flow cell, 3... Drain system, 5... Rade light source, 6... Light source lens system, 9... Scattering optical system, 10.11... Fluorescent optical system, 12... Fluorescent polarizing optical system, 13-16...
・Detector, 21...Analog 1jlNN (multi-parameter signal processing circuit), 22...Interface,
23...Microcomputer, 24...Display system,
161... Heat exchange block, 171... Oguti fiber, 181... Optical axis of irradiated laser beam, 18
2... Optical axis of fluorescent light, 191... Circular luminous flux, 192.
...Cylindrical lens, 193...elliptical luminous flux,
194...Pinhole mask. Applicant: JASCO Corporation Agent
Yukio Maruyama No. δ Figure No. 1 / No. /J Mr. Kazuo Wakasugi, Commissioner of the Patent Office 1. Indication of concerns 1982 Patent Application No. 190059 2 Name of the invention Microparticle analyzer 3 Relationship with the person making the amendment Patent applicant address 2967m5 Ishikawa-cho, Hachioji-shi, Tokyo Name 6r accumulation〒f% Representative director of a large company Examiner 1 Ikari T de 4, Agent 108 Address 3-25-27-1208 Takanawa, Minato-ku, Tokyo Telephone (03) 443-8886 - February 2, 1980 (Shipping date: same year) February 22nd) 6. Subject to correction
Power of attorney, 2 drawings of specification. Japanese Patent Publication No. 59-79835 (11)

Claims (1)

[Claims] 1.1 Microparticle suspension/l1ll liquid and the flow section where an inert fluid is flowed through the suspension. A jet nozzle part that injects as a central axis, maintains the confluence of the 1 suspension and the inert fluid in the 1a flow state, and causes it to pass through the detection area in the 1-flow state, to the detection area 11. The light emitting part detects the scattered light, fluorescence, and the degree of polarization of light emitted from the sample by the radar light irradiation. Analog performance that calculates the ratio, difference, sum, logarithm, or combination of one or more signals (height, time, area) of a linear signal! !
l-#% The output of the analog calculation section is converted to A/'D%7, and the microphone 0;
, Human power of the interface section 1') Frequency distribution of the height of the pulse-like signal from the output of the f-report, +OJ of the arm-shaped signal
*In a device consisting of a microconbeater section that displays the frequency distribution and the frequency distribution of the time width of a pulse-like signal, the A/1) converter output signal is sequentially read and recorded, and when it expires, the information is transferred to the next side. A series of signal readings of the moving train part consisting of a luxury number sequence that sequentially repeats the anti-θ action is performed by multiple 8' $ 16 & E, No. 1i, the hyperactive train 1aS, reads ω. After the expiry of the transfer, another signal reading J moving column performs f+' in the same manner, and it also reads the signal hL; What is claimed is: 1. A device for analyzing Fuko Miko, characterized by having one and both logic circuits for inputting information to a micro combinator. 2. Microparticle hanging i1! A flow pipe section through which an inert fluid flows through the liquid and the θl~ liquid, and microparticles are suspended in the middle of the flow pipe section.
, a jet nozzle part that injects the t-non-residue non-permanent fluid as the central axis, maintains the confluence of the suspension and the inert ηC body in a flow state, and causes it to pass through the detection area in the flow state; A radar source unit that irradiates the detection area, a light detection unit that detects the light intensity such as scattered light, fluorescence, and degree of fluorescence polarization from the sample due to radar light irradiation; /# An analog calculation unit that calculates the ratio, difference, sum, logarithm, or a combination thereof of one or more pieces of information of the 4-channel output signal (height, time width, area); An interface unit that converts the output of the analog calculation unit into the microcombi j-tani, and from the output of the input information of the interface unit, the frequency distribution of the height of the nozzle-like signal and the area frequency distribution of the pulse-like signal , when the signal is in the form of a pulse f) In a device consisting of a micro combinator unit that displays the frequency distribution of JJ Ig, the operation of storing the output signal of the D/D converter is independent of the time required and the storage location. It is equipped with a storage section capable of reading the signal and reading the signal t.
-Reading record 1. What is claimed is: 1. A microparticle analyzer, comprising a control logic circuit section for inputting information to a microcombination controller. 3. The scattered light detection unit that detects the scattered light from the sample by laser beam irradiation divides the fiber bundle into a plurality of fiber bundles and separates them into 7% fiber bundles. The cape of the patent claim is characterized in that it has a crotch TFL'L on the front side of the Enoki's detector. The microparticle analyzer described in Items 1 and 2. 4. Claim 441, characterized in that the fluorescence detection unit that detects the fluorescence from the sample due to radar light irradiation focuses the fluorescence at a selected angle with respect to the incident optical axis plane.
14'b The microparticle analyzer according to item 2. 5. Rade irradiation optics rtlζ that irradiates the sample with laser light
% characterized by a cross-combination of two cylindrical lenses that converge the laser beam into an arbitrary elliptical shape.
#'f Microparticle analyzer listed in 1st tomb and 2nd item 8C of scope of precision. 6. The microparticle separator described in item 4' and item 5 of the special fraud request, characterized in that a pinhole mask is installed in front of the sample. A small particle analyzer according to claims 1 and 2, characterized in that a temperature control means is provided. 8. A microparticle analyzer according to claims 1 and 612, characterized in that the particle i1 has a particle size of 1.