JPH0229185B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a microparticle analyzer, and particularly to a microparticle analyzer that automatically measures the number, shape, properties, etc. of microparticle samples in a continuously passing sample suspension. The present invention relates to a microparticle analyzer for analyzing and measuring sample particles according to their properties.

A device with this type of function is commonly known as a flow cytometer, and it is in demand in the fields of disease diagnosis and gene biology because it can quickly make decisions based on the properties of cells determined as a result of analysis. The current situation is that the number of people is increasing. A flow cytometer analyzes and measures cells at a rate of 5,000 or more cells per second and then outputs the analysis results.
In general, the following parts are involved, namely, the part where an inert liquid is pressurized and passed through the capillary tube as a laminar flow into which a test liquid such as a cell suspension is poured, immediately after the test liquid flows out of this capillary nozzle. a part that irradiates the laser beam with a laser beam, a scattered light detection part that detects scattered light from sample particles due to laser light irradiation, a fluorescence detection part that detects fluorescence, and a fluorescence depolarization degree detection part that detects the degree of fluorescence depolarization. Analog calculation part of the circuit that detects the output pulse height, pulse area, and pulse time width from the photodetection part, converts the output of the analog calculation part into A/D and inputs it to the microcomputer. It consists of an interface part that calculates and displays the frequency distribution of the pulse height, the frequency distribution of the pulse area, the frequency distribution of the pulse time width, etc.The outflow speed from the jet nozzle is approximately 10 m/s. The number and size of the cells are measured by detecting the scattered light generated by the irradiated laser beam on the cells in the sample solution, which has been fluorescently stained in advance, using a photomultiplier tube or the like.

Flow cytometers not only count cells and perform simple analysis of their properties, but also shape the laser beam with a cylindrical lens to determine the maximum and small diameters of cells, the position of the nucleus within the cell, or the cell diameter and nucleus. It is also possible to add functions such as determining the ratio to the diameter and preventing misjudgments due to changes in the orientation of cells with respect to laser light. In addition, when detecting fluorescent light from cells at a position slightly away from the jet nozzle tip at an angle different from the laser irradiation direction, the fluorescent-stained DNA in the cells is excited by the laser and returns to the ground state. In order to emit fluorescence during the transition process, the fluorescence emitted from cells is separated into two wavelength ranges, red and green, using a dichromic mirror, for example, and the fluorescence intensity at each wavelength is measured using a photomultiplier tube. The amount of DNA inside the cell is mainly measured from the intensity of the fluorescence. The phenomenon of fluorescence is complex and includes information about the intracellular location to which the fluorescent dye is attached and the surrounding conditions. Therefore, by measuring the polarization of fluorescence and the degree of depolarization of fluorescence, we can determine the energy between intracellular molecules. Detailed cell structure and properties will be elucidated, including analysis of transmission.

The multiple pieces of measurement information obtained in this way are each converted into an electrical signal by a detector and amplified, and then passed through a signal processing circuit to measure the signal strength with a pulse height analyzer or oscilloscope, or digitally converted to a minicomputer. Data is processed with etc. The signal processing circuit performs single-parameter analysis to measure the number of cells using scattered light, calculation of multiple fluorescence wavelength range signal ratios, calculation of fluorescence depolarization degree, control of cell sorting and collection, and detection of scattered light regarding the same droplet. It has functions such as delay operation control for time lag with fluorescence or fluorescence depolarization detection, correlation processing between scattered light and various fluorescence signals, and integration of various optical signals.

In general, information analyzed by a flow cytometer, such as wave height analysis, can be digitized and processed using a microprocessor or computer rather than an analog signal, but data processing using a microprocessor requires only a short time interval. It is not used in flow cytometers because there is a lag between the multiple signals generated and the time characteristics taken into the microprocessor, making it impossible to directly handle A/D converted signals. Conventional computers or mini-computers can match the time characteristics, and A/D-converted signals may be recorded on a data recorder and processed offline, but mini-computers It is well known that there is a strong desire to solve this problem because the operation is complicated and the device is large and expensive. Although it is possible to display measurement results in real time by observing analog signals with an oscilloscope or using a multichannel pulse height analyzer, it is also known that there are limits to the ability to measure multiple types.

Therefore, an object of the present invention is to solve these problems, and to provide a flow cytometer and an interface therefor that can perform advanced data processing using a commercially available mass-produced microcomputer. The interface according to the present invention can be assembled into a single board and can be installed inside a commercially available flow cytometer, so that it can be directly connected to a microcomputer via a bus line from the flow cytometer housing. Furthermore, the microcomputer used in the present invention is the most miniaturized, so-called hand-held type, which makes it possible to provide a popular, small-sized, and highly operable microparticle separation device. It is.

As already mentioned, the microparticle analyzer of the present invention is suitably used as a flow cytometer for the purpose of analyzing the number, size, properties, etc. of biological cell particles, and for separating and collecting them. It goes without saying that it can also be effectively used as a separation device for microparticles other than biological cells.

The present invention will be explained in more detail below with reference to the drawings, taking a flow cytometer as an example.

FIG. 1 is a block diagram of a cell sorting device that has an internal interface and is directly connected to a microcomputer. That is, in the drawing, 1 is a test liquid delivery and pressure adjustment section, 2 is a 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 systems, 8
is a dichromic mirror, 9 is a scattering optical system, 1
0 is a fluorescence optical system, 11 is a fluorescence optical system, 12 is a fluorescence polarization optical system, 13 is a scattered light detector, 14 is a fluorescence detector 2, 15 is a fluorescence detector 3, and 16 is a fluorescence polarization system. Detectors 4 and 17 are preamplifiers, 18 is a preamplifier 2, 19 is a preamplifier 3, 20 is a preamplifier 4, 21 is a multi-parameter signal processing circuit (analog operation section), 22 is an interface, 2
3 is a microcomputer, and 24 is a display system.

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

Reference numeral 21 is a multi-parameter signal processing circuit in FIG. 1, which converts the input signal to the interface into a single parameter from the scattering optical system, or any one of the scattering optical system signal and the fluorescence-related signal.
signal or a fluorescence-related signal. These signals are pulse outputs corresponding to any one cell, and 21 is an analog calculation unit that converts the 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., depending on the purpose of measurement. Pulse output A, pulse output B from 21
are the A/D converters in the interface, respectively,
digitized at 31 and 32. 35 and 36 are shift registers, and when the signal A and the signal B are alternately stored in one of them and the capacity of that shift register is filled, the other shift register starts storing the signals A and B in the same way. A shift register controller 39 controls the operation of the shift register. The filled shift register transfers data to the microcomputer via the input/output bus interface 41 under the action of the interrupt controller. Gate circuits 33, 34, and 3 open and close the flow of signals A and B to the two series of shift registers.
7,38 will do it. The output from 41 is processed in a microcomputer and output to a display system such as a printer or plotter. Software that requires high speed, such as processing interrupts to the computer, is written in assembly language, and other parts are written in assembly language.
Programmed in the BASIC language, a microcomputer controls the operation of the interface via bus lines 42 and input/output bus interface 41.

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

Among the outputs from each detector, the scattered light signal is selected as input A in FIG. 3 and the fluorescent signal is selected as input B in FIG. 3 and input to the multi-parameter signal processing circuit 21 shown in FIG. 2. . Signal A and signal B are digitized by A/D converters 51 and 52, respectively. If the A/D converter is 12 bits, the digital signals A and B are each 12 bits.
The signal passes through the main bus line to the next circuit, such as a buffer amplifier, and after processing is sent to the output cable end 70 to the microcomputer. Figure 3 schematically represents these bus lines.

Incidentally, each A/D converter outputs a signal EOC (End of Conversion) each time the A/D conversion is completed. Each A/D converter EOC signal is
It is inverted by the NAND circuit 53 and input to the one-shot circuit 56. 56 outputs an inverted output at the rising edge of the input signal. This signal is then input to the one-shot circuit 58 via the NOR circuit 57.
8 generates a pulse signal Q at the falling edge of the signal.
Here, the pulse widths of the one-shot circuits 56 and 58 are determined by the time constants of each one-shot circuit, and the pulse width of the one-shot circuit 58 is τ ₁
is made larger than the time constant τ ₂ of the one-shot circuit 56. These time relationships are as shown in the time chart of FIG. When the time determined by the time constant τ ₂ of the one-shot circuit 58 has elapsed, the pulse falls and this signal is input to the counter 63, which operates the counter Q _A to count one pulse.

Counter Q _A is inverted and becomes one shot circuit 5
Feed back to 8 and return 58 to its original state.
As a result, 58 generates a pulse with a time width of τ ₂ . The pulse generated at 58 operates the counter 63 to perform a carry. On the other hand, the output of the counter 63 enters buffer amplifiers 54 and 55 via inversion. That is, the output of 63 is sent to buffer amplifiers 54 and 5.
5 is operated to open and close gates alternately. That is, when the output of the counter 63 is positive, the buffer amplifier 50 is opened, and when the output is an inverted signal, the buffer amplifier 52 is opened. As a result, the shift register 60 alternately reads signal A and signal B from the A/D converter.

A hexadecimal counter is used in the example,
Therefore, 8 bits are counted for each pair of A and B. With the output of the maximum digit of the counter 63, the shift register 5
Switch between 9 and 60 operations.

Further, the output of one-shot circuit 58 is branched to one-shot circuit 64, which generates a pulse determined by a predetermined time constant τ ₃ . After a delay time of _.tau..sub.3 , the output of 64 operates a one-shot circuit 65, which likewise generates a pulse defined by a time constant _{of .tau..sub.4} . The output of the one-shot circuit 65 is input to shift registers 59 and 60, respectively, through a logic circuit 66 combined with AND and OR, and the data read into the shift registers are shifted. These shift registers 59 and 60 guide the output of the maximum digit of the counter 63 to a logic circuit 66 to decide which one to operate.

The data stored in the shift register is outputted to the microcomputer through a bus line 70 after receiving a command from the microcomputer from 69. Which of the two shift registers is to be read into the microcomputer is determined by the operating order of these shift registers and the data shift command signal, and is determined by the state of the maximum digit of the counter 63.

The flip-flops 67' and 67'' generate an interrupt operation command to send data to the microcomputer when the shift registers 59 and 60 operate alternately and data storage is completed.The interrupt command is sent to the microcomputer. 68 to the microcomputer from the interrupt command output 72.Flip-flops 67' and 67'' are connected to the shift register via logic circuit 66 and buffer amplifiers 61 and 62. In this embodiment, a shift register was used to temporarily store data, but a random access memory (RAN) may also be used.

FIG. 5 shows a block diagram when RAM is used. Compared to a shift register, the amount of data stored can be increased, and there is no need to alternately arrange data.

The signals digitized by AD converters 81 and 82 are passed through buffer amplifiers 83 and 84, respectively.
It enters the RAM 87 and consists of a counter.
Data is read into the address specified by the command from the RAM controller 85. RAM has multiple data storage units, data is read into one of them, and data read from the other is sent to the microcomputer. Data is read from the RAM by specifying the address to be read in response to a command from the RAM controller 85. Output to the microcomputer appears at output 42 by operation of interrupt controller 86.

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

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

Figure 6 is a diagram mainly showing the optical system of this device, in which the sample and inert liquid are fed under pressure from the liquid to be detected/pressure adjustment section 1 to the flow cell 2, and from the capillary nozzle in the flow cell, they are layered as described above. It flows out while maintaining the flow state. On the other hand, the irradiation light from the laser light source 5 is
A sample in a laminar state is irradiated through a light source lens system 6, and scattered light and fluorescent light from the sample are transmitted to a scattering optical system 9, fluorescent optical systems 10 and 11, and a fluorescent polarizing optical system 12, respectively.
The analysis is performed as described above.

In a preferred embodiment of the invention, an optical fiber 171 is used in the scattering optical system 9, as clearly shown in FIG. One end of the bundle of optical fibers is divided into a plurality of thin bundles, and the end surfaces thereof are divided into areas 1 to 5 as shown. and,
A fiber bundle corresponding to each fraction is positioned in front of each detector 172. In this way, the scattered light can be photometered by dividing it into arbitrary angular components, and in the illustrated example, cell identification processing can be performed as a five-dimensional vector. Alternatively, only a necessary section among the fractionated portions may be taken out.

FIG. 8 shows a condensing part in the fluorescence optical system, where 181 is the optical axis of the irradiated laser beam, and 182 is the optical axis of the fluorescence emitted from the sample. in general,
Scattered light generated at the capillary wall when the irradiated laser light passes through the flow cell 2 is scattered horizontally in a donut shape. Therefore, in order to prevent this scattered light from entering the measurement optical system, in the present invention, the fluorescent light is focused at an angle of θ with respect to a horizontal plane that includes the optical axis 181 of the irradiated light. The magnitude of θ is preferably θ, where the condensing angle (half angle) of the fluorescent light is taken as the angle.

FIGS. 9a and 9b show a light source lens system 6 for guiding the irradiated laser beam to the sample. Figure 9a, 19
1 is a circular light beam from the laser light source 5, 192 is a pair of cylindrical lenses, and 193 is an elliptical light beam after passing through the cylindrical lens. The two cylindrical lenses 192 are arranged in a cross pattern as shown. In this way, since the irradiation light to the sample can be focused into an arbitrary elliptical shape, the optimal laser irradiation light can be obtained according to the size of the cell to be measured.

However, if the cylindrical lens 193 is used, as shown at a, b, c, and d in FIG. 9b,
Scattered light is generated at the entrance/exit surface of the cylindrical lens 193. In order to prevent this scattered light from entering the measurement optical system, a pinhole mask 194 is placed between the cylindrical lens and the flow cell 2 in a further preferred embodiment of the invention.

In a preferred embodiment of the present invention, 16 in FIG.
As shown in 1, a heat exchange block for temperature adjustment is attached to the tip of the capillary nozzle in the flow cell 2, and the temperature of the sheath liquid and sample liquid is adjusted immediately before entering the flow cell. Since both liquids flow out into the flow cell 2 through the capillary nozzle, temperature changes are likely to occur due to high flow path resistance. However, in this implementation, the temperature of both liquids is always kept constant, and measurement is possible. stability is increased.

Furthermore, in a preferred embodiment of the present invention, a particle counter is provided in the measurement system, although not particularly shown. This particle counter is a meter that monitors how many particles of interest are being measured per certain period of time. This particle number monitor 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 scatter histogram of a mixed sample of mouse red blood cells and lymphocytes. 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]

FIG. 1 is a block diagram of the device, and FIG. 2 is a block diagram of the interface according to the invention.
Figure 3 is a schematic diagram of the interface circuit using a shift register, Figure 4 is a time chart of interface operation, Figure 5 is a block diagram of the interface using RAM, and Figure 6 mainly shows the details of this device. Figure 7 is a diagram showing the optical system, Figure 7 is a diagram showing the scattering optical system, Figure 8 is a diagram showing the condensing part of the fluorescent optical system, Figures 9a and b are diagrams each showing the light source lens system, FIG. 10 is a diagram showing an example of a forward scattering intensity histogram, FIG. 11 is a diagram showing an example of a fluorescence intensity histogram, and FIG. 12 is a diagram showing an example of outputting a two-dimensional distribution map. 1... Test liquid selection/pressure adjustment section, 2... Flow cell, 3... Drain system, 5... Laser light source, 6...
...Light source lens system, 9...Scattering optical system, 10, 11
... Fluorescence optical system, 12 ... Fluorescence polarization optical system, 13
~16...Detector, 21...Analog calculation unit (multi-parameter signal processing circuit), 22...Interface, 23...Microcomputer, 24...
...Display system, 161...Heat exchange block, 171...
...Optical fiber, 181... Optical axis of irradiated laser beam, 182... Optical axis of fluorescent light, 191...
Circular luminous flux, 192... Cylindrical lens, 1
93...Elliptical luminous flux, 194...Pinhole mask.

Claims (1)

[Claims] 1. A flow pipe section through which a microparticle suspension and an inert fluid flow through the suspension, and a flow pipe section in which the microparticle suspension is used as the central axis of the flow of the inert fluid in the middle of the flow pipe section. A jet nozzle part that injects the mixture of the suspension and the inert fluid to maintain it in a laminar flow state and passes it through the detection area in the laminar flow state, a laser light part that irradiates the detection area, and a sample that is irradiated with laser light. a light detection unit that detects optical information such as scattered light, fluorescent light, and degree of fluorescent light polarization;
an analog calculation unit that calculates the ratio, difference, sum, logarithm, or combination thereof of the height, time width, and area of the pulsed output signal from the photodetector and one or more pieces of information of the pulsed signal; An interface unit that A/D converts the output of the analog calculation unit and inputs it to the microcomputer, and from the output of the input information of the interface unit, the frequency distribution of the height of the pulsed signal, the area frequency distribution of the pulsed signal, and the pulse In a device consisting of a microcomputer section that calculates and displays the frequency distribution of the time width of a signal, the A/D converter output signal is sequentially read and recorded, and when the time period expires, the information is transferred to the next one, and the operation is repeated sequentially. A plurality of groups are provided with a series of information reading sections of a signal reading moving column section consisting of a plurality of columns, and after the signal reading moving column section completes reading, another signal reading moving column section reads the signal in the same manner, and also reads the signal already read. A microparticle analyzer characterized by having a control logic circuit for reading and inputting stored information from a stored signal reading movement train to a microcomputer. 2. A microparticle suspension and a flow tube section through which an inert fluid flows through the suspension, and a flow tube section in which the microparticle suspension is injected as the central axis of the flow of the inert fluid in the middle of the flow tube section, and the suspension is A jet nozzle section that maintains the confluence of the sample and the inert fluid in a laminar flow state and passes it through the detection region in the laminar flow state, a laser light section that irradiates the detection region, and scattered light and fluorescence from the sample due to laser light irradiation. , a light detection unit that detects optical information such as the degree of polarization of fluorescent light;
an analog calculation unit that calculates the ratio, difference, sum, logarithm, or combination thereof of the height, time width, and area of the pulsed output signal from the photodetector and one or more pieces of information of the pulsed signal; An interface unit that A/D converts the output of the analog calculation unit and inputs it to the microcomputer, and from the output of the input information of the interface unit, the frequency distribution of the height of the pulsed signal, the area frequency distribution of the pulsed signal, and the pulse A device comprising a microcomputer section that calculates and displays the frequency distribution of the time width of a signal in the form of a signal, wherein the operation of storing the A/D converter output signal is performed regardless of the time required and the storage location, and the storage section is readable. What is claimed is: 1. A microparticle analyzer comprising: a control logic circuit section for reading signals and inputting information already read and stored in the signals to a microcomputer. 3 A scattered light detection unit that detects scattered light from the sample by laser beam irradiation divides one end of the end surface of the optical fiber bundle into a plurality of thin bundles, and the fiber bundle of each fraction is sent to a plurality of detectors. Claims 1 and 2 are characterized in that they are installed on the front side.
The microparticle analyzer described in Section 1. 4. Claim 1, characterized in that the fluorescence detection unit that detects fluorescence from a sample by laser beam irradiation focuses the fluorescence at a selected angle with respect to the incident optical axis plane. and the microparticle analyzer according to item 2. 5. Claims 1 and 2, characterized in that two cylindrical lenses that converge the laser beam into an arbitrary elliptical shape are combined in a cross in the laser irradiation optical section that irradiates the sample with laser light. The described microparticle analyzer. 6. The microparticle separation device according to claim 5, characterized in that a pinhole mask is installed in front of the sample. 7. The microparticle analyzer according to claims 1 and 2, characterized in that a temperature control means is provided immediately before the sample and the inert liquid are discharged from the nozzle. 8. The microparticle analyzer according to claims 1 and 2, characterized in that it is equipped with a particle counter.