JPS6080764A - Method and device for identifying microparticle - Google Patents

Abstract

PURPOSE:To enable quick discrimination of microparticles by the light emission thereof with high accuracy even with a large amt. of sample by measuring the emission intensity of the change of the emission spectral with time at every specific wavelength by irradiation of strong pulse light as a strong ray for irradiation. CONSTITUTION:Electric charge electrodes 23 are provided with cell flow in- between in a place where the cell flow froms liquid drops. The liquid drop is charged according to the charge pulse thereof at the moment when the liquid drop is formed. The charge pulse receives the charge as the result of a relational operation when the cell passes through the charge electrode with delay after the cell is subjected to irradiation of pulse light, detection of fluorescence, etc. and the relational operation. A deflecting plate 24 is kept applied with + or -1,500- 2,000V voltage and the charged liquid drops are deflected by static electricity and are gathered in a cell receiver 25 below 4-5cm from the deflecting plate. The liquid drops which are not charged drop straightly without being deflected. The microparticles of the living body are classified and screened by the highly valuable information and the online classification and screening in short time with a large amt. of the sample is made possible.

Description

DETAILED DESCRIPTION OF THE INVENTION Technical Field The present invention relates to a method for identifying (sorting or sorting) cells, biological particles such as intracellular macromolecules, or microparticles such as organic macromolecules, and an apparatus for carrying out the method.

More specifically, microparticles in a continuously flowing suspension are irradiated with strong A' las light such as laser light, causing the particles to emit fluorescence, phosphorescence, etc. The present invention relates to a method and an apparatus for identifying microparticles by quickly and automatically measuring and calculating temporal changes in the emission intensity or emission spectrum of a particle.

BACKGROUND OF THE INVENTION In the field of cytology, there is an increasing demand for automated analysis and sorting of cells. Currently, screening of cell specimens, such as the detection of cancer cells or malignant cells, is mainly done by two or more methods. First, the cell sample is visually screened to look for cell samples that are suspected of containing abnormal cells.

These cell samples are stored and experts such as cytotechnologists or pathologists determine whether the cells are truly cancer cells or not. Although such methods currently work well, they have a number of drawbacks. In other words, it is time-consuming and expensive because it requires great skill, and the criteria for determining abnormality are mostly subjective and therefore not quantitative. Also, due to time and cost,
It is difficult to apply this method to a large number of samples.

In the case of analysis of biological microparticles, the flow system analysis method is called flow cytometry, and this method has the advantage of overcoming some of the above drawbacks. This is a method that has the potential to work. 70-Zytometry detects optical and electrical signals from cell suspensions to determine cell properties,
This is a method to elucidate the structure. A flow cytometer based on this is a device that irradiates light onto a cell suspension flowing at high speed within a small detection volume to detect and analyze optical and electrical signals from the cells.

According to this method, it is possible to distinguish between normal cells and abnormal cells by observing a large number of cells in a short time, and therefore it is possible to quickly perform automatic cell screening.〇The irradiation light is continuous. It is known that a single i4 meter, such as the size of a cell, is insufficient for observing cells. In addition to these, cellular light absorption, fluorescence, light scattering, or a combination thereof has come to be used as a parameter. Here, the parameter is, for example, cell size, because if the cell size is different between normal cells and abnormal + tlE cells, the cell size can be observed and the cells can be distinguished from normal and abnormal. It means to identify. In the case of cell types for which it is impossible to distinguish between normal and abnormal cells by comparing cell size alone, other parameters are used that allow for differentiation. Therefore, the accuracy of screening increases by using many parameters.

Furthermore, it not only detects and classifies optical and electrical signals from cells, but also has the function of dividing cells into groups, such as normal cells and abnormal cells, by signal analysis. The device is also called a cell sorter and has been put into practical use.

Regarding the above flow cytometry and cell sorter, for example, Electronic Technology Research Institute Business Bulletin Vol. 44 No. 3 (
Noguchi reported the cell sorter in 1980), and the cell sorter was published in Special Publication No. 46-27118 and Special Publication No. 56-13.
It is also described in Publication No. 266.

The flow system analysis method using a flow cytometer is
Unlike cell analysis under a microscope or in a sample cell, this method is superior in that it allows a large number of cells with specific properties to be identified in a short period of time while flowing the cells, and therefore enables statistical processing. However, there has not yet been a parameter that can be identified using high-value information that can be directly linked to cell abnormalities, such as information on cell morphology, molecular biology, or molecular chemistry. Such high-value information is directly related to the causes of cell abnormalities, so it not only increases clinical utility by improving screening accuracy based on increased parameters, but also has utility for basic medicine. It will be.

The present inventors have investigated the possibility of adding new parameters that can identify microparticles using the above-mentioned high-value information to the original excellent functions of the flow system analysis method.

On the other hand, in the field of laser technology, the technology of short pulse train tunable lasers is now being established. In recent years, with the advent of dye lasers that use a dye solution as a laser medium, it has become possible to obtain laser light that can be tuned to any wavelength over a wide range from ultraviolet to infrared. Pulsed light with a short time axis can now be obtained.

A mode locking method is a method for obtaining such short pulse light. There are various methods for achieving mode locking, but the most widely and easily used method is the synchronous mode locking method. In this method, short pulse light from a mode-locked argon ion laser, for example, is used as the excitation light source to excite the dye laser, and the gain of the dye laser is adjusted periodically by setting the cavity length of the dye laser corresponding to the period of the pulse train. This method generates an ultrashort pulse train of light by modulating the

By using this mode-locking method, a high-power, monochromatic, directional, tunable short pulse train laser with a pulse width τ on the order of several nanoseconds (io-9 seconds) to several picoseconds (10-12 seconds) can be produced. You can get light. The period can be varied in the range of several milliseconds to several nanoseconds by the cavity dump method.

The output of these laser beams is generally linearly polarized because the optical elements used in the laser resonator are set at the Priester angle and the excitation laser beam is linearly polarized. be.

When a substance emits light by irradiating it with light that changes at such a high speed, the time change of the light emission is also quite fast, so the observer of the time change of the light emission must also have a high degree of temporal precision. Must be.

If the time range of the change is longer than sub-nanoseconds, the time change of the light to be measured is received by a photoelectric detector such as a photodiode or photomultiplier tube, converted into an electrical signal, and the electrical pulse signal is converted into an electrical pulse signal. If necessary, there are ways to process the signal, such as inputting it to an oscilloscope and drawing it on a cathode ray tube.

However, when measuring pulsed light in the ultra-high-speed time domain of picoseconds or sub-picoseconds, these photoelectric detectors have problems in response speed or sensitivity, so a streak camera is used.

Among these, the synchronous scan streak camera method is suitable for measuring the temporal changes in weak, repetitive, and ultra-high-speed light emission. A streak camera is a device that accelerates and deflects photoelectrons from a light-receiving section of the light to be measured so that a change in time corresponds to a change in the spatial position of the electrons on the imaging phosphor surface.

The above-mentioned measurement of the temporal change in the transient emission intensity due to short Norse train laser excitation was carried out using photochemically reactive substances such as hemoglobin complexes, and further application to biopolymers was proposed.
Although it is expected that this method will be useful for analyzing its local structure, it is still limited to basic research in sample cells that cannot be used to check its usefulness.

The present inventor incorporated this into flow system analysis, and identified the local structure of microparticles such as biological particles or organic polymer particles at the molecular level based on the differences, that is, the classification of microparticles. We investigated the possibility of detecting microparticles or sorting them according to their classification.

Purpose of the Invention The purpose of the present invention is based on the idea of using strong pulsed light as a strong light beam for irradiation, and measuring the time change in the emission intensity or the time change in the emission spectrum for each specific wavelength by irradiation with /4' las light. The object of the present invention is to realize a method and apparatus that can quickly and accurately identify a large number of samples by using high-value identification information to identify microparticles by light emission.

Structure of the Invention In the present invention, as a basic form, a suspension of microparticles is formed into a particle stream containing substantially each of the particles at intervals, the particle stream is irradiated with strong light, and then an intense light beam is irradiated onto the particle stream. In a method in which the particle stream irradiated with a light beam is formed into a droplet, and particles in the droplet are identified by the intensity of light emission of the particles by irradiation with a strong light beam, strong pulsed light is used as the strong light beam, and the pulsed light is Provided is a method for identifying microparticles based on a time change in emission intensity or a time change in an emission spectrum for each specific wavelength due to irradiation.

Further, in the present invention, (1) a flow chamber, (2)
(3) means for generating intense pulsed light; (4) means for generating intense pulsed light in the flow chamber; means for irradiating the particle stream with the intense pulsed light; (5) means for converting the particle stream into droplets; (6) means for spectrally dispersing the emission of the particle stream due to the irradiation; and (7)
) A device for identifying microparticles is provided, which includes means for measuring temporal changes in emission intensity for each specific wavelength.

Further, in the present invention, (1) a flow chamber, (2)
) a means for feeding a suspension of microparticles into the flow chamber to form a particle stream containing substantially each particle at intervals, (3) a means for generating intense pulsed light, (4) a means for generating the flow chamber. means for irradiating the particle stream with the strong pulsed light; (5) means for converting the particle stream into droplets; (6)
means for spectrally emitting light from the particle stream caused by the irradiation, and
(7) A device for identifying microparticles is provided, which includes means for measuring temporal changes in emission spectra.

In the method according to the invention, the above-mentioned conventional flow system analysis techniques, techniques of tunable short pulsed light and techniques of measuring high-speed optical phenomena are combined by new high-speed real-time data processing techniques, i.e., for the identification of microparticles. Detection of classification of microparticles or sorting of particles according to the classification.

In the method according to the present invention, the microparticle itself or
A suspension of fine particles bound with dye is made into a trickle, and then the trickle is further ejected from a nozzle to form a jet and then into droplets, and the trickle or jet is injected with intense pulsed light tuned to the absorption band of the fine particles. irradiate and receive the resulting pulsed light such as fluorescent light, detect time changes in the intensity of a pre-selected specific wavelength of emitted light, or simultaneously detect each wavelength at high speed, and Compare the value of the division regarding the time change of the intensity, and generate an electrical signal of information regarding the pulse waveform according to the division of the degree of separation from the value of the division,
On the other hand, at the moment when the jet stream becomes a droplet, it is charged by a charging electrode, then passes through a deflection plate and is collected in a sample receiver, and at this time, the electrical signal is generated when the particles irradiated with pulsed light pass The particles are then sent to a charging electrode or deflection plate, where they are charged or deflected and sorted according to the classification.

In the method according to the present invention, compared to conventional flow system analysis methods and devices, intense pulsed light is used as the irradiation light, and the emitted light intensity of a preselected specific wavelength is changed over time or identified by irradiation with the pulsed light. Microparticles can be identified by simultaneously measuring each wavelength or by measuring temporal changes in the emission sheath.

The method and device according to the invention are based on the following facts.

First, particles that have a luminescent component within them, such as the particles themselves such as luminescent biological constituents, or microparticles bound with dyes for staining, are produced using strong light, that is, pulses, tuned to the absorption band of the molecules contained in the microparticles. Excited by receiving light,
It emits light such as fluorescence or phosphorescence and becomes inactive.

The light of a specific wavelength emitted from a photoexcited molecule exhibits a temporal change in intensity specific to the molecule or the interaction between the molecules. Furthermore, if specific wavelengths are selected successively and continuously, the temporal change in the emission spectrum will be unique to each molecule, and therefore unique to the microparticles containing it.

Here, "specific to each particle" means, for example, that it is unique to a group of most normal cells and a group of abnormal cells such as cancer cells or malignant cells. If temporal changes in luminescence intensity or luminescence spectra can be detected, it is possible to distinguish between normal cells and abnormal cells.

The absorption bands of molecules contained in microparticles generally include an absorption band in which the molecular skeleton, for example, the princely skeleton of DNA chlorine, absorbs light, and an absorption band in which pigments, such as acridine dye, absorb light. The information obtained differs depending on which absorption band the A) response light is tuned to.

FIG. 1 is an explanatory diagram of light emission and deactivation in response to light irradiation, with energy levels (E, L,) plotted on the vertical axis. Solid arrows represent laser light absorption and excitation (EX), curved arrows represent fluorescence (FL), and dashed arrows represent energy transfer (TR). (a) shows the case where pulsed light tuned to the absorption band of the molecular skeleton is irradiated, and different types of light are emitted from both the molecular skeleton itself and the dye. In (b), two dyes are bonded to the molecular skeleton,
This is the case when pulsed light tuned to the absorption band of a dye that serves as an energy donor is irradiated, and different types of light consisting of a donor dye and an acceptor dye are emitted. (c) is a case where two dyes (for example, a donor dye and an acceptor dye) are irradiated with pulsed light tuned to the absorption band of the bonded molecular skeleton, and different types of light are emitted from the molecular skeleton itself and the two dyes. Emitted.

The above-mentioned light emission is pulsed light, but in order to use it as a parameter for identifying microparticles, it is necessary to analyze the light emission and determine the 9-hour rise time and decay time of the light emission for a specific wavelength that characterizes particles. Alternatively, the time change of the luminous wave torque can be measured.

Should I irradiate more? When the lasing light is linearly polarized light, molecules having a transition moment in the direction of the electric vector of the light are selectively excited. This molecule rotates due to thermal motion, loses its anisotropy immediately after excitation, and becomes isotropic. This state of loose orientation > FI+ can be observed by observing the time change in the polarization of light emitted from excited molecules, such as fluorescence, for each specific wavelength, or the time change in the polarization spectrum when specific wavelengths are selected one after another. These time changes (hereinafter referred to as orientation relaxation time) are unique to each molecule and vary depending on the size of the molecule and the degree of interaction with the surrounding solvent and other molecules. If this is possible, it will be possible to identify the particles. To give an example of this case, since the cell membrane is sensitive to anisotropy, this /4' parameter is effective in identifying cells with respect to the cell membrane.

Note that the respective emission intensities occurring in the parallel direction (0°) and perpendicular direction (90°) to the polarization angle of the excitation pulse light are expressed as I(
0), I (90), the emission polarization ip for each wavelength is defined by ip. These temporal changes can be used as parameters for particle classification.

EXAMPLES The method and apparatus of the present invention will be described in more detail below, with an embodiment of distinguishing (sorting or sorting) between normal cells and abnormal cells.

FIG. 2 shows the identification of microparticles (
1 is a diagram showing the overall configuration of a device that performs sorting or sorting. FIGS. 3 and 4 are diagrams showing examples of the structure of the flow chamber in the apparatus shown in FIG. 2. FIG.

If necessary, the cell sample is first stained with an appropriate fluorescent dye. Suitable dyes are those that bind to cells, have pulsed light absorption bands, and preferably bind selectively to abnormal sites in abnormal cells. For example, Feulgen dye, acridine dye, etc. are used. It is also useful for selective binding to attach an antibody called a monoclonal antibody, which is unique to a specific site of a specific cell, to a cell and allow the antibody to adsorb a dye.

The cell sample or the cell sample stained with an appropriate fluorescent dye is then suspended in a solution such as physiological saline to form an aqueous suspension, filtered to remove large debris and clumps, and placed in a sample container. can be placed in The suspension from the sample container is conveyed under pressure through tube 5 to flow chamber 6 . An example of pressurizing means is pressurizing with a gas such as air, as shown by tube 32 in FIG. Another example is pong pressurization without pulsation or with elimination of pulsation. The suspension sent to the flow chamber 6 forms a trickle within the chamber. The rivulet is filled with a sheath fluid 63, e.g. It is injected from the outlet nozzle 62 of the flow chamber 6 while being concentrically surrounded by physiological saline.

Sheath fluid is conveyed from the container 1 through a pressurized tube 4 to a sheath tube coaxially surrounding the outlet nozzle in the flow chamber. The pressure adjustment of the sheath fluid and suspension is such that cells are passed through the exit nozzle substantially one at a time.

Among the types of flow chambers, there is a submerged measurement type having a light source projection port and an observation port, and an exit nozzle so as to be irradiated with Nollus light at the stage of a trickle flow as shown in FIG. Further, as shown in FIG. 4, the flow chamber forms a jet flow, and there is an aerial measurement type in which the jet flow ejected from the flow chamber/bar is irradiated with pulsed light. In the present invention, any of these types can be applied. In any case, the liquid falls as a droplet.

The upper part of the flow chamber is provided with mechanically connected vibration means 61, for example a piezoelectric type crystal, for regularly vibrating the outflowing liquid jet to produce uniform droplets. Not all droplets contain cells, and some droplets may contain multiple cells, but statistically the majority of cells are isolated to a single droplet. Ideally, the droplets would be slightly diluted, but in reality there would be one cell per several droplets.

These adjustments depend on the degree of pressurization of the cell suspension and sheath fluid;
This is done by controlling the frequency and voltage applied to the piezoelectric crystal, usually a frequency of 40 to 50 kHz and a voltage of 15V.

The cell flow, which has become a trickle or a jet as described above, is irradiated with pulsed light. The light source is, for example, an argon ion laser 1 equipped with a mechanism to generate pulsed light by mode-locking.
It is composed of a dye laser 11 that performs wavelength tuning from 0 to 0 and minimizes the pulse width, and a cavity damper 12 that controls the pulse train period.

Both use pulsed light as the light source, but the light beam used is the oscillation wavelength of the argon ion laser,
Mainly 488 nm or 514.5 nm, other wavelengths in the ultraviolet region, and wavelengths emitted by dye lasers, for example, 6 when rhodamine 6G is used as the dye.
Both wavelengths around 00 nm are used. If both of these wavelengths are used, it becomes a double beam light source, and it becomes easy to select the optimal excitation wavelength for cells.

This pulsed laser beam C, for example, a laser beam with a pulse width of 10 psec to 10 nsec, is
In order to improve the ratio, the light is focused so that its waist point is in the area of the cell flow, and irradiated almost perpendicular to the cell flow.

A photodiode 162 facing the irradiation light source on the side where the irradiation light passes through the cell flow detects that the cells have entered the detection range and outputs a signal to open or close the shutter of the streak camera 20, for example. used for. The light incident on the photodiode 162 normally operates in a pulsed manner, but when a cell enters the Noculus light, absorption occurs and the signal changes, which is used as a shutter open signal.

Cells are no! When the lasing light does not cross, no fluorescent light is emitted, so no signal other than base noise is output from the streak camera 20. However, in order to eliminate this base noise, the output signal of the photodiode 162 is input to the streak camera 2o. When the cells do not block the pulsed light, a shutter synchronization signal is generated to set the acceleration voltage of the streak camera 20 to 0 and the operating voltage of the channel grade to 0. After the shutter is opened and the accelerating voltage and channel rate start operating, the signal for closing the shutter is issued, for example, after 100 pulses have passed or after 1 μB has elapsed.

When cells flow to a point where the pulsed light and the cell flow intersect, biopolymers within the cells or dyes bound to them are excited, and pulsed fluorescence is emitted. This line of fluorescent light is
The cells are collected by a condensing lens 153 at a wide angle as much as possible in a direction substantially orthogonal to both the cell flow and the irradiation direction of the pulsed light, and are collected using a spectroscope 19 or a bandpass filter 173, 1 of a specific wavelength.
The streak camera 20 and/or photomultiplier tube or photodiode 163° 16
If necessary, a polarizing plate (not shown) is placed in front of these detection means.

However, if the flow chamber in the apparatus shown in Fig. 1 is not the one illustrated in Figs. 3 and 4, but the type in which the cell flow flows parallel to the incident optical axis, or the type in which the cell flow is incident obliquely, The detection of fluorescence does not need to be perpendicular to both the cell flow and the irradiation direction of the pulsed light, as long as the fluorescence is detected so as not to be affected by the incident light.

Photomultiplier tubes and photodiodes have large response time constants, so pulsed light shorter than that time constant cannot be detected. Conversely, the time constant of a streak camera may not be long. Therefore, it is sufficient to selectively use both of them as a detection means. For example, if the pulse width of the incident pulsed light is 0
．． If it is 1nsec and the repeat interval is 10nsec,
The number of pulses that cells receive while passing through the pulsed light is 100 to 1,000, depending on the cell flow rate.

The operation of the streak camera 20 will be explained with reference to FIG. The light emission pulse first enters the photocathode 201, and photoelectrons are emitted depending on the momentary intensity of the light I). The horizontal axis of the incident light is the wavelength. The momentary photoelectrons are accelerated by the accelerating electrode 2.
Channel plate 2 is accelerated with the same initial speed at 02
04, the electron beam is deflected vertically by a deflection electric field generated by a deflection electrode 203 perpendicular to the traveling direction of the electron beam.

A high-speed sweep voltage synchronized with the light emission pulse is applied to this deflection electrode 203 using a synchronized scan method, so that photoelectrons generated late are not incident on the lower side of the channel grating, for example. The electrons are then multiplied, collide with a fluorescent surface, and are converted into light again. Eventually, an image is given to this fluorescent surface in which the intensity of the fluorescent pulse is expressed as luminance (L) with time t on the vertical axis and wavelength λ on the horizontal axis. Although the photoelectron beam is generated for each fluorescent pulse and is swept at high speed, this optical image is an accumulation of pulses due to the afterimage effect of the fluorescent surface, that is, an image for each cell.

This optical image passes through an output relay lens, optical fiber, etc. 21 and forms an image on a light receiving element 22 such as an image pickup tube or a diode array.As a result, the electrical signal obtained by sweeping the light receiving element is equal to the intensity of the excitation emission pulse. It shows the time change or the time change of the emission spectrum.

The high-speed sweep voltage of the deflection electrode 203 that is synchronized with the light emission pulse is obtained by a sweep synchronization circuit that uses, as a synchronization source, an output when a portion of the irradiation pulse light is received by the photodiode 161.

The orientation relaxation time can be detected by a streak camera as follows. That is, there is a method in which a light emission pulse is divided into optical paths that pass through two polarizing plates at 0° and 90°, and each specific wavelength is detected with an IJ camera, and the polarization angles O0 and 90° are detected through the polarizing plates. Two polarized fluorescent light pulses of a certain wavelength are separately incident on the left and right halves of one streak camera, and the light images are separated on the left and right sides of the fluorescent surface behind the channel plate. In this method, the electrical signals of each optical image are obtained separately.

As mentioned above, when the excitation pulse light is a laser pulse, the pulse light is also generally linearly polarized light. To measure this, it is sufficient to observe through a polarizing plate whose polarization angle is set to 54.7 degrees, which is called the magic angle.

Photomultiplier tube and photodiode array 163°164
When used as a means for detecting luminescence pulses, it is possible to detect one of the many fluorescent pulses emitted by one cell and directly detect the rise time, decay time, and orientation relaxation time of luminescence. can. The average value of several i4 pulses may be taken to obtain high sensitivity.

The electric signal from the streak camera, photomultiplier tube, or photodiode is used to calculate the rise time, decay time, orientation relaxation time, or time change of the emission spectrum of light emission at a specific wavelength by a time constant timing circuit described later.

The configuration of the signal processing section 7 in the device shown in FIG. 2 is shown in FIG.

The arithmetic outputs of the time constant clock circuits 700a and 700b, which are the light emission rise time, decay time, and orientation relaxation time, are sent to the data multiplexer 71 and the sorting multiplexer 72.

The data multiplexer is a central processing unit (CPU)
) Based on the 740 command, the signals of each sensor are sequentially switched and written to the menu 73. The signals written in the memory 73 are displayed as measured values or data such as graphs on a display 81 such as a CRT according to instructions from the CPU 74 as an indication of the identification of microparticles, and these data are still printed on a printing device. , create a hard copy.

On the other hand, the sorting multiplexer 72 is configured with elements that should be used for sorting (separation), such as wavelength, startup time, decay time, orientation relaxation time, etc.
One or more element signal sources are selected by commands from 74 and one or more upper or lower limit comparators 75 .
Sent to 76. Here, the preset value or spectrum is compared with the measured value or the measured spectrum, and if it is within a predetermined range (for example, below the upper limit and below the lower limit), the load t/'' pulse generation circuit is controlled by the operation of the signal processing section 7. 82
outputs a deflection signal with a certain delay time, for example, about 500 to 1,000 μS.

In this way, the short rise time, decay time, time change of emission spectrum, or orientation relaxation time of fluorescence at a specific wavelength of one cell are set to these reference values (the above-mentioned set value or set spectrum) that a normal cell has. The deflection signal is compared to the reference value and identified according to the distance from the reference value.
It will be output for.

A charging electrode 23 is provided across the cell flow at the location where the cell flow forms droplets, and the charging electrode is charged with a 1.9 Lus width of 50 to 200 μB and a voltage of ±50 to 300 V. It is applied from the pulse generation circuit. Then, at the moment the droplet is formed, the droplet is charged according to the charging pulse. This charge pulse is delayed after the cell is irradiated with Mars light, fluorescence, etc. is detected and compared, so that when the cell just passes through the cart electrode, the It is adapted to receive a charge as a result of an operation.

Next, the droplets pass between two deflection plates 24 that are opposed to each other with an interval of 2 to 5 anns across the droplet flow. A voltage of ±1500 to 2000 V is applied to the deflection plate, and the charged droplets are deflected by electrostatic force and collected in the cell receiver 25 4 to 5 crn below the deflection plate. Uncharged droplets fall without being deflected.

Note that instead of outputting the deflection signal from the charge generator or the pulse generation circuit, the charge electrode may be kept in a constant charge state, and the strength of the deflection electric field may be varied using a circuit that drives the electric field of the deflection plate.

Figure 1 shows the waveforms of the particle flow in the device, the irradiated pulsed light, the emitted light pulses, the shutter gate of the streak camera, the sweep voltage of the streak camera, the charged electric field applied to the droplet, and the temporal relationship between them as shown in Figure 7. This will be explained with reference to the figures.

(1) in FIG. 7 shows the state of the particle flow, with the H level when the particles cross the pulsed light, and the L level when they do not. (2) in FIG. 7 shows the irradiated light, and (3) in FIG. 7 shows the generated light. (4) and (5) in Figure 7 show the shutter gate of the streak camera and the sweep voltage of the streak camera, respectively, and (6) in Figure 7 shows the voltage applied to the charged electrode at two levels. . Between (2) and (3) in FIG. 7, there is a slight delay in the rise time of the light emission, and after reaching the peak, the light attenuates slowly.

The period (5) in FIG. 7 causes a delay corresponding to the arrival time of the light from the light source to the streak camera, but this is ignored in the diagram. In (6) of FIG. 7, after the microparticle is irradiated with pulsed light, there is a delay by the time difference between when the microparticle reaches the charged electric field. Although the delay is incomparable compared to the delays in (2) to (5) in FIG. 7, which involve light delay, it is drawn slightly larger in the figure for the sake of convenience.

The configuration of the time constant clock circuit in the circuit of FIG. 6 is shown in FIGS. 8 and 9. When the time constant timing circuit calculates the rise time or decay time of a light emission pulse, the time constant timing circuit is configured as shown in FIG.

In the circuit shown in FIG. 8, the rise time of light emission is measured by first pre-amplifying the electrical signal S (OPT) converted from the optical image and performing pre-processing such as smoothing. Determine the standard level of
(703). The reference level of the rising edge υ, which is signal 5 (703), is set to an arbitrary value by the threshold setting device 5 (
702) It shall be possible. This threshold value is determined by taking into consideration the change point of the light emission signal from the time of no signal, noise in the optical system or the electrical system leading up to this circuit, and the like.

To measure the rise time, a signal when the signal reaches its peak value is required, and for this purpose, signal 5 (705) created by the second comparator 705 is used. That is, the light emission signal and the signal from its peak hold circuit are compared by the second comparator to obtain a signal at the peak point when the light emission signal becomes smaller. This 5 (703).

The rise time of light emission can be measured by measuring the time difference between the two signal generation points 8 (705) using a rise time clock circuit.

To measure the decay time of the light emission, the second freezing comparator 705 captures the time at which the peak point should occur.

To do this, if the light emission signal and the signal passed through the peak hold circuit are compared, the output of the second comparator 705 will be reversed when the light emission signal becomes smaller.
) is used as No. 46 of the hour meter S+ start of the time measuring circuit.

Decay time is usually a value of 1/e from the signal peak value.
(63%), but set this 1/e or other value to the attenuation ratio setter, input this and the emitted C signal to the third comparator 708, and calculate the magnitude. When reversing, output signal 5 (708) is obtained. The decay time is measured using this signal 5 (705).

5 (708) and is measured by a decay time measuring circuit. STT is used to start timing, and STP is used to stop timing.

Due to the clock completion signal COMPL of the falling time clock circuit,
(Reset the peak hold circuit and two clock circuits)
R8T. In this way, the rise time clock signal 5 (707) and the fall time clock signal 5 (70
9) is obtained.

The above is a time constant timing circuit for one specific wavelength.
If there are multiple specific wavelengths, similar processing may be performed for each wavelength depending on the number of specific wavelengths. In addition, when the circuit shown in FIG. 8 is used, the signal 5 (7
07) and signal 5 (709) can be omitted.

When the time constant clock circuit calculates the orientation relaxation time, the time constant clock circuit is configured as shown in FIG.

In the circuit shown in FIG. 9, after preamplifying electrical signals 8 (OPT) with polarization angles of 0° and 90° from the optical image detector, a converted value indicating the polarization, for example, the degree of polarization of light emission is calculated. let The output of the circuit is processed in the same manner as the decay time measurement described above. In this way, the orientation relaxation time clock signal 5 (7i6) is obtained by the circuit of FIG.

For a plurality of specific wavelengths, similar processing may be performed for each of them.

In the apparatus shown in FIG. 2, the parameters for classifying and sorting microparticles are based on differences in the local structure of the microparticles at the molecular level. Specifically, when it comes to biological microparticles, they are based on molecular biology or molecular chemistry at the cell level, chromosome level, and even down to the biomolecular level. More specifically, first of all, the proximity of each component molecule and site, such as the distance between a nucleic acid and a base bare, the distance between a staining dye molecule and a molecule skeleton, the distance between a donor dye molecule and an acceptor dye molecule, etc. The second is the size and type of particles such as nucleic acids and proteins, and the third is the structure of molecules, such as the pitch of double helices, internal vibrations of molecules, Brownian operation, chain bending, and local changes in molecules. This is information such as rotational movement.

The fourth is detection of transient intermediate products of biochemical reactions including photochemical reactions, and the fifth is information on cell regions, such as the state of binding between cell membranes and proteins and the thickness of the membrane.

Sixth, there is information regarding other properties of biological microparticles, such as the state of cell fusion and the fractional chromatography approach of surface lection receptors.

These are highly valuable information directly related to the different factors of biological microparticles. In the apparatus shown in FIG. 2, biological microparticles can be classified and sorted based on this highly valuable information, and moreover, it is possible to do this on-line on a large number of sandals in a short time. This means the clinical utility of the device shown in FIG. 2 in the medical field, its utility in substrate medicine, and also its utility in classifying and sorting other microparticles.

In implementing the present invention, various modifications can be made in addition to the embodiments described above. However, the method and apparatus of the present invention do not include a capacitive sensing system using a Coulter orifice as described in the above-mentioned Japanese Patent Publication No. 13266/1983, or pulsed fluorescence or scattered light from particles by pulsed light irradiation. Of course, it is possible to normalize the light as a continuous light, detect its intensity, and use these as parameters for particle classification and sorting, that is, to use conventional flow system analysis techniques in combination.

Effects of the Invention According to the present invention, identification of microparticles by light emission can be performed quickly and with high precision on a large amount of samples using high-value identification information.

[Brief explanation of the drawing]

FIG. 1 is a diagram explaining the state of light emission and deactivation of particles in response to light irradiation, and FIG. 2 is an overall diagram of an apparatus for identifying (sorting or sorting) microparticles as an embodiment of the present invention. Figures 3 and 4 are diagrams showing the structure of the flow chamber, Figure 5 is a diagram explaining the operation of the streak camera, and Figure 6 is a diagram showing the configuration of the signal processing section in the apparatus shown in Figure 2. FIG. 7 is a waveform diagram showing the temporal relationship of signals of each part in the device shown in FIG. 1, and FIGS.
It is a figure which shows the structure of the time constant clock circuit in a circuit of a figure. 1... Sheath liquid container, 2... Sample container, 4.5...
・Pipe, 6... Flow chamber, 7... Signal processing unit,
DESCRIPTION OF SYMBOLS 10... Argon ion laser, 11... Dye laser, 12... Cavity damper, 19... Spectrometer, 20... Streak camera, 23... Charged electrode,
24... Deflection plate, 25... Cell receiver, 32... Tube, 61... Vibration means, 62... Outlet nozzle, 63...
... Sheath liquid, 81 ... Display section, 82 ...
Load t pulse generator, 83...Sweep synchronization circuit, 84...
・Shutter synchronization circuit, 131-133... Half mirror 1134, 136... Guicroic mirror, 1
41゜142... Total reflection mirror, 151-158...
・Lens, 161.162...Photodiode, 1
63°164...B C electron multiplier tube or photodiode, 173-174...Band pass filter. Figure 1 DNA base Dye DNA base Donor dye Acceptor dye DNA base Donor dye Acceptor dye Figure 3 Figure 4

Claims (1)

[Claims] 1. A suspension of microparticles is formed into a particle stream containing substantially each of the particles at intervals, the particle stream is irradiated with strong light, and then the particle stream is irradiated with strong light. In a method in which the particle stream is formed into droplets and particles in the droplets are identified by the intensity of light emission of the particles by irradiation with strong light, strong pulsed light is used as the strong light and each specific wavelength is identified by irradiation with nosolous light. A method for identifying microparticles based on the time change in the luminescence intensity or the time change in the emission sheath. 2. (1) a fluidization chamber; (2) g. a means for feeding a suspension of small particles into the fluidization chamber to form a particle stream containing substantially each particle at intervals; (3) a strong pulse; (4) means for irradiating the particle stream with the intense pulsed light in the flow chamber; (5) means for converting the particle stream into droplets; (6) causing the particle stream to emit light due to the irradiation. A device for identifying microparticles, comprising: means for spectroscopy; and (7) means for measuring temporal changes in emission intensity for each specific wavelength. 3. (1) a fluidization chamber; (2) a means for feeding a suspension of microparticles into the fluidization chamber to form a particle stream containing substantially each particle at intervals; (3) a strong means for generating pulsed light; (4) means for irradiating the particle stream with the strong i4 las light in the flow chamber; (5) means for converting the particle stream into droplets; (6) reducing the particle stream by the irradiation. A device for identifying microparticles, comprising: means for spectrally emitting light; and (7) means for measuring changes over time in the emitted light quiescent.