JPH0792076A - Grain analyzing device - Google Patents

Abstract

PURPOSE:To measure the self-fluorescence of a grain to be analyzed, and perform a precise detailed analysis. CONSTITUTION:A flow part 2 is formed in the center of a flow cell 1, and a grain S to be analyzed is carried from above to below. A first optical beam generating light source 3 for generating a first optical beam B1 is arranged on the side of the flow cell 1, and a first optical detector 8 is arranged in the emitting direction of the flow cell 1. A second optical beam generating light source 9 for generating an optical beam B2 with a prescribed delay time after the detection of the grain to be analyzed obtained by the first detector 8 is arranged under the first optical detector 8, and a second optical detector 13 is arranged in the emitting direction of the flow cell. The second optical beam B2 has a pulse form, and the second optical detector 13 detects only the self- fluorescence of the scattered light and self-fluorescense generated from the grain to be analyzed.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a particle analyzer, such as a flow cytometer, which irradiates individual test particles in a sample with a light beam and analyzes the test particles by optical measurement. Is.

[0002]

2. Description of the Related Art A flow cytometer is a device for illuminating a cell suspension flowing at a high speed, that is, a sample solution with, for example, a laser beam and detecting a photoelectric signal due to scattered light or fluorescence to elucidate the properties and structure of the cell. And cell chemistry, immunology,
It is used in fields such as hematology, oncology, and genetics.

In a conventional particle analysis device used for this flow cytometer or the like, for example, the central part of the flow cell is
Irradiation light such as laser light is radiated to the test particles such as blood cells that are wrapped in the sheath liquid and pass through the circulation part having a minute rectangular cross section of 00 μm × 200 μm, and the resulting forward and side scattered light is generated. Due to the
It is possible to obtain particle properties such as size and refractive index. Further, for the test particles that can be stained with a fluorescent agent, by detecting the fluorescence of the test particles from the side scattered light in the direction substantially perpendicular to the irradiation light, important information for analyzing the test particles. Can be collected.

As a light source, a gas laser light source such as an argon laser having a high stability of light quantity and wavelength is generally used.

[0005]

In such a particle analyzer, the autofluorescence generated from the particles to be detected is discarded as noise, or it is used to detect whether autofluorescence is generated or not. It stayed. The latter case has the advantage of not requiring pretreatment such as staining, as compared with the case of using a fluorescent agent, but the amount of autofluorescence differs depending on the volume, shape, etc. of the particles to be examined, and the analysis is highly accurate. I can't.

Further, a method of measuring the time change of fluorescence from a fluorescent agent by periodically irradiating pulsed light instead of a continuous irradiation light source has been considered, but autofluorescence is also generated from dust or the like. Therefore, it is difficult to perform accurate analysis with this method.

An object of the present invention is to solve the above problems and to provide a particle analyzer capable of performing more detailed and more accurate autofluorescence analysis than before by detecting the time change of autofluorescence generated from a test particle. To provide.

[0008]

A particle analysis apparatus according to the present invention for achieving the above object comprises first detection means for detecting passage of test particles which are separated and sequentially flow one by one, The first detector is provided at a position downstream of the position of the first detector.
And a light beam generating means for generating a pulsed light beam for irradiating the particles to be detected in accordance with the passage of the particles to be inspected based on the output from the detection means, It is characterized by comprising a second detecting means for detecting the amount of fluorescent light emitted from the particles and a recording means for recording the amount of fluorescent light as a function of time.

[0009]

The particle analyzer having the above-mentioned configuration does not require complicated pretreatment and can detect the time change of the autofluorescence generated from the particles to be analyzed, which enables more detailed and accurate analysis of the particles to be analyzed. Becomes Furthermore, the time change of the fluorescence amount can be detected with high accuracy not only in the case of autofluorescence but also in the measurement of normal fluorescence, and a more detailed analysis than before can be performed.

[0010]

The present invention will be described in detail below with reference to the illustrated embodiments. FIG. 1 is a configuration diagram of an embodiment, in which a flow cell 1 is formed with a flow portion 2 at the center thereof, and particles to be tested S are wrapped in a sheath flow by a sheath flow method from above to below and flow. There is. A first light beam generation light source 3 that generates a first light beam B1 that is substantially continuous light is arranged on the side of the flow cell 1, and connects the first light beam generation light source 3 and the flow cell 1. An imaging lens 4 is provided in the optical path 01, and a stopper 5, a condenser lens 6, a bandpass filter 7, and a first photodetector 8 for detecting scattered light are provided on the side of the extension of the optical path 01 opposite to the flow cell 1. Are arranged in sequence. The light beam generating light source 3 to the first photodetector 8 constitute first detecting means for detecting passage of particles to be inspected.

A second light beam generating light source 9 for generating a pulsed second light beam B2 is provided below the first light detecting means.
Is provided, and an imaging lens 10 is provided on an optical path 02 connecting the light beam generating light source 9 and the flow cell 1, and a condenser lens 11, a band pass filter 12, and an extension line of the optical path 02 on the opposite side of the flow cell 1. Second photodetectors 13 for detecting the amount of autofluorescence emitted from the particles to be inspected are sequentially arranged,
The second detection means is configured.

The output of the first photodetector 8 is connected to the signal processing unit 14, and the output of the signal processing unit 14 is connected to the light beam generating light source 9 and the second photodetector 13.
Further, the output of the second photodetector 13 is connected to the analysis unit 16 via the recording unit 15.

As the test particles S, isolated floating cells of human lung tissue are used in the examples. A He-Ne laser light source having a wavelength of 633 nm is used as the first light beam generation light source 3, and the first light beam B1 generated from the first light beam generation light source 3 is focused on the center of the flow cell 1 by the imaging lens 4. Collected. The forward scattered light by the particles S to be tested flowing in the flow section 2 of the flow cell 1 is made incident on the first photodetector 8 through the condenser lens 6 and the bandpass filter 7 and converted into an electric signal, and the signal processing section. Sent to 14. The stopper 5 blocks the first light beam B1 traveling straight, and the bandpass filter 7 prevents external light having a wavelength other than 633 nm from entering the first photodetector 8.

The signal processing unit 14 determines whether the light is scattered light from the particles S to be detected or scattered light from dust or the like according to the level of the electric signal sent, and the particles S to be detected are detected.
When the scattered light from is an electric signal of a certain level, the light beam generating light source 9 is turned on after a predetermined delay time for the test particle S to reach the position where the light beam B2 is irradiated,
A light beam B2 of 1 pulse is generated.

This delay time is easily obtained if the distance between the irradiation position of the first light beam B1 and the irradiation position of the downstream pulsed light beam B2 and the velocity of the test particle S flowing through the flow section 2 are known. Desired. Further, the signal processing unit 14 turns on the second photodetector 13 after the irradiation of the one-pulse light beam B2.

In the embodiment, a die laser adjusted to have a wavelength of 530 nm and a pulse time of 100 pS is used as the light beam generating light source 9. The light beam B2 generated from the light beam generating light source 9 is focused on the center of the flow cell 1 by the imaging lens 10, and the test particle S is excited to generate autofluorescence.
This autofluorescence is collected by a condenser lens 11 and a bandpass filter 1.
Second photodetector 1 for detecting the amount of autofluorescent light via 2
3 and is converted into an electric signal, and the electric signal, that is, the amount of autofluorescence light is recorded as a function of time by the recording unit 15 and sent to the analyzing unit 16.

In the embodiment, a combination of a streak camera and a one-dimensional CCD is used as the second photodetector 13. The streak camera electrically bends the electrons generated by the incident light at a high speed, thereby enabling a rapid temporal change in the incident light amount to be spatially recorded. This streak camera is often used with a two-dimensional detector, but a one-dimensional CCD is sufficient in this embodiment. For example, a one-dimensional CCD with 512 pixels is used as compared with a two-dimensional CCD with 512 × 512 pixels. CCD
It is convenient for particle analysis because the memory capacity is reduced to 512 times by using, and high-speed analysis is possible.

The reason why the second light beam B2 is pulsed is that the light beam B2 is irradiated for a very short time until autofluorescence from the particles S to be detected is generated, and the second photodetector is used. 1
This is because it is possible to prevent the scattered light from the light beam B2 from entering the light source 3 and to detect only the autofluorescence. Here, the pulse time is 100 pS, but 500 pS is usually sufficient.

2 and 3 show examples of electric signals sent from the second photodetector 13 to the recording unit 15, which are from normal lung tissue cells and tumor lung tissue cells, respectively, and the horizontal axis represents time. The vertical axis represents the amount of autofluorescence proportional to the voltage in arbitrary units. The time of the fall portion of the amount of autofluorescence shown by the arrow in the figure appears long in the normal cells of FIG. 2 and short in the tumor cells of FIG.

By utilizing such a property that the amount of autofluorescence is a function of time, the analysis unit 16 can determine whether each test particle is a normal cell or a tumor cell. That is, for example, find the time when the peak value of the amount of autofluorescence becomes half,
If the time is longer than the set value, it may be determined that the cells are normal cells, and if the time is shorter than the set value, the cells may be tumor cells. By making a determination in such a time, it is possible to eliminate a large error due to a difference in volume or shape of individual cells, which is a problem when trying to make a determination based on a difference in autofluorescence amount.

In the embodiment, the first light beam generating light source 3, the first light detector 8 and the like constitute the first light detecting means for detecting the passage of the particles S to be inspected. The method may be a well-known method of detecting a change in electric resistance when the test particles pass through the pores.

Further, in the embodiment, the first light detecting means detects the test particles S by using the forward scattered light generated from the test particles S. However, the forward scattered light or the side scattered light or The fluorescence from the fluorescent agent bound to the particles to be detected is also detected from the particles S to be detected.
If it is used for analysis, the analysis parameter increases, and a more detailed analysis of the test particle S can be performed.

The second photodetector 13 for detecting the amount of autofluorescence emitted from the particles S to be detected detects the amount of fluorescence emitted from the fluorescent agent instead of the autofluorescence, and calculates the function of the time. It can also be used for analysis of the test particles S. In this case, since the passage of the test particles S is detected and the measurement is performed, the fluorescence from only the test particles S can be accurately measured.

[0024]

As described above, the particle analysis apparatus according to the present invention is the first detection means for detecting passage of individual test particles, and the light beam for generating the pulsed light beam for autofluorescence excitation. Providing a generation means, a second detection means for detecting the amount of autofluorescence, and a recording means for recording the amount of autofluorescence as a function of time, the time change of the amount of autofluorescence generated from each test particle is accurately detected Therefore, it is possible to perform a detailed analysis based on the autofluorescence of a large number of test particles such as cells in a short time without requiring complicated pretreatment. Further, not only autofluorescence but also normal fluorescence measurement can accurately detect the time change of the fluorescence amount, and there is an effect that a more detailed analysis than in the past can be performed.

[Brief description of drawings]

FIG. 1 is a configuration diagram of an embodiment.

FIG. 2 is a graph showing an example of signals from normal cells.

FIG. 3 is a graph of an example of signals from tumor cells.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Flow cell 2 Flow part 3 1st light beam generation light source 8 1st photodetector 9 2nd light beam generation light source 13 2nd photodetector 14 Signal processing part 15 Recording part 16 Analysis part

Claims (10)

[Claims] 1. A first detection means for detecting passage of test particles which are separated and sequentially flow one by one, and a first detection means from a position downstream of the position of the first detection means. Light beam generating means for generating a pulsed light beam for irradiating the test particle in accordance with passage of the test particle based on the output, and fluorescence generated from the test particle by the irradiation of the pulsed light beam A particle analysis device comprising: a second detecting means for detecting a light quantity; and a recording means for recording the fluorescent light quantity as a function of time. 2. The particle analysis device according to claim 1, wherein the fluorescence is autofluorescence. 3. The first detecting means irradiates a test particle with a continuous light beam that is substantially continuous light, and detects scattered light generated from the test particle to detect passage of the test particle. The particle analysis device according to claim 1, which detects the particle. 4. The particle analyzer according to claim 1, wherein the particles to be detected are caused to flow by a sheath flow method. 5. The particle analysis apparatus according to claim 1, wherein the pulsed light beam is a laser beam. 6. The particle analysis apparatus according to claim 1, wherein the pulse irradiation time of the pulsed light beam is 500 pS or less. 7. The particle analysis apparatus according to claim 3, wherein the continuous light beam is a laser beam. 8. The particle analysis device according to claim 3, wherein scattered light generated from the particles to be inspected by the continuous light beam is further used for analysis of the particles to be inspected. 9. The particle analysis apparatus according to claim 3, wherein the fluorescence generated from the particles to be inspected by the continuous light beam is further used for analysis of the particles to be inspected. 10. The analysis unit according to claim 1, further comprising an analysis unit that determines that the test particles are tumor cells when the temporal change in the amount of fluorescent light described in the recording unit is larger than a set value. Particle analyzer.