JP2009162660A - Detection method and detector - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a detection method and a detector capable of preventing clogging in a sample and detecting a plurality of samples having high concentration precisely and quickly. <P>SOLUTION: A sample liquid 2 containing a plurality of fine particles 4 is conducted to a flow cell 1 having a channel with a rectangular section vertical to an axial direction inside so that the sample liquid 2 is sandwiched by sheath liquids 3a, 3b from both the sides, and a strip of laminar flow where a section vertical to a conduction direction 5 is sidelong is formed. Then, laser beams 6 are applied to the sample liquid 2 in a laminar flow state while scanning in its width direction (w), and fluorescence and/or scattered light emitted from the fine particles 4 are detected. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for detecting a sample such as a microparticle and a detection apparatus using the method. More specifically, the present invention relates to a technique for forming a laminar flow with a sample liquid containing a sample and detecting the sample contained therein.

  In general, in order to identify biologically relevant microparticles such as cells, microorganisms, and liposomes, an optical measurement method using flow cytometry (flow cytometer) is used (for example, see Non-Patent Document 1). Flow cytometry irradiates laser light of a specific wavelength to minute particles that flow in a line in a flow path, and detects fluorescence or scattered light emitted from each minute particle, thereby detecting a plurality of minute particles. This is a method for identifying one by one.

  Specifically, in the flow cell, a laminar flow is formed by a sample liquid containing microparticles to be measured and a sheath (sheath) liquid flowing around the sample liquid, and a slight amount is further formed between the sample liquid and the sheath liquid. By creating a pressure difference, a plurality of microparticles contained in the sample liquid are arranged in a line. When the flow cell is irradiated with laser light in this state, fine particles pass one by one so as to cross the laser beam. At this time, fluorescence and / or scattered light emitted from each microparticle excited by the laser light is measured using an electro-optic detector.

  Conventionally, a flow cytometer for irradiating a plurality of laser beams having different wavelengths to a microparticle has also been proposed (see Patent Document 1). FIG. 12 is a diagram schematically showing a configuration of a conventional flow cytometer described in Patent Document 1. In FIG. As shown in FIG. 12, a flow cytometer 101 described in Patent Document 1 includes a fluid (flow) system 102 for arranging cells modified with a fluorescent dye in a row in a flow cell, and a wavelength of each cell. An optical system 103 for irradiating a plurality of different laser beams and detecting detection target light such as scattered light and fluorescence, and a signal processing device for controlling and processing electrical signals related to scattered light and fluorescence output from the optical system 103 104.

  In this conventional flow cytometer 101, a plurality of laser beams having different wavelengths are emitted from the light sources 106a, 106b, and 106c with a predetermined period and different phases, and the light guide member 107 further emits the plurality of laser beams. A plurality of fluorescence emitted from microparticles modified with a plurality of fluorescent dyes can be detected by guiding the laser light on the same incident optical path and condensing it on the cell 105.

  Furthermore, conventionally, there has been proposed a method using a substrate on which a fine flow path is formed without using a flow cell (see, for example, Patent Documents 2 and 3). FIG. 13 is a perspective view showing a configuration of a flat plate flow cell device described in Patent Document 2. As shown in FIG. In the flat flow cell apparatus 110 shown in FIG. 13, a sample liquid port 112 that stores a sample liquid containing cells and particles and sheath liquid ports 113 a and 113 b that store a sheath liquid are formed on a transparent substrate 111. In addition, flow paths 114, 115a, and 115b are connected to the sample liquid port 112 and the sheath liquid ports 113a and 113b, respectively.

  These flow paths 113, 115 a, and 115 b merge to form one flow path 117. As a result, the liquid flowing through each flow path merges so that the sample liquid is sandwiched between the sheath liquids at the confluence point 116, and the sample liquid forms a laminar flow. Then, laser light is irradiated in the flow path 117, and measurement is performed. Further, a waste liquid port 118 is formed at the end of the flow path 117.

  On the other hand, in the cell analysis / separation device described in Patent Document 3, the sample solution introduction flow path and the two sheath flow forming flow paths formed on both sides thereof once merge and then branch into two or more flow paths. is doing. In this cell analyzer, the merged portion serves as a measurement unit, and each branched flow channel serves as a fine particle sorting flow channel for separating and collecting trace particles.

JP 2007-046947 A JP 2003-302330 A JP 2004-85323 A Supervised by Hiromitsu Nakauchi, "Cell Engineering Separate Volume, Experimental Protocol Series, Flow Cytometry," Second Edition, Shujunsha Co., Ltd., August 31, 2006

  However, each of the above-described conventional techniques has a problem that the sample is easily clogged and the maintenance must be frequently performed because the flow path through which the liquid containing the sample to be measured flows is fine. is there. Further, in the conventional method, samples such as cells and fine particles are arranged in a line, and measurement is performed one by one. Therefore, as the concentration of the sample increases, the measurement time becomes longer and the flow path is clogged. There is also a problem that it is likely to occur.

  Furthermore, in the conventional method, when the sample liquid includes a plurality of samples, light emitted from the sample flowing in the vicinity leaks into the light emitted from the sample to be measured, and is detected in the detection signal. There is also a problem that crosstalk occurs and the detection accuracy of the sample decreases.

  SUMMARY OF THE INVENTION Accordingly, it is a main object of the present invention to provide a detection method and a detection apparatus capable of preventing a sample from being clogged and detecting a plurality of samples having a high concentration with high accuracy in a short time.

A detection method according to the present invention is a method of irradiating a sample flowing through a flow path with light and detecting light emitted from the sample, and forming a strip-shaped laminar flow with the sample liquid containing the sample Then, irradiation is performed while scanning light in the width direction of the sample liquid in the laminar flow state.
In the present invention, since the strip-shaped laminar flow is formed with the sample liquid, the sample is not easily clogged. Further, since the irradiation light is scanned in the width direction of the sample liquid in the laminar flow state, a plurality of samples can be detected by one scanning.
In this detection method, when the sample is a fine particle, it is desirable that the fine particle is two-dimensionally arranged in an in-plane direction in a sample liquid in a laminar flow state.
In addition, it is possible to irradiate a sample liquid in a laminar flow state with a plurality of lights having different wavelengths in a time division manner.
Furthermore, fluorescence and / or scattered light emitted from the sample may be detected.
Furthermore, the strip-shaped laminar flow can be formed, for example, by sandwiching the sample liquid with a sheath liquid and allowing the flow path to flow in that state.
Furthermore, the side wall of the channel may be irradiated with light, and the flow position of the sample may be specified based on detection light derived from the side wall.
In that case, the detection signal can be corrected based on the flow position information and the intensity distribution of the light applied to the sample.
Alternatively, the scanning speed of the light may be adjusted based on the flow position information and the flow velocity distribution of the sample liquid.
On the other hand, a plurality of auxiliary irradiation lights for correction are irradiated around the main irradiation light that irradiates the sample, and the respective detection signals are processed to remove leakage signals from the sample flowing through the surroundings. You can also.

Another detection method according to the present invention forms a laminar laminar flow, and detects a sample contained in the laminar flow while scanning a detecting means in the width direction of the laminar flow.
In the present invention, since a strip-shaped laminar flow is formed, clogging of the sample is unlikely to occur, and since detection is performed in the width direction of the laminar flow, a plurality of samples can be detected at one time.
Examples of the detection means in this detection method include a magnetic field or an electric field.
Further, when the sample is a microparticle, it is desirable that the microparticle is two-dimensionally arranged in the in-plane direction in the laminar flow sample liquid.
Furthermore, a strip-shaped laminar flow may be formed by sandwiching the sample liquid with a sheath liquid and allowing the flow path to flow in that state.

A detection apparatus according to the present invention is a detection apparatus that irradiates a sample flowing in a flow path with light and detects light emitted from the sample, and forms a laminar laminar flow with a sample solution containing the sample A laminar flow forming unit, a light irradiation unit that irradiates light while scanning the laminar sample liquid in the width direction, and a detection unit that detects light emitted from the sample. .
In the present invention, since the strip-shaped laminar flow is formed with the sample liquid, the sample is less likely to be clogged. In addition, since light is irradiated while scanning in the width direction of the sample liquid, a plurality of samples can be detected by a single scan.
In this detection apparatus, when the sample is a microparticle, the microparticle in the sample liquid is two-dimensionally arranged in the in-plane direction in the laminar flow forming portion.
In addition, a plurality of lights having different wavelengths can be irradiated in a time-sharing manner to a sample liquid in a laminar flow state.
Furthermore, the detection unit may detect fluorescence and / or scattered light emitted from the sample.
Furthermore, a strip-shaped laminar flow can be formed by sandwiching the sample liquid with a sheath liquid and allowing the flow path to flow in that state.
On the other hand, in this detection apparatus, it is possible to irradiate the side wall of the flow path with light, and in this case, the flow position of the sample can be specified based on the detection light derived from the side wall.
Then, the detection signal is corrected based on the flow position information and the intensity distribution of light applied to the sample, or the light scanning is performed based on the flow position information and the flow velocity distribution of the sample liquid. You can adjust the speed.
In addition, it is possible to irradiate a plurality of auxiliary irradiating lights for correction around the main irradiating light irradiating the sample. In this case, the sample flowing through the surroundings by calculating each detection signal Leakage signals from can be removed.

Another detection apparatus according to the present invention forms a laminar laminar flow, and detects a sample contained in the laminar flow while scanning a detection means in the width direction of the laminar flow.
In the present invention, since a laminar laminar flow is formed, the sample is hardly clogged. Moreover, since the detection is performed in the laminar flow width direction, a plurality of samples can be detected by one scan.
In this detection apparatus, a magnetic field or an electric field can be applied as the detection means.
When the sample is a microparticle, it is desirable that the microparticle is two-dimensionally arranged in the in-plane direction in the laminar sample liquid.
Furthermore, when forming a strip-shaped laminar flow, the sample liquid may be sandwiched between sheath liquids, and the flow path may be passed in that state.

  According to the present invention, a laminar laminar flow is formed and detected in the width direction, so that clogging of the sample can be prevented and a plurality of samples can be detected by one scan. As a result, the number of maintenance operations can be reduced and the measurement time can be greatly shortened. Further, more accurate detection signals can be obtained by removing leakage signals from a plurality of samples.

  The best mode for carrying out the present invention will be described below in detail with reference to the accompanying drawings. In addition, this invention is not limited to embodiment shown below.

  First, the detection method according to the first embodiment of the present invention will be described taking as an example the case of detecting fine particles such as cells and microbeads. FIG. 1A is a cross-sectional view showing a sample liquid flow state in the detection method of the present embodiment, and FIG. 1B is a cross-sectional view taken along line AA shown in FIG. As shown in FIGS. 1 (a) and 1 (b), in the detection method of the present embodiment, first, a plurality of flow cells 1 each having a rectangular cross-section perpendicular to the axial direction are provided in the flow cell 1. The sample liquid 2 containing the microparticles 4 is allowed to flow through the sheath liquids 3a and 3b so as to be sandwiched from both sides, thereby forming a strip-shaped laminar flow whose cross section perpendicular to the flow direction 5 is horizontally long.

  At this time, when a plurality of microparticles 4 are included in the sample liquid as in the present embodiment and these need to be detected individually, for example, the thickness d of the sample liquid 2 in a laminar flow state It is desirable to arrange the plurality of microparticles 4 two-dimensionally in the in-plane direction by adjusting In this way, in the sample liquid 2 that is in a laminar laminar flow, by preventing the microparticles 4 from overlapping in the thickness direction, the detection accuracy of the microparticles 4 in the detection process described later can be improved. it can.

  In the detection method of this embodiment, the laminar sample liquid 2 is irradiated with the laser beam 6 while scanning in the width direction w, and fluorescence and / or scattered light emitted from the microparticles 4. Is detected. FIG. 2 is a diagram schematically showing an example of the configuration of the optical system in the detection method of the present embodiment. As an optical system for realizing the detection method of the present embodiment, for example, as shown in FIG. 2, a light irradiation unit for irradiating one surface side of the flow cell 1 with the laser light 6 and the microparticles 4. A scattered light detection unit that detects the scattered light 10 emitted from the flow cell 1 and a fluorescence detection unit that detects the fluorescence 11 emitted from the microparticles 4 are provided on the other surface side of the flow cell 1. it can.

  The light irradiation unit in the optical system shown in FIG. 2 includes, for example, a laser oscillator, a mirror, a condenser lens 7 and the like. The laser beam 6 emitted from the laser oscillator and reflected by the mirror in the direction of the flow cell 1 is The light is condensed by the condenser lens 7 and irradiated to the sample liquid 2. At that time, the laser oscillator can be appropriately selected according to the measurement contents, and for example, a solid-state laser such as a YAG laser, a semiconductor laser, a femtosecond laser, or the like can be used. It is desirable that the size of the laser beam 6 to be irradiated is substantially the same as the size of the sample to be measured. Thereby, the leakage signal resulting from the sample flowing in the vicinity can be reduced.

  As a method of scanning the laser beam 6, for example, a method of operating a light beam by an acousto-optic modulator (AOM) or MEMS (micro electro mechanical systems), or only a condensing lens portion of the optical system. It is possible to apply a method of mechanically scanning the image.

  On the other hand, the fluorescence detection unit and the scattered light detection unit include, for example, a photodetection device such as a CCD (Charge Coupled Device) and a PMT (Photo-Multiplier Tube), a spectroscopic element, a mirror, and a condensing element. It can be set as the structure provided with the lens etc. In the case of the optical system shown in FIG. 2, a fly-eye lens 8 in which a plurality of condenser lenses 8a are arranged in a matrix and a condenser lens 9 having a diameter larger than that of the condenser lens 8a are the optical axes of the lenses. Are arranged in parallel with each other. Then, the fluorescence 11 emitted from the flow cell 1 is collected by one of the condenser lenses 8 a of the fly-eye lens 8, and then condensed by a condenser lens 9 disposed outside the fly-eye lens 8. Is done. With such a configuration, even when the optical axis changes in the width direction by scanning the laser light 6 in the width direction of the laminar flow, the fluorescence 11 can be efficiently collected.

  Furthermore, a dichroic filter 12 may be provided on the inner surface of the flow cell 1 on the condenser lens 8 side, that is, the inner surface on the side from which the fluorescence 11 is emitted. Thereby, since the wavelength of the laser beam 6 irradiated to the sample can be removed by the dichroic filter 12, only the light emitted from the sample can be incident on a photodetection device such as a CCD or PMT.

  FIG. 3 is a diagram showing the result of measuring the fluorescence intensity with the optical system shown in FIG. As shown in FIG. 3, when the fluorescence intensity of the microparticles 4 is measured by the detection apparatus having the above-described configuration, the fluorescence 11 emitted from the plurality of microparticles 4 can be detected by one scan. Then, by analyzing this measurement data, the size, shape, internal state, surface state, surface antibody reaction, etc. of each microparticle 4 can be identified.

  In the detection method of the present embodiment, a plurality of laser beams having different wavelengths may be irradiated in a time division manner. Thereby, since the microparticles 4 having different excitation wavelengths can be detected by one scanning, the measurement efficiency can be further improved.

  As described above, in the detection method of the present embodiment, the sample liquid 2 including the microparticles 4 to be measured forms a strip-shaped laminar flow whose cross section perpendicular to the flow direction 5 is horizontally long. The area of the cross section perpendicular to the axial direction (liquid flow direction) of the flow path is larger than that of the detection method. Thereby, it becomes difficult for the fine particles 4 to be clogged, and the number of maintenance can be reduced.

  Further, in the detection method of the present embodiment, the laser light irradiation position is not fixed as in the conventional detection method, but the laser light 6 is scanned in the width direction of the sample liquid 2, so that one scanning is performed. It is possible to measure a plurality of fine particles 4. And measurement efficiency can further be improved by irradiating a plurality of laser beams having different wavelengths in a time-sharing manner. As a result, the time required for measurement can be greatly reduced as compared with the conventional detection method.

  Furthermore, in the detection method of the present embodiment, the flow rate of the sample liquid can be increased as compared with the conventional detection method, so that the speed of sorting the fine particles performed based on the detection result is also improved. Furthermore, since the fluid pressure can be reduced by increasing the flow rate compared to the conventional detection method, the pressure applied to the microparticles can be reduced and the damage to the microparticles can be reduced. This effect is particularly effective when measuring biological materials such as cells.

  In the detection method of the present embodiment, a more accurate detection signal can be obtained by removing the leak signal from the sample caused by the sample flowing in the vicinity by various methods. Further, by using the flow position information obtained from the scanning irradiation light, it is possible to correct the detection signal intensity and adjust the flow of the sample. As a result, it is possible to detect the sample with higher accuracy and to perform analysis in more detail.

  In the detection method of the present embodiment, the case where the sample is a microparticle such as a cell and a bead has been described. However, the present invention is not limited to this, and detection of DNA, protein, blood, a tissue piece, and the like is performed. It is possible to apply to. The detection method of the present embodiment is particularly suitable for measuring DNA, protein, blood, and tissue fragments that are not sorted by size with a filter or the like.

  Also, the light applied to the sample is not limited to the laser light, and it is possible to irradiate light of various wavelengths such as visible light, infrared light, and ultraviolet light. And it can select suitably according to the characteristic of a sample. In particular, it is preferable to use light emitted from a laser or a light emitting diode (LED) as the irradiation light.

  Further, the shape of the flow path is not limited to the case where the cross-sectional shape perpendicular to the axial direction (flow direction) is rectangular. 4A and 4B are diagrams showing the cross-sectional shape of the flow path. Specifically, in addition to the channel whose cross-sectional shape perpendicular to the axial direction is rectangular, for example, the cross-sectional shape perpendicular to the axial direction is square like the flow cell 13 shown in FIG. As in the flow cell 14 shown in (b), the cross-sectional shape perpendicular to the axial direction may be circular, and furthermore, a flow path having an elliptical cross-sectional shape perpendicular to the axial direction may be applied.

  Furthermore, the method for detecting the sample flowing in the flow path is not limited to light irradiation, and for example, the sample can be detected by a magnetic field such as magnetic beads or magnetically modified cells. In this case, a method of detecting with a magnetic field detection element such as a Hall element or a magnetic optical element can be applied. In addition, when the sample can be detected by an electric field such as a minute conductor, a method of detecting a change in electric resistance, for example, by an electric field detection element such as a minute counter electrode element can be applied. FIG. 5 is a diagram schematically showing a method for detecting a sample by an electric field or a magnetic field. In this case, as shown in FIG. 5, a magnetic detection element array or electric field detection element array 33 in which a plurality of detection elements 33a each consisting of a magnetic detection element or an electric field detection element are arranged, and each detection element 33a is in the width direction of the flow path. By arranging so as to line up with each other, a magnetic field or an electric field can be scanned in the width direction of the flow path.

  Next, a detection method according to the second embodiment of the present invention will be described. In the detection method of the first embodiment described above, the sample is detected using the flow cell, but the present invention is not limited to this, and it is also possible to use a substrate or the like on which a fine channel is formed. it can. Therefore, in the detection method of the present embodiment, a strip-shaped laminar flow is formed in the fine channel formed on the substrate, and the sample contained therein is detected.

  FIG. 6 is a perspective view schematically showing an example of a channel structure for forming a strip-shaped laminar flow in the detection method of the present embodiment, and FIG. 7 is a sample solution flowing in the channel shown in FIG. It is a perspective view which shows a state typically. When a strip-shaped laminar flow is formed in a channel formed on a substrate, for example, a channel structure shown in FIG. 6 may be provided in the substrate. Specifically, the two sheath liquid introduction channels 22a and 22b from both sides of the sample solution introduction channel 21 merge into one channel in the substrate, and then the same in the sample solution introduction direction x. A first bent portion 23 bent in a direction y orthogonal to the plane, a second bent portion 24 bent in a direction z perpendicular to the direction x and the direction y, and a third bent portion 25 bent in the sample liquid introduction direction x. However, it is only necessary to form a flow path having a structure provided in this order. In the case of this flow path structure, the measurement unit 26 is provided at an arbitrary position downstream of the third bent portion 25.

  When the sample liquid 27 and the sheath liquids 28a and 28b are caused to flow through the flow path shown in FIG. 6, as shown in FIG. 7, at the junction between the sample liquid introduction flow path 21 and the sheath liquid introduction flow paths 22a and 22b. The sample liquid 27 is sandwiched between the sheath liquids 28a and 28b from both sides to form a belt-like flow. At this time, each liquid flows in the direction x, and flows in the order of the sheath liquid 28a, the sample liquid 27, and the sheath liquid 28b with respect to the direction y. Thereafter, the flow direction of the first bent portion 23 is rotated by approximately 90 ° on the same plane to become the direction y, and the second bent portion 24 is rotated by 90 ° in the vertical direction to become the direction z. Further, in the third bent portion 25, the flow direction is the same direction x as the sample liquid introduction channel 21, and the arrangement of the liquids is in the order of the sheath liquid 28a, the sample liquid 27, and the sheath liquid 28b toward the direction z. . Then, the sample liquid 27 that has passed through the third bent portion 25 becomes a strip-shaped laminar flow whose cross section perpendicular to the flow direction is horizontally long.

  Also in the detection method of the present embodiment, similarly to the detection method of the first embodiment described above, the sample liquid 27 in a laminar flow state is irradiated with light while scanning in the width direction. The light emitted from the sample contained in is detected. For example, when using the substrate provided with the flow path structure shown in FIG. 6, the measurement unit 26 emits light such as laser light while scanning in the width direction of the sample liquid 27, that is, the direction parallel to the direction y. Irradiate.

  Note that the structure of the flow path provided in the substrate is not limited to the structure shown in FIGS. 6 and 7, and any structure that can form a strip-like laminar flow may be used. FIG. 8A is a perspective view schematically showing another structural example of the flow path for forming a strip-shaped laminar flow, and FIG. 8B is a sample inside the flow path shown in FIG. It is a perspective view which shows the state through which a liquid flows typically. For example, in addition to the structures shown in FIGS. 6 and 7, a strip-shaped laminar flow can be formed even in a flow path having a structure having only one bent portion as shown in FIGS. 8 (a) and 8 (b). Is possible. In the case of this flow path structure, the measurement unit 26 is provided at an arbitrary position downstream of the bent portion 29.

  Also, a sheath liquid introduction channel is provided downstream of the third bent portion 25 of the flow channel shown in FIGS. 6 and 7 or the bent portion 29 of the flow channel shown in FIG. 8 to form a strip-shaped laminar flow. It is also possible to adopt a structure in which the sheath liquid is introduced so that the sample liquid is sandwiched from the width direction. This makes it possible to easily adjust the width of the strip-shaped laminar flow.

  Also in the detection method of the present embodiment, the sample liquid 27 forms a band-like laminar flow and irradiates light while scanning in the width direction, thereby preventing clogging of the sample and shortening a plurality of samples. Can be detected in time. As a result, the number of times of maintenance of the flow system can be reduced, and the measurement time can be greatly shortened. Moreover, in the detection method of this embodiment, since a laminar flow is formed in the flow path formed on the substrate, a strip-shaped laminar flow can be formed easily and stably.

  Furthermore, since the detection method of the present embodiment can make the incident direction of the laser light perpendicular to the substrate surface, the optical system can be easily installed, can be detected at points on a plurality of substrates, and an optical microscope, etc. It can be combined with other observation devices. Furthermore, in the detection method of the present embodiment, it is possible to further increase the speed by forming a plurality of the above-described flow paths on the substrate and further parallelizing them.

  The configuration and effects other than those described above in the detection method of the present embodiment are the same as those of the detection method of the first embodiment described above.

  On the other hand, as in the detection methods of the first and second embodiments described above, when a band-shaped laminar flow is formed and detection is performed by laser light irradiation, the incident position of light on the AOM element for scanning light As a result, an intensity distribution is generated in the laser beam, so that the amount of light of the laser spot after passing through the AOM is large at the center of the flow path and decreases as it approaches the end. The detection signal derived from the sample may be smaller than the detection signal derived from the sample flowing through the central portion. In such a case, it is desirable to correct the signal amount and sensitivity of detection light such as fluorescence and scattered light based on the flow position of each sample in the flow path and the distribution of the laser intensity after transmission through the AOM.

  FIG. 9A is a diagram showing the detection signal before correction, with the horizontal axis representing the position in the width direction in the flow path and the vertical axis representing the signal intensity, and FIG. 9B shows the width direction at that time. It is a figure which shows intensity distribution of the laser beam after AOM permeation | transmission in. FIG. 10 (a) is a diagram showing the corrected detection signal, with the horizontal axis representing the position in the width direction in the flow path and the vertical axis representing the signal intensity, and FIG. 10 (b) shows the signal after the AOM transmission. It is a figure which shows the correction coefficient of laser intensity | strength. Of the detection signals shown in FIGS. 9 (a) and 10 (a), the signal at the outermost position in the width direction is the detection signal from the side wall, and the signal at the inner side is the signal from the microparticle. Signal.

  Specifically, a laminar flow is formed with the sample liquid, and when irradiating laser light while scanning in the width direction, not only the sample liquid but also the side wall of the flow path is irradiated with laser light. Scattered light, diffracted light, reflected light or transmitted light from the part is detected. Then, based on the detection signal derived from the side wall of the flow path, the position of the laser spot in the flow path is calculated, the correction coefficient of the laser intensity after AOM transmission is read, and each detection signal shown in FIG. Is corrected with the correction coefficient shown in FIG. 10B based on the position information of the corresponding sample. Thereby, the detection signal shown in FIG.

  In addition, the flow velocity of the laminar flow is the fastest at the center due to the so-called Hagen-Poeisyu distribution, and slows toward the end, so that the detection signal derived from the sample flowing near the side of the flow path passes through the center. It may be larger than the detection signal derived from the flowing sample. In such a case, it is desirable to correct the scanning speed of the laser light in accordance with the flow speed of the sample in the flow path. Thereby, the intensity | strength, detection sensitivity, accuracy, etc. of a detection signal can be improved.

  Similarly in that case, when forming a laminar laminar flow with the sample liquid and irradiating the laser beam while scanning in the width direction, not only the sample liquid but also the side wall of the channel is irradiated with the laser beam, Fluorescence or scattered light emitted from this part is detected. Based on the detection signal derived from the flow channel side wall, the position of the laser spot in the flow channel is calculated, the flow rate coefficient of the sample in the flow channel is read, and each detection signal is converted to the corresponding sample position. Based on the information, correction may be made with a flow velocity correction coefficient.

  Further, as in the detection methods of the first and second embodiments described above, when a plurality of microparticles flow through the flow path, a plurality of samples that flow in the vicinity when there is one detection laser beam. Fluorescence or scattered light from each other may overlap, and crosstalk may occur in the detection signal. In this case, a plurality of auxiliary irradiation lights for correction are irradiated around the main irradiation light to be irradiated on the sample, and the detection signals are subjected to arithmetic processing to remove leakage signals from the sample flowing through the surroundings. It is desirable.

  Specifically, in addition to the main laser beam, a plurality of auxiliary laser beams are irradiated around it, and a leak signal is calculated by calculating a detection signal by the main laser beam and a detection signal by the auxiliary laser beam, respectively. Remove it. Thereby, the S / N ratio of the detection signal by the main laser beam can be improved.

  FIG. 11 is a diagram illustrating an example of the irradiation position of the auxiliary laser beam. Specifically, as shown in FIG. 11, a strip-like laminar flow is formed with the sample liquid 2, and the main laser beam 31 is irradiated while scanning in the width direction, and a plurality of auxiliary laser beams 32 are provided around it. Irradiation is performed, and fluorescence or scattered light emitted from each microparticle 4 is detected. At this time, not only the sample liquid 2 but also the side wall 30 of the flow path is irradiated with the laser beams 31 and 32. Then, the position of the laser spot in the flow path is calculated based on the detection signal derived from the flow path side wall, and each detection signal is added to the detection signal from the main laser beam 31 and the auxiliary based on the position information of the corresponding sample. What is necessary is just to calculate the detection signal by the laser beam 32, respectively.

  Various signal processing methods can be applied to the arithmetic processing method at that time. For example, the detection signal of the main laser beam is A, the detection signal of the auxiliary laser beam is B, and the adaptation coefficient is α (0 <α <1). ), The signal C after crosstalk correction can be obtained by the following formula 1.

  Although FIG. 8 shows a case where four auxiliary laser beams 32 are provided, the present invention is not limited to this, and the number, arrangement, intensity, and calculation method of the auxiliary laser beams are minute. It can be set as appropriate according to the density and flow rate of the particles.

(A) is sectional drawing which shows the flow state of the sample liquid in the detection method of the 1st Embodiment of this invention, (b) is sectional drawing by the AA line shown to (a). It is a figure which shows typically an example of a structure of the optical system in the detection method of 1st Embodiment of this invention. It is a figure which shows the result of having measured fluorescence intensity with the optical system shown in FIG. (A) And (b) is a figure which shows the cross-sectional shape of a flow path. It is a figure which shows typically the method of detecting a sample with an electric field or a magnetic field. In the detection method of the 2nd Embodiment of this invention, it is a perspective view which shows typically an example of the flow-path structure for forming a strip | belt-shaped laminar flow. FIG. 7 is a perspective view schematically showing a state in which a sample liquid flows in the flow path shown in FIG. 6. (A) is a perspective view which shows typically the other structural example of the flow path for forming a strip | belt-shaped laminar flow, (b) is a model which shows the state which a sample liquid flows through the flow path shown to (a) FIG. (A) is the figure which shows the position of the width direction in a flow path on a horizontal axis, and shows the detection signal before correction | amendment by taking the signal intensity on a vertical axis | shaft, (b) is after the AOM transmission in the width direction in that case It is a figure which shows laser intensity distribution. (A) is a figure which shows the detection signal after correction | amendment, taking the position of the width direction in a flow path on a horizontal axis, and a signal intensity on a vertical axis | shaft, (b) is a correction coefficient of the laser intensity after AOM transmission. FIG. It is a figure which shows an example of the irradiation position of an auxiliary | assistant laser beam. It is a figure which shows typically the structure of the conventional flow cytometer described in patent document 1. FIG. It is a perspective view which shows the structure of the flat flow cell apparatus described in patent document 2. FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1, 13, 14 Flow cell 2, 27 Sample liquid 3a, 3b, 28a, 28b Sheath liquid 4 Fine particle 5 Flow direction 6 Laser light 7, 8a, 9 Condensing lens 8 Fly eye lens 10 Scattered light 11 Fluorescence 12 Dichroic filter 21 Sample liquid introduction flow path 22a, 22b Sheath liquid introduction flow path 23, 24, 25, 29 Bending part 26 Measuring part 30 Side wall 31 Main laser light 32 Auxiliary laser light 33 Detection element array 33a Magnetic field detection element or electric field detection element 101 Flow Cytometer 102 Fluid 103 Optical system 104 Signal processing device 105 Cell 106a, 106b, 106c Light source 107 Light guide member 110 Flow cell device 111 Transparent substrate 112 Sample liquid port 113a, 113b Sheath liquid port 114, 115a, 115b, 117 flow path 11 6 Junction point 118 Waste liquid port

Claims (26)

A method of irradiating a sample flowing through a flow path with light and detecting light emitted from the sample,
A detection method in which a strip-shaped laminar flow is formed with a sample liquid containing the sample, and irradiation is performed while scanning light in the width direction of the sample liquid in the laminar flow state.   The detection method according to claim 1, wherein the sample is a microparticle, and the microparticle is two-dimensionally arranged in an in-plane direction in a laminar flow sample liquid.   The detection method according to claim 1 or 2, wherein the laminar sample liquid is irradiated with a plurality of lights having different wavelengths in a time-sharing manner.   The detection method according to any one of claims 1 to 3, wherein fluorescence and / or scattered light emitted from the sample is detected.   The detection method according to any one of claims 1 to 4, wherein a strip-shaped laminar flow is formed by sandwiching the sample liquid with a sheath liquid and allowing the flow path to flow in that state.   The detection method according to claim 1, wherein the side wall of the flow path is also irradiated with light, and the flow position of the sample is specified based on detection light derived from the side wall.   The detection method according to claim 6, wherein a detection signal is corrected based on the flow position information and an intensity distribution of light applied to the sample.   The detection method according to claim 6, wherein the scanning speed of the light is adjusted based on the flow position information and the flow velocity distribution of the sample liquid.   Irradiating a plurality of auxiliary irradiation lights for correction around the main irradiation light to irradiate the sample, calculating each detection signal, and removing leakage signals from the sample flowing through the surroundings The detection method according to claim 1, wherein:   A detection method in which a strip-shaped laminar flow is formed, and a sample included in the laminar flow is detected while scanning a detection means in the width direction of the laminar flow.   The detection method according to claim 10, wherein the detection means is a magnetic field or an electric field.   The detection method according to claim 10 or 11, wherein the sample is a microparticle, and the microparticle is two-dimensionally arranged in an in-plane direction in a sample liquid in a laminar flow state.   The detection method according to any one of claims 10 to 12, wherein a band-shaped laminar flow is formed by sandwiching the sample liquid with a sheath liquid and allowing the flow path to flow in that state. A detection device that irradiates light to a sample flowing in a flow path and detects light emitted from the sample,
A laminar flow forming part that forms a laminar laminar flow with a sample liquid containing a sample;
A light irradiation unit that irradiates light while scanning the sample liquid in a laminar flow state in the width direction;
A detection unit that detects light emitted from the sample.   The detection apparatus according to claim 14, wherein the sample is microparticles, and the microparticles in the sample liquid are two-dimensionally arranged in an in-plane direction in the laminar flow forming unit.   The detection apparatus according to claim 14 or 15, wherein the laminar sample liquid is irradiated with a plurality of lights having different wavelengths in a time-sharing manner.   The detection device according to any one of claims 14 to 16, wherein the detection unit detects fluorescence and / or scattered light emitted from the sample.   The detection apparatus according to any one of claims 14 to 17, wherein a band-shaped laminar flow is formed by sandwiching the sample liquid with a sheath liquid and allowing the flow path to flow in that state. .   The detection apparatus according to claim 14, wherein the side wall of the flow path is also irradiated with light, and the flow position of the sample is specified based on the detection light derived from the side wall.   The detection apparatus according to claim 19, wherein a detection signal is corrected based on the flow position information and an intensity distribution of light applied to the sample.   The detection apparatus according to claim 19, wherein a scanning speed of the light is adjusted based on the flow position information and a flow velocity distribution of the sample liquid.   A plurality of auxiliary irradiation lights for correction are irradiated around the main irradiation light irradiated to the sample, and leak signals from the sample flowing through the surroundings are removed by calculating each detection signal. The detection device according to claim 14.   A detection apparatus that forms a strip-shaped laminar flow and detects a sample contained in the laminar flow while scanning a detection means in the width direction of the laminar flow.   The detection device according to claim 23, wherein the detection means is a magnetic field or an electric field.   The detection apparatus according to claim 23 or 24, wherein the sample is a microparticle, and the microparticle is two-dimensionally arranged in an in-plane direction in a sample liquid in a laminar flow state. The detection apparatus according to any one of claims 23 to 25, wherein a band-like laminar flow is formed by sandwiching the sample liquid with a sheath liquid and allowing the flow path to flow in that state. .

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