JP2009270990A - Optical measurement method using passage and device therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new technique for simplifying an optical system and effectively performing measurement, in an optical system measurement method using a passage. <P>SOLUTION: A method for optically measuring a sample that passes in the passage by irradiating the sample with two or more laser beams with different wavelengths, at least includes the steps of: passing the sample in the passage, which is modified by two or more coloring matters that excitation spectra in the each wavelength are not duplicated, making each laser beam incident to the sample that passes in the passage concentrically and simultaneously, and detecting optical information from the coloring materials. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an optical measurement method using a flow path. More specifically, the present invention relates to an optical measurement method for optically detecting a sample flowing in a flow path, and an optical measurement device suitable for the optical measurement method.

  In recent years, with the development of analytical methods, biological microparticles such as cells and microorganisms, microparticles such as microbeads are passed through the flow path, and the microparticles are individually measured or measured in the flow process. A method for analyzing and sorting the fine particles is being developed. As a typical example of a technique for analyzing or sorting microparticles using such a flow path, technological improvement of an analysis technique called flow cytometry is rapidly progressing.

  The flow cytometry is generated from each microparticle by flowing microparticles to be analyzed into a fluid, forming a row of the microparticles, and irradiating the aligned microparticles with a laser beam or the like. This is an analysis method for analyzing microparticles by detecting fluorescence and scattered light, and further sorting microparticles based on the analysis results.

  Hereinafter, the flow cytometry will be specifically described with reference to FIG. FIG. 13 is a schematic diagram of a conventional flow cytometer. The process of flow cytometry can be roughly divided into (1) water flow system, (2) optical system, (3) electrical system, and (4) preparative system.

(1) Water flow system In the water flow system, fine particles to be analyzed are aligned in a line in a flow cell (flow channel) (see (1) in FIG. 13). More specifically, the sheath flow F102 is caused to flow into the flow cell at a constant flow rate, and in this state, the sample flow F101 containing microparticles is slowly injected into the center of the flow cell. At this time, due to the principle of laminar flow, the respective flows are not mixed with each other, and a laminar flow (laminar flow) is formed. Then, the inflow amounts of the sheath flow F102 and the sample flow F101 are adjusted according to the size of the microparticles to be analyzed, and the microparticles are allowed to flow in an aligned state.

(2) Optical system In the optical system, laser light is irradiated to fine particles to be analyzed, and fluorescence and scattered light emitted from the fine particles are detected (see (2) in FIG. 13). In the water flow system (1), the micro-particles are passed through the laser irradiation unit in a state where the micro-particles are aligned one by one, and each time the micro-particles pass, fluorescence and scattered light emitted from the micro-particles are emitted. Detect each parameter using an optical detector and analyze the characteristics of each microparticle.

(3) Electrical system In the electrical system, optical information detected by the optical system is converted into an electrical signal (voltage pulse) (see (3) in FIG. 13). The converted electrical signal is digitized, and a histogram is extracted by the computer and software for analysis based on the digitized data for analysis.

(4) Preparative system In the preparative system, fine particles that have been measured are separated and collected (see (4) in FIG. 13). As a typical sorting method, a plus or minus charge is added to a microparticle that has been measured, and the flow cell is sandwiched between two deflecting plates D having a potential difference. There is a method of sorting by being drawn to the deflection plate.

  There are various analysis and fractionation techniques for microparticles in the flow channel such as flow cytometry, such as medical field, drug discovery field, clinical laboratory field, food field, agricultural field, engineering field, forensic field, criminal field, etc. Widely used in various fields. Particularly in the medical field, it plays an important role in pathology, tumor immunology, transplantation, genetics, regenerative medicine, chemotherapy and the like.

  As described above, there is a need for a technique for analyzing and sorting microparticles in a flow path in a very wide field, and the techniques related to the processes (1) to (4) are being developed daily. ing. For example, in Patent Document 1, a measurement substance having a different size and color is immobilized on each of a plurality of types of measurement fine particles, and a fluorescently labeled substance bonded to the immobilized measurement substance is used with a laser beam. Thus, there has been proposed a method for measuring a substance using flow cytometry that makes it possible to identify the type in a two-dimensional matrix by measuring the size and color.

  In Patent Document 2, the intensity of the laser light is time-modulated at a predetermined frequency and irradiated to the labeled sample, and the fluorescence of the labeled sample at this time is received by a plurality of detection sensors having different light receiving wavelength bands, By collecting detection values including phase information from each detection sensor, etc., when obtaining each fluorescence intensity using detection values of fluorescence emitted by simultaneously irradiating a labeled sample containing a plurality of fluorescent dyes with laser light, Techniques have been proposed for increasing the number of types of fluorescent light that can be identified as compared to the conventional technique.

JP 2007-101212 A JP 2007-127415 A

  As described above, techniques for performing various measurements by modifying a sample to be measured with a plurality of dyes and optically obtaining the dye information are being developed.

  However, as shown in FIG. 14, when the excitation spectra X, Y, and Z of the respective dyes x, y, and z overlap in the wavelengths A, B, and C of the respective excitation lights a, b, and c, all the excitation lights Since a, b, and c could not be irradiated simultaneously, as shown in FIG. 15, it was necessary to irradiate each excitation light a, b, and c separately to the sample S flowing through the flow path 112. .

  Therefore, irradiation optical systems such as the condenser lens 117 are required for the number of excitation lights a, b, and c, and alignment of each laser spot by each irradiation optical system is also required, which is complicated.

  Further, as shown in FIG. 16, it is necessary to set the interval between the samples S to be longer than the total distance of the irradiation spots, and there is a problem that the detection time becomes longer.

  Furthermore, since it is necessary to perform spectrum analysis for each of the excitation lights a, b, and c, there is a problem that the analysis time is also prolonged.

  Therefore, the main object of the present invention is to provide a novel technique capable of simplifying the optical system and enabling more efficient measurement in an optical system measurement method using a flow path.

  In order to solve the above-mentioned object, the inventors of the present application have conducted intensive research on the structure of the dye and the optical system to be used. As a result, by paying attention to the relationship between the wavelength of the excitation light and the excitation spectrum of the dye, The present invention has been completed.

That is, in the present invention, first, a method of optically measuring the sample by irradiating the sample flowing through the channel with two or more laser beams having different wavelengths,
A flow step of flowing the sample modified with at least two or more dyes whose excitation spectra do not overlap at each wavelength through the flow path;
A light irradiating step in which the laser beams are incident on the sample passing through the flow path coaxially and simultaneously; and
An optical information detection step for detecting optical information from the dye;
An optical measurement method for performing at least the above is provided.
In the optical measurement method according to the present invention, a flow interval control step of controlling the flow interval of the sample in accordance with the irradiation spot of the laser light can be further performed.
In the optical information detection step, the method is not particularly limited as long as optical information from the dye can be detected. For example, so-called multi-color detection capable of detecting a plurality of dyes can be performed.

In the present invention, next, two or more light sources for irradiating the sample with laser light of different wavelengths,
A flow path through which the sample modified with at least two or more dyes whose excitation spectra do not overlap at each wavelength flows;
Optical path changing means for causing two or more laser beams from the light source to enter the sample coaxially and simultaneously; and
Optical information detection means for detecting optical information from the dye;
An optical measurement device comprising at least the above is provided.
The optical measurement apparatus according to the present invention may further include a flow interval control means for controlling the flow interval of the sample in accordance with the irradiation spot of the laser beam.
As long as the optical information detection means can detect optical information from the dye, the type of the detector is not particularly limited. For example, a so-called multichannel photodetector in which a plurality of photodetectors are arranged in an array. Can be used.

  Here, technical terms used in the present invention are defined. The “sample” in the present invention refers to a substance that can flow through a flow path, such as biologically relevant microparticles such as cells, microorganisms, liposomes, DNA, and proteins, or synthetic particles such as latex particles, gel particles, and industrial particles. If so, include all.

  In the optical measurement method according to the present invention, two or more laser beams having different wavelengths are simultaneously incident on the sample flowing through the flow path, so that the optical system can be simplified and the light detection time can be reduced. Since measurement can be shortened and analysis time can be shortened, efficient measurement is possible.

  DESCRIPTION OF EXEMPLARY EMBODIMENTS Hereinafter, preferred embodiments for carrying out the invention will be described with reference to the drawings. In addition, embodiment described below shows an example of typical embodiment of this invention, and, thereby, the range of this invention is not interpreted narrowly.

<Optical measurement method>
FIG. 1 is a flow diagram of an optical measurement method according to the present invention.
The optical measurement method according to the present invention irradiates two or more laser beams (L1, L2, L3) of different wavelengths (for example, λ1, λ2, λ3) to the sample S flowing in the flow path, It is a method for optically measuring the sample S, and is a method for performing at least the flow step (I), the light irradiation step (II), and the optical information detection step (III). Further, the flow interval control step (IV) can be performed as necessary. Hereinafter, each step will be described in detail.

(I) Flow process The flow process (I) is a dye in which excitation spectra E1, E2, and E3 in the wavelengths λ1, λ2, and λ3 of the laser beams L1, L2, and L3 to be irradiated do not overlap as shown in FIG. In this step, the sample S modified with P1, P2, and P3 is passed through the flow path 12 (see FIG. 4 described later).

  The dyes P1, P2, and P3 used in this embodiment are dyes corresponding to the excitation lights L1, L2, and L3 having wavelengths λ1, λ2, and λ3, respectively. In this embodiment, the sample S is modified with three dyes P1, P2, and P3. However, the number of dyes to be modified is not particularly limited as long as it is two or more, and excitation is performed at each wavelength of the irradiated laser light. If the spectra do not overlap, modification with four or more dyes is also possible.

  Further, as shown in FIG. 3, for example, a dye P4 corresponding to the excitation light L3 can be used as long as the excitation intensity is not exhibited at the wavelengths λ1 and λ2 simultaneously with the dye P3 corresponding to the excitation light L3.

  The kind of the dye that can be used in the optical measurement method according to the present invention is not particularly limited as long as the above conditions are satisfied, and any known dye can be selected and used. Examples include Cascade Blue, Pacific Blue, Fluorescein isothiocyanate (FITC), Phycoerythrin (PE), Propidium iodide (PI), Texas red (TR), Peridinin chlorophyll protein (PerCP), Allophycocyanin (APC), 4 ', 6- Diamidino-2-phenylindole (DAPI), Cy3, Cy5, Cy7 and the like.

  The sample S modified with these dyes P1, P2, and P3 is caused to flow through the flow channel 12 as shown in FIG. The method for passing the sample S through the flow path 12 is not particularly limited. For example, there is a method in which the sample S is conveyed while being sandwiched by a fluid medium (sheath flow) that promotes rectification. If transported in this way, a laminar flow of the sample flow F1 including the sample S can be formed, which is more preferable. The type of the fluid medium is not particularly limited as long as it has a function of promoting rectification of the sample flow F1 including the sample S. For example, when the sample S is a cell, physiological saline or the like can be used. .

  In addition, the form of the flow path 12 which can perform the optical measuring method which concerns on this invention is not specifically limited, It can design freely. For example, the detection method according to the present invention is performed not only in the form shown in FIG. 4 but also in the flow path 12 formed on a substrate such as two-dimensional or three-dimensional plastic or glass as shown in FIG. It is possible.

(II) Light Irradiation Step The light irradiation step (II) is a step in which the laser beams L1, L2, and L3 are incident on the sample S flowing through the flow channel 12 coaxially and simultaneously.

  For example, as shown in FIG. 5, the laser beams L1, L2, and L3 emitted from the light sources 11a, 11b, and 11c are collected on one condenser lens 17 using the mirrors 16a, 16b, and 16c. The sample S flowing through the flow channel 12 is coaxially and simultaneously incident.

  In the optical measurement method according to the present invention, in the flow step (I), the dyes P1, P2, and P3, P2, and P3 in which the excitation spectra E1, E2, and E3 do not overlap at the wavelengths λ1, λ2, and λ3 of the laser beams L1, L2, and L3, Since the sample S modified with P3 is allowed to flow through the flow path, it is possible to simultaneously irradiate excitation light (laser beams L1, L2, and L3) having different wavelengths λ1, λ2, and λ3. Therefore, the irradiation optical system such as the condenser lens 17 can be simplified.

  Further, since the irradiation laser spot can be made one, the alignment of the irradiation laser spot can be easily performed.

  Furthermore, since one irradiation laser spot can be provided, the interval in the flow path of the sample S to be measured can be reduced to the diameter of the irradiation laser spot, and the detection processing speed can be greatly reduced. .

(III) Optical Information Detection Step The optical information detection step (III) is a step of detecting optical information from the dyes P1, P2, and P3 that have modified the sample S after the light irradiation step (II). .

  In the optical information detection step (III), the detection method is not particularly limited as long as optical information from the dyes P1, P2, and P3 can be detected, and a known optical method can be freely adopted. For example, fluorescence measurement, scattered light measurement, transmitted light measurement, reflected light measurement, diffracted light measurement, ultraviolet spectroscopic measurement, infrared spectroscopic measurement, Raman spectroscopic measurement, FRET measurement, FISH measurement, other various spectrum measurements, and multiple dyes can be detected. Examples include so-called multi-color detection. Further, if detection is performed using an area imaging device such as a charge coupled device (CCD) or a complementary metal oxide semiconductor (CMOS), an image emitted from the sample S on the entire screen can be photoelectrically converted.

  In the present embodiment, the fluorescence emitted from the dyes P1, P2, and P3 is divided for each wavelength using the diffraction grating 18, and the fluorescence spectrum is measured by the multichannel photodetector 14 (optical information detection means 14). Yes.

  In the optical measurement method according to the present invention, in the light irradiation step (II), since there is one irradiation laser spot, optical information from each dye can be detected at a time, and the detection processing speed can be increased. It can be greatly shortened.

  Further, since the fluorescence spectrum from each detected dye can be analyzed at a time, the analysis time can be greatly shortened.

(IV) Flow interval control step Although not essential in the present invention, the flow interval control step (IV) can be performed. In the flow interval control step (IV), the flow interval of the sample S is controlled in accordance with the size of the irradiation laser spot.

  The flow interval control step (IV) may be performed at least before the light irradiation step (II). For example, as shown in the flow chart of FIG. 1, the flow interval control step (IV) may be performed before the flow step (I). You may carry out after performing flow process (I).

As a method of performing the flow interval control step (IV) before performing the flow step (I), for example, as shown in FIG. 6, the solution S1 containing the sample S before the flow step (I) is performed. By adjusting the concentration, the flow interval of the sample S can be controlled in accordance with the size of the irradiation laser spot. The method for adjusting the concentration is not particularly limited. For example, as shown in FIG. 6, when a storage unit for the solvent S ₀ is provided and the concentration is decreased, the solvent S ₀ is introduced and the concentration is increased. by discharging the solvent S _0, you are possible to perform density adjustment of S1.

In this case, although not shown, it is desirable to provide a filter or the like that does not allow the sample S to pass through the inlet / outlet portion (the arrow A portion in FIG. 6) of the storage portion of the solvent S ₀ . This is to prevent the sample S from being discharged when the solvent S ₀ is discharged.

  Although not shown in the figure, the flow path of the sample S is provided by providing a filter or an open / close cock or the like at the inlet of the channel 12 (arrow B portion in FIG. 6), and increasing or decreasing the inflow amount of the solution S1 containing the sample S. The interval can be controlled in accordance with the size of the irradiation laser spot.

As a method of performing the flow interval control step (IV) after performing the flow step (I), for example, as shown in FIG. 7, a storage unit for the solvent S ₀ is provided in the middle of the flow path 12, and the sample When the interval of S is too narrow, the solvent S ₀ flows into the flow channel 12, and when the interval of the sample S is too wide, the solvent S ₀ is discharged from the flow channel 12, thereby The flow interval can be controlled in accordance with the size of the irradiation laser spot.

In this case, although not shown, it is desirable to provide a filter or the like between not through the sample S in (in FIG arrow C portion) between the reservoir and the passage 12 of the solvent S _0. This is to prevent the sample S from being discharged when the solvent S ₀ is discharged from the flow path 12.

(V) Sorting step Although not essential in the present invention, the sorting step (V) can be performed after the optical information detection step (III). In the sorting step (V), the sample S is sorted based on the optical information of the sample S obtained in the optical information detection step (III).

  As a specific example, although not shown, based on information on the size, form, internal structure, etc. of the sample S obtained in the optical information detection step (III) Therefore, the sample S can be separated using the deflection plate D or the like.

<Optical measuring device>
Next, the structure of the optical measuring apparatus according to the present invention will be described with reference to FIG. FIG. 5 is a schematic view schematically showing an embodiment of the optical measuring device 1 according to the present invention. The optical measuring device 1 according to the present invention is roughly divided into two or more light sources 11a, 11b, and 11c, a flow path 12, an optical path changing means 13, and an optical information detecting means 14. Each will be described in detail below.

(1) Light sources 11a, 11b, 11c
In the optical measuring device 1 according to the present invention, two or more light sources 11a, 11b, and 11c are used to irradiate at least two or more laser beams L1, L2, and L3 having different wavelengths. The number of light sources used in the optical measuring apparatus 1 according to the present invention is three light sources as in this embodiment if two or more light sources are used to irradiate at least two laser beams having different wavelengths. 11a, 11b, 11c may be used, or four or more light sources may be used.

  Further, the types of the light sources 11a, 11b, and 11c that can be used in the optical measuring device 1 according to the present invention are not particularly limited, and any known light source can be selected and used. For example, two or more argon ion (Ar) lasers, helium-neon (He-Ne) lasers, die lasers, krypton (Cr) lasers, and the like can be used in any combination.

(2) Channel 12
The sample S flows through the flow path 12, and light irradiation and optical information detection are performed at the predetermined portion. Although not shown, the sample S flowing through the flow channel 12 has at least two or more excitation spectra that do not overlap in the wavelengths λ1, λ2, and λ3 of the laser beams L1, L2, and L3 emitted from the light sources 11a, 11b, and 11c. It is modified with a dye. Since the number, type, and the like of the dye P that can be modified to the sample S are the same as those in the optical measurement method described above, the description is omitted here.

  The form of the flow path 12 that can be used in the optical measuring device 1 according to the present invention is not particularly limited, and can be freely designed. For example, not only the form shown in FIG. 5 but also the flow path 12 formed on the substrate T such as two-dimensional or three-dimensional plastic or glass as shown in FIG. 8 is also included in the optical measuring device 1 according to the present invention. Can be used.

  Further, the channel width, the channel depth, and the channel cross-sectional shape of the channel 1 are not particularly limited as long as they can form a laminar flow, and can be freely designed. For example, even a micro flow channel having a flow channel width of 1 mm or less can be used in the optical measuring device 1 according to the present invention. In particular, if a microchannel having a channel width of 10 μm or more and 1 mm or less is used, the optical measurement method according to the present invention can be more suitably performed.

  In addition, when employ | adopting the flow path 12 formed on the board | substrate T, it is preferable to form the bottom face of the flow path 12 with a permeable material. As shown in FIG. 8, optical information detection means 14 to be described later is disposed on the side opposite to the light sources 11 a, 11 b, 11 c across the substrate T so that optical information from the bottom surface side of the flow path 12 can be detected. It is to make it.

(3) Optical path changing means 13
The optical path changing unit 13 is a unit that causes the laser beams L1, L2, and L3 to be incident on the sample S flowing through the flow path 12 coaxially and simultaneously.

  For example, as shown in FIGS. 5 and 8, the laser beams L1, L2, and L3 emitted from the light sources 11a, 11b, and 11c are collected on one condenser lens 17 using the mirrors 16a, 16b, and 16c. Thus, the sample S flowing through the flow channel 12 is incident on the same axis at the same time.

  In the optical measuring device 1 according to the present invention, the sample S modified with the dyes P1, P2, and P3 in which the excitation spectra E1, E2, and E3 do not overlap at the wavelengths λ1, λ2, and λ3 of the laser beams L1, L2, and L3 is flowed. Since it is made to flow through the path 12, it is possible to irradiate simultaneously the excitation lights (laser lights L1, L2, L3) having different wavelengths λ1, λ2, λ3. Therefore, the irradiation optical system such as the condenser lens 17 can be simplified.

  Further, since the irradiation laser spot can be made one, the alignment of the irradiation laser spot can be easily performed.

  Furthermore, since the irradiation laser spot can be made one, the interval in the flow path of the sample S to be measured can be reduced to the diameter of the irradiation laser spot, so that the detection processing speed can be greatly reduced. is there.

(4) Optical information detection means 14
The optical information detection unit 14 is a unit that detects optical information from the dyes P1, P2, and P3 obtained by modifying the sample S.

  If the optical information detection means 14 can detect the optical information from the pigments P1, P2, and P3, the type of the detector is not particularly limited, and a known optical detector can be freely adopted. For example, fluorescence measuring instrument, scattered light measuring instrument, transmitted light measuring instrument, reflected light measuring instrument, diffracted light measuring instrument, ultraviolet spectroscopic measuring instrument, infrared spectroscopic measuring instrument, Raman spectroscopic measuring instrument, FRET measuring instrument, FISH measuring instrument and others Examples include various spectrum measuring devices, so-called multi-channel photodetectors in which a plurality of photodetectors are arranged in an array. Further, if a detector using an area imaging device such as a charge coupled device (CCD) or a complementary metal oxide semiconductor (CMOS) is employed, an image emitted from the sample S on the entire screen can be photoelectrically converted.

  In the embodiment shown in FIG. 5 and FIG. 8, the fluorescence emitted from the dyes P1, P2, and P3 is divided for each wavelength using the diffraction grating 18, and the multichannel photodetector 14 (optical information detection means 14) is used. The fluorescence spectrum is measured.

  Further, the arrangement location of the optical information detection means 14 is not particularly limited as long as fluorescence emitted from the dyes P1, P2, and P3 can be detected. However, as shown in FIGS. , 11b and 11c are preferably arranged on the opposite side. This is because optical information can be detected simultaneously with the irradiation of the laser beams L1, L2, and L3 from the light sources 11a, 11b, and 11c, and efficient measurement can be performed.

  Moreover, the optical information detection means 14 can be arrange | positioned by a more free structure by arrange | positioning the optical information detection means 14 on the opposite side to light source 11a, 11b, 11c across the flow path 12. FIG. Furthermore, since it is not necessary to secure a space for the optical information detection means 14, the number of the light sources 11a, 11b, and 11c can be increased.

  In the case where the flow path 12 on the substrate T is employed as in the embodiment shown in FIG. 8, it is preferable to form the bottom surface of the flow path 12 with a permeable material.

  In the optical measuring device 1 according to the present invention, the optical path changing means 13 is provided, so that the irradiation laser spot can be made one. Therefore, the optical information from each dye can be detected at a time, and the detection is performed. Processing speed can be greatly reduced.

  Further, since the fluorescence spectrum from each detected dye can be analyzed at a time, the analysis time can be greatly shortened.

(5) Flow interval control means 15
Although not essential in the present invention, the optical measurement device 1 according to the present invention can be provided with a flow interval control means 15. The flow interval control means 15 is a means for controlling the flow interval of the sample S in accordance with the size of the irradiation laser spot.

  The flow interval control means 15 only needs to be provided at least upstream of the position where irradiation from the light sources 11 a, 11 b, 11 c of the flow channel 12 is performed, and the flow interval before the sample S flows through the flow channel 12. May be provided in the uppermost stream of the flow channel 12 or may be provided in the middle of the flow channel 12 so that the interval of the sample S flowing through the flow channel 12 can be controlled.

The flowing interval control unit 15, as a method of controlling the flows interval provided most upstream of the flow path 12, for example, as shown in FIG. 6, the most upstream of the flow path 12 a reservoir of solvent S ₀ By providing and adjusting the concentration of the solution S1 containing the sample S before flowing through the flow path 12, the flow interval of the sample S can be controlled in accordance with the size of the irradiation laser spot. Specifically, when it is desired to reduce the concentration, flow of solvent S _0, when it is desired to thicken the concentration, by discharging the solvent S _0, you are possible to perform density adjustment of S1.

In this case, although not shown, it is desirable to provide a filter or the like that does not allow the sample S to pass through the inlet / outlet portion (the arrow A portion in FIG. 6) of the storage portion of the solvent S ₀ . This is to prevent the sample S from being discharged when the solvent S ₀ is discharged.

  Further, in order to control the flow interval of the sample S according to the size of the irradiation laser spot by increasing / decreasing the inflow amount of the solution S1 containing the sample S, although not shown, the inlet (see FIG. 6 may be provided with a filter or an open / close cock.

The flowing interval control unit 15, as a method of controlling the flows intervals provided in the middle of the channel 12, for example, as shown in FIG. 7, in the middle of the channel 12, provided with a reservoir of solvent S ₀ When the interval between the samples S is too narrow, the solvent S ₀ flows into the flow channel 12, and when the interval between the samples S is too wide, the sample is obtained by discharging the solvent S ₀ from the flow channel 12. The flow interval of S can be controlled in accordance with the size of the irradiation laser spot.

In this case, although not shown, it is desirable to provide a filter or the like between not through the sample S in (in FIG arrow C portion) between the reservoir and the passage 12 of the solvent S _0. This is to prevent the sample S from being discharged when the solvent S ₀ is discharged from the flow path 12.

  In Example 1, as shown in Table 1 below, dyes corresponding to excitation light wavelengths of 405 nm, 473 nm, and 635 nm, (i) Alexa405 (registered trademark), (ii) Cascade Blue, (iii) Pacific Blue, (iv) Alexa532 (Vii) Alexa568 (registered trademark), (vi) Phycoerythrin (PE), (vii) Alexa633 (registered trademark), (viii) Alexa647 (registered trademark), (ix) Allophycocyanin (APC), (x ) When Cy7 was selected and each dye was combined, it was examined whether the excitation spectra overlapped at each excitation light wavelength.

  FIG. 9 shows excitation spectra when (1) (i) Alexa405 (registered trademark), (iv) Alexa532 (registered trademark), and (vii) Alexa633 (registered trademark) are combined. As shown in FIG. 9, (i) Alexa405 (registered trademark), (iv) Alexa532 (registered trademark), and (vii) Alexa633 (registered trademark) have an excitation spectrum having wavelengths of 405 nm and 473 nm of each excitation light, It was found that there was no overlap at 635 nm.

  Therefore, in the optical measurement method according to the present invention, (i) Alexa405 (registered trademark), (iv) Alexa532 (registered trademark), (vii) Alexa633 (registered trademark) may be used in combination. I understood.

  FIG. 10 shows an excitation spectrum when (2) (ii) Cascade Blue, (v) Alexa568 (registered trademark), and (viii) Alexa647 (registered trademark) are combined. As shown in FIG. 10, the excitation spectrum exhibited by the dyes of (ii) Cascade Blue, (v) Alexa568 (registered trademark), (viii) Alexa 647 (registered trademark) is shown in the wavelength of 405 nm, 473 nm, and 635 nm of each excitation light. I found that there was no overlap.

  Therefore, it was found that the optical measurement method according to the present invention can be used in combination with (ii) Cascade Blue, (v) Alexa568 (registered trademark), and (viii) Alexa647 (registered trademark).

  FIG. 11 shows the excitation spectrum when (3) (iii) Pacific Blue, (vi) Phycoerythrin (PE), and (ix) Allophycocyanin (APC) are combined. As shown in FIG. 11, the excitation spectra exhibited by the dyes of (iii) Pacific Blue, (vi) Phycoerythrin (PE), and (ix) Allophycocyanin (APC) overlap at wavelengths 405 nm, 473 nm, and 635 nm of each excitation light. I found out.

  Therefore, it was found that the optical measurement method according to the present invention can be used in combination with (iii) Pacific Blue, (vi) Phycoerythrin (PE), and (ix) Allophycocyanin (APC).

  (4) (i) Alexa405 (registered trademark), (ii) Cascade Blue, (iii) Pacific Blue, (iv) Alexa532 (registered trademark), (v) Alexa568 (registered trademark), (vi) Phycoerythrin (PE), FIG. 12 shows excitation spectra when (vii) Alexa633 (registered trademark), (viii) Alexa647 (registered trademark), (ix) Allophycocyanin (APC), and (x) Cy7 are combined. As shown in FIG. 12, the excitation spectra of (i) Alexa405 (registered trademark), (ii) Cascade Blue, and (iii) Pacific Blue corresponding to excitation light with a wavelength of 405 nm do not show excitation intensity at wavelengths of 473 nm and 635 nm. (Iv) Alexa532 (registered trademark), (v) Alexa568 (registered trademark), (vi) Phycoerythrin (PE) corresponding to excitation light having a wavelength of 473 nm does not exhibit excitation intensity at wavelengths of 405 nm and 635 nm, and has a wavelength of 635 nm. (Vii) Alexa633 (registered trademark), (viii) Alexa647 (registered trademark), (ix) Allophycocyanin (APC), and (x) Cy7 corresponding to the excitation light show no excitation intensity at wavelengths of 405 nm and 473 nm. It was.

  Therefore, the optical measurement method according to the present invention includes (i) Alexa405 (registered trademark), (ii) Cascade Blue, (iii) Pacific Blue, (iv) Alexa532 (registered trademark), (v) Alexa568 (registered trademark). ), (Vi) Phycoerythrin (PE), (vii) Alexa633 (registered trademark), (viii) Alexa647 (registered trademark), (ix) Allophycocyanin (APC), (x) Cy7 can be used in combination I found out.

It is a flowchart of the optical measuring method which concerns on this invention. It is a drawing substitute graph which shows the excitation spectrum of pigment | dye P1, P2, and P2 which can be used for the optical measuring method which concerns on this invention. It is a drawing substitute graph which shows the excitation spectrum of pigment | dye P1, P2, and P2 which can be used for the optical measuring method which concerns on this invention. It is a perspective schematic diagram of the flow path 12 for demonstrating the flow process (I) in the optical measuring method which concerns on this invention. It is a mimetic diagram showing typically one embodiment of optical measuring device 1 concerning the present invention. It is a schematic diagram which shows typically one Embodiment of the flow space control means 15 of the optical measuring device 1 which concerns on this invention. It is a schematic diagram which shows typically embodiment different from FIG. 6 of the flow space | interval control means 15 of the optical measuring device 1 which concerns on this invention. It is a schematic diagram which shows typically different embodiment from FIG. 5 of the optical measuring device 1 which concerns on this invention. In Example 1, it is a figure substitute graph which shows the excitation spectrum at the time of combining (i) Alexa405 (trademark), (iv) Alexa532 (trademark), (vii) Alexa633 (trademark). In Example 1, it is a drawing substitute graph which shows the excitation spectrum at the time of combining (ii) Cascade Blue, (v) Alexa568 (trademark), (viii) Alexa647 (trademark). In Example 1, it is a drawing substitute graph which shows the excitation spectrum at the time of combining (iii) Pacific Blue, (vi) Phycoerythrin (PE), and (ix) Allophycocyanin (APC). In Example 1, (i) Alexa405 (registered trademark), (ii) Cascade Blue, (iii) Pacific Blue, (iv) Alexa532 (registered trademark), (v) Alexa568 (registered trademark), (vi) Phycoerythrin (PE) ), (Vii) Alexa633 (registered trademark), (viii) Alexa647 (registered trademark), (ix) Allophycocyanin (APC), (x) A graph instead of a drawing showing an excitation spectrum when combining all of Cy7. It is a schematic diagram of the conventional flow cytometer. It is a drawing substitute graph which shows the excitation spectrum of the pigment | dye used for the conventional optical measuring method. It is a schematic diagram which shows the conventional optical measuring device typically. It is a schematic diagram which shows the relationship between the space | interval of the irradiation spot at the time of using the conventional optical measuring apparatus, and the space | interval of a sample.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Optical measuring device 11a, 11b, 11c Light source 12 Flow path 13 Optical path change means 14 Optical information detection means 15 Flow interval control means 16a, 16b, 16c Mirror 17 Condensing lens 18 Diffraction grating 111 Light source 112 Flow path 114 Light Detector 116 Condenser lens S Sample S1 Sample solution S ₀ Solvents L1, L2, L3 Laser light P1, P2, P3 Dye T Substrate D Deflector F1 Sample flow x, y, z Dye a, b, c Laser light F101 Sample Flow F102 Sheath flow

Claims (6)

A method for optically measuring the sample by irradiating the sample flowing through the flow path with two or more laser beams having different wavelengths,
A flow step of flowing the sample modified with at least two or more dyes whose excitation spectra do not overlap at each wavelength through the flow path;
A light irradiating step in which the laser beams are incident on the sample passing through the flow path coaxially and simultaneously; and
An optical information detection step for detecting optical information from the dye;
An optical measurement method that performs at least.   The optical measurement method according to claim 1, further comprising a flow interval control step of controlling a flow interval of the sample according to an irradiation spot of the laser beam.   The optical measurement method according to claim 1, wherein multi-color detection is performed in the optical information detection step. Two or more light sources for irradiating the sample with laser beams of different wavelengths;
A flow path through which the sample modified with at least two or more dyes whose excitation spectra do not overlap at each wavelength flows;
Optical path changing means for causing two or more laser beams from the light source to enter the sample coaxially and simultaneously; and
Optical information detection means for detecting optical information from the dye;
An optical measuring device comprising at least.   The optical measurement apparatus according to claim 4, further comprising a flow interval control unit that controls a flow interval of the sample according to an irradiation spot of the laser beam.   6. The optical measurement device according to claim 4, wherein the optical information detection means is a multi-channel photodetector.