WO2008007580A1 - Method for analyzing fine particles - Google Patents

Method for analyzing fine particles Download PDF

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Publication number
WO2008007580A1
WO2008007580A1 PCT/JP2007/063297 JP2007063297W WO2008007580A1 WO 2008007580 A1 WO2008007580 A1 WO 2008007580A1 JP 2007063297 W JP2007063297 W JP 2007063297W WO 2008007580 A1 WO2008007580 A1 WO 2008007580A1
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Prior art keywords
analysis
fine particles
scanning
region
beads
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PCT/JP2007/063297
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French (fr)
Japanese (ja)
Inventor
Tamiyo Kobayashi
Masayoshi Kusano
Morinao Fukuoka
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Olympus Corporation
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Publication of WO2008007580A1 publication Critical patent/WO2008007580A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1493—Particle size

Definitions

  • the present invention relates to a method for analyzing fine particles such as biological materials with high sensitivity and high accuracy.
  • Patent Literature 1 JP 2001-502062
  • Patent Document 2 JP 2001-502066 Publication
  • the obtained data is known to have a large variation. [0004] Therefore, even when analyzing large biological materials such as cell membrane fragments, cells themselves, etc. having a diameter of 0.3 / zm or more, the obtained data is highly variable and highly accurate. Analysis cannot be performed. In particular, when preparing cell membrane fragments by cell membrane fractionation etc., it is difficult to homogenize the size of the cell membrane fragments, and these cell membrane fragments are often spherical with a diameter of 0.1 to 0.5 m. 0. Contains large molecules or particles of 3 m or more.
  • FCS single-molecule measurement methods
  • FIDA fluorescence intensity does not change more than 1.3 times before and after the interaction, the interaction cannot be detected.
  • a method may be used in which one of the interacting molecules is fixed to, for example, the surface of polystyrene particle beads and the interaction is detected.
  • Patent Document 1 In order to obtain highly accurate data, there is a limit to the size of a biological substance that can be measured. There was great variation, and it was only possible to scan the same area in a donut shape.
  • Non-Patent Document 1 Patent Documents 1 and 2 do not describe appropriate conditions such as scan speed and scan area when measuring beads with a size of 0.3 / zm or more. I got it. Means for solving the problem
  • the present invention has been made in view of the above circumstances, and even when the analysis target includes a size of 0.3 ⁇ m or more, the size of the analysis target microparticles or the binding of the microparticles Another problem is to provide a method for analyzing the presence or absence of deviation with high sensitivity and high accuracy. And
  • the present invention relates to a fine particle analysis method for scanning a minute region in a confocal region and measuring the signal strength of the minute region, and analyzing the size of the fine particle or the presence or absence of binding or detachment of the fine particle.
  • Fine particles whose volume of one micro area of a plurality of micro areas to be measured is 1 FL or less and whose total area area of the scanning area plane of the plurality of micro areas is 500 ⁇ m 2 to 40000 ⁇ m 2 This is an analysis method.
  • the scanning speed of one minute region for measuring the signal intensity may be 2.5 mmZsec to 25 mmZsec.
  • the analysis may be performed by at least one of fluorescence correlation spectroscopy, fluorescence intensity distribution analysis, and fluorescence polarization intensity distribution analysis.
  • the fine particles may be one or more selected from cells, cell membranes, cell membrane fragments, organic particles, inorganic particles, and one or more complex forces of these.
  • the size force of the fine particles may be 0.3 / ⁇ ⁇ to 10 / ⁇ ⁇ .
  • an analysis object includes a particle having a size of 0.3 m or more
  • the size of the particle to be analyzed or the presence or absence of the binding or detachment of the particle is highly sensitive and high. It can be analyzed with accuracy.
  • FIG. 1 is an explanatory diagram illustrating a comparison between the size of a fine particle that can be analyzed and analysis conditions.
  • FIG. 2A is a diagram illustrating a scanning method in the prior art.
  • FIG. 2B is a diagram illustrating a scanning method in the prior art.
  • FIG. 2C is a diagram illustrating a scanning method in the present invention.
  • FIG. 2B is a diagram illustrating a scanning method in the present invention.
  • FIG. 2C is a diagram illustrating a scanning method in the present invention.
  • FIG. 3A is a graph showing the analysis results of Example 1.
  • FIG. 3B is a graph showing the analysis results of Example 1.
  • FIG. 3C is a graph showing the analysis results of Comparative Example 1.
  • FIG. 4A is a graph showing the results of five measurements of Example 2 and Comparative Example 2.
  • FIG. 4B is a graph showing five measurement results of Example 2 and Comparative Example 2.
  • FIG. 5A is a graph showing an average value of five measurement results of Example 2 and Comparative Example 2.
  • FIG. 5B is a graph showing an average value of five measurement results of Example 2 and Comparative Example 2.
  • FIG. 6 is a graph showing the analysis results of Example 3.
  • FIG. 7 is a graph showing the analysis results of Example 4.
  • FIG. 8 is a measurement image obtained in Example 5.
  • the volume of the minute region for measuring the signal intensity can be adjusted, for example, by changing the magnification of the objective lens to be used.
  • the volume of one minute region (each minute region in a plurality of minute regions) for measuring the signal intensity is set to 1 FL (humid torr) or less. If it is larger than 1FL, it may be difficult to detect the signal intensity with high sensitivity.
  • the total area value of the scanning region planes of the plurality of minute regions for measuring the signal intensity is set to 500 ⁇ m 2 to 40000 m 2 . Outside this range, the detection value of the signal intensity has a large variation, resulting in low reliability.
  • the scanning speed of one minute region may be adjusted as appropriate, but is preferably 2.5 mmZ seconds to 25 mmZ seconds. Within this range, the detected value of the signal intensity has a smaller variation, which is suitable for highly accurate analysis.
  • FCS fluorescence correlation spectroscopy
  • FIDA fluorescence intensity distribution analysis method
  • FIDA-polarization fluorescence polarization intensity distribution analysis method
  • the detected signal intensity changes depending on the size of the fine particles to be analyzed. Therefore, if the microparticles to be analyzed bind to other microparticles or dissociate into a plurality of microparticles, and the size changes, the binding or dissociation can be detected by the change in signal intensity.
  • the term “bond” as used herein refers to a bond based on an intermolecular attractive force such as a hydrogen bond or a hydrophobic bond that is not only a covalent bond.
  • the fine particles used as the analysis target are not particularly limited, and can be the analysis target by a conventional method, for example, a particle having a size of 0.1 m or less may be the analysis target.
  • a large size for example, a size of 0.3 m to 10 ⁇ m, which has been difficult to achieve, is suitable.
  • examples of such materials include biological substances, organic particles, inorganic particles, and one or more selected from complex forces that are one or more of these.
  • Preferred examples of the biological substance include cells, cell membranes and cell membrane fragments.
  • these fine particles those prepared by a conventionally known method or commercial products can be used. For example, as long as it is a bio-related substance, it is possible to use a sample extracted from the sample obtained by processing a sample obtained by collecting vital force by a conventionally known method.
  • Receptors present on the cell membrane such as GPCR, are thought to have many orphan receptors, but there are also important receptors that are targets for drug discovery. For this reason, recently, cell membrane fractions are often used to search for inhibitors or ligands. Thus, cells, cell membranes, cell membrane fragments and the like having important functions and large sizes are particularly suitable as the analysis target of the present invention.
  • the cell membrane usually exists as a ribosome in a solution.
  • membrane fractionation is performed by a conventionally known method, cell membrane fragments are obtained, and their sizes often vary by about 0.1 to 0.5 m in diameter.
  • Such items are conventionally labeled with radioisotopes (RI), spun down, and detected with a scintillation counter.
  • RI radioisotopes
  • analysis using RI is the mainstream.
  • the conventional single molecule measurement method is applied, only data with large variations can be obtained.
  • Fig. 1 shows an example of a comparison of the size of the fine particles that can be analyzed and the analysis conditions between the conventional method and the method of the present invention.
  • the scanning speed is approximated to a straight line because scanning is performed in a donut shape.
  • various biological substances fixed on the surface of beads having a diameter of 0.3 m to 10 m can be suitably used regardless of their sizes.
  • particle assembly of biological materials bound to such beads can be performed with high accuracy.
  • an antibody or antigen is immobilized on the bead surface and a biological substance is detected by forming a specific bond between the antigen and antibody
  • the larger the bead particle size the greater the number of antibodies or antigens that can be immobilized on the bead surface.
  • the present invention is particularly suitable when such large beads, particularly beads having a diameter of 0.3 ⁇ m to 10 ⁇ m are used.
  • beads having a large particle diameter is also preferable in terms of easy preparation and handling of beads having a biological substance immobilized thereon.
  • beads with a small particle size for example, those with a diameter of 0.3 / zm or less
  • the beads to be analyzed that have settled in the lower layer may spread immediately to the upper layer after centrifuge processing. For this reason, the beads to be analyzed are removed together with the upper layer during recovery and washing, and this causes the disadvantage that the concentration of biological substances cannot be measured accurately. This is done using beads with a diameter of 0.3 / zm or more. If the analysis method of the invention is applied, such inconvenience does not occur.
  • any material can be used as long as it does not affect the measurement of signal intensity such as fluorescence intensity.
  • various materials such as polystyrene are used.
  • Conventionally known ones such as plastic beads or metal beads may be used.
  • a conventionally well-known method may be applied also when immobilizing a biological substance or the like to be analyzed on beads. It should be noted that here, mainly bio-related substances are immobilized on the bead surface.
  • the force fixed to the bead surface is not limited to a biological substance as long as it is an analysis object of the present invention, and any material may be used.
  • conventionally known fluorescent dyes can be used, and examples thereof include fluorescein, rhodamine, alexa, ATTO dye and the like.
  • FIGS. 2A to 2E are diagrams illustrating scanning methods.
  • FIG. 2A is a point scan
  • FIG. 2B is a scan of the center of the prior art
  • a donut scan
  • FIGS. 2C to 2E are main scans.
  • Each preferred scan of the invention is shown.
  • a plurality of microscopic regions arranged in a straight line (a plurality of egg shapes in FIG. 2C) are sequentially scanned, and then, adjacent to the plurality of microscopic regions arranged in a straight line.
  • the confocal region is scanned by repeating the operation of sequentially scanning a plurality of minute regions arranged in a straight line.
  • the central force of the scan region is also directed toward the outer peripheral portion, and a plurality of minute regions arranged in a spiral shape are applied to the central portion.
  • a method of scanning forcefully as shown in Fig. 2E, a method of scanning a plurality of minute regions arranged in a spiral shape with the outer peripheral force of the scan area directed toward the central portion, and the outer peripheral force also moving toward the central portion.
  • These spiral scan methods can scan a desired area without changing the scan speed to zero or slowing down in order to change the scan direction. Therefore, it is more preferable because the scanning time can be shortened than when scanning in a straight line.
  • the beam scanner of the prior art uses MF20 (trade name; manufactured by Olympus Corporation), the confocal area is scanned in a donut shape, so the area where data can be acquired is as small as about 100 m 2. ( Figure 2B). Therefore, for example, cells with a size of 0.3 ⁇ m or more, such as cells, cell membranes, cell membrane fragments, or nanoparticles with a diameter of 0.3 / zm or more, have a very low probability of passing through the measurement area. Even if it is possible to measure, there is a large variation and data with high reliability cannot be obtained.
  • the measurement area is simply enlarged, for example, when the confocal area is enlarged by lowering the magnification of the objective lens, it is necessary to label the fine particles! Because the excitation energy is constant for any reason, the signal intensity per particle to be analyzed becomes small, making it impossible to detect with high sensitivity.
  • one confocal region for measuring the signal intensity is set to 1 FL or less, and the scan region is kept as a minimal region. The region is expanded from 500 ⁇ m 2 to 40000 ⁇ m 2 . Therefore, by using FID A, it is possible to detect fine particles having sufficient signal intensity. For example, a change in fluorescence intensity of 1.3 times or more due to the interaction of fine particles can be captured.
  • an analysis object having a diffusion speed of 0. or more with a slow diffusion rate has a sufficiently high probability of passing through the measurement area. Therefore, it is possible to measure with high sensitivity even those with a size exceeding 0.3 m, which was difficult to measure in the past.
  • the scan area is preliminarily set before the measurement is started.
  • the scan is terminated. You can also. In this way, the measurement time can be shortened.
  • one micro area for measuring signal intensity, the total area value of the scanning area plane, and the scanning speed of the micro area are as described above. Therefore, you can set the optimal conditions according to the situation.
  • Cell membrane fractionation was performed according to the following procedure, and cell membrane fragments were obtained as ribosomes having a variation of about 0.1 to 0.5 ⁇ m in diameter.
  • the cell membrane fraction solution 14 obtained above was mixed with 7 L of unlabeled PAF solution 7 adjusted to a concentration of 2 nM to 160 nM and reacted with TAMRA-labeled PAF solution 7 L at 25 ° C for 1 hour.
  • FIGS. 3A to 3C show a confocal laser microscope FV1000 (trade name; manufactured by Olympus Corporation) with a single molecule fluorescence analysis unit. Analysis was performed in the FIDA measurement mode under the following measurement conditions. The results are shown in FIGS. 3A to 3C.
  • Figure 3A shows a total scan area of 500 m 2 and
  • Figure 3B shows a total scan area of 25000 m 2 .
  • Measurement conditions Laser wavelength: 543 nm, laser power: 250 ⁇ W, measurement time: 30 seconds X 5 times, scanning speed: 25 mm / decrease, total scanning area: 500 ⁇ m 2 , 25000 ⁇ rn
  • Measurement conditions Laser wavelength: 543 nm, laser power: 250 ⁇ W, measurement time: 30 seconds x 5 times, beam scanner: 2. 85 mm / second, total scan area: 100 ⁇ rn
  • Example 1 From the results of Example 1 and Comparative Example 1, according to the present invention, if the total value of the scanned area is 500 ⁇ m 2 or more, stable data with small variations and high reproducibility can be obtained. It was. In other words, the data obtained in Example 1 was more reliable than the conventional method, which has no practical problem.
  • Polybead Carboxylate 10. Omicron microspheres, trade name, catalog number 18133; manufactured by Polyscienses) 250 ⁇ L was taken and centrifuged at 500 XG for 5 minutes to obtain purified beads. Next, PolyLink Protein Coupling Kit for COOH Microp Using a kit of articles (trade name, catalog number PL01N; manufactured by Bangs Laboratories), the purified beads were reacted with Alexa647—Albumin (1 ⁇ g), and 200 L of fluorescent bead solution with a diameter of 10 m to which Alexa647 was bound was added. Obtained. A 10-fold diluted solution was used for the following analysis.
  • Streptavidin Coated Microspheres (trade name, catalog number CP01N; manufactured by Bangs Laboratories) 0.49 m was diluted with PBS—0.05% t Ween20 and sonicated for 5 seconds. The washing operation for removing the supernatant was repeated three times. Then, 998 ⁇ L of PBS—0.05% tween20 was added to 10 / z L of PBS—0.05% tween20 solution of the obtained washed beads, followed by 2 ⁇ L of Biotin— ⁇ 065 5 ( After adding 10 m), the total liquid volume was 1000 ⁇ L, and the mixture was reacted at room temperature for 1 hour.
  • Tables 1 and 2 show the results when 0.5 m diameter fluorescent beads are used, and Table 2 shows the results when 10 m diameter fluorescent beads are used.
  • 4A and 4B are graphs showing the fluorescence intensity detection values for the first to fifth times, and FIG. 5A and FIG. 5B are graphs showing the average values of these detection values.
  • Figures 4A and 5A show the results when using fluorescent beads with a diameter of 0.5 m
  • Figures 4B and 5B show the results when using fluorescent beads with a diameter of 10 m.
  • Example 2 Analysis was performed in the FIDA measurement mode in the same manner as in Example 2 except that the single molecule fluorescence analysis system MF20 (trade name; manufactured by Olympus Corporation) was used and the measurement conditions were as follows. The results are shown in Tables 1 and 2, FIGS. 4A, 4B, 5A, and 5B. Table 1 shows the results when fluorescent beads with a diameter of 0.5 m were used, and Table 2 shows the results when fluorescent beads with a diameter of 10 m were used. 4A and 5A show the results when using fluorescent beads with a diameter of 0.5 / m, and FIGS. 4B and 5B show the results when using fluorescent beads with a diameter of 10 / z m.
  • Table 1 shows the results when fluorescent beads with a diameter of 0.5 m were used
  • Table 2 shows the results when fluorescent beads with a diameter of 10 m were used.
  • 4A and 5A show the results when using fluorescent beads with a diameter of 0.5 / m
  • FIGS. 4B and 5B show the results
  • Example 2 it was possible to detect fluorescent beads with a diameter of 0.5 ⁇ m and a diameter of 10 ⁇ m! On the other hand, in Comparative Example 2, fluorescent beads with a diameter of 0.5 / zm may be detected in some cases, but fluorescent beads with a diameter of 10 m that have no reproducibility are completely undetectable. That is, it was confirmed that the present invention has higher sensitivity and higher accuracy than the conventional method.
  • the fluorescent bead solution having a diameter of 10 m prepared in Example 2 was added to a 384-well microphone opening plate, and a confocal laser microscope FV1000 (trade name; manufactured by Olympus Corporation) was connected to a 1-molecule fluorescence analysis unit. Using this, we examined whether the difference in scanning speed would affect the detection value under the following measurement conditions. The measurement was performed 5 times per scanning speed.
  • FIG. Table 3 shows the detected values of the first to fifth fluorescence intensities
  • FIG. 6 shows a graph of the average of the detected values of the first to fifth fluorescence intensities.
  • Measurement conditions Laser wavelength: 633 nm, laser power: 41% (equivalent to about 300 ⁇ W), measurement time: 30 seconds x 5 times, measurement mode: FIDA, scan speed: 1, 2.5, 10, 20, 25 , 50m mZ second, total scanning area: 160 mX 160 / zm, objective lens magnification: X 60
  • Example 4 Confirmation of influence of difference in signal intensity acquisition region (magnification of objective lens) on detection accuracy Fluorescent bead solution with a diameter of 10 ⁇ m prepared in Example 2 was applied to a 384-well microphone mouthplate. Add a confocal laser microscope FV1000 (trade name; manufactured by Olympus Corporation) and connect a 1-molecule fluorescence analysis tube. We examined whether the volume of the sample affects the detection value.
  • FIG. Table 4 shows the detected values of the 1st to 5th fluorescence intensities
  • Fig. 7 shows a graph of the average value of the detected 1st to 5th fluorescence intensities.
  • Measurement conditions Laser wavelength: 633 nm, laser power: 41% (equivalent to about 300 ⁇ W), measurement time: 30 seconds x 5 times, measurement mode: FIDA, scanning speed: 20 mmZ seconds, total scan area: 160 m x 160 m, Objective lens magnification: X 10, X 20, X 40, X 60
  • the signal intensity can be detected with high sensitivity when the volume of the confocal region is 1FL or less, that is, when the magnification of the objective lens used is X20 to X60.
  • the fluorescent bead solution with a diameter of 10 m prepared in Example 2 was analyzed under the following measurement conditions using a confocal laser microscope FV 1000 (trade name; manufactured by Olympus Corporation) with a single molecule fluorescence analysis unit connected. Then, the number of fluorescent beads was confirmed. The results are shown in Fig. 8. Measurement conditions: Laser wavelength: 633 nm, laser power: 41% (equivalent to about 300 ⁇ W), scanning speed: 20 mm / sec, total scanning area: 25 ⁇ 25 ⁇ m to 200 ⁇ m X 200 ⁇ m, used Objective lens magnification: X 60
  • the present invention can be used in the fields of clinical testing, hygiene testing, biochemical research, and the like, and is useful for analysis of biological samples.

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Abstract

In a method for analyzing fine particles, signal strength in a fine region in a confocal region is measured by scanning the fine region and analyzing the sizes of the fine particles or existence of particle bonding or particle divergence. The volume of one fine region among a plurality of fine regions of which the signal strengths are to be measured is 1FL or less, and a total area of scanning region flat surfaces of the fine regions is 500μm2-40,000μm2.

Description

明 細 書  Specification
微粒子の解析方法  Analysis method of fine particles
技術分野  Technical field
[0001] 本発明は、生体関連物質等の微粒子を、高感度かつ高精度に解析する方法に関 する。 本願は、 2006年 7月 13日に出願された特願 2006— 192742号に対し優先 権を主張し、その内容をここに援用する。  The present invention relates to a method for analyzing fine particles such as biological materials with high sensitivity and high accuracy. This application claims priority to Japanese Patent Application No. 2006-192742 filed on July 13, 2006, the contents of which are incorporated herein by reference.
背景技術  Background art
[0002] 蛍光相関分光解析等、信号強度を検出し、生体関連物質の大きさ、あるいは生体 関連物質の結合または乖離の有無を高感度に解析する方法として、例えば、標準位 置を中心として振動するように配列された対物レンズ側平面偏光ミラーを有する偏向 ミラー配列が、共焦点顕微鏡に配置されている装置を用いる方法が開示されている（ 非特許文献 1、特許文献 1および 2参照)。このミラーは、標準位置を中心として振動 または回転するようになっており、ビームスキャナーと通常よばれている。そして、蛍 光強度分布解析に用いられるこのミラー技術により、レーザなどの光による解析対象 物の褪色が防止され、与えられた測定時間内において共焦点領域内の解析対象物 の数を増カロさせ、データ取得時間が相当に節約されると記載されている。  [0002] As a method of detecting signal intensity and analyzing the magnitude of a biological substance or the presence or absence of binding or detachment of a biological substance with high sensitivity, such as fluorescence correlation spectroscopy analysis, for example, vibration centered on a standard position A method of using an apparatus in which a deflecting mirror array having objective lens side plane polarizing mirrors arranged in such a manner is arranged in a confocal microscope has been disclosed (see Non-Patent Document 1, Patent Documents 1 and 2). This mirror is designed to vibrate or rotate around a standard position and is usually called a beam scanner. This mirror technology used for fluorescence intensity distribution analysis prevents the analysis object from fading due to light such as a laser, and increases the number of analysis objects in the confocal region within a given measurement time. The data acquisition time is saved considerably.
非特許文献 l : Pro. Natl. Acad. Sci USA, vol. 96, 1375 - 1378, 1999 特許文献 1：特表 2001 - 502062号公報  Non-Patent Literature l: Pro. Natl. Acad. Sci USA, vol. 96, 1375-1378, 1999 Patent Literature 1: JP 2001-502062
特許文献 2：特表 2001— 502066号公報  Patent Document 2: JP 2001-502066 Publication
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0003] しかし、これら文献には、ビームスキャナーを用いることが生体関連物質の解析に 有効であることが述べられているだけで、例えば、測定対象である生体関連物質の 大きさによる適切なスキャン速度や領域などのスキャン方法に関する詳細は一切述 ベられていない。 [0003] However, these documents only mention that the use of a beam scanner is effective for the analysis of a biological substance. For example, an appropriate scan according to the size of the biological substance to be measured is used. No details about scanning methods such as speed or area are given.
そして、例えば、 0. 3 m以上の大きさを有する対象物を解析した場合、得られる データはばらつきの大き 、ものであることが知られて 、る。 [0004] したがって、大きな生体関連物質、例えば、細胞膜断片、細胞そのものなど、直径 0 . 3 /z m以上の大きさを有するものを解析する場合も、得られるデータはばらつきが大 きいので精度の高い解析が行えない。特に、細胞の膜分画などにより細胞膜断片を 調製する際は、細胞膜断片の大きさの均一化が難しぐこれら細胞膜断片は直径 0. 1〜0. 5 mの球状であることが多ぐ直径 0. 3 m以上の大きな分子または粒子を 含んでいる。 For example, when an object having a size of 0.3 m or more is analyzed, the obtained data is known to have a large variation. [0004] Therefore, even when analyzing large biological materials such as cell membrane fragments, cells themselves, etc. having a diameter of 0.3 / zm or more, the obtained data is highly variable and highly accurate. Analysis cannot be performed. In particular, when preparing cell membrane fragments by cell membrane fractionation etc., it is difficult to homogenize the size of the cell membrane fragments, and these cell membrane fragments are often spherical with a diameter of 0.1 to 0.5 m. 0. Contains large molecules or particles of 3 m or more.
[0005] すなわち、従来技術のビームスキャナーを用いる方法では、測定できる生体関連物 質の大きさには上限があり、上限を超える大きさの生体関連物質を解析した場合に は、標準偏差の小さな安定したデータを得ることが困難であるという問題点があった。  [0005] That is, in the method using the conventional beam scanner, there is an upper limit to the size of the biological substance that can be measured, and when analyzing a biological substance that exceeds the upper limit, the standard deviation is small. There was a problem that it was difficult to obtain stable data.
[0006] また、 FCSや FIDAなどの 1分子測定法により分子間の相互作用を検出する場合、 精度の高いデータを得るためには、 FCSでは相互作用前後で 4倍力 8倍以上の分 子量の変化を必要とする。一方、 FIDAでは、相互作用前後で蛍光強度が 1. 3倍以 上変化しなければ、それらの相互作用を検出することができない。このような場合、相 互作用する一方の分子を、例えば、ポリスチレン製パーティクルビーズ表面に固定し て、その相互作用を検出する方法をとることがある。  [0006] In addition, when detecting interactions between molecules by single-molecule measurement methods such as FCS and FIDA, in order to obtain highly accurate data, in FCS, the molecular force is more than 4 times 8 times before and after the interaction. Requires a change in quantity. On the other hand, in FIDA, if the fluorescence intensity does not change more than 1.3 times before and after the interaction, the interaction cannot be detected. In such a case, a method may be used in which one of the interacting molecules is fixed to, for example, the surface of polystyrene particle beads and the interaction is detected.
[0007] この場合、従来法のようにビームスキャナーを使用して精度の高いデータを得るた めには、解析対象の分子の大きさがたとえ 0. 3 m以下であっても、パーティクルサ ィズすなわちビーズの直径の上限は 0. 3 mであり、それ以上の大きさではデータ のばらつきが大きくなるという問題点があった。  [0007] In this case, in order to obtain highly accurate data using a beam scanner as in the conventional method, even if the size of the molecule to be analyzed is 0.3 m or less, the particle size In other words, the upper limit of the diameter of the beads, which is 0.3 m, has a problem that the variation of data becomes larger at larger diameters.
[0008] 以上のように従来法では、精度の高いデータを得るためには、測定できる生体関連 物質の大きさに限界があり、 0. 3 m以上の大きさのもの力 得られるデータには、 大きなばらつきがあり、ドーナツ状に同じ領域をスキャンすることしかできな力つた。そ して、非特許文献 1、特許文献 1および 2には、大きさが 0. 3 /z m以上のビーズを測 定する際のスキャン速度やスキャン領域などの適切な条件が記載されていな力つた。 課題を解決するための手段  [0008] As described above, in the conventional method, in order to obtain highly accurate data, there is a limit to the size of a biological substance that can be measured. There was great variation, and it was only possible to scan the same area in a donut shape. Non-Patent Document 1, Patent Documents 1 and 2 do not describe appropriate conditions such as scan speed and scan area when measuring beads with a size of 0.3 / zm or more. I got it. Means for solving the problem
[0009] 本発明は上記事情に鑑みてなされたものであり、解析対象物として 0. 3 μ m以上 の大きさのものが含まれる場合でも、解析対象の微粒子の大きさ、あるいは微粒子の 結合または乖離の有無を、高感度かつ高精度に解析する方法を提供することを課題 とする。 [0009] The present invention has been made in view of the above circumstances, and even when the analysis target includes a size of 0.3 μm or more, the size of the analysis target microparticles or the binding of the microparticles Another problem is to provide a method for analyzing the presence or absence of deviation with high sensitivity and high accuracy. And
[0010] すなわち、上記課題を解決するため、  [0010] That is, in order to solve the above problems,
本発明は、共焦点領域内の微少領域を走査して微少領域の信号強度を測定し、 微粒子の大きさ、あるいは微粒子の結合または乖離の有無を解析する微粒子の解析 方法であって、信号強度を測定する複数の微少領域のうちの一つの微少領域の容 積が 1FL以下であり、前記複数の微少領域の走査領域平面の面積合計値が、 500 μ m2〜40000 μ m2である微粒子の解析方法である。 The present invention relates to a fine particle analysis method for scanning a minute region in a confocal region and measuring the signal strength of the minute region, and analyzing the size of the fine particle or the presence or absence of binding or detachment of the fine particle. Fine particles whose volume of one micro area of a plurality of micro areas to be measured is 1 FL or less and whose total area area of the scanning area plane of the plurality of micro areas is 500 μm 2 to 40000 μm 2 This is an analysis method.
本発明において、信号強度を測定する一つの微少領域を走査する速度が 2. 5m mZ秒〜 25mmZ秒であっても良 ヽ。  In the present invention, the scanning speed of one minute region for measuring the signal intensity may be 2.5 mmZsec to 25 mmZsec.
本発明において、蛍光相関分光法、蛍光強度分布解析法および蛍光偏光強度分 布解析法の少なくとも 、ずれか一つで前記解析を行っても良 ヽ。  In the present invention, the analysis may be performed by at least one of fluorescence correlation spectroscopy, fluorescence intensity distribution analysis, and fluorescence polarization intensity distribution analysis.
本発明において、前記微粒子が、細胞、細胞膜、細胞膜断片、有機物粒子、無機 物粒子およびこれらのうちの一種以上力 なる複合体力 選ばれる一種以上であつ ても良い。  In the present invention, the fine particles may be one or more selected from cells, cell membranes, cell membrane fragments, organic particles, inorganic particles, and one or more complex forces of these.
本発明において、前記微粒子の大きさ力 0. 3 /ζ πι〜10 /ζ πιであっても良い。 発明の効果  In the present invention, the size force of the fine particles may be 0.3 / ζ πι to 10 / ζ πι. The invention's effect
[0011] 本発明により、解析対象物として 0. 3 m以上の大きさのものが含まれる場合でも、 解析対象の微粒子の大きさ、あるいは微粒子の結合または乖離の有無を、高感度か つ高精度に解析することができる。  [0011] According to the present invention, even when an analysis object includes a particle having a size of 0.3 m or more, the size of the particle to be analyzed or the presence or absence of the binding or detachment of the particle is highly sensitive and high. It can be analyzed with accuracy.
図面の簡単な説明  Brief Description of Drawings
[0012] [図 1]図 1は、微粒子の解析可能な大きさと解析条件とを比較して説明した説明図で ある。  [0012] FIG. 1 is an explanatory diagram illustrating a comparison between the size of a fine particle that can be analyzed and analysis conditions.
[図 2A]図 2Aは、従来技術におけるスキャン方法を例示する図である。  FIG. 2A is a diagram illustrating a scanning method in the prior art.
[図 2B]図 2Bは、従来技術におけるスキャン方法を例示する図である。  FIG. 2B is a diagram illustrating a scanning method in the prior art.
[図 2C]図 2Cは、本発明におけるスキャン方法を例示する図である。  FIG. 2C is a diagram illustrating a scanning method in the present invention.
[図 2D]図 2Bは、本発明におけるスキャン方法を例示する図である。  FIG. 2B is a diagram illustrating a scanning method in the present invention.
[図 2E]図 2Cは、本発明におけるスキャン方法を例示する図である。  FIG. 2C is a diagram illustrating a scanning method in the present invention.
[図 3A]図 3Aは、実施例 1の解析結果を示すグラフである。 [図 3B]図 3Bは、実施例 1の解析結果を示すグラフである。 FIG. 3A is a graph showing the analysis results of Example 1. FIG. 3B is a graph showing the analysis results of Example 1.
[図 3C]図 3Cは、比較例 1の解析結果を示すグラフである。  FIG. 3C is a graph showing the analysis results of Comparative Example 1.
[図 4A]図 4Aは、実施例 2および比較例 2の 5回の測定結果を示すグラフである。  [FIG. 4A] FIG. 4A is a graph showing the results of five measurements of Example 2 and Comparative Example 2.
[図 4B]図 4Bは、実施例 2および比較例 2の 5回の測定結果を示すグラフである。  FIG. 4B is a graph showing five measurement results of Example 2 and Comparative Example 2.
[図 5A]図 5Aは、実施例 2および比較例 2の 5回の測定結果の平均値を示すグラフで ある。  FIG. 5A is a graph showing an average value of five measurement results of Example 2 and Comparative Example 2.
[図 5B]図 5Bは、実施例 2および比較例 2の 5回の測定結果の平均値を示すグラフで ある。  FIG. 5B is a graph showing an average value of five measurement results of Example 2 and Comparative Example 2.
[図 6]図 6は、実施例 3の解析結果を示すグラフである。  FIG. 6 is a graph showing the analysis results of Example 3.
[図 7]図 7は、実施例 4の解析結果を示すグラフである。  FIG. 7 is a graph showing the analysis results of Example 4.
[図 8]図 8は、実施例 5で得られた測定画像である。  FIG. 8 is a measurement image obtained in Example 5.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0013] 以下、本発明について、詳しく説明する。 Hereinafter, the present invention will be described in detail.
信号強度を測定する微小領域の容積は、例えば、用いる対物レンズの倍率を変え ることで調整することができる。そして本発明においては、信号強度を測定する一つ の微小領域 (複数の微少領域における夫々の微少領域)の容積は、 1FL (フ ムトリツ トル)以下とする。 1FLよりも大きいと、信号強度の検出を高感度に行うことが困難とな る場合がある。  The volume of the minute region for measuring the signal intensity can be adjusted, for example, by changing the magnification of the objective lens to be used. In the present invention, the volume of one minute region (each minute region in a plurality of minute regions) for measuring the signal intensity is set to 1 FL (humid torr) or less. If it is larger than 1FL, it may be difficult to detect the signal intensity with high sensitivity.
[0014] 信号強度を測定する複数の微小領域の走査領域平面の面積合計値は、 500 μ m2 〜40000 m2とする。この範囲外では、信号強度の検出値がばらつきの大きいもの となり、信頼'性が低くなつてしまう。 [0014] The total area value of the scanning region planes of the plurality of minute regions for measuring the signal intensity is set to 500 μm 2 to 40000 m 2 . Outside this range, the detection value of the signal intensity has a large variation, resulting in low reliability.
[0015] 本発明にお 、ては、一つの微小領域を走査する速度は、適宜調整すれば良!、が、 2. 5mmZ秒〜 25mmZ秒であることが好ましい。この範囲内であると信号強度の検 出値が、よりばらつきの小さいものとなり、高精度に解析を行うのに好適である。  [0015] In the present invention, the scanning speed of one minute region may be adjusted as appropriate, but is preferably 2.5 mmZ seconds to 25 mmZ seconds. Within this range, the detected value of the signal intensity has a smaller variation, which is suitable for highly accurate analysis.
[0016] 本発明においては、信号強度の測定には各種の方法を適用できる力 蛍光色素で 標識した微粒子の蛍光を検出する方法が好適である。この場合、例えば、蛍光相関 分光法 (以下、 FCSと略記する）、蛍光強度分布解析法 (以下、 FIDAと略記する）、 蛍光偏光強度分布解析法 (FIDA— polarization)等が好ま 、方法として挙げられ る。これらは単独で行っても良いし、複数の方法を組み合わせて行っても良い。これ らの測定は、例えば、共焦点レーザー顕微鏡 FV1000 (商品名；ォリンパス株式会社 製)へ 1分子蛍光分析ユニットを接続したものを用いて行うことができる。 In the present invention, various methods can be applied to the measurement of signal intensity. A method of detecting fluorescence of fine particles labeled with a fluorescent dye is preferable. In this case, for example, fluorescence correlation spectroscopy (hereinafter abbreviated as FCS), fluorescence intensity distribution analysis method (hereinafter abbreviated as FIDA), fluorescence polarization intensity distribution analysis method (FIDA-polarization), etc. are preferred. Is The These may be performed singly or in combination of a plurality of methods. These measurements can be performed using, for example, a confocal laser microscope FV1000 (trade name; manufactured by Olympus Corporation) connected to a single molecule fluorescence analysis unit.
[0017] 前記のような信号強度測定法によれば、解析対象である微粒子の大きさに依存し て、検出される信号強度が変化する。したがって、解析対象である微粒子が、他の微 粒子と結合したり、複数の微粒子に解離したりして大きさが変化すれば、信号強度の 変化によって、これら結合または解離を検出することができる。なお、ここでいう結合と は、共有結合だけでなぐ水素結合あるいは疎水結合等の分子間引力に基づく結合 なども指す。 [0017] According to the signal intensity measurement method as described above, the detected signal intensity changes depending on the size of the fine particles to be analyzed. Therefore, if the microparticles to be analyzed bind to other microparticles or dissociate into a plurality of microparticles, and the size changes, the binding or dissociation can be detected by the change in signal intensity. . The term “bond” as used herein refers to a bond based on an intermolecular attractive force such as a hydrogen bond or a hydrophobic bond that is not only a covalent bond.
[0018] 本発明において、解析対象として用いる微粒子は特に限定されず、従来法で解析 対象とすることができた、例えば、 0. 1 m以下の大きさのものでも良いが、解析対象 とすることが困難であった大きいサイズのもの、例えば、 0. 3 m〜10 μ mの大きさ のものが好適である。このようなものとして、例えば、生体関連物質、有機物粒子、無 機物粒子およびこれらのうちの一種以上力 なる複合体力 選ばれる一種以上のも のが挙げられる。そして、生体関連物質としては、例えば、細胞、細胞膜および細胞 膜断片等が好ましいものとして挙げられる。これら微粒子は、従来公知の方法で調製 したものあるいは巿販品等を用いることができる。例えば、生体関連物質であれば、 従来公知の方法により、生体力 採取したサンプルを処理し、該サンプルより抽出し たものを用いることができる。  [0018] In the present invention, the fine particles used as the analysis target are not particularly limited, and can be the analysis target by a conventional method, for example, a particle having a size of 0.1 m or less may be the analysis target. A large size, for example, a size of 0.3 m to 10 μm, which has been difficult to achieve, is suitable. Examples of such materials include biological substances, organic particles, inorganic particles, and one or more selected from complex forces that are one or more of these. Preferred examples of the biological substance include cells, cell membranes and cell membrane fragments. As these fine particles, those prepared by a conventionally known method or commercial products can be used. For example, as long as it is a bio-related substance, it is possible to use a sample extracted from the sample obtained by processing a sample obtained by collecting vital force by a conventionally known method.
[0019] 細胞膜上に存在する受容体、たとえば GPCRなどは、ォーファンレセプターが多い とされている力 一方で創薬のターゲットとなる重要なレセプターも存在する。このた め最近では、阻害剤あるいはリガンドの探索に細胞膜分画がつかわれるケースが多 い。このように、重要な機能を有しかつサイズの大きい細胞、細胞膜および細胞膜断 片等は、本発明の解析対象として特に好適である。  [0019] Receptors present on the cell membrane, such as GPCR, are thought to have many orphan receptors, but there are also important receptors that are targets for drug discovery. For this reason, recently, cell membrane fractions are often used to search for inhibitors or ligands. Thus, cells, cell membranes, cell membrane fragments and the like having important functions and large sizes are particularly suitable as the analysis target of the present invention.
[0020] 例えば、細胞膜は、通常溶液中ではリボソームとなって存在している。そして従来 公知の方法により膜分画処理を行うと、細胞膜断片が得られ、その大きさは直径 0. 1 〜0. 5 m程度でばらついていることが多い。このようなものは、従来、ラジオアイソト ープ (RI)標識をし、遠心して沈殿させて、シンチレーシヨンカウンターで検出するな ど、 RIを用いた解析が主流である。また、従来の 1分子測定法を適用した場合では、 ばらつきの大きいデータしか得られない。しかし本発明によれば、 0. 3 mを超える 大きさのものが解析対象物として含まれていても、危険な RIを用いることなぐ高精度 に解析を行うことができる。解析可能な微粒子の大きさと解析条件とを、従来法と本 発明の方法とで比較した一例を図 1に示す。なお従来法では、ドーナツ状にスキャン するため、走査速度は直線に近似して算出している。 [0020] For example, the cell membrane usually exists as a ribosome in a solution. When membrane fractionation is performed by a conventionally known method, cell membrane fragments are obtained, and their sizes often vary by about 0.1 to 0.5 m in diameter. Such items are conventionally labeled with radioisotopes (RI), spun down, and detected with a scintillation counter. However, analysis using RI is the mainstream. In addition, when the conventional single molecule measurement method is applied, only data with large variations can be obtained. However, according to the present invention, even if a thing with a size exceeding 0.3 m is included as an analysis object, analysis can be performed with high accuracy without using a dangerous RI. Fig. 1 shows an example of a comparison of the size of the fine particles that can be analyzed and the analysis conditions between the conventional method and the method of the present invention. In the conventional method, the scanning speed is approximated to a straight line because scanning is performed in a donut shape.
[0021] また、直径 0. 3 m〜10 mのビーズ表面に固定された各種生体関連物質等も、 その大きさによらず好適に用いることができる。例えば、このようなビーズに結合させ た生体関連物質のパーティクルアツセィなども高精度に行うことができる。ビーズ表面 に抗体あるいは抗原を固定して、生体関連物質を抗原 抗体間の特異的結合形成 によって検出する場合には、ビーズの粒径が大きいほど、ビーズ表面に固定できる抗 体あるいは抗原の数を増やすことができ、一つのビーズにより多くの生体関連物質を 結合させることができる。すなわち、用いるビーズが大きいほど、測定時に生体関連 物質の信号強度を増幅することができる。そして本発明は、このような大きなビーズ、 特に直径 0. 3 μ m〜10 μ mのビーズを用いる場合に、特に好適である。  [0021] In addition, various biological substances fixed on the surface of beads having a diameter of 0.3 m to 10 m can be suitably used regardless of their sizes. For example, particle assembly of biological materials bound to such beads can be performed with high accuracy. When an antibody or antigen is immobilized on the bead surface and a biological substance is detected by forming a specific bond between the antigen and antibody, the larger the bead particle size, the greater the number of antibodies or antigens that can be immobilized on the bead surface. It is possible to increase the number of biological substances by one bead. That is, the larger the bead used, the more the signal intensity of the biological substance can be amplified during measurement. The present invention is particularly suitable when such large beads, particularly beads having a diameter of 0.3 μm to 10 μm are used.
[0022] また、粒径の大きなビーズを用いることは、生体関連物質を固定したビーズの調製 および取り扱いが容易である点でも好ましい。粒径の小さいビーズ、例えば、直径 0. 3 /z m以下のものを用いた場合には、生体関連物質のビーズへの固定力 反応、測 定の各手順に付随する遠心処理にぉ 、て、下層に沈殿した解析対象のビーズが遠 心処理後、すぐに上層へ広がることがある。そのため、回収および洗浄時に、解析対 象のビーズが上層とともに除去されてしまい、生体関連物質の濃度を正確に測定で きないという不都合が生じる力 直径 0. 3 /z m以上のビーズを用いて本発明の解析 方法を適用すれば、このような不都合も生じることが無い。  [0022] The use of beads having a large particle diameter is also preferable in terms of easy preparation and handling of beads having a biological substance immobilized thereon. When beads with a small particle size, for example, those with a diameter of 0.3 / zm or less, are used for the fixation force reaction of biologically related substances to the beads and the centrifugation process associated with each measurement procedure. The beads to be analyzed that have settled in the lower layer may spread immediately to the upper layer after centrifuge processing. For this reason, the beads to be analyzed are removed together with the upper layer during recovery and washing, and this causes the disadvantage that the concentration of biological substances cannot be measured accurately. This is done using beads with a diameter of 0.3 / zm or more. If the analysis method of the invention is applied, such inconvenience does not occur.
[0023] 本発明でビーズを用いる場合には、その材質は、蛍光強度等の信号強度の測定に 影響を与えないものであれば、如何なるものも用いることができ、例えば、ポリスチレ ン等の各種プラスチックビーズあるいは金属ビーズ等、従来公知のものを用いれば 良い。そして、解析対象の生体関連物質等をビーズに固定する場合も、従来公知の 方法を適用すれば良い。なお、ここでは主に、ビーズ表面に生体関連物質を固定し た場合について説明している力 ビーズ表面に固定するものは、本発明の解析対象 物であれば、生体関連物質に限定されず如何なるものでも良い。 [0023] When using beads in the present invention, any material can be used as long as it does not affect the measurement of signal intensity such as fluorescence intensity. For example, various materials such as polystyrene are used. Conventionally known ones such as plastic beads or metal beads may be used. In addition, a conventionally well-known method may be applied also when immobilizing a biological substance or the like to be analyzed on beads. It should be noted that here, mainly bio-related substances are immobilized on the bead surface. The force fixed to the bead surface is not limited to a biological substance as long as it is an analysis object of the present invention, and any material may be used.
[0024] 本発明において蛍光色素は、従来公知のものを用いることができ、例えば、フルォ ロセイン、ローダミン、アレクサ、 ATTO色素等を挙げることができる。  In the present invention, conventionally known fluorescent dyes can be used, and examples thereof include fluorescein, rhodamine, alexa, ATTO dye and the like.
[0025] 図 2A〜図 2Eは、スキャン方法を例示する図であり、図 2Aはポイントスキャン、図 2 Bは従来技術の中心部をスキャンしな 、ドーナツ状スキャン、図 2C〜図 2Eは本発明 の好ましいスキャンをそれぞれ示している。本発明では、図 2Cで記載されているよう に、直線状に並ぶ複数の微少領域（図 2Cにおける複数の卵形状)を順次スキャンし 、その後、前記直線状に並ぶ複数の微少領域に隣接して直線状に並ぶ複数の微少 領域を順次スキャンする、との動作を繰り返すことにより、共焦点領域のスキャンを実 施する。また、本発明における他のスキャンの方法としては、図 2Dに示すような、スキ ヤン領域の中心部力も外周部へ向力つて渦巻状に並ぶ複数の微少領域を、中心部 力も外周部へ向力つてスキャンする方法、図 2Eに示すような、スキャン領域の外周部 力も中心部へ向力つて渦巻状に並ぶ複数の微少領域を、外周部力も中心部へ向か つてスキャンする方法が挙げられる。これらの渦巻状のスキャン方法は、スキャンの方 向を変えるために、スキャン速度をゼロにしたり、遅くしたりすることなぐ所望の領域 をスキャンできる。したがって、直線状にスキャンする場合よりもスキャンの時間を短縮 できるので、より好ましい。従来技術のビームスキャナ一は、例えば、 MF20 (商品名 ；ォリンパス株式会社製)を用いた場合、共焦点領域をドーナツ状にスキャンするため 、データを取得できる領域が 100 m2程度と非常に小さい（図 2B)。したがって、例 えば、細胞、細胞膜、細胞膜断片、直径 0. 3 /z m以上のナノパーティクルなど、 0. 3 μ m以上の大きさのものは、測定領域を通過する確率が非常に小さぐ信号強度を 測定できた場合でもばらつきが大きく信頼性の高いデータは得られない。 [0025] FIGS. 2A to 2E are diagrams illustrating scanning methods. FIG. 2A is a point scan, FIG. 2B is a scan of the center of the prior art, a donut scan, and FIGS. 2C to 2E are main scans. Each preferred scan of the invention is shown. In the present invention, as described in FIG. 2C, a plurality of microscopic regions arranged in a straight line (a plurality of egg shapes in FIG. 2C) are sequentially scanned, and then, adjacent to the plurality of microscopic regions arranged in a straight line. The confocal region is scanned by repeating the operation of sequentially scanning a plurality of minute regions arranged in a straight line. In addition, as another scanning method in the present invention, as shown in FIG. 2D, the central force of the scan region is also directed toward the outer peripheral portion, and a plurality of minute regions arranged in a spiral shape are applied to the central portion. A method of scanning forcefully, as shown in Fig. 2E, a method of scanning a plurality of minute regions arranged in a spiral shape with the outer peripheral force of the scan area directed toward the central portion, and the outer peripheral force also moving toward the central portion. . These spiral scan methods can scan a desired area without changing the scan speed to zero or slowing down in order to change the scan direction. Therefore, it is more preferable because the scanning time can be shortened than when scanning in a straight line. For example, when the beam scanner of the prior art uses MF20 (trade name; manufactured by Olympus Corporation), the confocal area is scanned in a donut shape, so the area where data can be acquired is as small as about 100 m 2. (Figure 2B). Therefore, for example, cells with a size of 0.3 μm or more, such as cells, cell membranes, cell membrane fragments, or nanoparticles with a diameter of 0.3 / zm or more, have a very low probability of passing through the measurement area. Even if it is possible to measure, there is a large variation and data with high reliability cannot be obtained.
[0026] 単に測定領域を拡大した場合、例えば、対物レンズの倍率を下げることで共焦点領 域を拡大するような場合では、微粒子を標識して!/ヽる蛍光色素の褪色を防ぐためな どの理由で励起エネルギーを一定とするため、解析対象の微粒子一つあたりの信号 強度が小さくなり、感度良く検出することが不可能になる。これに対して本発明では、 信号強度を測定する一つの共焦点領域を 1FL以下と 、う極小領域としたまま、スキヤ ン領域を 500 μ m2〜40000 μ m2に拡大している。したがって、 FID Aを用いることで 、十分な信号強度を有する微粒子を検出することが可能になり、例えば、微粒子同 士の相互作用による 1. 3倍以上の蛍光強度の変化を捉えることができる。さらに、ス キャン領域を拡大することにより、解析対象物として、拡散速度が遅い 0. 以上 の大きさのものも、測定領域を通過する確率が十分に高くなる。したがって、従来測 定が困難であった、このような 0. 3 mを越える大きさのものも、高感度に測定するこ とがでさる。 [0026] When the measurement area is simply enlarged, for example, when the confocal area is enlarged by lowering the magnification of the objective lens, it is necessary to label the fine particles! Because the excitation energy is constant for any reason, the signal intensity per particle to be analyzed becomes small, making it impossible to detect with high sensitivity. In contrast, in the present invention, one confocal region for measuring the signal intensity is set to 1 FL or less, and the scan region is kept as a minimal region. The region is expanded from 500 μm 2 to 40000 μm 2 . Therefore, by using FID A, it is possible to detect fine particles having sufficient signal intensity. For example, a change in fluorescence intensity of 1.3 times or more due to the interaction of fine particles can be captured. Furthermore, by enlarging the scan area, an analysis object having a diffusion speed of 0. or more with a slow diffusion rate has a sufficiently high probability of passing through the measurement area. Therefore, it is possible to measure with high sensitivity even those with a size exceeding 0.3 m, which was difficult to measure in the past.
[0027] 通常、スキャンを行う場合は、測定開始前にスキャン領域をあら力じめ設定しておく 力 あら力じめ設定した数の微粒子のシグナルの取得が終了した時点で、スキャンを 終了することもできる。このようにして、測定時間を短縮することができる。  [0027] Normally, when scanning is performed, the scan area is preliminarily set before the measurement is started. When the acquisition of the signals of the number of fine particles that have been preliminarily set is completed, the scan is terminated. You can also. In this way, the measurement time can be shortened.
[0028] 本発明にお 、ては、信号強度を測定する一つの微小領域、走査領域平面の面積 合計値、微小領域の走査速度は、ここまで述べた通りであるが、その他の測定条件 につ 、ては、適宜状況に応じて最適な条件に設定すれば良 、。  [0028] In the present invention, one micro area for measuring signal intensity, the total area value of the scanning area plane, and the scanning speed of the micro area are as described above. Therefore, you can set the optimal conditions according to the situation.
実施例  Example
[0029] 以下、具体的実施例により、本発明についてさらに詳しく説明する。ただし、本発明 は以下の実施例に何ら限定されるものではない。  Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples.
[実施例 1]細胞膜分画を用いたレセプタ一一リガンド結合阻害実験 (PAFの阻害剤 ： PAF受容体アンタゴ-スト WEB2086の検討）（細胞膜の分画処理）  [Example 1] Receptor monoligand binding inhibition experiment using cell membrane fractionation (PAF inhibitor: PAF receptor antagonist WEB2086 study) (cell membrane fractionation treatment)
以下の手順にしたがって細胞膜の分画処理を行い、細胞膜断片を、直径 0. 1〜0 . 5 μ m程度のばらつきを有するリボソームとして得た。  Cell membrane fractionation was performed according to the following procedure, and cell membrane fragments were obtained as ribosomes having a variation of about 0.1 to 0.5 μm in diameter.
(1)チューブ内のヒト PAF発現 CHO細胞を、約 10倍量 (wZw)の HEPES緩衝液を 用いて、氷上でホモジナイズした。  (1) Human PAF-expressing CHO cells in the tube were homogenized on ice using approximately 10 times (wZw) of HEPES buffer.
(2) 4°C、 800 X Gで 20分間遠心分離し、その後、上清を注意深くとり別のチューブ に移した。  (2) Centrifugation was performed at 4 ° C and 800 X G for 20 minutes, and then the supernatant was carefully removed and transferred to another tube.
(3)さらに、 4°C、 100000 X Gで 60分間遠心分離し、その後、上清を除去した。 (3) Further, the mixture was centrifuged at 4 ° C, 100000 X G for 60 minutes, and then the supernatant was removed.
(4) HEPES緩衝液にて沈殿をピペッティングした。 (4) The precipitate was pipetted with HEPES buffer.
(5)ホモジナイザーを用いて、氷上にて 10回ストロークで懸濁させた。  (5) Using a homogenizer, the suspension was suspended 10 times on ice.
(6) 4°C、 100000 X Gで 60分間遠心分離し、その後、上清を除去した。 (7)細胞と等量の HEPES緩衝液で沈殿をほぐした。 (6) Centrifugation was performed at 4 ° C and 100000 XG for 60 minutes, and then the supernatant was removed. (7) The precipitate was loosened with the same amount of HEPES buffer as the cells.
(8)ホモジナイザーを用いて、氷上にて 10回ストロークで懸濁させた。  (8) Using a homogenizer, the suspension was suspended 10 times on ice.
(9)タンパク質の量を測定して、 0. 5mgZmlに調製した。  (9) The amount of protein was measured and adjusted to 0.5 mgZml.
[0030] (反応） [0030] (Reaction)
上記で得た細胞膜分画溶液 14 レ 2nM〜160nMの濃度に調整した未標識 PA F溶液 7 レ TAMRA標識 PAF溶液 7 Lを混合し、 25°Cで 1時間反応させた。  The cell membrane fraction solution 14 obtained above was mixed with 7 L of unlabeled PAF solution 7 adjusted to a concentration of 2 nM to 160 nM and reacted with TAMRA-labeled PAF solution 7 L at 25 ° C for 1 hour.
[0031] (FIDA解析） [0031] (FIDA analysis)
共焦点レーザー顕微鏡 FV1000 (商品名；ォリンパス株式会社製)へ 1分子蛍光分 析ユニットを接続したものを用いて、下記の測定条件で、 FIDAの測定モードにより 解析を行った。結果を図 3 A〜図 3Cに示す。図 3Aは、合計スキャン面積が 500 m 2の場合、図 3Bは、合計スキャン面積が 25000 m2の場合である。 Using a confocal laser microscope FV1000 (trade name; manufactured by Olympus Corporation) with a single molecule fluorescence analysis unit, analysis was performed in the FIDA measurement mode under the following measurement conditions. The results are shown in FIGS. 3A to 3C. Figure 3A shows a total scan area of 500 m 2 and Figure 3B shows a total scan area of 25000 m 2 .
測定条件；レーザー波長： 543nm、レーザーパワー： 250 μ W、測定時間： 30秒 X 5回、走査速度： 25mm/禾少、合計スキャン面積： 500 μ m2、 25000 μ rn Measurement conditions: Laser wavelength: 543 nm, laser power: 250 μW, measurement time: 30 seconds X 5 times, scanning speed: 25 mm / decrease, total scanning area: 500 μm 2 , 25000 μ rn
[0032] [比較例 1]従来法によるレセプタ一一リガンド結合阻害実験 [0032] [Comparative Example 1] Receptor-one-ligand binding inhibition experiment by conventional method
1分子蛍光分析システム MF20 (商品名；ォリンパス株式会社製)を用いたこと、測 定条件が下記の通りであること以外は、実施例 1と同様に、 FIDAの測定モードにより 解析を行った。結果を図 3Cに示す。  Analysis was performed in the FIDA measurement mode in the same manner as in Example 1 except that the single molecule fluorescence analysis system MF20 (trade name; manufactured by Olympus Corporation) was used and the measurement conditions were as follows. The results are shown in Fig. 3C.
測定条件；レーザー波長： 543nm、レーザーパワー： 250 μ W、測定時間： 30秒 X 5回、ビームスキャナー： 2. 85mm/秒、合計スキャン面積： 100 μ rn  Measurement conditions: Laser wavelength: 543 nm, laser power: 250 μW, measurement time: 30 seconds x 5 times, beam scanner: 2. 85 mm / second, total scan area: 100 μ rn
[0033] 実施例 1および比較例 1の結果から、本発明により、スキャンした面積の合計値が 5 00 μ m2以上であれば、ばらつきが小さく再現性の高い安定したデータを得ることが できた。すなわち、実施例 1得られたデータは、実用上全く問題なぐ従来法よりも信 頼'性の高!、ものであった。 [0033] From the results of Example 1 and Comparative Example 1, according to the present invention, if the total value of the scanned area is 500 μm 2 or more, stable data with small variations and high reproducibility can be obtained. It was. In other words, the data obtained in Example 1 was more reliable than the conventional method, which has no practical problem.
[0034] [実施例 2]異なる大きさのビーズを用いた場合の検出感度の確認 (直径 10 μ mの蛍 光ビーズの作製） [0034] [Example 2] Confirmation of detection sensitivity when beads of different sizes are used (production of fluorescent beads with a diameter of 10 μm)
Polybead Carboxylate 10. Omicron microspheres、商品名、カタログ番 18133 ; Polyscienses社製）を 250 μ Lとり、 500 X Gで 5分間遠心処理して精製ビ ーズを得た。次いで、 PolyLink Protein Coupling Kit for COOH Microp articles (商品名、カタログ番号 PL01N ; Bangs Laboratories社製)のキットを用い 、前記精製ビーズと Alexa647— Albumin (1 μ g)とを反応させ、 Alexa647が結合 した直径 10 mの蛍光ビーズ溶液 200 Lを得た。そして、これを 10倍希釈したもの を以下の解析に供した。 Polybead Carboxylate 10. Omicron microspheres, trade name, catalog number 18133; manufactured by Polyscienses) 250 μL was taken and centrifuged at 500 XG for 5 minutes to obtain purified beads. Next, PolyLink Protein Coupling Kit for COOH Microp Using a kit of articles (trade name, catalog number PL01N; manufactured by Bangs Laboratories), the purified beads were reacted with Alexa647—Albumin (1 μg), and 200 L of fluorescent bead solution with a diameter of 10 m to which Alexa647 was bound was added. Obtained. A 10-fold diluted solution was used for the following analysis.
[0035] (直径 0. 5 μ mの蛍光ビーズの作製） [0035] (Preparation of fluorescent beads with a diameter of 0.5 μm)
ビーズの凝集を防止するため、 Streptavidin Coated Microspheres (商品名、 カタログ番号 CP01N ; Bangs Laboratories社製） 0. 49 mを、 PBS— 0. 05%t Ween20で希釈し、 5秒間超音波処理してから上清を除去する洗浄操作を 3回繰り返 した。そして、得られた洗浄済みビーズの PBS— 0. 05%tween20溶液 10 /z Lにさ らに PBS— 0. 05%tween20を 998 μ L添加し、続いて 2 μ Lの Biotin— ΑΤΤ065 5 (10 m)を添カ卩して、液量の合計を 1000 μ Lとしてから、室温で 1時間反応させた 。反応終了後、 20000 X Gで 5秒間遠心分離し、上清を除去した後、 PBS— 0. 05 %tween20を lmL添カ卩して、再度 20000 X Gで 5秒間遠心分離して上清を除去し た。そして、 PBS— 0. 05%tween20を lmL添カ卩してよく懸濁したものを、直径 0. 5 μ mの蛍光ビーズ溶液とし、以下の解析に供した。  To prevent bead aggregation, Streptavidin Coated Microspheres (trade name, catalog number CP01N; manufactured by Bangs Laboratories) 0.49 m was diluted with PBS—0.05% t Ween20 and sonicated for 5 seconds. The washing operation for removing the supernatant was repeated three times. Then, 998 μL of PBS—0.05% tween20 was added to 10 / z L of PBS—0.05% tween20 solution of the obtained washed beads, followed by 2 μL of Biotin—ΑΤΤ065 5 ( After adding 10 m), the total liquid volume was 1000 μL, and the mixture was reacted at room temperature for 1 hour. After completion of the reaction, centrifuge at 20000 XG for 5 seconds, remove the supernatant, add 1 mL of PBS—0.05% tween20, and centrifuge again at 20000 XG for 5 seconds to remove the supernatant. It was. Then, PBS-0.05% tween20 was added in 1 mL and well suspended to obtain a fluorescent bead solution having a diameter of 0.5 μm, which was subjected to the following analysis.
[0036] (FIDA解析） [0036] (FIDA analysis)
共焦点レーザー顕微鏡 FV1000 (商品名；ォリンパス株式会社製)へ 1分子蛍光分 析ユニットを接続したものを用いて、下記の測定条件で、 FIDAの測定モードにより、 二種の前記蛍光ビーズ溶液の解析を行った。  Using a confocal laser microscope FV1000 (trade name; manufactured by Olympus Corporation) with a single-molecule fluorescence analysis unit connected, analysis of the two types of fluorescent bead solution is performed under the following measurement conditions, using the FIDA measurement mode. Went.
測定は 5回行い、それぞれの蛍光強度検出値 (Hz)と、その平均値および標準偏 差を算出した。その結果を表 1および 2に示す。表 1は直径 0. 5 mの蛍光ビーズを 用いた場合、表 2は直径 10 mの蛍光ビーズを用いた場合の結果である。また、 1〜 5回目の蛍光強度の検出値をグラフ化したものを図 4A、図 4Bに、さらにこれらの検 出値の平均値をグラフ化したものを図 5A、図 5Bに示す。図 4A、図 5Aは直径 0. 5 mの蛍光ビーズを用いた場合、図 4B、図 5Bは直径 10 mの蛍光ビーズを用いた 場合の結果である。  The measurement was performed 5 times, and the detected fluorescence intensity (Hz), the average value, and the standard deviation were calculated. The results are shown in Tables 1 and 2. Table 1 shows the results when 0.5 m diameter fluorescent beads are used, and Table 2 shows the results when 10 m diameter fluorescent beads are used. 4A and 4B are graphs showing the fluorescence intensity detection values for the first to fifth times, and FIG. 5A and FIG. 5B are graphs showing the average values of these detection values. Figures 4A and 5A show the results when using fluorescent beads with a diameter of 0.5 m, and Figures 4B and 5B show the results when using fluorescent beads with a diameter of 10 m.
測定条件；レーザー波長： 543nm、レーザーパワー： 250 μ W、測定時間： 30秒、 走査速度： 25mmZ秒、合計スキャン面積： 25000 μ m2 [0037] [比較例 2]従来法による検出感度の確認 Measurement conditions; laser wavelength: 543 nm, laser power: 250 μW, measurement time: 30 seconds, scanning speed: 25 mmZ seconds, total scanning area: 25000 μm 2 [0037] [Comparative Example 2] Confirmation of detection sensitivity by conventional method
1分子蛍光分析システム MF20 (商品名；ォリンパス株式会社製)を用いたこと、測 定条件が下記の通りであること以外は、実施例 2と同様に、 FIDAの測定モードにより 解析を行った。結果を表 1および 2、図 4A、図 4B、図 5A、および、図 5Bに示す。表 1は直径 0. 5 mの蛍光ビーズを用いた場合、表 2は直径 10 mの蛍光ビーズを用 いた場合の結果である。そして、図 4A、図 5Aは直径 0. 5 / mの蛍光ビーズを用いた 場合、図 4B、図 5Bは直径 10 /z mの蛍光ビーズを用いた場合の結果である。  Analysis was performed in the FIDA measurement mode in the same manner as in Example 2 except that the single molecule fluorescence analysis system MF20 (trade name; manufactured by Olympus Corporation) was used and the measurement conditions were as follows. The results are shown in Tables 1 and 2, FIGS. 4A, 4B, 5A, and 5B. Table 1 shows the results when fluorescent beads with a diameter of 0.5 m were used, and Table 2 shows the results when fluorescent beads with a diameter of 10 m were used. 4A and 5A show the results when using fluorescent beads with a diameter of 0.5 / m, and FIGS. 4B and 5B show the results when using fluorescent beads with a diameter of 10 / z m.
測定条件；レーザー波長： 543nm、レーザーパワー： 250 μ W、測定時間： 30秒、 走査速度： 2. 85mmZ秒、合計スキャン面積： 100 m2 Measurement conditions; laser wavelength: 543 nm, laser power: 250 μW, measurement time: 30 seconds, scanning speed: 2. 85 mmZ seconds, total scanning area: 100 m 2
[0038] 実施例 2では、直径 0. 5 μ mおよび直径 10 μ mの!、ずれの蛍光ビーズも感度良く 検出できた。一方、比較例 2では、直径 0. 5 /z mの蛍光ビーズは検出できる場合もあ るが再現性が無ぐ直径 10 mの蛍光ビーズは全く検出できな力つた。すなわち、本 発明は従来法よりも高感度かつ高精度であることが確認された。  [0038] In Example 2, it was possible to detect fluorescent beads with a diameter of 0.5 μm and a diameter of 10 μm! On the other hand, in Comparative Example 2, fluorescent beads with a diameter of 0.5 / zm may be detected in some cases, but fluorescent beads with a diameter of 10 m that have no reproducibility are completely undetectable. That is, it was confirmed that the present invention has higher sensitivity and higher accuracy than the conventional method.
[0039] [表 1]  [0039] [Table 1]
¾光ピーズの直径 0 . 5 rn  ¾ diameter of light peas 0.5 rn
Figure imgf000012_0001
Figure imgf000012_0001
[0040] [表 2] 蛍光ビーズの直径 10 am [0040] [Table 2] Fluorescent bead diameter 10 am
Figure imgf000013_0001
Figure imgf000013_0001
[0041] [実施例 3]走査速度が検出精度に与える影響の確認 [0041] [Example 3] Confirmation of influence of scanning speed on detection accuracy
実施例 2で調製した直径 10 mの蛍光ビーズ溶液を、 384ウェルマイク口プレート に添加し、共焦点レーザー顕微鏡 FV1000 (商品名；ォリンパス株式会社製)へ 1分 子蛍光分析ユニットを接続したものを用いて、下記の測定条件により、走査速度の違 いが検出値に影響を与えるかどうかを検討した。測定は、一つの走査速度につき 5回 行った。  The fluorescent bead solution having a diameter of 10 m prepared in Example 2 was added to a 384-well microphone opening plate, and a confocal laser microscope FV1000 (trade name; manufactured by Olympus Corporation) was connected to a 1-molecule fluorescence analysis unit. Using this, we examined whether the difference in scanning speed would affect the detection value under the following measurement conditions. The measurement was performed 5 times per scanning speed.
結果を表 3および図 6に示す。表 3には 1〜5回目の蛍光強度の検出値を示し、図 6 には 1〜5回目の蛍光強度の検出値の平均値をグラフ化したものを示す。  The results are shown in Table 3 and FIG. Table 3 shows the detected values of the first to fifth fluorescence intensities, and FIG. 6 shows a graph of the average of the detected values of the first to fifth fluorescence intensities.
測定条件；レーザー波長： 633nm、レーザーパワー：41% (約 300 μ Wに相当）、 測定時間： 30秒 X 5回、測定モード: FIDA、走査速度： 1、 2. 5、 10、 20、 25、 50m mZ秒、合計スキャン面積： 160 mX 160/z m、使用対物レンズ倍率： X 60  Measurement conditions: Laser wavelength: 633 nm, laser power: 41% (equivalent to about 300 μW), measurement time: 30 seconds x 5 times, measurement mode: FIDA, scan speed: 1, 2.5, 10, 20, 25 , 50m mZ second, total scanning area: 160 mX 160 / zm, objective lens magnification: X 60
[0042] 2. 5、 10、 20、 25mmZ秒の場合は、検出値がより安定していたのに対して、 lm mZ秒および 50mmZ秒ではデータのバラツキ（エラーバー）が見られた。このことか ら、本発明における走査速度は、 2. 5〜25mmZ秒が好ましいことが確認された。  [0042] 2. The detected values were more stable for 5, 10, 20, and 25 mmZ seconds, whereas data variations (error bars) were observed for lm mZ seconds and 50 mmZ seconds. From this, it was confirmed that the scanning speed in the present invention is preferably 2.5 to 25 mmZ seconds.
[0043] [表 3] mm/秒 SO 25 20 10 2.5 1  [0043] [Table 3] mm / sec SO 25 20 10 2.5 1
1回目 1256.1 1796 2149. β 2123 2489.6 554.7  1st 1256.1 1796 2149.β 2123 2489.6 554.7
2回目 1724.1 2436.2 2741.7 190t.9 3279.1 2879.1  2nd 1724.1 2436.2 2741.7 190t.9 3279.1 2879.1
3回目 2452.8 2140.1 1858.1 2834.6 2639.9  3rd 2452.8 2140.1 1858.1 2834.6 2639.9
4回目 2480.3 Z1S7.1 17C4.1 2083.5 3I9B.4 1749.4  4th 2480.3 Z1S7.1 17C4.1 2083.5 3I9B.4 1749.4
5回目 2466.6 2197.4 2161.6 E534.3 3315.2 321.6 平均値 2071.66 2207.9 2179.46 2100.16 3023.18 1628.94 標準偏差 556.3787 266.669 369- 0626 j 267.9444 354.1296 1168.608 [0044] [実施例 4]信号強度取得領域の違い (対物レンズの倍率)の検出精度への影響の確 実施例 2で調製した直径 10 μ mの蛍光ビーズ溶液を、 384ウェルマイク口プレート に添加し、共焦点レーザー顕微鏡 FV1000 (商品名；ォリンパス株式会社製)へ 1分 子蛍光分析ュュットを接続したものを用いて、下記の測定条件により、使用する対物 レンズの倍率の違いすなわち共焦点領域の容積が検出値に影響を与えるかどうかを 検討した。 5th 2466.6 2197.4 2161.6 E534.3 3315.2 321.6 Average 2071.66 2207.9 2179.46 2100.16 3023.18 1628.94 Standard deviation 556.3787 266.669 369- 0626 j 267.9444 354.1296 1168.608 [0044] [Example 4] Confirmation of influence of difference in signal intensity acquisition region (magnification of objective lens) on detection accuracy Fluorescent bead solution with a diameter of 10 μm prepared in Example 2 was applied to a 384-well microphone mouthplate. Add a confocal laser microscope FV1000 (trade name; manufactured by Olympus Corporation) and connect a 1-molecule fluorescence analysis tube. We examined whether the volume of the sample affects the detection value.
測定は、一つの対物レンズ倍率につき 5回行った。結果を表 4および図 7に示す。表 4には 1〜5回目の蛍光強度の検出値を示し、図 7には 1〜5回目の蛍光強度の検出 値の平均値をグラフ化したものを示す。  The measurement was performed five times for each objective lens magnification. The results are shown in Table 4 and FIG. Table 4 shows the detected values of the 1st to 5th fluorescence intensities, and Fig. 7 shows a graph of the average value of the detected 1st to 5th fluorescence intensities.
測定条件；レーザー波長： 633nm、レーザーパワー：41% (約 300 μ Wに相当）、 測定時間： 30秒 X 5回、測定モード： FIDA、走査速度： 20mmZ秒、合計スキャン 面積： 160 m X 160 m、使用対物レンズ倍率： X 10、 X 20、 X 40、 X 60  Measurement conditions: Laser wavelength: 633 nm, laser power: 41% (equivalent to about 300 μW), measurement time: 30 seconds x 5 times, measurement mode: FIDA, scanning speed: 20 mmZ seconds, total scan area: 160 m x 160 m, Objective lens magnification: X 10, X 20, X 40, X 60
[0045] 高感度に信号強度を検出できるのは、共焦点領域の容積が 1FL以下、すなわち使 用対物レンズの倍率が X 20〜 X 60の場合であることが確認された。  [0045] It was confirmed that the signal intensity can be detected with high sensitivity when the volume of the confocal region is 1FL or less, that is, when the magnification of the objective lens used is X20 to X60.
[0046] [表 4]  [0046] [Table 4]
Figure imgf000014_0001
Figure imgf000014_0001
[0047] [実施例 5]蛍光観察による検出対象の粒子数の確認 [0047] [Example 5] Confirmation of the number of particles to be detected by fluorescence observation
実施例 2で調製した直径 10 mの蛍光ビーズ溶液を、共焦点レーザー顕微鏡 FV 1000 (商品名；ォリンパス株式会社製)へ 1分子蛍光分析ユニットを接続したものを 用いて、下記の測定条件により解析し、蛍光ビーズの数の確認を行った。結果を図 8 に示す。 測定条件；レーザー波長： 633nm、レーザーパワー：41% (約 300 μ Wに相当）、 走査速度： 20mm/秒、合計スキャン面積 : 25 μ ηι Χ 25 μ m〜200 ^ m X 200 μ m 、使用対物レンズ倍率： X 60 The fluorescent bead solution with a diameter of 10 m prepared in Example 2 was analyzed under the following measurement conditions using a confocal laser microscope FV 1000 (trade name; manufactured by Olympus Corporation) with a single molecule fluorescence analysis unit connected. Then, the number of fluorescent beads was confirmed. The results are shown in Fig. 8. Measurement conditions: Laser wavelength: 633 nm, laser power: 41% (equivalent to about 300 μW), scanning speed: 20 mm / sec, total scanning area: 25 μηιΧ 25 μm to 200 ^ m X 200 μm, used Objective lens magnification: X 60
[0048] 細胞と同等の大きさの蛍光ビーズが 10数個〜 20個程度、ゆっくりと移動していく様 子を観察できた。すなわち、 10数個〜 20個程度の蛍光ビーズを検出対象として、十 分に FIDA解析を行うことが可能であること、この時のスキャン領域は、 25 m X 25 μ m〜200 m X 200 mの範囲が適切であることが確認された。  [0048] About 10 to 20 fluorescent beads of the same size as the cells were observed to move slowly. In other words, it is possible to perform FIDA analysis sufficiently with about 10 to 20 fluorescent beads as detection targets, and the scan area at this time is 25 m X 25 μm to 200 m X 200 m The range was confirmed to be appropriate.
[0049] 本実施例では、解析対象物としてビーズを用いた力 これはフローサイトメトリーで は通常細胞の標準化測定方法として行われている手法である。そして、測定対象物 として細胞等を用いても、同様の結果を得ることができる。  [0049] In the present example, force using beads as an analysis target. This is a technique usually used as a standardized measurement method for cells in flow cytometry. The same result can be obtained even if a cell or the like is used as the measurement object.
産業上の利用可能性  Industrial applicability
[0050] 本発明は、臨床検査、衛生検査および生化学研究の分野等で利用可能であり、生 体試料の解析に有用である。 [0050] The present invention can be used in the fields of clinical testing, hygiene testing, biochemical research, and the like, and is useful for analysis of biological samples.

Claims

請求の範囲 The scope of the claims
[1] 共焦点領域内の微少領域を走査して微少領域の信号強度を測定し、微粒子の大 きさ、ある 、は微粒子の結合または乖離の有無を解析する微粒子の解析方法であつ て、  [1] A fine particle analysis method that scans a small region within a confocal region and measures the signal intensity in the small region to analyze the size of the fine particle, whether or not there is a binding or detachment of the fine particle,
信号強度を測定する複数の微少領域のうち一つの微少領域の容積が 1FL以下で あり、前記複数の微少領域の走査領域平面の面積合計値が、 500 /ζ πι2〜40000 m2である微粒子の解析方法。 Fine particles in which the volume of one micro area of the plurality of micro areas for measuring signal intensity is 1 FL or less, and the total area value of the scanning area planes of the plurality of micro areas is 500 / ζ πι 2 to 40000 m 2 Analysis method.
[2] 信号強度を測定する一つの微少領域を走査する速度が 2. 5mmZ秒〜 25mmZ 秒である請求項 1に記載の微粒子の解析方法。 [2] The method for analyzing fine particles according to [1], wherein the scanning speed of one minute region for measuring the signal intensity is 2.5 mmZ second to 25 mmZ second.
[3] 蛍光相関分光法、蛍光強度分布解析法および蛍光偏光強度分布解析法の少なく ともいずれか一つで前記解析を行う請求項 1または 2に記載の微粒子の解析方法。 [3] The method for analyzing fine particles according to claim 1 or 2, wherein the analysis is performed by at least one of fluorescence correlation spectroscopy, fluorescence intensity distribution analysis, and fluorescence polarization intensity distribution analysis.
[4] 前記微粒子が、細胞、細胞膜、細胞膜断片、有機物粒子、無機物粒子およびこれ らのうちの一種以上力 なる複合体力 選ばれる一種以上である請求項 1〜3のいず れか一項に記載の微粒子の解析方法。 [4] The microparticle according to any one of claims 1 to 3, wherein the microparticle is a cell, a cell membrane, a cell membrane fragment, an organic particle, an inorganic particle, or one or more selected from these complex forces. The microparticle analysis method described.
[5] 前記微粒子の大きさ力 0. 3 m〜10 μ mである請求項 1〜4のいずれか一項に 記載の微粒子の解析方法。 [5] The method for analyzing fine particles according to any one of [1] to [4], wherein a size force of the fine particles is 0.3 m to 10 μm.
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