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
region
particles
scanning
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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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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • G01N21/645Specially adapted constructive features of fluorimeters
    • G01N21/6456Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458Fluorescence microscopy
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10Investigating individual particles
    • G01N15/14Electro-optical investigation, e.g. flow cytometers
    • G01N15/1456Electro-optical investigation, e.g. flow cytometers without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • GPHYSICS
    • G02OPTICS
    • G02BOPTICAL ELEMENTS, SYSTEMS, OR APPARATUS
    • G02B21/00Microscopes
    • G02B21/06Means for illuminating specimens
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10Investigating individual particles
    • G01N15/14Electro-optical investigation, e.g. flow cytometers
    • G01N2015/1493Particle size

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

Specification

The method of analysis fine

Technical field

[0001] The present invention, regarding the fine particles of the biological substance or the like, a method of analyzing a high sensitivity and high accuracy. This application claims priority to the Japanese Patent Application No. 2006- 192742, filed on July 13, 2006, which is incorporated herein by reference.

BACKGROUND

[0002] The fluorescence correlation spectroscopy analysis, etc., as a method for detecting the signal strength, analyzing the size of the biological substance, or the presence or absence of binding or dissociation of the biological substance with high sensitivity, for example, vibration around the standard position location deflecting mirror arrangement having an objective lens side flat polarizing mirror arranged to have, a method using a device located on a confocal microscope is disclosed (non-patent Document 1, Patent Document 1 and 2). The mirror, the normal position being adapted to oscillate or rotate about a, are called beam scanner and normal. By this mirror technique used in fluorescence intensity distribution analysis, it is prevented discoloration of light by the object to be analyzed, such as laser, to increase the number of analysis objects confocal region within a given measurement time Caro It has been described as data acquisition time can be considerably saved.

... Non-Patent Literature l: Pro Natl Acad Sci USA, vol 96, 1375 - 1378, 1999 Patent Document 1:. Kohyo 2001 - 502062 Patent Publication

Patent Document 2: JP-T 2001- 502066 JP

Disclosure of the Invention

Problems that the Invention is to you'll solve

[0003] However, these documents only it is stated that the use of a beam scanner is effective for the analysis of biological substances, for example, a suitable scan by the size of the biological substance to be measured for more information on scanning methods, such as speed and the area it is not at all been mentioned base.

Then, for example, when analyzing the object having a 0. 3 m or more in size, the data of the variation magnitude obtained, in which it has been known one, Ru. [0004] Accordingly, a large biological substances, e.g., cell membrane fragments, such as cells themselves, high accuracy when analyzing those having a diameter 0. 3 / zm or more size, the data obtained so heard Baratsukigadai analysis can not be performed. In particular, when preparing a cell membrane fragments due membrane fraction of a cell, it is tagged diameter these cells membrane fragments uniform in size is difficult instrument of the cell membrane fragments are spherical diameter 0. 1~0. 5 m it includes more large molecules or particles 0. 3 m.

[0005] That is, the method using a beam scanner of the prior art, there is an upper limit to the size of measurable bio-related substances, when analyzing the biological substance of a size greater than the upper limit, it small standard deviation had stable problem data is difficult to obtain a.

[0006] In addition, FCS and when detecting the interaction between molecules by a molecular assays such as FIDA, in order to obtain accurate data, quadruple force eight times or more molecular before and after the interaction in the FCS It requires a change in the amount. On the other hand, in FIDA, if the fluorescence intensity is changed on the 1.3 more than double before and after the interaction, it is impossible to detect their interactions. In such a case, one of the molecules that act mutual, for example, fixed to the polystyrene particles bead surface may take the method of detecting the interaction.

[0007] In this case, Meniwa where obtain accurate data using a beam scanner as in the conventional method, the size of the molecules to be analyzed are even though 0. 3 m or less, the particle Sa I the upper limit of the diameter of's i.e. beads are 0. 3 m, there is a problem that variation of data is large at higher magnitude.

The [0008] above conventional methods as, in order to obtain accurate data, there is a limit to the size of measurable biological substance, the data obtained force ones 0. 3 m or more in size is , there is a large variation, such can only scan the same area in a donut shape ChikaraTsuta. Their to Non-Patent Document 1, Patent Documents 1 and 2, such contains the appropriate conditions, such as scan speed, scan area when the magnitude to measure the 0. 3 / zm or more bead force ivy. Means for Solving the Problems

[0009] The present invention has been made in view of the above circumstances, even if include the 0. 3 mu m or more in size as the analysis object, the size of the particles to be analyzed, or binding of the microparticles or the presence or absence of the offset, and to provide a method of analyzing a high sensitivity and high accuracy.

[0010] That is, to solve the above problems,

The present invention, the signal strength of the small region by scanning the minute region of the confocal region is measured, a method of analyzing particles for analyzing the size, or the presence or absence of binding or dissociation of the microparticles in the microparticle, the signal strength capacity product of one minute region of the plurality of small areas of measuring is not more than 1FL, the area total value of the scanning area plane of said plurality of micro region is 500 μ m 2 ~40000 μ m 2 particles it is a method of analysis.

In the present invention, even the speed of scanning one minute area of ​​measuring the signal strength 2. 5 m mZ sec ~ 25MmZ seconds good ヽ.

In the present invention, fluorescence correlation spectroscopy, at least of the fluorescence intensity distribution analysis and fluorescence polarization intensity distribution analysis, even if the analysis in one or misalignment good ヽ.

In the present invention, the fine particles, cells, cell membranes, cell membrane fragments, organic particles, inorganic compound particles and may be filed with one or more force becomes complex fitness least one selected from among these.

In the present invention, the size force of said particulate 0. 3 / ζ πι~10 / ζ may be Paiiota. Effect of the invention

[0011] The present invention, even if include the 0. 3 m or more in size as the analysis object, the size of the particles to be analyzed, or the binding or the existence of the gap of the fine particles, high sensitivity or Tsu high it is possible to analyze the accuracy.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] [FIG 1] FIG 1 is an explanatory diagram for explaining by comparing the parsable size and analysis conditions of the microparticles.

[Figure 2A] Figure 2A is a diagram illustrating the scanning method in the prior art.

[Figure 2B] Figure 2B is a diagram illustrating the scanning method in the prior art.

[Figure 2C] FIG 2C is a diagram illustrating the scanning method of the present invention.

FIG 2D] FIG. 2B is a diagram illustrating the scanning method of the present invention.

FIG 2E] FIG. 2C is a diagram illustrating the scanning method of the present invention.

[Figure 3A] Figure 3A is a graph showing the analysis results of Example 1. [Figure 3B] Figure 3B is a graph showing the analysis results of Example 1.

FIG 3C] Figure 3C is a graph showing the analysis results of Comparative Example 1.

[Figure 4A] Figure 4A is a graph showing the five measurement results of Example 2 and Comparative Example 2.

[Figure 4B] Figure 4B is a graph showing the five measurement results of Example 2 and Comparative Example 2.

[Figure 5A] Figure 5A is a graph showing the average of five measurements results of Example 2 and Comparative Example 2.

[Figure 5B] Figure 5B is a graph showing the average of five measurements results of Example 2 and Comparative Example 2.

FIG. 6 is a graph showing the analysis results of Example 3.

[7] FIG. 7 is a graph showing the analysis results of Example 4.

[8] FIG. 8 is a measurement image obtained in Example 5.

BEST MODE FOR CARRYING OUT THE INVENTION

[0013] The present invention will be described in detail.

Volume of micro area of ​​measuring the signal strength, for example, can be adjusted by Rukoto changing the magnification of the objective lens used. And in the present invention, the volume of one minute regions measuring the signal strength (small area of ​​each of the plurality of minute areas) is less 1FL (full Mutoritsu torr). If greater than 1FL, sometimes that Do is difficult to detect the signal strength with high sensitivity.

[0014] area total value of the scanning area plane of the plurality of microscopic regions of measuring signal strength, and 500 μ m 2 ~40000 m 2. Outside this range, next to those detected values ​​of the signal intensity variation large, reliability 'property resulting in summer low.

[0015] Contact with the present invention, Te is the speed of scanning one minute area is good be suitably adjusted !, but 2. is preferably 5mmZ sec ~ 25MmZ seconds. Test detection value of the signal strength is within this range, it becomes smaller ones variation, it is preferable to perform the analysis with high precision.

[0016] In the present invention, the measurement of signal strength method for detecting the fluorescence of the labeled particles with a force fluorescent dye applied by these methods is preferred. In this case, for example, fluorescence correlation spectroscopy (hereinafter abbreviated as FCS), fluorescence intensity distribution analysis (hereinafter, abbreviated as FIDA), fluorescence polarization intensity distribution analysis (FIDA- polarization) or the like is preferred, mentioned as a method is Ru. These may be performed alone or may be performed by combining a plurality of methods. These measurements may, for example, a confocal laser microscope FV1000; can be carried out using those connected with 1 molecule fluorescence analysis unit to (trade name Orinpasu Co., Ltd.).

[0017] According to the signal strength measurement method as described above, depending on the size of the particles to be analyzed, the signal intensity detected changes. Thus, fine particles to be analyzed is, or combined with other micro-particles, if the size and or dissociate into a plurality of particles changes, the change in signal intensity, it is possible to detect these binding or dissociation . Herein, the term bond and also refers to such bonds based on intermolecular attractions such as hydrogen bonds or hydrophobic bonds Nag only covalent bonds.

[0018] In the present invention, fine particles used as an analysis target is not particularly limited, could be analyzed by conventional methods, for example, it may but be of 0. 1 m or less in size, and analyzed what is of large size is difficult, for example, it is preferable that a size of 0. 3 m~10 μ m. As such, for example, biological substances, organic particles, one or more kinds of selected non-aircraft material particles and force becomes complex physical one or more of these may be mentioned are. Then, as the biological substances, for example, as a cell, cell membrane and cell membrane fragments and the like are preferable. These fine particles may be used or 巿販 Hinto those prepared by a conventional method. For example, if the biological substance, by a conventionally known method, processing the samples raw physical strength taken, it can be used extracted from the sample.

[0019] receptors present on cell membranes, e.g. GPCR etc., also important receptor that is the target of drug discovery, while the force is that there are many O over fan receptor exists. This was recently because a case the cell membrane fraction is used in the search for inhibitors or ligands have many. Thus, it has important functions and the size of the large cell, cell membrane and cell Makudan piece or the like is particularly suitable as an analysis target of the present invention.

[0020] For example, the cell membrane, in the normal solution is present in a ribosomal. When the performing membrane fractions treated by a conventional method, the cell membrane fragments are obtained, the magnitude of which is often vary in diameter from 0.1 to 0. About 5 m. Such may, conventionally, a radioisotope-loop (RI) label by centrifugation sedimentation, etc. Do detected by scintillation counter analysis using RI is the mainstream. Further, in the case of applying the conventional one molecule measurements, obtained only variations in the large data. However, according to the present invention, even if of a size of more than 0. 3 m is contained as the analysis object, it is possible to analyze with high precision Nag be used dangerous RI. The size and analysis conditions analysis microparticles, shown in Figure 1 an example of a comparison between the method of the prior art and the present invention. Note In the conventional method, to scan in a donut shape, scanning speed is calculated by approximation to a straight line.

[0021] The diameter 0. 3 M to m various biological substance immobilized on the bead surface and the like also can be suitably used regardless of its size. For example, it is possible to perform such a biological-related substance bound to the beads particle mediation Si also high accuracy like. And the antibody or antigen fixed to the bead surface, in the case where the biological substance is detected by a specific binding formation between antigen antibody, as the particle size of the beads is large, the number of antibody or antigen can be fixed on the bead surface can be increased, it is possible to combine many biological substance by a single bead. That is, it is possible to amplify the larger beads, the signal strength of the biomaterial at the time of measurement to be used. The present invention, such a big bead, particularly when using beads having a diameter of 0. 3 μ m~10 μ m, are particularly preferred.

[0022] In addition, the use of large beads of particle size, is preferable from the viewpoint preparation and handling of the beads with a fixed biological substance is easy. Having a small particle diameter beads, for example, when using the following diameter 0. 3 / zm, the fixed force reaction to the beads of biological substances, centrifuging associated with each procedure of the measurement Nio Te, after analyzed the beads precipitated in the lower layer is centrifugal processing, sometimes immediately spread to the upper layer. This Therefore, when the recovery and washing, cause analysis Target beads are removed with the upper layer, using a force diameter 0. 3 / zm or more beads inconvenience of not come concentrations of biological substances in accurately measuring by applying the analysis method of the invention, it is not also occur such a disadvantage.

[0023] In the case of using the beads in the present invention, the material may be any one which does not affect the measurement of the signal strength such as fluorescence intensity, any can also be used as, for example, polystyrene emissions of various plastic beads or metal beads may be used conventionally known ones. Even if the bio-related substances, etc. to be analyzed is fixed to the bead may be applied to conventionally known methods. Here, mainly, the ones to be fixed to the force the bead surface that describes the case where the biological substance is fixed on the bead surface, if the analysis object of the present invention comprises what is not limited to a bio-related substance it may be one.

[0024] fluorescent dye in the present invention may be a conventionally known, for example, can be cited Furuo Rosein, rhodamine, Alexa, and ATTO dyes.

[0025] FIG 2A~ Figure 2E is a diagram illustrating the scanning method, FIG. 2A point scan, scan Shinano the center of FIG. 2 B the prior art, a donut-shaped scan, FIG 2C~-2E are present the preferred scanning invention are shown, respectively. In the present invention, as described in FIG. 2C, sequentially scan a plurality of minute areas arranged in a straight line (a plurality of ovoid in FIG. 2C), then adjacent to a plurality of small regions arranged in the straight line by repeating the operations of the sequentially scanning a plurality of minute areas arranged in a straight line Te, scanning confocal region real Hodokosuru. Further, as another scanning method of the present invention, as shown in FIG. 2D, the center force-scan region a plurality of minute areas arranged countercurrently force connexion spiral to the outer peripheral portion, the central portion force also to the outer peripheral portion toward power connexion as shown scanning method that, in FIG. 2E, a plurality of minute areas arranged on the outer periphery force also directed force connexion spiral to the center of the scan area, and a method of connexion scan suited outer periphery force also to the center . These spiral scanning method, in order to change the the direction of scan, or a scan speed to zero, can scan a desired region Nag possible or slower. Therefore, it is possible to shorten the scan time than when scanning in a straight line, and more preferred. Beam scanner One prior art, for example, MF20; the case of using (trade name Orinpasu Ltd.), to scan the confocal region in a donut shape, area that can retrieve data is very small as about 100 m 2 (Figure 2B). Thus, example embodiment, cells, cell membranes, cell membrane fragments, such as the diameter 0. 3 / zm or more nanoparticles, 0. 3 mu m or more in size ones, very small sag signal strength probability of passing through the measurement region large reliable data can not be obtained variation even when able to measure.

[0026] If you simply enlarged measurement area, for example, in the case so as to enlarge the confocal area by lowering the magnification of the objective lens, it order to prevent discoloration of the indicator to! / Ru fluorochrome microparticles what reason for the excitation energy is constant, the signal intensity per one particle analyzed is reduced, it becomes impossible to sensitively detected. In the present invention, on the other hand, a 1FL following one confocal region for measuring the signal strength, while the cormorants minimum area and expands sukiya emission region 500 μ m 2 ~40000 μ m 2 . Therefore, by using the FID A, it is possible to detect fine particles having a sufficient signal strength, for example, it is possible to catch a change in the 1.3 times or more in fluorescence intensity due to interaction of the fine particles the workers. Further, by enlarging the scan region, as an analysis object, even those of a low diffusion rate 0.5 or more in size, the probability of passing through the measurement region is sufficiently high. Accordingly, the conventional measurement is difficult, others such 0.3 exceeding m size, leaving measured child transgression with high sensitivity.

[0027] Usually, when performing scanning, when the acquisition of the signal of the force rough force Ji number of fine particles because set to keep fit set Ji roughness force a scan region before start of measurement has been completed, and ends the scan it is also possible. In this way, it is possible to shorten the measurement time.

[0028] Contact with the present invention, Te is a minute area of ​​measuring the signal strength, the area total value of the scanning area plane, the scanning speed of the micro-region is the same as described so far, the other measurement conditions One, Te is good by setting the optimum conditions depending on the appropriate conditions.

Example

[0029] Hereinafter, the specific examples will be described in more detail the present invention. However, the invention is not intended to be limited to the following Examples.

[Example 1] Cell membrane fractions receptor eleven ligand binding inhibition experiments using (PAF inhibitors: PAF receptor antagomir - Study of stringent WEB 2086) (fractionation process of the cell membrane)

Perform fractionation process of the cell membrane according to the following procedure, the cell membrane fragments, was obtained as a ribosome with variation of diameter of about 0. 1~0. 5 μ m.

(1) Human PAF-expressing CHO cells in the tube, using a HEPES buffer of about 10 times (WZw), and homogenized on ice.

(2) 4 ° C, centrifuged at 800 XG 20 minutes, then carefully transferred to take a separate tube supernatant.

(3) In addition, 4 ° C, centrifuged for 60 minutes at 100000 XG, then the supernatant was removed.

(4) was pipetted precipitated with HEPES buffer.

(5) using a homogenizer, it was suspended in 10 strokes on ice.

(6) 4 ° C, centrifuged for 60 minutes at 100000 XG, then the supernatant was removed. (7) was loosened precipitated with HEPES buffer cell and an equal volume.

(8) with a homogenizer, it was suspended in 10 strokes on ice.

(9) by measuring the amount of protein, it was adjusted to 0. 5mgZml.

[0030] (reaction)

Mixing the unlabeled PA F solution 7 Les TAMRA labeled PAF solution 7 L adjusted to a concentration of cell membrane fraction solution 14 Les 2nM~160nM obtained above was allowed to react for 1 hour at 25 ° C.

[0031] (FIDA analysis)

Confocal laser microscope FV1000 (trade name; Orinpasu Co., Ltd.) using a material obtained by connecting a single molecule fluorescence analysis unit to, in the following measuring conditions, was analyzed by measuring mode FIDA. The results are shown in Figure 3 A to FIG 3C. Figure 3A, if the total scan area of 500 m 2, FIG. 3B is a case where the total scan area of 25000 m 2.

Measurement conditions: laser wavelength: 543 nm, laser power: 250 mu W, measurement time: 30 seconds X 5 times, scanning rate: 25 mm /禾少, total scan area: 500 μ m 2, 25000 μ rn

[0032] [Comparative Example 1] receptor eleven ligand binding inhibition assay according to the conventional method

Single molecule analysis system MF20 (trade name; Orinpasu Co., Ltd.) that was used, measurement conditions, except that as follows, in the same manner as in Example 1, was analyzed by measuring mode FIDA. The results are shown in Figure 3C.

Measurement conditions: laser wavelength: 543 nm, laser power: 250 mu W, measurement time: 30 seconds X 5 times, the beam scanner: 2. 85 mm / sec, the total scan area: 100 mu rn

[0033] From the results of Example 1 and Comparative Example 1, the present invention, if the total value of the scanned area is 5 00 μ m 2 or more, dispersion can be achieved a small highly reproducible stable data It was. That is, the data obtained in Example 1, practically no problem Nag confidence 'of higher than conventional methods! Were those.

[0034] Confirmation of detection sensitivity when using the Example 2 different sizes of beads (Preparation of fluorescent beads having a diameter of 10 mu m)

Polybead Carboxylate 10. Omicron microspheres, product name, catalog number 18133; Polyscienses Co., Ltd.) was taken up 250 μ L, to obtain a purified bi-over's and centrifuged for 5 minutes at 500 XG. Then, PolyLink Protein Coupling Kit for COOH Microp articles (trade name, catalog number PL01N; Bangs Laboratories, Inc.) using a kit, is reacted with the purified beads and Alexa647- Albumin (1 μ g), diameter Alexa647 bound to obtain a fluorescent bead solution 200 L of 10 m. And it subjected those which was diluted 10-fold in the following analysis.

[0035] (Preparation of fluorescent beads of diameter 0. 5 mu m)

To prevent agglomeration of the beads, Streptavidin Coated Microspheres (trade name, catalog number CP01N; Bangs Laboratories, Inc.) and 0. 49 m, PBS- 0. diluted with 05% t Ween20, from 5 seconds sonicated It was repeated three times a cleaning operation to remove the supernatant. The resulting washed PBS-0. 05% of the beads tween20 solution 10 / z L to be al in PBS-0. 05% tween20 added 998 mu L, followed by 2 μ L Biotin- ΑΤΤ065 5 ( 10 m) by 添Ka 卩 and the total amount of liquid from a 1000 mu L, and reacted at room temperature for 1 hour. After completion of the reaction, 20000 and centrifuged for 5 seconds at XG, after removing the supernatant, PBS- 0. 05% tween20 in lmL 添Ka卩 and the supernatant was removed by centrifugation for 5 seconds again 20000 XG It was. Then, the PBS- 0. 05% tween20 those well suspended with lmL 添Ka卩, the fluorescent bead solution having a diameter of 0. 5 mu m, was subjected to the following analysis.

[0036] (FIDA analysis)

Confocal laser microscope FV1000 (trade name; Orinpasu Co., Ltd.) using a material obtained by connecting a single molecule fluorescence analysis unit to, in the following measuring conditions, the measurement mode FIDA, two of the analysis of the fluorescent bead solution It was carried out.

The measurement was performed 5 times, each of the fluorescence intensity detected value (Hz), and the average value was calculated and standard deviations. The results are shown in Tables 1 and 2. Table 1 in the case of using fluorescent beads with a diameter of 0. 5 m, Table 2 shows the results obtained by using fluorescent beads with a diameter of 10 m. Further, FIG. 4A is a graph of a detected value of the fluorescence intensity of one to fifth, in 4B, 5A those further graph of the average value of the test detection value, shown in Figure 5B. FIGS. 4A, 5A in the case of using fluorescent beads with a diameter of 0. 5 m, FIG. 4B, FIG. 5B is the results obtained by using fluorescent beads with a diameter of 10 m.

Measurement conditions: laser wavelength: 543 nm, laser power: 250 mu W, measurement time: 30 seconds, scanning speed: 25MmZ seconds, the total scan area: 25000 μ m 2 [0037] [ Comparative Example 2] Confirmation of detection sensitivity by the conventional method

Single molecule analysis system MF20 (trade name; Orinpasu Co., Ltd.) that was used, measurement conditions, except that as follows, in the same manner as in Example 2, was analyzed by measuring mode FIDA. Results Table 1 and 2, 4A, 4B, 5A and shown in Figure 5B. Table 1 in the case of using fluorescent beads with a diameter of 0. 5 m, Table 2 shows the results when had use fluorescent beads having a diameter of 10 m. Then, 4A, 5A in the case of using fluorescent beads with a diameter of 0. 5 / m, FIG. 4B, FIG. 5B is the results obtained by using fluorescent beads with a diameter of 10 / zm.

Measurement conditions: laser wavelength: 543 nm, laser power: 250 mu W, measurement time: 30 seconds, the scanning speed: 2. 85MmZ seconds, the total scan area: 100 m 2

[0038] In Example 2,! Diameter 0. 5 mu m and diameter 10 mu of m, fluorescent beads deviation also be sensitively detected. On the other hand, in Comparative Example 2, a possible fluorescent beads totally detection mower Ru Reproducibility is Mugu diameter 10 m when fluorescent beads having a diameter of 0. 5 / zm is capable of detecting ChikaraTsuta. That is, the present invention was confirmed to be a highly sensitive and accurate than the conventional method.

[0039] [Table 1]

Diameter of ¾ light Peas 0. 5 rn

[0040] [Table 2] fluorescent beads having a diameter of 10 am

[0041] Confirmation of Example 3 Effect scanning speed to provide the detection accuracy

Those connecting the 1 minute child fluorescence analysis unit to; (manufactured by Orinpasu Ltd. trade name) fluorescence bead solution having a diameter 10 m prepared in Example 2, was added to 384-well microphone port plate, a confocal laser microscope FV1000 using, by the following measuring conditions, it was examined whether affect differences bur detection value of the scanning speed. The measurement was carried out 5 times for one of the scanning speed.

The results are shown in Table 3 and Figure 6. Table 3 shows the detection value of the fluorescence intensity of the fifth time, in FIG. 6 shows a graph of the mean value of the detected value of the fluorescence intensity of 1-5 time.

Measurement conditions: laser wavelength: 633 nm, laser power: 41% (corresponding to about 300 mu W), measuring time: 30 seconds X 5 times, measurement mode: FIDA, scanning speed: 1, 2.5, 10, 20, 25 , 50 m mZ seconds, the total scan area: 160 mX 160 / zm, using an objective lens magnification: X 60

[0042] 2.5, 10, 20, when the 25mmZ sec, whereas the detected value was more stable, variation in the data (error bars) was observed in lm mZ seconds and 50mmZ seconds. This either et al., The scanning speed in the present invention, 2. 5~25MmZ sec is preferably confirmed.

[0043] [Table 3] mm / sec SO 25 20 10 2.5 1

1st 1256.1 1796 2149. β 2123 2489.6 554.7

Second time 1724.1 2436.2 2741.7 190t.9 3279.1 2879.1

Third 2,452.8 2,140.1 1,858.1 2,834.6 2,639.9

4th 2480.3 Z1S7.1 17C4.1 2083.5 3I9B.4 1749.4

Fifth 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] The difference of the signal intensity acquisition area of ​​(the magnification of the objective lens) fluorescent beads solution of diameter 10 mu m prepared in Make example 2 Effect of the detection accuracy, and added to 384-well microphone port plate, a confocal laser microscope FV1000 (trade name; Orinpasu Ltd.) to 1 minute child using that connects the fluorescence analysis Yuyutto by the following measuring conditions, the volume of the magnification difference i.e. confocal region of the objective lens to be used it was examined whether affect detection value.

The measurement was carried out 5 times for one of the objective lens magnification. The results are shown in Table 4 and Figure 7. The Table 4 shows the detection value of the fluorescence intensity of the fifth time, in FIG. 7 shows a graph of the mean value of the detected value of the fluorescence intensity of 1-5 time.

Measurement conditions: laser wavelength: 633 nm, laser power: 41% (corresponding to about 300 mu W), measuring time: 30 seconds X 5 times, measurement mode: FIDA, scanning speed: 20MmZ seconds, the total scan area: 160 m X 160 m, using an objective lens magnification: X 10, X 20, X 40, X 60

[0045] can detect the signal strength in the high sensitivity, the volume of the confocal region 1FL or less, that the magnification of the objective lens used was confirmed to be the case for X. 20 to X 60.

[0046] [Table 4]

[0047] Confirmation of Example 5 the number of particles detected by fluorescence observation

Fluorescent beads solution of diameter 10 m prepared in Example 2, confocal laser microscopy FV 1000; using those connected with 1 molecule fluorescence analysis unit to (trade name Orinpasu Ltd.), analyzed by the following measuring conditions and it was carried out to confirm the number of fluorescent beads. The results are shown in Figure 8. Measurement conditions: laser wavelength: 633 nm, laser power: 41% (corresponding to about 300 mu W), the scanning speed: 20 mm / sec, the total scan area: 25 μ ηι Χ 25 μ m~200 ^ m X 200 μ m, using objective lens magnification: X 60

[0048] cells and fluorescent beads of the same size as 10 several to 20 or so, was able to observe a child like that moves slowly. That is, the detection target 10 several to 20 or so fluorescent beads, it is possible to perform the FIDA analysis ten minutes, the scan region at this time, 25 m X 25 μ m~200 m X 200 m it was confirmed that the scope of it is appropriate.

[0049] In this example, the force was used beads as the analysis object which is a technique that has been performed as a normal standardized method of measuring cells by flow cytometry. Then, as a measurement object even using a cell like, it is possible to obtain the same results.

Industrial Applicability

[0050] The present invention, laboratory tests are available in the fields such as sanitary inspection and biochemical studies are useful in the analysis of the raw body samples.

Claims

The scope of the claims
[1] to measure the signal strength of the small region by scanning the minute region of the confocal region, atmospheric of the particles, some, an alien in the analysis method of the fine particles of analyzing the binding or absence of the offset of the fine particles,
Volume of one minute region of the plurality of small areas for measuring the signal strength is less 1FL, the area total value of the scanning area plane of said plurality of micro region is 500 / ζ πι 2 ~40000 m 2 particles method of analysis.
[2] Analysis method of fine particles according to claim 1 scanning speed of one minute area of ​​measuring the signal strength 2. a 5mmZ sec ~ 25MmZ seconds.
[3] Fluorescence correlation spectroscopy, analysis method of fine particles according to claim 1 or 2 carries out the analysis in any one also less of the fluorescence intensity distribution analysis and fluorescence polarization intensity distribution analysis.
[4] the fine particles, cells, cell membranes, cell membrane fragments, organic particles, the inorganic particles and the claims 1-3, whichever is one wherein these at least one force becomes complex fitness least one selected from among the analysis method of the fine particles of the described.
[5] Analysis method of fine particles according to the size force 0. 3 m~10 μ m any one of claims 1 to 4, of the fine particles.
PCT/JP2007/063297 2006-07-13 2007-07-03 Method for analyzing fine particles WO2008007580A1 (en)

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