公开(公告)号：US09134234B2

公开(公告)日：2015-09-15

申请号：US14496177

申请日：2014-09-25

Applicant: OLYMPUS CORPORATION

Inventor： Takuya Hanashi

IPC: G01N21/64 ,  G01N15/14

Abstract: There is provided a single particle detection technique based on the scanning molecule counting method which individually detects single particles using light measurement with a confocal or multiphoton microscope, where the existences of a non-light-emitting particle and a light-emitting particle can be detected while being discriminated from one another in a sample solution. The inventive technique of detecting a single particle detects light from a light detection region during moving the position of the light detection region of the microscope in a sample solution containing a non-light-emitting particle and a light-emitting particle to generate time series light intensity data; and detects in the time series light intensity data a light intensity increase relative to background light intensity as a signal indicating the existence of the light-emitting particle and a light intensity reduction relative to the background light intensity as a signal indicating the existence of the non-light-emitting particle.

公开(公告)号：US08958066B2

公开(公告)日：2015-02-17

申请号：US13789111

申请日：2013-03-07

Applicant: Olympus Corporation

Inventor： Takuya Hanashi ,  Tetsuya Tanabe ,  Mitsushiro Yamaguchi ,  Hidetaka Nakata

IPC: G01N21/00 ,  G01N21/64

CPC classification number: G01N21/6486 ,  G01N21/6428 ,  G01N21/6452 ,  G01N21/6458

Abstract: There is provided an optical analysis technique enabling identification of a kind of light-emitting particle corresponding to a signal on a time series light intensity data or identification of a signal corresponding to light-emitting particles other than a particle to be observed in an optical measurement using a confocal microscope or a multiphoton microscope. The inventive optical analysis technique measures simultaneously and separately intensities of lights of two or more wavelength bands from a light detection region in a sample solution containing light-emitting particles of two or more kinds to generate time series light intensity data of the respective wavelength bands; detects signals simultaneously generated on the time series light intensity data of at least two wavelength bands; and identifies the simultaneously generated signals as signals of a light-emitting particle of at least one specific kind.

Abstract translation: 提供了一种光学分析技术，其能够对与光学测量中要观察的颗粒以外的发光粒子相对应的时间序列光强度数据或对应于发光粒子的信号的识别相对应的一种发光粒子 使用共焦显微镜或多光子显微镜。 本发明的光学分析技术测量来自含有两种或更多种发光粒子的样品溶液中的光检测区域的两个或更多个波长带的光的同时且分开的强度，以产生各波长带的时间序列光强度数据; 检测同时产生的至少两个波长带的时间序列光强数据的信号; 并将同时生成的信号识别为至少一种特定种类的发光粒子的信号。

公开(公告)号：US20140356969A1

公开(公告)日：2014-12-04

申请号：US14465208

申请日：2014-08-21

Applicant: OLYMPUS CORPORATION

Inventor： Kazutaka Nishikawa ,  Takuya Hanashi

IPC: G01N33/53 ,  G01N21/64 ,  G01N33/52 ,  G01N21/76

CPC classification number: G01N33/5308 ,  G01N15/1434 ,  G01N21/6486 ,  G01N21/76 ,  G01N33/52 ,  G01N33/54313 ,  G01N2201/12 ,  G02B21/00 ,  G02B21/0024 ,  G02B21/0076 ,  Y10T436/143333

Abstract: Provided is a method for detecting a target particle that is a method for detecting a non-luminescent target particle dispersed and randomly moving in a sample solution using an optical system of a confocal microscope or multi-photon microscope, having: (a) preparing a sample solution containing target particles, and labeling particles of which the average outer diameter is less than 15% of the diameter of a photodetection region of the optical system, binding two or more molecules of the labeling particles per molecule of the target particles in the sample solution, and forming a non-luminescent complex of which the outer diameter is 15% or more of the diameter of the photodetection region; and, (b) calculating the number of molecules of the complex in the sample solution prepared in the (a) using an inverse scanning molecule counting method.

Abstract translation: 本发明提供一种检测作为使用共聚焦显微镜或多光子显微镜的光学系统在样品溶液中分散并随机移动的非发光靶颗粒的方法的目标颗粒的检测方法，其特征在于：（a） 含有目标颗粒的样品溶液，并且标记其平均外径小于光学系统的光电探测区域的直径的15％的颗粒，每个分子中的每个分子的靶标颗粒结合两个或更多个分子的样品 并且形成外径为光电检测区域的直径的15％以上的非发光性络合物; 和（b）使用逆扫描分子计数法计算在（a）中制备的样品溶液中的络合物的分子数。

公开(公告)号：US20140162378A1

公开(公告)日：2014-06-12

申请号：US14179174

申请日：2014-02-12

Applicant: OLYMPUS CORPORATION

Inventor： Takuya Hanashi

IPC: G01N33/543

CPC classification number: G01N33/543 ,  G01N15/1456 ,  G01N21/6408 ,  G01N21/6428 ,  G01N21/645 ,  G01N21/6452 ,  G01N21/6458 ,  G01N33/54326 ,  G01N2021/6432 ,  G01N2021/6439 ,  G01N2021/6441 ,  G02B21/0032 ,  G02B21/0048 ,  G02B21/0076 ,  G02B21/26

Abstract: This method for detecting a target particle comprises (a) preparing a solution containing a target particle, a luminescent probe that binds to the target particle and a particle for separation and recovery, or containing the target particle bound to the luminescent probe, the luminescent probe and the particle for separation and recovery, and forming a complex composed of the target particle, the luminescent probe and the particle for separation and recovery in the solution, (b) recovering the particle for separation and recovery from the solution by solid-liquid separation treatment after the (a) and preparing a sample solution containing the particle for separation and recovery, and (c) calculating the number of the complex present in the sample solution according to a scanning molecule counting method, wherein the particles for separation and recovery bind to a complex composed of the target particles and the luminescent probe.

Abstract translation: 这种检测目标颗粒的方法包括（a）制备含有目标颗粒的溶液，与目标颗粒结合的发光探针和用于分离和回收的颗粒，或包含与发光探针结合的靶颗粒，发光探针 和用于分离和回收的颗粒，并且形成由靶颗粒，发光探针和溶液中的分离和回收颗粒组成的络合物，（b）通过固液分离从溶液中回收分离和回收颗粒 （a）之后的处理，并制备含有用于分离和回收的颗粒的样品溶液，和（c）根据扫描分子计数法计算样品溶液中存在的络合物的数量，其中用于分离和回收的颗粒结合 涉及由目标颗粒和发光探针组成的复合物。

公开(公告)号：US20190271027A1

公开(公告)日：2019-09-05

申请号：US16382532

申请日：2019-04-12

Applicant: OLYMPUS CORPORATION ,  RIKEN

Inventor： Takuya Hanashi ,  Tetsuya Tanabe ,  Takeshi Hanami ,  Yoshihide Hayashizaki

IPC: C12Q1/6818 ,  C12Q1/6876 ,  G01N21/64

Abstract: A method for detecting a target nucleic acid molecule of the present invention includes a step of associating a first and third probes labeled with a first fluorescent substance which is an energy donor with a second probe labeled with a second fluorescent substance which is an energy acceptor to form an associate in a nucleic acid molecule; and a step of emitting light with an excitation wavelength of the first fluorescent substance to the associate to detect the target nucleic acid molecule using fluorescence released from the second fluorescent substance in the associate as an indicator, wherein a region associating with the second probe is between a region associating with the first probe and a region associating with the third probe in the target nucleic acid molecule.

公开(公告)号：US09435727B2

公开(公告)日：2016-09-06

申请号：US13969830

申请日：2013-08-19

Applicant: OLYMPUS CORPORATION

Inventor： Takuya Hanashi ,  Mitsushiro Yamaguchi ,  Tetsuya Tanabe

IPC: G01N15/14 ,  G01N21/64 ,  G01N21/51 ,  G02B21/00

CPC classification number: G01N15/1429 ,  G01N21/51 ,  G01N21/6408 ,  G01N21/645 ,  G01N21/6458 ,  G02B21/0076 ,  G02B21/0084

Abstract: There is provided a structure to make the setting of a criterion for eliminating noises easy in the scanning molecule counting method. In the inventive optical analysis technique of detecting light of a light-emitting particle in a sample solution, time series light intensity data of light from a light detection region detected with moving the position of the light detection region in the sample solution is generated, and a signal of a light-emitting particle individually is detected in the time series light intensity data, wherein a signal having a light intensity in a light intensity range set based upon a signal generation frequency integrated value distribution which is a distribution, obtained by using as a variable an intensity of a signal, of integrated values of generation frequencies of signals having an intensity not lower than the variable is extracted as the signal of the light-emitting particle.

Abstract translation: 提供了一种结构，用于设置扫描分子计数方法中消除噪声的标准。 在本发明的用于检测样品溶液中的发光粒子的光的光学分析技术中，产生了通过移动样品溶液中的光检测区域的位置而检测到的光检测区域的光的时间序列光强度数据， 在时间序列光强度数据中检测出发光粒子的信号，其中，根据作为分配获得的作为分布的信号生成频率积分值分布设定的光强度的光强度的信号， 提取具有不低于该变量的信号的信号的产生频率的积分值的信号强度的变量作为发光粒子的信号。

公开(公告)号：US20160252456A1

公开(公告)日：2016-09-01

申请号：US15151524

申请日：2016-05-11

Applicant: OLYMPUS CORPORATION

Inventor： Takuya Hanashi ,  Tetsuya Tanabe

IPC: G01N21/64

CPC classification number: G01N21/64 ,  G01N15/14 ,  G01N15/1429 ,  G01N15/1434 ,  G01N21/51 ,  G01N21/6408 ,  G01N21/645 ,  G01N21/6452 ,  G01N21/6458 ,  G02B21/0032 ,  G02B21/0076 ,  G02B21/008

Abstract: There is provided a single particle detection technique based on a scanning molecule counting method, enabling individual detection of a single particle using light measurement with a confocal or multiphoton microscope, and quantitative observation of conditions or characteristics of the particle. The inventive technique of detecting a single particle in a sample solution detects light containing substantially constant background light from a light detection region with moving the position of the light detection region of the microscope in a sample solution to generate time series light intensity data; and detects individually a light intensity reduction occurred when a single particle which does not emit light (or a particle whose emitting light intensity in a detected wavelength band is lower than the background light) enters in the light detection region in the time series light intensity data as a signal indicating the existence of each single particle.

Abstract translation: 提供了基于扫描分子计数方法的单粒子检测技术，能够使用共聚焦或多光子显微镜进行光测量来单个检测单个颗粒，并定量观察颗粒的条件或特性。 本发明的检测样品溶液中的单个颗粒的技术通过在样品溶液中移动显微镜的光检测区域的位置来检测来自光检测区域的基本上恒定的背景光的光，以产生时间序列光强度数据; 并且单独检测当不发光的单个颗粒（或检测波长带中的发光强度低于背景光的颗粒）在时间序列光强数据中的光检测区域中时发生的光强度降低 作为指示每个单个颗粒的存在的信号。

公开(公告)号：US20130242307A1

公开(公告)日：2013-09-19

申请号：US13888868

申请日：2013-05-07

Applicant: OLYMPUS CORPORATION

Inventor： Takuya Hanashi ,  Tetsuya Tanabe ,  Mitsushiro Yamaguchi

IPC: G01J1/16

CPC classification number: G01J1/16 ,  G01C21/02 ,  G01J1/58 ,  G01N15/1463 ,  G01N21/6452 ,  G01N21/6458 ,  G01N2021/6421 ,  G02B21/0032 ,  G02B21/0076 ,  G02B21/26

Abstract: The inventive technique of detecting and analyzing light from a light-emitting particle in accordance with the scanning molecule counting method using an optical measurement with a confocal microscope or a multiphoton microscope is characterized by detecting intensities of components of two or more wavelength bands of light from a light detection region of an optical system with moving the position of the light detection region in a sample solution by changing the optical path of the optical system of the microscope; detecting individually signals of the light from each light-emitting particle in the intensities of the components of the two or more wavelength bands of the detected light; and identifying a kind of light-emitting particle based on the intensities of the components of the two or more wavelength bands of the signals of the light of the detected light-emitting particle.

Abstract translation: 根据使用共聚焦显微镜或多光子显微镜的光学测量的扫描分子计数法检测和分析来自发光粒子的光的本发明的技术的特征在于检测两个或更多个波长带的光的分量的强度， 光学系统的光检测区域，通过改变显微镜的光学系统的光路来移动样品溶液中的光检测区域的位置; 在检测到的光的两个或更多个波长带的分量的强度中单独地检测来自每个发光粒子的光的信号; 并且基于检测到的发光粒子的光的信号的两个或更多波长带的分量的强度来识别发光粒子的种类。

公开(公告)号：US20180259457A1

公开(公告)日：2018-09-13

申请号：US15983412

申请日：2018-05-18

Applicant: OLYMPUS CORPORATION

Inventor： Tetsuya Tanabe ,  Takuya Hanashi ,  Hidetaka Nakata

IPC: G01N21/64 ,  C12Q1/6816

CPC classification number: G01N21/6428 ,  C12Q1/6816 ,  G01N21/64 ,  G01N21/6408 ,  G01N21/6458 ,  G01N21/78 ,  G01N2021/6439 ,  C12Q2565/101 ,  C12Q2565/601

Abstract: In the scanning molecule counting method of measuring light intensity from a light detection region while moving the position of the light detection region of a confocal or multiphoton microscope in a sample solution containing light-emitting particles, generating time series light intensity data and detecting each of signals of the light-emitting particles individually in the data, wherein the light-emitting particles are formed by binding to a particle to be observed a light-emitting probe which emits light through binding to the particle to be observed and in which a stochastic transition between a non-light-emitting state and a light-emitting state occurs in the unbound state, the moving speed of the position of the light detection region is adjusted to make the time during which the unbound probe is encompassed by the moving light detection region longer than an average lifetime during which the probe is in the light-emitting state.

公开(公告)号：US09488578B2

公开(公告)日：2016-11-08

申请号：US14162142

申请日：2014-01-23

Applicant: OLYMPUS CORPORATION

Inventor： Takuya Hanashi ,  Tetsuya Tanabe

IPC: G01N21/64 ,  G01N15/14 ,  G01N21/51 ,  G02B21/00

CPC classification number: G01N21/64 ,  G01N15/14 ,  G01N15/1429 ,  G01N15/1434 ,  G01N21/51 ,  G01N21/6408 ,  G01N21/645 ,  G01N21/6452 ,  G01N21/6458 ,  G02B21/0032 ,  G02B21/0076 ,  G02B21/008

Abstract: There is provided a single particle detection technique based on a scanning molecule counting method, enabling individual detection of a single particle using light measurement with a confocal or multiphoton microscope, and quantitative observation of conditions or characteristics of the particle. The inventive technique of detecting a single particle in a sample solution detects light containing substantially constant background light from a light detection region with moving the position of the light detection region of the microscope in a sample solution to generate time series light intensity data; and detects individually a light intensity reduction occurred when a single particle which does not emit light (or a particle whose emitting light intensity in a detected wavelength band is lower than the background light) enters in the light detection region in the time series light intensity data as a signal indicating the existence of each single particle.

Abstract translation: 提供了基于扫描分子计数方法的单粒子检测技术，能够使用共聚焦或多光子显微镜进行光测量来单个检测单个颗粒，并定量观察颗粒的条件或特性。 本发明的检测样品溶液中的单个颗粒的技术通过在样品溶液中移动显微镜的光检测区域的位置来检测来自光检测区域的基本上恒定的背景光的光，以产生时间序列光强度数据; 并且单独检测当不发光的单个颗粒（或检测波长带中的发光强度低于背景光的颗粒）在时间序列光强数据中的光检测区域中时发生的光强度降低 作为指示每个单个颗粒的存在的信号。