# US 8,541,759 B2 · Optical analysis device, optical analysis method and computer program for optical analysis > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,541,759 B2 - Document ID: US8541759B2 - Status: Granted - Kind: B2 - Application number: 13/596,280 - Filed: 2012-08-28 - Published: 2012-12-20 - Granted: 2013-09-24 - Earliest claimed: 2010-03-01 - Assignees: Olympus Corporation - Applicants: Mitsushiro Yamaguchi; Seiji Kondo; Tetsuya Tanabe; Kunio Hori - Inventors: Mitsushiro Yamaguchi; Seiji Kondo; Tetsuya Tanabe; Kunio Hori - Web page: https://patentgrep.com/patent/US8541759B2 ## CPC - G01N 21/6452: {Individual samples arranged in a regular 2D-array, e.g. multiwell plates} - G01N 15/1456: {without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals} - G01N 21/6408: {with measurement of decay time, time resolved fluorescence} - G02B 21/0076: {arrangements using fluorescence or luminescence} - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - G02B: OPTICAL ELEMENTS, SYSTEMS OR APPARATUS ## Abstract The inventive optical analysis technique uses an optical system capable of detecting light from a micro region in a solution, such as an optical system of a confocal microscope or a multiphoton microscope, to detect the light from the light-emitting particle to be observed while moving the position of the micro region in the sample solution (while scanning the inside of the sample solution with the micro region); generates time series light intensity data, computes a characteristic value of the light intensity indicating the presence or absence of the light from a single light-emitting particle in every time section of a predetermined width in the light intensity data; and detects the light-emitting particle crossing the inside of the micro region individually using the characteristic value, thereby enabling the counting of the light-emitting particle(s) or the acquisition of the information on the concentration or number density of the light-emitting particle. ## Claims ### Claim 1 An optical analysis device which detects light from a light-emitting particle dispersed and moving at random in a sample solution by using an optical system of a confocal microscope or a multiphoton microscope, wherein the device comprises: a light detection region moving mechanism moving a position of a light detection region of the optical system in the sample solution; a light detector detecting light from the light detection region; and a signal processor generating time series light intensity data of the light from the light detection region detected with the light detector during moving the position of the light detection region in the sample solution, and detects individually a light signal from each light-emitting particle in the time series light intensity data; wherein the signal processor computes a characteristic value of light intensity indicating a presence or an absence of the light from a single light-emitting particle in every time section of a predetermined width in the time series light intensity data, and detects individually a light signal from each light-emitting particle, using the characteristic value. ### Claim 2 (depends on 1) The device of claim 1, wherein the signal processor counts the number of the light-emitting particle(s) detected during the moving of the position of the light detection region by counting the number of the time section(s) of the predetermined width having a light signal from a light-emitting particle. ### Claim 3 (depends on 1) The device of claim 1, wherein the light detector detects the light from the light detection region by photon counting, and the time series light intensity data is time series photon count data. ### Claim 4 (depends on 3) The device of claim 3, wherein the characteristic value of the light intensity is a value selected from a group of a total value of photon counts, a center value of photon counts, an average value of photon counts, a standard deviation of photon counts, a variance of photon counts, an entropy of photon counts, a maximum value of photon counts and the number of particles computed from an autocorrelation function value of photon counts with a correlation time being set to 0, in the time section of the predetermined width. ### Claim 5 (depends on 1) The device of claim 1, wherein the light detection region moving mechanism moves the position of the light detection region at a velocity faster than a diffusional moving velocity of the light-emitting particle. ### Claim 6 (depends on 1) The device of claim 1, wherein the characteristic value of the light intensity in the time section of the predetermined width in which light from a single light-emitting particle exists is larger than the characteristic value of the light intensity in the time section of the predetermined width in which no light from a single light-emitting particle exist, and it is judged that the light signal from the light-emitting particle exists in the time section of the predetermined width when the characteristic value of the light intensity is larger than a predetermined threshold value. ### Claim 7 (depends on 1) The device of claim 1, wherein the predetermined width is set to be substantially longer than a time width required for an interval from a time of one light-emitting particle's entering into the light detection region to a time of the one light-emitting particle's exiting therefrom, and also substantially shorter than a time width from a time of one light-emitting particle's entering into the light detection region to a time of a different light-emitting particle's entering into the light detection region. ### Claim 8 (depends on 1) The device of claim 1, wherein the signal processor determines a number density or a concentration of the light-emitting particle in the sample solution based on the number of the detected light-emitting particle(s). ### Claim 9 An optical analysis method of detecting light from a light-emitting particle dispersed and moving at random in a sample solution by using an optical system of a confocal microscope or a multiphoton microscope, said method comprising steps of: moving a position of a light detection region of the optical system in the sample solution; detecting light from the light detection region while moving the position of the light detection region in the sample solution and generating time series light intensity data; computing a characteristic value of light intensity indicating a presence or an absence of the light from a single light-emitting particle in every time section of a predetermined width in the time series light intensity data; and detecting individually a light signal from each light-emitting particle, using the characteristic value of light intensity in each of the time section(s) of the predetermined width. ### Claim 10 (depends on 9) The method of claim 9, further comprising a step of counting the number of the light-emitting particle(s) detected during the moving of the position of the light detection region by counting the number of the time sections of the predetermined width having a light signal from a light-emitting particle. ### Claim 11 (depends on 9) The method of claim 9, in that, in the step of detecting light from the light detection region and generating time series light intensity data, the light from the light detection region is detected by photon counting, and the time series light intensity data is time series photon count data. ### Claim 12 (depends on 11) The method of claim 11, wherein the characteristic value of the light intensity is a value selected from a group of a total value of photon counts, a center value of photon counts, an average value of photon counts, a standard deviation of photon counts, a variance of photon counts, an entropy of photon counts, a maximum value of photon counts and a the number of particles computed from an autocorrelation function value of photon counts with a correlation time being set to 0, in the time section of the predetermined width. ### Claim 13 (depends on 9) The method of claim 9, wherein, in the step of moving the position of the light detection region, the position of the light detection region is moved at a velocity faster than a diffusional moving velocity of the light-emitting particle. ### Claim 14 (depends on 9) The method of claim 9, wherein the characteristic value of the light intensity in the time section of the predetermined width in which light from a single light-emitting particle exists is larger than the characteristic value of the light intensity in the time section of the predetermined width in which no light from a single light-emitting particle exist, and it is judged that the light signal from the light-emitting particle exists in the time section of the predetermined width when the characteristic value of the light intensity is larger than a predetermined threshold value. ### Claim 15 (depends on 9) The method of claim 9, wherein the predetermined width is set to be substantially longer than a time width required for an interval from a time of one light-emitting particle's entering into the light detection region to a time of the one light-emitting particle's exiting therefrom, and also substantially shorter than a time width from a time of one light-emitting particle's entering into the light detection region to a time of a different light-emitting particle's entering into the light detection region. ### Claim 16 (depends on 9) The method of claim 9, further comprising a step of determining a number density or a concentration of the light-emitting particle in the sample solution based on the number of the detected light-emitting particle(s). ### Claim 17 A computer readable storage device having a computer program product including programmed instructions for optical analysis for detecting light from a light-emitting particle dispersed and moving at random in a sample solution by using an optical system of a confocal microscope or a multiphoton microscope, said programmed instructions causing a computer to perform steps of: moving a position of a light detection region of the optical system in the sample solution; detecting light from the light detection region while moving the position of the light detection region in the sample solution and generating time series light intensity data; computing a characteristic value of light intensity indicating a presence or an absence of the light from a single light-emitting particle in every time section of a predetermined width in the time series light intensity data; and detecting individually a light signal from each light-emitting particle, using the characteristic value of light intensity in each of the time sections of the predetermined width. ### Claim 18 (depends on 17) The computer readable storage device of claim 17, further comprising a step of counting the number of the light-emitting particle(s) detected during the moving of the position of the light detection region by counting the number of the time section(s) of the predetermined width having a light signal from a light-emitting particle. ### Claim 19 (depends on 17) The computer readable storage device of claim 17, wherein, in the step of detecting light from the light detection region and generating time series light intensity data, the light from the light detection region is detected by photon counting, and the time series light intensity data is time series photon count data. ### Claim 20 (depends on 19) The computer readable storage device of claim 19, wherein the characteristic value of the light intensity is a value selected from a group of a total value of photon counts, a center value of photon counts, an average value of the photon counts, a standard deviation of photon counts, a variance of photon counts, an entropy of photon counts, a maximum value of photon counts and the number of particles computed from an autocorrelation function value of photon counts with a correlation time being set to 0, in the time section of the predetermined width. ### Claim 21 (depends on 17) The computer readable storage device of claim 17, wherein, in the step of moving a position of a light detection region of the optical system, the position of the light detection region is moved at a velocity faster than a diffusional moving velocity of the light-emitting particle. ### Claim 22 (depends on 17) The computer readable storage device of claim 17, wherein the characteristic value of the light intensity in the time section of the predetermined width in which light from a single light-emitting particle exists is larger than the characteristic value of the light intensity in the time section of the predetermined width in which no light from a single light-emitting particle exist, and it is judged that the light signal from the light-emitting particle exists in the time section of the predetermined width when the characteristic value of the light intensity is larger than a predetermined threshold value. ### Claim 23 (depends on 17) The computer readable storage device of claim 17, wherein the predetermined width is set to be substantially longer than a time width required for an interval from a time of one light-emitting particle's entering into the light detection region to a time of the one light-emitting particle's exiting therefrom, and also substantially shorter than a time width from a time of one light-emitting particle's entering into the light detection region to a time of a different light-emitting particle's entering into the light detection region. ### Claim 24 (depends on 17) The computer readable storage device of claim 17, further comprising a step of determining a number density or a concentration of the light-emitting particle in the sample solution based on the number of the detected light-emitting particle(s).