# US 11,994,468 B2 · Fluorescence intensity correcting method, fluorescence intensity calculating method, and fluorescence intensity calculating apparatus > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,994,468 B2 - Document ID: US11994468B2 - Status: Granted - Kind: B2 - Application number: 17/732,681 - Filed: 2022-04-29 - Published: 2022-08-18 - Granted: 2024-05-28 - Earliest claimed: 2010-04-28 - Assignees: Sony Corporation - Applicants: Sony Corporation - Inventors: Yasunobu Kato; Yoshitsugu Sakai - Web page: https://patentgrep.com/patent/US11994468B2 ## CPC - G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)} - G01N 2021/6421: {Measuring at two or more wavelengths} - G01N 2021/6439: {with indicators, stains, dyes, tags, labels, marks} - 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) ## Abstract A fluorescence intensity calculating apparatus, includes a measuring section configured to receive fluorescences generated from plural fluorescent dyes excited by radiating a light to a microparticle multiply-labeled with the plural fluorescent dyes having fluorescence wavelength bands overlapping one another by photodetectors which correspond to different received light wavelength bands, respectively, and whose number is larger than the number of fluorescent dyes, and obtain measured spectra by collecting detected values from the photodetectors, and a calculating section configured to approximate the measured spectra based on a linear sum of single-dyeing spectra obtained from the microparticle individually labeled with the fluorescent dyes, thereby calculating intensities of the fluorescences generated from the fluorescent dyes, respectively. ## Claims ### Claim 1 A flow cytometer system comprising: a plurality of detectors configured to receive a light from each of microparticles labeled with a plurality of fluorescent dyes, and the detectors corresponding to different received light wavelength bands; a plurality of laser light sources configured to radiate laser beams through a flow cell where the microparticles flow; and a processor circuitry configured to calculate fluorescence intensity for each of the fluorescent dyes using a linear sum of single-dyeing spectra, wherein a number of single-dyeing spectra being equal or less than a number of the detectors, and output a plot diagram of the calculated fluorescence intensity, wherein the laser light sources include at least one of a 488 nm laser or a 640 nm laser. ### Claim 2 (depends on 1) The flow cytometer system according to claim 1, further comprising: a sorting device configured to sort a microparticle having desired characteristics in the microparticles. ### Claim 3 (depends on 1) The flow cytometer system according to claim 1, wherein the processor circuitry is configured to calculate the fluorescence intensity by approximating measured spectra based on the linear sum of the single-dyeing spectra. ### Claim 4 (depends on 1) The flow cytometer system according to claim 1, wherein the approximation of the measured spectra based on the linear sum of the single-dyeing spectra is carried out by using a least-squares method. ### Claim 5 (depends on 1) The flow cytometer system according to claim 1, wherein the plot diagram represents the calculated fluorescence intensity of two different fluorescence dyes. ### Claim 6 (depends on 1) The flow cytometer system according to claim 1, wherein the processor circuitry is configured to output a plurality of dot diagrams representing the calculated fluorescence intensity of different fluorescent dyes for each respective fluorescent dye. ### Claim 7 (depends on 1) The flow cytometer system according to claim 1, wherein the microparticles are cells. ### Claim 8 (depends on 1) The flow cytometer system according to claim 1, wherein the number of the fluorescent dyes is more than five. ### Claim 9 (depends on 1) The flow cytometer system according to claim 1, wherein the detectors include first detectors configured to receive the light excited by the 488 nm laser and second detectors configured to receive the light excited by the 640 nm laser. ### Claim 10 (depends on 1) The flow cytometer system according to claim 1, wherein the detectors are configured to receive a light from each of microparticles labeled with a single fluorescent dye of the fluorescent dyes, and wherein the processor circuitry is configured to store single-dyeing spectra obtained by the detectors. ### Claim 11 (depends on 1) The flow cytometry system according to claim 1, wherein the processor circuitry is configured to read out single-dyeing spectra obtained by preparing microparticles labeled with a single fluorescent dye from measured spectra obtained by the detectors. ### Claim 12 (depends on 1) The flow cytometer system according to claim 1, wherein the plot diagram represents the calculated fluorescence intensity of two different fluorescence dyes. ### Claim 13 (depends on 1) The flow cytometer system according to claim 1, wherein the fluorescent dyes include at least one of FITC, PE, PerCP, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, AF488, PE, PI, Alexa500, Alexa514, Alexa532, PE-TR, PI, Alexa600, or PE-Cy5. ### Claim 14 (depends on 2) The flow cytometer system according to claim 2, wherein the sorting device comprises a vibrating element configured to vibrate sample liquid including the microparticles; a charging section configured to charge a droplet generated by the vibrating element with electric charge; and paired electrodes configured to change a movement direction of the charged droplet. ### Claim 15 (depends on 8) The flow cytometer system according to claim 8, wherein the number of the fluorescent dyes is more than twelve. ### Claim 16 (depends on 13) The flow cytometer system according to claim 13, wherein the fluorescent dyes include PE, PE-Cy7, PE-Cy5, and APC. ### Claim 17 (depends on 14) The flow cytometer system according to claim 14, wherein the approximation of the measured spectra based on the linear sum of the single-dyeing spectra is carried out by using a linear least-squares method. ### Claim 18 (depends on 14) The flow cytometer system according to claim 14, wherein the approximation of the measured spectra based on the linear sum of the single-dyeing spectra is carried out by using a weighted least-squares method. ### Claim 19 A flow cytometer system comprising: a plurality of detectors configured to receive a light from each of microparticles labeled with a plurality of fluorescent dyes, and the detectors corresponding to different received light wavelength bands; and a processor circuitry configured to calculate fluorescence intensity for each of the fluorescent dyes using a linear sum of single-dyeing spectra, wherein a number of single-dyeing spectra being equal or less than a number of the detectors, and output a plot diagram of the calculated fluorescence intensity, wherein the microparticles are synthetic particles. ### Claim 20 (depends on 19) The flow cytometer system according to claim 19, wherein the fluorescent dyes include PE, PE-Cy7, PE-Cy5, and APC. ### Claim 21 A flow cytometer system comprising: a plurality of detectors configured to receive a light from each of microparticles labeled with a plurality of fluorescent dyes, and the detectors corresponding to different received light wavelength bands; and a processor circuitry configured to calculate fluorescence intensity for each of the fluorescent dyes using a linear sum of single-dyeing spectra, wherein a number of single-dyeing spectra being equal or less than a number of the detectors, and output a plot diagram of the calculated fluorescence intensity, wherein the fluorescent dyes include at least one of FITC, PE, PerCP, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, AF488, PE, PI, Alexa500, Alexa514, Alexa532, PE-TR, PI, Alexa600, or PE-Cy5. ### Claim 22 (depends on 21) The flow cytometer system according to claim 21, wherein the fluorescent dyes include PE, PE-Cy7, PE-Cy5, and APC. ### Claim 23 A flow cytometer system comprising: a plurality of detectors configured to receive a light from each of microparticles labeled with a plurality of fluorescent dyes, and the detectors corresponding to different received light wavelength bands; and a processor circuitry configured to calculate fluorescence intensity for each of the fluorescent dyes using a linear sum of single-dyeing spectra, wherein a number of single-dyeing spectra being equal or less than a number of the detectors, and output a plot diagram of the calculated fluorescence intensity, wherein the processor circuitry is configured to read out single-dyeing spectra that was previously stored. ### Claim 24 (depends on 23) The flow cytometer system according to claim 23, wherein the fluorescent dyes include PE, PE-Cy7, PE-Cy5, and APC. ### Claim 25 A method comprising: receiving, by a plurality of detectors, a light from each of microparticles labeled with a plurality of fluorescent dyes, and the detectors corresponding to different received light wavelength bands; calculating, by a processor, fluorescence intensity for each of the fluorescent dyes using a linear sum of single-dyeing spectra by approximating measured spectra based on the linear sum of the single-dyeing spectra, wherein a number of single-dyeing spectra being equal or less than a number of the detectors, and the approximation of the measured spectra based on the linear sum of the single-dyeing spectra is carried out by using a least-squares method; and outputting, by the processor, a plot diagram of the calculated fluorescence intensity. ### Claim 26 (depends on 25) The method according to claim 25, further comprising: vibrating sample liquid including the microparticles; charging a droplet generated by the vibrating element with electric charge; and changing a movement direction of the charged droplet. ### Claim 27 (depends on 25) The method according to claim 25, wherein the approximation of the measured spectra based on the linear sum of the single-dyeing spectra is carried out by using a linear least-squares method. ### Claim 28 (depends on 25) The method according to claim 25, wherein the approximation of the measured spectra based on the linear sum of the single-dyeing spectra is carried out by using a weighted least-squares method. ### Claim 29 (depends on 25) The method according to claim 25, wherein the number of the fluorescent dyes is more than twelve. ### Claim 30 (depends on 25) The method according to claim 25, wherein the fluorescent dyes include PE, PE-Cy7, PE-Cy5, and APC.