US20120123722A1 - Fluorescent spectrum correcting method and fluorescent spectrum measuring device - Google Patents

Fluorescent spectrum correcting method and fluorescent spectrum measuring device Download PDF

Info

Publication number
US20120123722A1
US20120123722A1 US13/287,459 US201113287459A US2012123722A1 US 20120123722 A1 US20120123722 A1 US 20120123722A1 US 201113287459 A US201113287459 A US 201113287459A US 2012123722 A1 US2012123722 A1 US 2012123722A1
Authority
US
United States
Prior art keywords
fluorescent
spectrum
fluorescent spectrum
micro
particles
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US13/287,459
Other languages
English (en)
Inventor
Masaya Kakuta
Koji Futamura
Yoshitsugu Sakai
Yasunobu Kato
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sony Corp
Original Assignee
Sony Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sony Corp filed Critical Sony Corp
Assigned to SONY CORPORATION reassignment SONY CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KATO, YASUNOBU, SAKAI, YOSHITSUGU, FUTAMURA, KOJI, KAKUTA, MASAYA
Publication of US20120123722A1 publication Critical patent/US20120123722A1/en
Priority to US16/100,884 priority Critical patent/US10908075B2/en
Priority to US17/142,632 priority patent/US11454588B2/en
Priority to US17/897,916 priority patent/US11726031B2/en
Priority to US18/349,234 priority patent/US20230349819A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
    • G01N21/274—Calibration, base line adjustment, drift correction
    • 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
    • G01N15/1429—Signal processing
    • 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
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • 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
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells

Definitions

  • the present disclosure relates to a fluorescent spectrum correcting method and a fluorescent spectrum measuring device. More particularly, the present disclosure relates to a technique for separating the fluorescent spectrum obtained from micro-particles labeled with a plurality of fluorescent pigments, for each pigment.
  • flow cytometry flow cytometer
  • the flow cytometry is a method of irradiating micro-particles flowing in a flow path in one row with laser light (excitement light) of a specific wavelength and detecting fluorescent light or diffused light emitted by the micro-particles to analyze the plurality of micro-particles one by one.
  • the light detected by an optical detector is converted into an electrical signal to be a value and statistical analysis is performed to determine types, sizes, structures, and the like of individual micro-particles.
  • a spectral deconvolution method of previously registering the light emission spectrum of used fluorescent labels in advance in a computer, separating the light emission spectrum of a measurement target into the light emission spectrum of the fluorescent label using the data, and determining an existence ratio of the fluorescent labels is proposed (see Japanese Unexamined Patent Application Publication No. 2005-181276).
  • spectrum absorption light measurement such as an infrared spectrum method
  • correction or analysis of the measured spectrum is performed on the basis of a standard spectrum or reference spectrum (e.g., see Japanese Unexamined Patent Application Publication Nos. 2005-195586 and 2009-162667).
  • the stray fluorescent light detected by detectors other than the desired detector is a big problem, as well as it being necessary to prepare high-sensitivity detectors corresponding to the number of desired fluorescent pigments.
  • the fluorescent correction is not performed due to stray fluorescent light.
  • each of the fluorescent pigments has a special spectrum, and the spectrum information represents the characteristics of the fluorescent pigment itself to be important data.
  • the spectrum information represents the characteristics of the fluorescent pigment itself to be important data.
  • data of a single stain sample is necessary.
  • the number of operations of fluorescent correction is in proportion to substantially the square of the number of fluorescent pigments used, and it is troublesome to the observer.
  • the volume of a test target object such as collectible blood is finite, and thus there is a case where it is difficult to produce the single stain sample for each fluorescent pigment.
  • a fluorescent spectrum correcting method including: comparing the fluorescent spectrum obtained from micro-particles labeled with a plurality of fluorescent pigments with a reference spectrum to separate the fluorescent spectrum into a fluorescent spectrum for each pigment, wherein previously measured spectrum data is used as the reference spectrum.
  • spectrum data in which an error from a single stain sample is equal to or less than 8% may be used as the reference spectrum.
  • the measurement date, the potential of a detector, the type of coupled antibody, and any spectrum data of a different type of cell when the micro-particles are cells are used as the reference spectrum.
  • fluorescent spectrum data measured using cells may be used as the reference spectrum.
  • a fluorescent spectrum measuring device including: a detection unit that simultaneously detects fluorescent light emitted from micro-particles in an arbitrary wavelength region; an analysis unit that separates the data detected by the detection unit into a fluorescent spectrum for each pigment; and a memory unit that stores the fluorescent spectrum data separated by the analysis unit, wherein the analysis unit uses the previously measured fluorescent spectrum data stored in the memory unit as the reference spectrum to perform separation of a fluorescent spectrum.
  • the detection unit may be provided with a multi-channel photo-multiplier tube.
  • the single stain sample is not necessary, the overlap between each spectrum can be dissolved with high precision, and further the single stain sample is not necessary.
  • FIG. 1 is a block diagram illustrating a configuration of a fluorescent spectrum measuring device according to a first embodiment of the present disclosure.
  • FIG. 2A is a graph illustrating a relationship between a measurement date and fluorescent spectrum in which a horizontal axis is a channel number of a detector and a vertical axis is a fluorescent intensity
  • FIG. 2B is a graph illustrating an error in each wavelength (FITC:CD 14 ).
  • FIG. 3A is a graph illustrating a relationship between a measurement date and a fluorescent spectrum in which the horizontal axis is a channel number of a detector and the vertical axis is a fluorescent intensity
  • FIG. 3B is a graph illustrating an error in each wavelength (PE:CD 3 ).
  • FIG. 4A is a graph illustrating a relationship between a measurement date and a fluorescent spectrum in which the horizontal axis is a channel number of a detector and the vertical axis is a fluorescent intensity
  • FIG. 4B is a graph illustrating an error in each wavelength (spectrum corresponding to FITC of BD 7-Color Setup Beads).
  • FIG. 5A is a graph illustrating a relationship between a measurement date and a fluorescent spectrum in which the horizontal axis is a channel number of a detector and the vertical axis is a fluorescent intensity
  • FIG. 5B is a graph illustrating an error in each wavelength (spectrum corresponding to PE of BD 7-Color Setup Beads).
  • FIG. 6A is a graph illustrating a relationship between potential of a detector and a fluorescent spectrum in which the horizontal axis is a channel number (wavelength dependent number) of a detector and the vertical axis is a fluorescent intensity
  • FIG. 6B is a graph illustrating an error in each wavelength.
  • FIG. 7A is a graph illustrating a relationship between a coupled antibody and a fluorescent spectrum in which the horizontal axis is a channel number (wavelength dependent number) of a detector and the vertical axis is a fluorescent intensity
  • FIG. 7B is a graph illustrating an error in each wavelength (FITC:CD 45 vs FITC:CD 45 RA).
  • FIG. 8A is a graph illustrating a relationship between a coupled antibody and a fluorescent spectrum in which the horizontal axis is a channel number (wavelength dependent number) of a detector and the vertical axis is a fluorescent intensity
  • FIG. 8B is a graph illustrating an error in each wavelength (PE:CD 8 vs PE:CD 3 ).
  • FIG. 9 is a density plot of blood cells for managing precision in which the horizontal axis is data of an antibody CD 45 of a fluorescent pigment FITC and the vertical axis is data of an antibody CD 8 of a fluorescent pigment PE.
  • FIG. 10 is an analysis result in which the horizontal axis is data of an antibody CD 45 RA of a fluorescent pigment FITC and the vertical axis is data of an antibody CD 3 of a fluorescent pigment PE.
  • FIG. 11A is a graph illustrating a relationship between a type of micro-particle and a fluorescent spectrum in which the horizontal axis is a channel number (wavelength dependent number) of a detector and the vertical axis is a fluorescent intensity
  • FIG. 11B is a graph illustrating an error in each wavelength.
  • FIG. 12A is a graph illustrating a relationship between a type of micro-particle and a fluorescent spectrum in which the horizontal axis is a channel number (wavelength dependent number) of a detector and the vertical axis is a fluorescent intensity
  • FIG. 12B is a graph illustrating an error in each wavelength.
  • FIG. 13 is an analysis result in which the horizontal axis is data of polystyrene beads containing a fluorescent pigment FITC of BD 7-Color Setup Beads and the vertical axis is data of polystyrene beads containing a fluorescent pigment PE of BD 7-Color Setup Beads.
  • a fluorescent spectrum correcting method (hereinafter, merely referred to as a correction method) according to a first embodiment of the present disclosure will be described.
  • previously measured fluorescent spectrum is used as reference spectrum when fluorescent spectrum obtained from micro-particles labeled with a plurality of fluorescent pigments is separated for each pigment.
  • the “micro-particles” widely include bionic micro-particles such as cells, microorganisms, and liposomes, or synthetic particles such as latex particles, gel particles, and industrial particles.
  • the bionic micro-particles include chromosomes constituting various cells, liposomes, mitochondria, organelles (cell organelles), and the like.
  • the cells include vegetable cells, animal cells, blood corpuscle cells, and the like.
  • the microorganisms include bacilli such as colon bacilli, viruses such as tobacco mosaic viruses, germs such as yeast, and the like.
  • the bionic micro-particles may include bionic polymers such as hexane, protein, and complexes thereof.
  • the industrial particles may be formed of, for example, organic polymer materials, inorganic materials, or metal materials.
  • Polystyrene, styrene divinyl benzene, polymethyl methacrylate, and the like may be used as the organic polymer materials.
  • Glass, silica, magnetic materials, and the like may be used as the inorganic materials.
  • gold colloid, aluminum, and the like may be used as the metal materials.
  • the shape of the micro-particles is generally spherical, but may be non-spherical, and the size, mass, and the like are not particularly limited.
  • an error from spectrum of a single stain sample of measurement target micro-particles is preferably 8% or less, and more preferably 3% or less. Accordingly, a matching error from the measurement data is small, and it is possible to perform fluorescent correction with high precision.
  • the reference spectrum of each pigment for example, the measurement date, the potential of the detector, the output of laser, the flux of micro-particles, the type of coupled antibodies, or data (fluorescent spectrum) of a different type of cells when the micro-particles are cells may be used. Since such conditions do not have a great influence on the fluorescent spectrum, it is possible to dissolve the overlap with high precision even when such spectrum data is used in the reference spectrum to perform correction.
  • the results obtained from the measurement using beads are not used as the reference spectrum, for example, even when they are labeled with the same fluorescent pigment, and the opposite case is the same.
  • the reference spectrum even when there is a difference in type between cells, they may be used as the reference spectrum.
  • the reference spectrum even when there is a difference in type between beads, they may be used as the reference spectrum.
  • the spectrum data in which the error from the single stain sample of the previously measured measurement target micro-particles is 8% or less is used as the reference spectrum, it is not necessary to prepare the single stain sample at the stage of measurement. Accordingly, the burden on the worker is reduced, and thus work efficiency is also improved. Even when the amount of a test target object is small like a small animal such as a rat, it is possible to perform analysis without decreasing accuracy.
  • the fluorescent spectrum correcting method of the embodiment is applicable irrespective of processes before and after it when the method is a method having a process of separating the fluorescent spectrum obtained from the micro-particles labeled with the plurality of fluorescent pigments for each pigment using the reference spectrum.
  • FIG. 1 is a block diagram illustrating a configuration of the fluorescent spectrum measuring device of the embodiment.
  • the fluorescent spectrum measuring device 1 of the embodiment includes at least a detection unit 2 , a memory unit 3 , and an analysis unit 4 , and performs the correction method of the first embodiment.
  • the fluorescent spectrum measuring device 1 shown in FIG. 1 may further include a liquid transmitting unit.
  • the detection unit 2 may have a configuration in which fluorescent light emitted from the analysis target micro-particles can be simultaneously detected in an arbitrary wavelength region.
  • a plurality of independent sensors capable of detecting the wavelength region for each wavelength region are disposed, or one or more detectors capable of simultaneously detecting a plurality of light such as a multi-channel photo-multiplier tube (PMT) may be provided.
  • the number of wavelength regions detected by the detector 2 that is, the number of channels or sensors provided in the detector 2 is preferably equal to or more than the number of used pigments.
  • the fluorescent spectrum measuring device 1 of the embodiment may have a configuration in which the detector 2 is provided with a spectroscope, and the fluorescent light emitted from the micro-particles is dispersed by the spectroscope and then enters a detector such as the multi-channel PMT.
  • the detection unit 2 may be provided with an object lens, a condensing lens, a pinhole, a band cutoff filter, a dichroic mirror, and the like, as necessary.
  • the light of each wavelength region detected by the detection unit 2 is quantified to acquire total fluorescent light quantity (intensity) using an electronic calculator or the like. Fluorescent spectrum correction using the reference spectrum is performed as necessary. The result (fluorescent spectrum data) is stored in the memory unit 4 .
  • the memory unit 4 stores the fluorescent spectrum data processed by the analysis unit 3 .
  • the fluorescent spectrum data of the single stain sample may be stored in the memory unit 4 , as well as the previously measured fluorescent spectrum data.
  • the micro-particles analyzed by the fluorescent spectrum measuring device 1 of the embodiment are not particularly limited, but may be, for example, cells or micro-beads.
  • the type or number of fluorescent pigments modifying the micro-particles is not particularly limited, but existing pigments such as FITC (fluorescein isothiocynate: C 21 H 11 NO 5 S), PE (phycoerythrin), PerCP (peridinin chlorophyll protein), and PE-Cy5, and PE-Cy7 may be appropriately selected and used as necessary.
  • the micro-particles may be modified by the plurality of fluorescent pigments.
  • the micro-particles are optically analyzed using the fluorescent spectrum measuring device 1 of the embodiment, first, excitement light is output from a light source and the micro-particles flowing in a flow path are irradiated with the excitement light. Then, the fluorescent light output from the micro-particles is detected by the detection unit 2 . Specifically, only light (desired fluorescent light) of a specific wavelength is separated from the light output from the micro-particles using a dichroic mirror, a band pass filter, or the like, and the light is detected by a detector such as a 32-channel PMT. In this case, the fluorescent light is dispersed using, for example, a spectroscope, and light of different wavelengths is detected in each channel of the detector. Accordingly, it is possible to obtain the spectrum information of the detection light (fluorescent light).
  • the information of several detectors acquired in the detection unit 2 are converted into digital signals in, for example, a conversion unit (not shown), and is further quantified in the analysis unit 3 .
  • the fluorescent correction is performed using the previously measured fluorescent spectrum data stored in the memory unit 4 as the reference spectrum.
  • fluorescent spectrum data in which an error from the spectrum of the single stain sample of the micro-particles is 8% or less is used, for example, measurement date, potential of the detector, type of coupled antibody, or different type of cells when the micro-particles are cells.
  • the fluorescent spectrum data after correction is stored in the memory unit 4 .
  • the fluorescent spectrum measuring device of the present disclosure since the spectrum data in which the error from the spectrum of the single stain sample of the measurement target micro-particles is 8% or less is used as the reference spectrum, it is possible to perform the correction with high precision even when the single stain sample is not used.
  • the fluorescent spectrum data that is the reference spectrum is sequentially accumulated in the memory unit 4 , and thus it is possible to construct a database suitable for a real use situation.
  • the measurement data, the potential of the detector, the type of coupled antibody, and the type of micro-particles were changed, the fluorescent spectrum was compared, and the difference thereof was examined.
  • an Immuno-TROL made by Beckman Coulter, Co., Ltd.
  • a Multi-Check made by Becton Dickinson, Co., Ltd.
  • They are positive process controls for flow cytometry (whole blood control examination target object), and represent diffused light, distribution of cell groups, fluorescent intensity, and antigen density since a positive rate of a particular surface antigen and an absolute number are calibrated in a monocyte.
  • a product available on the market (made by made by Beckman Coulter, Co., Ltd. or Becton Dickinson, Co., Ltd.) was used as an antibody labeled with a fluorescent pigment.
  • Dyeing of the sample was performed according to a titration method. Specifically, the temperature of the sample was kept at room temperature, then the antibody labeled with the desired fluorescent pigment was dropped into a dedicated plastic tube, blood of 50 ⁇ L was dropped therein to be smoothly infiltrated, and the antibody and the cell were made to react. It was left for 20 minutes at a dark place at room temperature. Then, a hemolytic agent (FACS Lyse solution: ammonium chloride solution, Beckman Coulter, Co., Ltd.) of 1 ml was dropped into it. Accordingly, red blood corpuscles were hemolyzed, granulocyte, monocyte, and lymphocyte remain. It was centrifuged and washed by an appropriate solution, and thus a high purity sample solution was obtained.
  • FACS Lyse solution ammonium chloride solution, Beckman Coulter, Co., Ltd.
  • the cell solution (sample solution) adjusted by the method described above was introduced into a special measurement cell for cell analysis formed of plastic, 3-dimensional focus was performed by a sheath solution for flow cytometer, and then it was irradiated with the excitement light.
  • Laser beams with wavelengths of 488 nm and 640 nm were used as an excitement source.
  • the fluorescent light emitted from each cell was dispersed by a prism spectroscope or the like, and then was detected by the 32ch PMT.
  • the 32ch PMT was used as the detector, but two laser beams were used as the excitement light. Accordingly, the spectrum data of 64 channels as the amount of information were transmitted to the analysis unit and the memory unit.
  • FIG. 2A , FIG. 3A , FIG. 4A , and FIG. 5A are graphs in which the horizontal axis is a channel number (wavelength dependent number) of the detector and the vertical axis is fluorescent intensity
  • FIG. 2B , FIG. 3B , FIG. 4B , and FIG. 5B are graphs illustrating an error in each wavelength.
  • the florescent spectrum shown in FIG. 2A and FIG. 2B is data measured using FITC as the florescent pigment and CD 14 as the antibody
  • the florescent spectrum shown in FIG. 3A and FIG. 3B is data measured using PE as the florescent pigment and CD 3 as the antibody. The same lot was used at any date.
  • FIG. 4A and FIG. 4B are fluorescent spectrum of polystyrene beads containing the florescent pigment FITC of BD 7-Color Setup Beads.
  • FIG. 5A and FIG. 5B are fluorescent spectrum of polystyrene beads containing the fluorescent pigment PE of BD 7-Color Setup Beads.
  • the PMT was used as all the detectors, and application voltage was 630 V.
  • FIG. 6A is a graph illustrating a relationship between the potential of the detector and the florescent spectrum in which the horizontal axis is the channel number (wavelength dependent number) of the detector and the vertical axis is the fluorescent intensity
  • FIG. 6B is a graph illustrating an error in each wavelength.
  • the fluorescent spectrum shown in FIG. 6A and FIG. 6B is data measured using the PE as the fluorescent pigment, the CD 3 as the antibody, and the PMT as the detector.
  • PMTV150 is 525V
  • PMTV160 is 560V
  • PMTV170 is 595V
  • PMTV180 is 630V
  • PMTV190 665V
  • PMTV200 is 700V.
  • the error of the spectrum was 3% or less even when the potential of the detector was changed. Accordingly, it was confirmed that the fluorescent spectrum data with the different potential of the detection was usable as the reference spectrum.
  • FIG. 7A and FIG. 8A are graphs illustrating a relationship between the coupled antibody and the fluorescent spectrum in which the horizontal axis is the channel number (wavelength dependent number) of the detector and the vertical axis is the fluorescent intensity
  • FIG. 7B and FIG. 8B are graphs illustrating an error in each wavelength.
  • the fluorescent spectrum shown in FIG. 7A and FIG. 7B is data measured using A: the FITC as the fluorescent pigment and the CD 45 as the antibody and B: the FITC as the fluorescent pigment and the CD 45 RA as the antibody.
  • the fluorescent spectrum shown in FIG. 8A and FIG. 8B is data measured using A: the PE as the fluorescent pigment and the CD 8 as the antibody and B: the FE as the fluorescent pigment and the CD 3 as the antibody.
  • the PMT was used as the detector, and all the application voltages were 525 V.
  • FIG. 9 is a density plot illustrating a result thereof. As shown in FIG. 9 , the analysis was performed using the reference spectrum generated by the single stain, and it could be divided into three cell groups. Each group indicates that the fluorescent correction is satisfactorily performed at an orthogonal position. The number of existence in a region throughout a gate was FITC+PE+:278 and PICT+PE ⁇ :750, and a ratio thereof was 0.37:1.
  • FIG. 10 is a density plot illustrating a result thereof. As shown in FIG. 10 , in the 2-dimensionally developed plot based on the FITC and PE as the fluorescent pigment, three cell groups are clearly classified, and each of them was positioned at the orthogonal position. Comparing distribution throughout the gate, the number of was FITV+PE+:280 and PITC+PE ⁇ :750, and the ratio thereof was 0.37:1 and was equal to the existence ratio of the data shown in FIG. 9 .
  • FIG. 11A and FIG. 12A are graphs illustrating a relationship between the type of the micro-particles and the fluorescent spectrum in which the horizontal axis is the channel number (wavelength dependent number) and the vertical axis is the fluorescent intensity
  • FIG. 11B and FIG. 12B are graphs illustrating an error in each wavelength.
  • FIG. 11A and FIG. 11B are A: fluorescent spectrum when the FITC was used as the pigment and the CD 45 was used as the antibody
  • B fluorescent spectrum when polystyrene beads containing the fluorescent pigment FITC of BD 7-Color Setup Beads were used.
  • A fluorescent spectrum when the PE was used as the pigment and the CD 8 was used as the antibody
  • B fluorescent spectrum when polystyrene beads containing the fluorescent pigment PE of BD 7-Color Setup Beads were used. All the application voltages were 630 V.
  • FIG. 13 is a density plot illustrating the result thereof. As shown in FIG. 13 , the cell groups are classified into three, and each of them was positioned at the orthogonal position. Comparing distribution throughout the gate, the number of was FITV+PE+:272 and PITC+PE ⁇ :213, and the ratio thereof was 1.28:1 and was not equal to the existence ratio of the data shown in FIG. 9 and FIG. 10 .

Landscapes

  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Signal Processing (AREA)
  • Dispersion Chemistry (AREA)
  • Optics & Photonics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Mathematical Physics (AREA)
  • Theoretical Computer Science (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
US13/287,459 2010-11-11 2011-11-02 Fluorescent spectrum correcting method and fluorescent spectrum measuring device Abandoned US20120123722A1 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US16/100,884 US10908075B2 (en) 2010-11-11 2018-08-10 Fluorescent spectrum correcting method and fluorescent spectrum measuring device
US17/142,632 US11454588B2 (en) 2010-11-11 2021-01-06 Fluorescent spectrum correcting method and fluorescent spectrum measuring device
US17/897,916 US11726031B2 (en) 2010-11-11 2022-08-29 Fluorescent spectrum correcting method and fluorescent spectrum measuring device
US18/349,234 US20230349819A1 (en) 2010-11-11 2023-07-10 Fluorescent spectrum correcting method and fluorescent spectrum measuring device

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2010-252863 2010-11-11
JP2010252863A JP5937780B2 (ja) 2010-11-11 2010-11-11 蛍光スペクトル補正方法及び蛍光スペクトル測定装置

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US16/100,884 Continuation US10908075B2 (en) 2010-11-11 2018-08-10 Fluorescent spectrum correcting method and fluorescent spectrum measuring device

Publications (1)

Publication Number Publication Date
US20120123722A1 true US20120123722A1 (en) 2012-05-17

Family

ID=46048572

Family Applications (5)

Application Number Title Priority Date Filing Date
US13/287,459 Abandoned US20120123722A1 (en) 2010-11-11 2011-11-02 Fluorescent spectrum correcting method and fluorescent spectrum measuring device
US16/100,884 Active US10908075B2 (en) 2010-11-11 2018-08-10 Fluorescent spectrum correcting method and fluorescent spectrum measuring device
US17/142,632 Active US11454588B2 (en) 2010-11-11 2021-01-06 Fluorescent spectrum correcting method and fluorescent spectrum measuring device
US17/897,916 Active US11726031B2 (en) 2010-11-11 2022-08-29 Fluorescent spectrum correcting method and fluorescent spectrum measuring device
US18/349,234 Pending US20230349819A1 (en) 2010-11-11 2023-07-10 Fluorescent spectrum correcting method and fluorescent spectrum measuring device

Family Applications After (4)

Application Number Title Priority Date Filing Date
US16/100,884 Active US10908075B2 (en) 2010-11-11 2018-08-10 Fluorescent spectrum correcting method and fluorescent spectrum measuring device
US17/142,632 Active US11454588B2 (en) 2010-11-11 2021-01-06 Fluorescent spectrum correcting method and fluorescent spectrum measuring device
US17/897,916 Active US11726031B2 (en) 2010-11-11 2022-08-29 Fluorescent spectrum correcting method and fluorescent spectrum measuring device
US18/349,234 Pending US20230349819A1 (en) 2010-11-11 2023-07-10 Fluorescent spectrum correcting method and fluorescent spectrum measuring device

Country Status (3)

Country Link
US (5) US20120123722A1 (ja)
JP (1) JP5937780B2 (ja)
CN (1) CN102539397B (ja)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140158913A1 (en) * 2012-12-06 2014-06-12 Sony Corporation Microparticle analysis apparatus and microparticle analysis method
US20150160133A1 (en) * 2013-12-06 2015-06-11 Azbil Corporation Particle detecting device and particle detecting method
US20170045436A1 (en) * 2015-08-12 2017-02-16 Bio-Rad Laboratories, Inc. Multi-spectral filter profiling and quality control for flow cytometry

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103063628A (zh) * 2012-12-13 2013-04-24 公安部第一研究所 一种多荧光染料的激发方法
JP7022670B2 (ja) * 2018-09-10 2022-02-18 株式会社日立ハイテク スペクトル校正装置及びスペクトル校正方法
JP7528087B2 (ja) 2018-12-28 2024-08-05 ベクトン・ディキンソン・アンド・カンパニー サンプルの蛍光物質をスペクトル的に分解する方法及びシステム
CN110081975B (zh) * 2019-04-04 2021-06-04 深圳和而泰数据资源与云技术有限公司 光谱仪校准治具及系统
CN110208251B (zh) * 2019-06-20 2022-01-21 安徽创谱仪器科技有限公司 等离子体发射光谱干扰校正方法
WO2021153192A1 (ja) * 2020-01-31 2021-08-05 ソニーグループ株式会社 情報処理装置、粒子分析装置、情報処理方法、及びプログラム
WO2021159479A1 (zh) * 2020-02-14 2021-08-19 深圳华大智造科技股份有限公司 基于图像分析液滴的方法、计算机装置及存储介质

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6201241B1 (en) * 1996-09-17 2001-03-13 Kabushiki Kaisha Topcon Organic substance analyzer
US20020125136A1 (en) * 1998-09-16 2002-09-12 Sharaf Muhammad A. Spectral calibration of fluorescent polynucleotide separation apparatus
US20050046835A1 (en) * 2003-08-27 2005-03-03 Leica Microsystems Heidelberg Gmbh Method for setting a fluorescence spectrum measurement system for microscopy
US20110153226A1 (en) * 2009-12-18 2011-06-23 General Electric Company Spectral searching method for substance identification
US8649008B2 (en) * 2010-02-04 2014-02-11 University Of Southern California Combined spectral and polarimetry imaging and diagnostics

Family Cites Families (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6649414B1 (en) * 1999-08-17 2003-11-18 Luminex Corporation Microparticles with multiple fluorescent signals and methods of using same
AU2002246811A1 (en) * 2000-12-29 2002-08-06 Chromagen, Inc. Scanning spectrophotometer for high throughput fluorescence detection
US6539323B2 (en) * 2001-05-04 2003-03-25 Electronics For Imaging, Inc. Methods and apparatus for correcting spectral color measurements
US7122384B2 (en) * 2002-11-06 2006-10-17 E. I. Du Pont De Nemours And Company Resonant light scattering microparticle methods
WO2005036143A1 (ja) * 2003-10-10 2005-04-21 Hamamatsu Photonics K.K. 蛍光色素の濃度を定量する方法およびシステム
JP4021414B2 (ja) 2003-11-26 2007-12-12 オリンパス株式会社 スペクトラルデコンボリューション法及びスペクトラルブラインドデコンボリューション法
JP4313166B2 (ja) 2003-12-02 2009-08-12 セントラル硝子株式会社 光学活性β−トリフルオロメチル−β−ヒドロキシカルボニル化合物の製造方法
JP2005181726A (ja) 2003-12-19 2005-07-07 Seiko Precision Inc プロジェクタ装置および投影画像調整方法
EP1550855A2 (en) 2003-12-30 2005-07-06 Rohm And Haas Company Method for detecting contaminants
US7625712B2 (en) 2004-05-21 2009-12-01 Beckman Coulter, Inc. Method for a fully automated monoclonal antibody-based extended differential
EP2369326B1 (en) * 2005-02-15 2013-09-04 Mitsui Engineering & Shipbuilding Co., Ltd. Fluorescence detecting device and fluorescence detecting method
JP4649231B2 (ja) 2005-02-28 2011-03-09 株式会社カネカ フローサイトメータ、細胞の解析方法、細胞解析プログラム、蛍光検出器の感度設定方法および陽性率判定法における基準ゲート設定方法
JP4537231B2 (ja) * 2005-03-07 2010-09-01 独立行政法人理化学研究所 多重蛍光からの蛍光色素濃度の推定方法および多重蛍光からの蛍光強度の推定方法
WO2007016517A1 (en) * 2005-08-02 2007-02-08 Luminex Corporation Methods, data structures, and systems for classifying microparticles
US20070098594A1 (en) * 2005-11-03 2007-05-03 Roche Molecular Systems, Inc. Analytical multi-spectral optical detection system
US8360574B2 (en) * 2006-03-20 2013-01-29 High Performance Optics, Inc. High performance selective light wavelength filtering providing improved contrast sensitivity
US8244021B2 (en) * 2006-12-20 2012-08-14 Ventana Medical Systems, Inc. Quantitative, multispectral image analysis of tissue specimens stained with quantum dots
JP4509163B2 (ja) * 2007-10-26 2010-07-21 ソニー株式会社 微小粒子の測定方法
JP4389991B2 (ja) * 2007-10-26 2009-12-24 ソニー株式会社 微小粒子の光学的測定方法及び光学的測定装置
CN104122191B (zh) * 2007-10-29 2020-02-18 希森美康株式会社 细胞分析仪及细胞分析方法
JP2009115672A (ja) 2007-11-08 2009-05-28 Sony Corp 微小粒子の光学的測定方法及び分取方法、並びに前記光学的測定方法及び分取方法に用いる流路、光学的測定装置及びフローサイトメータ
JP5345785B2 (ja) 2008-01-08 2013-11-20 Ｓｕｍｃｏ Ｔｅｃｈｘｉｖ株式会社 分光吸収測定方法及び分光吸収測定装置
JP5148387B2 (ja) * 2008-06-30 2013-02-20 浜松ホトニクス株式会社 分光測定装置、分光測定方法、及び分光測定プログラム
US20100050737A1 (en) * 2008-09-01 2010-03-04 Andrew Mark Wolters Separation technology method and identification of error
WO2010033383A1 (en) * 2008-09-16 2010-03-25 Beckman Coulter, Inc. Interactive tree plot for flow cytometry data
CA2647953A1 (en) * 2008-12-29 2010-06-29 Sqi Diagnostics Systems Inc. Multiplex analyte detection
DE102009018141A1 (de) * 2009-04-08 2010-10-21 Karl Storz Gmbh & Co. Kg Vorrichtung zur Fluoreszenzdiagnose
FR2956207B1 (fr) * 2010-02-10 2012-05-04 Horiba Abx Sas Dispositif et procede de mesures multiparametriques de microparticules dans un fluide
JP5841315B2 (ja) * 2010-04-28 2016-01-13 ソニー株式会社 微小粒子分析装置

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6201241B1 (en) * 1996-09-17 2001-03-13 Kabushiki Kaisha Topcon Organic substance analyzer
US20020125136A1 (en) * 1998-09-16 2002-09-12 Sharaf Muhammad A. Spectral calibration of fluorescent polynucleotide separation apparatus
US20050046835A1 (en) * 2003-08-27 2005-03-03 Leica Microsystems Heidelberg Gmbh Method for setting a fluorescence spectrum measurement system for microscopy
US20110153226A1 (en) * 2009-12-18 2011-06-23 General Electric Company Spectral searching method for substance identification
US8649008B2 (en) * 2010-02-04 2014-02-11 University Of Southern California Combined spectral and polarimetry imaging and diagnostics

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140158913A1 (en) * 2012-12-06 2014-06-12 Sony Corporation Microparticle analysis apparatus and microparticle analysis method
US20150160133A1 (en) * 2013-12-06 2015-06-11 Azbil Corporation Particle detecting device and particle detecting method
US20170045436A1 (en) * 2015-08-12 2017-02-16 Bio-Rad Laboratories, Inc. Multi-spectral filter profiling and quality control for flow cytometry
US10180385B2 (en) * 2015-08-12 2019-01-15 Bio-Rad Laboratories, Inc. Multi-spectral filter profiling and quality control for flow cytometry
US10429291B2 (en) * 2015-08-12 2019-10-01 Bio-Rad Laboratories, Inc. Multi-spectral filter profiling and quality control for flow cytometry

Also Published As

Publication number Publication date
US20200217784A9 (en) 2020-07-09
JP2012103159A (ja) 2012-05-31
CN102539397B (zh) 2016-04-06
US20180348120A1 (en) 2018-12-06
US20220412881A1 (en) 2022-12-29
US11454588B2 (en) 2022-09-27
US10908075B2 (en) 2021-02-02
US11726031B2 (en) 2023-08-15
US20230349819A1 (en) 2023-11-02
US20210123859A1 (en) 2021-04-29
CN102539397A (zh) 2012-07-04
JP5937780B2 (ja) 2016-06-22

Similar Documents

Publication Publication Date Title
US11726031B2 (en) Fluorescent spectrum correcting method and fluorescent spectrum measuring device
Welsh et al. FCMPASS software aids extracellular vesicle light scatter standardization
US20200150023A1 (en) Instrument and Method for Optical Particle Sensing
Wang et al. Standardization, calibration, and control in flow cytometry
Maciorowski et al. Basic multicolor flow cytometry
US20220082488A1 (en) Methods of forming multi-color fluorescence-based flow cytometry panel
EP2630492B1 (en) Internal focus reference beads for imaging cytometry
JPWO2017126170A1 (ja) 微小粒子測定装置、情報処理装置及び情報処理方法
FR2873813A1 (fr) Procede et dispositif de caracterisation des composants cellulaires d'un liquide biologique
EP3978902B1 (en) Maturity classification of stained reticulocytes using optical microscopy
Pierzchalski et al. Introduction A: Recent advances in cytometry instrumentation, probes, and methods
US20240027457A1 (en) High parameter reagent panel and reagent kit for effective detection of aberrant cells in acute myeloid leukemia
Siddiqui et al. Principles of Advanced Flow Cytometry: a practical guide
Wang et al. Flow cytometer performance characterization, standardization, and control
US20240210397A1 (en) High parameter flow cytometric assay to identify human myeloid derived suppressive cells
EP4083606B1 (en) Information processing device, particle measurement system, and information processing method
US8716026B2 (en) Methods and systems for determining composition and completion of an experiment
US20240027447A1 (en) Methods and aparatus for a mouse surface and intracellular flow cytometry immunophenotyping kit
US20240027448A1 (en) B cell monitoring reagent panel and reagent kit for analyzing b cell subsets in anti-cd20 treated autoimmune patients
US20240337581A1 (en) Methods and aparatus for a twenty-five-color fluorescence-based assay and flow cytometry panel
WO2023171463A1 (ja) 情報処理装置及び情報処理システム
Ortolani Standards, Setup, Calibration, and Control Techniques
Pink et al. Antibody titrations are critical for microflow cytometric analysis of extracellular vesicles
WO2023240165A2 (en) Methods and apparatus for a twenty-five-color fluorescence-based assay and flow cytometry panel
CN105334191A (zh) 单个红细胞的血红蛋白浓度、体积修正方法及装置

Legal Events

Date Code Title Description
AS Assignment

Owner name: SONY CORPORATION, JAPAN

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:KAKUTA, MASAYA;FUTAMURA, KOJI;SAKAI, YOSHITSUGU;AND OTHERS;SIGNING DATES FROM 20110922 TO 20110928;REEL/FRAME:027599/0532

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION