US 10,295,466 B2Granted

Fluorescence intensity correcting method, fluorescence intensity calculating method, and fluorescence intensity calculating apparatus

AssigneeSony Corporation

InventorsYasunobu Kato, Yoshitsugu Sakai

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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.

Claims15

Dependent claims

A microparticle processing method, comprising:

radiating a light in a range from 488 nm to 640 nm, by a laser light source in a cytometer apparatus, to a microparticle;
detecting, by photodetectors in the cytometer apparatus, multicolor measurements of fluorescences excited by the light;
receiving the multicolor measurements of the fluorescences, which are generated from plural fluorescent dyes excited by radiating the light to the microparticle, which is multiply-labeled with said plural fluorescent dyes by the photodetectors which correspond to different received light wavelength bands, respectively;
correcting fluorescence intensities of the multicolor measurements of the fluorescences in the cytometer apparatus by determining intensities of the multicolor measurements of the fluorescences generated from the fluorescent dyes by obtaining a parameter ak (k=1 to m) at which an evaluation function expressed by the following Expression gets a minimum value:
χ
2
≡
∑
i
=
1
N
⁢
[
y
i
-
∑
k
=
1
M
⁢
a
k
⁢
X
k
⁡
(
X
i
)
σ
i
]
2
where Xk(xi) represents a detected value from the i-th photodetector in a single dyeing spectrum of the k-th fluorescent dye, wherein the single-dyeing spectrum is obtained from the microparticle individually labeled with the fluorescent dyes, yi represents a detected value from the i-th photodetector in the measured spectra, and σi represents a reciprocal number of a weight for the measured value from the i-th photodetector;
and
separating a plurality of microparticles into groups based on corrected fluorescence intensities.

37 more elements

A microparticle processing method, comprising:

radiating a light in a range from 488 nm to 640 nm, by a laser light source in a cytometer apparatus, to a microparticle;
detecting, by photodetectors in the cytometer apparatus, multicolor measurements of fluorescences excited by the light;
receiving the multicolor measurements of the fluorescences, which are generated from plural fluorescent dyes excited by radiating the light to the microparticle, which is multiply-labeled with said plural fluorescent dyes by the photodetectors which correspond to different received light wavelength bands, respectively;
correcting fluorescence intensities of the multicolor measurements of the fluorescences in the cytometer apparatus by determining intensities of the fluorescences generated from the fluorescent dyes by obtaining a parameter ak (k=1 to m) at which an evaluation function expressed by the following Expression gets a minimum value:
X
k
′
⁡
(
x
i
)
=
X
k
⁡
(
x
i
)
⁢
(
k
=
1
~
M
k
i
=
1
~
N
1
)
X
k
′
⁡
(
x
i
)
=
0
⁢
⁢
(
k
=
1
~
M
k
i
=
N
1
+
1
~
N
)
χ
2
≡
∑
i
=
1
N
⁢
[
y
i
-
∑
k
=
1
M
⁢
a
k
⁢
X
k
⁡
(
X
i
)
σ
i
]
2
where Xk(xi) represents a detected value from the i-th photodetector in a single-dyeing spectrum of the k-th fluorescent dye, wherein the single-dyeing spectrum is obtained from the microparticle individually labeled with the fluorescent dyes, yi represents a detected value from the i-th photodetector in the measured spectra, σi represents a reciprocal number of a weight for the measured value from the i-th photodetector, an invalid detected value is taken to be yi (i=“N1+1” to N), and a valid detected value is taken to be yi (i=1 to N1);
and
separating a plurality of microparticles into groups based on corrected fluorescence intensities.

96 more elements

A microparticle processing apparatus, comprising:

a laser light source in a cytometer apparatus configured to radiate a light in a range from 488 nm to 640 nm to a microparticle;
photodetectors in the cytometer apparatus, multicolor measurements of configured to detect fluorescences excited by the light;
and
a data processing device that includes circuitry configured to:
receive the multicolor measurements of the fluorescences, which are generated from plural fluorescent dyes excited by the laser light source radiating the light to the microparticle, which is multiply-labeled with said plural fluorescent dyes by the photodetectors which correspond to different received light wavelength bands, respectively;
correct fluorescence intensities of the multicolor measurements of the fluorescences in the cytometer apparatus by determine intensities of the fluorescences generated from the fluorescent dyes by obtaining a parameter ak (k=1 to m) at which an evaluation function expressed by the following Expression gets a minimum value:
χ
2
≡
∑
i
=
1
N
⁢
[
y
i
-
∑
k
=
1
M
⁢
a
k
⁢
X
k
⁡
(
X
i
)
σ
i
]
2
where Xk(xi) represents a detected value from the i-th photodetector in a single dyeing spectrum of the k-th fluorescent dye, wherein the single-dyeing spectrum is obtained from the microparticle individually labeled with the fluorescent dyes, yi represents a detected value from the i-th photodetector in the measured spectra, and σi represents a reciprocal number of a weight for the measured value from the i-th photodetector;
and
separate a plurality of microparticles into groups based on corrected fluorescence intensities.

39 more elements

Dependencies are inferred from the claim text.

Facts

Application: 14/452,085Filed 5 Aug 2014 · continuation
Priority: JP 2010-10456628 Apr 2010
Published: US 2014/0365159 A111 Dec 2014
Granted: 21 May 2019
Term ends: Not estimatedThe source records don’t support an estimate.

Classification4 codes

G01N 21/6428
Fluorescence; Phosphorescence: measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g.…
G01N 2021/6421
Spectrofluorimetric devices: measuring at two or more wavelengths
G01N 2021/6439
Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects,…:…
G01N
Investigating or analysing materials by determining their chemical or physical properties

Family

Application13/089,961Parent of this continuation · filed 19 Apr 2011
US 2014/0365159 A1Pre-grant publication · published 11 Dec 2014
US 8,825,431 B2Parent of this continuation
US 2012/0016616 A1Published application
US 10,295,466 B2This patent

From the US filing history. Foreign family members aren’t listed.