Fluorescence intensity compensation method and fluorescence intensity calculation device

摘要

A fluorescence intensity compensation method, includes: receiving, with photodetectors having different input wavelength bands, fluorescence emitted from fluorochromes excited by irradiating light on microparticles multiply-labeled by a plurality of fluorochromes with overlapping fluorescence wavelengths; collecting detected values for the photodetectors; and obtaining a measurement spectrum, by approximating, from the linear sum of single-stain spectrums obtained from microparticles individually labeled with the fluorochromes; wherein approximation of the measurement spectrum by the linear sum of the single-stain spectrums is performed using the restricted least-square method.

主权项

1. A fluorescence intensity compensation method, comprising:
receiving, with photodetectors having different input wavelength bands, fluorescence emitted from fluorochromes excited by irradiating light on microparticles multiply-labeled by a plurality of fluorochromes with overlapping fluorescence wavelengths; collecting detected values for the photodetectors; and obtaining a measurement spectrum, by approximating, from a linear sum of single-stain spectrums obtained from microparticles individually labeled with the fluorochromes; wherein approximation of said measurement spectrum by the linear sum of said single-stain spectrums is performed using a restricted least-square method; wherein the intensity of fluorescence emitted from each fluorochrome is calculated by obtaining a parameter xj (j=1 through M) where an evaluation function shown in the following Expression (1) satisfies the following Expression (2) and also is a smallest valueχ2=∑i=1N⁢(pi-∑j=1M⁢sij·xjσi)2(1)xj≥Uj(2)where Sij represents a detected value of the i'th photodetector in the single-stain spectrum of the j'th fluorochrome, pi represents a detected value of the i'th photodetector in the measurement spectrum, σi represents an inverse number of a weight as to a detected value of the i'th photodetector, and Uj represents a lower limit value of the fluorescence intensity of each fluorochrome to be calculated;
wherein said parameter xj (j=1 through M) is obtained by solving the quadratic programming problem in the following Expressions (3) through (5)Minimize⁢⁢Sx-p2(3)subject⁢⁢to⁢⁢Ax≤b⁢(4)x≥0(5)A=(-10…00-10⋮⋱⋮00…-1)(6)b=(-U1-U2⋮-UM)(7)where S represents an N×M order matrix with sij as elements, x represents an M order matrix with xj as elements, p represents an N'th order vector with pi as elements, A is set as an M×M order matrix and b as an M×1 order matrix, and U1 through UM represent said lower limit value;
wherein in said Expression (2), an undyed average value Vj of the j'th fluorochrome obtained from an average value of detection values from each of said photodetectors, obtained by irradiating light on microparticles not marked with the j'th fluorochrome, is used as said lower limit value Uj.