Fluorescence Intensity Compensation Using Restricted Least-Square Method
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Solution Overview
Problem
Current fluorescence intensity measurement methods in multicolor analysis face errors due to fluorescence leakage between overlapping wavelength bands, leading to negative intensity calculations and incorrect population representation on cytograms.
Innovation Solution
The method employs a restricted least-square approach to approximate the measurement spectrum as a linear sum of single-stain spectrums, using a lower limit constraint to ensure non-negative fluorescence intensity calculations, thereby correcting measurement errors and accurately representing populations on cytograms.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescence compensation is performed using a compensation matrix to correct measurement errors from wavelength overlap, then measurement precision is improved, but fluorescence intensity may become negative due to noise in detection values
Solution Approach 1:
The patent changes the mathematical parameters of the compensation calculation by introducing inequality constraints (x ≥ 0) on the fluorescence intensity variables. This transforms the ordinary least squares problem into a constrained optimization problem, ensuring that the solution parameters (fluorescence intensities) remain non-negative while still minimizing the measurement error.
Solution Approach 2:
The patent introduces an iterative computational approach where the compensation calculation is performed repeatedly with adjustments. The algorithm dynamically adjusts the fluorescence intensity values through multiple calculation cycles, incorporating constraints at each iteration to ensure non-negativity while converging toward the optimal compensation result.
2Measurement precision
If fluorescence compensation is performed using a compensation matrix, then measurement precision is improved, but population representation on cytograms becomes incorrect due to negative intensity values
Solution Approach 1:
By imposing non-negativity constraints on the fluorescence intensity parameters, the patent ensures that population data plotted on cytograms reflects actual physical reality. This prevents the loss of population information that would occur if negative intensities caused subpopulations to be excluded from display.
3Adaptability or versatility
If multiple fluorochromes with overlapping wavelength bands are used for multicolor measurement, then analysis capability is improved, but fluorescence leakage between wavelength bands causes measurement error
Solution Approach 1:
The patent extracts and separates the contribution of each fluorochrome from the composite fluorescence signal by using spectral unmixing. It takes out the individual fluorochrome spectra as reference components and mathematically separates their contributions from the overlapping measurement signals, enabling accurate quantification of each fluorochrome despite wavelength overlap.
Solution Approach 2:
The patent introduces a mathematical compensation model as an intermediary between the raw fluorescence measurements and the final intensity values. This intermediary calculation process accounts for the leakage between wavelength bands by using reference spectra and solving a system of equations, thereby correcting the measurement errors introduced by spectral overlap.
4Reliability
If noise is present in detection values, then measurement reliability deteriorates, but fluorescence compensation calculations become inaccurate leading to negative intensity values
Solution Approach 1:
The patent applies non-negativity constraints as a form of prior cushioning against the harmful effects of noise. By pre-establishing the physical constraint that fluorescence intensity cannot be negative, the method protects the calculation from producing unrealistic results even when noise is present in the detection values, thereby maintaining both reliability and precision.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach effectively prevents negative fluorescence intensity calculations, ensuring accurate representation of populations on cytograms and reducing measurement errors, thereby improving the reliability of multicolor analysis results.
Implementation Method 1
fluorescence emitted from fluorochromes excited by irradiating light on microparticles multiply-labeled by a plurality of fluorochromes
Implementation Method 2
measuring fluorescence intensities and patterns emitted from these fluorochromes
Data Source
Figure 1
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Figure 3(A)~3(C)
AI summary
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.