Fluorescence Intensity Compensation Using Non-Negative Constraints
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Solution Overview
Problem
Current fluorescence intensity compensation methods in multi-color measurements can result in errors due to negative intensity values and variations in autofluorescence among subpopulations, leading to inaccurate plotting in two-dimensional correlation diagrams.
Innovation Solution
A method that computes fluorescence intensities by approximating the measured spectrum with a linear sum of simple staining and autofluorescence spectra, using a least-squares method and imposing constraints to ensure non-negative intensity values, thereby preventing measurement errors and accurately representing population sizes in cytograms.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescence compensation is performed using a compensation matrix with negative values allowed, then the mathematical compensation can be applied, but the computed fluorescence intensity may become negative which is physically impossible and causes measurement errors
Solution Approach 1:
The patent changes the parameter space from allowing negative values to constraining values to be non-negative. By introducing non-negativity constraints on the fluorescence intensity parameters during the compensation calculation, the method ensures physically valid results while maintaining measurement precision through constrained optimization techniques.
2Ease of manufacture
If the same average autofluorescence value is subtracted from all populations, then the compensation process is simplified, but measurement errors increase due to variations in autofluorescence among subpopulations
Solution Approach 1:
The patent segments the population into distinct subpopulations and calculates separate average autofluorescence values for each subpopulation. This segmentation allows the method to account for autofluorescence variations among different cell types or states, thereby improving measurement precision while maintaining computational feasibility through structured processing.
Solution Approach 2:
The patent applies local quality by calculating and using subpopulation-specific autofluorescence values rather than a single global average. Each subpopulation receives customized compensation based on its specific autofluorescence characteristics, improving measurement accuracy for heterogeneous samples while keeping the overall process manageable through systematic organization.
3Productivity
If negative fluorescence intensity values are produced, then the compensation calculation is straightforward, but the population size in two-dimensional correlation diagrams is misrepresented
Solution Approach 1:
The patent changes the parameter domain from negative to non-negative values for fluorescence intensities. By implementing non-negativity constraints during compensation calculation, the method ensures that population sizes represented in two-dimensional correlation diagrams remain accurate and physically meaningful, preventing information loss while maintaining efficient computation through constrained optimization.
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 ensures accurate computation of fluorescence intensities for each fluorochrome and autofluorescence, preventing errors caused by negative values and autofluorescence variations, and providing a more accurate representation of microparticle populations in multi-color measurements.
Implementation Method 1
emits a laser beam onto the fluorochrome to excite the fluorochrome, and measures the intensity or pattern of the fluorescence emitted from the excited fluorochrome
Implementation Method 2
receiving fluorescence generated by the excited fluorochromes using photodetectors
Data Source
AI summary
A fluorescence intensity compensation method includes emitting light onto microparticles multiply labeled with a plurality of fluorochromes having fluorescence wavelength ranges that overlap each other in order to excite the fluorochromes and receiving fluorescence generated by the excited fluorochromes using photodetectors having different reception wavelength ranges, and computing fluorescence intensities of the fluorochromes by compensating detection values of the photodetectors under a predetermined constraint condition imposed on the computed fluorescence intensities.