Spectral Unmixing for Overlapping Fluorophore Sorting
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Solution Overview
Problem
Flow-type particle sorting systems struggle to accurately sort particles based on their fluorescence characteristics due to overlapping fluorescence spectra of fluorophores, which complicates the identification and separation of particles.
Innovation Solution
The method involves detecting light from a sample using a light detection system, calculating a spectral unmixing matrix to resolve light from each fluorophore, and using this information to estimate the abundance of fluorophores and sort particles accordingly. This is achieved through algorithms such as weighted least squares, Newton-Raphson iteration, and Sherman-Morrison iterative inverse updater.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple fluorophores with overlapping fluorescence spectra are used to label different particles, then the multiplicity of particles or components that can be simultaneously detected increases, but the measurement precision of individual fluorophore signals deteriorates due to spectral overlap
Solution Approach 1:
The patent segments the overlapping fluorescence spectrum into distinct contributions from individual fluorophores by dividing the spectral range into multiple wavelength channels. This allows the total fluorescence signal at each wavelength to be decomposed into separate fluorophore components through mathematical modeling, enabling precise measurement of multiple fluorophores simultaneously despite spectral overlap.
Solution Approach 2:
The patent introduces a spectral unmixing algorithm as an intermediary between the detected total fluorescence signal and the individual fluorophore abundances. This computational mediator processes the mixed spectral signals and calculates the contribution of each fluorophore based on reference spectra, thereby resolving the measurement precision issue caused by spectral overlap.
2Measurement precision
If spectral unmixing algorithms are applied to resolve overlapping fluorescence spectra, then the measurement precision of individual fluorophores improves, but the device complexity increases due to computational requirements
Solution Approach 1:
The patent performs preliminary action by pre-calculating and storing reference fluorescence spectra for each fluorophore under different excitation wavelengths. These reference spectra are obtained in advance and stored in a lookup table, eliminating the need for complex real-time calculations during actual measurements. The spectral unmixing process then becomes a straightforward comparison and interpolation task.
Solution Approach 2:
The patent replaces complex real-time computational algorithms with a simplified lookup table approach. Instead of performing intensive spectral deconvolution calculations during data acquisition, the system uses pre-computed reference spectra and basic mathematical operations to determine fluorophore abundances, significantly reducing computational complexity while maintaining measurement precision.
3Productivity
If real-time spectral unmixing is performed for each particle, then the sorting speed increases, but the computational resources required increase
Solution Approach 1:
The patent applies preliminary action by pre-computing reference fluorescence spectra and storing them in a lookup table before actual sorting operations. During real-time sorting, the system only needs to perform simple spectral matching and interpolation using these pre-stored references, dramatically reducing the computational energy required per particle while maintaining high sorting speeds.
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 enables precise identification and sorting of particles by effectively resolving overlapping fluorescence spectra, allowing for real-time sorting of particles based on their fluorophore abundance.
Implementation Method 1
the light scattering and fluorescence properties of the particles are measured
Implementation Method 2
Particles or components thereof can be labeled with fluorescent dyes to facilitate detection
Implementation Method 3
Droplets are passed through an electrostatic field and are deflected based on polarity and magnitude of charge on the droplet
Data Source
AI summary
Aspects of the present disclosure include methods for spectrally resolving light from fluorophores having overlapping fluorescence spectra in a sample. Methods according to certain embodiments include detecting light with a light detection system from a sample having a plurality of fluorophores having overlapping fluorescence spectra and spectrally resolving light from each fluorophore in the sample. In some embodiments, methods include estimating the abundance of one or more of the fluorophores in the sample, such as on a particle. In certain instances, methods include identifying the particle in the sample based on the abundance of each fluorophore and sorting the particle. Methods according to some embodiments includes spectrally resolving the light from each fluorophore by calculating a spectral unmixing matrix for the fluorescence spectra of each fluorophore. Systems and integrated circuit devices (e.g., a field programmable gate array) for practicing the subject methods are also provided.