RF-Multiplexed Fluorescence Unmixing for High-Throughput Cell Sorting
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Solution Overview
Problem
Conventional cell sorting methods, such as FACS and MACS, lack sub-cellular resolution and are not suitable for high-throughput applications due to their latency in making sorting decisions.
Innovation Solution
A method and system that utilizes a laser beam with multiple optical frequencies to elicit fluorescence emissions, processes these emissions through Fourier transforms and unmixing matrices to generate phase-corrected images, enabling sorting decisions based on detailed cell characteristics.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional fluorescence-activated cell sorting (FACS) is used to separate cell subpopulations, then cell sorting capability is achieved, but sub-cellular resolution is lost and sorting decisions are based only on average cell parameters
Solution Approach 1:
The patent segments the cell analysis process into multiple stages: acquiring multiple images at different time points, transforming each to frequency space, and performing spectral unmixing on corresponding pixels across images. This segmentation allows extraction of both spatial (sub-cellular) and spectral information, resolving the contradiction between measurement precision and device complexity
Solution Approach 2:
The patent transitions from temporal space to frequency space through Fourier transformation, adding a spectral dimension to the analysis. By performing spectral unmixing in frequency space and then transforming back to temporal space, the system extracts sub-cellular resolution information without requiring complex hardware modifications
2Productivity
If conventional imaging-based cell sorting methods are used, then sub-cellular resolution can be achieved, but high latency in making sorting decisions prevents high throughput application
Solution Approach 1:
The patent performs spectral unmixing and sorting decision analysis while the cell is still in the imaging region, before the cell exits the analysis zone. By pre-calculating sorting decisions based on frequency-space analysis of multiple time-point images, the system eliminates post-processing delays and achieves high throughput without sacrificing sub-cellular resolution
Solution Approach 2:
The patent replaces conventional temporal sequential analysis with parallel frequency-space processing. By transforming images to frequency space and performing spectral unmixing simultaneously across multiple channels, the system reduces processing time and eliminates the latency inherent in sequential temporal analysis
3Measurement precision
If multiple fluorophores are detected simultaneously in conventional methods, then spectral overlap causes mixing that reduces measurement precision
Solution Approach 1:
The patent uses periodic modulation of excitation frequencies to encode different fluorophore signals at distinct temporal frequencies. By detecting fluorescence emissions at multiple excitation frequencies and transforming to frequency space, the system separates overlapping spectral signals through their unique temporal frequency signatures, achieving precise fluorophore detection without complex unmixing algorithms
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
Enables high-resolution, high-throughput cell sorting by accurately determining cell characteristics through spectral unmixing, improving the precision and efficiency of cell separation.
Implementation Method 1
illuminating a cell of a sample comprising a plurality of cells with a laser beam having a plurality of different optical frequencies to elicit fluorescence emissions of a plurality of fluorophores associated with the cell
Data Source
AI summary
Disclosed herein include embodiments of a system, a device, and a method for sorting a plurality cells of a sample. A plurality of raw images comprising pixels of complex values in a frequency space can be generated from a plurality of channels of fluorescence intensity data of fluorescence emissions of fluorophores, the fluorescence emissions being elicited by fluorescence imaging using radiofrequency-multiplexed excitation in a temporal space. Spectral unmixing can be performed on the raw images prior to a sorting decision being made.