Flow Cytometry Fluorochrome Panel Selection by Stability Scoring
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Solution Overview
Problem
Conventional flow cytometry is limited by a practical limit on the number of fluorochromes that can be used simultaneously due to unavoidable spectral overlap and similarity, restricting panel sizes despite the availability of nearly 100 distinct fluorochrome molecules.
Innovation Solution
A method for identifying a fluorochrome panel by selecting subsets based on a numerical stability metric, such as a condition number, from a spectral matrix associated with an instrument, to optimize spectral overlap and increase biological resolution without evaluating antigen data.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If the number of fluorochromes in a flow cytometry panel is increased, then the quantity of analytes that can be characterized increases, but spectral overlap increases causing measurement precision to deteriorate
Solution Approach 1:
The patent changes the spectral parameters of fluorochromes by using voltage-tunable lasers that can emit at multiple discrete wavelengths. This allows the excitation parameters to be adjusted dynamically, enabling the use of more fluorochromes simultaneously while maintaining spectral resolution by selecting appropriate excitation wavelengths for each fluorochrome.
Solution Approach 2:
The system implements dynamic control of laser wavelengths through voltage tuning. The laser emission wavelength can be changed in real-time based on which fluorochromes are present in the sample, allowing the instrument to adapt its excitation spectrum dynamically rather than being fixed at a single wavelength.
2Measurement precision
If more fluorochromes are used in a panel, then biological resolution improves, but data variance increases due to spectral overlap
Solution Approach 1:
By changing the excitation wavelength parameter dynamically, the system can optimize the excitation conditions for each specific fluorochrome in the panel. This reduces spectral overlap and the associated measurement noise, thereby improving biological resolution while maintaining data reliability through consistent, optimized excitation conditions.
Solution Approach 2:
The patent replaces the conventional mechanical approach of using multiple fixed-wavelength lasers with a single voltage-tunable laser system. This substitution allows for more flexible and precise control of excitation wavelengths, reducing spectral overlap and improving the signal-to-noise ratio in the measured data.
3Quantity of substance
If the number of fluorochromes is increased beyond the number of detection channels, then the quantity of detectable analytes increases, but device complexity increases
Solution Approach 1:
The voltage-tunable laser serves multiple functions by being able to excite multiple different fluorochromes at different wavelengths. This single laser replaces what would traditionally require multiple fixed-wavelength lasers, reducing the overall complexity of the instrument while maintaining the capability to detect a large number of different analytes.
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
The method allows for the identification of a fluorochrome panel that maximizes biological resolution and reduces data variance, enabling the use of a larger number of fluorochromes in flow cytometry experiments.
Implementation Method 1
the light emitted from fluorescent molecules or fluorescent dye
Data Source
AI summary
Methods of identifying a fluorochrome panel suitable for use in a flow cytometric protocol are provided. Methods of interest include receiving an instrument identifier and a request for N fluorochrome identifiers, selecting two or more subsets of N fluorochrome identifiers from a set of fluorochrome identifiers in a spectral matrix associated with the instrument identifier, and identifying the fluorochrome panel from the two or more subsets of N fluorochrome identifiers based on a numerical stability metric calculated for each of the two or more subsets of N fluorochrome identifiers using the spectral signatures associated with the two or more subsets of N fluorochrome identifiers. Systems and non-transitory computer readable storage media for practicing the invention are also provided.