Cyclic Flow Cytometry with Optical Identifiers
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Solution Overview
Problem
Current flow cytometry is limited by the number of simultaneously usable fluorescence probes due to spectral overlap, making it challenging to measure multiple markers accurately, especially beyond 30-40 markers, which requires time-consuming and costly panel design and validation processes, and often necessitates the use of more expensive tools like single-cell mass spectrometry or sequencing.
Innovation Solution
The method involves using optical identifiers, such as micro-laser particles, to tag cellular entities, allowing for multiple passes through a flow cytometer with different sets of fluorescent probes, enabling the combination of attributes from each pass to determine a unique identification for each cell, thereby overcoming spectral overlap issues and increasing the number of measurable markers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If the number of fluorescence probes is increased to measure more markers, then measurement precision is improved, but spectral overlap increases making it difficult to distinguish individual probes
Solution Approach 1:
The measurement process is divided into multiple sequential passes, with each pass measuring a subset of markers using a distinct set of fluorescence probes. This segmentation allows each probe set to be measured with minimal spectral overlap, while the unique optical identifiers enable data integration across all passes to achieve comprehensive multi-marker analysis.
2Measurement precision
If spectral compensation is performed to discriminate fluorophores with spectral overlap, then measurement precision is improved, but data quality degrades and interpretation becomes difficult
Solution Approach 1:
Instead of attempting to discriminate all fluorophores simultaneously through spectral compensation, the system segments the measurement into multiple passes where each pass uses a limited set of probes with minimal spectral overlap. This eliminates the need for complex spectral compensation while maintaining data quality and interpretability.
3Adaptability or versatility
If multiple passes through the flow channel are performed to measure different marker sets, then the number of measurable markers increases, but time consumption increases
Solution Approach 1:
Each cellular entity is pre-tagged with stable optical identifiers before the multi-pass measurement process. This preliminary action enables rapid re-identification of cells across passes without time-consuming search or matching procedures, significantly reducing the time penalty associated with multiple measurement passes.
Solution Approach 2:
The system uses highly stable optical identifiers that maintain their signal强度 over multiple passes, effectively accelerating the measurement process by eliminating signal degradation issues that would require repeated cell sampling or re-staining.
4Measurement precision
If panel design and validation are performed to optimize fluorescence probe selection, then measurement precision is improved, but the process becomes time-consuming and costly
Solution Approach 1:
The marker panel is segmented into multiple smaller subsets, each measured in a separate pass with a limited set of fluorescence probes. This reduces the complexity of panel design and validation for each individual pass, while the optical identifier system enables seamless integration of results across passes, achieving comprehensive multi-marker analysis with simplified panel 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 allows for the measurement of a large number of markers per cell with improved accuracy and reduced costs by eliminating the need for complex spectral unmixing and reducing reagent requirements, enabling high-throughput, low-cost analysis of cellular entities.
Implementation Method 1
causing each cellular entity in the population to be tagged with an optical identifier... each cellular entity in the population has been tagged with an optical identifier
Implementation Method 2
Modern flow cytometry uses fluorescent probes to detect specific molecules or molecular complexes... a laser beam that excites the fluorophores present on each cell. The fluorescence emission from each cell is collected and analyzed
Data Source
AI summary
Method of and apparatus for performing cyclic flow cytometry analysis on a sample population of cellular entities including: causing each cellular entity to be labeled with an optical identifier; for each cellular entity, performing a first pass of flow cytometry measurement over a flow channel with respect to a first set of parameters, under conditions of determining an identification for the cellular entity for which values of the first set of parameters are being obtained, and storing the values of the first set in association with the identification; and performing a second pass of flow cytometry measurement over the flow channel with respect to a second set of parameters, under conditions of separately determining an identification for the cellular entity for which values of the second set of parameters are being obtained, and storing the values of the second set in association with the identification.